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African Journal of<br />
Biotechnology<br />
Volume10 Number 20 16 May, 2011<br />
ISSN 1684-5315
ABOUT AJB<br />
The African Journal of Biotechnology (AJB) is published bi-weekly (one volume per year) by <strong>Academic</strong><br />
<strong>Journals</strong>.<br />
African Journal of Biotechnology (AJB) a new broad-based journal, is an open access journal that was founded<br />
on two key tenets: To publish the most exciting research in all areas of applied biochemistry, industrial<br />
microbiology, molecular biology, genomics and proteomics, food and agricultural technologies, and metabolic<br />
engineering. Secondly, to provide the most rapid turn-around time possible for reviewing and publishing, and<br />
to disseminate the <strong>articles</strong> freely for teaching and reference purposes. All <strong>articles</strong> published in AJB are peerreviewed.<br />
Submission of Manuscript<br />
Submit manuscripts as e-mail attachment to the Editorial Office at: ajb@acadjournals.org. A manuscript<br />
number will be mailed to the corresponding author shortly after submission.<br />
The African Journal of Biotechnology will only accept manuscripts submitted as e-mail attachments.<br />
Please read the Instructions for Authors before submitting your manuscript. The manuscript files should be<br />
given the last name of the first author.
Editors<br />
George Nkem Ude, Ph.D<br />
Plant Breeder & Molecular Biologist<br />
Department of Natural Sciences<br />
Crawford Building, Rm 003A<br />
Bowie State University<br />
14000 Jericho Park Road<br />
Bowie, MD 20715, USA<br />
N. John Tonukari, Ph.D<br />
Department of Biochemistry<br />
Delta State University<br />
PMB 1<br />
Abraka, Nigeria<br />
Prof. Dr. AE Aboulata<br />
Plant Path. Res. Inst., ARC, POBox 12619, Giza,<br />
Egypt<br />
30 D, El-Karama St., Alf Maskan, P.O. Box 1567,<br />
Ain Shams, Cairo,<br />
Egypt<br />
Dr. S.K Das<br />
Department of Applied Chemistry<br />
and Biotechnology, University of Fukui,<br />
Japan<br />
Prof. Okoh, A. I<br />
Applied and Environmental Microbiology Research<br />
Group (AEMREG),<br />
Department of Biochemistry and Microbiology,<br />
University of Fort Hare.<br />
P/Bag X1314 Alice 5700,<br />
South Africa<br />
Dr. Ismail TURKOGLU<br />
Department of Biology Education,<br />
Education Faculty, Fırat University,<br />
Elazığ,<br />
Turkey<br />
Prof T.K.Raja, PhD FRSC (UK)<br />
Department of Biotechnology<br />
PSG COLLEGE OF TECHNOLOGY (Autonomous)<br />
(Affiliated to Anna University)<br />
Coimbatore-641004, Tamilnadu,<br />
INDIA.<br />
Dr. George Edward Mamati<br />
Horticulture Department,<br />
Jomo Kenyatta University of Agriculture<br />
and Technology,<br />
P. O. Box 62000-00200,<br />
Nairobi, Kenya.<br />
Dr Helal Ragab Moussa<br />
Bahnay, Al-bagour, Menoufia,<br />
Egypt.<br />
Dr VIPUL GOHEL<br />
Flat No. 403, Alankar Apartment, Sector 56, Gurgaon-<br />
122 002,<br />
India.<br />
Dr. Sang-Han Lee<br />
Department of Food Science & Biotechnology,<br />
Kyungpook National University<br />
Daegu 702-701,<br />
Korea.<br />
Dr. Bhaskar Dutta<br />
DoD Biotechnology High Performance Computing<br />
Software Applications<br />
Institute (BHSAI)<br />
U.S. Army Medical Research and Materiel Command<br />
2405 Whittier Drive<br />
Frederick, MD 21702<br />
Dr. Muhammad Akram<br />
Faculty of Eastern Medicine and Surgery,<br />
Hamdard Al-Majeed College of Eastern Medicine,<br />
Hamdard University,<br />
Karachi.<br />
Dr. M.MURUGANANDAM<br />
Departtment of Biotechnology<br />
St. Michael College of Engineering & Technology,<br />
Kalayarkoil,<br />
India.<br />
Dr. Gökhan Aydin<br />
Suleyman Demirel University,<br />
Atabey Vocational School,<br />
Isparta-Türkiye,<br />
Dr. Rajib Roychowdhury<br />
Centre for Biotechnology (CBT),<br />
Visva Bharati,<br />
West-Bengal,<br />
India.<br />
Dr.YU JUNG KIM<br />
Department of Chemistry and Biochemistry<br />
California State University, San Bernardino<br />
5500 University Parkway<br />
San Bernardino, CA 92407
Editorial Board<br />
Dr. Takuji Ohyama<br />
Faculty of Agriculture, Niigata University<br />
Dr. Mehdi Vasfi Marandi<br />
University of Tehran<br />
Dr. FÜgen DURLU-ÖZKAYA<br />
Gazi Üniversity, Tourism Faculty, Dept. of Gastronomy<br />
and Culinary Art<br />
Dr. Reza Yari<br />
Islamic Azad University, Boroujerd Branch<br />
Dr. Zahra Tahmasebi Fard<br />
Roudehen branche, Islamic Azad University<br />
Dr. Tarnawski Sonia<br />
University of Neuchâtel – Laboratory of Microbiology<br />
Dr. Albert Magrí<br />
Giro Technological Centre<br />
Dr. Ping ZHENG<br />
Zhejiang University, Hangzhou,<br />
China.<br />
Prof. Pilar Morata<br />
University of Malaga<br />
Dr. Greg Spear<br />
Rush University Medical Center<br />
Dr. Mousavi Khaneghah<br />
College of Applied Science and<br />
Technology-Applied Food Science, Tehran,<br />
Iran.<br />
Prof. Pavel KALAC<br />
University of South Bohemia,<br />
Czech Republic.<br />
Dr. Kürsat KORKMAZ<br />
Ordu University, Faculty of Agriculture,<br />
Department of Soil Science and Plant nutrition<br />
Dr. Tugay AYAŞAN<br />
Çukurova Agricultural Research Institute, PK:01321,<br />
ADANA-TURKEY.<br />
Dr. Shuyang Yu<br />
Asistant research scientist, Department of Microbiology,<br />
University of Iowa<br />
Address: 51 newton road, 3-730B BSB bldg.Tel:+319-335-<br />
7982, Iowa City, IA, 52246,<br />
USA.<br />
Dr. Binxing Li<br />
E-mail: Binxing.Li@hsc.utah.edu<br />
Dr Hsiu-Chi Cheng<br />
National Cheng Kung University and Hospital.<br />
Dr. Kgomotso P. Sibeko<br />
University of Pretoria,<br />
South Africa.<br />
Dr. Jian Wu<br />
Harbin medical university ,<br />
China.
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Pseudomonad. Biochem. J. 174:<br />
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Alexander M (1965). Biodegradation: Problems of<br />
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and Microbial Fallibility. Adv. Appl. Microbiol. 7: 35-80.<br />
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.<br />
African Journal of Biotechnology<br />
International Table of Contents: Journal Volume of Medicine 10 Number and Medical 20 16 May, Sciences 2011<br />
ences<br />
Research Articles<br />
GENETICS AND MOLECULAR BIOLOGY<br />
ARTICLES<br />
Highly heterogeneous Ty3/Gypsy-like retrotransposon sequences in the<br />
genome of cassava (Manihot esculenta Crantz) 3951<br />
Michael A. Gbadegesin and John R. Beeching<br />
Genetics of trichomes and its association with fibre and agronomic traits<br />
in cotton 3964<br />
Nausherwan Nobel Nawab, Qamar Shakil, Shahid Niaz, Muhammad<br />
Munir Iqbal, Muhammad Ahsan Asif and Iftikhar Ahmad Khan<br />
Quick and sensitive determination of gene expression of fatty acid synthase<br />
in vitro by using real-time polymerase chain reaction amplification (PCR) 3973<br />
Zuo-Hua Liu, Jin-Long Yang, Fei-Yun Yang, Ding-Biao Long, Ren-Yong Jia,<br />
Kang-Cheng Pan, and Dai-Wen Chen<br />
Characterization of a chestnut FLORICAULA/LEAFY homologous gene 3978<br />
Tao Liu, Yun-qian Hu and Xiao-xian Li<br />
Overexpression of a foxtail millet Acetyl-CoA carboxylase gene in maize<br />
increases sethoxydim resistance and oil content 3986<br />
Zhigang Dong, Huji Zhao,Junguang He, Junling Huai, Heng Lin, Jun Zheng,<br />
Yunjun Liu and Guoying Wang
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
ences<br />
ARTICLES<br />
Variation in nodulation and growth of groundnut (Arachis hypogaea L.)<br />
on oxisols from land use systems of the humid forest zone in southern<br />
Cameroon 3996<br />
Laurette Ngo Nkot, Dieudonné Nwaga, Albert Ngakou, Henri Fankem and<br />
François-Xavier Etoa<br />
Comparison of some anthropometric and biologic parameters in two<br />
groups of Tunisian infants (0 to 2 years) 4005<br />
M. Kamel, A. Barkia, M. Hamdaoui, H. Ketata, M. Kassis, M. Nasri and<br />
A. Aouidet<br />
Isolation and molecular characterization of RcSERK1: A Rosa caninagene<br />
transcriptionally induced during initiation of protocorm-like bodies 4011<br />
Xu Kedong, Liu Qinglin, Yang Huifang, Zeng Li, Dong Lili, Liu Fengluan,<br />
Bi Ling, Ma Nan and Zhao Liangjun<br />
Molecular cloning and expression of the luciferase coding genes of<br />
Vibrio fischeri 4018<br />
Golnaz Asaadi Tehrani, Sina Mirzaahmadi, Mojgan Bandehpour, Faramarz Laloei,<br />
Akram Eidi, Toraj Valinasab and Bahram Kazemi<br />
Study of polymorphism of leptin gene receptor in Mazandaran fowls 4024<br />
H. A. Abbasi, S. Gharahveysi and R. Abdullahpour<br />
Detection of genomic instability in hypospadias patients by random<br />
amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method 4029<br />
Adnan A. Alsulaimani, Nabil S. Awad and Adel E. El-Tarras<br />
cDNA, genomic sequence cloning and overexpression of cytochrome c<br />
oxidase gene (COX6b1) from the Ailuropoda melanoleuca 4033<br />
Jun Li, Yiling Hou, Wanru Hou, Bing Sun and Xiulan Su
ences<br />
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
PLANT AND AGRICULTURAL TECHNOLOGY<br />
ARTICLES<br />
Effect of plant growth regulators (PGRs) on micropropagation of a<br />
vulnerable and high value medicinal plant Hedychium spicatum 4040<br />
Dinesh Giri and Sushma Tamta<br />
Genotype-dependent responses of wheat (Triticum aestivum L.) seedlings<br />
to drought, UV-B radiation and their combined stresses 4046<br />
Liheng He, Xiaoyun Jia, Zhiqiang Gao and Runzhi Li<br />
The effects of the regulated deficit irrigation on yield and some yield<br />
components of common bean (Phaseolus vulgaris L.) under semi-arid<br />
conditions 4057<br />
Mehmet Simsek, Nuray Comlekcioglu and Irfan Ozturk<br />
Molecular cloning of a novel GSK3/shaggy-like gene from Triticum<br />
monococcum L. and its expression in response to salt, drought and other<br />
abiotic stresses 4065<br />
Yang Xian-Guang, Fan Jin-Yu and Deng Chuan-Liang<br />
Some quality traits and neurotoxin β-N-oxalyl-L-α,β-diaminopropionic acid<br />
(β-ODAP) contents of Lathyrus sp. cultivated in Turkey 4072<br />
Ugur Basaran, Ozlem Onal Asci, Hanife Mut, Zeki Acar, Ilknur Ayan<br />
Optimization of a plant regeneration protocol for broccoli 4081<br />
Ke. Huang, Qiuyun Wu, Juncheng Lin and Jingui Zheng<br />
Evaluation of bread wheat genotypes for salinity tolerance under saline<br />
field conditions 4086<br />
Munir Ahmad, Muhammad Munir, Armghan Shahzad, M. Shahid Masood<br />
and Muhammad Iqbal
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
ences<br />
ARTICLES<br />
Antioxidant enzymes activities during secondary somatic embryogenesis in<br />
Persian walnut (Juglans regia L.) 4093<br />
M. Jariteh, H. Ebrahimzadeh, V. Niknam, K. Vahdati and R. Amiri<br />
Sugar beet genotype effect on potential of bioethanol production using<br />
Saccharomyces cerevisiae fermentation 4100<br />
Parviz Mehdikhani, Hrachya Hovsepyan and Mahmood Rezazadeh Bari<br />
Modeling and experiment to threshing unit of stripper combine 4106<br />
Xu Lizhang and Li Yaoming<br />
ENVIRONMENTAL BIOTECHNOLOGY<br />
Yield responses of forage sorghums to salinity and irrigation frequency 4114<br />
Siti Aishah, H. A. R. Saberi, R. A. Halim and A. R. Zaharah<br />
Isolation and characterization of a novel sulfur-oxidizing<br />
chemolithoautotroph Halothiobacillus from Pb polluted paddy soil 4121<br />
Jiyan Shi, Huirong Lin, Xiaofeng Yuan and Yidong Zhao<br />
Biomonitoring of some heavy metal contaminations from a steel plant by<br />
above ground plants tissue 4127<br />
M. Ataabadi, M. Hoodaji and P. Najafi<br />
Isolation, purification and identification of bacteria from the shoes worn<br />
by children 4133<br />
Hui Li, Changqing Zhao, Jin Zhou, Huanhuan Shao and Wuyong Chen
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
ences<br />
ARTICLES<br />
Analyzing the two-dimensional plot of the interannual climate variability<br />
for detection of the climate change in the Large Karoun River Basin, Iran 4138<br />
Narges Zohrabi, Ali Reza Massah Bavani, Hossein Sedghi and Abdol RasolTelvari<br />
INDUSTRIAL MICROBIOLOGY<br />
Screening and optimization of extracellular lipases by Acinetobacter species<br />
isolated from oil oil-contaminated soil in South Korea 4147<br />
Periasamy Anbu, Myoung-Ju Noh, Da-Hye Kim, Jun-Seok Seo, Byung-Ki Hur<br />
and Kyeong Ho Min<br />
Antifungal activity of selected plant leaves crude extracts against a pepper<br />
anthracnose fungus, Colletotrichum capsici (Sydow) butler and bisby<br />
(Ascomycota: Phyllachorales) 4157<br />
Lucy Johnny, Umi Kalsom Yusuf and R. Nulit<br />
Isolation, identification and application in lignin degradation of an<br />
ascomycete GHJ-4 4166<br />
Huiju Gao, Yanwen Wang, Wenting Zhang, Weile Wang and Zhimei Mu<br />
Improved taxol production in Nodulisporium sylviforme derived from<br />
inactivated protoplast fusion 4175<br />
Kai Zhao, Lixin Sun, Xi Ma, Xiuliang Li, Xin Wang, Wenxiang Ping, Dongpo Zhou<br />
Production of ethanol from mango (Mangifera indica L.) peel by<br />
Saccharomyces cerevisiae CFTRI101 4183<br />
Lebaka Veeranjaneya Reddy, Obulam Vijaya Sarathi Reddy and<br />
Young-Jung Wee
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
ences<br />
ARTICLES<br />
Secretory expression of Rhizopus oryzae α-amylase in Kluyveromyces lactis 4190<br />
Song Li, Wei Shen, Xianzhong Chen, Guiyang Shi and Zhengxiang Wang<br />
FOOD TECHNOLOGY<br />
Volatile compounds of maari, a fermented product from baobab (Adansonia<br />
digitata L.) seeds 4197<br />
Charles Parkouda, Brehima Diawara, Samuel Lowor, Charles Diako, Firibu Kwesi<br />
Saalia, Nana T Annan, Jan S. Jensen, Kwaku Tano-Debrah and Mogens Jakobsen<br />
Antioxidant activities of Rosmarinus officinalis L. essential oil obtained by<br />
hydro-distillation and solvent free microwave extraction 4207<br />
Okoh O. O., Sadimenko A. P. and Afolayan A. J.<br />
APPLIED BIOCHEMISTRY<br />
Effects of bagging on sugar metabolism and the activity of sugar metabolism<br />
related enzymes during fruit development of Qingzhong loquat 4212<br />
Zhaojun Ni, Zhen Zhang, Zhihong Gao, Linping Gu and Leifang Huang<br />
Anti-inflammatory evaluation of immature fruit and seed aqueous extracts<br />
from several populations of Tunisian Citrullus colocynthisSchrad<br />
Belsem Marzouk, Zohra Marzouk, Ehsen Haloui, Manel Turki,<br />
Abderrahman Bouraoui, Mahjoub Aouni and Nadia Fenina<br />
MEDICAL AND PHARMACEUTICAL BIOTECHNOLOGY<br />
Effects of artemether on the plasma and urine concentrations of some<br />
electrolytes in rats 4226<br />
R.O. Akomolafe, I.O. Adeoshun, J.B. Fakunle, E.O. Iwalewa, A.O. Ayoka,<br />
O.E. Ajayi, O.M. Odeleye and B.O. Akanji
ences<br />
ARTICLES<br />
Expression of biological active VHH camelid single domain antibody in<br />
transgenic tobacco 4234<br />
Behnaz Korouzhdehy, Mehdi Dadmehr ,Issa Piri, Fatemeh Rahbarizadeh and<br />
Mahmood Solouki<br />
Aloe arborescens aqueous gel extract alters the activities of key hepatic<br />
enzymes and blood concentration of triglycerides, glucose and insulin in<br />
alloxan-induced diabetic rats 4242<br />
Motetelo Alfred Mogale, Sogolo Lucky Lebelo, Leshweni Jeremia Shai and<br />
Jacobus Nicholaas Eloff<br />
Increased vascular endothelial growth factor (VEGF) expression in rats with<br />
spinal cord injury by transplantation of bone marrow stromal cells 4249<br />
Deshui Yu, Libo Liu, Xiaodong Zhi, Yang Cao and Gang lv<br />
In-ovo evaluation of the antiviral activity of methanolic root-bark extract<br />
of the African Baobab (Adansonia digitata Lin) 4256<br />
Sulaiman, Lanre K., Oladele, Omolade A., Shittu, Ismaila A., Emikpe,<br />
Benjamin O., Oladokun, Agnes T. and Meseko, Clement A<br />
ENTOMOLOGY<br />
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
Effect of gamma radiation on different stages of Indian meal moth Plodia<br />
interpunctella Hübner (Lepidoptera: Pyralidae) 4259<br />
Hosseinzadeh Abbas, Shayesteh Nouraddin, Zolfagharieh Hamid Reza,<br />
Bernousi Iraj, Babaei Mohammad, Zareshahi Hasan, Ahari Mostafavi Hossein,<br />
Fatollahi Hadi<br />
FISHERY SCIENCE<br />
Body composition of freshwater Wallago attu in relation to body size,<br />
condition factor and sex from southern Punjab, Pakistan 4265<br />
Muhammad Yousaf, Abdus Salam and Muhammad Naeem
Table of Contents: Volume 10 Number 20 16 May, 2011<br />
ences<br />
BIOTECHNIQUES<br />
ARTICLES<br />
Rhizopus stolonifer exhibits dimorphism 4269<br />
C. O. Omoifo<br />
Effect of extracellular calcium chloride on sporangiospore-yeast<br />
transformation of Rhizopus stolonifer 4276<br />
C. O. Omoifo<br />
ANIMAL SCIENCE<br />
Genetic variation and bottleneck in Japanese quail (Coturnix japonica)<br />
strains using twelve microsatellite markers 4289<br />
Hossein Emrani, Cyrus Amirinia and Mohammad Ali Radjaee Arbabe<br />
Evaluation of the effects of bovine herpesvirus-1 VP22 gene adjuvant in<br />
the inhibin DNA vaccine for improving follicular development and litter size<br />
in mice 4296<br />
Han Li, Zhen YanHong, Sang Lei, Bai LiYa, Cai KaiLai, Guo AiZhen and Yang LiGuo<br />
Productive performance of laying hens fed wheat-based diets included olive<br />
pulp with or without a commercial enzyme product 4303<br />
M. Zarei, M. Ehsani and M. Torki<br />
ENVIRONMENTAL RADIOACTIVITY<br />
An investigation on natural radioactivity from mining industry # 4313<br />
E. Esmeray and M.E. Aydin
African Journal of Biotechnology Vol. 10(20), pp. 3951-3963, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1315<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Highly heterogeneous Ty3/Gypsy-like retrotransposon<br />
sequences in the genome of cassava (Manihot<br />
esculenta Crantz)<br />
Michael A. Gbadegesin 1 * and John R. Beeching 2<br />
1 Department of Biochemistry, University of Ibadan, Ibadan 200005, Nigeria.<br />
2 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U. K.<br />
Accepted 2 March, 2011<br />
The use of PCR has enabled the survey of transposable elements in many plants; thereby making the<br />
study of their diversity and applications possible in species where the full genome sequence data are<br />
not yet available. In the present study, we used PCR primers anchored on the conserved domain of<br />
reverse transcriptase and endonuclease to amplify the Ty3/Gypsy-like polyprotein fragment from the<br />
genome of cassava (Manihot esculenta Crantz). The PCR product was cloned and sequenced.<br />
Sequence analysis of individual clones clearly identified the conserved domain of the polyprotein<br />
enzymes and showed the cassava Ty3/Gypsy-like retrotransposon, Megyp (for Manihot esculenta<br />
gypsy-like), sequences to be highly heterogeneous. Some Megyps clustered with other plants’<br />
Ty3/Gypsy-like retrotransposons, while some clustered with Gypsy of Drosophila melanogaster and<br />
Ty3-2 of Saccharomyces cerevisiae in the comparative multiple sequence analysis. This suggests that<br />
the later belong to the retrovirus lineage of this group of elements. Southern analysis showed that, the<br />
Megyps and analogues were highly repeated within the genomes of cassava cultivars.<br />
Key words: Cassava, transposable-elements, retrotransposons, retroviruses, Manihot esculenta, Ty3/Gypsy.<br />
INTRODUCTION<br />
There are two major super-families of transposable<br />
elements (TEs) based on their transposition intermediate<br />
and transposition mechanisms (Finnegan, 1992). DNA<br />
TEs (Class II elements) move by excision and reintegration<br />
via a DNA intermediate. They transpose by a ‘cut<br />
and paste’ mechanism mediated by a transposase that<br />
recognises their short terminal inverted repeated<br />
sequences (TIRs). On the other hand, retrotransposons<br />
or retro-elements (Class I elements) move and amplify<br />
through RNA intermediates, which are reverse transcribed<br />
before their integration into the nuclear genome.<br />
They have been divided into two principal groups, the<br />
long terminal repeat (LTR) retrotransposons and the non-<br />
LTR retrotransposons.<br />
Non-LTR retrotransposons lack LTRs and are trans-<br />
cribed from an internal promoter. They are subdivided<br />
*Corresponding author. E-mail: magbadegesin@yahoo.com.<br />
Tel: +234 2 7504769. Fax: +44 8712 564876.<br />
into long interspersed nuclear elements (LINEs) and<br />
short interspersed nuclear elements (SINEs). The LTR<br />
retrotransposons are further divided into two groups<br />
Ty1/copia and Ty3/gypsy. These were so named after the<br />
elements first described in Saccharomyces cerevisiae<br />
(Ty1 and Ty3) and Drosophila melanogaster (Copia and<br />
Gypsy). Transcription of LTR retrotransposons starts at<br />
the 5’ LTR and ends at the 3’ LTR. The LTRs usually<br />
contain the regulatory sequences for promoting and<br />
terminating transcription of the element.<br />
The use of PCR primers based on the highly conserved<br />
amino acid sequence of enzymes domains has proved<br />
highly successful in the survey of transposons in many<br />
plants (Flavell et al., 1992; Hirochika and Hirochika,<br />
1993; Suoniemi et al.,1998; Vershhinin et al., 2002;<br />
Staginnus et al., 2001). It is making the study of transposable<br />
elements diversity, abundance and applications<br />
possible in species where full genome sequence data are<br />
not yet available.<br />
Although Ty1/copia-like elements have been reported<br />
in many higher plants, fewer Ty3/gypsy-like retrotran-
3952 Afr. J. Biotechnol.<br />
pol<br />
LTR gag pr<br />
RT RNaseH endo LTR<br />
PBS ORF3 PPT<br />
Figure 1. Structural organisation of Ty3/gypsy-like<br />
retrotransposons. They are bounded at their termini by long<br />
terminal repeats (LTRs). The primer binding site (PBS) and<br />
polypurine tract (PPT) is represented as grey rectangles. In<br />
between PBS and PPT (coloured boxes), are the two open<br />
reading frames (ORFs) with coding potential for the structural<br />
and enzymatic proteins needed for the retrotransposition<br />
cycle: the group antigenic glycoprotein (gag) domain coding<br />
for the protein that forms the nucleocapsid core; the protease<br />
(pr) domain encoding the proteins necessary for the<br />
maturation of the different proteins; the reverse transcriptase<br />
(RT) domain encoding the enzyme responsible for the<br />
creation of a DNA copy from the genomic RNA template; the<br />
ribonuclease H (RNaseH) domain encoding the enzyme for<br />
degradation of RNA hybridised to the first strand DNA; the<br />
endonuclease (endo) domain, encoding proteins necessary<br />
for the integration of the DNA copy into the host genome.<br />
Most of these proteins are encoded as polyproteins (pol)<br />
sometimes with overlapping ORFs and are processed into<br />
individual components by pr. In addition, a third open reading<br />
frame (ORF3) encoding an env-like activity is frequently found<br />
in Ty3/gypsy retrotransposons. The block arrow heads indicate<br />
the position of the forward and reverse primers for the<br />
PCR.<br />
sposon sequences or elements have been identified in<br />
plant species (Su and Brown, 1997). Ty3/gypsy-like<br />
retrotransposons share common features with Ty1/copialike<br />
elements but the order of the domains between the<br />
two long terminal repeats (LTRs) in Ty3/gypsy–like<br />
elements resembles those of the retroviruses (LTR-gagpr-rt-RNaseH-endo-LTR)<br />
(Figure 1). Some members of<br />
Ty3/gypsy superfamily also sometimes contain an<br />
additional open reading frame (ORF3) encoding an envlike<br />
gene.<br />
Cassava (Manihot esculenta Crantz) is the world’s sixth<br />
most important crop in terms of production (Mann, 1997)<br />
and the staple food of over 500 million people in the<br />
tropical regions of the world. It however, has been grossly<br />
understudied. In this study we isolated, cloned, sequenced<br />
and analysed cassava polyprotein fragment unique<br />
to Ty3/gypsy-like retrotransposons using degenerate<br />
PCR primers. Cassava Ty3/gypsy-like retrotransposons<br />
have been named Megyp for M. esculenta gypsy-like.<br />
The diversity and organization of Megyp within the<br />
cassava genome and their relationship to those of other<br />
plants are also analyzed. The nucleotide sequences<br />
described here have been submitted to the Genbank<br />
database and given the accession numbers AY946154 -<br />
AY946199.<br />
MATERIALS AND METHODS<br />
Plant material and DNA isolation<br />
Using the method of Dellaporta et al. (1983), DNA was extracted<br />
from young leaf samples of cassava cultivars grown in the tropical<br />
glasshouse at the University of Bath. The growth conditions include<br />
temperature at 22 to 28°C, relative humidity of 40 to 80% and a<br />
minimum light period of 12 h per day under day light, supplemented<br />
with 400 W Phillips high-pressure sodium lights when necessary.<br />
PCR Amplification of polyprotein fragment of Megyp<br />
sequences and cloning<br />
The PCR method used was as described by Suoniemi et al. (1998)<br />
with some modifications as described by Gbadegesin et al. (2008).<br />
Amplified DNA bands were gel purified (Qiagen, ‘Qiaquik’), ligated<br />
into pGEM ® -T Easy vector (Promega) and used to transform<br />
competent Escherichia coli DH5α according to standard procedures<br />
(Sambrook et al., 1989).<br />
DNA gel blot analysis<br />
Restriction digestions of genomic DNA (5 µg each) were carried out<br />
using buffer and reaction conditions specified by the manufacturer<br />
(Promega). Blotting and hybridisation were performed using<br />
standard procedures (Sambrook et al., 1989).<br />
Sequence and phylogenetic analyses<br />
DNA molecules were sequenced on an ABI 337 automated dye<br />
primer sequencer using universal primers for the cloning vector.<br />
The first line of sequence identification was by using BLASTN and<br />
TBLASTX searches against the GenBank non-redundant database<br />
at the default parameters (Altschul et al., 1990). The sequence<br />
fragments were assembled using the Vector NTI program.<br />
Consensus sequence data were aligned using CLUSTAL W<br />
(version 1.82) (Higgins et al., 1994). The PHYLIP program package<br />
version 3.63 (Felsenstein, 2004), available from the author at<br />
Department of Genetics, University of Washington, Seattle,<br />
Washington, was used for phylogenetic analysis. Consensus<br />
NEIGHBOR–joining trees (Saitou and Nei, 1987) were derived from<br />
equally parsimonious trees using the extended majority rule in the<br />
CONSENSE. Unless otherwise stated, distance matrices for<br />
phylogenetic analyses based on nucleotide sequences data were<br />
computed using DNADIST according to the Kimura 2-parameter<br />
model (Kimura, 1980). Trees were drawn using TREEVIEW program<br />
version 1.6.6 available from the author, Roderic D.M. Page of<br />
the Taxonomy Unit, Department of Zoology, University of Glasgow.<br />
RESULTS<br />
PCR amplification of cassava Ty3/Gypsy-like<br />
retrotransposon polyprotein fragment, cloning and<br />
sequence analysis<br />
PCR was carried out as described in the materials and<br />
methods section. The amplified products were analysed<br />
by electrophoresis on ethidium bromide stained 0.8%
Figure 2. PCR amplification of Ty3/gypsy-like polyprotein<br />
fragment from cassava genomic DNA. PCR product was run<br />
on a 0.8% agarose gel stained with ethidium bromide. The<br />
size marker (lane M) is bioline DNA 100 bp ladder, while the<br />
PCR product is shown in the right lane<br />
agarose gel. Approximately 1.6 kb cassava DNA was<br />
amplified (Figure 2). Amplified DNA was purified and<br />
cloned as described earlier.<br />
A clone was selected at random and sequenced from<br />
both ends using T7 and SP6 primers. The sequence was<br />
then submitted to BLASTN and TBLASTX searches<br />
against the GenBank non-redundant database using the<br />
default parameters. The searches confirmed that, a<br />
Ty3/gypsy-like polyprotein fragment had been amplified<br />
in the PCR experiments. The cassava element was 67%<br />
identical (within the region of the alignment) to the<br />
Ty3/gypsy-like retrotransposon polyprotein of Olea<br />
europaea at the amino acid sequence level (Figure 3).<br />
Thirty-six (36) clones (named Megyp1, Megyp2….<br />
Megy36) in total were randomly selected.<br />
Sequence and phylogenetic analysis<br />
The selected clones were partially sequenced in both<br />
directions using the T7 and SP6 primers. The sequences<br />
were assembled using the Vector NTI contig assembly<br />
program. The NTI contig assembly allowed visualization<br />
and removal of vector sequences. The vector free sequence<br />
data were submitted to BLASTN and TBLASTX<br />
searches as before. Sequencing from the 5’ end gave 26<br />
Megyp clones with good sequences of which 20 (77%)<br />
showed clear homology to the polyprotein of Ty3/gypsylike<br />
retrotransposons. However, sequencing from the 3’<br />
end gave 28 clones with good sequences of which 26<br />
(93%) showed clear homology to the polyprotein of<br />
Ty3/gypsy-like retrotransposons (in most cases E-value<br />
Gbadegesin and Beeching 3953<br />
were in the region of e -63 ). These data show that, cassava<br />
Ty3/gypsy-like retrotransposons are more diverged at the<br />
5’ end of the amplified polyprotein fragment compared<br />
with the 3’ end. Overall, the use of PCR primers<br />
anchored on RT and endonuclease domains proved<br />
useful and efficient for the isolation and characterisation<br />
of this group of cassava retroelements.<br />
The deduced translations of the Megyps left and right<br />
nucleotide sequences were obtained using ORF finder<br />
(www.ncbi.nlm.nih.gov/gorf/) (data not shown). Twelve<br />
(70.6%) of the clones having good left and right sequences<br />
and clear homology to Ty3/gypsy-like retrotransposon,<br />
contain neither a frame shift nor a nonsense<br />
mutation, while five (29.4%) have these mutations within<br />
the sequences analysed. While it is possible to say that<br />
the latter group could be defective enzymes, full<br />
sequence data would be necessary to conclude that the<br />
former code for functional enzymes.<br />
Two of the clones with uninterrupted open reading<br />
frames within the left and right sequences, Megyp5 and<br />
Megyp28, were fully sequenced. They contain no stop or<br />
frame shift mutations within the RT-RNaseH-endonuclease<br />
sequences analysed. The nucleotide sequences<br />
and deduced translation of these clones are shown in<br />
Figure 4a, b. The two shared 88% sequence identity at<br />
the nucleotide sequence level and 89% identity at the<br />
level of amino acid sequence. The 5’ (RT) ends of the<br />
Megyps are more diverged than the 3’<br />
(ENDONUCLEASE) ends and the Megyp5 and Megyp28<br />
nucleotide sequences did not align in the first 15<br />
nucleotide base positions (data not shown). However, the<br />
presence of the highly conserved block YAKFSKCEF of<br />
the RT domain characteristic of Ty3/gypsy retrotransposons<br />
(highlighted grey in Figures 4a, b) is a quick<br />
check and provides strong evidence for it being part of<br />
the polyprotein sequence in all of the cassava Ty3/gypsylike<br />
retrotransposons.<br />
To determine the relatedness of the cassava Ty3/<br />
gypsy–like retrotransposons to each other the nucleotide<br />
sequences (with the primer regions removed) for the 17<br />
Megyps (left and right fragments for 15; full ~1.6 kb<br />
fragment sequences for Megyps 5 and 28) were aligned<br />
using CLUSTAL W) (Higgins et al., 1994). The aligned<br />
nucleotide sequences were used to compute a distance<br />
matrix using DNADIST of the PHYLIP package version<br />
3.63 (Felsenstein, 2004), according to the Kimura 2parameter<br />
model (Kimura, 1980). Trees were then produced<br />
using the neighbor-joining method.<br />
This method is based on all pairwise comparisons in<br />
which positions for which there was no sequence data,<br />
for example, the central regions for all sequences other<br />
than Megyp5 and 28, were treated as missing data rather<br />
than as gaps (Felsenstein, 2004).<br />
Using an extended majority rule in the CONSENSE<br />
program from the PHYLIP package, a consensusunrooted<br />
tree was derived from 100 equally parsimonious<br />
trees. The consensus tree was drawn using TREEVIEW
3954 Afr. J. Biotechnol.<br />
Me: 55 CKIYQRVKLEHQKPAGMLNPLPIPEWKWENVVMDFVVGLPATSNRLNSIWVIVDRLTKSA 234<br />
C + Q+VK+EHQKPAG LNPL IPEWKWEN+ MDFVVG P ++ N+IWV+VDRLTKSA<br />
Oe: 1535 CMVCQQVKVEHQKPAGWLNPLDIPEWKWENITMDFVVGFPKSAIGNNAIWVVVDRLTKSA 1714<br />
Me: 235 HFIPVRSGYSVDKLAQVYVEEIIRLHGAPVSIVSDRRLQFTSRSWRSLQNAMGTRLDLST 414<br />
HF+PV+ +S+D+LAQ+Y+++++RL G PVSIVSDR L+FTS+ W+SLQ AMGT+L+ ST<br />
Oe: 1715 HFLPVKMTFSLDQLAQLYIKDVVRLCGVPVSIVSDRDLRFTSKFWKSLQGAMGTKLNFST 1894<br />
Me: 415 AFHPQTDGQSER 450<br />
A+HPQTDGQSER<br />
Oe: 1895 AYHPQTDGQSER 1930<br />
Figure 3. Alignment of cassava (Me) amino acid sequence with Olea europaea (Oe) amino acid sequence of partial<br />
polyprotein gypsy-like retrotransposon (gi, 7283091). The two sequences show 67% identity and 86% homology.<br />
(version 1.6.6) as shown in (Figure 5). Three families of<br />
Megyps, I, II and III emerged from the phylogenetic<br />
analysis (Figure 5). There are seven, six and four clones,<br />
respectively in these families.<br />
The predicted amino acid sequences of the plant<br />
Ty3/gypsy-like polyprotein listed in Table 1 and that of<br />
Gypsy were aligned with those of Megyps (representative<br />
cassava Ty3/gypsy-like retrotransposons) using the<br />
CLUSTAL W programme (1.82) and colour shaded in<br />
GENDOC as shown in Figure 6. The alignment reveals<br />
blocks of residues previously identified as highly conserved<br />
(Barber et al., 1990; Kulkosky et al., 1992; Springer<br />
and Britten, 1993; Xiong and Eickbush, 1990). There is<br />
highly conserved block YAKFSKCEF (box a) that<br />
includes the invariant lysine (underlined) of reverse transcriptase<br />
(Barber et al., 1990).<br />
The conserved TDAS motif that defined the RNase H<br />
region in most other Ty3/gypsy-like retrotransposons<br />
(Springer and Britten, 1993) is present in most cassava<br />
elements as CDAS (box b). In both cases, a key activesite<br />
aspartate (Campbell and Ray, 1993) is conserved.<br />
Also, conserved in the two fully sequenced cassava<br />
Ty3/gypsy POL fragments, Megyp5 and 28, is the motif<br />
N-3-DXL (box c) known to be essential in RNase H<br />
catalysis (Campbell and Ray, 1993).<br />
The N-terminal DNA-binding domain of integrase<br />
(Kedar and Khan, 1990) is revealed as a conserved X-6-<br />
H-29-C-2-C motif (box d), from which all the cassava<br />
elements lack the first four upstream amino acids, a<br />
feature shared with many other published sequences of<br />
Ty3/gypsy POL (box d, Figure 6). The highly conserved<br />
N-terminal GLLQPLPI motif (box e) of integrase is<br />
present in all the Megyps as homologous GMLNPLPI.<br />
Also present in the aligned Megyps is a D-60-D-35-E<br />
motif of integrase domain, where E is part of the 3’ primer<br />
sequence (not included in the alignment). The D,D-35-E<br />
motif is completely conserved in retroviral and retrotransposon<br />
integrases and is essential for enzymatic<br />
activity (Baker and Luo, 1994; Kulkosky et al., 1992).<br />
Overall, the amino acid sequences of the predicted<br />
translation of cassava Ty3/gypsy-like POL compared well<br />
with other Ty3/gypsy elements. This therefore confirmed<br />
them again as authentic Ty3/gypsy-like polyprotein sequences.<br />
Three families of Megyps and other plants Ty3/gypsylike<br />
retrotransposons emerged from the subsequent<br />
phylogenetic analysis (Figure 7). The cassava elements<br />
on the tree are indicated with arrowheads. These<br />
analyses revealed a high level of heterogeneity of<br />
Megyps among the reported plant Ty3/gypsy group retrotransposon<br />
using a PCR based assay. There are two<br />
monophyletic families (I and II) consisting of cassava<br />
Ty3/gypsy-like retrotransposons. The two clades were<br />
supported by bootstrap values of 49 and 45%,<br />
respectively, in the extended majority rule consensus tree<br />
(Figure 7). The third clade (III) supported by 100%<br />
bootstrap value consists of Gypsy of Drosophila<br />
melanogaster and the Ty3/gypsy-like retrotransposons of<br />
Arabidopsis thaliana rAt1, Ananas comosus, Oryza<br />
sativa, Hordeum vulgare rHv1, Lilium henryi del and one<br />
cassava element, Megyp18 and at 63% bootstrap, a<br />
second cassava element (Megyp 22) is included in this<br />
clade. The association within the sequences in this clade<br />
is very robust as shown by the high values of the<br />
bootstrap. Surprisingly, Megyp18 associated closely with<br />
Gypsy and Ty3-2 in clade III (Figure 7) Gypsy, like other<br />
retrovirus-like Ty3/gypsy retrotransposons, is known to<br />
encode env-like activity. Further studies would be<br />
required to classify the Megyps in this grouping as<br />
members of these endogenous retroviruses.<br />
Study on the genomic organization and diversity of<br />
Ty3/Gypsy-like retrotransposons in cassava cultivars<br />
A representative cassava Ty3/gypsy-like polyprotein<br />
fragment, Megyp5, was used to probe Southern blots of<br />
restriction digests of genomic DNA from a range of
A<br />
Gbadegesin and Beeching 3955<br />
1 ctggggttggtcttgcagactttgagggaacatggcttgtatgccaagttctctaaatgt<br />
L G L V L Q T L R E H G L Y A K F S K C<br />
61 gagttctggctgaggagcatttcgttcttggggcatgtagtgtcagagaatggtattgag<br />
E F W L R S I S F L G H V V S E N G I E<br />
121 gtagaccccaagaagacaaaaactgtggctaactggcctagacccacttcagtaacagag<br />
V D P K K T K T V A N W P R P T S V T E<br />
181 attagaagtttcttgggtttggcaggttactacaggaggttcgttcaggacttctcaaag<br />
I R S F L G L A G Y Y R R F V Q D F S K<br />
241 atagtagctcctctgaccagactgaccaggaagaatcagaagtttctgtggaccgacctg<br />
I V A P L T R L T R K N Q K F L W T D L<br />
301 tgcgaggagagtttcgaagagcttaagaagaggttgacttcagcaccagtgttagctctg<br />
C E E S F E E L K K R L T S A P V L A L<br />
361 ccatctagtgatgaggactttacagtcttttgtgatgcgtcccatatgggactgggttgt<br />
P S S D E D F T V F C D A S H M G L G C<br />
421 gtactgatgcagaatgagagggtgatcgcttatgcttctaggcagctgaagaagcatgag<br />
V L M Q N E R V I A Y A S R Q L K K H E<br />
481 ttgaattaccccacacatgaccttgagatggcagcagtaatctttgtactcaagatgtgg<br />
L N Y P T H D L E M A A V I F V L K M W<br />
541 aggcattacctctatggggtgaaatgtgagatctttacagatcataagagcctgcagtac<br />
R H Y L Y G V K C E I F T D H K S L Q Y<br />
601 atcttgagtcagagggatctgaatctgaggcagaggaggtgggtggagctgctgagtgac<br />
I L S Q R D L N L R Q R R W V E L L S D<br />
661 tatgattgcaagattcagtatcatccgggtaaggcgaatgtcgtggcagacgccctaagc<br />
Y D C K I Q Y H P G K A N V V A D A L S<br />
721 cggaagtcactaggcagtctatcccacatcgcggcagagaggagaccagtggtgaaggaa<br />
R K S L G S L S H I A A E R R P V V K E<br />
781 ttctacaagcttattgaggaaggtctacagttggagttgtctggtacaggtgccttagtg<br />
F Y K L I E E G L Q L E L S G T G A L V<br />
841 gcccagatgagagtagcacccatgtttctggagcaggtggctcagaaacagcatgaggac<br />
A Q M R V A P M F L E Q V A Q K Q H E D<br />
901 ccggagttagtgaaggttgccaggactgttcagtcaggcaaggatagcgagtacagattc<br />
P E L V K V A R T V Q S G K D S E Y R F<br />
961 gacagtaaagggatcctccgctatgggagcagactatgtgtaccagatgacattgggcta<br />
D S K G I L R Y G S R L C V P D D I G L<br />
1021 aaaggagacattatgagagaggctcataatgcaagatacagcattcaccctggagccact<br />
K G D I M R E A H N A R Y S I H P G A T<br />
1081 aagatgtatcaagatttgaagaaagtttattggtggccagcgatgaagaaagaagtggca<br />
K M Y Q D L K K V Y W W P A M K K E V A<br />
1141 cagttcgtgtcagcctgcgaagtgtgtcagagggtgaagctggaacatcagaagccggct<br />
Q F V S A C E V C Q R V K L E H Q K P A<br />
1201 ggaatgcttaacccgctacctatcccagaatggaaatgggagaatatagctatggacttc<br />
G M L N P L P I P E W K W E N I A M D F<br />
1261 gtagtggggttaccggcggcgtccaacagagtggactccatatgggtgattgtggacaga<br />
V V G L P A A S N R V D S I W V I V D R<br />
1321 ctcaccaaatctgctcacttcattcctgtcaggagtggctactctgtagacaagttggcg<br />
L T K S A H F I P V R S G Y S V D K L A<br />
1381 caggtgtatgtagatgagatcgtcaggctgcatggggttcctgtttcgatagtgtcagat<br />
Q V Y V D E I V R L H G V P V S I V S D<br />
1441 agagggccccagttcacctccagattttggcggagtctgcagaatgccatgggtactagg<br />
R G P Q F T S R F W R S L Q N A M G T R<br />
1501 ttggatttcagtactgccttc 1521<br />
L D F S T A F<br />
Figure 4. (A) Nucleotide sequence and deduced translation of Megyp5. The primer sequences are<br />
omitted. The highly conserved amino acid sequence block YAKFSKCEF of RT domain characteristic of<br />
Ty3/gypsy retrotransposons is highlighted grey. Recognition enzyme sequences are shown in bold<br />
face for Eco RI (underlined), Hind III (oval) and Bgl II (box) used in Southern analysis of cassava; (B)<br />
Nucleotide sequences and deduced translations of Megyp28. Primer sequences are omitted. The<br />
highly conserved amino acid sequence block YAKFSKCEF of RT domain characteristic of Ty3/gypsy<br />
retrotransposons is highlighted grey. Restriction enzymes sequences are shown as detailed in Figure<br />
4a.
3956 Afr. J. Biotechnol.<br />
B<br />
1 ctgaggataatattacagaccttgagggaacatggcttgtatgccaagttctccaagtgt<br />
L R I I L Q T L R E H G L Y A K F S K C<br />
61 gagttctggttaaggagcatatcattcttggggcatatagtgtcagagaatggaatagag<br />
E F W L R S I S F L G H I V S E N G I E<br />
121 gtagaccccaagaagatagaagctgtgactaactggccaagacccacctcagtgacagag<br />
V D P K K I E A V T N W P R P T S V T E<br />
181 atcagaagcttcttgggtttggctggctactacaggaggttcgttcaggacttctctaag<br />
I R S F L G L A G Y Y R R F V Q D F S K<br />
241 attgcagctcctttaaccagattaaccagaaagaatcagagattcgagtggaccgatcag<br />
I A A P L T R L T R K N Q R F E W T D Q<br />
301 tgtgaagaaagtttcgaagagcttaagaagaggttgacttcagcaccagtgttagctctg<br />
C E E S F E E L K K R L T S A P V L A L<br />
361 ccaaacagcaatgaggatttcacagtgttctgtgatgcatccagagtaggcctgggttgt<br />
P N S N E D F T V F C D A S R V G L G C<br />
421 gtgttgatgcagaatggtaaggtgatcgcttatgcttctagacagccgaagaggcatgag<br />
V L M Q N G K V I A Y A S R Q P K R H E<br />
481 ttgaattaccccacacacgacctggaaatggcagcagttatctttgccctcaagatgtgg<br />
L N Y P T H D L E M A A V I F A L K M W<br />
541 aggcattacctctatggggtaaaatgtgagatcttcacagatcataagagcctgcagcac<br />
R H Y L Y G V K C E I F T D H K S L Q H<br />
601 atcttgaaccagagagagctgaacttgaggcagaggagatgggtagaactgttgagtgac<br />
I L N Q R E L N L R Q R R W V E L L S D<br />
661 tacgattgcaagatccagtaccatccgggtaaggctaatgtagtagctgatgccttaagc<br />
Y D C K I Q Y H P G K A N V V A D A L S<br />
721 cggaaatcacttggcagtctatcccacatcacggcagagaggagaccggtggtgaaggag<br />
R K S L G S L S H I T A E R R P V V K E<br />
781 ttttataagctcattgaggagggtctacagatggagttgtctggtacaggtgctttgatt<br />
F Y K L I E E G L Q M E L S G T G A L I<br />
841 gcacagatgaaagtaacccccgtgtttctggagcaagtggctcagaaacagcacgaggac<br />
A Q M K V T P V F L E Q V A Q K Q H E D<br />
901 ccagagttagtgaagattgccaggactgttcagtcaggcaaagatagtgagttcagattt<br />
P E L V K I A R T V Q S G K D S E F R F<br />
961 gatgataaggggatcctccgctatgggaacagactatgtgtaccagatgacatcgggcta<br />
D D K G I L R Y G N R L C V P D D I G L<br />
1021 aaaggagacattatgagagaggctcataatgcaaggtacagtgttcaccctggagccacc<br />
K G D I M R E A H N A R Y S V H P G A T<br />
1081 aagatgtaccaggatctgaagggagtgtattggtggccagctatgaagagggaagtggca<br />
K M Y Q D L K G V Y W W P A M K R E V A<br />
1141 cagttcgtgtcagcctgcgaaatatgtcagagggtgaagctggaacatcagaagccggct<br />
Q F V S A C E I C Q R V K L E H Q K P A<br />
1201 ggaatgcttaacccactgccgattccagagtggaaatgggagaacatagctatggatttt<br />
G M L N P L P I P E W K W E N I A M D F<br />
1261 gtagtggggttaccggcaacatccaacagactagactccatatgggtgattgtggacaga<br />
V V G L P A T S N R L D S I W V I V D R<br />
1321 ctcaccaaatctgctcacttcatccctgttaggagcaactactctgtggataagttagcg<br />
L T K S A H F I P V R S N Y S V D K L A<br />
1381 caggtttatgtggatgaagttgtcaggctgcatggggtcccagtttctatagtgtcagat<br />
Q V Y V D E V V R L H G V P V S I V S D<br />
1441 agagggccccagttcacctccaggttttggcggagtctgcagaatgctatgggtaccagg<br />
R G P Q F T S R F W R S L Q N A M G T R<br />
1501 ttggatttcagtactgccttc 1521<br />
L D F S T A F<br />
Figure 4. Contd.
Gbadegesin and Beeching 3957<br />
Figure 5. Phylogenetic analysis of 17 cassava Ty3/gypsy-like retrotransposons (Megyps). The tree is based on 17<br />
nucleotide sequences of pol gene fragments (Megyps): 15 are partial sequences from the two ends of the ~1.6 kb gene<br />
fragments, while Megyps 5 and 28 were full 1.6 kb length. This is a consensus neighbor-joining unrooted tree<br />
constructed with the PHYLIP package from the distance matrix following the Kimura 2-parameter model (Kimura, 1980).<br />
Bootstrap values (100 replicates) are shown.<br />
Table 1. Sources of polyprotein amino acids sequences used in comparative<br />
phylogenetic analysis with the 16 cassava Megyps amino acid sequences.<br />
Locus or sequence name Source species Gi number<br />
A. comosus Ananas comosus 2995405<br />
O. sativa Oryza sativa 37532428<br />
Del Lilium henryi 19442<br />
rHv1 Hordeum vulgare 3413486<br />
rAt1 right Arabidopsis thaliana 3413430<br />
rAt1 left Arabidopsis thaliana 3413431<br />
Ty3-2 Saccharomyces cerevisiae 1084606<br />
Gypsy Drosophila melanogaster 130583<br />
The table shows the name of Ty3/gypsy retrotransposons and the GI (Geneinfo<br />
identifier) number of corresponding polyprotein as well as the name of the source<br />
organisms. The romani elements are rHv1 and rAt1.
3958 Afr. J. Biotechnol.<br />
Figure 6. Alignment of the predicted amino acid sequences for 16 polyprotein fragments of cassava Ty3/gypsy-like retrotransposons<br />
with those of eight other plants. Ty3-2 and Gypsy are included for reference (detail of the polyproteins in Table 4). Each of the clones<br />
was represented by either translation of full ~1.6 kb or partial sequences from the two ends of the POL fragments. Colour blocking<br />
indicates sequence conservation. Black = 100% identity, deep grey = > 80% identity, light grey = > 60% identity and non-shaded = <<br />
60% identity. Letters in bold orange colour below the alignment and boxed regions labelled with small letter a-e indicate the key residues<br />
conserved in all related enzymes as explained in the text.<br />
cassava cultivars. Following high stringency washes (0.2<br />
X SSC, 0.1% SDS, 65°C), strong signals were observed<br />
in all the digests (Figure 8) and the autoradiograph<br />
required a short exposure time. This showed that, the
Figure 6. Contd.<br />
Megyp5 sequence and its homologues were highly<br />
repeated within these genomes. The probe contained<br />
one each of the Bgl II, Eco RI and Hind III recognition<br />
sites (Figure 4a), which could explain the presence of<br />
Gbadegesin and Beeching 3959<br />
two bands in the DNA digestions by each of these<br />
enzymes. However, multiples of two hybridising bands<br />
were observed for each of the three enzymes (Figure 8),<br />
indicating that multiple copies of Megyp5 and relatives
3960 Afr. J. Biotechnol.<br />
Figure 6. Contd.<br />
were integrated in the genome. Many of the bands are<br />
very strong and distinct but there are few weak ones<br />
suggesting that Megyp5 is cross hybridising with<br />
sequences highly homologous to the probe, represented<br />
by the strong major bands, as well as related diverged<br />
fragments, seen as weak signals. The cultivars showed<br />
no clear polymorphism of hybridisation fragments with<br />
Megyp5 probe used (Figure 8).<br />
DISCUSSION<br />
The detection of Ty3/gypsy-like retrotransposons using<br />
heterologous primers based on conserved domain of RT<br />
in PCRs has not been efficient due to the relatively high<br />
sequence heterogeneity among these elements (Su and<br />
Brown, 1997). The main problem with the use of these<br />
primers alone has been in the frequent amplification of<br />
other sequences (other retroelements, transposons and<br />
non-transposons), in addition to the desired Ty3/gypsy<br />
sequences. For instance, of forty four sequenced clones<br />
following genomic DNA PCR amplification using primers<br />
based on the conserved RT domain in Brassica sp, only<br />
twenty were similar to any of the known transposon types<br />
and just fifteen were Ty3/gypsy-like of all known lineages<br />
(Alix and Heslop-Harrison, 2004). In contrast, the use of<br />
degenerate PCR primers anchored on both the integrase<br />
and reverse transcriptase (Suoniemi et al., 1998) has<br />
proved a more robust and reliable tool, as the design of<br />
these primers exploited the differences in the domainorder<br />
between the Ty1/copia and Ty3/gypsy groups of<br />
retrotransposons. The usefulness of these primer sets<br />
has been confirmed here by the isolation and characterisation<br />
of the polyprotein fragment diagnostic of<br />
Ty3/gypsy-like retrotransposon in cassava. This has<br />
enabled a study of diversity of this group of retrotran-
Gbadegesin and Beeching 3961<br />
Figure 7. Comparative phylogenetic analysis of 16 cassava Ty3/gypsy-like retrotransposons (Megyps) with other eight<br />
from other organisms. The tree is based on predicted amino acid sequences of pol gene fragments. This is a consensus<br />
neighbor-joining unrooted tree constructed with PHYLIP package. Distance matrix used the Jones-Taylor-Thornton model<br />
(Jones et al., 1992). Three groups of Ty3/gypsy-like retrotransposons were revealed. The cassava elements are indicated<br />
with arrowheads. Fourteen of them clustered into two monophyletic groups but Megyp18 and Megyp22 associated with<br />
Gypsy and Ty3-2 group. The identities of other sequences used in comparative analyses with cassava’s are as in Table 2.<br />
Bootstrap values (100 replicates) = > 45% is shown.
3962 Afr. J. Biotechnol.<br />
23.1<br />
9.4<br />
6.56<br />
4.36<br />
2.3<br />
2.0<br />
0.56<br />
M 1 2 3 4 5 6 7 8 9 10 11 12 M 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 M<br />
Bgl II<br />
EcoR I<br />
Hind III<br />
Figure 8. Southern blot analysis of Ty3/gypsy-like polyprotein of 12 cassava cultivars. 10 µg of genomic DNA from each<br />
of the cassava cultivars; lanes 1 (MGA1); 2 (MNGA2); 3 (MDOM5); 4 (MNGA19); 5 (MCOL22); 6 (CMC40); 7 (MVEN77);<br />
8 (CG402); 9 (SM627); 10 (SM985); 11 (SM1088); 12 (CM2177) were digested with Bgl II, Eco RI or Hind III. The<br />
digested DNAs were separated on 0.8% agarose gels, transferred to nylon membrane and hybridised with the Megyp5<br />
probe. Hind III-digested lambda DNA was used as a DNA size marker (M).<br />
sposons in this important food crop. This approach has<br />
also provided better information from all the conserved<br />
domains for better resolution of the Ty3/gypsy group than<br />
could be afforded by the use of individual enzymatic<br />
domains (Springer and Britten, 1993; Wright and Voytas,<br />
1998; Xiong and Eickbush, 1990).<br />
Alignments of the nucleotide sequences of cassava<br />
Ty3/gypsy clones (Megyps) and subsequently, the<br />
inferred phylogenetic tree led to the identification of<br />
diverse members of this group of elements in cassava. In<br />
addition, the predicted translation of Megyps, aligned with<br />
other plant Ty3/gypsy sequences, revealed the presence<br />
of conserved residues established to be critical for<br />
enzymatic activity of integrase, reverse transcriptase and<br />
RNase H (Campbell and Ray, 1993; Kedar and Khan,<br />
1990; Baker and Luo, 1994; Kulkosky et al., 1992) and<br />
proving that they represent authentic Ty3/gypsy-like<br />
retrotransposon sequences and suggesting that they<br />
were probably derived from recently active elements.<br />
Phylogenetic analysis of cassava and other plant<br />
Ty3/gypsy polyproteins revealed a level of heterogeneity<br />
in cassava elements that has not been reported in many<br />
of plant Ty3/gypsy group retrotransposons. Most cassava<br />
Ty3/gypsy-like retrotransposons are clustered into<br />
monophyletic sub-groups (Figure 7). The groupings were<br />
supported by bootstrap values of 49 and 45%. The low<br />
bootstrap values are most probably due to the<br />
heterogeneity of the cassava sequences. Megyp18<br />
clustered closely with Ty3-2 retroelements (Figure 7)<br />
suggesting that, Megyp18 represents the retrovirus<br />
lineage of Ty3/gypsy retrotransposons in the genome of<br />
cassava.<br />
The findings in this study also support the suggestion<br />
23.1<br />
9.4<br />
6.56<br />
4.36<br />
2.3<br />
2.0<br />
0.56<br />
that, the use of primers based on conserved domains of<br />
RT, which have proved inefficient for the isolation of<br />
plants Ty3/gypsy-like retrotransposons, could be the<br />
limiting factor in the study of the diversity and heterogeneity<br />
among plants Ty3/gypsy-like retrotransposons. In<br />
fact, the availability of the whole genome sequence has<br />
revealed the presence of seven families of Ty3/gypsy-like<br />
retrotransposons in A. thaliana (Wright and Voytas, 2002).<br />
Availability of more Ty3/gypsy-like sequences from other<br />
plants may give a better picture of the diversity of this<br />
group of retrotransposons among plants. In their study,<br />
Wright and Voytas (2002) further used primers specific<br />
for the conserved domains of RT of endogenous retroviruses<br />
lineage of Ty3/gypsy-like retrotransposons to<br />
make a survey of this family of retrotransposons among<br />
plants. The PCR assay revealed that, they are almost<br />
universally present in genome of dicots and old-world<br />
monocots (Wright and Voytas, 2002). Their ubiquitous<br />
nature and potential for horizontal transfer by infection<br />
implicates these retrotransposons as important vehicles<br />
for plant genome evolution (Wright and Voytas, 2002).<br />
Also of interest is the fact that, Ty3/gypsy-like<br />
retrotransposons from a single or related plant species<br />
were clustered in a subfamily indicating that, sequence<br />
divergence during vertical transmission has a major<br />
influence on the evolution of this group of retrotransposons<br />
in plants. The presence of more than one family<br />
of Ty3/gypsy-like retrotransposons in one plant species<br />
indicates that, the retrotransposons of a family could<br />
evolve independently within a species without affecting<br />
the evolution of the members of other families. Southern<br />
hybridisation supports the diversity identified by sequencing<br />
and highlights that, multiple copies of Megyps are
integrated in the genome of all cassava cultivars tested.<br />
However, these cultivars have the same pattern of<br />
hybridisation with the three different restriction enzymes<br />
(Bgl II, Eco RI or Hind III) digestion of the genomic DNA.<br />
This suggests that, there are no recent retrotransposition<br />
activities among these cassava elements. However, a<br />
unique distribution of Ty3/gypsy-like sequences was<br />
found for each of the four basic genomes of Hordeum<br />
genus except for the subspecies H. vulgare and H.<br />
spontaneum that were reported to show no polymorphism<br />
of hybridisation fragments with all the Ty3/gypsy clones<br />
used as probe (Vershinin et al., 2002). Also, study on the<br />
genomic organization of the Ty3/gypsy-like retrotransposons<br />
of different oil palm species and accessions by<br />
southern hybridisation revealed minor differences (Kubis,<br />
et al., 2003).<br />
ACKNOWLEDGEMENTS<br />
The first author would like to acknowledge funding from<br />
the Commonwealth Scholarship Commission, UK. This<br />
publication is part of an output from a research project<br />
funded by the United Kingdom, Department for International<br />
Development (DFID) for the benefit of developing<br />
countries: R8156 Crop Post-Harvest Programme.<br />
The views expressed are not necessarily those of DFID.<br />
This work has been carried out in compliance with the<br />
current laws governing genetic experimentation in the<br />
U.K.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 3964-3972, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1752<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Genetics of trichomes and its association with fibre and<br />
agronomic traits in cotton<br />
Nausherwan Nobel Nawab 1* , Qamar Shakil 1 , Shahid Niaz 2 , Muhammad Munir Iqbal 1 ,<br />
Muhammad Ahsan Asif 1 and Iftikhar Ahmad Khan 1<br />
1 Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan.<br />
2 Vegetable Research Institute, Faisalabad, Pakistan.<br />
Accepted 1 April, 2011<br />
Hairiness/trichomes act as an important insect non-preference trait. The inheritance of this trait was<br />
studied in three cross combinations involving a common pilose parent with three sparse hairy parents<br />
which showed a theoretical monohybrid ratio of 1:2:1, showing incomplete dominance in F2. The<br />
positive correlation of number of trichomes with lint percentage and micronaire suggested that the<br />
selection for pilose hairiness would be helpful in enhancing the lint percentage and fibre fineness. The<br />
number of trichomes had no effect on the expression of the traits like number of monopodial branches,<br />
number of sympodial branches, number of bolls, boll weight and seed cotton yield which proved no<br />
chance of undesirable linkage of these traits with number of trichomes.<br />
Key words: Gossypium hirsutum L., trichomes, inheritance, fibre traits, agronomic traits.<br />
INTRODUCTION<br />
The sustainability of cotton production worldwide has<br />
been affected due to the piercing, sucking insect pests<br />
and bollworms which are a serious threat to the cotton<br />
crop. Insect pests constitute a major factor in production<br />
in all over the cotton growing areas of the world. In recent<br />
times, insect control has been totally based on the use of<br />
chemical insecticides. During the 1970’s and 1980’s, the<br />
use of insecticides increased tremendously in almost all<br />
cotton producing countries of the world including<br />
Pakistan.<br />
In Pakistan, during 2007- 2008 a total of 28 thousand<br />
tonnes of insecticides was imported which measures the<br />
foreign exchange of 74.5 million US dollars (GOP, 2007-<br />
08). Little emphasis was placed on the plant genetic<br />
resistance as a means of suppressing insect pests.<br />
Painter (1951) noted that there were no efforts made to<br />
develop genetic resistance to cotton insect pests. Genetic<br />
resistance in the form of resistant varieties is an effective<br />
means of minimizing yield losses caused by insect pests<br />
and also leads to reduction in the use of insecticides<br />
(Vaden Bosch, 1972; Van Dinther, 1972; Maxwell et al.,<br />
1972; Bhatti et al., 1976). In the present scenario, by failing<br />
*Corresponding author . E-mail: nnnawab24a@gmail.com. Tel:<br />
0092-41-2652518 or 0092-321-766-2112.<br />
to cope with all other possible means for insect control,<br />
research has been accelerated on the host plant<br />
resistance.<br />
Hairiness/trichomes act as an important insect nonpreference<br />
trait. The role of trichomes in plant defence<br />
was evaluated by Levin (1973). It is clear that trichomes<br />
play a role in plant defence, especially with regard to<br />
phytophagous insects. Hairiness has been reported to<br />
have a resistance against the sucking insect pests of cotton<br />
(Lee, 1985). The degree of hair or trichome density on<br />
the leaves of Gossypium species and cultivars is related<br />
to varying degrees of resistance/susceptibility to sucking<br />
pests, like whiteflies (Meagher et al., 1997), aphids and<br />
jassids (Jenkins, 1989; Watson, 1989), or the boll weevil<br />
(Thomson and Lee, 1980; Percy and Kohel, 1999). The<br />
degree of jassid resistance had definite correlation with<br />
the pilosity of the plant. The more tufted types were less<br />
prone to jassid attack (Sikka et al., 1966).<br />
The genetics of the insect resistance traits in cotton<br />
have been reported to be governed by oligogenes<br />
(Endrizzi et al., 1984). Knight (1952) identified two genes<br />
for hairiness in cotton whereas, Niles (1980) reported that<br />
the increase in the plant hairiness is governed by two<br />
major genes and a complex of modifier genes. The major<br />
gene was designated as H1 for sparse hairiness. A<br />
second major gene, H2 controlled the finely dense<br />
pubescence in an upland mutant designated as ‘pilose’.
In the F1 populations, both H1 and H2 showed incomplete<br />
dominance (Simpson, 1947; Niles, 1980; Rahman and<br />
Khan, 1998).<br />
From industrial point of view, agronomic and fibre<br />
quality traits hold a special position. The effect of pilosity<br />
on agronomic traits was studied by Lee (1984) but no,<br />
significant inferences were inferred for the trichome count<br />
per cm, lint percentage and boll weight. The physical<br />
parameters required for good spinning performance of<br />
cotton fibre is a challenge for breeders. The pilose<br />
condition is associated with decreased fibre length and<br />
increased micronaire (Simpson, 1947; Lee, 1964, 1984).<br />
But Kloth (1993) discovered a pilose like plant with<br />
unexpectedly low micronaire among the homozygous<br />
pilose plants.<br />
Breeding through conventional tools has not lost its<br />
significance even in the presence of modern tools of<br />
science. Keeping in view, the extensive use and consumption<br />
of cotton and its products, there is a need for<br />
breeding varieties by incorporating the gene for hairiness<br />
into promising cotton genotypes, which in turn will not<br />
only minimize possible insect pest attack and insecticide<br />
usage but also helps to improve yield and fibre quality<br />
attributes. The information reported herein, would be<br />
useful in determining the genetics for trichomes and its<br />
effects on agronomic and fibre traits.<br />
MATERIALS AND METHODS<br />
Four cotton genotypes, Acala 63-74, FH 1000, CIM 446 and HRVO-<br />
1 were selected and selfed for four generations by growing twice a<br />
year, in a glasshouse and field during 2003 to 2004. The selected<br />
parents with contrasting traits (Table 1) were planted in 300 × 300<br />
mm earthen pots, containing a mixture of equivalent proportion of<br />
sand, soil and farmyard manure, during November, 2004 in a<br />
glasshouse. Temperature in the glasshouse was maintained at 30 ±<br />
2°C during the day and 25 ± 2°C at night by using built in steam<br />
heaters. The plants were exposed to natural sunlight supplemented<br />
with artificial lighting, for a photoperiod of 16 h (ICAC, 2007). Three<br />
crosses were attempted to obtain F0 seed during February to<br />
March, 2005. The selfed seed of the parents was obtained by<br />
covering their floral buds with butter paper bags. The crossing<br />
scheme is given in Table 2. The F1 and their parents were sown<br />
during the normal crop season of 2006 in two different sets planted<br />
at two different locations. The F1 plants of each cross were divided<br />
in three groups for developing BC1, BC2 and F2 for each<br />
combination. The fresh F0, BC1 and BC2 seed was developed by<br />
manual crossing, while the seed for F2 generations was produced<br />
for each of the four combinations through manual selfing. The<br />
experimental field was fertilized with N-P-K at the rate of 100-75-00<br />
Kg/ha. The experiment in the field was laid out in a randomized<br />
complete block design with three replications of each of the six<br />
generations of the three crosses. The length of the plot was<br />
maintained at 4.5 m, accommodating approximately 15 plants<br />
spaced 300 mm apart.<br />
The distance between the rows was 750 mm. A single plot per<br />
replication was assigned to each of the parents and their respective<br />
F1, while four plots per replication were assigned to each of the<br />
backcrosses and eight plots per replication were assigned to raise<br />
the F2 population of each cross. Ten guarded plants were selected<br />
randomly in the parents and their F1, while fifty and thirty plants in<br />
each replication were selected in F2 and backcross generations,<br />
Nawab et al. 3965<br />
respectively, to record the data during 2006. At maturity, the data<br />
on various agronomic traits viz; plant height, number of monopodial<br />
branches, number of sympodial branches, number of bolls, seed<br />
cotton yield, boll weight and lint percentage were recorded. Fibre<br />
quality characteristics like fibre length, fibre strength, fibre fineness,<br />
fibre elongation percentage and fibre uniformity ratio of each plant<br />
of a generation were measured using Spin lab high volume<br />
instrument (HVI-900-A), M/S Zellweger Uster, Switzerland,<br />
available in the Department of Fibre Technology, University of<br />
Agriculture, Faisalabad, Pakistan. HVI measures fibre quality<br />
characteristics according to the international trading standard. A<br />
minimum of 10 g sample of lint from each of the guarded plants in<br />
each generation was pre-conditioned to moisture applicability for at<br />
least four to five hours prior to testing in the HVI. Fibre length was<br />
measured, on the basis of the fibrograph. The samples were<br />
prepared at fibro sampler in the form of fibre comb and the<br />
fibrograph-910 brushed the sample fibres automatically by vacuum<br />
action, and optical density of the sample was displayed on the<br />
screen. The mean fibre length was derived according to the ASTM<br />
(1977a) standard through the procedure laid down by Hunter<br />
(1991). The average fibre length was calculated in mm for further<br />
analysis. Fibre bundle strength was determined by Pressley<br />
strength tester using the flat bundle method as specified by ASTM<br />
standard (1977b). The fibre elongation was measured on HVI-900-<br />
A. When a sample was moved into an optical sensor, where the<br />
test for length and strength were performed, the percentage<br />
increase in the length before fibre breakage was measured<br />
(Anonymous, 1992a, b). For fibre uniformity ratio, the sample was<br />
moved into the optical sensor of HVI-900-A and the reading of<br />
optical density of the sample was displayed on the screen. The<br />
uniformity ratio of fibre was measured according to ASTM standard<br />
(1977a). Fibre fineness of micronaire value was measured on<br />
fibrofine-920 device of HVI-900- A. When lid of the chamber<br />
containing a sample is closed, the sample is compressed to a fix<br />
and known volume. The sample was weighed on an electric<br />
balance before placing in the test chamber. This balance<br />
transmitted the mass through the control processor. This mass was<br />
accepted if the weight was between 8.5 and 11.5 g of cotton. The<br />
measured values of mass and pressure calculated were in fineness<br />
value according to ASTM standard (1977c).<br />
Trichome density studies<br />
Trichomes represent the presence of small hairs on the cotton plant<br />
(Figure 1). The trichome density on leaves was estimated following<br />
the method proposed by Bourland et al. (2003). Three leaves at<br />
random, each from upper, middle and lower portion of five plants in<br />
parents and their F1’s and thirty and fifty plants were selected in<br />
BC’s and F2’s in all the three replications were used to assess for<br />
the quantitative measure of trichomes on the abaxial leaf surface.<br />
Observations pertaining to the number of trichomes were recorded<br />
with the help of an index card within an area of 1 mm 2 (Figure 2) lay<br />
over the abaxial side of each leaf from three different positions and<br />
averaged. The resultant mean values for number of trichomes from<br />
three different portions of the plant were worked out as the final<br />
reading for average number of trichomes per unit area. Trichomes<br />
in the 1 mm 2 area were counted with the aid of high magnifying<br />
power microscope (Olympus Z61).<br />
On the basis of the trichome counts made for the leaves, a rating<br />
scale was developed to categorize the trichome density on abaxial<br />
surface of leaves by using a scale of 1 for sparsely (non) hairiness,<br />
2 for moderate number of trichomes, 3 for (pilose) hairiness (Lee,<br />
1968; Wright et al., 1999; Bourland et al., 2003; Stiller et al., 2004;<br />
Lacape and Nguyen, 2005).<br />
Chi-squared values and probabilities of goodness of fit of the<br />
segregation ratios of F2 and backcross generations were tested<br />
against theoretical ratio (Harris, 1912). Phenotypic and genotypic
3966 Afr. J. Biotechnol.<br />
Table 1: Distinctive morphological features of the upland cotton accessions<br />
S.No. Variety/ Accession Parentage Hairiness state Origin<br />
1 Acala 63-74 - Glabrous USA.<br />
2 CIM 446 CP 15/2 × S 12 Lightly hairy CCRI, Multan, Pakistan.<br />
3 FH 1000 S 12 × CIM 448 Lightly hairy CRI, Faisalabad, Pakistan.<br />
4 HRVO-1 B-557/2/Gambo Okra/Rajhans/3/Rajhans Pilose hairiness CRI, Faisalabad, Pakistan.<br />
* CCRI = Central Cotton Research Institute, Multan. Pakistan. * CRI = Cotton Research Institute, Faisalabad. Pakistan.<br />
Table 2: Scheme of crossing<br />
S.No. Cross Trait Considered<br />
1 HRVO-1 × FH 1000 Pilose hairiness × Lightly hairy<br />
2 HRVO-1 × CIM 446 Pilose hairiness × Lightly hairy<br />
3 HRVO-1 × Acala 63-74 Pilose hairiness × Glabrous<br />
(a) Pilose hairiness (P1)<br />
H2H2<br />
Figure 1. Inheritance of leaf trichome trait<br />
(c) Intermediate class of hairiness (F1)<br />
H2h2<br />
(b) Normal hairiness (P2)<br />
h2h2
correlation coefficients between trichome counts and agronomic<br />
and fibre traits were determined using the F2 data. Phenotypic<br />
correlation coefficients were calculated following Dewey and Lu<br />
(1959) using Minitab computer programme. The genetic<br />
correlations (rg) between two characters X and Y were calculated<br />
following Falconer (1981).<br />
RESULTS<br />
Genetic studies<br />
The mean squares from the analysis of variance for<br />
agronomic/fibre and number of trichomes for six generations<br />
in three cotton crosses were tested at two different<br />
locations and are presented in Table 3. Phenotypic<br />
classes were developed as evident from Table 4, on the<br />
basis of the trichome count ratings on the abaxial leaf<br />
surface in the three crosses involving pilose hairy and<br />
normal/sparse hairy plants. The inheritance pattern for<br />
trichomes/hairiness in three different cross combinations<br />
was studied on the basis of the F2 and test/back cross<br />
population data.<br />
In all the three cross combinations, leaf trichomes<br />
segregated into three distinct classes (Figure 3). Nonsignificant<br />
chi-squared values were observed for the<br />
segregating ratios in F2 and backcross generations of the<br />
three crosses. Observations of 1 sparse hairiness : 2<br />
intermediate class of hairiness : 1 pilose hairiness on<br />
leaves were observed in the F2 populations of the three<br />
crosses. In the backcrosses with parent-I, ratios of 1 pilose :<br />
1 intermediate, leaf hairiness were obtained. Similarly, in<br />
the backcrosses with parent-II, ratios of 1 sparse : 1<br />
Figure 2 Trichome counts on the abaxial side of cotton leaf with an<br />
area of 1 mm2 of an index card.<br />
intermediate, leaf hairiness were observed.<br />
Phenotypic and genotypic correlations<br />
Nawab et al. 3967<br />
In all the three crosses (Table 5), there existed positive<br />
and significant correlation of number of trichomes with lint<br />
percentage. For fibre length and fibre strength, there was<br />
highly significant and negative correlation with number of<br />
trichomes. A significant and positive correlation of<br />
number of trichomes with micronaire value meant a<br />
negative correlation of number of trichomes with fibre<br />
fineness in all of the three cross combinations. In the<br />
case of fibre uniformity ratio and fibre elongation, a<br />
negative association with number of trichomes was<br />
observed in the crosses.<br />
The study of number of trichomes were negatively but<br />
significantly correlated with plant height in the cross<br />
HRVO-1 × CIM 446. Non-significant correlations of<br />
number of trichomes with number of monopodial<br />
branches, number of sympodial branches, number of<br />
bolls, seed cotton yield and boll weight were recorded.<br />
DISCUSSION<br />
Genetic studies<br />
Highly significant differences among different generations<br />
for all characters studied (Table 3) were observed in all<br />
the three crosses. Significant differences among different<br />
generations indicated the existence of genetic variability.
3968 Afr. J. Biotechnol.<br />
Table 3. Mean squares from analysis of variance along with the calculated F-values in parenthesis for agronomic/fibre and number of trichomes for six generations in three cotton crosses<br />
tested at two different locations.<br />
Source D.F<br />
Replication 2 1.62<br />
(1.62)<br />
Generation 5 5.46<br />
(5.44)<br />
Location 1 0.40<br />
(0.40)<br />
G × L 5<br />
1.37<br />
(1.37)NS<br />
HRVO-1 × FH 1000 Probability (F-value)<br />
PH NMB NSB NBP BWt SCY L% FL FS FE U % Mic T 0.05 0.01<br />
0.010<br />
(0.47)<br />
0.113<br />
(5.33)<br />
0.002<br />
(0.12)<br />
0.022<br />
(1.06)NS<br />
0.38<br />
(1.05)<br />
56.96<br />
(156.7)<br />
0.092<br />
(0.25)<br />
0.298<br />
(0.82)<br />
NS<br />
1.20<br />
(3.37)<br />
66.64<br />
(187.3)<br />
0.059<br />
(0.17)<br />
0.144<br />
(0.40)<br />
NS<br />
0.004<br />
(0.93)<br />
0.88<br />
(190.7)<br />
0.004<br />
(0.82)<br />
0.002<br />
(0.47)<br />
NS<br />
1.02<br />
(3.39)<br />
2561.12<br />
(8485.1)<br />
1.28<br />
(4.23)<br />
0.15<br />
(0.49)<br />
NS<br />
0.91<br />
(3.03)<br />
40.95<br />
(135.6)<br />
1.00<br />
(3.33)<br />
0.044<br />
(0.15)<br />
NS<br />
0.003<br />
(0.16)<br />
5.56<br />
(357.9)<br />
0.010<br />
(0.66)<br />
0.009<br />
(0.58)<br />
NS<br />
0.02<br />
(0.06)<br />
2.98<br />
(9.84)<br />
0.25<br />
(0.82)<br />
0.26<br />
(0.87)<br />
NS<br />
0.004<br />
(0.45)<br />
0.082<br />
(9.4)<br />
0.001<br />
(0.02)<br />
0.001<br />
(0.09)<br />
NS<br />
1.39<br />
(1.21)<br />
82.78<br />
(72.25)<br />
0.001<br />
(0.00)<br />
0.002<br />
(0.001)<br />
NS<br />
0.009<br />
(3.0)<br />
0.352<br />
(140.7)<br />
0.000<br />
(0.04)<br />
0.001<br />
(0.25)<br />
NS<br />
96.68<br />
(1.70)<br />
28576.09<br />
(504.4)<br />
40.19<br />
(0.71)<br />
48.58<br />
(0.85)<br />
NS<br />
Error 22 1.00 0.021 0.364 0.356 0.005 0.302 0.302 0.016 0.30 0.009 0.146 0.003 56.65<br />
HRVO-1 × CIM 446<br />
Replication 2 1.12<br />
(0.55)<br />
Generation 5 62.98<br />
(31.10)<br />
Location 1 7.70<br />
(3.80)<br />
G × L 5 1.42<br />
(0.70)<br />
NS<br />
0.00<br />
(0.03)<br />
0.023<br />
(24.04)<br />
0.004<br />
(4.20)<br />
0.001<br />
(0.56)<br />
NS<br />
0.18<br />
(0.07)<br />
17.82<br />
(7.26)<br />
5.53<br />
(2.25)<br />
6.45<br />
(6.10)<br />
NS<br />
0.235<br />
(0.64)<br />
25.95<br />
(71.3)<br />
0.340<br />
(0.93)<br />
0.280<br />
(0.57)<br />
NS<br />
0.00<br />
(0.22)<br />
0.650<br />
(335.66)<br />
0.009<br />
(4.25)<br />
0.003<br />
(1.75)<br />
NS<br />
3.29<br />
(3.33)<br />
1210.5<br />
(1215.8)<br />
3.32<br />
(3.36)<br />
1.26<br />
(1.27)<br />
NS<br />
0.16<br />
(1.30)<br />
9.74<br />
(78.97)<br />
0.82<br />
(6.66)<br />
0.175<br />
(1.42)<br />
NS<br />
0.24<br />
(4.70)<br />
12.54<br />
(3.2)<br />
0.032<br />
(0.42)<br />
0.071<br />
(0.95)<br />
NS<br />
0.43<br />
(3.08)<br />
6.30<br />
(45.70)<br />
0.25<br />
(1.80)<br />
0.23<br />
(0.17)<br />
NS<br />
0.035<br />
(1.12)<br />
0.60<br />
(19.32)<br />
0.051<br />
(1.61)<br />
0.036<br />
(1.14)<br />
NS<br />
0.188<br />
(3.36)<br />
4.64<br />
(83.65)<br />
0.095<br />
(1.71)<br />
0.025<br />
(0.45)<br />
NS<br />
0.00<br />
(0.08)<br />
0.69<br />
(362.8)<br />
0.00<br />
(0.18)<br />
0.001<br />
(0.38)<br />
NS<br />
4.62<br />
(0.98)<br />
39653.05<br />
(8433.6)<br />
20.00<br />
(4.25)<br />
0.78<br />
(0.17)<br />
NS<br />
Error 22 2.02 0.001 2.45 0.364 0.002 0.99 0.123 0.075 0.14 0.031 0.056 0.002 4.70<br />
HRVO-1 × Acala 63-74<br />
Replication 2 4.02 0.09 0.112 0.34 0.005 0.55 0.098 0.006 0.03 0.02 0.04 0.001 158.12<br />
(0.73) (1.38) (0.71) (0.34) (1.23) (0.13) (0.22) (0.19) (0.74) (0.69) (0.75) (0.45) (3.29)<br />
Generation 5 71.26 1.87 33.19 12.93 1.55 1268.54 8.80 0.136 0.36 0.42 0.88 0.36 33179.38<br />
(12.9) (28.7) (210.5) (13.21) (419.9) (301.9) (20.10) (4.54) (8.48) (14.45) (16.35) (181.3) (691.7)<br />
Location 1 92.18<br />
(16.79)<br />
G × L 5 3.36<br />
(0.61)<br />
NS<br />
0.06<br />
(0.90)<br />
0.001<br />
(0.02)<br />
NS<br />
0.67<br />
(4.24)<br />
0.29<br />
(1.85)<br />
NS<br />
4.16<br />
(4.24)<br />
1.19<br />
(1.22)<br />
NS<br />
0.002<br />
(0.63)<br />
0.006<br />
(1.57)<br />
NS<br />
0.16<br />
(0.04)<br />
2.00<br />
(0.47)<br />
NS<br />
0.63<br />
(1.43)<br />
0.204<br />
(6.80)<br />
0.10<br />
(2.40)<br />
0.001<br />
(0.02)<br />
0.14<br />
(2.63)<br />
0.041<br />
(20.30)<br />
Error 22 5.49 0.065 0.16 0.97 0.004 4.20 0.44 0.030 0.04 0.029 0.05 0.002 47.96<br />
0.162<br />
(0.37)<br />
NS<br />
0.022<br />
(0.74)<br />
NS<br />
0.02<br />
(0.51)<br />
NS<br />
0.029<br />
(0.98)<br />
NS<br />
0.04<br />
(0.78)<br />
NS<br />
0.00<br />
(0.21)<br />
NS<br />
30.14<br />
(0.62)<br />
3.95<br />
(0.08)<br />
NS<br />
3.44 5.72<br />
2.66 3.99<br />
4.30 7.95<br />
2.66 3.99<br />
3.44 5.72<br />
2.66 3.99<br />
4.30 7.95<br />
2.66 3.99<br />
3.44 5.72<br />
2.66 3.99<br />
4.30 7.95<br />
2.66 3.99<br />
PH = Plant height, NMB = no. of monopodial branches/plant, NSB = no. of sympodial branches/plant, NBP = no. of bolls/plant, SCY = seed cotton yield, BWt = boll weight, L% = lint percentage, FL =<br />
fibre length, FS = fibre strength, FE = fibre elongation, U% = fibre uniformity ratio, FF = fibre fineness.
Number of plants<br />
Number of plants<br />
Number of plants<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
0<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
P 2<br />
a) HRVO x FH 1000<br />
P 2<br />
B 2<br />
F 1<br />
F 2<br />
40 60 80 100 120 140 160 180 200 220 240<br />
b) HRVO x CIM 446<br />
B 2<br />
F 1<br />
F 2<br />
40 60 80 100 120 140 160 180 200 220 240<br />
c) HRVO x Acala 63-74<br />
P 2<br />
B 2<br />
40 60 80 100 120 140 160 180 200 220 240<br />
Figure 3. Frequency distributions for number of leaf trichomes in F2 generations of three crosses..<br />
B 1<br />
B 1<br />
F 2<br />
F 1<br />
B 1<br />
Nawab et al. 3969<br />
P 1<br />
P 1<br />
P 1
3970 Afr. J. Biotechnol.<br />
Table 4: Chi-Squared values and probabilities of goodness of fit of segregation ratios of F2 and backcross generations in a study of inheritance of leaf<br />
trichomes trait<br />
Cross Gen. Expected<br />
Ratios<br />
HRVO-1 × FH 1000<br />
HRVO-1 × CIM 446<br />
HRVO-1 × Acala 63-74<br />
Sparse<br />
Hairy<br />
Observed value Expected value<br />
Intermediate<br />
Hairy<br />
Pilose<br />
Velvet<br />
Sparse<br />
Hairy<br />
Intermediate<br />
Hairy<br />
Pilose<br />
Velvet<br />
χ 2 Prob.<br />
F2 1: 2:1 34 79 37 37.5 75 37.5 0.55 0.90-0.75<br />
BC1 1:1 - 53 37 - 45 45 2.84 0.10-0.05<br />
BC2 1:1 36 54 - 45 45 - 3.60 0.10-0.05<br />
F2 1: 2:1 41 69 40 37.5 75 37.5 0.97 0.75-0.50<br />
BC1 1:1 - 37 53 - 45 45 2.84 0.10-0.05<br />
BC2 1:1 48 42 - 45 45 - 0.40 0.75-0.50<br />
F2 1: 2:1 35 74 41 37.5 75 37.5 0.50 0.90-0.75<br />
BC1 1:1 - 47 43 - 45 45 0.18 0.75-0.50<br />
BC2 1:1 52 38 - 45 45 - 2.18 0.25-0.10<br />
Table 5: Genotypic (upper value) and phenotypic (lower value) correlations along with the probability values given in parenthesis for<br />
number of trichomes and agronomic/fibre traits in three cotton crosses.<br />
Trait 1 2 3<br />
Number of Trichomes<br />
L %<br />
0.85**<br />
(0.00)<br />
0.81**<br />
(0.00)<br />
FL<br />
-0.96**<br />
(0.00)<br />
-0.71**<br />
(0.00)<br />
FS<br />
-0.98**<br />
(0.00)<br />
-0.86<br />
(0.00)<br />
FE<br />
-0.83**<br />
(0.00)<br />
-0.71**<br />
(0.00)<br />
U% -0.83**<br />
(0.00)<br />
-0.73**<br />
(0.00)<br />
FF<br />
0.69*<br />
(0.040)<br />
0.56*<br />
(0.048)<br />
0.92**<br />
(0.00)<br />
0.91**<br />
(0.00)<br />
-0.95**<br />
(0.00)<br />
-0.95**<br />
(0.00)<br />
-0.98**<br />
(0.00)<br />
-0.96**<br />
(0.00)<br />
-0.95**<br />
(0.00)<br />
-0.74**<br />
(0.00)<br />
-0.97**<br />
(0.00)<br />
-0.92**<br />
(0.001)<br />
0.62*<br />
(0.042)<br />
0.60*<br />
(0.040)<br />
0.90**<br />
(0.00)<br />
0.88**<br />
(0.00)<br />
-0.75**<br />
(0.00)<br />
-0.72**<br />
(0.00)<br />
-0.90**<br />
(0.00)<br />
-0.80**<br />
(0.00)<br />
-0.88**<br />
(0.00)<br />
-0.72**<br />
(0.00)<br />
-0.86**<br />
(0.00)<br />
-0.82**<br />
(0.00)<br />
0.63*<br />
(0.045)<br />
0.60*<br />
(0.040)<br />
Trait 1 2 3<br />
Number of Trichomes<br />
PH<br />
-0.35<br />
(0.064)<br />
-0.34<br />
(0.063)<br />
0.16<br />
NMB<br />
(0.133)<br />
0.15<br />
(0.149)<br />
NSB -0.18<br />
(0.105)<br />
-0.18<br />
(0.105)<br />
NBP -0.29<br />
(0.07)<br />
-0.28<br />
(0.068)<br />
SCY 0.27<br />
(0.22)<br />
0.26<br />
(0.18)<br />
BWt<br />
0.17<br />
(0.12)<br />
-0.18<br />
(0.11)<br />
-0.88**<br />
(0.00)<br />
-0.84**<br />
(0.00)<br />
-0.07<br />
(0.32)<br />
-0.06<br />
(0.34)<br />
0.38<br />
(0.07)<br />
0.30<br />
(0.10)<br />
-0.34<br />
(0.09)<br />
-0.23<br />
(0.058)<br />
0.22<br />
(0.06)<br />
0.18<br />
(0.10)<br />
0.15<br />
(0.14)<br />
0.12<br />
(0.20)<br />
L% = Lint percentage, FL = Fibre length, FS = Fibre strength, FE = Fibre elongation, U% = Fibre uniformity ratio,<br />
FF = Fibre fineness, PH = Plant height, NMB = No. of monopodial branches/plant, NSB = No. of sympodial branches/plant,<br />
NBP = No. of bolls/plant, SCY = Seed cotton yield, BWt = Boll weight.<br />
1 = HRVO-1 × FH-1000, 2 = HRVO-1 × CIM-446, 3 = HRVO-1 × Acala 63-74.<br />
*P < 0.05 = Significant, **P < 0.01 = Non-significant.<br />
However, the non-significant values of the generation ×<br />
location interactions (G × L) in the three cross combina-<br />
-0.12<br />
(0.20)<br />
-0.11<br />
(0.22)<br />
-0.29<br />
(0.08)<br />
-0.28<br />
(0.09)<br />
-0.32<br />
(0.08)<br />
-0.29<br />
(0.061)<br />
-0.26<br />
(0.10)<br />
-0.21<br />
(0.07)<br />
-0.35<br />
(0.25)<br />
-0.32<br />
(0.19)<br />
-0.22<br />
(0.072)<br />
-0.24<br />
(0.081)<br />
tions further confirmed the authenticity of the results as<br />
these results were independent of the environmental
influence. The intermediate (H2h2) progeny in F1 was due<br />
to incomplete dominance. The two homozygous extremes<br />
for trichomes/hairiness : pilose hairy (H2H2) and<br />
sparse/normal hairy (h2h2) were easily distinguishable<br />
(Figure 3). The F2 data regarding number of trichomes<br />
were categorized into three main classes (Table 4):<br />
pilose hairy (H2H2), sparse/normal hairy (h2h2) and intermediate<br />
hairy (H2h2) (Simpson, 1947). The major gene is<br />
designated as H1 for sparse hairing. A second major<br />
gene, H2 controls the finely dense pubescence in an<br />
upland mutant designated as ‘pilose’. In the F1 populations,<br />
both H1 and H2 showed incomplete dominance<br />
(Niles, 1980; Rahman and Khan, 1998). The segregation<br />
in the backcrosses with parent-I and parent-II also fit to<br />
the theoretical ratio of 1:1 which further confirmed the<br />
incomplete dominance pattern of inheritance. The nonsignificant<br />
chi-square values in F2 for trichomes in all the<br />
crosses fit well against the theoretical monohybrid ratio of<br />
1:2:1.This ratio is in proximity to the understanding gotten<br />
from Figure 3a, suggesting that inheritance of this trait<br />
can be manipulated easily in a breeding programme.<br />
However, a very small proportion of plants fell in another<br />
interme-diately resembling hairiness category. This<br />
phenotypic expression of the intermediate hairy state in<br />
hetero-zygous condition was probably affected by the<br />
genetic background of the parents indicating, modifying<br />
gene effects (Falconer and Mackay, 1996; Rieseberg et<br />
al., 1996; Xu et al., 1997; Rahman and Khan, 1998;<br />
Schwarz-Sommer et al., 2003).<br />
Phenotypic and genotypic correlations<br />
Correlation in plant breeding is a useful tool of indirect<br />
selection of the secondary trait with the improvement in<br />
the primary trait. Correlation coefficient is a statistical<br />
measure which is used to find out the degree and<br />
direction of the relationship between two or more variables.<br />
A positive value gives the indication of the same direction<br />
of the two variables in question and vice-versa. Fibre<br />
quality traits hold a special position from the cotton<br />
spinning industry. It was therefore, necessary to study the<br />
effect of this insect non-preference trait (trichomes) on<br />
the fibre quality determining traits. Genotypic correlations<br />
indicated correlation between the two characters (Table<br />
5). Similar type of magnitude was observed by Dhanda et<br />
al. (1984) and Tyagi (1987) for genetic correlations. The<br />
higher values of phenotypic correlation coefficients than<br />
the genotypic correlation coefficients where ever present,<br />
indicating that the correlation between the two characters<br />
was not only due to genes, but environment also played<br />
its role in the expression of the character. The fibre<br />
fineness is recorded in micronaire value. This means that<br />
the higher the magnitude of micronaire value, the lesser<br />
will be the fineness of the fibre and vice-versa. Significant<br />
correlation between the two characters gave dependency<br />
of the two characters, while non-significant correlation<br />
indicated the independent nature of the two characters<br />
Nawab et al. 3971<br />
under study as suggested by Singh and Narayanan<br />
(2000).<br />
The positive correlation of number of trichomes with lint<br />
percentage in all of three crosses (Table 4) suggested<br />
that the selection for pilose hairiness would be helpful in<br />
enhancing the lint percentage. This result is in line with<br />
that of Lee (1984) and Kloth (1993). The negative<br />
correlation of fibre length, fibre strength, fibre uniformity<br />
ratio, fibre elongation and micronaire value (positive<br />
correlation of number of trichomes with fibre fineness)<br />
with number of trichomes in the crosses corroborated to<br />
the findings of Lee (1964, 1984). The negative correlation<br />
of hairiness/trichomes with these mentioned traits determining<br />
fibre quality gave an understanding that the<br />
presence of the genes controlling these traits might be<br />
present on the same chromosome. Reports are also<br />
available regarding the pilosity with shorter fibre (Kohel et<br />
al., 1967, Lee, 1964, 1984). Association of hairiness with<br />
short fibre length was supposed to be gene pleiotropy as<br />
it was proposed by Simpson (1947) but the later studies<br />
by Kloth (1993) proved this association to be a linkage<br />
between the genes affecting hairiness and fibre quality<br />
determining traits.<br />
The negative correlation of trichomes with plant height<br />
in HRVO-1 × CIM 446 suggest that increase in hairiness<br />
state would decrease the plant height but otherwise,<br />
there would be no effect on other morphological traits.<br />
Yield determining traits like number of monopodial<br />
branches, number of sympodial branches, number of<br />
bolls, boll weight and seed cotton yield are components<br />
of an efficient breeding programme. Non-significant<br />
correlations of these traits with number of trichomes/<br />
hairiness revealed that hairiness or number of trichomes<br />
had no effect on the expression of these agronomic/yield<br />
related traits. In contrast to the effect of number of<br />
trichomes on fibre related traits, a well documented proof<br />
is available but the effect of the same on yield<br />
determining traits was proved in the present manuscript<br />
as a step forward in the research on trichomes. Incorporation<br />
of the gene for hairiness into commercial cotton<br />
cultivars will not only limit the population of the sucking<br />
insects but also result in the improvement of the fibre<br />
attributes without disturbing the major traits relating to<br />
yield (number of monopodial branches, number of<br />
sympodial branches, number of bolls, boll weight and<br />
seed cotton yield).<br />
Keeping in view, the bio-safety requirements of the<br />
emerging era and the available knowledge of the<br />
naturally conferring resistance against insects, the<br />
present studies were performed to investigate the genetics<br />
of hairiness/trichomes in relation with the agronomic and<br />
fibre related traits in cotton. The main conclusions from<br />
this work are the non-significant chi-square values in F2<br />
for trichomes in all the crosses which fit well against the<br />
theoretical monohybrid ratio of 1:2:1. The positive<br />
correlation of number of trichomes with lint percentage<br />
and micronaire (negative correlation of number of<br />
trichomes with fibre fineness) in all the three crosses
3972 Afr. J. Biotechnol.<br />
suggested that the selection for pilose hairiness would be<br />
helpful in enhancing the lint percentage and fibre<br />
fineness.<br />
The present findings elaborated the novelty of the<br />
research with a confidence that incorporation of the gene<br />
for pilosity had no effect on the expression of these<br />
agronomic/yield related traits.<br />
Acknowledgement<br />
The authors are thankful to Dr. Muhammad Shafique<br />
Tahir, Assistant Botanist and Dr. Noor-Ul-Islam, Director,<br />
Cotton Research Institute, Ayub Agricultural Research<br />
Institute, Faisalabad, Pakistan for providing the<br />
germplasm as well as support in basic field<br />
operations.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 3973-3977, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2304<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Quick and sensitive determination of gene expression<br />
of fatty acid synthase in vitro by using real-time<br />
polymerase chain reaction amplification (PCR)<br />
Zuo-Hua Liu 1,2 , Jin-Long Yang 1,2 , Fei-Yun Yang 1 , Ding-Biao Long 1 , Ren-Yong Jia 2 , Kang-<br />
Cheng Pan 2 * and Dai-Wen Chen 3<br />
1 Chongqing Academy of Animal Science, Chongqing, 402460, Chongqing China.<br />
2 College of Veterinary Medicine of Sichuan Agricultural University, Yaan 625014, Sichuan Province, China.<br />
3 Institute of Animal Nutrition, Sichuan Agricultural University, Yaan 625014, Sichuan Province, China.<br />
Accepted 17 March, 2011<br />
Obesity results from an imbalance between energy intake and energy expenditure, which leads to a<br />
pathological accumulation of adipose tissue, but the underlying mechanism at gene level, is far from<br />
being elucidated. The objective of this study was to investigate the correlation between mRNA express<br />
from fatty acid synthase (FAS) with a different glucose level in primary adipocytes by real-time<br />
polymerase chain reaction amplification (PCR), which can aid in the understanding of the mechanism of<br />
obesity in vitro. By using the following formula, this study was able to quantify the mRNA expression of<br />
FAS of unknown samples: Y = -3.156X + 41.21 (Y = threshold cycle, X = log starting quantity). The high<br />
concentrations of glucose group significantly improved the mRNA expression of FAS (P < 0.01) rather<br />
than 0.25 and 0% concentrations of glucose. These results provide significant data that confirm an<br />
association between different glucose level and FAS expression in preadipocytes. The glucose<br />
concentration of the high group substantially augmented the mRNA expression of FAS.<br />
Key words: Expression, fatty acid synthase, lipid deposition, real-time polymerase chain reaction amplification<br />
(PCR).<br />
INTRODUCTION<br />
Excessive weight gain can be attributed to the interactions<br />
among environmental factors (dietary intake and<br />
physical activity), genetic predisposition and the<br />
individual behaviors (Campion et al., 2009; Lomba et al.,<br />
2010). Thus, genetic predisposition is an important determinant<br />
of the increasing prevalence of metabolic<br />
syndrome and associated complications such as obesity<br />
(Stone et al., 2002; Speakman, 2007; Dorn et al., 2010).<br />
Obesity is strongly associated with the metabolic<br />
syndrome and related to many diseases such as<br />
hypertension, heart disease and diabetes. Obesity results<br />
from an imbalance between energy intake and energy<br />
expenditure, leading to a pathological accumulation of<br />
adipose tissue (Kopelman, 2000). It has a heterogeneous<br />
*Corresponding author. E-mail: 391556428@qq.com. Tel: +86-<br />
23-46792362. Fax: +86-23-46792362.<br />
phenotype with the involvement of multiple genes and<br />
their interactions with non-genetic factors (Vladimir et al.,<br />
1997). The accumulation of fat is caused by hypertrophy<br />
and hyperplasia of adipocytes. Therefore, an understanding<br />
of the molecular basis of hyperplasia as well as<br />
hypertrophy would contribute to the establishment of<br />
medical treatments for preventing health risks that can<br />
cause serious illnesses and death (Hishida et al., 2007).<br />
One explanation for the increase in the number of<br />
adipocytes is that, the uptake of exogenously derived<br />
fatty acids (FA) and de novo of biosynthesis of FA are<br />
increased and the hydrolysis of acylglycerols is<br />
decreased. Among the different mechanisms that could<br />
lead to fat deposition and obesity, the epigenetic<br />
regulation of gene expression has emerged in recent<br />
years as a potentially important contributor (Campion et<br />
al., 2009; Lomba et al., 2010). Recent studies have revealed<br />
that, several proteins such as fatty acid synthase<br />
(FAS), a key enzyme regulating lipid metabolism, play
3974 Afr. J. Biotechnol.<br />
Table 1. Details of the primer pairs and cDNA concentration used for the FQ-PCR.<br />
Name 5′ sequence 3′ Amplicon size (bp) cDNA (µM)<br />
Forward(FAS -F) AGCCTAACTCCTCGCTGCAAT 196 1<br />
Reverse(FAS -R) TCCTTGGAACCGTCTGTGTTC 1<br />
TaqMan probe(FAS -P) (FAM) TCCTGCGGCATCCACGAGACCACC (Eclipse) 1.2<br />
The cDNA concentration refers to the final concentration of cDNA equal to reverse-transcribed RNA in each PCR.<br />
important roles in adipocyte metabolism (Lomba et al.,<br />
2010; Marie et al., 2010). The events occurring during the<br />
stages of adipocyte meta-bolism are relatively well<br />
characterized, but the molecular mechanisms underlying<br />
the stages of adipogenesis remain unknown.<br />
This study reported a fluorescent quantitative real-time<br />
polymerase chain reaction amplification (FQ-PCR) assay<br />
for quantifying FAS gene expression at different glucose<br />
levels in primary adipocytes. The results of this study<br />
provide interesting data that may help understand the<br />
molecular mechanisms underlying the stages of adipogenesis.<br />
MATERIALS AND METHODS<br />
Isolation of adipocyte precursors from pig adipose tissue<br />
Epididymal fat pads of healthy pigs (age, 18 to 20 days; Landrace)<br />
were removed under sterile conditions and transferred to ice-cold<br />
Dulbecco's modified eagle medium containing penicillin, streptomycin<br />
and fungizone (Sangon Biological Engineering Technology<br />
and Services Co., Ltd, Shanghai, China). Preadipocytes were<br />
isolated as described by Janke et al. (2002).<br />
Cells were cultured with different glucose contents<br />
Preadipocytes were incubated at 37°C in a humidified atmosphere<br />
with 5% CO2 as described by Janke et al. (2002). The cells were<br />
stimulated to proliferate over 24 h and then, glucose was added at<br />
various concentrations to the culture medium (glucose-free<br />
adipocyte medium, supplemented with 1 µM insulin; 0.25% glucose<br />
adipocyte medium, supplemented with 1 µM insulin and 0.5%<br />
glucose adipocyte medium, supplemented with 1 µM insulin).<br />
Gene expression analyzed using FQ-PCR<br />
RNA isolation and FQ-PCR<br />
Preadipocytes (1 ml) were trypsinized with 1 ml digestive, washed<br />
once with PBS, and pelleted (380 g, 10 min) after glucose was<br />
added at various concentrations to the culture medium at 2, 4, 8, 16<br />
and 32 h. Total RNA was extracted with TRIzol (Invitrogen,<br />
Carlsbad, CA) according to the manufacturer’s instructions. An ABI<br />
PRISM 7000 sequence detection system (Applied Biosystems,<br />
Foster City, CA) was used for FQ-PCR. The reaction mixture was<br />
prepared using the universal PCR master mix (Applied Biosystems)<br />
according to the manufacturer’s instructions. The mixture was<br />
incubated at 50°C for 5 min and at 94°C for 40 s and then, the PCR<br />
was performed by 34 cycles at 94°C for 40 s and at 59°C for 40 s,<br />
72°C for 40 s and an additional extension period at 72°C for 2 min.<br />
Melting curve from 65 to 95°C read every 0.5°C hold 5 s. Relative<br />
standard curves were generated in each experiment to calculate<br />
the input amounts of the unknown samples. Sensitivity and<br />
specificity analysis of the assay was as described by Yang et al.<br />
(2009).<br />
Specific RT-PCR primer and probe design<br />
Expression analysis was performed for the genes encoding FAS<br />
published sequences at GenBank. The oligonucleotide primer set<br />
for the gene was designed using Primer Premier 5.0 software. The<br />
sequences and concentrations of primers and cDNA are shown in<br />
Table 1. The specificity of all the primers was tested by searching<br />
the homologous DNA sequences against the GenBank BLAST<br />
database.<br />
Preparation of standard templates<br />
The conventional RT-PCR was performed for standard RNA using<br />
the primers (FAS-F, FAS-R) mentioned earlier. The standard template<br />
was prepared as described by Yang et al. (2008).<br />
Establishment of the FQ-PCR standard curve<br />
Standard plasmids (1 × 10 9 copies/µl) were used to establish the<br />
standard curve .The Primers (FAS-F, FAS-R and FAS-P) were used<br />
for this amplification and their concentrations ranged from 1 × 10 8 to<br />
1 × 10 2 gene copies/µl. A regression curve was constructed by<br />
plotting the threshold cycle (Ct) values versus the logarithm of the<br />
RNA copy number. Analysis of the measures was undertaken after<br />
each run to verify identical amplification efficiencies and conditions<br />
between runs.<br />
Finally, based on the data for generating the standard curve with<br />
the software of the iCycler IQ Detection System (Bio-Rad, USA), Ct<br />
values for each sample were determined from the point at which<br />
fluorescence breached the threshold fluore-scence line (Yang et al.,<br />
2008; Yang et al., 2009).<br />
Statistical analysis<br />
Data were expressed as means ± SDs. Statistical significance of<br />
the differences was determined using Student’s t-test. P values less<br />
than 0.01 were considered statistically significant (Kang et al.,<br />
2010).<br />
RESULTS<br />
Standard curve of the established FQ-PCR<br />
The standard curve of FQ-PCR was constructed using
Liu et al. 3975<br />
Figure 1. FAS gene FQ-PCR standard curve graph. A, standard curve of FAS by the FQ-PCR: B, dissociation curve for FAS. Ten-fold<br />
dilutions of standard DNA ranging from 1×10 8 to 1×10 2 copies/µl were used, as indicated in the x-axis, whereas the corresponding cycle<br />
threshold (Ct) values are presented on the y-axis. Each dot represents the result of triplicate amplification of each dilution. The correlation<br />
coefficient and the slope value of the regression curve were calculated and are indicated.<br />
serially diluted plasmids with tenfold dilution, ranging from<br />
10 8 to 10 2 copies as templates. The correlation coefficients<br />
of the standard curve was 0.9998 and the PCR<br />
efficiencies was greater than 0.95 (Figure 1). The high<br />
PCR reaction efficiencies indicated that, the FQ-PCR<br />
assay was suitable for genomic DNA quantification. The<br />
good linearity between DNA quantities and fluorescence<br />
values (Ct) indicated that, the assay was well suitable for<br />
quantitative measurements. By using the following<br />
formula, this study could quantify the mRNA expression<br />
of FAS of unknown samples: Y = -3.156X + 41.21 (Y =<br />
threshold cycle, X = log starting quantity).<br />
Sensitivity, specificity analysis of the established FQ-<br />
PCR<br />
Ten-fold dilutions of the standard templates were tested<br />
by the established FQ-PCR assay to evaluate the sensitivity<br />
of the system and the detection limit was found to<br />
be 1.0 × 10 1 copies/reaction (data no shown).<br />
The test was performed using DNA from standard RNA<br />
and several other bacteria as templates to examine its<br />
specificity; the result of this analysis showed that, none of<br />
the bacteria yielded any amplification signal, suggesting<br />
that the established FQ-PCR assay was highly specific<br />
(data no shown).<br />
Dynamic changes in FAS gene expression in<br />
adipocyte<br />
The dynamic changes in FAS gene expression in adipocytes<br />
after glucose was added at various concentrations<br />
to the culture medium were intermittently determined over<br />
a 32 h period by FQ-PCR. The study results revealed<br />
that, the copy numbers of FAS of samples peaked at 2 h<br />
when the glucose concentration was 0, 0.25 and 0.5%,<br />
they were 4.05 ± 0.86, 4.84 ± 0.62, 5.75 ± 0.88 (log<br />
copies/µl), respectively. The glucose concentration of the<br />
high group substantially augmented the mRNA<br />
expression of FAS (P < 0.01) (Figure 2).<br />
DISCUSSION<br />
FQ-PCR has become a potentially powerful alternative<br />
for quantifying mRNA expression because it is simpler,<br />
more rapid, more reproducible and more sensitive than<br />
other methods (Shimazu et al., 2009; Brym et al., 2007;<br />
Calikoglu et al., 2006). In this study, the applicability of<br />
FQ-PCR for the quantification of FAS mRNA expression
3976 Afr. J. Biotechnol.<br />
Figure 2. Dynamic changes in FAS gene expression in adipocytes with different glucose concentration in different<br />
time.<br />
was clearly established by showing that, it has remarkable<br />
sensitivity and high-throughput potential, which are<br />
beyond the scope of other assay methods.<br />
FAS catalyze the last step in the fatty acid biosynthesis<br />
and thus, it is believed to be a major determinant to<br />
generate fatty acids by de novo lipogenesis (Dorn et al.,<br />
2010). It is clear that some genes would show an<br />
increase or decrease in response to glucose. The aim of<br />
this study was to analyze the expression of FAS, which is<br />
a central enzyme of lipogenesis with a different glucose<br />
level. This study revealed a significant correlation of FAS<br />
expression with the high degree of glucose in primary pig<br />
preadipocytes in vitro. Surprisingly, the copy numbers of<br />
FAS peaked at 2 to 8 h at various concentrations of<br />
glucose. At 0.5% concentration, glucose significantly augmented<br />
the mRNA expression of FAS (P < 0.01) when<br />
compared with 0.25 and 0% concentrations. These<br />
results finding is just an initiation of FAS in obesity to be<br />
elucidated. Further studies are required to unravel the<br />
relationship between nutrient and hormone and other<br />
gene such as hormone-sensitive lipase.<br />
Conclusion<br />
In conclusion, the study results confirm the association<br />
between different glucose levels and FAS expression in<br />
preadipocytes. At 0.5% concentrations, glucose significantly<br />
augmented the mRNA expression of FAS (P <<br />
0.01) rather than at concentrations of 0.25 and 0%. However,<br />
further research is needed to validate this conclusion.<br />
ACKNOWLEDGEMENTS<br />
This study was supported by The Chongqing Natural<br />
Science Funds (No. 2008BA1013); The Chongqing<br />
Natural Science Funds (No. 2009BA1078).<br />
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African Journal of Biotechnology Vol. 10(20), pp. 3978-3985, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2334<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Characterization of a chestnut FLORICAULA/LEAFY<br />
homologous gene<br />
Tao Liu 1 *, Yun-qian Hu 2 and Xiao-xian Li 2<br />
1 Faculty of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan, 650201, China.<br />
2 Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan, 650204, China.<br />
Accepted 24 March, 2011<br />
The FLORICAULA/LEAFY (FLO/LFY) homologues’ genes are necessary for normal flower development<br />
and play a key role in diverse angiosperm species. In this paper, an orthologue of FLORICAULA/LEAFY,<br />
CmLFY (chestnut FLO/LFY), was isolated from the inflorescence of chestnut trees. Its expression was<br />
detected in various tissues. Furthermore, the flowering effectiveness of the gene was assessed with<br />
transgenic Arabidopsis. CmLFY protein showed a high degree of similarity to PEAFLO (78%), which is a<br />
homologue of FLO/LFY from pea. RT-PCR analysis showed that, CmLFY expressed at high levels in<br />
inflorescences, but not in young leaves, fruits or stems. The transgenic Arabidopsis with<br />
over-expressed CmLFY showed accelerated flowering, which supports that CmLFY encodes a functional<br />
orthologue of the FLORICAULA/LEAFY genes of angiosperms despite its sequence divergence. These<br />
results suggest that CmLFY is involved in inflorescence development in chestnut.<br />
Key words: Chestnut, homologue, FLORICAULA/LEAFY.<br />
INTRODUCTION<br />
Two developmental programmes can be distinguished<br />
during the life cycle of flowering plants. During the initial<br />
vegetative phase, the apical meristem produces leaves<br />
and lateral shoot. After floral induction, the plant enters<br />
into a reproductive phase and the apical meristem chan-<br />
ges its developmental pattern and initiates the production<br />
of flowers. In Antirrhinum and Arabidopsis this transition<br />
from vegetative to reproductive development pattern<br />
requires the establishment of floral meristem identity in<br />
the lateral meristems of the inflorescence, a process<br />
which has been demonstrated to be controlled by the<br />
homo- logous genes FLORICAULA (FLO) and LEAFY<br />
(LFY), respectively (Ma, 1998; Pidkowich et al., 1999).<br />
LFY is a transcriptional regulation gene thought to play a<br />
primary role in determining flower meristem identity.<br />
FLORICAULA (FLO), a LFY homologue in snapdragon,<br />
has been shown to have almost the same role. Loss func-<br />
* Corresponding author. E-mail: beixue2007@gmail.com. Tel:<br />
+86-871 5227721.<br />
Abbreviations: PCR, Polymerase chain reaction; RT-PCR,<br />
reverse transcriptase-PCR; RACE, rapid amplified cDNA ends.<br />
tion mutants of these genes result in the conversion of<br />
flowers into indeterminate secondary shoots (Coen et al.,<br />
1990; Weigel et al., 1992). The over-expression of LFY<br />
under a constitutive promoter in Arabidopsis caused early<br />
flowering and converted all lateral shoots into solitary<br />
flowers. In heterologous plants such as aspen, LFY has<br />
been shown to have effects on the acceleration of flower-<br />
ing and induction of ectopic flowers (Weigel and Nilsson,<br />
1995; Rottman et al., 2000), suggesting a conservation of<br />
LFY function across long phylogenetic distances within<br />
angiosperms. These reports strongly suggest that, the<br />
ability to control the expression of LFY or of its orthologs<br />
from other plants, could make it possible to artificially<br />
induce various plants to blossom and even skip or shorten<br />
the juvenile phase of woody plants.<br />
Flowering is an essential stage for fruit production and<br />
thus, an understanding of the genetic mechanisms under-<br />
lying the flowering event is important for efficient fruit<br />
production. During the last decade, molecular mecha-<br />
nisms of flowering have been studied extensively in<br />
herbaceous ‘‘model’’ plants such as Arabidopsis and<br />
Antirrhinum. In contrast, studies on the molecular<br />
mechanisms of flowering in fruit trees have just begun.<br />
Although, several flowering-related FLO/LFY orthologs<br />
have been cloned from kiwifruit (Actinidia deliciosa)
(Walton et al., 2001), grapevine (Vitis vinifera) (Carmona<br />
et al., 2002), and apple (Malus domestica) (Kotoda et al.,<br />
2002), their specific role in the induction of the<br />
characteristic features of tree reproductive development is<br />
still being elucidated.<br />
Trees are characterized by an extended adolescence.<br />
This is particularly important for commercial fruit/nut tree<br />
growers and breeders, because prolonged juvenile<br />
periods delay harvesting and the evaluation/breeding of<br />
new strains. Chestnut (Castanea mollissima) is one of the<br />
most commercially valuable fruit trees, with production<br />
once only annually. Chestnut has an extended juvenile-<br />
vegetative phase, during which vegetative growth is<br />
maintained. Attempts to exploit existing knowledge of the<br />
genetic control of flower development in Arabidopsis to<br />
engineer nut trees could have significant economic and<br />
scientific implications for the tree fruit industry. As yet, no<br />
studies have reported on the molecular mechanism<br />
underlying the development of chestnut flowers. Recently,<br />
MADS-box genes have been cloned and characterized in<br />
chestnut by us (Liu et al., 2006). Our results suggest that<br />
these chestnut MADS genes may be involved in floral<br />
organ and fruit development in chestnut. In this paper, we<br />
identified a chestnut FLORICAULA/LEAFY homologous<br />
gene involved in floral development. We described the<br />
isolation and characterization of CmLFY (DQ270548).<br />
The function of CmLFY was demonstrated by transgenic<br />
Arabidopsis, as expected for normal plants, transgenic<br />
plants showed earlier flowering phenotype, indicating that<br />
CmLFY may play a similar role of promoting flowering as<br />
the FLORICAULA/LEAFY genes in flower development.<br />
MATERIALS AND METHODS<br />
Plant materials<br />
The chestnut and Arabidopsis ecotype Columbia (Col) were used in<br />
this study. Chestnut examples were collected from Kunming,<br />
Yunnan, People’s Republic of China and were frozen in liquid<br />
nitrogen immediately and stored at -80°C.<br />
RNA extraction and cDNA cloning<br />
Total RNA was extracted from the inflorescences using a modified<br />
CTAB method (Zeng and Yang, 2002). The cDNA was prepared<br />
using Smart cDNA library construction kit (BD Biosciences Clontech,<br />
American). Full-length CmLFY cDNA was obtained by the 5 and 3<br />
rapid amplified cDNA ends (RACE) method (Chenchik et al., 1996).<br />
The first amplified chestnut cDNA contained about 440 bp between<br />
primers CmS and CmA, designed from Arabidopsis thaliana LFY<br />
(Weigel et al., 1992) and Antirrhinum majus FLO (Coen et al., 1990)<br />
cDNA sequences. The 3 RACE was carried out between cassette<br />
CDSIII/3’ primer and CmS2 primer. The DNA fragment amplified<br />
was cloned into PMD18-T vector (Takara, Japan). The 5 RACE was<br />
carried out between cassette CDSIII/5’ primer and CmA2 primer.<br />
Full-length cDNAs were amplified with the sense primer LFY-ORF5<br />
and antisense primer CDSIII/3’ primer. These clones were sequenced<br />
completely by the dideoxy method using ABI3730 automated<br />
sequencer (ABI Company, USA).<br />
Phylogenetic comparisons<br />
Liu et al. 3979<br />
Protein sequences of different LFY homologs were retrieved from<br />
GenBank and aligned with CLUSTAL. Phylogenetic relationships<br />
among these genes were inferred by NJ analysis (MEGA Ver.3.1).<br />
Plant transformation<br />
The full length CmLFY (including start codon ATG) was amplified<br />
between LFY-ORF5 and LFY-ORF3 (Figure 1). Then, the amplified<br />
cDNA was blunted and KpnI and SalI linkers were ligated both ends,<br />
cloned into a pCambia2301-101 binary vector in a sense-oriented<br />
manner under the CaMV 35S promoter. An Agrobacterium<br />
tumifaciens GV3101 strain (Van-Larebeke et al., 1974) was used for<br />
the transformation of the A. thaliana Columbia ecotype by the<br />
floral-dip method (Clough and Bent, 1998). For the selection of<br />
transformed plants, resultant seeds were planted on a 1/2MS culture<br />
medium containing kanamycin (50 g mL --1 ) as selective antibiotics<br />
and then, transferred to 22°C under LD conditions (16 h light, 8 h<br />
dark). Transformants were identified as kanamycin-resistant when<br />
seedlings in the medium produced green leaves and well-<br />
established roots. Resistant transformants were transplanted to<br />
moistened potting soil composed of vermiculite and perlite (1:1<br />
(v/v --1 )) after two to four adult leaves had developed and their flower<br />
phenotypes were observed under long-day conditions.<br />
RNA analysis of transgenic plants by RT-PCR<br />
Detection of CmLFY transcripts was performed by RT-PCR. RNA<br />
was isolated from whole plants of Arabidopsis by a method using<br />
CTAB described previously described. CmLFY specific transcripts<br />
were identified using 1 g of total RNA as a template and the<br />
following primers: a sense primer LFY-ORF5 and an antisense<br />
primer LFY-ORF3 for CmLFY (Table 1), giving rise to about 1300 bp<br />
long PCR product. PCR reactions were run for 35 cycles at 58°C for<br />
CmLFY. The PCR products were run on 1.5% (w/v --1 ) agarose gels<br />
stained with ethidium bromide.<br />
RESULTS<br />
Cloning and sequence analyses of CmLFY<br />
A comparison of Arabidopsis LFY (Weigel et al., 1992)<br />
and Antirrhinum FLO (Coen et al., 1990) showed the<br />
presence of several conserved regions. Two of these<br />
regions, CmS and CmA, were used to design degenerate<br />
oligo- nucleotide primers for RT-PCR. Using these<br />
primers with cDNA prepared from an inflorescence of<br />
chestnut, we obtained a PCR amplified fragment<br />
containing both primers and other conserved sequences.<br />
This fragment was named CmLFY (chestnut<br />
FLORICAULA/LEAFY homologue). To obtain a full-length<br />
CmLFY cDNA, several primers for 5’/3’ RACE were<br />
designed. Primer sites and the nucleotide sequences of<br />
primers used for cloning were shown in Figure 1 and<br />
Table 1, respectively.<br />
The coding region of CmLFY is 1,161 bp, encoding<br />
386 amino acids. The deduced amino acid sequence of<br />
CmLFY cDNA has 78% homology with PEAFLO, 79%<br />
with PtFL and 59.9% with PlaraLFY. NLF, PRFLL, PEAFLO,
3980 Afr. J. Biotechnol.<br />
Table 1. Primer sequences used in PCR cloning of CmLFY.<br />
Primer Oligonucleotide<br />
Primers for an internal fragment<br />
CmS sense primer<br />
CmA antisense primer<br />
Cassette primers<br />
CDSIII/5’ primer<br />
CDSIII/3’ primer<br />
3’ RACE primer<br />
CmS2 primer<br />
5’ RACE primer<br />
CmA2 primer<br />
CmLFY specific primers<br />
LFY-ORF5<br />
LFY-ORF3<br />
5’- GGGAGCACCCGTTCATTGTGACTG -3’<br />
5’-GA/CAGCTTG/TGTG/TGGGACATACCAGAC-3’<br />
5’-AAGCAGTGGTATCAACGCAGAGT-3’<br />
5’-ATTCTAGAGGCCGAGGCGGCCGACATG-3’<br />
5’-CGGCCTTGATTACCTCTTCCATCTC -3’<br />
5’- CACCGCGCTCCTTGGCAATGTTCTGT -3’<br />
5’-GGTACCAAGCTAGCTTCATTGATG -3’<br />
5’-CGGTCGACTAGAAATGCAAATTTTTCTC-3’<br />
Figure 1. Nucleotide and deduced amino sequences (single-letter code) of CmLFY cDNA. The asterisk (*) indicates a stop<br />
codon. Arrows above the sequences indicate the primers used for RACE-PCR.<br />
ELF1 and CmLFY lacked a proline-rich region in<br />
the variable region (roughly the first 40 amino acids). To<br />
more closely determine the evolutionary relationship<br />
between FLO/LFY-like proteins, a phylogenetic tree was<br />
con- structed (Figure 2b). This tree showed that, the<br />
topology of these genes seems to be concordant with the<br />
topology of the species phylogeny and suggests that<br />
CmLFY is an angiosperm ortholog of FLO/LFY-like genes.
a<br />
Liu et al. 3981
3982 Afr. J. Biotechnol.<br />
b<br />
Expression patterns of CmLFY<br />
Figure 2. (a) Sequence comparison of FLO/LFY-like proteins (accession numbers in<br />
parentheses): NLY and PRFLL from P. radiata (U76757 and U92008, respectively); CmLFY<br />
from chestnut (DQ270548); BOFH from B. oleracea (Z18362); FLO from A. majus (M55525);<br />
LFY from A. thaliana (M91208); NFL1 and NFL2 from N. tabacum (U16172 and U16174,<br />
respectively); ALF from P. hvbrida (AF030171); PtFL from P. balsamifera (U93196); RFL from<br />
O. sativa (AB005620); TOFL from L.esculentum (AF197934); PEAFLO from P. sativum<br />
(AF010190); ClLFY from C. glaziovii (AY633622); ELF1 from Eucalyptus globules (AF034806);<br />
JunefLFY from J. effusus (AF160481) and PlaraLFY from P. hispanica (AF106842). Black<br />
boxes indicate identical amino acids, shaded boxes indicate amino acids with similar<br />
properties, and dots indicate gaps introduced to optimize alignment. c1 and c2, conserved<br />
regions; v1 and v2, variable regions; (b) Protein sequence comparisons of CmLFY to other<br />
LFY/FLO homologs. The deduced amino acid sequence of CmLFY was compared with some<br />
published LFY homologs. Box characters represent chestnut protein.<br />
CmLFY mRNA expression was analyzed in various<br />
tissues by RT-PCR. Total RNA was isolated and PCR<br />
primers specific to CmLFY were used to detect the<br />
expression patterns in several tissues. The result showed<br />
that CmLFY mRNA was expressed in inflorescence<br />
tissues, but not in young leaves, fruits and stems (Figure<br />
3). The RT-PCR of CmLFY -specific primers resulted in<br />
amplification of a single band. Increasing the PCR cycle<br />
did not change these expression patterns. The amplified<br />
DNA fragments were the expected length.<br />
Ectopic expression in Arabidopsis<br />
To further verify the function of CmLFY, we constructed a<br />
binary vector pCambia2301-101-CmLFY containing full-<br />
length CmLFY coding area inserted in the sense-oriented<br />
direction under the control of the Cauliflower 35S<br />
promoter. The pCambia2301-101-CmLFY was introduced<br />
into wild- type Arabidopsis plants by<br />
Agrobacterium-mediated transformation. Fourteen<br />
independent transgenic plants that survived on kanamycin<br />
were identified (Table 2). Five (lines one-five) of them<br />
flowered earlier than the wild-type plants by five-seven
Figure 3. Expression pattern of CmLFY gene revealed by RT-PCR<br />
analysis. Lane 1: inflorescences; Lane 2: stem; Lane 3: tender leaves with<br />
no flowers; Lane 4: tender leaves with flowers; Lane 5: fruit. rRNA is used<br />
as RNA standard. Amplification of CmLFY was performed for 30 cycles.<br />
Table 2. Comparison of flowering time, number of rosette leaves in T1 transgenic and wild-type Arabidopsis (Col)<br />
plants in LD conditions.<br />
Transgenic line Days to flowering a Rosette leaves at time of flowering b Note<br />
1 18 4 Early flowering<br />
2 16 3 Early flowering<br />
3 18 4 Early flowering<br />
4 20 5 Early flowering<br />
5 20 4 Early flowering<br />
6 24 7<br />
7 24 8<br />
8 25 9<br />
9 22 8<br />
10 24 8<br />
11 25 8<br />
12 26 9<br />
13 26 10<br />
14 25 8<br />
wt c 25.5 d 9.5 d (n=10) e<br />
v-wt f 26 d 9.5 d (n=8) e<br />
a Days to flowering is defined as the time when flower primordial were first visible to the naked eye; b rosette leaves were<br />
counted on the day that flower primordial were first visible; c w wild-type Arabidopsis Columbia plants; d Mean number; e<br />
Number of plants;<br />
f vector-transformed plants.<br />
days and produced only four to five rosette leaves when<br />
they flowered (Figure 4 and Table 2). Most of the T1<br />
transgenic plants could self-pollinate and grow siliques<br />
normally and the resultant seeds also had the ability to<br />
germinate except for the lines 1, which is so small that it<br />
was difficult to obtain its seeds. The early flowering were<br />
inherited in the next generation and co- segregated with<br />
the kanamycin-resistant genes. The expression of the<br />
CmLFY mRNA in T1 plants was confirmed by RT-PCR, all<br />
putative transformed lines showed the expected products<br />
of CmLFY. No amplifi- cation was observed for the cDNA<br />
prepared from non-transformed Arabidopsis plants and<br />
vector-trans- formed Arabidopsis plants. The transgenic<br />
plants are stably transformed and the siblings which<br />
segregate without the transgene had no amplification of<br />
CmLFY and flowered at the same time as wild-type<br />
plants.<br />
DISCUSSION<br />
Liu et al. 3983<br />
Despite the importance of understanding the regulation of<br />
the flowering process in woody perennials for the<br />
management and improvement of woody species, very<br />
little is known about the underlying molecular<br />
mechanisms. Regulation of flowering in woody perennials<br />
shows remarkable differences in contrast to herbaceous<br />
species, that is, long juvenile phases, bud dormancy and<br />
the alternating vegetative and reproductive development<br />
according to the season.<br />
To investigate the molecular mechanism in chestnut<br />
flowering, we cloned CmLFY, a putative homologue<br />
of Arabidopsis LFY. A comparison (Figure 2a) of amino<br />
acid sequences with Arabidopsis LFY (Weigel et al.,<br />
1992), Antirrhinum FLO (Coen et al., 1990) and other<br />
FLO/LFY homologs (Brassica oleracea. BOFH.
3984 Afr. J. Biotechnol.<br />
Figure 4. Transgenic and wild-type Arabidopsis plants grown under LD<br />
photoperiods for 20 days. Arabidopsis plants were grown in one-half strength of<br />
medium for 9 days and transferred to potting soil. (a) wild-type Arabidopsis<br />
Columbia. (b) Transgenic Arabidopsis carrying a 35S::CmLFY gene.<br />
Anthony et al., 1993; Nicotiana tabacum. NFL. Kelly et al.,<br />
1995; Oraza sativa. RFL. Kyozuka et al., 1998; Pinus<br />
radiata. NLY. Mouradov et al., 1998 ; Petunia hybrida. ALF.<br />
Souer et al., 1998; Eucalyptus globulus, ELF1, Southerton<br />
et al., 1998 ; Lycopersicon esculentum. TOFL.<br />
Molinero-Rosales et al., 1999; Juncus effusus. JunefLFY.<br />
Frohlich and Parker., 2000; Platanus hispanica. PlaraLFY.<br />
Frohlich and Parker., 2000; Populus balsamifera. PTFL.<br />
Rottmann et al., 2000; Cedrela glaziovii. ClLFY. Marcelo<br />
and Adriana, 2006) showed the presence of conserved<br />
regions including two large conserved regions (c1 and c2)<br />
and two shorter regions of lower similarity (variable<br />
regions v1 and v2). These domains are typical markers for<br />
transcriptional acti- vators and may be functionally<br />
important for FLO/LFY-like proteins (Weigel et al., 1992;<br />
Coen et al., 1990). The phylogenetic tree showed that the<br />
topology of these genes seems to be concordant with the<br />
topology of the species phylogeny and suggests that<br />
CmLFY is an angiosperm ortholog of FLO/LFY-like genes<br />
(Figure 2b).<br />
The expression of CmLFY was only found in<br />
inflorescences, but not in tissues such as fruits, stems and<br />
tender leaves (Figure 3). It supports that the CmLFY is a<br />
functional homologous gene of chestnut. In the species<br />
such as apple, kiwifruit and grape, the expression levels<br />
of their FLO/LFY homologs increase in the proliferating<br />
inflorescence meristems generating inflorescence<br />
branches, with the highest levels detected in young floral<br />
meristems (Walton et al., 2001; Carmona et al., 2002).<br />
Therefore, the high levels of CmLFY expression in inflo-<br />
rescence suggests that CmLFY plays a role during chest-<br />
nut reproductive development as it has been suggested<br />
for most FLO/LFY-like genes studied.<br />
To further demonstrate the function of CmLFY, we<br />
have produced transgenic Arabidopsis plants expressing<br />
CmLFY. They flowered earlier and had a shorter<br />
inflorescence and reduced number of rosette leaves<br />
compared with the controls (Figure 3). Based on the result<br />
that several transgenic Arabidopsis with 35S::CmLFY<br />
flowered earlier than the controls, the expression of<br />
CmLFY should have some relationship with the early<br />
flowering phenotypes, although, the mechanism of<br />
flower-bud formation in chestnut might be different from<br />
that in Arabidopsis. Of course, to be more certain that the<br />
CmLFY gene functions similarly to LFY, LFY mutant-<br />
rescue experiments will be required. The CmLFY gene<br />
was not expressed strongly in transformants showing<br />
early flowering relative to those that were wild type in<br />
appearance, which suggests that the severity of pheno-<br />
type in transformants does not depend solely on the level<br />
of gene expression in this case, although the exact cause<br />
is still unknown. Some reports are available that describe<br />
transgenic Arabidopsis plants expressing LFY homologs<br />
from other plant species. For example, Arabidopsis<br />
transgenic lines expressing NEEDLY from Radiata pine<br />
and ELF1 from Eucalyptus showed early flowering<br />
(Mouradov et al., 1998; Southerton et al., 1998). Until now,<br />
however, our ability to manipulate fruit/nut tree strains<br />
through genetics has been limited by the extended<br />
maturation period of these plants. Both taking long time to<br />
flowering and the fact that, no characterized flowering<br />
mutants have been described in this genus make genetic<br />
studies in chestnut more difficult. Nevertheless, advances<br />
in the establishment of transformation protocols for
chestnut may allow us to use reverse genetic approaches<br />
and to define more clearly the role played by CmLFY in<br />
the reproductive development.<br />
The breeding of fruit trees of chestnut often requires<br />
more than 20 years, including periods of cross pollination,<br />
seedling selection and regional trials, to produce varieties<br />
that meet the demands of consumers. One of the limit<br />
factors of chestnut breeding is the long juvenile phase of<br />
at last several years. It had been found that transgenic<br />
approaches of LFY introducing could reduce the juvenile<br />
phase of Populus and the transgenic poplar flowered in 5<br />
months after regeneration (Weigel and Nilsson, 1995).<br />
Therefore, probably we can anticipate that these techni-<br />
ques are likely applicable to fruit trees of chestnut in future<br />
years and believed that, the transgenic approach would<br />
be a useful breeding strategy for reducing the time<br />
required for generation among woody plants. However,<br />
the efficiency of producing early flowering transgenic lines<br />
with Arabidopsis LFY gene seems to be low in woody<br />
plants in some case (Pena et al., 2001). This suggests<br />
that regulatory genes such as LFY do not always function<br />
beyond species as well as expected. Since environmental<br />
and genetic factors controlling flower development in<br />
chestnut have not been made clear so far, the CmLFY<br />
gene could at least, be one of the tools available for<br />
studying the mechanism of flower development in tree<br />
fruits such as the chestnut.<br />
ACKNOWLEDGMENTS<br />
This research was financially supported by the Faculty of<br />
Agronomy and Biotechnology (SJCX09012 to T Liu) and<br />
the National Natural Science Foundation of China (NSFC,<br />
30800062 to XX Li).<br />
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African Journal of Biotechnology Vol. 10(20), pp. 3986-3995, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB11.053<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Overexpression of a foxtail millet Acetyl-CoA<br />
carboxylase gene in maize increases sethoxydim<br />
resistance and oil content<br />
Zhigang Dong 1,2# Huji Zhao 1# Junguang He 1 , Junling Huai 1 , Heng Lin 1 , Jun Zheng 3 , Yunjun<br />
Liu 3 and Guoying Wang 1,3*<br />
1 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.<br />
2 Pomology Research Institute, Shanxi Academy of Agricultural Sciences, Taigu 030815, China.<br />
3 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China.<br />
Accepted 11 April, 2011<br />
Acetyl-coenzyme A carboxylase (ACCase) plays a crucial role in fatty acid metabolism in plants,<br />
catalyzing the carboxylation of acetyl-CoA to produce malonyl-CoA. In grasses, the plastids ACCases<br />
are critical determinants of plant sensitivity to herbicide classes cyclohexanediones (CHDs) and<br />
aryloxyphenoxy propionates (APPs). We transformed the full-length cDNA of a plastid ACCase gene<br />
from foxtail millet (Setaria italica) line Chum BC6-1, which is highly resistant to sethoxydim, into maize<br />
(Zea mays L.) drived by a ubiquitin promoter. The substantial results indicated that the sethoxydim<br />
resistance of transgenic plants is increased, with a damage index of 33%, much lower than that of 97%<br />
in the untransformed plants. Additionally, the oil content of transgenic maize seeds was 24 to 65%<br />
higher than that of untransformed seeds. These results indicate that the ACCase transgenic plants can<br />
be used to develop new maize hybrids that are tolerant to herbicide CHDs with high oil content.<br />
Key words: Zea mays, Acetyl-coenzyme A carboxylase (ACCase), oil content, transgenic plants,<br />
Agrobacterium tumefaciens<br />
INTRODUCTION<br />
Acetyl-coenzyme A carboxylase (ACCase) was first<br />
discovered nearly 50 years ago and has been studied<br />
extensively over the years (Wakil et al., 1983). Two forms<br />
of ACCase exist in plants. One is located in the plastids,<br />
the primary sites of plant fatty acid synthesis. This form is<br />
a “prokaryotic-type” multi-subunit enzyme and the other<br />
form, usually located in the cytoplasm, is a “eukaryotictype”<br />
multifunctional enzyme. In the grass family (including<br />
wheat, rice and maize), however, the “eukaryotic-type” is<br />
located in both the cytosol and plastids “(Alban et al.,<br />
1994; Konishi and Sasaki, 1994; Gornicki et al., 1997;<br />
*Corresponding author. E-mail: gywang@caas.net.cn.<br />
Abbreviations: ACCase, Acetyl-CoA carboxylase; CHDs,<br />
cyclohexanediones; APPs, aryloxyphenoxy propionates; HR,<br />
high resistance; HS, high sensitivity.<br />
#These authors contributed equally.<br />
Zhao et al., 2004; Tong L, 2005).<br />
Plant ACCases have attracted particular attention<br />
because the grass plastid ACCase is the active site of<br />
three chemically distinct classes of herbicides,<br />
aryloxyphenoxypropionates (APPs, also known as<br />
FOPs), cyclohexanediones (CHDs, also known as DIMs)<br />
and phenylpyrazolin. These classes of compounds<br />
reduce ACCase activity in meristematic tissue and kill<br />
sensitive plants by inhibiting their fatty acid biosynthesis.<br />
It has been reported that, grass ACCase in plastids is<br />
sensitive to these herbicides, but dicot and nongraminaceous<br />
monocot ACCases exhibit less sensitivity<br />
(Herbert et al., 1997; Christoffers et al., 2002). When<br />
these herbicides are used to kill weed grasses, they also<br />
kill the crop grasses, thus, greatly limiting their<br />
application. An alternative approach would be to produce<br />
grass crops highly tolerant to these herbicides.<br />
There are some reports indicating that the sensitive<br />
form of the enzyme has an Ile residue, while the resistant<br />
form has a Leu residue at the putative herbicide-binding
site (Zagnitko et al., 2001). Additionally, a single Ile to<br />
Leu replacement at an equivalent position changes the<br />
wheat plastid ACCase from sensitive to resistant<br />
(Zagnitko et al., 2001). Furthermore, the cDNA (foxACC-<br />
R gene) of the foxtail millet (Setaria italica) line Chum<br />
BC6-1, which has been reported to show high resistance<br />
to sethoxydim, has the same Leu reside in this position<br />
compared with an Ile residue in sensitive lines (Wang T<br />
et al., 1997; Zhao et al., 2004).<br />
In this study, we transformed the foxACC-R gene into<br />
the maize inbred line Zong 3 using Agrobacterium<br />
tumefaciens mediated transformation (Zhang et al.,<br />
2001). Southern blot assay, RT-PCR detection and the<br />
test of sethoxydim tolerance on transgenic plants indicated<br />
that the foxACC-R gene was stably integrated into<br />
the maize genome; also, all these plants with the higher<br />
oil contents. Our objective was to develop maize lines<br />
that were highly resistant to sethoxydim; therefore, these<br />
transgenic lines can be incorporated into maize breeding<br />
programs in the future.<br />
MATERIALS AND METHODS<br />
Plasmid construction<br />
The binary vector pCAMBIA3301 (Center for the Application of<br />
Molecular Biology to International Agriculture, Canberra, ACT,<br />
Australia) was used as a backbone for the construction of a plant<br />
expression vector. The bar gene, 35S promoter and GUS reporter<br />
gene were replaced by the foxACC-R (AY219175) driven by the<br />
ubiquitin promoter from maize to construct a marker-free p3301-<br />
ACCase vector (Figure 1).<br />
Maize transformation<br />
The approach used to generate transgenic maize plants was<br />
adapted from that described by Yang et al., 2001 and Zhang et al.,<br />
2001 The size of immature embryos are 1.0 to 2.0 mm, embryos<br />
were isolated from the maize inbred line Zong 3 and put into liquid<br />
infection medium (D basal medium (Duncan et al.,1985), 68.5 gl –1<br />
Suc, 36.0 gl –1 Glc, 100 µM AS (Sigma), pH 5.2) in 2-ml Eppendorf<br />
tubes (20 to 100 embryos per tube). Then, they were washed twice<br />
with this medium. The final wash was removed and 1.5 ml of A.<br />
tumefaciens suspension was added. The tubes were gently<br />
inverted 20 times and then, rested upright for five minutes with the<br />
embryos submerged. After infection, the embryos were transferred<br />
to the surface of co-cultivation medium (D basal medium, 20 gl –1<br />
Suc, 10 gl –1 Glc, 0.85 mgl –1 silver nitrate, 100 µM AS, 8 gl –1 agar, pH<br />
5.8). The embryos were incubated in the dark for three days at<br />
25°C, after which they were transferred to resting medium (D basal<br />
medium, 20 gl –1 Suc, 10 gl –1 Glc, 0.85 mgl –1 silver nitrate, 250 mgl –1<br />
cefotaxime, 8 gl –1 agar, pH 5.8) for seven days. Then, the embryos<br />
were moved to selection medium which was identical to resting<br />
medium with the addition of 5 µM sethoxydim and maintained for<br />
two weeks in the dark at 25°C. The concentration of sethoxydim<br />
was then increased to 10 µM for another three successive<br />
selections. Sethoxydim-resistant calli were placed on MS medium<br />
for regeneration. The shoots of 2 to 3 cm in height were moved to<br />
1/2 MS rooting medium (Murashige and Skoog, 1962). Calli for<br />
regeneration and regenerated plantlets were grown in a growth<br />
chamber at 28°C under fluorescent white light in 16:8 h light: dark<br />
cycle.<br />
PCR analysis of transgenic maize plants<br />
Dong et al. 3987<br />
Genomic DNA was isolated from leaves of transgenic maize and<br />
untransformed plants by the SDS method for PCR analysis (Sabelli<br />
and Shewry, 1995). Primers for the ACCase gene (ACCase-S 5’-<br />
CAATGTGTATGCTACATGTTTTTGT-3’ and ACCase-A 5’-<br />
ACCCCTGCAAAACCAGA-3’) were used for PCR analysis, it is<br />
specific for foxtail millet and there was no corresponding band in<br />
the maize genome (Zhao et al., 2004). The PCR products<br />
were subjected to 0.8% agarose gel electrophoresis and stained by<br />
EtBr to visualize the expected band.<br />
Southern blotting<br />
Southern blot analysis was carried out by standard procedures as<br />
described by Sambrook and Russell (2001). DNA samples isolated<br />
from maize leaves of transgenic and untransformed plants were<br />
purified, concentrated and quantified. About 50 µg of genomic DNA<br />
was digested with 40 U of EcoRI (Promega) at 37°C overnight. The<br />
digested DNA was separated by electrophoresis on a 0.8% agarose<br />
gel and blotted on Hybond- N+ nylon membrane (Amersham<br />
Pharmacia) in 0.4 mol/l NaOH. The probe (ACCase-S 5’-<br />
CAATGTGTATGCTACATGTTTTTGT-3’ and ACCase-A 5’-<br />
ACCCCTGCAAAACCAGA-3’) was random -primed labeled with ( -<br />
32P) dCTP. Southern hybridization was carried out overnight at<br />
42°C. The hybridized filters were washed in 2 × SSC with 0.5%<br />
SDS at 42°C for 30 min, twice in 1×SSC with 0.1% SDS at 65°C for<br />
30 min and twice in 2×SSC at 65°C for 10 min then exposed to xray<br />
film (Fuji) at -70°C.<br />
Semi-quantitative RT-PCR analysis of transgenic maize plants<br />
Total RNA of the transgenic and untransformed plants was<br />
extracted according to the Trizol reagent (Invitrogen) protocol. All<br />
preparations of total RNA were evaluated by denatured agaroseglyoxal<br />
gel electrophoresis. First-strand cDNAs were synthesized<br />
using ImProm-II TM reverse transcriptase (Promega) and the<br />
ACCase transcript was amplified using foxtail millet ACCase gene<br />
primers (ACCase-S 5’- CAATGTGTATGCTACATGTTTTTGT-3’<br />
and ACCase-A 5’- ACCCCTGCAAAACCAGA-3’). At the same time,<br />
maize tubulin cDNA (Tubulin-S 5’-<br />
GCTATCCTGTGATCTGCCCTGA-3’ and Tubulin-A 5’-<br />
CGCCAAACTTAATAACCCAGTA-3’) was also amplified as a<br />
control.<br />
Determination of oil content in transgenic maize seeds<br />
For the oil content measurements, the transgenic and<br />
untransformed plants were grown in the same environment and<br />
harvested at the same time. Using a near infrared reflectance<br />
spectroscopy (NIRS) instrument (BRUKER, VECTOR22/N), 20<br />
intact seeds in a revolving sample with a diameter of 50 mm were<br />
scanned 64 times at a wavelength region of 80 to 240 nm to<br />
calculate the average spectrum. In order to eliminate the influence<br />
of seed size and seed uniformity on the spectrum, each sample<br />
was replicated three times. Means for transgenic and<br />
untransformed plants were compared by using the two-sample ttest<br />
at the 5% confidence level as detected by LSD tests. The T0<br />
plants of PCR positive were self-pollinated to produce T1 seeds,<br />
and T1, T2 plants that were highly resistant to sethoxydim were selfpollinated<br />
to produce T3 seeds.<br />
Sethoxydim spraying test<br />
On a sunny day (>25°C), six-leaf stage T1 plants were painted with
3988 Afr. J. Biotechnol.<br />
Figure 1. Structure of the expression vector p3301-ACCase. ACCase. The ubiqutin promoter was fused<br />
with the foxACC-R R gene. Horizontal lines represent short pieces of polylinker DNA that connects<br />
the gene elements. The plasmid vector pCAMBIA3301 was as the backbone, and for the future<br />
application, ation, the bar gene, the 35S promoter and GUS reporter gene were deleted. Relevant<br />
restriction endonuclease sites are indicated.<br />
a 625 mg/l solution of sethoxydim (12.5%; Nisso, Japan). Four Four- to<br />
six-leaf stage T2 plants were sprayed in the field with a<br />
concentration of 1250 mg/l sethoxydim. The reaction of the plants<br />
to sethoxydim was recorded one week later. The degree of<br />
herbicide damage was evaluated according to the following criteria:<br />
level 0, no obvious chemical ical damage symptoms; level I, leaves<br />
show temporary chemical damage spots or slight growth<br />
suppression; level II, leaves show heavy chemical damage<br />
spots,loss loss of green coloration, shrinkage, abnormal growth or<br />
obvious growth suppression, but normal growth may be restored;<br />
level III, the growing point dies or continued growth is seriously<br />
suppressed; level IV, partial or the complete plant death.<br />
The damage index was s calculated according the following<br />
formula: damage degree index = ( (damage ge level * number)/ total<br />
number * highest level) * 100% (Jiang, 1987). ). The resistance of<br />
maize to the herbicide was divided into five grades according to the<br />
damage degree index: HR (highly resistant) (index 20); R<br />
(resistant) (20
Comparison of oil content of transgenic and wildtype<br />
plants<br />
The oil content of seeds was not determined for T0 plants<br />
of PCR positive lines due to the potential epigenetic<br />
variation generated during plant tissue culture. Starting<br />
from T1 plants, oil content was measured and compared<br />
with control plants. From the results of the comparison of<br />
T1, T2 and T3 generation plants (Table 2; Figure 7), all<br />
positive plants of each line had significantly higher oil<br />
content than controls. Lines 40 and 46 had 54 to 65%<br />
higher oil content than the controls in the T3 generation.<br />
Sethoxydim tolerance assay of transgenic plants<br />
To determine the response of the transgenic plants to<br />
Figure 2. Alignment of the amino acid sequence of<br />
the CT domain of ACCase genes. The isoleucine<br />
/leucine is indicted in bold. ACCase sequences were<br />
obtained from GeneBank, left is the accession no.<br />
The species is listed in Table 1.<br />
Dong et al. 3989<br />
sethoxydim, the fifth leaf of seedlings at the six-leaf stage<br />
was painted with sethoxydim at a concentration of 625<br />
mg/l. Seven days after treatment, the transgenic<br />
seedlings showed normal growth (Figure 8a), while the<br />
untransformed plants wilted, turned brown and died in<br />
two weeks (Figure 8b).<br />
T2 plants were sprayed with 1250 mg/l sethoxydim (the<br />
commercial concentration) in the field. Seven days later,<br />
the transgenic plants all showed normal growth, while the<br />
untransformed plants wilted (Figure 8c, d and e). The<br />
degree of herbicide damage was evaluated for every<br />
plant of the T2 generation (Table 3). The damage index of<br />
lines 40, 46 and 59 was lower than 20% and that of<br />
lines6, 17 and 76 were between 20 and 33% and the<br />
control had a damage index of 97%.<br />
These results showed that the foxACC-R transgenic<br />
plants were more resistant than the control.
3990 Afr. J. Biotechnol.<br />
DISCUSSION<br />
Table 1. The species chosen for the analysis and their accession number.<br />
Accession number Species name Special residue<br />
AF025469 Caenorhabditis elegans L<br />
U39321 Triticum aestivum (bread wheat)-Acc2 L<br />
AF029895 Triticum aestivum (bread wheat)-Acc1 I<br />
U19183 Zea mays I<br />
AY219175 Setaria italica (foxtail millet)-R L<br />
AY219174 Setaria italica (foxtail millet)-S I<br />
AF330145 Toxoplasma gondii L<br />
AF072737 Avena sativa (oat) I<br />
AF359513 Lolium rigidum-S I<br />
AF359514 Lolium rigidum-R L<br />
AJ132890 Bos taurus (cattle) L<br />
NM-198839 Homo sapiens (human)-ACC1 L<br />
NM001093 Homo sapiens (human)-ACC2 L<br />
J03541 Gallus gallus (chicken) L<br />
J03808 Rattus norvegicus (Norway rat) L<br />
L20784 Cyclotella cryptica L<br />
L25042 Medicago sativa L<br />
L42814 Glycine max (soybean) L<br />
M92156 Saccharomyces cerevisiae L<br />
P78820 Schizosaccharomyces pombe L<br />
X77576 Brassica napus (rape) L<br />
X99102 Hordeum vulgare subsp. vulgare L<br />
Y15996 Emericella nidulans L<br />
Z46886 Ustilago maydis L<br />
CM000142 Oryza sativa Japonica I<br />
EU660897 Aegilops tauschii I<br />
EU660891 Aegilops tauschii L<br />
CM000147 Oryza sativa Japonica L<br />
EQ973783 Ricinus communis (castor bean) L<br />
AF062308 Arabidopsis thaliana-ACC1 L<br />
AF062308 Arabidopsis thaliana-ACC2 L<br />
CM000338 Populus trichocarpa L<br />
CM000341 Populus trichocarpa L<br />
In plants, ACCase is a proven target for herbicide action.<br />
Genetic analyses have identified several single mutation<br />
sites in the CT domain that determine the herbicide<br />
resistance (Nikolau et al., 2003). A single-site Ile-to-Leu<br />
mutation at the position equivalent to residue 1705 in<br />
yeast ACC which represents an extremely subtle change<br />
in the side chain of the amino acid, is sufficient to confer<br />
resistance to both APPs and CHDs in the ACC enzyme<br />
of many grasses, while those that are resistant to these<br />
herbicides have a Leu residue at this position (Zagnitko<br />
et al., 2001; Delye et al., 2002). The second single-site<br />
mutation that confers resistance to herbicides is an Ile-to-<br />
Asn change (equivalent to residue 1967 in yeast ACC),<br />
again in the CT domain of ACC (Delye et al. 2003, 2004).<br />
Mutations at three additional sites in the CT domain have<br />
been identified from resistant weeds (Delye et al., 2005).<br />
An Asp/Gly change (equivalent to residue 2004 in yeast<br />
ACC) confers resistance to both APPs and CHDs,<br />
whereas a Trp/Cys and a Gly/Ala change (1953 and 2022<br />
in yeast ACC, respectively) confer resistance to APPs<br />
only. From our results and analysis, we concluded that<br />
the change from Leu to Ile at residue 1705 is critical for<br />
the interaction of ACCase with CHDs. Interestingly, our<br />
phylogenetic analysis (Figure 9) of ACCase genes of
Figure 3. Production of transgenic maize plants. (A) Sethoxydimresistant<br />
calli with the white growing point; (B) plantlet regeneration<br />
from the sethoxydim-resistant calli; (C) plantlets in 1/2 MS rooting<br />
medium; (D) regenerated plantlets in a growth chamber.<br />
Dong et al. 3991<br />
Figure 4. PCR detection of ACCase band (195 bp) in the posterity. M: DNA marker; lane 1, 6, 9, 17, 25, 33,40, 46,<br />
59 and 76 represents the transgenic lines; lane CK- represent the untransformed plant Zong3; lane CK+ represent<br />
the plasmid p3301-ACCase.<br />
Table 2. The oil contents of T1, T2 and T3 generation plants.<br />
Line<br />
Oil content of T1<br />
generation (%)<br />
Average oil content of T2 generation<br />
(%)<br />
Average oil content of T3<br />
generation (%)<br />
6 3.56±0.05 de 3.61±0.08 c 4.29±0.21 c<br />
17 3.86±0.13 cd 4.30±0.25 b 4.47±0.35 bc<br />
40 5.03±0.43 b 5.49±0.36 a 5.34±0.64 a<br />
46 5.62±0.30 a 5.98±0.44 a 5.71±0.20 a<br />
59 4.10±0.29 c 5.52±0.32 a 5.07±0.31 ab<br />
76 5.09±0.18 b 4.54±0.39 b 4.58±0.43 bc<br />
Z3 3.39±0.19 e 3.51±0.37 c 3.47±0.21 d<br />
The oil contents of T2 and T3 generation are the mean value of four to five plants derived from one T1 plant. The number 6, 17,<br />
40, 46, 59 and 76 represented independent transgenic lines, Z3 is untransformed inbred line. Means with standard errors were<br />
calculated from three replicates. Different letters represent significant difference at 5% confidence level as detected by LSD tests
3992 Afr. J. Biotechnol.<br />
Figure 5. Southern blot analysis of TT2<br />
transgenic maize plants.<br />
Aliquots of 50 µg of EcoRI -digested total maize leaf DNA was<br />
fractionated on 0.8% agarose gel, blotted to nylon membrane and<br />
probed with<br />
plasmid p3301<br />
plant.<br />
32 P-labeled 195bp ACCase gene fragment. CK+ is the<br />
plasmid p3301-ACCase (16.37 kb) and CK- is the untransformed<br />
Figure 6. Detection of levels of the foxtail millet ACCase<br />
transcripts by semi semi-quantitative RT-PCR. Upper bands are the<br />
PCR fragments specific for Accase Accase. Lower bands are the maize<br />
actin PCR fragments used as the control. 6, 17, 40, 46, 59 and 76<br />
are the transgenic lines, Z3 is the untransformed plant.<br />
Figure 7. The ratio of oil content of the TT1,<br />
T2 and T3 generation plants. The data ata bars<br />
indicate the ratio of oil content of transgenic lines to untransformed control.
Table 3. The damage index of T3 generation transgenic plants by herbicide.<br />
Parameter<br />
Line<br />
The damage index (%) 30.00 22.22 16.67 14.28 13.33 32.14 96.67<br />
The damage index is the mean value of fourteen to fifteen plants plants.<br />
Figure 8. The sethoxydim resistance evaluation in the field. The fifth leaf of seedlings with 4 to 6 true leaves was pained by<br />
625 mg/l of sethoxydim. Seven days after treatment, the WT plants turned wilt and brown (B), while the transgenic seedlings<br />
grown normal (A). The T2 plants were sprayed with 1250 mg/l of se sethoxydim thoxydim in the field, (C) is the transgenic plant, (D) is the<br />
untransformed control. (E) Left line is the transgenic plants and right line is untransformed plants.<br />
these eukaryotic and prokaryotic types indicates that, all<br />
types that hat have an Ile residue at this position are grouped<br />
into one subfamily, except for AF359514 and AY219175,<br />
which show a leu/Ile substitution at this position. These<br />
results suggest that the CT domain of ACCases in this<br />
subfamily has a special structure that t can bind the<br />
herbicide and that this particular mutation may interfere<br />
with herbicide binding, leading to herbicide resistance.<br />
However, the exact mechanism is not clear. clear.ACCase has<br />
been identified as an important regulatory enzyme for<br />
plant fatty acid synthesis by three in vivo approaches.<br />
Analysis of substrate and product pool sizes has<br />
implicated ACCase in the light/dark regulation of fatty<br />
acid synthesis in spinach leaves (Post-Beittenmiller<br />
et<br />
al.,1992) and chloroplasts (Post-Beittenmiller Beittenmiller et al.,<br />
1991). The total oil content of seeds is increased<br />
approximately 5% by the expression of Arabidopsis<br />
ACCase in rapeseed plastids (Keith et al., 1997). In this<br />
study, over-expression of the plastid foxACC foxACC-R gene<br />
from a foxtail millet increased oil content from 24 to 65%<br />
in the T3 generation over that of the control maize inbred<br />
Transgenic line<br />
6 17 40 46 59 76<br />
Dong et al. 3993<br />
Control<br />
line Zong3 (Table 2). This result is consistent with that<br />
found in tobacco suspension cells, where ACCase is the<br />
apparent site of feedback inhibition of fatty acid synthesis<br />
when supplemented with exogenous fatty acids (Shintani<br />
and Ohlrogge, 1995).<br />
Sethoxydim is a post-emergence emergence herbicide used to<br />
eliminate weeds in dicotyledonous crops. It is absorbed<br />
by the leaves and transported throughout the phloem<br />
towards the apical meristem. stem. Systemic herbicides require<br />
from several days to a few weeks to move throughout the<br />
vascular system of a treated plant. In monocots, necrotic<br />
zones develop in newly formed leaves, resulting in<br />
growth inhibition and cell death in one or two weeks. In<br />
dicots, effects of post-emergence emergence herbicides have been<br />
reported under field conditions (Griffin and Habetz, 1989;<br />
Hart and Roskamp, 1998), but plants usually recover<br />
from injury, depending on growth conditions and season<br />
(Kapusta et al., 1986).<br />
Our results s indicate that the full-length full cDNA of<br />
foxACC-R, when introduced duced into maize, leads to high<br />
resistance to the herbicide sethoxydim; the damage indices<br />
Z3
3994 Afr. J. Biotechnol.<br />
Figure 9. Phylogenetic analysis of the ACCase genes. The amino acid sequences of the<br />
ACCase gene were aligned by ClustalX1.83 and the phylogenetic tree was constructed<br />
using the NJ method in MEGA 3.1. All the types which have Ile residue were grouped into<br />
one subfamily, except for the AF359514 and AY219175 which is leucine /isoleucine<br />
substitution in this position.<br />
of all transgenic lines were lower than 33%, while that of<br />
the control was 97%.<br />
In summary, over-expression of the full-length cDNA of<br />
the foxACC-R gene in maize leads to increased<br />
resistance to sethoxydim. These lines might be used to<br />
develop new maize hybrids with herbicide tolerance in<br />
the near future.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 3996-4004, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB11.106<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Variation in nodulation and growth of groundnut<br />
(Arachis hypogaea L.) on oxisols from land use<br />
systems of the humid forest zone in southern<br />
Cameroon<br />
Laurette Ngo Nkot 1 *, Dieudonné Nwaga 2 , Albert Ngakou 3 , Henri Fankem 1 and François-Xavier<br />
Etoa 4<br />
1 Department of Plant Biology, Faculty of Science, University of Douala. P.O. Box 24157, Douala, Cameroon.<br />
2 Laboratory of Soil Microbiology, Biotechnology Centre, University of Yaoundé I. P.O. Box 812 Yaoundé, Cameroon.<br />
3 Department of Biological Sciences, Faculty of Science, University of Ngaoundéré. P.O. Box 454, Ngaoundéré,<br />
Cameroon.<br />
4 Department of Biochemistry, Faculty of Science, University of Yaoundé I. P.O. Box: 812 Yaoundé, Cameroon.<br />
Accepted 11 April, 2011<br />
Improving biological nitrogen fixation through legume nodulating bacteria (LNB) inoculation requires<br />
knowledge on the abundance and effectiveness of indigenous population in the ferralsols. Nodulation<br />
of groundnut was examined under pots experiment in four location sites of the Humid-forest zone:<br />
Bertoua in the East; Ebolowa in the South; Bokito and Yaoundé in the Centre Regions of Cameroon and<br />
within each of the locations, in four land use systems (LUSs) of different levels of disturbance: mixed<br />
farming (1 to 3 years old); fallows (3 to 5 years old); cocoa plantation (> 20 years old); forest (> 30 years<br />
old). Results indicate that, soils under investigation are acidic with pH ranging from 3.68 in the Ebolowa<br />
forest to 6.92 in mixed farming at Bokito. Groundnut formed nodules in all the four LUSs. Soils from<br />
plantations and forests were poorly nodulated, whereas those from mixed farming and fallows were<br />
highly nodulated, with a positive and significant correlation (r 0.406; p < 0.0001) observed between<br />
nodulation and plant biomass in all the studied sites. These results suggest a high variation in<br />
groundnut nodulating bacteria density in soils from diverse LUSs of the humid forest zone of<br />
Cameroon; although, there was a site effect.<br />
Key words: Acid soil, Arachis hypogaea, biomass, land use systems, multilocal, nodulation.<br />
INTRODUCTION<br />
Many soils in the tropics are fragile, with high acidity and<br />
aluminium toxicity, high phosphorus fixation and low soil<br />
biodiversity (Cardoso and Kuyper, 2006). Nitrogen and<br />
phosphorus are the most limiting nutrients for crop<br />
production in these ferral soils (Dogbe et al., 2002). In<br />
Cameroon, acid soils cover more than 80% of arable<br />
lands (The, 2000). Synthetic fertilizers for improving soil<br />
*Corresponding author. E-mail: lnkot@yahoo.fr. Tel: (00 237) 99<br />
92 77 16 or (00 237) 33 30 31 07. Fax: (00 237) 33 42 76 12.<br />
fertility are rarely available to most farmers. In addition,<br />
these fertilizers may induce soil acidification and become<br />
less efficient after many cropping years (Bado, 2002),<br />
leading to a high dependence of soil to N fertilizer for<br />
optimum yield (Fening and Danso, 2002). Since fallow<br />
practice to restore soil fertility is no longer possible<br />
because of land scarcity, there is a need of more efficient<br />
practices.<br />
Four land use systems (LUSs) are common in humid<br />
forest zone in Cameroon: forest; cocoa or coffee plantation;<br />
fallow and mixed farming. Forest conversion to<br />
farms or plantations is increasing in response to popula-
tion growth (Van Noordwijk et al., 2002). Cocoa<br />
(Theobroma cacao) plantation, is one of the dominant<br />
LUS, has served in the past as an economically-attractive<br />
land use but has been rejected by many farmers because<br />
of low and unpredictable prices on the world markets.<br />
Hence, farmers were forced to abandon cocoa or coffee<br />
plantations toward other income-generating opportunities<br />
such as food crops. Forest and some old plantations are<br />
used for annual crops such as groundnut in a mixed<br />
farming system. Under conditions of low external input,<br />
the use of biological nitrogen fixation appears to be a<br />
cheaper and environmentally friendly source of N for<br />
plants (Peoples et al., 1995). According to Bogino et al.<br />
(2006) a high degree of nodulation and nitrogen fixation<br />
in groundnut is provided by indigenous LNB in some soils<br />
from Argentina. Groundnut (Arachis hypogaea L.) is an<br />
important legume crop that provides food for human<br />
subsistence and other products. In Cameroon, groundnut<br />
is the major legume crop in slash and burn, traditional<br />
agricultural food production system. It is grown on<br />
250.000 ha with more than half of the production area in<br />
the northern part of the country (Ntoukam et al., 1996).<br />
The yield is low, with an average 0.85 t.ha -1 compared<br />
with 1.05 t.ha -1 in Senegal and 1.03 t.ha -1 in Nigeria<br />
(Anonymous, 2000). In sub-Saharan Africa, cropping<br />
systems, slash and burn agriculture and acid soil infertility<br />
could be major limiting factors for this crop yield. In<br />
addition, the density of indigenous LNB in diverse land<br />
use systems may affect nodulation and legume performances.<br />
According to Nwaga et al. (2010), land use<br />
changes and cropping practices such as burning may<br />
affect soil functioning in the humid forest, along with<br />
diversity and occurrence of beneficial micro-organisms.<br />
When comparing indigenous versus selected rhizobia<br />
from USA, yield diversity of 0.33 to 1.48 t/ha was noticed<br />
according to groundnut genotypes and 0.27 to 0.76 t/ha<br />
according to rhizobia x genotype interactions in northern<br />
Cameroon low rainfall regions (670-1068 mm)<br />
(Mekontchou et al., 2007). It is therefore, important to<br />
select acid LNB strains with high symbiotic effectiveness<br />
to improve legume production in tropical acid soils. One<br />
of the major problems to improve A. hypogaea in this<br />
region is ‘flat pod phenomenon’ or ‘empty seeds<br />
phenomenon’ of groundnut. Few data are available on<br />
the nodulation and plant growth of legumes such as A.<br />
hypogaea in the Congo Basin zone in Central Africa.<br />
Therefore, the objective of this study was to assess under<br />
greenhouse conditions, nodulation and plant growth of A.<br />
hypogaea on acidic soils from diverse land use systems<br />
in the humid forest zone of southern Cameroon.<br />
MATERIALS AND METHODS<br />
Study sites, experimental design and treatments<br />
Experiments were carried out in soil samples (2 kg plastic bags)<br />
collected from four location sites of the humid forest zone: Bertoua<br />
in the East; Ebolowa in the South; Bokito and Yaoundé in the<br />
Ngo Nkot et al. 3997<br />
Centre regions of Cameroon (Figure 1). Within each site, soils were<br />
collected in four replicates in the four different land use systems<br />
(LUSs): mixed farming, fallow (Chromolaena odorata), cocoa or<br />
coffee plantation and forest. The experimental design was thus, a<br />
split plot (4 x 4) x 4 with the 4 different land uses representing the<br />
main factors and the 4 study sites the sub-factors.<br />
Soil sampling and analysis<br />
A total of 64 soil samples were collected in four location sites<br />
according to Swift et al. (2001). A composite soil sample was a<br />
mixture of ten sub-samples all collected from a LUS of the same<br />
location site. Aseptic precautions were taken during sampling and<br />
handling of each soil to avoid contamination. Soil sample were<br />
taken from sites that had no previous history of LNB inoculation.<br />
The 64 composite samples were taken from the top 0 to 20 cm soil<br />
depth, from which the un-decomposed plant material were removed<br />
by hand. The chemical analysis of the soil was conducted using<br />
standard methods (Anderson and Ingram, 1993).<br />
Assessment of nodulation and growth of A. hypogaea<br />
Groundnut seeds of the A26 variety were obtained from the Institute<br />
of Agricultural Research and Development (IRAD). Nodulation was<br />
assessed by growing germinated seed in perforated plastic bags<br />
containing more than 2 kg of soil samples. Seeds were surface<br />
sterilized in 3.3% calcium hypochlorite solution for 3 min and<br />
washed thoroughly with sterile water, re-suspended in 70% alcohol<br />
for 3 min and washed thoroughly with sterile water. The seeds were<br />
incubated to germinate in sterile Petri dishes containing 0.9% (w/v)<br />
water agar for 2 to 3 days at 28°C. After germination, six seedlings<br />
were sown in each plastic bag. Two weeks after germination, the<br />
number of plants was thinned to four per plastic bag, thus, sixteen<br />
per treatment.<br />
Plants were watered and harvested 6 weeks after sowing. The<br />
root systems of individual plants were washed separately, their<br />
nodules picked and counted from sixteen plants per treatment.<br />
Plant shoot and roots systems were let to dry at 70°C for more than<br />
72 h in an air dry oven and then weighed until constant mass (Athar<br />
and Johnson, 1996).<br />
Statistical analysis<br />
Data were subjected to analysis of variance (ANOVA). Means were<br />
separated between treatments with the least significant difference<br />
(LSD), using the Statgraphic plus, version 5.0 (SIGMA PLUS)<br />
computer package. Comparisons were made among treatments of<br />
the same location site. A p-value < 0.05 was generally used to<br />
evaluate significance, although, higher levels were considered for<br />
p-value < 0.0001. The statistical package for social sciences<br />
(SPSS) was used to assess the correlation between the nodulation<br />
and growth parameters.<br />
RESULTS<br />
Differences in the chemical components of studied<br />
soils<br />
The chemical properties of soils under study are shown in<br />
Table 1. The soil aluminium content was consistently<br />
lower in the fallow (0.34 meq/100 g) than in other LUSs at<br />
Bertoua (p = 0.025), whereas at Bokito, it was
3998 Afr. J. Biotechnol.<br />
Figure 1. Localisation of Cameroon in Africa and distribution of the investigated sites in<br />
humid forest zones.<br />
significantly lower (p = 0.013) in the forest (0.28 meq/100<br />
g) than the plantation (0.49 meq/100 g). At Ebolowa, the<br />
soil aluminium content was significantly lower in the<br />
fallow (0.32 meq/100 g; p = 0.002). In contrast, no significant<br />
difference was found between the soil aluminium<br />
contents of different LUSs in Yaoundé. The lower<br />
aluminium values was 0.20 meq/100 g and the higher<br />
values was 0.52 meq/100 g.<br />
Soil pH of the four sites of the humid forest zone<br />
ranged from 3.96 in the forest at Ebolowa to 6.00 in<br />
mixed farming at Bertoua and did not show significant<br />
differences between the LUSs in any of the studied sites.<br />
Ebolowa soils were more acid than those of Bertoua,<br />
Bokito and Yaoundé. Soil organic carbon ranged from<br />
0.88 to 2.18%, while soil organic matter varied from 1.51<br />
to 3.03%, with a mean value of 2.59%. At Bokito, the soil<br />
organic carbon was similar in forest, mixed farming and<br />
fallow, but was significantly higher (p = 0.017) than in<br />
plantation. Similarly, the soil organic matter did not significantly<br />
vary between the Ebolowa mixed farming and<br />
forest, but was significantly higher (p = 0.05) than those<br />
of the fallow and plantation. The total mean soil N value<br />
was low and ranged between 0.11 and 0.15% with little or<br />
no site variations. Plantation had the most acid LUS soil<br />
with an average of 4.85.<br />
Nodulation of groundnut in different LUSs<br />
All the soils used for nodulation experiment had<br />
indigenous rhizobia that were able to nodulate groundnut.<br />
The mean nodule number varies from 13 in the<br />
plantations to 94 nodules in the fallow, depending on site<br />
and LUSs. A highly significant correlation was found<br />
between nodule number and groundnut growth.<br />
The number of nodules per plant was significantly<br />
higher (p < 0.0001) in the fallow than in other LUSs in all<br />
the experimental sites, with the maximum number of<br />
nodules observed at Bokito (94 nodules). This nodulation<br />
status (>75 nodules/root system) is characterized as<br />
good according to Amijee and Giller (1998). There was<br />
no significant difference between the number of nodules<br />
formed in the plantation and forest at Bertoua, mixed<br />
farming, plantation and forest at Bokito and Yaoundé,<br />
mixed farming and plantation at Ebolowa. The number of<br />
nodules formed by A. hypogaea in all the LUSs at<br />
Ebolowa was lower than that of any of the sites, with the<br />
lowest accounting for plantation (13 nodules). This nodu-
Ngo Nkot et al. 3999<br />
Table 1. Variation of the soil chemical parameters under different land uses of the experimental sites in the humid forest zone of<br />
southern Cameroon.<br />
Site Chemical parameter<br />
Bertoua<br />
Bokito<br />
Ebolowa<br />
Yaoundé<br />
Farm Fallow<br />
Land use<br />
Plantation Forest<br />
p-value LSD (5%)<br />
Al 3+ (meq/100 g) 0.47 b 0.34 a 0.40 ab 0.39 ab 0.025 0.127*<br />
C/N (ratio) 8.60 a 12.04 a 9.40 a 8.65 a 0.561 ns<br />
pH (H2O) 6.00 a 5.97 a 5.71 a 5.54 a 0.285 ns<br />
N (%) 0.12 a 0.11 a 0.11 a 0.11 a 0.846 ns<br />
OM (%) 1.77 a 2.05 a 1.78 a 1.60 a 0.549 ns<br />
OC (%) 1.03 a 1.20 a 1.03 a 0.93 a 0.535 ns<br />
Al 3+ (meq/100 g) 0.45 ab 0.45 ab 0.49 b 0.28 a 0.013 0.20**<br />
C/N (ratio) 8.14 a 8.70 a 7.99 a 9.54 a 0.736 ns<br />
pH (H2O) 5.68 a 5.71 a 5.15 a 5.68 a 0.830 ns<br />
N (%) 0.125 a 0.112 a 0.115 a 0.130 a 0.450 ns<br />
OM (%) 1.72 ab 1.67 ab 1.51 a 2.12 b 0.097 0.61*<br />
OC ( %) 1.00 ab 0.97 ab 0.88 a 1.22 b 0.017 0.34**<br />
Al 3+ (meq/100 g) 0.52 c 0.32 a 0.37 ab 0.44 bc 0.002 0.122**<br />
C/N(ratio) 11.62 a 18.89 b 15.37 ab 17.92 b 0.041 6.29*<br />
pH(H2O) 4.09 a 4.02 a 4.14 a 3.96 a 0.783 ns<br />
N (%) 0.14 c 0.11 a 0.13 bc 0.12 ab 0.005 0.01**<br />
OM (%) 2.93 bc 2.15 a 2.23 ab 3.03 c 0.05 0.77*<br />
OC (%) 1.68 a 2.15 a 2.02 a 2.18 a 0.222 ns<br />
Al 3+ (meq/100 g) 0.21 a 0.20 a 0.25 a 0.20 a 0.488 ns<br />
C/N (ratio) 12.14 a 8.94 b 8.93 b 8.52 b 0.081 3.20*<br />
pH (H2O) 4.75 a 5.15 a 4.39 a 5.26 a 0.325 ns<br />
N (%) 0.125 a 0.127 a 0.145 a 0.135 a 0.369 ns<br />
OM (%) 2.55 a 1.95 a 2.24 a 2.04 a 0.468 ns<br />
OC (%) 1.48 a 1.13 a 1.30 1.17 a 0.463 ns<br />
Values in a row followed by the same letter within a studied site are not significantly different at 5% level (ns: non-significant; *,<br />
significant; **, highly significant).<br />
lation status (25 nodules/root system) was characterized<br />
as poor according to Amijee and Giller (1998). Poor<br />
nodulation occurs only in plantation and forest, while<br />
moderate nodulation (25 to 75 nodules/root system were<br />
obtained in fallow and mixed farming. The number of<br />
nodules per plant was higher at Bokito and the lowest<br />
one at Ebolowa.<br />
The dry weight of nodules harvested from plants at<br />
Bokito was significantly higher than that of any other<br />
studied site (Figure 2). Although, there were some<br />
differences in the nodules dry weight between LUSs,<br />
which was not statistically significant between the sites.<br />
Growth of A. hypogaea in different LUSs<br />
The highest shoot dry weights of A. hypogaea were<br />
observed in Bokito soils, followed by Bertoua soils;<br />
whereas the shoot dry weights of plants in plantations<br />
were lower than those of other LUSs (Table 2). Fallow<br />
significantly improved the fresh and dry weight of root<br />
and shoot systems of groundnut in Bertoua (p < 0.0006),<br />
Bokito (p < 0.0009) and Ebolowa (p < 0.007) compared<br />
with mixed farming, plantation and forest. In Yaoundé in<br />
contrast, whereas the fresh weight of shoot system was<br />
significantly greater in the forest (p < 0.0001) than in<br />
other land uses, the fresh weight of the root system was<br />
similar in mixed farming and fallow, but was at the same<br />
time significantly enhanced (p < 0.0001) than in<br />
plantation and forest.<br />
There was no significant difference (p = 0.321) between<br />
the land uses as far as the dry weight of root system is<br />
concerned. The greatest plant fresh weight system was<br />
registered in a forest of Yaoundé (11.16 g), while the<br />
smallest was observed in a plantation of Ebolowa (4.03<br />
g). The dry weight of the root system was generally not<br />
high, ranging from 0.11 g in plantations (Bokito) to 0.32 g<br />
in mixed farming (Yaoundé).<br />
At Bokito, highly significant correlations were found
4000 Afr. J. Biotechnol.<br />
Table 2. Effect of LUSs on the biomass of A. hypogaea in four sites in humid forest zone of southern<br />
Cameroon.<br />
Study site Parameter<br />
Bertoua<br />
Bokito<br />
Ebolowa<br />
Yaoundé<br />
Land use<br />
Farm Fallow Plantation Forest<br />
p-value LSD 5%<br />
Fwss 8.50 b 9.08 b 5.77 a 5.90 a
Table 3. Correlations between number of nodules per plant and the shoot and root biomass of groundnut<br />
from humid forest soils in southern Cameroon.<br />
Experimental site<br />
Bertoua<br />
Bokito<br />
Ebolowa<br />
Yaoundé<br />
Shoot and root biomass<br />
Fwrs Fwss Dwrs Dwss<br />
r = 0.579*** r = 0.774*** r = 0.055 ns r = 0.635***<br />
p < 0.0001 p < 0.0001 p = 0.665 p < 0.0001<br />
r = 0.468*** r = 0.692 *** r = 0.461*** r = 0.474***<br />
p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001<br />
r = 0.081 ns r = 0.503*** r = - 0.010 ns r = 0.406***<br />
p = 0.522 p < 0.0001 p = 0.935 p< 0.001<br />
r = -0.133 ns r = 0.698*** r = -0.069 ns r = 0.558***<br />
p = 0.296 p < 0.0001 p = 0.589 p < 0.0001<br />
Fw, Fresh weight; Dw, dry weight; ns, non significant; rs, root system; ss, shoot system; ***, very highly<br />
significant.<br />
between nodule number and respectively the fresh<br />
weight root system (r = 0.468; p < 0.0001) and the dry<br />
weight root system (r = 0.461; p < 0.0001). Other significant<br />
and positive correlations were observed between<br />
the number of nodules and the dry weight of shoot<br />
system (r = 0.474; p < 0.0001) (Table 3). In contrast,<br />
there was no significant correlation between the number<br />
of nodules and the dry weight of root system at Bertoua,<br />
between the number of nodules and the dry weight of<br />
root system at Ebolowa and between the number of<br />
nodules and the dry weight of root system at Yaoundé.<br />
The positive correlation observed between nodulation<br />
and dry weight shoot growth of groundnut suggests that<br />
the two parameters evolve together.<br />
DISCUSSION<br />
The soils of all the four LUSs were acidic with high<br />
aluminium content in most sites except Yaoundé. Since<br />
these mixed farming and fallow soils generally have<br />
never received inorganic fertilizer, the low pH value is<br />
assigned to growing lands. Except in Yaoundé, LUSs<br />
significantly influence the soil aluminium. The lower<br />
aluminium values, 0.20 meq/100 g is from the fallow and<br />
the higher values, 0.52 meq/100 g is from the mixed<br />
farming. In Bertoua and Ebolowa, the soil aluminium<br />
content was significantly lower in the fallow when<br />
compared with other LUSs. The low aluminium content in<br />
fallow could be due to the residual effect of the wood ash<br />
from the slash and burn practice. Agoumé and Birang<br />
Ngo Nkot et al. 4001<br />
(2009) also reported that, aluminium saturation was<br />
significantly affected by the LUSs with the lowest<br />
aluminium saturation obtained in soils of C. odorata<br />
fallows. They noted that, soils of fallows presented a<br />
higher fertility level when compared with those of the<br />
secondary forests and cocoa plantations.<br />
The different LUSs did not significantly influence soil<br />
acidity in all the experimental sites. Soils under<br />
investigation have pH values ranging from 3.96 to 6.00.<br />
Ebolowa soil was the most acidic of all (pH 4.07), in<br />
agreement with other results from which the soils of the<br />
southern region of Cameroon were acid with pH ranging<br />
from 3.69 to 4.12 (Fankem et al., 2006).<br />
All the groundnut plants examined formed effective root<br />
nodules, suggesting that all these soils contain<br />
indigenous LNB able to nodulate A. hypogaea. The cross<br />
section of some nodules showed the red coloration,<br />
suggesting the presence of leghemoglobin, related to<br />
nitrogen efficiency (Abdel-Wahab et al., 2002). In all the<br />
experimental soils, no rhizobia inoculant strains were<br />
used. Nodules were thus, formed by indigenous LNB.<br />
These results are in accordance with other studies that<br />
showed that, nodulation failure is rare for many tropical<br />
legumes such as A. hypogaea, a promiscuous species<br />
belonging to cowpea miscellany group (Alwi et al., 1989).<br />
Moderate to higher number of nodules, from 30 to 94<br />
per plant was formed in the mixed farming and fallows in<br />
soils from humid forest zone in southern Cameroon. But<br />
can we consider that, natural nodulation in these LUSs is<br />
adequate? Assessments of more mixed farming<br />
nodulation and inoculation experiments are needed to
4002 Afr. J. Biotechnol.<br />
confirm this. Some results reported that, nodule number<br />
from indigenous rhizobia was higher in farm previously<br />
planted with groundnut than in other farms where<br />
groundnut was not previously planted (Bogino et al.,<br />
2006). The density of LNB in these soils can be<br />
compared with nodulation response of groundnut in the<br />
different LUSs to explain the results. The comparison<br />
supports the observation that, when LNB density in these<br />
LUSs is high, the nodulation is also high. LNB density is<br />
generally lower in cocoa or coffee plantations and forest<br />
soils than in fallows or mixed farming of southern<br />
Cameroon (Ngo Nkot, 2009). This could be the<br />
consequence of farming practices management involving<br />
pesticides in plantations and a legume for LNB in mixed<br />
farming and fallows. Thakuria et al. (2009) also observed<br />
a gradual increase in nitrogenase activities in roots of rice<br />
and pea under Azospirillum/Rhizobium alone integrated<br />
nutrient management (INM) or Azospirillum/Rhizobium<br />
plus phosphate-solubilizing bacteria dual INM plots in<br />
acidic soils, which could be a result of either population<br />
build-up of introduced bio-inoculants in the rhizosphere or<br />
better soil environment for the N fixers. The highest level<br />
of rhizobia genetic diversity was recorded in soils under<br />
cocoa plantation when compared with soils under mixed<br />
farming with groundnut (Ngo Nkot et al., 2008).<br />
Soils from cocoa or coffee plantations and forests in<br />
southern Cameroon had the lowest number of<br />
groundnuts nodules, respectively 32 and 42 per plant.<br />
This suggests that, LNB were less abundant in these<br />
LUSs. The lack of response to inoculation has been<br />
previously demonstrated to be attributed to other factors<br />
such as inadequate nodulation, unfavourable conditions<br />
for survival of introduced LNB strains and inability of<br />
inoculant strains to compete with indigenous ones for<br />
nodule sites (Bogino et al., 2006). The mean number of<br />
nodules per LUS was very low in cocoa/coffee plantations,<br />
probably because these soils had been under cocoa or<br />
coffee plantations for the past 20 years at least, excluding<br />
legumes but regularly using fungicides and insecticides<br />
for disease and pest control. Ciani and Diriye (1995)<br />
noted the absence of LNB in soils under banana and<br />
papaya plantations compared with soil were leguminous<br />
plants such as groundnut and cowpea were cultivated.<br />
Soils under plantations were the most acid of all the<br />
LUSs (pH 5.83 in average). By decreasing the LNB<br />
population density, acidity may delay the initiation of<br />
infection process and hence, the appearance of<br />
subsequent nodules. The decline of LNB in plantation<br />
may be the consequence of the lower soil pH. These<br />
findings are similar to other results reported by Cheng<br />
(2003), who showed that, Medicago murex grown in acid<br />
soil (pH 4.3) produced fewer nodules (2.3/plant) than<br />
plants grown at pH 7.0 (8.2/plant) 41 days after sowing.<br />
According to Brady and Weil (2002), the low pH in acid<br />
soils leads to an increased availability of aluminium and<br />
manganese which become toxic, as well as a low<br />
concentration of phosphate, calcium and molybdenum.<br />
The lack of adequate number of Bradyrhizobium<br />
japonicum in Nigerian soils was shown to limit nodulation<br />
and nitrogen fixation of soybean, thus, decreasing the<br />
yield (Broomfiel and Ayanaba, 1980). The frequently low<br />
number of nodules observed in forest soils has been<br />
associated with a low requirement for N, which is<br />
considered not to be a limiting factor under these<br />
conditions (Zilli et al., 2004). Tematio et al. (2001)<br />
showed that, soils in the forest are not very fertile. The<br />
fertility is limited by soil acidity, the low amount of cations<br />
exchanged capacity, the aluminium toxicity and the low<br />
available phosphorus. In such conditions, LNB growth<br />
was reduced (Hussein, 2000; Zahran, 1999). Selection of<br />
acid tolerance rhizobial strains of Phaseolus vulgaris L.<br />
done by Gutierrez and Barraquio (2010) yielded that only<br />
36 acid-tolerant strains over 189 (19%) were isolated in<br />
Philippines. Some local strains of rhizobia have been<br />
previously shown to be more acid tolerant than a Chinese<br />
one (Nwaga and Ngo Nkot, 1998). Further studies are<br />
needed to characterize, assess LNB density and select<br />
A. hypogaea strains adapted for acidic oxisols. According<br />
to Lindström et al. (2010) a challenge for agriculture is to<br />
match rhizobia and legume crops for optimal performance<br />
either by having plant genotypes adapted to local<br />
rhizobial populations or by inoculating effective strains<br />
adapted to prevailing environmental conditions and good<br />
competitive ability against local, less effective strains.<br />
Nodules were present in large number at Bokito grey<br />
soils compared with Ebolowa reddish soil sites. This poor<br />
nodulation at Ebolowa clay soils might indicate its<br />
contribution for poor plant growth and low yield of A.<br />
hypogaea. Alemayehu (2010) also find poor nodulation of<br />
Vicia faba in Ethiopian soils and conclude that, poor BNF<br />
may partially be responsible for reduction of faba bean<br />
nodulation.<br />
In Bokito sandy loam soils, the cropping systems are<br />
more diversified including A. hypogaea in the farms<br />
together with other legumes such as Vigna unguiculata,<br />
while in fallows, Crotalaria juncea was present. The<br />
improved soil fertility of Bokito site, due to the presence<br />
of more local LNB can account for the large density of<br />
nodules observed in this site. Remarkable increases in<br />
groundnut yield have been achieved through inoculation<br />
with Bradyrhizobium sp. mainly in areas cultivated with<br />
groundnut for the first time (Lanier et al., 2005).<br />
Inoculation of groundnut by LNB provided contrasting<br />
responses on two sites in central region of Cameroon;<br />
since yield increases of 169% on a relatively fertile clayey<br />
loam soil from Yaoundé and of only 5% on a mixed<br />
farming low-fertility sandy loam soil from Bokito (Mandou<br />
et al., 2002; Nwaga et al., 2010). A synergistic effect of<br />
inoculation and molybdenum seed treatment was also<br />
noticed, since this dual treatment resulted in a 288% yield<br />
increase in the Yaoundé site and a 21% increase in the<br />
Bokito site, when compared with the untreated control.<br />
The contrasting response to inoculation in the two study<br />
sites may be related to the density of indigenous LNB<br />
populations, since the Bokito soil contained 17,000<br />
cells/g and the Yaoundé soil only 170. Increase yield of
82% has been observed for A. hypogaea after rhizobia<br />
inoculation (Betsama, 1999). An inoculation response is<br />
extremely unlikely when native LNB soil bacteria density<br />
is more than 1,000 cells g -1 (Thies et al., 1991; Mafongoya<br />
et al., 2004). Some preliminary results indicate that, soil<br />
infertility and low density of LNB could be one of the<br />
causes of low pod filling of groundnut.<br />
The ability of A. hypogaea to nodulate in all the soils<br />
examined is an indication that the compatible LNB were<br />
present in these soils, but in diverse density. The growth<br />
of groundnut expressed in terms of shoot and root weight<br />
basis was better at Bokito and Bertoua than Yaoundé<br />
and Ebolowa soils, confirming the relation with nodulation<br />
results. Once again, growth was more improved in fallow<br />
than mixed farming, plantation and forest in all the<br />
experimental sites. The positive correlation observed<br />
between nodulation and dry weight, shoot growth of<br />
groundnut reflect that the LNB symbiosis enhance the<br />
plant response by providing increase nitrogen for the<br />
growth. Similar results were reported in sub Saharan<br />
Africa mixed farming inoculated with strains of LNB for<br />
cowpea (Ngakou et al., 2007), soybean (Megueni et al.,<br />
2006) and many other tropical legumes (Nwaga et al.,<br />
2010). Alemayehu (2010) also reported a positive corre-<br />
lation between nodulation and shoot height of V. faba.<br />
According to Lindström et al. (2010) inoculation is<br />
recommended if the field has no history of legume<br />
cultivation, especially if the plant is exotic to a new<br />
environment or if the soil is acidic, saline or otherwise<br />
hostile to rhizobia. In this study, inoculation is more likely<br />
to provide significant increase in nodulation, nitrogen<br />
fixation and seed yield in plantations and forest where<br />
soils are acidic.<br />
Conclusion<br />
Various soils of the humid forest zone of Cameroon<br />
contain LNB able to nodulate groundnut, but, are this<br />
enough for optimal nitrogen fixation and seeds production<br />
of A. hypogaea? Nodulation and growth of A. hypogaea<br />
closely depend on the land use system type and much<br />
more on the sites. An important variation was noticed on<br />
groundnut nodulation and growth according to site and<br />
land use. Further studies are needed to characterize<br />
major factors involved such as rhizobia density and select<br />
acidity tolerant strains adapted to environmental<br />
conditions of the humid forest zone of Cameroon.<br />
Investigations on these isolates could provide additional<br />
information on their symbiotic effectiveness with the aim<br />
of identifying very effective indigenous nitrogen fixing<br />
strains for local production of a specific groundnut<br />
inoculant.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4005-4010, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.861<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Comparison of some anthropometric and biologic<br />
parameters in two groups of Tunisian infants (0 to 2<br />
years)<br />
M. Kamel 1 *, A. Barkia 1,2 , M. Hamdaoui 3 , H. Ketata¹, M. Kassis 4 , M. Nasri 2 and A. Aouidet 3<br />
1 Ecole Supérieure des Sciences et Techniques de la Santé, de Sfax, boite postale 1099 Sfax 3038, Tunisie.<br />
2 Laboratoire de Génie Enzymatique et de Microbiologie, Ecole des Ingénieurs, Sfax, Tunisie.<br />
3 Ecole Supérieure des Sciences et Techniques de la Santé, Tunis, Tunisie<br />
4 Faculté de Médecine, Sfax, Tunisie.<br />
Accepted 17 December, 2010<br />
Malnutrition still remains a preoccupying health problem that affects the most vulnerable age group<br />
(less than five years). The aim of this research is to establish the nutritional profile of the Tunisian<br />
infants less than 2 years, to specify the principal deficiencies and the possible origins of these<br />
deficiencies. In our transverse exploratory study carried out in a period of 12 months, two groups of<br />
infants less than 2 year old; a control group (n = 18) and a malnourished group (n = 18) were used. Our<br />
data consolidate the impact of pregnant women’s nutritional state, breastfeeding on the infant and the<br />
infant growth. In comparison with the control group, the malnourished group showed a significant<br />
reduction in weight/age, weight/height (p < 0.001), a significant reduction of the average values of<br />
cholesterol associated with high density lipoproteins (Chol-HDL) and the ratio of cholesterol associated<br />
with high density lipoproteins to cholesterol associated with low density lipoproteins (Chol-HDL / Chol-<br />
LDL). This shrinkage showed a positive correlation with the weight/height ratio which is an indicator of<br />
malnutrition gravity. The results of this study confirmed the existence of a remarkable change of the<br />
biological profile among malnourished infants. In addition, the study shows the necessity to target and<br />
develop means to fight against factors favoring malnutrition.<br />
Key words: Malnutrition, infants, breastfeeding, protein, lipoprotein.<br />
INTRODUCTION<br />
In Tunisia, protein energy malnutrition (PEM) in preschool<br />
children is quite common, especially in rural areas (El Ati<br />
et al, 2002). According to the available epidemiological<br />
data, it was noticed that the prevalence of PEM varied<br />
between 11.2 and 23.4% in rural areas and 10.4 and<br />
6.2% in urban areas (El Ati et al., 2002). PEM affects<br />
various significant functions such as the physical growth,<br />
mental development, training capacity, the organism deence<br />
capacity, in particular the immunizing response and<br />
*Corresponding author. E-mail : kamelmed2006@yahoo.fr.<br />
Abbreviations: PEM, Protein energy malnutrition; EDTA,<br />
ethylenediamine tetra-acetic acid; apo, apolipoproteins; TG,<br />
triglycerides.<br />
antioxidant processes (Ahmed et al., 2009; Chen et al.,<br />
2009; Moynihan et al., 2009). The danger of malnutrition<br />
is especially serious among the young population, particularly<br />
children younger than 5 years.<br />
A well regulated diet must satisfy not only the protein<br />
energetic needs, but also those various micro nutriments<br />
(vitamins, zinc, etc.), which interact with the pathogenneses<br />
of various diseases that are established. For<br />
example, it was reported that a deficit in zinc, selenium,<br />
iron, vitamins A, E and acid folic is able to alter the<br />
immunizing responses (Makonnen et al., 2003; Rubhana<br />
et al., 2004). Similarly, it was shown that there existed a<br />
significant relation between diarrhoea, measles, anaemia<br />
and deficit in zinc, vitamin A and iron, respectively<br />
(Rahman et al., 2002; Muller et al., 2003).<br />
Malnutrition is also associated with biological disturbance.<br />
It is particularly accompanied by a reduction in
4006 Afr. J. Biotechnol.<br />
various proteins serum (albumin, prealbumin, retinol binding<br />
and apolipoproteins) and a reduction in the total<br />
cholesterol (chol), Chol-high density lipoprotein (HDL)<br />
and the ratio of Chol-HDL to Chol-low density lipoprotein<br />
(LDL).<br />
The aim of this study is to look for ways of establishing<br />
the nutritional profile of the Tunisian infants of less than<br />
two years, examine the effect of malnutrition on this<br />
profile and specify the principal deficiencies affecting the<br />
young infants.<br />
MATERIALS AND METHODS<br />
Subjects<br />
In our transverse exploratory study carried out during a period of 12<br />
months (from January to September, 2007), thirty six infants under<br />
2 years were recruited and divided into two groups: The first group<br />
is a control (n = 18), aged 11.3 ± 9.4 months, they were recruited<br />
among those frequenting the services of University Hospital Centre<br />
(UHC) of Sfax (large city in the south of Tunisia) through the regular<br />
postnatal control. They had a Z score for both their weight and<br />
height (between – 1 DS and + 1 DS).<br />
The second group (n = 18), malnourished infants aged 7.8 ± 6.6<br />
months, was recruited among those hospitalized in the service of<br />
paediatrics of this hospital. All the malnourished cases had a Z<br />
score < -2 D S (Pelletier et al., 1995). The protocol of research was<br />
made and approved by the ethical committee of the centre (UHC)<br />
and accepted by parents who gave written consent to the inclusion<br />
of their children in the study.<br />
Socio-economic, anthropometric and dietary assessments<br />
In the two groups, epidemiological characteristics (age and daily<br />
food consumption), clinics and anthropometrics parameters (height<br />
and weight) were noted. A socioeconomic survey, using a type<br />
closed standard questions was investigated. Moreover, the daily<br />
food intake of mothers was recorded according to the 24 h dietary<br />
recall method. The team also measured the index of body weight<br />
mass and the brachial perimeter.<br />
Blood collection<br />
Blood samples were taken from malnourished infants before any<br />
treatment. In both groups, fasting blood was drawn in vacationer<br />
tubes containing ethylenediamine tetra-acetic acid (EDTA) and<br />
centrifuged at 3000 rpm for 10 min, then the plasma was removed<br />
and conserved at 80°C until the analysis of different biological<br />
parameters were done.<br />
Plasma biological parameters<br />
Total proteins were quantified using the Biuret method (Biuret and<br />
Layne, 1957). The albumin, prealbumin, apolipoproteins AI (apo-AI)<br />
and B (apo-B) were determined by immunoturbidimetry on automates<br />
COBAS INTEGRA 400 (Roche Diagnostic Gmb H,<br />
Mannheim, Germany). The serum zinc and iron were determined<br />
after dilution of the sample in ultra-pure water by atomic absorption<br />
spectrophotometry using titrisol standard solution (Merck, Darmstadt,<br />
Germany) and PERKIN EL MER MODEL 305 B.<br />
Vitamins<br />
The serum vitamins A and E were realized using reversed-phase<br />
height-pressure liquid chromatography (RP-HPLC) according to the<br />
method of Jacob and Elmadfa (1995). Briefly, plasma proteins were<br />
precipitated with ethanol and lipids were extracted with n-hexane.<br />
After evaporation, the dry residue was redissolved with 150 µl of<br />
methanol-dichlormethane (85:15 v/v), mixed and then an amount of<br />
100 µl of this solution was injected into a guard column (Merck<br />
LiChrospher 100 RP18 (10 µm), 250 x 4 mm). Samples were run at<br />
a flow rate of 1.0 ml/min on a Dionex HPLC system (Summit TM<br />
HPLC, USA). Absorption was monitored at 325 nm for retinol, at<br />
295 nm for tocopherols, at 450 nm for carotenoids and at 270 nm<br />
for coenzyme Q10. Concentrations were calculated from areas<br />
under the curve using an external calibration curve. The vitamins<br />
were determined by spectrophotometer detector in 340 nm for the<br />
retinol and the retinal- acetate and in 292 nm for the alphatocopherol.<br />
Lipoproteins<br />
The fraction of HDL lipoproteins was obtained after precipitation<br />
with phosphotungstic acid containing the apo B (LDL), by using the<br />
case marketed by Bio Merieux (France). The total cholesterol,<br />
triglycerides (TG) and HDL were determined by enzymatic methods<br />
by using the automat RA 1000 analyser (Bayer Diagnostic, München,<br />
Germany). The cholesterol LDL were calculated by using the<br />
formula of Friedward:<br />
Chol LDL = CT- (Chol LDL + Chol VLDL)<br />
The very low density lipoproteins were calculated by dividing the<br />
rates of TG by 5. All our subjects had concentrations of TG lower<br />
than 3 g/l, which is a necessary condition for the application of Friedewald<br />
et al., (1972) formula.<br />
Statistical assessment of the data<br />
The average and standard deviation of the different parameters<br />
studied were calculated. Students’ t-test was used to assess the<br />
differences between the average values of these studied parameters<br />
in the groups. Statistical significance was considered for p<br />
values
Table 1. Comparison to the average values of the anthropometric parameters in the two groups of<br />
infants.<br />
Parameter Control (C) Malnourished (M) Significances (P)<br />
Age (months) 11.32 ± 9.37 7.83 ± 6.62 NS<br />
Weight at birth ( kg) 3.14 ± 0.28 2.74 ± 0.45 P < 0.05<br />
Weight (kg) 8.06 ± 2.86 5.13 ± 2.23 P < 0.02<br />
Weight of reference (kg) 7.62 ± 2.53 7.62 ± 2.54 NS<br />
Height at birth (cm) 50.39 ± 0.92 49.89 ± 0.96 NS<br />
Height (cm) 66.43 ±10.10 61.08 ± 9.56 NS<br />
Height of reference (cm) 67.39 ± 9.98 67.79 ± 9.54 NS<br />
Weight/height 0.12 ± 0.03 0.08 ± 0.02 P < 0.001<br />
Height/age 18.17 ±17.52 16.56 ±15.67 NS<br />
ns: No significant difference<br />
Table 2. Comparison of the mean values of the biological parameters in control and malnourished<br />
groups.<br />
Parameter Control (C) Malnourished (M) Significances(P)<br />
Apolipoprotein AI (g/l) 1.00 ± 0.19 0.77± 0.13 P < 0.001<br />
Apolipoprotein B (g/l) 0.59 ± 0.27 0.54 ± 0.09 NS<br />
Proteins (g/l) 60.71±10.09 49.25 ± 9.85 P < 0.001<br />
Albumin (g/l) 30.80 ± 5.86 24.62 ± 5.22 P < 0.001<br />
Prealbumin (g/l) 0.14 ± 0.07 0.11 ± 0.05 NS<br />
Vitamin A (µg/ml) 0.34 ± 0.16 0.21 ± 0.09 P < 0.01<br />
Vitamin E (µg/ml) 10.07 ± 3.79 4.72 ± 4.14 P < 0.001<br />
Iron (mg/ l) 40.91 ± 3.40 23.55 ± 3.65 P
4008 Afr. J. Biotechnol.<br />
Table 3. Correlate coefficients of weight/height ratio with other<br />
parameters.<br />
Parameter Weight/height<br />
Age 0.69 (***)<br />
Weight at birth 0.37 (*)<br />
Apolipoprotein AI ns<br />
Apolipoprotein B ns<br />
Proteins 0.68 (***)<br />
Albumin 0.49 (***)<br />
Prealbumin 0.70 (***)<br />
Vitamin A 0.66 (***)<br />
Vitamin E 0.77 (***)<br />
Zinc ns<br />
Iron 0.67 (***)<br />
Total cholesterol 0.37 (*)<br />
Triglycerides 0.48 (***)<br />
Chol- HDL ns<br />
Chol-LDL 0.37 (*)<br />
Chol-HDL /Chol-LDL ratio -0.04<br />
Chol-HDL; Cholesterol associated with high density lipoproteins;<br />
Chol-LDL: cholesterol associated with low density lipoproteins;<br />
Chol-HDL /Chol-LDL: ratio of Chol-HDL to Chol-LDL.<br />
*p < 0.05, ** p < 0.01, ***p < 0.001, ns: no significant difference.<br />
decrease in vitamin A, zinc (p < 0.01), Chol-HDL / Chol-<br />
LDL ratio (p < 0.02), Apo-AI, total proteins, albumin,<br />
vitamin E, HDL and iron (p < 0.001).<br />
In our study, we examined the relationship between the<br />
ratio weight/height (at the moment of recruitment) with<br />
the various examined parameters. Our data (Table 3)<br />
show that, some parameters correlated significantly with<br />
the ratio weight/height at the moment of recruitment,<br />
except the Apo AI, Apo-B, zinc and Chol-HDL.<br />
Our results show that the malnutrition of the infants<br />
induced a delay of the growth and perturbation of the<br />
biological parameters. Nutritional status was assessed<br />
using weight-for-height as an indicator to the present<br />
state of nutrition and height-for-age as an indicator of<br />
past nutrition. The growth of our groups was evaluated<br />
from the study of the ratios weight/height and height/age.<br />
The decrease in the first ratio is due to a fast thinning,<br />
whereas the decrease in the second ratio can be<br />
explained by a delay in the height growth. Only the first<br />
ratio has been found to be significantly decreased in the<br />
malnourished group, comparatively with the control<br />
group. The non variation of the second ratio can be explained<br />
by the age of our subjects which was of few<br />
months (7.83 ± 6.62 months).<br />
The borderline grade weight/height ratio among malnourished<br />
infants is probably attributable to the less<br />
privileged socioeconomic conditions of the community<br />
they belonged to. In addition, the abnormal state of our<br />
patients would be, in part, due to a prenatal origin as it is<br />
suggested by their weight at birth, less than that found in<br />
the control group. This hypothesis is confirmed by the<br />
existence of a positive correlation between their weight at<br />
birth and the weight/height relationship at the time of their<br />
admission in the hospital.<br />
The interaction between the nutritional state of the<br />
mother and that of the foetus has been established. It has<br />
been notably reported that a mother’s iron deficiency is<br />
explained, within the newborn baby, by an increasing<br />
anaemia (Sibeko et al., 2004). Besides, it has been<br />
proved that the satisfaction of the needs of the foetus for<br />
calcium requires an adequate amount of calcium and<br />
vitamin D by pregnant women. In the default case, the<br />
infant would have been born with a deficit of calcium and<br />
a weak reserve of vitamin D. This relationship between<br />
mother-infant also appeared clearly after the quantifycations<br />
of vitamins A and E in the mother’s blood as well<br />
as in that of the umbilical cord (Schulpis et al., 2004).<br />
The alimentary survey realized in our subjects revealed<br />
that, many postnatal factors were thought to aggravate<br />
the delay of the growth and biological disturbances. The<br />
absence of breast-feeding in our patients (94%) and the<br />
contribution of an insufficient need of artificial milk clearly<br />
showed a nutritional deficit. Besides, the great loss in<br />
weight and height should also be taken into consideration<br />
since our patients suffered from gastro-intestinal<br />
problems. These problems could result from inadequate<br />
complementary foods taken by babies in/before due time<br />
(before 4 to 6 months), due to the insufficient amounts of<br />
milk they have had. Other factors could interfere in the<br />
growth, especially the problems of infection as a result of<br />
the bad hygienic conditions. Such state was also likely to<br />
occur in underfed babies who are protected by various<br />
defensive factors existing in the mother’s milk (IgA,<br />
lysozyme, macrophages and granulocytes) (Hanson and<br />
Soderstrom, 1981).<br />
Therefore, it was of much importance to study parameters<br />
relating to oxidative stress (antioxidant vitamins<br />
and lipid peroxidation products). In fact, in malnourished<br />
infants, levels of plasma antioxidant vitamins A and E did<br />
significantly differ from levels observed in a similar control<br />
group. In addition, plasma levels of antioxidants were<br />
significantly lower in these malnourished infants. These<br />
observations are in agreement with other studies (Golden<br />
and Ramadath, 1987; Houssaini et al., 1977; Mc Laren et<br />
al., 1969). From the course of this study, it is evident that<br />
there is a significant correlation between enzymatic antioxidants<br />
and the antioxidant vitamin levels that reflect the<br />
pathological features of nutritional program rehabilitation.<br />
Therefore, there should be special emphasis on supplying<br />
antioxidant agents during any nutritional rehabilitation<br />
programme such as vitamins (A, E and C) and trace elements<br />
(calcium, magnesium, potassium and iron). Consideration<br />
should also be given to supplying therapeutic<br />
enzymatic antioxidants in order to reduce free radical
generation and enhance antioxidant status. These measures<br />
may help to reduce the mortality and morbidity<br />
associated with malnutrition.<br />
Other studies attempted to investigate the combination<br />
of several antioxidants as supplements (zinc, copper,<br />
selenium, vitamin A and E, etc.). It appears that, anti-oxidants<br />
work together to help protect the body from<br />
potentially harmful effects of free radicals (Gauche and<br />
Hausswirth, 2006). On the other hand, the malnourished<br />
group compared to the control group, showed a significant<br />
reduction of other various plasmatic parameters as<br />
vitamins A and E, proteins, apo-AI and trace element<br />
(Table 3). In accordance with our findings, a reduction of<br />
serum concentrations of albumins and vitamin E has<br />
been observed in Moroccan infants suffering from severe<br />
under nutrition (Squali et al., 2001). However, the<br />
concentration of TG which increases in these babies was<br />
found to be stable in our patients. A study on young<br />
Tunisian population suffering from stagnation reported a<br />
reduction of the apo-AI but not in the cholesterol, triglycerides,<br />
chol-LDL or chol-HDL concentrations (Sibeko et<br />
al., 2004).<br />
The alteration of the biological profile observed in the<br />
malnourished group can result from the insufficient food,<br />
the over loss and an unsatisfactory absorption. This can<br />
be the result, notably of a quantitative imbalance of feeding.<br />
A deficit in vitamin D (a favourable factor for calcium<br />
absorption) or an excess in phosphorus (accelerating<br />
calcium) can, for example, lead to a hypocalcaemia<br />
despite the resulted sufficient calcium. The resulted irons<br />
under a form of weak absorptions (such as vitamin C)<br />
can produce hyposideremy, even if the resulted iron is<br />
sufficient.<br />
Other markers of nutritional status, the haemoglobin<br />
and albumin levels were also reduced in malnourished<br />
group. At this stage, it is interesting to notice that, the<br />
mothers of these infants consume a lot of tea, which can<br />
aggravate anaemia, as reported in other surveys<br />
(Cournot and Hercberg, 1993; Hamdaoui et al., 1995).<br />
The change in the iron rate, observed in the malnourished<br />
group would then have started since the foetal<br />
stage as a result of an interaction of the mother’s state<br />
with that of foetus. This hypothesis could be seen as<br />
decrease in the rates of haemoglobins found in the<br />
mothers of the malnourished young.<br />
Our results show the existence of the relationship between<br />
the growth delay and the biological disturbance.<br />
Indeed, in the malnourished group, the ratio of weight/<br />
height correlated positively with some biological parameters<br />
as protein, albumin, prealbumin, vitamin A, vitamin<br />
E, iron, total Chol, TG and LDL Chol. Such effects were<br />
reported by other studies concerning young malnourished<br />
subjects. These people particularly showed a low rate of<br />
apo-AI (13 and 23) and a stable level of these parameters<br />
(Sibeko et al., 2004; Slimane et al., 1992; Truswell,<br />
1975). However, their apo-B remained unchanged<br />
(Sibeko et al., 2004).<br />
Conclusion<br />
Kamel et al. 4009<br />
The results of this study confirmed the impact of pregnant<br />
women nutritional state and breastfeeding on the infant<br />
and infant growth. Our data showed a significant alteration<br />
in the level of several anthropometric and biological<br />
parameters in the young malnourished when compared<br />
to the control. Such effects show the necessity of establishing<br />
a program to fight against the factors favouring<br />
malnutrition in our country. These results also show that,<br />
it is necessary to take the supplementation of antioxidants<br />
agents such as vitamins (A, E and C) and trace<br />
elements (calcium, magnesium, potassium and iron)<br />
during any nutritional rehabilitation program into consideration.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4011-4017, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1520<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Isolation and molecular characterization of RcSERK1: A<br />
Rosa canina gene transcriptionally induced during<br />
initiation of protocorm-like bodies<br />
Xu Kedong 1 , Liu Qinglin 2 , Yang Huifang 1 , Zeng Li 3 , Dong Lili 1 , Liu Fengluan 1 , Bi Ling 1 , Ma<br />
Nan 1 and Zhao Liangjun 1 *<br />
1 Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, 2 Yuanmingyuan<br />
West Road, Haidian District, Beijing 100193, People’s Republic of China.<br />
2 Department of Landscape Architecture, Sichuan Agricultural University, Ya'an, Sichuan 625014, People’s Republic of<br />
China.<br />
3 Agricultural and Biological College of Shanghai Jiaotong University, Shanghai 200240, People’s Republic of China.<br />
Accepted 20 January, 2011<br />
A somatic embryogensis receptor-like kinase (SERK) gene was isolated from protocorm-like bodies<br />
(PLBs) of Rosa canina by a rapid amplification of cDNA ends (RACE) approach and was designated as<br />
RcSERK1. The RcSERK1 encodes a protein of 626 amino acid residues with a calculated molecular<br />
mass of 68.79 kDa and theoretical isoelectric point of 5.65. The amino acid sequence of RcSERK1<br />
shares all the characteristic features of a SERK protein, including the signal peptide (SP), the leucine<br />
zipper (LZ), the five leucine-rich repeats (LRRs), the pro-rich domain containing the so-called Ser-Pro-<br />
Pro (SPP) motif, the transmembrane domain (TM), the kinase domain and the C-terminal domain. The<br />
transcripts of RcSERK1 were more enriched in PLBs than in rhizoids and callus, but not detected in<br />
leaflets (incubated under dark and before producing callus) and the regenerated shoots. Subcellular<br />
localization indicated that the fluorescence of RcSERK1-GFP was recorded in the plasma membrane.<br />
We argue that RcSERK1 is a Leu-rich repeat receptor-like kinase (LRR-RLK) and plasma membrane<br />
localization protein.<br />
Keywords: somatic embryogensis receptor-like kinase (SERK)1, protocorm-like bodies (PLBs), Rosa canina,<br />
RACE, RcSERK1.<br />
INTRODUCTION<br />
Somatic embryogensis receptor-like kinase (SERK)<br />
genes encode leucine-rich repeat receptor-like kinases<br />
*Corresponding author. E-mail: zhaolj5073@sina.com. Tel: +86-<br />
10-62733315. Fax: +86-10-62733603.<br />
Abbreviations: EX, Extracellular domain; LRRs, leucine-rich<br />
repeats; LRR-RLK, leucine-rich repeat receptor-like kinase; LZ,<br />
leucine zipper; PLBs, protocorm-like bodies; UTR, untranslated<br />
region; SE, somatic embryogenesis; SERK, somatic<br />
embryogenesis receptor-like kinase; SP, signal peptide; SPP,<br />
serine-proline-proline; TDZ, thidiazuron; TM, transmembrane<br />
domain; RACE, rapid amplification of cDNA ends; CaMV,<br />
cauliflower mosaic virus; PCR, polymerase chain reaction; RT-<br />
PCR, reverse transcriptase-PCR.<br />
(LRR-RLKs) (Schmidt et al., 1997; Hecht et al., 2001),<br />
and SERKs share the canonical structure of LRR-RLKs<br />
but have a limited number of leucine-rich repeat (LRR)<br />
motifs (Colcombet et al., 2005). The first SERK gene<br />
identified was reported in carrot (Daucus carota) suspension<br />
cultures where it was specifically expressed in<br />
cells which developed into somatic embryos (Schmidt et<br />
al., 1997; Nolan et al., 2009). SERK genes have been<br />
isolated from several plant species including Arabidopsis<br />
thaliana (Hecht et al., 2001), Zea mays (Baudino et al.,<br />
2001), Medicago truncatula (Nolan et al., 2003),<br />
Helianthus annuus (Thomas et al., 2004), Ocotea<br />
catharinensis (Santa-Catarina et al., 2004), Dactylis<br />
glomerata (Somleva et al., 2000), Citrus unshiu (Shimada<br />
et al., 2005), Oryza sativa (Hu et al., 2005) and<br />
Theobroma cacao (Santos et al., 2005). Ectopic expres-
sion of the full-length AtSERK1 cDNA under the control of<br />
the cauliflower mosaic virus (CaMV) 35S promoter did<br />
not result in any altered plant phenotype. However, seedlings<br />
that overexpressed the AtSERK1 mRNA exhibited a<br />
3- to 4-fold increase in efficiency for initiation of somatic<br />
embryogenesis (Hecht et al., 2001). AtSERK1 and<br />
AtSERK2 receptor kinases function together as an<br />
important control point for sporophytic development controlling<br />
male gametophyte production (Colcombet et al.,<br />
2005). Fradin et al. (2009) indicated that tomato<br />
SERK3/BAK1 physically associates with the RLP Ve1 to<br />
initiate verticillium immunity.<br />
Somatic embryogensis receptor-like kinase (SERKs)<br />
form a sub-group among LRR-RLKs, which comprise the<br />
largest sub-family of RLKs in plants, and are involved in<br />
key plant developmental processes. SERK genes were<br />
isolated in several plant species suggesting the ubiquitous<br />
presence of a small SERK gene family in all plants,<br />
and moreover, their functional conservation with a<br />
specific role in embryogenesis, and possibly other developmental<br />
processes (Nolan et al., 2009).<br />
We have successfully established a high efficiency<br />
somatic embryogenesis (SE) system of Rosa canina,<br />
which is an important ornamental plant widely grown in<br />
the world, whose protocorm-like bodies (PLBs) were<br />
induced from rhizoids by thidiazuron (TDZ) using leaflets<br />
of tissue cultured seedlings. PLBs were also considered<br />
to be somatic embryos, with an intermediary callus formation,<br />
which sometimes is a prerequisite for PLBs<br />
formation (Tian et al., 2008). We have studied the<br />
development process, morphological characteristics and<br />
microscopic structure of PLBs when compared with that<br />
of somatic embryos from other plants, and we found that<br />
PLBs were embryo aggregates, which were coated by<br />
cellular tissues (Tian et al., 2008).<br />
In this study, we isolated and characterized a LRR-RLK<br />
gene, designated as RcSERK1. And our analyses suggested<br />
that RcSERK1 was orthologous to other plant<br />
SERKs, and the possible role of RcSERK1 would be<br />
crucial for the explanation of somatic embryogenesis<br />
(SE) of R. canina.<br />
MATERIALS AND METHODS<br />
Plant materials<br />
Leaflets, callus, rhizoids, PLBs and the regenerated shoots of R.<br />
canina were collected separately. All samples of plants were frozen<br />
immediately in liquid nitrogen and stored at -80°C for RNA extraction.<br />
Isolation of the RcSERK1 gene<br />
Polymerase chain reaction (PCR) was performed to obtain a partial<br />
sequence of RcSERK1 by using the first strand cDNA of R. canina<br />
as a template. With multiple sequence alignment of related SERK<br />
base sequences, two degenerate primers corresponding to the<br />
amino acid sequences, FKS1 sequence (5'-GTGAAY(C/T)<br />
Kedong et al. 4012<br />
CCTTGCACATGGTTY(C/T)CATGT-3') and RKS1 sequence (5'-<br />
ATGGAR(A/G)TACAAGGAR(A/G)ACCCAR(A/G)GTH(A/T/C)ACA-<br />
3') were used for the PCR of partial sequence. The AUAP primer<br />
(5’-GGCCACGCGTCGACTAGTAC-3’) and another primer<br />
corresponding to the FKS2 sequence (5'-TAATGGCAGCGTT<br />
GCCTCAGTT-3') were used for 3’ RACE. Primers for 5' RACE<br />
were: AAP sequence, 5'-GGCCACGCGTCGACTAGTA CGGGII<br />
GGGIIGGGIIG-3' and RK5-1 sequence, 5'-TGGTGCTGCTTTA<br />
CTATTTGCTGCC-3', for the first polymerase chain reaction (PCR).<br />
And the second PCR primers were: AUAP and RK5-1.<br />
The RACE reactions were performed according to the<br />
manufacturer’s protocol (Invitrogen RACE cDNA amplification kit,<br />
USA). We obtained a single full-length cDNA sequence by combining<br />
the 5'-RACE fragment, a partial fragment and C-terminal<br />
fragment. Finally, a pair of primers (FK1: 5'-TGGGGTGGTGGT<br />
GAGAACAGGCTTTGG-3' and RK1: 5'-ACCGCCGCAAATGA<br />
TACAAC TTGC-3') were then designed from the putative 5' and 3'<br />
untranslated region (UTR) of the full-length cDNA sequence. An<br />
1881bp putative RcSERK1 fragment was generated. The nucleotide<br />
sequences of RcSERK1 reported in this paper have been<br />
submitted to the GenBank under accession numbers: HM802242.<br />
The resultant DNA fragments and RACE products were gel purified<br />
and cloned into the pMD18-T vector (Takara) and sequenced<br />
(Invitrogen, Beijing).<br />
RNA isolation, DNase treatment and semi-quantitative reverse<br />
transcription polymerase chain reaction (RT-PCR) assay<br />
Total RNA from various R. canina tissues was extracted using<br />
RN09-EASY spin Kit (Biomed, Beijing, China) according to the<br />
manufacturer’s instructions. Total RNA preparations were subjected<br />
to an on-column DNase digestion, while simultaneously performing<br />
RNA clean-up, using a Qiagen RNase-Free DNase-Set and Qiagen<br />
RNeasy RNA Clean-up Midi Kit (Qiagen, Germany). The first strand<br />
cDNA was synthesized with 1 µg total RNA and 1 µl superscriptII<br />
enzyme (Invitrogen, USA) according to the manufacturer’s protocol.<br />
As a control, the 18s rRNA gene (Genbank accession number:<br />
FM164424.1) was amplified from various R. canina tissues. The<br />
primers used for detecting RcSERK1 gene expression were: forward<br />
primer 5'-CGTCGCTCATCCCTTATGGATCAT-3' and reverse<br />
primer 5'-AGAATTCGGATGAGGAGCTAATTC-3'. The PCR was<br />
performed as follow: pre-denaturation at 94°C for 5 min, followed by<br />
35 cycles of 45 s at 94°C, 45 s at 55°C, 2 min at 72°C for<br />
RcSERK1, 28 cycles for 18s rRNA and a final extension of 10 min<br />
at 72°C. The amplified products were resolved on a 1.2% agarose<br />
gel and then detected by agarose gel electrophoresis. All RT-PCR<br />
experiments were repeated at least three times.<br />
Sequence alignment and phylogenetic tree analysis<br />
The sequence alignment of RcSERK1 and other SERK amino acid<br />
sequences were compared by DNAMAN (ver 5.2.2) and the<br />
phylogenetic tree was constructed by neighbor-joining method with<br />
MEGA program (ver 4.0).<br />
Subcellular localization<br />
The RcSERK1 open reading frame (ORF) were cloned into the Hind<br />
III and SmaI sites of the pSAT6-GFP-N1 vector. This vector<br />
contains a modified red-shifted (green fluorescent protein, GFP) at<br />
NcoI-XbaI sites. The RcSERK1-GFP construct was transformed<br />
into onion epidermal cells by particle bombardment as described<br />
earlier (Wang and Fang, 2002). The transient expression of the<br />
RcSERK1-GFP fusion protein was observed using confocal<br />
microscopy.
4013 Afr. J. Biotechnol.<br />
RESULTS<br />
Figure 1. Nucleotide and deduced amino acid sequences of RcSERK1 (GenBank No. HM802242). The canonical<br />
structure of the RcSERK1 proteins is the presence of the extracellular SPP motif in combination with precisely five<br />
LRRs, which is underlined.<br />
Isolation of the RcSERK1 gene from PLBs of R.<br />
canina<br />
Using the homologous regions of the SERK1 genes, a<br />
partial cDNA was isolated from PLBs of R. canina by<br />
using degenerate PCR. The full-length cDNAs were ob-<br />
tained by employing rapid amplification of the 3'-cDNA<br />
end (3'-RACE) and the 5'-cDNA end (5'-RACE), and were<br />
designated as RcSERK1 (Genbank accession No.<br />
HM802242). Sequence analysis showed that the<br />
RcSERK1 cDNA was 2438 bp in length, including a complete<br />
ORF of 1881 bp flanking with a 5'-UTR of 301 bp<br />
and a 3'-UTR of 256 bp (Figure 1). The predicted protein<br />
of RcSERK1 comprises 626 amino acids with a calculated
Kedong et al. 4014<br />
Figure 2. The alignment of the identified RcSERK1 with other plant SERK proteins including the SP, LZ, five LRRs, SPP motif,<br />
TM, kinase domain (DI to DXI) and the C-terminal domain. Positions containing identical residues are shaded in navy blue,<br />
while conservative residues are shaded in pink.<br />
molecular mass of 68.79 kDa and its theoretical isoelectric<br />
point was 5.65.<br />
As shown in the Figure 2 alignment, RcSERK1 belongs<br />
to the LRR-type cell surface RLKs, which possess a<br />
number of characteristic domains. These include an<br />
extracellular domain (EX) containing a variable number of<br />
LRR units immediately followed by a single transmembrane<br />
domain (TM) and an intracellular kinase<br />
domain responsible for phosphorylating downstream<br />
proteins (Hecht et al., 2001). The amino acid sequence of<br />
RcSERK1 shows a high percentage of identity with<br />
AtSERK1 (88.52%) and DcSERK (77.32%). The<br />
predicted protein structure of the RcSERK1 protein<br />
shares all the characteristic features of that protein, and<br />
starts at the N-terminus with a signal peptide (SP) followed<br />
by a LZ, the five LRRs, a pro-rich domain called the<br />
serine-proline-proline (SPP), a single TM, the 11<br />
conserved subdomains of a Ser-Thr kinase and a Cterminal<br />
leu-rich domain (Hanks et al., 1988). The hallmark<br />
of the SERK proteins is the presence of the extracellular<br />
Ser-Pro-Pro motif in combination with precisely<br />
five LRRs (Albrechta et al., 2005). Upon comparison of
4015 Afr. J. Biotechnol.<br />
Figure 3. Phylogenetic tree analysis of RcSERK1 and other plant SERK proteins. The tree<br />
was constructed by neighbor-joining method with MEGA program (ver 4.0). Branch numbers<br />
represent percentage of bootstrap values in 1000 sampling replicates and scale indicates<br />
branch lengths. The accession numbers are as follows: CsSERK (Citrus sinensis)<br />
(FJ851422), CuSERK (Citrus unshiu) (AB115767), RcSERK1 (R. canina) (HM802242),<br />
StSERK (Solanum tuberosum) (EF175215), AtSERK1 (Arabidopsis thaliana) (NM_105841),<br />
AtSERK2 (A. thaliana) (AF384969), AtSERK3 (A. thaliana) (AF384970), DcSERK (Daucus<br />
carota) (DCU93048), CnSERK (Cocos nucifera) (AY791293), ZmSERK1 (Zea mays)<br />
(NM_001111662), ZmSERK2 (Z. mays) (NM_001111663), OsSERK1 (Oryza sativa)<br />
(AY652735) and PtSERK (Populus tomentosa) (DQ680855).<br />
the amino acid sequences with other species, it was<br />
found that RcSERK1 had very high identity including the<br />
LZ, LRRs, SPP motif, TM, kinase domain and the Cterminal<br />
domain, except for the SP (Figure 2).<br />
We chose herbaceous and woody dicots and monocots,<br />
with different families and genera, to investigate the<br />
evolutionary relationship among plant SERK proteins. A<br />
phylogenetic tree (Figure 3) was constructed using the<br />
neighbor-joining method with the full-length amino acid<br />
residues. The results showed that RcSERK1 was tightly<br />
clustered with CsSERK and CuSERK, and StSERK were<br />
grouped into a cluster, and henceforth, designated as<br />
RcSERK1, whereas AtSERK1 and AtSERK2 formed<br />
another cluster.<br />
Expression analysis of RcSERK1<br />
The expression profiles of RcSERK1 gene in various R.<br />
canina tissues were investigated using a semi-quantitative<br />
RT-PCR assay. Various tissues were respectively<br />
collected, as described in materials and methods.<br />
RcSERK1 mRNA was detected in PLBs, callus and<br />
rhizoids, but not in leaflets and the regenerated shoots,<br />
and mainly in the PLBs but weak in the callus and rhizoids<br />
(Figure 4).<br />
Localization of RcSERK1 in the plasma membrane<br />
To examine subcellular localization of RcSERK1 protein,<br />
the RcSERK1-GFP fusion protein was introduced into<br />
onion epidermal cells by particle bombardment. As<br />
shown in Figure 5, the RcSERK1-GFP fusion protein was<br />
recorded in the plasma membrane, whereas the control<br />
GFP alone was distributed throughout the cytoplasm.<br />
These results show that the RcSERK1 protein is a plasma<br />
membrane localized protein.<br />
DISCUSSION<br />
In this study, a LRR-RLK gene RcSERK1 has been<br />
isolated from PLBs of R. canina, and its expression has<br />
been investigated in leaflets, callus, rhizoids, PLBs and<br />
the regenerated shoots. To our knowledge, this is the first
Figure 4. Expression patterns of RcSERK1 in different tissues.<br />
Expression patterns of RcSERK1 in leaflets (LEA), callus (CAL), rhizoids<br />
(RHI), PLBs (PLB) and the regenerated shoots (REG). Ethidium bromide<br />
staining of PCR products using RcSERK1-specific primers with (top) and<br />
without (middle) prior reverse transcription and the RT-PCR products with<br />
18S rRNA-specific primers (bottom).<br />
Kedong et al. 4016<br />
Figure 5. RcSERK1 localizes to the plasma membrane. Onion epidermal cells were transformed with 35S::GFP and<br />
35S::RcSERK1-GFP. Transformed cells in the bright light (A and E), in the dark for cell nucleuses stained with DAPI (B and F),<br />
in the dark for the GFP-fusion proteins (C and G) and the merge of A, B and C (D) / E, F and G (H) were visualized, after<br />
incubation for 20 h.<br />
Report of cloning of a SERK gene in R. canina and the<br />
study of its expression.<br />
Sequence analysis of RcSERK1 revealed high levels of<br />
similarity to other plant species of SERKs, and it contains<br />
the signal peptide (SP), the leucine zipper (LZ), the five<br />
leucine-rich repeats (LRRs), the pro-rich domain containing<br />
two tandemly repeated SPP sequences, the TM,<br />
the kinase domain and the C-terminal domain. Its sub-<br />
cellular location was verified as a membrane protein,<br />
consistent with previous reports (Shah et al., 2001), and<br />
implied the role of RcSERK1 as a functional gene. The<br />
sequence alignment and phylogenetic tree analysis both<br />
were consistent with RcSERK1 being a functional SERK<br />
orthologue, for the hallmark of the SERK proteins is the<br />
presence of the extracellular SPP motif in combination<br />
with precisely five LRRs.
4017 Afr. J. Biotechnol.<br />
Furthermore, RcSERK1 clustered most closely with<br />
SERK gene family members such as CsSERK1, CuSER-<br />
K1, AtSERK1 and StSERK, which is implicated in<br />
evoking somatic embryogenesis. Monitoring of SERK1<br />
expression during progression of R. canina SE revealed<br />
RcSERK1 expression not only in PLBs, but also in callus<br />
and rhizoids, however, not detected in leaflets and the<br />
regenerated shoots. It showed that RcSERK1 was<br />
expressed at the beginning of the callus of leaves in vitro,<br />
but under artificial stress-induced, such as dark and 2,4dichloro-phenoxyacetic<br />
acid (2,4-D), and then<br />
disappeared at the stage of protocorm-like bodies<br />
(PLBs). So expression analysis suggests that the isolated<br />
RcSERK1 could be a functional SERK orthologue and an<br />
important embryogenic correlation factor. Transformation<br />
of the RcSERK1 into plants and further analysis should<br />
reveal its possible functions in SE of R. canina.<br />
ACKNOWLEDGEMENTS<br />
This research was supported by the Surface Project from<br />
National Natural Science Foundation of China (Grant No.<br />
30871733)<br />
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Santos MO, Romanoa E, Yotokoc KSC, Tinoco MLP, Diasa BBA,<br />
Araga˜oa FJL (2005). Characterisation of the cacao somatic<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4018-4023, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2363<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Molecular cloning and expression of the luciferase<br />
coding genes of Vibrio fischeri<br />
Golnaz Asaadi Tehrani 1 , Sina Mirzaahmadi 1 , Mojgan Bandehpour 2 , Faramarz Laloei 3 , Akram<br />
Eidi 1 , Toraj Valinasab 3 and Bahram Kazemi 2,4 *<br />
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.<br />
2 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.<br />
3 Iranian Fisheries Research Organization, Ecology research center of the Caspian Sea, Sari, Iran.<br />
4 Department of Biotechnology Shahid Beheshti University of Medical Sciences, Tehran, Iran.<br />
Accepted 7 April, 2011<br />
Vibrio fischeri is a symbiotic marine bacterium and a nonpathogenic member of the Vibrionaceae which<br />
produces luminescence by expressing the lux operon. The lux operon encoding luciferase (luxAB) and<br />
proteins required to synthesize the aldehyde substrate (luxCDE), is controlled with luxR and luxI. In this<br />
study, we amplified the chromosomal fragment contains luxAB of V. fischeri, amplified fragment cloned<br />
into the pTZ57R vector and sequencing to confirmed the fragment. The sub cloning of luxAB gene was<br />
carried out in the pETDuet-1 expression vector and expression procedures were performed in<br />
Escherichia coli strain Nova blue. As a result, a 2046 bp fragment which contains the whole fragment of<br />
luciferase coding genes and intergenic sequences were cloned in pETDuet-1 expression vector.<br />
pETDuet luxAB recombinant plasmid was confirmed by restriction analysis; subsequently 76 kD<br />
expressed protein was detected using SDS–PAGE and western blot using specific polyclonal antibody.<br />
In this study, cloning of the luciferase coding genes was performed successfully, in which the<br />
synthesized construct can be applied as a reporter cassette in prokaryotic systems and as a marker or<br />
tag in the manipulation, and the control of gene expression in the fields of research, production, control<br />
of microorganism and other biotechnological applications.<br />
Key words: Vibrio fischeri, lux operon, luciferase, luminescence.<br />
INTRODUCTION<br />
Vibrio fischeri is a marine bioluminescence bacterium and<br />
a nonpathogenic member of the Vibrionaceae, a large<br />
family of marine γ –proteobacteria, (Thompson et al.,<br />
2004) it lives both as a free living organism and also as a<br />
symbiotic in the light-emitting organs of the Hawaiian<br />
bobtail squid, Euprymna scolopes, where it produces<br />
luminescence by expressing the lux operon (Nyholm and<br />
Mcfall-Ngai, 2004; Ruby, 1999; Visick and Mcfall-Ngai,<br />
2000). In seawater and free-living form, V. fischeri exists<br />
at low cell densities and appear to be non-luminescent,<br />
while in light organ symbiosis with fish and squid, where<br />
the density of V. fischeri cells is high, it is usually lumine-<br />
*Corresponding author. E-mail: Bahram_14@yahoo.com. Tel:<br />
+9821 22439957. Fax: +98 21 22439956.<br />
scent (Sitnikov et al., 1995). In the best studied luminous<br />
bacterium, V. fischeri, there are at least eight lux genes<br />
encoding the proteins essential for luminescence. The lux<br />
regulon is organized in two operons with a divergent<br />
transcription pattern. The seven genes (luxICDABEG),<br />
followed by a transcriptional terminator comprise the<br />
rightward operon, which encode the enzymes required for<br />
the synthesis of the autoinducer (luxI) and the alpha<br />
(luxA) and beta (luxB) subunits of the enzyme luciferase,<br />
the luxC, luxD and luxE genes encode the enzymes<br />
participating in the formation of the long-chain aldehyde,<br />
luxG is not essential for luminescence but is believed to<br />
increase the capacity of the cell to synthesize flavin<br />
mononucleotide (FMN). The leftward operon consists of a<br />
single gene, LuxR, encoding the transcriptional regulatory<br />
protein (Figure 1) (Visick et al., 2000). Bacterial luciferase<br />
is a heterodimeric enzyme of 77 kDa comprising α and β
Tehrani et al. 4019<br />
Figure 1. Luciferase gene organization in v. fischeri: Structural genes contain, luxA and luxB, luxC, luxD, luxE and<br />
regulatory genes contain, luxI and luxR.<br />
Figure 2. The reaction of bacterial luciferase.<br />
subunits with approximate molecular size of 40 and 37<br />
kDa, respectively. Amino acid sequence alignment<br />
between the two subunits reveals that, they share 32%<br />
sequence identity. The α subunit contains 29 additional<br />
amino acid residues inserted between residues 258 and<br />
259 of the β subunit, indicating that the two genes have<br />
arisen by gene duplication through the course of<br />
evolution. There is a single active center in the luciferase<br />
heterodimer that resides on α subunit and binds one<br />
reduced flavin molecule. It seems that, β subunit is<br />
essential for a high quantum yield reaction (Baldwin et<br />
al., 1995; Fisher et al., 1996).<br />
In the luminescence reaction, luciferase converts<br />
aliphatic-aldehyde substrate, oxygen and reduced FMN<br />
(FMNH2) into the corresponding aliphatic acid, water and<br />
FMN, with the concomitant production of light (Figure 2).<br />
In the absence of the aldehyde substrate, luciferase<br />
catalyzes a reaction that yields no light and produces<br />
oxygen radicals rather than water (Nelson et al., 2007).<br />
Luminescence in V. fischeri is controlled by a<br />
population density- responsive regulatory mechanism<br />
called quorum sensing (Dunlap, 1999). At low cell<br />
population density, LuxI synthesizes only basal levels of<br />
the signal, 3-oxohexanoyl L-homoserine lactone (N3-oxo-<br />
C6-HSL); however, with increasing cell density the signal<br />
molecule accumulates to reach a threshold concentration<br />
that can bind to LuxR. The LuxR-AHL complex directly<br />
activates transcription of the luxICDABEG genes and<br />
resulting light production. V. fischeri cells also synthesize<br />
a second autoinducer molecule, octanoyl L-homoserine<br />
lactone (C8- HSL), that under some conditions it may<br />
stimulate transcription of the lux genes (Fuqua et al.,<br />
1994; Milton, 2006; Ulitzur, 1998; Waters and Bassler,<br />
2005). The V. fischeri transcriptional regulator, LitR also<br />
participates in luminescence regulation by inducing the<br />
transcription of luxR, particularly at low cell densities<br />
(Lupp et al., 2003).<br />
The aim of the current study was to clone the alpha<br />
(luxA) and beta (luxB) subunits of the enzyme luciferase<br />
gene in a suitable prokaryotic expression vector in order<br />
to express and produce the desired protein in Escherichia<br />
coli and it can be used as a reporter gene construct for<br />
further biological and biotechnological applications.<br />
Moreover, purification and testing the biological activities<br />
along with comparison with other reporter gene systems<br />
or other constructs which carry luciferase genes will be<br />
the next goals of this research work.<br />
MATERIALS AND METHODS<br />
Bacterial strains and plasmids<br />
V. fischeri strain, ATCC 7744 was kindly provided by Iranian<br />
Research Organization for Science and Technology (IROST) which<br />
was grown at 20 to 25°C. Nutrient media used for the growth of V.<br />
fischeri was DSMZ medium 246 that contained artificial seawater<br />
(750.0 ml), peptone (10.0 g), beef extract (10.0 g) and water (250.0<br />
ml). The ingredients of artificial sea water were NaCl (28.13 g), KCl<br />
(0.77 g), CaCl2 × 2H20(1.60 g), MgCl2 × 6H20(4.80 g), NaHCO3<br />
(0.11 g), MgSO4 × 7H20(3.50 g) in 1000.0 ml distilled water. Cloning<br />
vector pTZ57R (2.8 kb) and expression vector pETDuet-1(5.4 kb)<br />
were obtained from Fermentase and Novagene, respectively.<br />
Genomic DNA extraction, PCR and sequencing<br />
Genomic DNA extraction was carried out by Phenol-chloroform<br />
procedure and precipitated in ethanol (Sambrook and Russell,<br />
2001). Sense and antisense oligonucleotide primers were designed<br />
(Table 1) based on the nucleotide sequence data of luxA and luxB<br />
obtained from Gene Bank (NC_006841.1 for luxA and<br />
NC_006841.2 for luxB). PCR reaction mixture (30 µl) contained, 1<br />
µg DNA 0.1 mM dNTPs, 1.5 mM MgCl2, 1X PCR buffer, 20 pmol of<br />
each primers and 1.25 units Taq DNA polymerase (CinnaGen,<br />
Iran).<br />
PCR amplification was performed under the following condition:<br />
denaturation step; 5 min at 94°C followed by 30 cycles of 40 s at<br />
94°C, 60 s at 51°C in the annealing step and 60 s at 72°C for the<br />
extension step. The resulting PCR product was extended for a<br />
further 5 min at 72°C. The PCR product was analyzed on a 1.5%<br />
agarose gel and purified by using DNA extraction kit (Fermentase<br />
Lithuania) according to the manufacturer’s instructions and
4020 Afr. J. Biotechnol.<br />
Table 1. Primers for amplifying lux genes by PCR.<br />
subjected to sequencing using dideoxynucleotide chain termination<br />
method.<br />
Construction of recombinant plasmids pETDuet-luxAB<br />
To construct luminescent E. coli, purified PCR product of luxAB<br />
gene was ligated to a 3´ T tailed EcoRV digested pTZ57R (Gaastra<br />
and Hansen, 1984) and transformed in E. coli TOP10 competent<br />
cells as described previously (Hanahan, 1983). Recombinant<br />
clones were confirmed by universal PCR and restriction enzyme<br />
digestion analysis .The luxAB gene was released by BamH1 and<br />
Kpn1 digestion and subcloned in to the pETDuet-1 expression<br />
vector.<br />
Protein expression<br />
PETDuet-luxAB was transformed in E. coli Nova blue strain and<br />
selected on LB agar containing 50 µg/lm of ampicilin. A bacterial<br />
colony was inoculated into a medium (1.2% Bacto tryptone, 2.4%<br />
yeast extract, 0.04% glycerol, 1% M9 salts (6.4% Na2HPO4-7H20,<br />
1.5% KH2PO4, 0.025% NaCl, 0.05% NH4Cl)) and incubated<br />
overnight at 37°C in a shaker incubator at 200 rpm. The overnight<br />
cultured bacteria was inoculated into a 50 ml flask and incubated at<br />
37°C on orbital shaker incubator at 200 rpm. Cultures in logarithmic<br />
phase (at OD600 of 0.6) were induced with IPTG (0.5 mM) and<br />
samples were collected before and after induction (0, 3 and 5 h),<br />
cells were lysed in 2x sample buffer (100 mM Tris-HCl pH 8, 20%<br />
glycerol, 4% SDS, 2% beta mercaptoethanol, 0.2% bromo phenol<br />
blue) and separated by 12% SDS-PAGE. The gel was stained by<br />
Coomassie brilliant blue R250 and gene expression analyzed in<br />
comparison with uninduced control samples in parallel (Smith,<br />
1984).<br />
Western blot analysis using antibody<br />
For western blot analysis, cell lysate was separated on 12% (v/v)<br />
SDS-PAGE and electrophoretically transferred onto nitrocellulose<br />
membrane, after UV cross linking for protein fixation; the membrane<br />
was blocked with 3% BSA at room temperature. The bacterial<br />
luciferase antibody (HRP) (Abcam, UK) was used as specific<br />
antibody in 1:1000 of the protein band was performed by DAB<br />
(Diamino benzoic acid) and H202 (Shewry and Fido, 1998).<br />
RESULTS<br />
A DNA fragment encoding the luxAB part of lux operon<br />
was amplified by PCR using sense and antisense<br />
primers, specific restriction sites for BamH1 and Kpn1<br />
were introduced into 5´ end of forward and reverse<br />
primer, respectively. The PCR product was analyzed<br />
in1.5% agarose gel after electrophoresis (Figure 3).<br />
LuxAB Primer<br />
Forward (FluxA) GGATCCATGAAGTTTGGAAATATTTG<br />
Reverse (RluxB) GGTACCTTAAGGCAGATTCTTTTC<br />
The primers are presented from 5ʹ to 3ʹ ends; the introduced restriction<br />
sites are underlined.<br />
The PCR product of luxAB was purified from agarose gel<br />
using Fermentase kit and ligated into cleaved pTZ57R<br />
cloning vector. Following the confirmation of the cloned<br />
luciferase coding genes by sequencing, using BamH1,<br />
Kpn1 restriction enzymes pTZ57R/luxAB plasmid was<br />
digested, a 2046 bp DNA fragment purified, ligated into<br />
the BamH1, Kpn1 sites of an expression vector<br />
(pETDuet-1) and transformed into E. coli TOP10;<br />
recombinant plasmid was confirmed through restriction<br />
digestion using BamH1 and Kpn1 enzymes and named<br />
pETDuet -1/luxAB, (Figure 4); subsequently, transformation<br />
procedure continued into E. coli expression<br />
strains including BL21, Nova blue and JM109.<br />
After induction with IPTG (1 mM), produced proteins<br />
were run onto 12% SDS- PAGE gel, the three protein<br />
bands 37, 40 and 76 kD were detected, which indicates<br />
protein expression of luxB, luxA and luxAB genes,<br />
respectively as shown in Figure 5. The best protein<br />
expression results was obtained after transformation of<br />
the recombinant plasmid onto Nova blue cells in comparision<br />
with two other E. coli strains JM109 and BL21. The<br />
gene segment was under the control of T7 promoter in<br />
the expression vector and the expressed proteins were<br />
accumulated in the cytoplasm.<br />
The protein identity was verified by western blot<br />
analysis. The assay revealed specifically recognition of<br />
luciferase coding genes (luxAB) by bacterial luciferase<br />
antibody (a rabbit polyclonal antibody to bacterial luciferase,<br />
IgG HRP conjugated), whereas no reactivity was observed<br />
in control sample (Figure 6).<br />
DISCUSSION<br />
Gene screening with an easily assayable product,<br />
reporter genes, amplify the signal from the cell surface to<br />
produce a rapid, highly sensitive, reproducible and easily<br />
detectable response. The variety of reporter genes<br />
available including β-galactosidase (lacZ), chloramphenicol<br />
acetyltransferase (CAT), insect luciferase (luc),<br />
bacterial luciferase (lux), alkaline phosphates (phoA), βlactamase<br />
(bla), β-glucuronidase uidA (gusA, gurA),<br />
green fluorescent protein (GFP) and their applications are<br />
very broad in both in vitro and in vivo assays (Jiang et al.,<br />
2008; Köhler et al., 2000; Naylor, 1999). Among all<br />
studied reporter genes, bacterial luciferase are the most<br />
abundant, widely distributed, extensively studied and the<br />
best understood of all types of bioluminescent genes. It is
Figure 3. PCR amplification of the luxAB coding<br />
region using FluxA and RluxB primers. Lane 1and<br />
2 are the same PCR products; lane 3 is a DNA<br />
ladder marker.<br />
Figure 4. lane 1 and 3; the products of digestion with BamH1 and Kpn1; lane 2 and 4 uncut recombinant<br />
plasmid; lane 5 molecular size marker 10000, the released fragment migrated below the 2500 bp fragment<br />
of the DNA ladder.<br />
an α/β heterodimer, a flavine monooxygenize which is<br />
homologous in all bacterial luciferase, catalyzes the<br />
oxidation of a long-chain aldehyde and releases energy<br />
in the form of visible light. Yet luciferase coding genes<br />
have been isolated and cloned from different bacterial<br />
strains like Vibrio harveyi, V. fischeri, Photobacterium<br />
phosphoreum, Photobacterium leiognathi and<br />
Xenorhabdus luminescens in separate or fusion forms<br />
(Meighen, 1994; Wilson and Hasting, 1998). The lux<br />
Tehrani et al. 4021<br />
genes have been transferred into E. coli and a multitude<br />
of different prokaryotic species by transformation,<br />
transduction, conjugation or even bacterial genome<br />
integration, using a variety of different plasmid vectors<br />
(Meighen, 1991).<br />
Previous studies indicate that, the genes encoding<br />
luciferase subunits can generate heterodimers in various<br />
forms (AB1, AB2, BA1, BA2 and A+ B), but the enzyme<br />
activity is decreased in comparison with wild type binary
4022 Afr. J. Biotechnol.<br />
A+B construct, especially in the case of BA1 and BA2<br />
forms which their activity reported to be about 2% of the<br />
wild type (Olsson et al., 1989). In contrast, the current<br />
synthesized construct pETDuet. LuxAB not only simplify<br />
the use of lux system as a reporter enzyme in prokaryotic<br />
cells, but also can provide a special condition in which<br />
both lux genes express simultaneously, from a same<br />
promoter (T7). Furthermore, it has been shown that<br />
obtaining the high expression of the lux genes requires a<br />
strong promoter and ribosome binding site (RBS) on the<br />
expression plasmid that can increase the amount of<br />
product, in which both are available in this effort using<br />
pETDuet-1 vector.<br />
In the previous researches, expression of luciferase<br />
(luxAB) component of the lux system using multiple<br />
plasmids (PB, pGMC12, pFIT001, Plx, pRS1105, pCVG)<br />
about V. harveyi and in the fusion form (pCK218) about<br />
V. fischeri have been reported (Greer and Szalay, 2002),<br />
but in this study, we constructed a polycistronic lux<br />
system by cloning the luxA, luxB and intergenic sequence<br />
Figure 5. SDS PAGE analysis, lane 1 control cell lysate without<br />
recombinant palsmid; Lane 3 and 4 cell lysate of E. coli Nova blue<br />
containing pETDuet-1/luxAB in different sampling times (3 and 5 h,<br />
respectively) after induction with IPTG; lane 2 before induction with<br />
IPTG.<br />
Figure 6. Western blot analysis of luxAB protein. Lane<br />
1 induced culture containing Nova blue bacteria<br />
harboring pETDuet-1 recombinant plamsids after 3 h<br />
induction; lane 2 after 5 h induction; lane 3 extracted<br />
protein from culture containing Nova blue strain, as<br />
control.<br />
as a 2046 bp fragment into a TA cloning vector<br />
(pTZ57R), followed by subcloning the fragment into the<br />
expression vector pETDuet-1 and successfully expressed<br />
in Nova blue cells that accordingly, not have already<br />
been expressed and reported. In addition to luxAB<br />
protein (76 kD), free luxA and luxB subunits (40 and 37<br />
kD) were also detected in different extracts. The amount<br />
of free subunits, moreover the 76 kD protein, increased<br />
with IPTG induction which confirm that these subunits<br />
were also translated from the lux transcripts.<br />
Finally, pETDuet-luxAB construct can now be applied<br />
as a reporter cassette in prokaryotic systems, resulting in<br />
luminous phenotype on addition of a fatty aldehyde<br />
(decanal) to the cells. The expression of the lux genes in<br />
different bacterial species provided a simple and<br />
sensitive system for monitoring the growth and distribution<br />
of the bacteria in the environment. By using the lux<br />
genes as reporters of gene expression, the strength and<br />
regulation of transcription from various promoters can be<br />
readily monitored and also light emission can easily be
detected and measured. Furthermore, this reporter<br />
cassette can be used as a marker or tag in the manipulation<br />
and the control of gene expression in the fields of<br />
research, production, control of microorganism and other<br />
biotechnological applications.<br />
ACKNOWLEDGEMENTS<br />
This study was financially supported by Iranian Fisheries<br />
Research Organisation, Ecology research center of the<br />
Caspian Sea and was done in Cellular and Molecular<br />
Biology Research Center- Shahid Beheshti University of<br />
Medical Sciences. The authors are thankful for the<br />
support and cooperation of the directors.<br />
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The 1.5-A resolution crystal structure of bacterial luciferase in low salt<br />
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Bacteria: the LuxR-LuxI Family of Cell Density-Responsive<br />
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methods in Molecular Biology vol 2 Nucleic Acids. Edited by Walker<br />
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expression of luciferase in living cells and organisms : a review.<br />
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Köhler S, Belkin S, Schmid RD (2000). Reporter gene bioassays in<br />
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Meighen EA (1991). Molecular Biology of Bacterial Bioluminescence.<br />
Microbiological Reviews, 55: 123-142.<br />
Milton DL (2006). Quorum sensing in vibrios: complexity for<br />
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Naylor LH. (1999). Reporter gene technology: the future looks bright.<br />
Biochemical Pharmacology 58: 749-757.<br />
Nelson EJHS, Tunsjo PM, Fidopiastis HS, Ruby EG (2007). A novel lux<br />
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1825-1833.<br />
Nyholm SV, Mcfall-Ngai MJ (2004). The winnowing: establishing the<br />
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Olsson O, Escher A, Sandberg G, Schell J, Konez C, Szalay AA (1989).<br />
Engineering of monomeric bacterial luciferases by fusion of luxA and<br />
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Ruby EG (1999). The Euprymna scolopes-Vibrio fischeri symbiosis: a<br />
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manual in Molecular Biomethods Handbook. Humana press.<br />
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pp: 435-444.<br />
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Microbiology, 17: 801-812.<br />
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Ulitzur S (1998). LuxR Controls the Expression of Vibrio scheri<br />
luxCDABE Clone in Escherichia coli in the Absence of luxI. Genet. J.<br />
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fischeri lux genes play an important role in colonization and<br />
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the Vibrio fischeri-Euprymna scolopes partnership. J. Bacteriol. 182:<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4024-4028, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2466<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Study of polymorphism of leptin gene receptor in<br />
Mazandaran fowls<br />
H. A. Abbasi*, S. Gharahveysi and R. Abdullahpour<br />
Department of Animal Sciences, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran<br />
Accepted 25 March, 2011<br />
In chickens, leptin is expressed mainly in the liver and adipose tissue. In Iran, Mazandaran native fowls<br />
are under recording and breeding programs, but according to the action modes and importance of the<br />
leptin receptor, its polymorphisms can be related to economical traits such as body weight. In this<br />
study, in order to identify allelic polymorphism in leptin gene receptor, a restriction fragment length<br />
polymorphism (RFLP) method was used. Blood samples were collected randomly from 100 individuals.<br />
The DNA extraction was based on a salting-out method, while an amplified polymerase chain reaction<br />
technique was used. The quantity and quality of extracted DNA were examined using<br />
spectrophotometric and agarose gel electrophoresis. A strategy, employing polymerase chain reaction,<br />
was used to amplify a 374 bp fragment of 9 to 11 exon leptin gene receptor. Digestion of amplicons with<br />
HaeIII revealed leptin gene receptor. The obtained results from restriction digestion showed none of the<br />
polymorphism in leptin receptor gene, so all samples were monomorph due to the fact that there was<br />
no mutation that was related to polymorphism.<br />
Key words: Leptin gene receptor, PCR- RFLP, polymorphism, fowl, HaeIII.<br />
INTRODUCTION<br />
In farm animals, the control and prediction of fatness is of<br />
a high economic interest. The exaggerated adipose<br />
tissue development negatively affects the whole body<br />
metabolism, production efficiency, reproduction and meat<br />
quality. The word leptin comes from the Greek leptos,<br />
meaning thin, referring to the anti-obesity effect which<br />
was believed to be the primary physiological function of<br />
the hormone. Leptin is the product of the ob gene,<br />
discovered by Zhang et al. (1994) using the positional<br />
cloning technique.<br />
Leptin was first identified as the gene product found<br />
deficient in the obese ob/ob mouse. The gene is located<br />
on chromosome 6 in the mouse and chromosome 7 in<br />
humans, and encodes a protein that shows a high degree<br />
of homology between species. Mutations in this ob gene<br />
* Corresponding author. E-mail: h.abbasi1389@gmail.com.<br />
Abbreviations: PCR, Polymerase chain reaction; RFLP,<br />
restriction fragment length polymorphism; LEP, leptin; LEPR,<br />
leptin receptor; NPY, neuropeptide Y.<br />
revealed the pivotal role of leptin in energy balance<br />
(Zhang et al., 1994). The full coding sequence contains<br />
167 amino acids (a.a.) and is composed of a 21-amino<br />
acid signal peptide and a 146-amino acid circulating,<br />
bioactive hormone. However, leptin is a 16-kDa hormone<br />
that has been shown to play an important role in the<br />
regulation of food intake, energy expenditure and hypothalamus<br />
endocrine function in response to nutritional<br />
changes (Friedman and Halaas, 1998; Elmquist et al.,<br />
1999). Recent studies have demonstrated that leptin is<br />
produced by other tissues, such as brain, pituitary gland,<br />
skeletal muscles and stomach. In mammals, leptin is<br />
expressed primarily in adipose tissue (Zhang et al., 1994)<br />
and at a lower level in the placenta and stomach<br />
(Masuzaki et al., 1997; Bado et al., 1998). Thus, it<br />
represents an excellent candidate gene for polymorphism<br />
investigation and association with economic traits in<br />
livestock species. Organization of this gene is conserved<br />
among mouse, human and bovine, presenting three<br />
exons and two introns (Taniguchi et al., 2002). In bovine,<br />
polymorphisms on LEP gene has been associated with<br />
body fat, feed intake and milk yield (Buchanan et al.,<br />
2002; Liefers et al., 2002; Nkrumah et al., 2005). Unlike
mammals, little is known about the avian LEP gene<br />
function. In chicken, only its coding sequence was identified<br />
and sequenced by Taouis et al. (1998). The leptin<br />
signal is mediated through a species receptor localized in<br />
the target tissues, and the leptin receptor (LEPR) belongs<br />
to the class I cytokine receptor superfamily that shares<br />
common structural features and signal transduction<br />
pathways (Tartaglia et al., 1995). Leptin mediated its<br />
central effect through specific receptors located in the<br />
hypothalamus. Leptin receptors have been located on<br />
neurons producing neuropeptide Y (NPY), and when<br />
activated by leptin binding, it is hypothesized to function<br />
in part by down regulating the production of hypohypothalamic<br />
NPY (orexigenic effector) to inhibit ingestive<br />
behavior (Schwartz et al., 1997).<br />
Mammalian adipocytes produce and secrete more<br />
leptin in bloodstream as fat storage increases (Maffei et<br />
al., 1995), signalling the brain via leptin receptor<br />
(Hakansson et al., 1996; Banks et al., 1996) and modulating<br />
the hypothalamic neuropeptide system to suppress<br />
appetite and increase energy expenditure (Rohner et al.,<br />
1996; Kristensen et al., 1998). More interestingly, it has<br />
been shown that chicken leptin gene expression is sensitive<br />
to hormonal treatment in liver, but not in adipose<br />
tissue (Ashwell et al., 1999). These observations are<br />
thought to be due to the role of the avian liver as the<br />
primary source of lipogenesis (Goodridge and Ball, 1967;<br />
Leveille et al., 1968). The peripheral action of leptin in the<br />
chicken is poorly documented except in the pancreas,<br />
where it has recently been demonstrated that leptin has a<br />
profound inhibitory influence upon insulin secretion in the<br />
perfused chicken pancreas (Benomar et al., 2003).<br />
Several studies have showed that exogenous administration<br />
of leptin decreased feed intake in chicks, which<br />
was similar to the one described in mammals, but the<br />
anorexigenic effect within chicken hypothalamus was<br />
mediated via selective neuropeptides, such as NPY and<br />
orexin (Dridi et al., 2005).<br />
In Iran, Mazandaran native fowls are under recording<br />
and breeding programs, but according to the action<br />
modes and importance of leptin gene receptor, its polymorphisms<br />
can be related to economical traits such as<br />
body weight. The aim of the present study is to investigate<br />
the polymorphisms of leptin receptor gene in<br />
Mazandaran native fowls using PCR-RFLP methodology.<br />
Association between different allelic and genotypic forms<br />
of gene and economical important traits can be found if<br />
the polymorphisms are seen. Finding an association<br />
between these caused an improved accuracy and genetic<br />
gain in fowls.<br />
MATERIALS AND METHODS<br />
Experimental population<br />
The study, which was conducted at the Native fowls breeding<br />
station of Mazandaran located in the North of Iran, have been<br />
established in 1988 with the objective of conserving the endan-<br />
gered population of native fowls in rural areas. The location is<br />
Abbasi et al. 4025<br />
typically hot and semi-arid with yearly minimum and maximum<br />
temperature ranges between 4 and 34°C, respectively. The station<br />
has two main activities, namely extension and genetic improvement.<br />
Genetic improvement is done by selecting the best 100 cocks<br />
and 800 hens as parents of the next generations. Parents of each<br />
generation are selected among 7000 pedigreed and performance<br />
recorded birds produced by each generation. The extension part is<br />
continuously producing and distributing 8 weeks old chicks among<br />
rural communities with the aim of increasing the population of<br />
native fowls in Northern provinces of Iran. Rearing chicks in a 30 to<br />
60 days period and distributing them in rural areas to enhance meat<br />
and egg production are quantitative goals of Mazandaran native<br />
fowl breeding station.<br />
Sample collection and DNA isolation<br />
A total of 100 blood samples (5 ml) were collected in EDTA (1 mg<br />
ml -1 ) treated tubes as an anticoagulant from randomly chosen<br />
individuals. The chicks were sampled randomly from four rearing<br />
salons, and each contains chicks with same hatching. Samples<br />
were transferred to the laboratory with ice flask and stored at -20°C<br />
for further analysis. DNA was isolated by standard salt procedure<br />
described by Miller et al. (1988). The quality and quantity of the<br />
extracted DNA was checked by spectrophotometer and agarose gel<br />
electrophoreses.<br />
DNA samples were adjusted to a concentration of 25 µl reaction<br />
mixture, containing 200 ng/µl, and exactly 1/5 µl of the DNA samples<br />
were used as template for polymerase chain reaction.<br />
Primers design<br />
One set of primers was designed using Primer3 primer design<br />
software [(Rozen and Skaletzky, 2000) - www.frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi].<br />
These primers were designed based<br />
on the chicken sequence leptin gene receptor (GenBank–<br />
NC006095.2), Gallus gallus chromosome 8 and reference assembly<br />
(based on Gallus_gallus-2.1). The primers were analyzed on<br />
NetPrimer (http://www.premierbiosoft.com/netprimer/netprlaunch/<br />
netprlaunch.html), in order to avoid secondary structures, such as<br />
hairpins and loops and primer dimmer. Specific primer pairs were<br />
prepared from the Sina gene (Iran) company in a lyophilized form<br />
and were solved in double steriled water and stored at -20°C. Blood<br />
purified DNA showed better quantity and quality.<br />
Leptin gene receptor amplification<br />
The design primer, primer premier 3(2000) software, was used for<br />
the DNA sequence in exons 9 to 11 leptin gene receptor, while the<br />
Net Primer was used to compare the produced sequence and the<br />
saved sequence. The sequences of the forward and reverse<br />
primers for the amplification of the leptin gene receptor were: F 5´-<br />
GTGTGATAGCTTTGAATGTTGGTG -3´ and R 5´- CTCTTCTG<br />
TTGCCAGCTGTGAT -3´. The polymerase chain reaction for the<br />
leptin gene receptor was performed in a 25 µl reaction mixture,<br />
containing 3 mM MgCl 2 , 200 µM of each dNTPs, 0.4 µM of each<br />
primers, 1X PCR buffer, 1U Taq polymerase (Cinagen, Iran) and<br />
100 ng of genomic DNA template. The reaction mixture was placed<br />
in a DNA thermal cycler (Perkin Elmer 9700). Thermal cycling<br />
conditions included an initial denaturation step at 93°C for 3 min<br />
followed by 33 cycles of 93°C for 45 sec, 57°C for 45 s, 72°C for 45<br />
s and a final extension at 72°C for 4 min.<br />
Restriction digest<br />
For the PCR- RFLP assays, 15 U of the PCR products were digested
4026 Afr. J. Biotechnol.<br />
Figure 1. Various samples obtained from PCR.<br />
Figure 2. Schematic of the 374 bp sequence of leptin gene receptor.<br />
with 5 units of HaeIII (Gibco BRL, life Technologies, USA) at 37°C<br />
for at least 24 h, respectively. Digested products were separated by<br />
electrophoresis on 1% agarose gel in 1×TBE (Tris-Boric acid-<br />
EDTA) buffer at 85 V for 1 h. The 100 bp DNA ladder (Fermentas<br />
SM0331) was used in each gel as a molecular size standard. The<br />
gels were stained with ethidium bromide and the fragments were<br />
visualized using UV transilluminator.<br />
RESULTS AND DISCUSSION<br />
One of the challenges of the poultry industry is to improve<br />
chicken's carcass quality and reduce fat content, without<br />
prejudicial effects on the genetic gains already obtained.<br />
In poultry, the animal’s rapid growth rate has led to excessive<br />
body fat associated with impairment of the total<br />
body metabolism, disorders in the reproductive functions<br />
and muscular development resulting in low performance<br />
with high mortality.<br />
Using an appropriate thermal program in PCR, the 374<br />
400 bp<br />
300 bp<br />
bp segment was amplified without unspecific bands<br />
(Figure 1). The specific primer pairs were designed from<br />
sequence of leptin receptor gene in genbank NCBI site.<br />
Using these primers, a 374 bp segment from exons 9 to<br />
11, which started from nucleotide 55 to 429, was amplified<br />
(Figure 2). The HaeIII restriction enzyme has a<br />
restriction site of ((GGCC)) in the amplified segment and<br />
this site was cut after the second ((G)) base. If the<br />
enzyme cuts the segment, then two bands with lengths of<br />
241 and 133 are seen after electrophoresis on the gel<br />
(Figure 3). However, if the mutation did not occur in the<br />
amplified segment, the HaeIII can not cut the segment<br />
and as such, only one allele (allele B) would be seen, but<br />
if the mutation occurs, two alleles (A and B) are seen<br />
after restriction of digestion. It is shown in the samples<br />
with two bands that the DNA was cut at both strands after<br />
digestion. The digestion products showed that<br />
Mazandaran native fowls were monomorph at the leptin<br />
receptor gene, but the entire samples showed one band
Abbasi et al. 4027<br />
300 bp<br />
100 bp<br />
Figure 3. Results of PCR-RFLP analysis for leptin gene receptor by restriction enzyme HaeIII on 1% agarose<br />
gel and on ladder 100 bp (fermentas).<br />
on agarose gel; therefore, all had BB genotype in this<br />
locus. To confirm the accuracy of digestion, this process<br />
was performed twice, and the cloning study revealed that<br />
the leptin receptor gene has six isoforms such as the long<br />
isoform. Other cloning studies showed three exons and<br />
two introns in the leptin gene. Investigation of the mRNA<br />
of the leptin receptor gene showed eighteen exons in it<br />
(Almeida et al., 2003).<br />
Nevertheless, the cloning study revealed an expression<br />
of leptin gene in the chicken’s liver (Taose et al., 1998).<br />
The polymorphisms of leptin receptor gene were<br />
surveyed in Khoozestan native fowl population using<br />
RFLP-PCR at 2009. It showed that change in the<br />
restriction site of HaeIII generated different restricted segments.<br />
Consequently, three genotypes and two alleles<br />
were seen in Khoozestan fowls. The allelic frequencies of<br />
leptin gene receptor in Khoozestan fowls were 31.19 and<br />
61.81 for alleles A and B, and the genotypic frequencies<br />
were 18.81, 24.75 and 54.44 for genotypes AA, AB and<br />
BB, respectively. Results showed deviation from Hardy-<br />
Weinberg equilibrium in the population of Khoozestan<br />
fowls. Furthermore, the association study revealed that<br />
allele A had positive effect on economical traits than<br />
allele B. The observed and expected heterozygosities<br />
were 0.243 and 0.568, respectively. However, the obtained<br />
results from Mazandaran native fowls were<br />
contrary to those of Khoozestan fowls. The frequency of<br />
the wild type allele is higher than the mutant allele in<br />
Mazandaran native fowls. This can be due to the<br />
physiological role of allele B in Iranian fowls. Inbreeding<br />
and family selection can be one of the major factors that<br />
enhance the BB genotype in Mazandaran native fowls.<br />
Reared chickens in the breeding station of Mazandaran<br />
native fowls are prepared from the state native fowls'<br />
breeding center, and in addition, it is a closed population<br />
and therefore is open for disequilibrium factors.<br />
Inbreeding coefficient is high in the closed population<br />
which, in turn, causes a decrease of diversity in the population.<br />
Increasing effective population size, controlling<br />
mating and preparing independent populations with large<br />
number of primitive individuals are necessary for preventing<br />
decrease of diversity in Mazandaran native fowls.<br />
Nonetheless, designed primers in this study were first<br />
used in Iranian chickens.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4029-4032, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2563<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Detection of genomic instability in hypospadias<br />
patients by random amplified polymorphic DNApolymerase<br />
chain reaction (RAPD-PCR) method<br />
Adnan A. Alsulaimani 1 , Nabil S. Awad 2 * and Adel E. El-Tarras 2<br />
1 College of Medicine, Taif University, Taif, Kingdom of Saudi Arabia.<br />
2 College of Medicine, Biotechnology and Genetic Engineering Unit, Taif University, Taif, Kingdom of Saudi Arabia.<br />
Accepted 18 March, 2011<br />
Hypospadias is a urogenital malformation, and it is a common inborn disorder in male individuals. The<br />
etiology of hypospadias is still unsolved. The present study is aimed to identify the genetic instability in<br />
hypospadias patients. Random amplified polymorphic DNA (RAPD), a polymerase chain reaction (PCR)<br />
based technique, was adopted using ten random primers in twelve cases and twelve controls. The<br />
primer detectability on genomic instability in 12 samples ranged from 25% with primer OPA-01 to 66%<br />
with OPA-08. Case 2 showed the highest genomic instability (80%). The lowest genomic instabamility<br />
was (10%) case 6. The results determined numbers of genomic instabilities among hypospadias<br />
patients. In addition, the RAPD-PCR technique is a powerful tool for detection of genomic instability in<br />
hypospadias patients. Further larger studies are needed, which include low and high grade of patients<br />
to: 1) Obtain RAPD markers useful for hypospadias early diagnosis; 2) investigate different genes<br />
directly involved in the etiology of hypospadias; 3) analyze chromosomal instability among hypospadia<br />
patients.<br />
Key words: Hypospadias, random amplified polymorphic DNA (RAPD), genomic instability.<br />
INTRODUCTION<br />
Hypospadias is one of the most common congenital<br />
disorders (Sutherland et al., 1996) characterized by<br />
opening on the ventral side of the penis (Manson and<br />
Carr 2003). Hypospadias occurs in 1 out of 300 live male<br />
births (Kalfa et al., 2008). Different studies on the etiology<br />
of hypospadias were conducted and most of them have<br />
focused on a number of candidate genes that control<br />
androgen action and metabolism including the SRD5A2,<br />
HSD17B3 and the AR genes (Manson and Carr., 2003)<br />
and CXorf6 gene mutations (Fukami et al., 2006; Kalfa et<br />
al., 2008).<br />
Measurement of genomic instability has been<br />
performed by techniques like flow cytometry, fluorescent<br />
*Corresponding author. E-mail: nabilfaris151@yahoo.com.<br />
Abbreviations: CGH, Comparative genomic hybridization;<br />
RAPD, random amplified polymorphic DNA; PCR, polymerase<br />
chain reaction; EDTA, ethylenediaminetetraacetic acid.<br />
in situ hybridization, comparative genomic hybridization<br />
(CGH) and allelotyping, which, although informative, are<br />
cumbersome to perform and hence, impractical in the<br />
assessment of clinical cases (Basik et al., 1997).<br />
Random amplified polymorphic DNA (RAPD) is a<br />
polymerase chain reaction (PCR) based fingerprinting<br />
technique that amplifies random DNA fragments with<br />
single short primers of arbitrary nucleotide sequence<br />
under low annealing stringency (Ong et al., 1998;<br />
Williams et al., 1990). The applications of RAPD<br />
technique have been found among several kinds of<br />
organism including bacteria (Sahoo et al., 2010), fungi<br />
(Motlagh and Anvari, 2010), plants (El-Tarras et al., 2004,<br />
animals (Güneren et al., 2010), insects (Awad et al.,<br />
2010) and humans (Saleh et al., 2010). The usefulness of<br />
the RAPD technique for the detection of genomic<br />
instability in various types of human tumors has been<br />
widely documented (Singh and Roy, 2001; Papadopoulos<br />
et al., 2002; Zhang et al., 2004; Xian et al., 2005; Saleh<br />
et al., 2010). No evidence for an association between<br />
genomic instability and hypospadias has been
4030 Afr. J. Biotechnol.<br />
investigated. So the specific aims of this study are to<br />
investigate the association between genomic instability<br />
and hypospadias, as well as to evaluate the usefulness of<br />
the RAPD technique for the detection of genomic<br />
instability in hypospadias patients.<br />
MATERIALS AND METHODS<br />
Blood samples collection and DNA extraction<br />
Whole blood samples of 12 hypospadias patients and 12 healthy<br />
individuals were drawn from a peripheral vein into<br />
ethylenediaminetetraacetic acid (EDTA) tubes and stored at -20°C<br />
until DNA extraction step. Genomic DNA was extracted from blood<br />
samples according to instructions of Blood DNA Preparation Kit<br />
(Jena Bioscince; Germany).<br />
DNA amplification<br />
Total reaction volume of 25 ul of 2x superhot PCR Master Mix<br />
(Bioron; Germany) was used and contains 10 Pmol of each 10<br />
different arbitrary 10-mer primers and 25 to 50 ng of genomic DNA.<br />
The names and sequences of these oligoprimers are listed in Table<br />
1. The RAPD-PCR amplification reactions were performed in<br />
Eppendorf ® thermal cycler using the following PCR program: 1<br />
cycle at 94°C, 4 min; 35 additional cycles consisting of 94°C 5 s,<br />
37°C 20 s and 72°C 20 s. After the amplification, the PCR reaction<br />
products were electrophoresed with 100 bp ladder marker<br />
(Fermentas, Germany) on 10 x 14 cm 1.5%-agarose gel (Bioshop;<br />
Canada) for 30 min using Tris-borate- EDTA Buffer. The gel was<br />
stained with 0.5 μg/ml of ethidium bromide (Bioshop; Canada).<br />
Data analysis<br />
All gels were visualized and documented using a GeneSnap 4.00-<br />
Gene Genius Bio Imaging System (Syngene; Frederick, Maryland,<br />
USA). Each RAPD-PCR amplified DNA fragment was assumed to<br />
represent a single locus. The digital image files were analyzed<br />
using Gene Tools software from Syngene. The densitometric<br />
scanning of each based on its three characteristic dimensions was<br />
carried out. Each band was recognized by its length, width and<br />
intensity. Accordingly, the relative amount of each band was<br />
measured and scored. The absence or addition of an amplified<br />
product in at least one hypospadias sample and its corresponding<br />
control sample was used as a criterion of genomic instability.<br />
RESULTS AND DISCUSSION<br />
During the present study, 12 hypospadias patients and<br />
corresponding normal individuals were examined using<br />
RAPD-PCR method to detect genomic instability which<br />
appears as banding pattern changes between patients<br />
and normal amplified DNA with ten different RAPD<br />
primers. Results of genomic instability detected by<br />
RAPD-PCR analysis are shown in Table 1 and Figure 1.<br />
Among all studied cases, genomic instability was demonstrated<br />
with at least one primer. Among all studied cases<br />
with all primers, the detectability of genomic instability<br />
ranged from 25% with primer OPA-01 to 66.6% with<br />
OPA-08 primer. With all used primers, case 2 showed the<br />
highest genomic instability (80%), whereas, t he lowest<br />
genomic instability was (10) with case 6.<br />
There are three types of hypospadias glandular or<br />
coronal (mild), penile (moderate), scrotal and perineal<br />
(severe) which are also called first, second and third<br />
grade, respectively (Ghirri et al., 2009). The powerful<br />
technique that detects genomic alteration correlated with<br />
human tumor is microsatellites analysis (Odenthal et al.,<br />
2009; Janavicius et al., 2010). However, this methodology<br />
is time consuming and can only detect base-pair<br />
expansion or contraction in specific microsatellite loci<br />
(Ong et al., 1998). On the contrary, for genomic instability<br />
analysis, it is important to investigate genetic alterations<br />
in the entire genome besides microsatellite loci. In<br />
contrast, the RAPD method can simply and rapidly detect<br />
genetic alterations in the entire genome without<br />
knowledge of specific DNA sequence information<br />
(Papadopoulos et al., 2002; Ibrahim et al., 2010). In the<br />
RAPD method, genetic alterations appeared as either<br />
loss or gain of a band, shift of a band, or decrease or<br />
increase of intensities of a band of cancer tissue DNA<br />
relative to the corresponding normal tissue DNA (Maeda<br />
et al., 1999). Obtained results indicated that RAPD-PCR<br />
is an effective tool for identifying genetic alteration and<br />
genomic instability which is in agreement with several<br />
various studies (Ibrahim et al., 2010; Wang, 2001). Figure<br />
1 shows the banding profiles of hypospadias and<br />
corresponding normal DNAs and demonstrate the<br />
detected genetic alteration by RAPD technique among<br />
hypospadias patients in comparison with normal control<br />
group. Banding shifts, missing bands and/or banding<br />
intensity changes, which indicate genomic instability,<br />
were demonstrated in this figure.<br />
These results might be due to mutations that occurred<br />
at the primer - template interaction sites (Maeda et al.,<br />
1999). The summarized results which are illustrated in<br />
Table 1 indicated that, there are differences in genetic<br />
instability among the studied cases which ranged from<br />
10% with case number 6 to 80% for case number 2.<br />
These differences might be due to differences in studied<br />
hypospadias types (mild, moderate or sever). Data in<br />
Table 1 and Figure 1 reflects the ability of each primer to<br />
detect genomic instability which range from 25% with<br />
primer OPA-01 to 66.6% with OPA-08 primer. This<br />
finding might be due to the fact that some loci in the DNA<br />
or chromosome were apt to changes of nucleotide<br />
sequences like the sequences which amplified by primer<br />
OPA-08, while the sequences amplified by primer OPA-<br />
01 would tend to remain stable among hypospadias<br />
patients (Jianxun et al., 2002). Although, the number of<br />
hypospadias samples used in this study is relatively<br />
small, these results seem to indicate that there is a close<br />
relationship between genomic instability detected by RAPD<br />
analysis and this is in agreement with (Ong et al., 1998).<br />
Conclusion<br />
From the obtained results, it could be concluded that,
Alsulaimani et al. 4031<br />
Table 1. Primer code, their sequences and % of primer detectability of genetic instability among hypospadias cases in comparison<br />
with controls.<br />
Primer<br />
code<br />
Primer sequence Samples Primer<br />
detectability (%)<br />
1 2 3 4 5 6 7 8 9 10 11 12<br />
OPA-01 CAGGCCCTTC - + - - - - - - + - - + 25<br />
OPA-02 TGCCGAGCTG - + - - + - - + + + - - 41.6<br />
OPA-03 AGTCAGCCAC + + - - - - - + - + - - 33.3<br />
OPA-04 AATCGGGCTG + - - + + + - - + + - - 50<br />
OPA-05 AGGGGTCTTG + - - + - - - - + - + - 33.3<br />
OPA-06 GGTCCCTGAC - + + + - - - + + - - + 50<br />
OPA-07 GAAACGGGTG - + - + + - + - - + + - 50<br />
OPA-08 GTGACGTAGG + + + + - - + + - + - + 66.6<br />
OPA-09 GGGTAACGCC - + - + - - - + - - - + 33.3<br />
OPA-10 GTGATCGCAG - + - + - - - + - - - + 33.3<br />
Total<br />
40 80 20 70 30 10 20 60 50 50 20 40<br />
+: Genomic instability detected; -: no genomic instability detected.<br />
Figure 1. Genetic alteration revealed from RAPD-PCR analysis of hypospadias (h) and corresponding<br />
control (n). A, with primer OPA-02; B, with primer OPA-03 and C, with OPA-05.<br />
among hypospadias patients, a number of genetic insta-<br />
bilities represented as loss or gain, shift and/or decrease<br />
or increase of intensities of a DNA bands were identified.<br />
RAPD-PCR method is suitable for detecting genomic<br />
changes among hypospadias patients. Further, deep<br />
molecular genetic case control studies with high and low<br />
hypospadias grade are needed to: 1) Determine and<br />
identify specific DNA markers that could be used in early<br />
detection and molecular diagnosis programs of<br />
hypospadias; 2) investigate different genes directly<br />
involved in the etiology of hypospadias and 3) analyze<br />
chromosomal instability among hypospadias patients.<br />
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Yamada G, Morohashi K & Ogata T (2006). CXorf6 is a causative
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Güneren G, Akyüz B, Ertugrul O (2010). Use of RAPD-PCR for genetic<br />
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Jianxun W, Qianwen W, Feng Y (2002). Genetic instability in cancer<br />
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Chi.med.J. 111(3):430-432.<br />
Kalfa N, Liu B, Ophir K, Audran F, Wang 1M, Mei C, Sultan C, Baskin L<br />
(2008). Mutations of CXorf6 are associated with a range of severities<br />
of hypospadias. Europ. J. Endocrino. 159 (4): 453-458.<br />
Maeda T, Jikko A, Hiranuma H, Fuchihata H (1999). Analysis of<br />
genomic instability in squamous cell carcinoma of the head and neck<br />
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Manson J, Carr C (2003). Molecular Epidemiology of Hypospadias:<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4033-4039, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2564<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
cDNA, genomic sequence cloning and overexpression<br />
of cytochrome c oxidase gene (COX6b1) from the<br />
Ailuropoda melanoleuca<br />
Jun Li 1,2 , Yiling Hou 1,2 , Wanru Hou 1,2 *, Bing Sun 1,2 and Xiulan Su 1,2<br />
1 College of Life Science, China West Normal University, 637009, Nanchong, Sichuan, People’s Republic of China.<br />
2 Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), People’s Republic of<br />
China.<br />
Accepted 7 April, 2011<br />
Cytochrome c oxidase (COX) is a component of the mitochondria respiratory chain. COX6b1 is one of<br />
the COX small subunits encoded by nuclear genes. In currently study, the cDNA and the genomic<br />
sequence of COX6b1 were successfully cloned from the Ailuropoda melanoleuca with the RT-PCR<br />
technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned is 341 bp in size,<br />
containing an open reading frame (ORF) of 261 bp encoding 86 amino acids. The length of the genomic<br />
sequence is 4657 bp, which was found to possess 3 exons and 2 introns. Alignment analysis indicated<br />
that the nucleotide sequence and the deduced amino acid sequence are highly conserved with other six<br />
species studied. Topology prediction showed that there are one N-glycosylation site, one protein kinase<br />
C phosphorylation site, two casein kinase II phosphorylation sites, two N-myristoylation sites and one<br />
microbodies C-terminal targeting signal site in the COX6b1 protein of the A. melanoleuca. The COX6b1<br />
gene was also overexpressed in Escherichia coli and the result indicated that COX6b1 fusion with the<br />
HIS-tagged gave rise to the accumulation of an expected 13.46 kDa polypeptide. The expression<br />
product obtained could be used to purify the protein and study its function further.<br />
Key words: Cytochrome c oxidase subunit Vib polypeptide 1 (COX6b1), clone, overexpression, Ailuropoda<br />
melanoleuca (Giant Panda), sequences analysis.<br />
INTRODUCTION<br />
Cytochrome c oxidase (COX; EC 1.9.3.1), the terminal<br />
enzyme complex of the mitochondria respiratory chain, is<br />
a multisubunit enzyme located in the mitochondrial inner<br />
membrane. It oxidizes cytochrome c and transfers<br />
electrons to molecular oxygen. Meanwhile, COX is an<br />
important energy-generating enzyme critical for the<br />
proper functioning of most cells, especially those of highly<br />
oxidative organs, such as the brain (Capaldia, 1990). In<br />
mammals, it is comprised of 13 protein subunits of which<br />
three large subunits (subunit I to III) are encoded by the<br />
mitochondrial genome (mtDNA), constituting the catalytic<br />
core of the enzyme, and the remaining 10 subunits (IV,<br />
*Corresponding author. E-mail: hwr168@yahoo.com.cn.<br />
Tel/Fax: +86-0817-2568653.<br />
Va, b, VIa, b, c, VIIa, b, c and VIII) are nuclear genes<br />
(nDNA) products, which are synthesized on cytoplasmic<br />
ribosomes and are imported into the mitochondria by<br />
leading sequences (Cao et al., 1988; Barrientos et al.,<br />
2002).<br />
Study had confirmed that all 13 subunits are essential<br />
for a functional holoenzyme (Liang et al., 2006). The<br />
mtDNA-encoded subunits provide the catalytic core of<br />
cytochrome-c oxidase, and the nuclear-encoded subunits<br />
may be involved in the regulation and assembly of the<br />
complex (Yanamura et al., 1988). As for COX6b1 gene,<br />
Massa et al. (2008) found that it is associated with severe<br />
infantile encephalomyopathy. Cui et al. (2010) also<br />
pointed out that the loss of COX6b1 genes leads to<br />
mitochondria dysfunction that could cause apoptosis of<br />
the blastocyst-stage embryos.<br />
Ailuropoda melanoleuca, a rare and endangered<br />
species currently found only in China, has a very high
4034 Afr. J. Biotechnol.<br />
ecological, scientific, economic, cultural and aesthetic<br />
value. At present, the COX genes have been extensively<br />
characterized in mammals (Bachman, 1995; Sacconi et<br />
al., 2005; Wu et al., 2007; Du et al., 2007), whereas there<br />
is a handful of report about the COX6b1 genes of A.<br />
melanoleuca.<br />
In this study, RT-PCR technique was used for<br />
amplifying the cDNA of COX6b1 gene from the total RNA.<br />
Touchdown-PCR technique was employed to amplify the<br />
genomic sequence of the COX6b1 from extraction DNA<br />
with the skeleton muscle of the A. melanoleuca. The<br />
characteristics of this gene was identified by homologous<br />
analysis according to the related sequences in GenBank,<br />
and the overexpression of the gene was conducted in<br />
Escherichia coli using pET28a plasmids. The research<br />
can provide a scientific data to enrich the species<br />
database.<br />
MATERIALS AND METHODS<br />
Skeletal muscle was collected from a dead A. melanoleuca at the<br />
Wolong Conservation Center of the A. melanoleuca, Sichuan,<br />
China. The collected skeletal muscle was frozen in liquid nitrogen<br />
for DNA and RNA isolation.<br />
DNA and RNA extraction<br />
Approximately 500 mg muscle tissue of A. melanoleuca was ground<br />
to fine powder in liquid nitrogen, and the powder was then<br />
immediately transferred into a 2 ml centrifuge tube and carefully<br />
added to 1 ml cold lysis buffer (100 mM Tris-HCl, pH 8.0, 100 mM<br />
EDTA and 0.5% SDS). The mixture was incubated at 65°C at least<br />
1 h with gently shaking occasionally. After incubation, the mixture<br />
was centrifuged with 10000 rpm for 10 min at 4°C. Subsequently,<br />
the supernatant was carefully transferred to a new 2 ml centrifuge<br />
tube and an equal volume of chloroform : isoamylalcohol (24:1) was<br />
added and mixed gently by inversion for 1 min, followed by keeping<br />
for 10 min at room temperature and centrifugation at 10000 rpm for<br />
10 min at 4°C. The aqueous phase was carefully transferred to a<br />
fresh tube, and two third volumes of ice-cold isopropanol were<br />
added. Finally, the DNA obtained was then dissolved in TE buffer<br />
and kept at -20°C.<br />
Total RNAs were isolated from the muscle tissue of A.<br />
melanoleuca using the Total Tissue/Cell RNA Extraction Kits (Waton<br />
Inc., Shanghai, China) according to the manufacturer's instructions.<br />
RNA sample was dissolved in RNase-free water, and kept at -70°C.<br />
DNA and RNA sample quality was determined by electrophoresis<br />
on 1.0% agarose gel and quantification was performed spectrophotometrically.<br />
Primers design, RT-PCR and cloning of cDNA sequence<br />
The PCR primers were designed by Primer Premier 5.0, according<br />
to the mRNA sequence of COX6b1 from Homo sapiens<br />
(NM_001863), Bos taurus (NM_176675), Pongo abelii<br />
(NM_001131741), Sus scrofa (NM-001097497), Mus musculus<br />
(NM_025628) and Rattus norvegicus (NM_001145273). The<br />
specific primers of cDNA sequence are as follows: COX6b1-F:<br />
CTTTGCTGAGGGTCACATTG; COX6b1-R: GAGAGGTG [G/A]<br />
GTGGAGCCAG.<br />
Total RNAs were utilized to synthesis the first-stranded cDNAs<br />
using a reverse transcription kit with Oligo dT as the primers<br />
according to the manufacturer’s instructions (Promega). The 20 µl<br />
of first-strand cDNA synthesis reaction system included 1 µg of total<br />
RNAs, 5mM of MgCl2, 1mM of dNTPs, 0.5µg of Oligo dT15, 10U/µL<br />
of RNase inhibitor, and 15 U of AMV reverse transcriptase, and was<br />
incubated at 42°C for 60 min.<br />
The first-strand cDNA synthesized was used as a template for<br />
cds sequence amplification. The total reaction volume for DNA<br />
amplification was 25 l. The reaction mixtures contained 1.5 mM of<br />
MgCl2, 200 M of each of dATP, dGTP, dCTP and dTTP (Omega),<br />
0.3 M of each primer, 5.0 units of Taq plus DNA polymerase<br />
(Sangon Co, Shanghai, China). DNA amplification was performed<br />
using a MJ Research thermocycler, Model PTC-200 (Watertown,<br />
MA) with a program of 4 min at 94.0°C, followed by 30 cycles at<br />
94.0°C for 30 s, 45°C for 30 s, and 72.0°C for 1 min, then ended<br />
with the final extension for 10 min at 72.0°C. After amplification,<br />
PCR products were separated by electrophoresis in 1.5% agarose<br />
gel with 1× TAE (Tris-acetate-EDTA) buffer, stained with ethidium<br />
bromide and visualized under UV light. The expected fragments of<br />
PCR products were harvested and purified from gel using a DNA<br />
estraction kit (Omega, China), and then ligated into a pET28a<br />
vector at 16°C for 12 h. The recombinant molecules were<br />
transformed into E. coli complete cells (JM109), and then spread on<br />
the LB-plate containing 50 g/ml ampicillin, 200 mg/ml IPTG<br />
(isopropyl-beta- D-thiogalactopyranoside) and 20 mg/ml X-gal.<br />
Plasmid DNA was isolated and digested by PstI and ScaII to verify<br />
the insert size. Plasmid DNA was sequenced by Huada<br />
Zhongsheng Scientific Corporation (Beijing, China).<br />
Cloning the genomic sequence of COX6b1<br />
The PCR primers were designed based on the cDNA sequence of<br />
the COX6b1 from the A. melanoleuca obtained earlier and complete<br />
genomic sequences of the COX6b1 from four mammals studied<br />
including H. sapiens, M. musculus, R. norvegicus and B. taurus.<br />
The specific primers of genomic sequence are as follows: COX6b1-<br />
F: AGAGTCAGCACCATGGCAG; COX6b1-R:<br />
GAGAGGTGGGTGGAGCCAG.<br />
The genomic sequence of the COX6b1 gene was amplified using<br />
Touchdown-PCR with the following conditions: 94°C for 30 s, 62°C<br />
for 45 s, 72°C for 2 min in the first cycle and the annealing<br />
temperature deceased 1°C per cycle; after 10 cycles conditions<br />
changed to 94°C for 30 s, 52°C for 45 s, 72°C for 2 min for another<br />
25 cycles. The fragment amplified was also purified, ligated into the<br />
clone vector and tansformed into the E. coli competent cells. Finally,<br />
the recombinant fragment was sequenced by Sangon (Shanghai,<br />
China).<br />
Construction of the expression vector and overexpression of<br />
recombinant COX6b1<br />
PCR fragment corresponding to the COX6b1 polypeptide was<br />
amplified from the COX6b1 cDNA clone with the forward primer, 5’-<br />
CAGAATTCATGGCAGAAGACA (EcoR I)-3’ and reverse primer, 5’-<br />
CGAAGCTTCAGATCTTCCCA (Hind III)-3’, respectively. The PCR<br />
was performed at 94°C for 3 min; 35 cycles of 30 s at 94°C, 45 s at<br />
55°C and 1 min at 72°C; 8 min at 72°C. The amplified PCR product<br />
was cut and ligated into corresponding site of pET28a vector<br />
(Stratagen). The resulting construct was transformed into E. coli<br />
BL21 (DE3) strain (Novagen) and used for the induction by adding<br />
IPTG at an OD600 of 0.6 and culturing further for 4 h at 37°C, using<br />
the empty vector transformed BL21 (DE3) as a control. The<br />
recombinant protein samples were induced after 0, 0.5, 1.5, 2, 2.5,<br />
3, 3.5 and 4 h and then separated by SDS-PAGE and stained with<br />
Commassie blue R250.
Data analysis<br />
The sequence data were analyzed by GenScan<br />
(http://genes.mit. edu/GENSCAN.html). Homology research of the<br />
A. melanoleuca COX6b1 gene as compared with the gene<br />
sequences of other species was performed using Blast 2.1<br />
(http://www.ncbi.nlm.nih.gov/blast/). Open reading frame (ORF) of<br />
the DNA sequence was searched by ORF finder<br />
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Protein structure of the<br />
COX6b1 sequence cloned was deduced using predict protein<br />
software (http:// cubic. Bioc. columbia.edu/predictprotein/). Multiple<br />
sequence alignment was performed by DNAMAN 6.0. The<br />
prediction of protein functional sites and biochemical characteristics<br />
depend on the software ExPASy Proteomics Server. Protein tertiary<br />
structure was simulated by SWISS MODEL<br />
(http://swissmodel.expasy.org/). The construction of evolutionary<br />
tree relies on MEGA4.0RESULTS<br />
RESULTS<br />
Analysis of the cDNA of COX6b1 from the A.<br />
melanoleuca<br />
The electrophoresis showed that the amplified cDNA<br />
fragment was about 500 bp in size (Figure not shown).<br />
The sequencing result indicated that the length of the<br />
cDNA cloned is 436 bp, corresponding to the<br />
eletrophoretic result. Blast research analysis revealed<br />
that the COX6b1 cDNA sequence cloned share high<br />
similarities with other mammals reported, including H.<br />
sapiens, B. taurus, S. scrofa, P. abelii, M. musculus and<br />
R. norvegicus, especially the similarity is up to 95.79%<br />
with B. Taurus (Taber 1). On the basis of the high identity,<br />
we concluded that we had cloned the cDNA encoding the<br />
COX6b1 protein of A. melanoleuca. The COX6b1 cDNA<br />
sequence has been submitted to Genbank (accession<br />
number: HQ326486), which contained a 261-bp-length<br />
ORF encoding 86 amino acids (Figure 1).<br />
Analysis of the genomic sequence of COX6b1<br />
The electrophoresis showed that about 5000-bp-length<br />
DNA fragment was amplified with primers COX6b1 -F<br />
and COX6b1 –R (Figure not shown). The sequencing<br />
indicated that the length of the DNA fragment cloned is<br />
4657 bp. The genomic sequence of the COX6b1 has<br />
been submitted to Genbank (accession number:<br />
HQ326485). The comparison of nucleotides sequence<br />
between the genomic and cDNA sequences indicated<br />
that genomic sequence cloned contains three exons and<br />
two introns with software Lasergene. The distribution of<br />
three exons is as follows: 13 to 118 bp; 1487 to 1587 bp;<br />
4584 to 4637 bp (Table 2).<br />
Comparison of the genomic sequences with some<br />
vertebrates (including H. sapiens, B. taurus, M. mulatta,<br />
P. abelii, M. musculus and X. tropicalis) was performed by<br />
DNAMAN 6.0. The result demonstrated that their<br />
sequences have high similarity. Nevertheless, the number<br />
Li et al. 4035<br />
and length of exons are different among the species<br />
when they are compared (Table 2).<br />
Prediction and analysis of protein functional sites in<br />
COX6b1 protein of the A. melanoleuca<br />
The results indicated that the COX6b1 protein sequence<br />
of A. melanoleuca share over 85% similarities with six<br />
species when they are compared, especially high<br />
similarities with B. Taurus (96.51%) and S. scrofa<br />
(97.67%). Primary structure analysis revealed that the<br />
molecular weight of the putative COX6b1 protein is 10.16<br />
kDa with a theoretical pI of 8.83. Topology prediction<br />
showed that there are one N-glycosylation site, one<br />
protein kinase C phosphorylation site, two casein kinase<br />
II phosphorylation sites, two N-myristoylation sites and<br />
one microbodies C-terminal targeting signalsite in<br />
COX6b1 protein of A. melanoleuca (Figure 2). There was<br />
complete consistent in the comparison of functional sites,<br />
except that B. taurus has one more protein kinase C<br />
phosphorylation site than other species related.<br />
Overexpression of the COX6b1 gene in E. coli<br />
The COX6b1 gene was overexpressed in E. coli, using<br />
pET28a plasmids carrying strong promoter and<br />
terminator sequences derived from phage T7. Therefore,<br />
the COX6b1 gene was amplified individually by PCR and<br />
cloned in a pET28a plasmid, resulting in a gene fusion<br />
coding for a protein bearing a HIS-tag extension at the N<br />
terminus. The product of expression was tested by SDS-<br />
PAGE analysis of protein extracted from recombinant E.<br />
coli BL21 strains (Figure 4). The results indicated that the<br />
protein COX6b1 fusion with the N-terminally HIs-tagged<br />
gave rise to the accumulation of an expected 13.46 kDa<br />
polypeptide that formed inclusion bodies. Apparently, the<br />
recombinant protein was expressed after an hour of<br />
induction, and it reached the highest level at 3 h after<br />
induction. These results suggested that the protein is<br />
active and it is just the protein encoded by the COX6b1<br />
from the A. melanoleuca. The expression product<br />
obtained could be used to purify the protein and study its<br />
function further.<br />
DISCUSSION<br />
COX is a key enzyme in the process of oxidative<br />
phosphorylation of mitochondria respiratory chain, and its<br />
damage can directly affect mitochondria function. Studies<br />
has also shown that mutation in a nulear-encoded COX<br />
subunit, COX6b1, will cause drial encephalomyopathy<br />
due to complex IV deficiency (Massa et al., 2008). In this<br />
study, we cloned successfully, COX6b1 gene from the A.<br />
melanoleuca, which share high identity on the nucleotide<br />
sequence and the deduced amino acid sequence with H.
4036 Afr. J. Biotechnol.<br />
Figure 1. Nucleotide sequence and putative amino acid sequence of COX6b1 cDNA from the<br />
A. melanoleuca. The asterisk (*) represents termination codon.<br />
Figure 2. Comparison of functional sites based on the COX6b1 amino acid sequences among<br />
the different species. : N-glycosylation site; : protein kinase C phosphorylation site; :<br />
casein kinase II phosphorylation site; N-myristoylation site; : microbodies C-terminal<br />
targeting signal; polymorphic site.
Li et al. 4037<br />
Table 1. The comparison of gene sequence and encoding sequence with Cox6b1 among the A. melanoleuca, human and other<br />
four species (%).<br />
Parameter H. sapiens B. taurus P. abelii S. scrofa M. musculus R. norvegicu<br />
cds similarity (100%) 90.42 95.79 89.27 95.02 91.57 91.19<br />
aa similarity (100%) 86.05 96.51 84.88 97.67 95.35 95.35<br />
Molecular<br />
(kD)<br />
weight<br />
10.19 10.16 10.19 10.19 10.07 10.07<br />
Isoelectric point (PI) 6.85 8.51 6.85 8.51 8.81 8.81<br />
Table 2. The length comparision of the Cox6b1 genomic sequence between A. melanoleuca and other six.<br />
Species Size (bp) Number of exons Join site in the CDS Accession number<br />
A. melanoleuca 4657 3 13-118; 1487-1587; 4584-4637 HQ326485<br />
H. sapiens 10562 3 3022..3127, 6349..6449, 10372..10425 NG_012193<br />
B. taurus 8879 2 1644..1749, 8690..8744 NC_007316<br />
M. mulatta 11268 3 2866..2974, 7119..7219, 11081..11134 NC_007876<br />
P. abelii 9889 2 5274..5375,9697..9750 NC_012610<br />
M. musculus 9178 3 1547..1652, 2637..2737, 8990..9043 NC_000073<br />
X. tropicalis 10357 2 8856..>8957, 10160..10213 NW_003164399<br />
Figure 3. ME evolution tree constructed based on cds sequence of variety species.<br />
sapiens, B. taurus, P. abelii, S. scrofa, R. norvegicus and<br />
M. musculus as well as on the deduced amino acid<br />
sequence. The pI and molecular weight with COX6b1 of<br />
A. melanoleuca are close to the compared six species<br />
(Table 1). These results suggested that the gene we<br />
cloned is COX6b1 gene of A. melanoleuca.<br />
Schmidt et al. (2002) examined the amino acid<br />
replacements for 16 genes that encode the proteins of<br />
the COX holoenzyme in eight vertebrate species, and<br />
found that COX6b1 have undergone acceleration in<br />
amino acid replacement rates. They thought the mature<br />
portions of these genes are likely to have undergone a<br />
functionally significant change to adapt in nature. In this<br />
study, a variety of polymorphic sites were found on the<br />
nucleotide sequence, but they belong mostly to<br />
synonymous mutation. Furthermore, a lot of amino acid<br />
variation was also found by the comparison of amino acid<br />
sequence (Figure 2). The result shows that the change of<br />
A. melanoleuca and B. Taurus, S. scrofa in amino acid<br />
appeared largely consistency. We also analyzed the<br />
function sites of COX6b1 and found that the function sites<br />
are greatly consistent in seven mammal species when<br />
they are compared, except that the B. Taurus has one<br />
more casein kinase II phosphorylation site (Figure 2).<br />
These results are in agreement with the results obtained<br />
by the ME (minimum evolution) evolutionary tree of<br />
COX6b1 (Figure 3). In addition, Lu et al. (2010) found<br />
that the untranslated region (UTR) polymorphisms of<br />
COX6b1 subunit gene is pervasive and considered COX<br />
subunit gene heterogeneity may mediate COX functional<br />
variation. In this paper, the genomic sequence of<br />
COX6b1 was compared by DNAMAN 6.0, and UTR
4038 Afr. J. Biotechnol.<br />
Figure 4. Protein extracted from recombinant E. coli BL21 strains were analyzed by SDS-PAGE gel<br />
stained with Commassie blue R250. Numbers on the right shows the molecular weight and the arrow<br />
indicates the recombinant protein bands induced by IPTG with 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h<br />
(lanes 2 to 9), respectively. Lane 1 represents the products of the E. coli strains with the empty vectors.<br />
polymorphisms of COX6b1 subunit gene were also<br />
found.<br />
As is well known, the biogenesis of cytochrome c<br />
oxidase is a complex process controlled by two distinct<br />
genomes. Meanwhile, NRF (nuclear respiratory factor)<br />
also act as an important mediator of coordinated<br />
regulation of all ten nuclear-encoded COX subunit genes<br />
(Scarpulla, 2002; Shilpa et al., 2009). COX6b1 is one of<br />
COX small subunits encoded by nuclear genes. Similarly,<br />
NRF will also have an effect on COX6b1. Furthermore,<br />
Taanman’s experiment in 1990 have demonstrated that<br />
the steady-state levels of the COX6b transcript were<br />
different in the tissues examined in human liver, skeletal<br />
muscle, MOLT-4 cells and fibroblasts. In this paper, we<br />
achieved only efficient express in a prokaryotic organism.<br />
The gene’s transcription level needs further research.<br />
The cDNA and the genomic sequence of COX6b1 gene<br />
were cloned successfully from the skeleton muscle of the<br />
A. melanoleuca, respectively, which were sequenced and<br />
analyzed preliminarily. The cDNA of the COX6b1 gene<br />
was also overexpressed in E. coli. The data will enrich<br />
and supplement the information about COX6b1. In<br />
addition, it will contribute to the protection for gene<br />
resources and the discussion of the genetic<br />
polymorphism.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by the Key Chinese National<br />
Natural Science Foundation (30470261), Application<br />
Technology Project in Sichuan Province (2006J13-057),<br />
Key Scientific Research Foundation of Educational<br />
Committee of Sichuan Province (07ZA120), Key<br />
Discipline Construction Project in Sichuan Province<br />
(SZD0420), Sichuan Key Discipline Zoology Construction<br />
Funds Subsidization Project (404001), Application<br />
Infrastructure Projects in Sichuan Province (2009JY0061)<br />
and Education Department Youth Fund Project of<br />
Sichuan Province (092B088).<br />
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mitochondrial biogenesis. Biochim. biophys acta. 1576(1-2):1-14.<br />
Schmidt TR, Goodmana M, Grossman LI (2002). Amino acid<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4040-4045, 16 May, 2011<br />
Available online at http://www.academicjournals.org<br />
DOI: 10.5897/AJB10.857<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Effect of plant growth regulators (PGRs) on<br />
micropropagation of a vulnerable and high value<br />
medicinal plant Hedychium spicatum<br />
Dinesh Giri 1 * and Sushma Tamta 2<br />
1 Plant Tissue Culture Laboratory, Department of Botany, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand,<br />
263002, India.<br />
2 Department of Biotechnology, Bhimtal Campus, Kumaun University, Nainital, Uttarakhand, India.<br />
Accepted 15 March, 2011<br />
A complete micropropagation protocol was developed by applying different plant growth regulators<br />
(PGRs) of a vulnerable and high value aromatic medicinal plant, Hedychium spicatum. Three cytokinins,<br />
6-benzyladenine (BA), kinetin (KN) and thidiazuron (TDZ) were used and among these, the lower<br />
concentration of TDZ (1.0 µM) was found to be the most effective treatment in relation to induction of<br />
high frequency shoot multiplication (83.33%), number of shoots per explant (3.86 shoots) and average<br />
number of shoots per flask (19.33 shoots). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-<br />
naphthalene acetic acid (NAA) were the used auxins in this study for in-vitro rooting. Among these used<br />
auxins, the lower concentration of IBA (2.5 µM) was the prominent plant growth regulator regarding in<br />
vitro rooting. Well rooted and healthy plantlets were obtained after 2 months of hardening and<br />
transferred to the field (1990 m) with 90.0% survival. On the basis of available literature, this is the first<br />
and significant study regarding the comparative effect of different PGRs on in-vitro propagation study<br />
of H. spicatum. This significant study could be useful for large scale propagation and ex-situ<br />
conservation of this vulnerable Himalayan species.<br />
Key words: Cytokinins, thidiazuron, shoot multiplication, in-vitro propagation, ex-situ conservation.<br />
INTRODUCTION<br />
Hedychium is one of the plant genus which is commonly<br />
used in the preparation of indigenous medicine. There<br />
are 50 species of Hedychium in tropical Asia (Kirtikar and<br />
Basu, 1984), 37 species in India and 8 species in<br />
Western Himalaya. Hedychium spicatum (Zingiberaceae),<br />
commonly known as kapoor kachri, van- haldi or ginger<br />
lilly, is distributed in subtropical Himalayas in the states of<br />
Assam, Arunanchal Pradesh and Uttarakhand at an<br />
altitude of 1000 to 3000 m. The root stock (rhizome) is<br />
useful in asthma, pain, foul breath, hiccough, vomiting<br />
and laxative, stomachache, carminative, stimulant tonic to<br />
the brain, in liver complaints, diarrhea and pains (Kirtikar<br />
and Basu, 1984).<br />
Rhizome extracts showed antimicrobial activity against<br />
*Corresponding author. E-mail: giri_gld@rediffmail.com.<br />
gram-positive and negative bacteria (Bisht et al., 2006).<br />
Recently, the crude extract of the rhizome has been used<br />
in the preparation of an anticancerous drug, PADMA 28<br />
(Nayab et al., 2004). Samant et al. (2007) reported that<br />
H. spicatum is commercially exploited from its natural<br />
area. According to the threat status of IUCN criteria, H.<br />
spicatum has become vulnerable (Samant and Pant,<br />
2006). It is also listed in near threatened category of the<br />
essential oil bearing plants (Samant and Palni, 2000).<br />
Hence, prioritization of this species needs to be done for<br />
propagation and conservation. Therefore, the present<br />
study was conducted to see the effect of different PGRs<br />
on micropropagation of this species.<br />
MATERIALS AND METHODS<br />
Seeds of H. spicatum were collected in the month of October to<br />
November, 2008, from Nainital catchment forest (1990 m) and
Table 1. Effects of cytokinins on in vitro shoot multiplication of H. spicatum using excised shoot tips in MS<br />
medium.<br />
Treatment (µM)<br />
Number of<br />
shoot/ flask<br />
Number of<br />
shoot/ explant<br />
Percentage shoot<br />
multiplication<br />
Giri and Tamta 4041<br />
Average shoot<br />
length (cm)<br />
Control (0.0) 05.00±0.00 - - 1.80±0.65<br />
BA (0.1) 05.66±0.88 1.13±0.17 53.33±6.66 3.92±0.30<br />
(1.0) 07.00±0.57 1.40±0.11 53.33±6.66 3.653±0.52<br />
(2.5) 08.66±1.45 1.73±0.29 43.33±23.33 3.214±0.41<br />
(5.0) 07.00±1.54 1.40±0.23 40.00±11.54 2.568±0.07<br />
(10.0) 05.66±0.33 1.13±0.06 40.00±11.54 2.632±0.36<br />
(20.0) 05.66±0.66 1.13±0.13 40.00±11.54 2.236±0.34<br />
KN (0.1) 07.66±1.76 1.46±0.40 76.66±8.81 4.94±0.36<br />
(1.0) 07.33±0.88 1.46±0.17 53.33±6.66 4.00±0.35<br />
(2.5) 07.00±1.52 1.40±0.30 73.33±13.33 4.54±0.95<br />
(5.0) 07.66±1.45 1.46±0.35 56.66±20.27 3.90±0.17<br />
(10.0) 07.33±0.33 1.13±0.26 40.00±11.54 3.52±0.23<br />
(20.0) 05.33±0.33 1.15±0.07 26.66±6.66 3.59±0.83<br />
TDZ (0.1) 10.66±1.20 2.13±0.24 83.33±12.01 1.52±0.08<br />
(1.0) 19.33±4.91 3.86±0.98 83.33±12.08 1.10±0.15<br />
(2.5) 16.66±0.88 2.66±0.54 63.33±14.52 2.01±0.34<br />
(5.0) 14.00±6.50 2.80±1.13 50.00±20.81 1.41±0.08<br />
(10.0) 08.33±1.76 1.16±0.35 56.66±20.27 1.53±0.03<br />
(20.0) 10.33±2.90 2.06±0.58 40.00±11.54 1.99±0.16<br />
Values are mean ± standard error, each treatment consisted of 15 explants (5 in each flask, 3 flasks per<br />
treatment). Data were recorded after 4 weeks of inoculation. - = no shoot multiplication.<br />
brought to the laboratory. Seeds were separated from spikes, air<br />
dried for 3 days at room temperature and stored at 4°C in the dark.<br />
After 65 days, seeds were taken out and washed under running tap<br />
water along with a mild detergent and treated with a systemic<br />
fungicide (Bavistin, 1% w/v, 15 min). Following washing with double<br />
distilled water, seeds were transferred to laminar air flow cabinet.<br />
Surface sterilization was carried out by using mercuric chloride<br />
(HgCl2, 0.1% w/v, 3 min) followed by washing with sterile distilled<br />
water (5 times). Seeds were inoculated (10 seeds per flask) on MS<br />
(Murashige and Skoog, 1962) basal medium containing agar (0.8%<br />
w/v) and transferred to the culture room. Three weeks after<br />
inoculation, shoot tips (size approximately 1.5 cm) were excised<br />
from in vitro germinated seedlings and inoculated on MS medium<br />
supplemented with different concentrations (0.1 to 20 µM) of 6benzyladenine<br />
(BA), kinetin (KN) and thidiazuron (TDZ) (Table 1)<br />
for shoot multiplication. In vitro generated microshoots were used to<br />
induce rooting. These microshoots (2.5 to 5.0 cm in length) were<br />
inoculated on MS medium supplemented with different concentrations<br />
of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and<br />
α- naphthalene acetic acid (Table 2).<br />
All cultures were raised in 250 ml erlenmeyer flasks (100 ml<br />
medium in each flask). Sucrose (3.0%, w/v) was added as a carbon<br />
source and medium was gelled with agar (0.8%, w/v). The pH of the<br />
medium was adjusted to 5.8 before autoclaving (1.05 kg cm -2 ,<br />
121°C and 20 min). Cultures were maintained in a culture room at<br />
25 ± 1°C in 16/8 h light/dark photoperiodic cycle, with irradiance by<br />
cool fluorescent tubes (Philips 40 W).<br />
After rooting, the microshoots with well developed roots were<br />
taken out from the culture flasks and roots were then washed<br />
thoroughly with tap water to remove the adhering medium.<br />
Following washing, these plantlets were transferred to thermocole<br />
cups (8 cm height and 4 cm diameter) containing soil and farmyard<br />
manure (3:1, v/v). Transparent polythene bags were inverted over<br />
these thermocole cups (8 cm height and 4 cm diameter) to maintain<br />
the humidity and watered on alternate days. Initially, thermocole<br />
cups were kept inside a culture room for first four weeks and<br />
gradually removed to polyhouse (25 ± 1°C) for further growth. After<br />
2 months, these hardened plantlets were transferred to field (near<br />
department nursery) to study the survival rate of these in vitro<br />
raised plantlets.<br />
Statistical analysis<br />
Data were subjected to statistical analysis and standard error (SE)<br />
and ANOVA were calculated following Snedecor and Cochran<br />
(1967).<br />
RESULTS AND DISCUSSION<br />
Seeds started germinating within 15 days of inoculation<br />
on MS basal medium. After one week of germination (3<br />
weeks after inoculation), the explants (shoot tips, size<br />
approximately, 1.5 cm) from in-vitro germinated seedlings<br />
were excised and inoculated on MS medium supplemented<br />
with different concentrations of BA, KN and TDZ<br />
which resulted in multiple shoot formation. However,<br />
number of shoots per explant, as well as number of<br />
shoots per flask varied with the treatments (Table 1).<br />
Each shoot tip when cultured on MS basal medium<br />
(without PGRs), developed into a complete plantlet with<br />
profuse root system. MS medium when supplemented
4042 Afr. J. Biotechnol.<br />
Figure 1. Micropropagation of H. spicatum. A and B, Lower concentration of TDZ (1.0 µM) showed high frequency of shoot<br />
multiplication after 4 weeks of inoculation in MS medium; C, effect of cytokinins on shoot length after 8 weeks of inoculation in<br />
MS medium: II (KN) > I (BA) > III (TDZ); D, effect of auxins on rooting after 4 weeks of inoculation in MS medium; E, rooting in<br />
lower concentration of IBA (2.5 µM) after 8 weeks of root initiation; F, hardening of plantlets in a culture room; G, well rooted<br />
plants after two months of hardening (photograph taken just before field transfer); H, field transfer of plantlets (at department<br />
nursery); I, well grown plant in field after 3 months of field transfer.<br />
with different concentrations of BA had best response in<br />
2.5 µM in terms of the number of shoots per flask (8.66)<br />
and number of shoots per explant (1.73). However,<br />
percent shoot multiplication was maximum (53.33%) with<br />
0.1 and 1.0 µM BA, and the average shoot length was<br />
highest (3.92 cm) in lower concentration (0.1 µM) of BA.<br />
When KN was used for shoot multiplication, all the used<br />
concentrations (0.1 to 20 µM) had more or less same<br />
effect for three parameters (number of shoots per flask,<br />
number of shoots per explant and average shoot length)<br />
(Table 1), but percent shoot multiplication was maximum<br />
(76.66%) in lower concentration of KN (0.1 µM). Among<br />
various concentrations of TDZ tested, 1.0 µM TDZ<br />
produced maximum number of shoots (19.33 shoots per<br />
flask, 3.86 shoots per explant) within four weeks (Table 1<br />
and Figure 1A, B). Although other concentrations of TDZ<br />
also gave good shoot multiplication, the percent shoot<br />
multiplication was highest (83.33%) in lower concen-<br />
tration of TDZ (0.1 and 1.0 µM). The shoots formed in<br />
TDZ supplemented medium were short in length but the<br />
shoot length was quite good in KN supplemented MS<br />
medium (Figure 1C). Data were analyzed by using<br />
ANOVA which showed that among the used cytokinins,<br />
average number of shoots per flask, number of shoots<br />
per explant and average length of shoots were highly<br />
significant (p < 0.01 at 1%). However, only percent shoot<br />
multiplication was insignificant (p < 0.01 at 1%) within<br />
used concentrations of cytokinins (Table 2).<br />
Effect of different auxins on in vitro induction of rooting<br />
of microshoots of H. spicatum is summarized in Table 3.<br />
All the used auxins (concentration ranges from 0.1 to 10<br />
µM) were able to induce rooting in microshoots, including<br />
control (auxin free MS medium) also. However, rooting<br />
percentage in control was only 29.62% and it took 21<br />
days to root in comparison with other treatments where<br />
the differentiation of roots on the elongated shoots
Table 2. ANOVA summary.<br />
Source Dependent variable df Mean square F-value<br />
Cytokinins<br />
Concentration<br />
Average number of shoots per flask 2 215.73 16.25*<br />
Average number of shoots per explant 2 7.39 12.40*<br />
Percentage shoot multiplication 2 1220.63 2.51ns<br />
Average shoot length (cm) 2 22.46 37.39*<br />
Average number of shoots per flask 6 42.75 3.22 *<br />
Average number of shoots per explant 6 4.77 8.02*<br />
Percentage shoot multiplication 6 5039.68 10.37*<br />
Average shoot length (cm) 6 2.775 4.62*<br />
*Significant at 1% (p < 0.01) and ns, not significant; no, number; df, degree of freedom<br />
Table 3. Effect of different auxins on in vitro rooting of micro-shoots of H. spicatum on MS medium.<br />
Treatment<br />
(µM)<br />
Percentage<br />
rooting<br />
Time taken for root<br />
emergence (days)<br />
Average number of<br />
root per shoot<br />
Giri and Tamta 4043<br />
Average length<br />
of root (cm)<br />
Control (0.0) 29.62±3.70 21.00±1.15 1.15±0.03 1.86±0.03<br />
IAA (0.1) 40.73±3.70 14.00±0.57 2.74±0.41 2.20±0.20<br />
(1.0) 51.84±3.70 15.33±0.66 3.15±0.26 3.30±0.52<br />
(2.5) 66.66±0.00 10.66±0.88 3.40±0.87 3.15±0.30<br />
(5.0) 55.55±6.41 16.33±1.45 2.54±0.05 2.56±0.46<br />
(10.0) 33.33±0.00 15.00±0.57 2.17±0.12 1.78±0.33<br />
NAA (0.1) 44.44±6.41 11.66±1.45 3.12±0.43 2.94±0.10<br />
(1.0) 62.95±3.70 10.35±0.47 3.07±0.29 3.74±0.19<br />
(2.5) 81.47±3.70 10.5±0.57 3.83±0.16 3.92±0.18<br />
(5.0) 85.17±3.70 10.44±0.59 3.78±0.18 3.42±0.46<br />
(10.0) 37.03±3.70 11.77±0.39 2.59±0.02 2.54±0.22<br />
IBA (0.1) 59.25±3.70 11.00±0.57 3.86±0.16 3.30±0.20<br />
(1.0) 70.36±3.70 10.33±0.88 4.07±0.27 4.31±0.38<br />
(2.5) 96.29±3.70 10.24±0.58 5.25±0.08 4.87±0.39<br />
(5.0) 92.58±3.70 10.00±0.57 5.06±0.47 5.26±0.44<br />
(10.0) 55.55±6.41 12.00±0.57 3.27±0.46 3.01±0.25<br />
Values are mean ± standard error (SE). There are nine explants (three microshoots per flask) in each treatment.<br />
occurred over a period of 1 to 2 weeks (Table 3). Trend<br />
for percent rooting was similar in all the three used<br />
auxins, which means percent rooting increased with<br />
increasing concentration of auxins up to certain extent<br />
and then it decreased with increasing concentration of<br />
auxin. In case of NAA, rooting percentage increased with<br />
increasing concentration up to 5.0 µM, and 5.0 µM NAA<br />
was able to induce 85.17% rooting, then rooting percentage<br />
decreased to 37.03% as the concentration increased<br />
to 10.0 µM. Similar trend was observed in microshoots<br />
which were inoculated in IAA and IBA supplemented MS<br />
medium. In these two auxin treatments, percentage<br />
rooting increased only up to 2.5 µM concentration and<br />
then it decreased with increasing concentration (Table 3).<br />
Among different concentration of IAA, 2.5 µM concentration<br />
enhanced maximum rooting percentage (66.66%)<br />
within 10 days of inoculation. The average number of<br />
roots per shoot was highest (3.40 roots/shoot) in 2.5 µM<br />
IAA, while 1.0 µM showed maximum average length of<br />
roots (3.3 cm). Percent rooting was improved up to<br />
85.0% by using 5.0 µM of NAA within 10 days of inoculation,<br />
however, the lower concentration of NAA (2.5 µM )<br />
showed best response in relation to the average number<br />
of roots per shoot (3.83 roots) and average length of<br />
roots (3.92 cm). Among various concentrations of IBA,<br />
the lower concentration (2.5 µM) showed maximum
4044 Afr. J. Biotechnol.<br />
Table 4. ANOVA summary.<br />
Source<br />
Percentage rooting<br />
Time taken for root<br />
emergence (in day)<br />
Average number of<br />
root/shoot<br />
Average length of<br />
root (cm)<br />
DF MS F-ratio DF MS F-ratio DF MS F-ratio DF MS F-ratio<br />
Concentration 5 3791.0 75.37* 5 128.0 57.80* 5 10.3 52.9* 5 10.39 36.67*<br />
Auxins 2 1982.0 39.40* 2 49.3 22.26* 2 7.40 38.0* 2 7.54 26.61*<br />
Concentration x auxins 10 160.4 3.19* 10 6.0 2.74** 10 0.60 3.1* 10 0.64 2.28**<br />
Error 36 50.29 36 2.2 36 0.19 36 0.28<br />
*Significant at 0.01 level (at 1%); ** significant at 0.05 level (5%).<br />
percent rooting (96.29%) and average number of roots<br />
per shoot (5.25 roots), while the average length of roots<br />
was 5.26 cm in 5.0 µM IBA supplemented medium. Out<br />
of these three used auxins, IBA showed best response in<br />
relation to percent rooting; average number of roots per<br />
shoot and average length of roots (Table 3 and Figure<br />
1D, E). Mean significance difference (at the 5% level)<br />
computed by applying ANOVA showed a significant<br />
difference among various parameters (percent rooting,<br />
time taken for root emergence (in days), average number<br />
of roots per shoot and average length of roots) (Table 4).<br />
One month after rooting, these plantlets were hardened<br />
under growth chamber for four weeks (Figure 1F). Well<br />
rooted and healthy plantlets were obtained after 2 months<br />
of hardening (Figure 1G) and transferred to the field condition<br />
(1990 m) with ninety percent survival rate (Figure<br />
1H and I).<br />
Therefore, on the basis of results obtained in this<br />
experiment, TDZ was found to be the most effective<br />
cytokinin for shoot multiplication, however, shoot lengths<br />
was better in KN supplemented medium. On the basis of<br />
literature, it is known that TDZ act as potent regulator for<br />
in vitro propagation system and as an effective mean of<br />
induction of adventitious shoots in a number of plant<br />
species (Huettman and Preece, 1993; Lu, 1993). It has<br />
been known to promote cytokinins-like activity similar to<br />
that of N 6 substituted adenine derivatives and has been<br />
used in tissue culture studies since 1982 (Mok et al.,<br />
1982; Siddique and Anis, 2007). TDZ stimulate cytokinins<br />
activity and it is a better substitute of other cytokinins like<br />
BA and KN that are generally used in tissue culture work<br />
(Kannan et al., 2007; Vinayak et al., 2009). Sujatha and<br />
Reddy (1998), also reported that a higher shoot<br />
multiplication rate was obtained on TDZ supplemented<br />
media. However, it concomitant with a mark reduction in<br />
shoot length. Mok et al. (1987) showed that lower concentration<br />
of TDZ (0.3 µM) was very effective in<br />
multiplication of Broccoli (Brassica oleracea) and higher<br />
concentration (10.0 µM) induced abnormal growth and<br />
vitrification. TDZ stimulated shoot regeneration in a<br />
concentration-dependent manner, and higher concentration<br />
significantly reduced the height of newly formed<br />
shoot (Tomsone et al., 2004). Present study indicates<br />
that by using lower concentration of TDZ (1.0 µM), the<br />
shoot multiplication frequency was the best. The high<br />
efficiency of TDZ may be attributed to its ability to induce<br />
cytokinin accumulation (Victor et al., 1999) or enhance<br />
the accumulation and translocation of auxin within the<br />
tissue (Murthy et al., 1998). The growth promoting activity<br />
of thidiazuron was accompanied by high acid-phosphate<br />
level (Mok et al., 1987) and is stable and biologically<br />
active at low concentration than other cytokinins (Genkov<br />
and Ivanova, 1995).<br />
This significant study concluded that the lower concentration<br />
of IBA was the prominent PGRs regarding in vitro<br />
rooting. In the present study, the superiority of IBA over<br />
other auxins regarding in vitro rooting is supported by the<br />
result of other studies like Sreekumar et al. (2000) in<br />
Hemidesmus indicus, Mala and Bylinsky (2004) in<br />
Daphne cneorum, Kannan et al. (2006) in Caesalpinia<br />
bonduc and Gantait et al. (2009) in Dendrobium<br />
chrysotoxum. The present investigation suggests that the<br />
IAA showed least response in root formation. In comparison<br />
with other auxins, similar observation was reported<br />
by Kannan et al. (2007). However, it was clear that<br />
increasing the concentration of these auxins did not<br />
responded well in the present study. The variation in<br />
effectiveness of different auxin sources may be attributed<br />
to their differential affinity to auxin receptors involved in<br />
the rooting process, which may be cultivar specific<br />
(Tereso et al., 2008) and depend on the plant type<br />
(Nandgopal and Kumari, 2007). A disadvantage of micropropagation<br />
is the high mortality rate during the transfer<br />
of in vitro plantlets to ex vitro (Lovato et al., 1999). But in<br />
the present study, a very good survival rate (90.0%) was<br />
achieved in comparison to earlier studies like Badoni et<br />
al. (2010), where they got only 40 to 50% survival rate,<br />
and Koul et al. (2005) that reported 80 to 85% survival<br />
rate. There were no previous literatures available on the<br />
comparative study that applied these three different<br />
auxins on in vitro rooting of this species. This significant<br />
study shows that the lower concentration of IBA was the<br />
prominent plant growth regulator regarding in vitro<br />
rooting.<br />
On the basis of available literature regarding micropropagation<br />
study of H. spicatum (Koul et al., 2005;<br />
Badoni et al., 2010), there was no such type of comparative<br />
study using different plant growth regulators and
this would be the first report on this species. This<br />
significant study concluded that, by using lower concentrations<br />
of TDZ (1.0 µM) and IBA (2.5 µM), it could be<br />
able to achieve a number of in vitro raised plantlets within<br />
a limited period of time. Beside propagation, this study<br />
would also become an ex situ conservation strategy of<br />
this vulnerable Himalayan species.<br />
ACKNOWLEDEGMENT<br />
Authors are thankful to the head of department for<br />
providing necessary facility in the department.<br />
REFERENCES<br />
Badoni A, Bisht C, Chauhan JS (2010). Micropropagation of Hedychium<br />
spicatum Smith using in-vitro shoot tip. Stem Cell. 1(1): 11-13.<br />
Bisht GS, Awasthi AK, Dhole TN (2006). Antimicrobial activity of<br />
Hedychium spicatum. Fitoterapia, 77: 240-242.<br />
Gantait S, Mandal N, Das PK (2009). Impact of auxins and activated<br />
charcoal on in-vitro rooting of Dendrobium chrysotoxum Lindl. Cv.<br />
Golden Boy. J. of Tropical Agric. 47(1-2): 84-86.<br />
Genkov T, Ivanova I (1995). Effect of cytokinin-active phenyl urea<br />
derivatives on shoot multiplication, peroxidase and superoxide<br />
dismutase activities of in-vitro cultured Carnation. Bulg. J. Plant<br />
Physiol. 21(1)73:83.<br />
Huettman MJ, Preece JE (1993). Thidiazuron: a potent cytokinin for<br />
woody plant tissue culture. Plant Cell Tissue Organ Cult. 33: 105-<br />
119.<br />
Kannan P, Premkumar A, Ignacimuthu S (2006). Organogenesis from<br />
stem explants of Caesalpinia bonduc. J. Trop. Med. Plants. 7: 95-<br />
100.<br />
Kannan P, Premkumar A, Ignacimuthu S (2007). Thidiazuron induced<br />
shoot regeneration in the endangered species, Exacum<br />
travancoricum Beedi. Ind. J. Biotechnol. 6: 564-566.<br />
Kirtikar KR, Basu BD (1984). Indian medicinal plants. Bishan Singh,<br />
Mahendra Pal Singh, Dehradun. pp. 824-826.<br />
Koul S, Raina V, Sharma SK (2005). Conservation and propagation of<br />
high altitude medicinal and aromatic plant: Hedychium spicatum. J.<br />
Plant Biochem. Biotechnol. 14: 57-59.<br />
Lovato PE, Schuepp H, Trouvelot A, Gianinazzi S (1999). In Mycorrhiza<br />
(eds Varma A and Hook B). Springer-Verlag. pp. 443-468.<br />
Lu CY (1993). The use of thidiazuron in tissue culture. In Vitro Cell Dev.<br />
Biol. 29, 92-96.<br />
Mala J, Bylinsky V (2004). Micropropagation of endangered species<br />
Daphne cneorum. Biol/ Plant. 48: 633-636.<br />
Mok MC, Mok DWS, Armstrong DJ, Shudo K, Isogai Y, Okamoto T<br />
(1982). Cytokinin activity of N-phenyl-N-1,2,3-thiadiazol-5-ylurea<br />
(thidiazuron). Phytochem. 21: 1509-1511.<br />
Giri and Tamta 4045<br />
Mok MC, Mok D, Turner J, Mujer C (1987). Biological and biochemical<br />
effects of cytokinins-active phenylurea derivatives in tissue culture<br />
systems. In Chemical Regulation in Tissue Culture: An Overview.<br />
Hort. Sci. 22(6): 1194-1197.<br />
Murashige T, Skoog F (1962). A revised medium for rapid growth and<br />
bio assays with tobacco tissue culture. Physiol. Plant. 15: 473-497.<br />
Murthy BNS, Murch SJ, Saxena PK (1998). Thiadiazuron: A potent invitro<br />
plant morphogenesis. In Vitro Cell Dev Biol. Plant, 34: 267-275.<br />
Nandgopal S, Kumari R (2007). Effectiveness of auxin induced in vitro<br />
root culture in Chicory. J. Central Eur. Agric. 8(1): 73-80.<br />
Nayab R, Aingorn H, Fallavollita L, Sallon S, Mechoulam R, Ginsburg I,<br />
Vlodavsky I, Brodt P (2004). PADMA-28, a traditional Tibetan herbal<br />
preparation, blocks cellular responses to bFGF and IGF-I.<br />
Inflammopharmacol. 12(4): 373-89.<br />
Samant SS, Palni LMS (2000). Diversity, distribution and indigenous<br />
uses of essential oil yielding medicinal plants of Indian Himalayan<br />
region. J. Med. Arom. Plant Sci. 22: 671-684.<br />
Samant SS, Pant S (2006). Diversity, Distribution, Pattern and<br />
conservation status of the plants used in liver Diseases/Ailments in<br />
Indian Himalaya region. J. Mountain Sci. 3: 28-47.<br />
Samant SS, Butola JS, Sharma A (2007). Assessment of Diversity,<br />
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Mountain Sci. 4(1): 034-056.<br />
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basilicum using shoot tip explants pre-cultured in thidiazuron<br />
supplemented liquid medium. Biol. Plant. 51: 787-790.<br />
Snedecor GW, Cochran WG (1967). Statistical methods. Oxford and<br />
IBH Publishing. New Delhi, India.<br />
Sreekumar S, Seeni S, Pushpangadan P (2000). Micropropagation of<br />
Hemidesmus indicus for cultivation and production of 2-hydroxy-4methoxybenzaldehyde.<br />
Plant Cell Tissue Org. Cult. 62: 211-218.<br />
Sujatha M, Reddy TP (1998). Differential cytokinins effect on the<br />
stimulation of in-vitro shoot proliferation from meristematic exlant of<br />
Castor (Ricinus communis L). Plant Cell Rep. 17: 561-566.<br />
Tereso S, Miguel CM, Mascarenhas M, Roque A, Trindade H, Maroco<br />
J, Oliveira MM (2008) .Improved in vitro rooting of Prunus dulcis Mill.<br />
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thidiazuron on shoot regeneration and proliferation of Rhododendron<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4046-4056, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1968<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Genotype-dependent responses of wheat (Triticum<br />
aestivum L.) seedlings to drought, UV-B radiation and<br />
their combined stresses<br />
Liheng He 1 , Xiaoyun Jia 1 , Zhiqiang Gao 1 * and Runzhi Li 2<br />
1 Agronomy College, Shanxi Agricultural University, Taigu 030801, China.<br />
2 Department of Plant and Soil Science, University of Kentucky, Lexington, KY 40546, USA.<br />
Accepted 8 April, 2011<br />
Experiments were conducted under controlled conditions to investigate the growth and physiologicalbiochemical<br />
responses of wheat (Triticum aestivum L.) seedlings to UV-B, drought, and their combined<br />
stresses. Both UV-B and drought treatments retarded seedling growth with UV-B having worse impact<br />
on wheat plants. Chlorophyll content and relative water content (RWC) in leaves, as stress makers,<br />
were significantly affected by UV-B and drought, respectively. The increased rate and amount of H2O2<br />
were stress-different and genotype-dependent. Likely, the temporary expression patterns of antioxidant<br />
enzymes (superoxide dismutase, SOD, and catalase, CAT) and compounds (proline, and ascobate acid,<br />
AsA) exhibited differences under the tested stressful conditions in the two genotypes, indicating that<br />
they play significant roles in plant responses to these stresses. Pre-application of either stress<br />
reduced the damage caused by subsequent application of the other stress, and this induced defense<br />
was greater by UV-B than by drought. Compared to the stress applied separately, the combined<br />
application of drought and UV-B at the same time resulted in more adverse effects on the wheat<br />
seedlings of the susceptible variety, and more positive effects on the tolerant wheat genotype. These<br />
results provide novel insights into understanding the cellular and molecular mechanisms responsible<br />
for plant tolerance to various stresses and their interactions.<br />
Key words: Wheat (Triticum aestivum L.), UV-B radiation, drought, combined stress, antioxidant system,<br />
genotype.<br />
INTRODUCTION<br />
The ongoing depletion of stratosphere ozone layer is<br />
resulting in an increase of solar ultraviolet-B (UV-B)<br />
radiation (280 to 320 nm) reaching the earth’s surface, as<br />
measured in many geographic regions (McKenzie et al.,<br />
2003). The enhanced levels of UV-B radiation has been<br />
shown to have deleterious effects on biological organisms.<br />
Plants exhibit different responses to UV-B irradiation<br />
with respect to growth, production of dry matter and<br />
biochemical changes (Kramer et al., 1991). Some plant<br />
*Corresponding author. E-mail: gsshlh@hotmail.com or<br />
gaozhiqiang1964@126.com. Tel: +86 354 6289789.<br />
Abbreviations: SOD, Superoxide dismutase activity; CAT,<br />
catalase activity.<br />
species are unaffected by UV-B irradiation and several<br />
are apparently stimulated in their growth, but most<br />
species are sensitive and damaged (Teramura, 1983).<br />
UV-B irradiation can also influence the physiological<br />
responses of plants to the other environmental factors.<br />
Elevated UV-B irradiation limited the ability of wheat, rice<br />
and soybean to take advantage of the elevated CO2 in<br />
photosynthesis (Teramura et al., 1990), but however,<br />
enhanced heat tolerance of cucumber seedlings<br />
(Teklemariam and Blake, 2003).<br />
On the other hand, the effect of enhanced UV-B<br />
radiation on plants can be modified by other co-occurring<br />
stresses or by simply changing environmental factors like<br />
atmospheric CO2 (Bjorn et al., 1997). Temperature<br />
affected the extent of growth inhibition of cucumber<br />
cotyledons irradiated with UV-B (Takeuchi et al., 1993).<br />
Sensitivity of crop plants to UV-B is also influenced by
water regime, ambient levels of visible radiation and<br />
nutrient status (Manetas et al., 1997; Balakumar et al.,<br />
1993; Levizou and Manetas, 2001).<br />
Water stress or soil drought is an important restricting<br />
factor, which limits the productivity of many crops and<br />
affects both quality and quantity of the yield. Drought<br />
stress brings about a reduction in growth rate, stem<br />
elongation, leaf expansion and stomatal movements<br />
(Hsiao, 1973). It also affect various physiological and biochemical<br />
processes governing plant growth and<br />
productivity (Daie, 1988). Under field conditions plants<br />
usually experience several stresses simultaneously. The<br />
stresses may cause a variety of plant responses which<br />
can be additive, synergistic or antagonistic.<br />
In many cases, UV-B irradiation appears companying<br />
with drought stress during crop plant growth seasons.<br />
Elucidation of the interaction between drought and UV-B<br />
stresses and their effects on plant growth and development<br />
would help in understanding the mechanism<br />
responsible for plant adaptation to changing environmental<br />
conditions and developing agronomic systems for<br />
crop productions. Although the responses of plants to<br />
UV-B or drought have been intensively investigated,<br />
evidence of study in interaction between UV-B exposure<br />
and drought stress in plants had just emerged in the<br />
latest years. The mechanisms of sensitivity or tolerance<br />
of crop plants, either in growth and yield, to combined<br />
stresses remain unknown. Particularly, genotypic effects<br />
on plant responses to the two-stress combination are still<br />
unclear.<br />
Wheat (Triticum aestivum L.), as one of the crucially<br />
important crops in the global food supply, is mainly<br />
distributed in the mid-latitude regions in the northern<br />
hemisphere (Lantican et al., 2005) where UV-B radiation<br />
and drought naturally occur simultaneously or subsequently<br />
during wheat growth seasons. Therefore, the<br />
purpose of this study was to investigate and compare<br />
their effect and interaction on some biochemical stress<br />
markers and stress defense enzyme systems in seedlings<br />
of winter wheat. We also evaluated the genotypic<br />
differences in physiological response of wheat to UV-B<br />
radiation and drought combined stress.<br />
MATERIALS AND METHODS<br />
Drought and UV-B treatments<br />
Wheat (Triticum aestivum L.) varieties, Jinmai 47(drought-resistant)<br />
and Shunmai 1718 (drought-susceptible) were used in this<br />
experiment. After surface sterilization, healthy seeds were sown in<br />
plastic pots (15 x 15 cm) containing vermiculite, and irrigated with ½<br />
strength Hoagland’s solution. Wheat seedlings were grown in a<br />
growth chamber (14/10 h photoperiod; 25/20°C day/night). 7 dayold<br />
wheat seedlings were subjected to drought stress (+D) by 10%<br />
polyethylene glycol (PEG6000), which provoked moderate water<br />
stress (-0·5 MPa), to UV-B radiation (+UV), or to a combination (+D<br />
+ UV), respectively. All stresses were applied throughout 20 days<br />
for both genotypes.<br />
As a source of UV-B radiation, a mercury lamp with a charac-<br />
He et al. 4047<br />
teristic emission in the range of 280 to 320 nm (HPQ 125 W,<br />
Phillips, Eindhoven, The Netherlands) was used. The lamp<br />
irradiation gave a photon flux density of 64·4 μmol m -2 s -1 and the<br />
UV-B irradiation was about 80% of the total light emission. The<br />
seedlings were transferred daily under the HPQ lamp for 2 h in the<br />
middle of the day. The distance between the lamp and plants was<br />
25 cm. The plants without UV-B irradiation treatment were kept<br />
under a polyester film (0.13 mm Mylar Type D) (Du Pont Co,<br />
Newton, CT, USA), which absorbs radiation below 320 nm. For the<br />
treatments with UV-B irradiation, a cellulose acetate filter (0.13 mm)<br />
was used to cut off the radiation below 280 nm, and plants received<br />
49 kJm -2 d -1 biologically effective UV-B radiation.<br />
Measurements of wheat seedling growth parameter<br />
All measurements for seedling growth parameter were conducted at<br />
the end of each stress treatment. Fresh weight of the above-ground<br />
parts was measured, and after that plants were dried at 105°C to<br />
constant weight for the determination of dry weight. Relative leaf<br />
water content (RWC) was calculated according to the equation of<br />
Fletcher et al., (1988). Leaf area was measured according to<br />
Tsonev and Sergiev (1993) using a planar scanner and image plot<br />
software.<br />
Determination of proline, H2O2, chlorophyll and ascorbate<br />
The fresh plant materials collected at various time points were<br />
immediately used for the extraction and assay according to the<br />
appropriate methods listed here.<br />
Free proline was extracted, derivatized with acid ninhydrin, and<br />
absorbance was read according to Bates et al. (1973) method using<br />
L-proline as a standard. Content of proline was expressed as<br />
µmol/g fresh weight. Total chlorophylls were extracted with 80%<br />
acetone and were estimated according to Arnon (1949). Content of<br />
the chlorophylls was expressed as mg/g fresh weight. Hydrogen<br />
peroxide (H2O2) was measured according to Alexieva et al. (2001).<br />
H2O2 was measured spectrophotometrically after reaction with KI.<br />
The reaction mixture consisted 0.5 ml 0.1% trichloroacetic acid<br />
(TCA) leaf extract supernatant, 0.5 ml of 100 mM K-phosphate<br />
buffer and 2 ml reagent (1 M KI w/v in fresh double-distilled water<br />
H2O). The blank probe consisted of 0.1% TCA in the absence of<br />
leaf extract. The reaction was developed for 1 h in darkness, and<br />
absorbance was measured at 390 nm. The amount (µmol/g fresh<br />
weight) of hydrogen peroxide was determined using a given H2O2<br />
standard curve.<br />
Ascorbate content was determined according to the method<br />
described by Foyer et al. (1983). Plant materials (0.5 g) were<br />
ground in liquid nitrogen and then 2 mL of 2.5 M perchloric acid was<br />
added. The crude extract was centrifuged at 4°C for 15 min at<br />
15,000 g, and the supernatant was neutralized with saturated<br />
K2CO3 using methyl orange as an indicator. Insoluble KClO4 was<br />
removed by centrifugation and aliquots of the supernatant were<br />
used for measuring ascorbate and dehydroascorbic acid (DHA)<br />
contents. The reduced ascorbate (ascorbic acid, AsA) was<br />
determined spectrophotometrically at 265 nm in 0.1 M NaH2PO4<br />
buffer (pH 5.6), with 0.1 units of ascorbate oxidase. The total<br />
ascorbate was determined after incubation in the presence of<br />
30 mM DTT. The standard curve was prepared with AsA. DHA level<br />
was obtained as the difference between AsA and total ascorbate.<br />
The content was calculated as µmol/g fresh weight.<br />
Enzyme extraction and assay<br />
For enzymatic activities, assays were carried out using the crude<br />
extract of the leaves as the enzyme source. Leaves were
4048 Afr. J. Biotechnol.<br />
Table 1. Effect of drought and UV-B applied alone or in combination on growth parameters of wheat seedlings.<br />
Parameter Control UV-B (+U) Drought (+D)<br />
UV-Bdrought<br />
(preU+D)<br />
Drought-<br />
UV-B<br />
(preD+U)<br />
Drought-<br />
UV-B<br />
(U+D)<br />
Resistant genotype<br />
Plant height (cm) 16.6±1.5 14.3±0.9** 16.1±1.3 15.5±1.2 * 15.1±0.8* 15.9±1.4*<br />
Fresh weight (mg) 102.2±3.5 89.1±5.2 ** 99.1±3.4 96.3±3.9* 93.4±4.5* 97.2±3.6*<br />
Dry weight (mg) 17.1±1.4 14.8±0.9** 16.6±0.5 16.1±0.6* 15.6±0.8* 16.5±0.5*<br />
Leaf area (cm 2 ) 6.94±0.53 5.98±0.31** 6.59±0.48 6.46±0.65 6.26±0.40* 6.50±0.37<br />
RWC (%) 95.08±1.85 93.70±2.03 74.25±3.46** 85.70±1.57* 78.69±2.82* 84.03±2.91*<br />
Chlorophyll content (mg/g fresh weight) 2.49±0.09 2.02±0.13** 2.40±0.10 2.20±0.11 2.43±0.15 2.11±0.15*<br />
Susceptible genotype<br />
Plant height (cm) 17.9±1.3 14.8±1.1** 17.6±1.1 16.3±1.3* 15.6±1.0** 13.6±1.2**<br />
Fresh weight (mg) 113.4±3.9 93.4±5.7** 109.6±3.5* 100.2±4.4 * 98.5±4.9** 87.3±4.2**<br />
Dry weight (mg) 18.3±1.5 15.2±1.1** 17.6±0.5* 16.4±0.7* 15.8±0.8** 14.6±0.6**<br />
Leaf area (cm 2 ) 7.28±0.46 5.24±0.12** 5.79±0.28* 5.68±0.39 5.44±0.21** 5.03±0.10**<br />
RWC (%) 96.04±1.76 94.09±2.12 70.10±3.77** 80.30±1.62* 73.88±3.11** 78.20±2.89*<br />
Chlorophyll content (mg/g fresh weight) 3.07±0.21 2.69±0.20* 2.97±0.19 2.74±0.26 2.99±0.24 2.82±0.23<br />
Values in brackets are percentage of control. The data are mean±SE (n=20). * and **, indicate significant difference between control and UV-B<br />
radiation, Drought or their combined stress at P
Hundred percent of control(%)<br />
300<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0 2 4 6 8 10 2 4 6 8 10<br />
Time of treatment (d)<br />
Figure 1. The level of H2O2 in wheat seedlings under the successive stress (pred+u, preu+d) of UV-B<br />
and drought. 100 percent (%) corresponds to the H2O2 level in the control plants without any stress.<br />
Samples were collected at 0 d, 2 d, 4 d, 6 d, 8 d and 10 d after the first stress (preD or preU) and then<br />
the second stress (+U or +D), respectively. R1, drought-resistant genotype treated by preD+U<br />
stresses; S1, drought-susceptible genotype treated by preD+U stresses; R2, drought-resistant<br />
genotype treated by preU+D stresses; S2, drought-susceptible genotype treated by preU+D stresses.<br />
Resistant genotype (R) and susceptible genotype (S) were indicated. Vertical bars are standard<br />
errors (S.E.) of means.<br />
reduction of chlorophyll content were considerable in<br />
comparison with the control in Jinmai 47 (drought-tolerant<br />
genotype). These data indicated that the stress<br />
pretreatment induced plant defense response which<br />
subsequently alleviated the negative effects of the other<br />
stress. Moreover, plant responses to drought, UV-B and<br />
the combined stress were genotype-dependent.<br />
Time course of H2O2 content in wheat seedlings<br />
under the stresses<br />
H2O2 concentrations were maintained around 1.1 to 1.3<br />
µmol g −1 FW in the control leaves at the beginning and<br />
end of the experiment. However, the content of H2O2<br />
increased significantly under stressful conditions following<br />
seedling growth (Figure 1 and 2) compared to the<br />
control plants without any stress. Moreover, the peak<br />
value and time course of H2O2 level were different for<br />
different stresses and genotypes.<br />
In the pretreatments, UV-B induced H2O2 up to its peak<br />
level at days 2 and 4 for Jinmai 47 and Shunmai 1718,<br />
respectively; two days earlier than drought stress. The<br />
maximum amount of H2O2 under UV-B was 31 and 47%<br />
higher than that under drought stress for the two<br />
genotypes, respectively. After the peak time, H2O2<br />
R1<br />
S1<br />
R2<br />
S2<br />
He et al. 4049<br />
content declined till the end (day 10) of the pretreatments,<br />
at which H2O2 level was higher in the UV-B-stressed<br />
plants than the drought-stressed plants for both varieties.<br />
In the following second treatments, H2O2 was induced to<br />
elevate again, and then reduced. The enhancement in<br />
the drought-pretreated seedlings by UV-B was greater<br />
than the UV-B-pretreated seedlings by drought. At the<br />
end of the second stress, all the plants showed a lower<br />
H2O2 level (P>0.01) than at the end of the pretreatments.<br />
Furthermore, H2O2 level in the tolerant genotype was<br />
significantly lower (P>0.01) than in the susceptible one.<br />
Particularly, the pre-UV-B and following drought<br />
treatments led to lower H2O2 compared to the pre-drought<br />
and consequent UV-B stresses for both varieties.<br />
Under the combined stress of UV-B and drought<br />
applied simultaneously (Figure 2), H2O2 level quickly increased<br />
to its maximum and then reduced through to the<br />
end of the treatments. Comparison between the two<br />
genotypes showed that the peak level and time were<br />
37% lower and were 2 days earlier in the tolerant<br />
genotype than in the susceptible one. At the end of the<br />
combined stress, H2O2 level was 62% higher in the<br />
susceptible variety than in the tolerant variety. Overall,<br />
UV-B radiation caused more effects on H2O2 than the<br />
drought treatment. Pretreatment of either stress reduced<br />
the impact of subsequent application of the other stress.
4050 Afr. J. Biotechnol.<br />
Hundred percent of control(%)<br />
300<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
R<br />
S<br />
0 2 4 6 8 10<br />
Time of treatment (d)<br />
Figure 2. The level of H2O2 in wheat seedlings under the combined stress (U+D) of UV-B and drought.<br />
100% corresponds to the H2O2 level in the control plants without any stress. Samples were collected at<br />
0, 2, 4, 6, 8 and 10 days after the combined stress. R,Resistant genotype; S, Susceptible genotype.<br />
Vertical bars are standard errors (S.E.) of means.<br />
For susceptible genotype, the combined stress produced<br />
more H2O2 than the drought or UV-B treatment alone,<br />
while the H2O2 level induced by the combined stress was<br />
higher than by the drought and lower than by UV-B stress<br />
in the tolerant variety.<br />
Temporary expression of antioxidative enzymatic<br />
activity induced by the stresses<br />
The activity of SOD and CAT experienced a slight<br />
change in the control plants during the 20 days of growth,<br />
and this variation was not significant. However, the three<br />
enzymes displayed distinct patterns of activity expression<br />
under the stressful conditions.<br />
Following the pretreatment of drought (preD) and<br />
subsequent UV-B stress (+U), SOD activity (Figure 3A)<br />
increased to its first peak on day 2 and day 4 post preD in<br />
Jinmai 47 and Shunmai 1718, respectively. The second<br />
higher peak occurred on day 2 post +U for both genotypes.<br />
After the second peak, SOD level slowly increased<br />
till the end of the treatment. However, only one peak of<br />
SOD activity was induced on day 2 and day 4 post<br />
pretreatment of UV-B (preU) in Jinmai 47 and Shunmai<br />
1718, respectively. Unlike in the preD+U treatments, the<br />
subsequent drought stress (+D) did not significantly<br />
enhance the additional SOD activity to the end of the<br />
preU+D treatments. As shown in Figure 1A, SOD levels<br />
at the peak and the end of the preU+D treatments were<br />
both higher in the susceptible variety ( Shunmai, 1718)<br />
than in the tolerant one (Jinmai 47).<br />
CAT activity (Figure 3B) was also changed greatly<br />
following the treatments. The first peak of CAT activity<br />
occurred at day 2 or day 4 after the pretreatment of either<br />
UV-B or drought. The subsequent stress of the other induced<br />
more enhancement of CAT activity and resulted in<br />
the second peak at different time for different stresses<br />
and genotypes. In contrast to SOD, the increased CAT<br />
level was much higher in the tolerant variety than in the<br />
susceptible one, particularly at the peak time and the end<br />
of the treatments. Moreover, CAT activity post the peak<br />
was reduced rapidly in the susceptible variety while the<br />
activity kept stable or just slightly declined in the tolerant<br />
genotype. Again, UV-B radiation exhibited more effects<br />
on CAT activity than drought stress.<br />
The activity expression of SOD and CAT under the<br />
combine stresses (Figure 4) was mostly like the pattern<br />
under UV-B radiation alone. For tolerant genotype, there<br />
were a higher level of CAT and a lower level of SOD in<br />
the double-stress treatments.<br />
Conversely, higher SOD and lower CAT were detected<br />
in the double stresses for the susceptible variety.
SOD activity<br />
Units/ mg protein<br />
Catalase activity<br />
(nmol H2O2 /mg protein min)<br />
45<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
70<br />
65<br />
60<br />
55<br />
50<br />
45<br />
40<br />
35<br />
30<br />
25<br />
A<br />
0 2 4 6 8 10 2 4 6 8 10<br />
B<br />
Time of treatment(d)<br />
0 2 4 6 8 10 2 4 6 8 10<br />
Time of treatment (d)<br />
Figure 3. Changes in activities of antioxidant enzymes under the successive stresses (preD+U,<br />
preU+D) of UV-B and drought. Samples were collected at 0, 2, 4, 6, 8 and 10 days after the first<br />
stress (preD or preU) and then the second stress (+U or +D), respectively. R1, drought-resistant<br />
genotype treated by preD+U stresses; S1, drought-susceptible genotype treated by preD+U<br />
stresses; R2, drought-resistant genotype treated by preU+D stresses. S2: drought-susceptible<br />
genotype treated by preU+D stresses. Resistant genotype (R) and susceptible genotype (S)<br />
were indicated. Vertical bars are standard errors (S.E.) of means.<br />
Changes in contents of antioxidative metabolites<br />
under the stresses<br />
Free proline in the drought-pretreated plants were observed<br />
to increase by 1.16 and 0.5 folds greater than that<br />
R1<br />
S1<br />
R2<br />
S2<br />
R1<br />
S1<br />
R2<br />
S2<br />
He et al. 4051<br />
in the control at the end of the pretreatment for tolerant<br />
and susceptible genotype, respectively (Figure 5). However,<br />
pretreatment of UV-B just enhanced proline by a<br />
smaller amount, and there was no significant difference<br />
between the tolerant and susceptible genotypes. Sub-
4052 Afr. J. Biotechnol.<br />
SOD activity<br />
(Units/ mg protein)<br />
Catalase activity<br />
(nmol H2O2 /mg protein min)<br />
45<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
A<br />
0 2 4 6 8 10<br />
B<br />
Time of treatment (d)<br />
0 2 4 6 8 10<br />
Time of treatment (d)<br />
Figure 4. Changes in activities of antioxidant enzymes under the combined stress of UV-B<br />
and drought. Samples were collected at 0, 2, 4, 6, 8 and 10 days after the combined stress.<br />
R3, drought-resistant genotype treated by the combined stresses; S3, drought-susceptible<br />
genotype treated by the combine stresses. Vertical bars are standard errors (S.E.) of means.<br />
sequent UV-B stress only induced proline level which<br />
additionally accumulated a little in the drought-pretreated<br />
plants, while subsequent drought stress resulted in a<br />
larger enhancement of proline content in the UV-Bpretreated<br />
plants. The combination of both stresses led to<br />
a slightly lower proline content in comparison with the<br />
drought application alone, and this depressed effect on<br />
proline content was greater in the susceptible genotype<br />
than in the tolerant one (Figure 5). The data showed that<br />
drought stress had a much stronger impact than UV-B<br />
R3<br />
S3<br />
R3<br />
S3<br />
stress in relation to proline accumulation regardless<br />
applied together, separately, and successively.<br />
All stresses increased ascorbic acid (AsA) concentration,<br />
and did not cause much change in dehydroascorbic<br />
acid (DHA) content compared to the control<br />
(data not shown). As a result, a higher ASA/DHA ratio<br />
was observed following the treatments (Figure 6). No<br />
significant difference was detected between the two<br />
genotypes. The percent increment of ASA/DHA ratio was<br />
lower in the pretreatment of either UV-B (36%) or drought
AsA/DHA<br />
Proline (µmol/g FW)<br />
10<br />
9<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
R S<br />
control D preD+U U preU+D U+D<br />
Treatment<br />
Figure 5. Free proline content in winter wheat seedlings under UV-B, drought, and the<br />
combined stresses. Samples were collected at the end of each treatment. D, Drought treatment<br />
alone; U, UV-B treatment alone; U+D, UV-B and drought treatments applied simultaneously;<br />
preD+U, drought treatment applied firstly and then UV-B treatment was applied; preU+D, UV-B<br />
treatment was applied firstly and then drought treatment was applied. Values are means of six<br />
replicates and standard errors (S.E.).<br />
4.5<br />
4<br />
3.5<br />
3<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
0<br />
R S<br />
control D preD+U U preU+D U+D<br />
Treatment<br />
Figure 6. The reduced-to-oxidized ascorbate ratio (AsA/DHA) in winter wheat seedlings under UV-B,<br />
drought, and the combined stresses. Samples were collected at the end of each treatment. D, Drought<br />
treatment alone; U, UV-B treatment alone; U+D, UV-B and drought treatments were applied simultaneously;<br />
preD+U, Drought treatment was applied firstly and then UV-B treatment was applied; preU+D, UV-B<br />
treatment was applied firstly and then drought treatment was applied. Values are means of six replicates<br />
and standard errors (S.E.).<br />
He et al. 4053
4054 Afr. J. Biotechnol.<br />
(38%) than in the subsequent application of drought (81%)<br />
or UV-B (77%) stress. This enhancement was more pronounced<br />
under the combined stresses (Figure 6) so that<br />
ASA/DHA ratio at the end of the treatment was higher in<br />
the plants (3.82) by the combined stresses than those<br />
when UV-B or drought stress was applied alone (2.33<br />
and 2.36) and successively (3.16 and 3.14), indicating<br />
that the combined stress exceeded the effect of a single<br />
stress factor on ASA concentration.<br />
DISCUSSION<br />
With increasing depletion of stratospheric ozone and acelerating<br />
global warming, UV-B irradiation and drought<br />
stresses are becoming much more detrimental to<br />
ecological systems and crop production in the world. The<br />
mechanisms of crop plants to the combined stress of<br />
these two factors remain unknown although the<br />
interaction between UV-B and drought stress in plants<br />
has emerged these years. Therefore, the present study<br />
was conducted to investigate physiological responses of<br />
different genotypes of winter wheat to the combined<br />
stresses of UV-B and drought applied simultaneously and<br />
successively.<br />
Our data (Table 1) of wheat growth parameters measured<br />
under the stresses tested displayed that pretreatment of<br />
either UV-B or drought reduced the damage caused by<br />
subsequent application of the other stress, conforming<br />
the viewpoint that exposure of plants to a moderate<br />
stress can induce a tolerance to a more severe stress<br />
and such treatment also can improve tolerance to other<br />
stresses (Wang et al., 2003). Moreover, the present study<br />
showed that the tolerant variety exhibited better performance<br />
than the susceptible one in this aspect, and the<br />
UV-B-induced wheat plant protection against drought was<br />
stronger than the drought-induced tolerance to UV-B<br />
radiation. This result indicated that the expression of the<br />
so-called cross-acclimation to stresses was related to the<br />
plant genotypes and stress factors.<br />
In comparison to the inhibited effects on plant growth of<br />
UV-B or drought stress alone, the effect of the combination<br />
of the two stress factors could be additive (more<br />
damage) (Tian and Lei, 2007) or antagonistic (reduced<br />
damage) (Sullivan and Teramura, 1990; Alexieva et al.,<br />
2001). Our data further indicated that this combined<br />
effect was genotype-dependent. For the tolerant variety,<br />
the combined stress of UV-B and drought resulted in the<br />
moderated injury to wheat seedlings, which was less than<br />
the injury caused by the stresses applied individually.<br />
However, the combined stress caused more severe<br />
damage to wheat seedlings than stress factors applied<br />
separately for the susceptible variety. Thus, the combined<br />
application of drought and UV-B had more strong<br />
adverse effects on wheat seedlings of the susceptible<br />
genotype, but more positive effects on the tolerant wheat<br />
genotype.<br />
UV-B and drought stresses seemed to act in two different<br />
ways, as it could be expected, drought signifi-cantly<br />
influenced the RWC, and UV-B largely affected<br />
chlorophyll of the treated leaves (Table 1). This finding<br />
further support that chlorophyll content and RWC were<br />
proposed as a typical maker for plant response to UV-B<br />
and drought stresses, respectively (Teramura et al., 1991;<br />
Correia et al., 1999; Santos et al. 2004). When UV-B<br />
irradiation and drought stresses were applied simultaneously<br />
or successively, the specific effects of UV-B on<br />
chlorophyll and drought on RWC were slightly reduced<br />
compared to the single stress, again indicating that<br />
interaction between UV-B and drought alleviated, to<br />
some extent, the negative effect of UV-B or drought alone.<br />
Generation of reactive oxygen species (ROS) including<br />
H2O2 is recognized as one of the early effects of various<br />
biotic and abiotic stresses on plants (Bowler and Fluhr,<br />
2000), and high accumulation of ROS can result in<br />
oxidative damage in the absence of effective protective<br />
mechanism (Alexieva et al., 2001). In our case, an<br />
increase in H2O2 was observed in all the treated plants<br />
compared to the control. However, the increase was<br />
more evident in the UV-B treated plants than in the<br />
drought-stressed plants (Figure 1), and thus UV-B<br />
caused a more severe damage than the drought stress<br />
on wheat seedlings measured as there was more obvious<br />
reduction in growth (Table 1). Comparatively, the interaction<br />
of UV-B and drought led to less damage on wheat<br />
seedlings, which was evidenced by a lower level of the<br />
peak value of H2O2 in the plants measured at the end of<br />
the combined stresses applied simultaneously or successively<br />
(Figure 2).<br />
On the other hand, H2O2 received much attention as a<br />
signal molecule in response to different stresses (Prasad<br />
et al., 1994; Gong et al. 2001; Aroca et al., 2003). H2O2<br />
mediated the regulation of transcription in response to<br />
UV-B exposure as an important early upstream signal<br />
(Brosche and Strid, 2003). Moreover, H2O2 was also<br />
implicated in the gene expression related to cold<br />
acclimation to resist freezing stress (Foyer et al. 1997).<br />
Activation of endogenous protective mechanisms can in<br />
turn tolerate or delete excess ROS burst. We found that<br />
the enhanced H2O2 level under the stresses was followed<br />
by the up-regulation of the enzyme activities (Figure 3).<br />
This suggests that H2O2 may act more as a signal<br />
molecule than directly inducing oxidative damage. In<br />
other words, the increased H2O2 concentration by UV-B<br />
or drought may trigger the cross-acclimation to tolerant<br />
the subsequent stresses in winter wheat through stimulation<br />
of the antioxidant defense systems.<br />
SOD, the first antioxidant enzyme for scavenging ROS,<br />
catalyzes the dismutation of superoxide into oxygen and<br />
H2O2, while CAT reacts with H2O2 to produce water and<br />
oxygen. In this study, it was observed that activities of the<br />
two key antioxidant enzymes increased following all the<br />
treatments compared to the control plants, but their<br />
expression patterns were different for different genotypes
and stress conditions. For example, under the successive<br />
stress conditions of first UV-B radiation and then drought,<br />
SOD expression was “rapid up to the peak and then kept<br />
stable” in both genotypes, whereas the peak level was<br />
higher and occurred two days later in the susceptible<br />
genotype than in the tolerant one (Figure 3A). Consequently,<br />
SOD level was greater in the susceptible<br />
genotype than in the tolerant one at the end of the<br />
treatment. CAT expression pattern, however, was “rapid<br />
up to peak, sustaining roughly stable, and then increasing<br />
again” for the tolerant genotype or “decreasing” for the<br />
susceptible one (Figure 3B). As a result, the CAT level<br />
was higher in tolerant genotype than in the susceptible<br />
one at the end of the treatments. Similarly, SOD and CAT<br />
activities also increased following the successive<br />
stresses of first drought and then UV-B except that the<br />
enhancement rate was lower in the first stress than in the<br />
second stress. Therefore, different levels of H2O2 under<br />
all stresses, particularly higher H2O2 content in the susceptible<br />
genotype than in the other may be due to the<br />
differential expressions of activities of those enzymes<br />
following the stress. Our inference is in agreement with<br />
the report that cellular H2O2 concentration is the result of<br />
the balance between its production and utilization (Bowler<br />
et al., 1992).<br />
Under the combined stresses of UV-B and drought<br />
applied simultaneously, the two enzyme activities (Figure<br />
4) were expressed like in the case under UV-B stress<br />
alone, but SOD activity was lower and CAT was higher<br />
than that under drought and UV-B stress alone for the<br />
tolerant variety. Again, our data of the two enzyme<br />
activities indicated that the interaction of UV-B and<br />
drought stresses alleviated the negative effect of the<br />
stress factors on the tolerant genotype, but the combined<br />
stress led to more damage on the susceptible one.<br />
Notably, many contradictory results about antioxidant<br />
enzyme response to different stresses have emerged due<br />
to the fact that the levels of enzyme responses depend<br />
on the plant species, the developmental stage, the<br />
organs, as well as on the duration and severity of the<br />
stress (Wilson et al., 1993; Caldwell et al., 1983;<br />
Chappell et al., 1994). In addition, the presence of<br />
different enzyme isoforms expressed to a different extent<br />
in different seedling growth conditions (stressed and unstressed),<br />
and with different substrate affinity could be<br />
not excluded.<br />
In addition to the activation of antioxidant enzymes, the<br />
enhancement of antioxidant metabolites such as proline<br />
and ascorbate acid (AsA) in cells is also another defense<br />
mechanism against the ROS burst caused by various<br />
stresses (Monk et al., 1989). In many plants, free proline<br />
accumulates in response to biotic and abiotic stresses,<br />
including water stress (Day et al. 1993), extreme temperatures<br />
(Strid et al., 1992), heavy metal toxicity (Long<br />
et al., 1983), and UV-B irradiation (Schreiber et al., 1986).<br />
Our data showed that drought stress had a much<br />
stronger impact than UV-B stress in relation to proline<br />
He et al. 4055<br />
accumulation regardless applied together, separately,<br />
and successively (Figure 5), confirming that proline is a<br />
typical marker for osmotic stress, which is well-described<br />
in water and salt stresses (Heuer, 1994; Fedina et al.,<br />
2006). However, proline may exert a protective action on<br />
UV-B stress because drought-treated wheat seedlings<br />
accumulated higher level of proline, and appeared less<br />
damaged by UV-B radiation compared to the control<br />
plants (Table1). Consistence with our results, a positive<br />
effect of proline accumulation on the reduction of the UV-<br />
B induced damage is proposed in other reports<br />
(Kurkdjian and Guern, 1989; Alexieva et al., 2001).<br />
Ascorbate is a major primary antioxidant, reacting<br />
directly with hydroxyl radicals, superoxide and singlet<br />
oxygen, and is also a powerful secondary antioxidant, reducing<br />
the oxidized form of a-tocopherol (Agrawal and<br />
Rathore, 2007). In our study, foliar ascorbic acid<br />
concentration was found to increase in all the stressful<br />
conditions. Moreover, the rate of increase was lower in<br />
the pretreatments of ether UV-B or drought than in the<br />
subsequent stresses (Figure 6), suggesting that<br />
enhancement of ASA content was not a rapid response<br />
to the single stress of UV-B or drought. However, under<br />
the combined stress of the two factors applied together,<br />
ASA increase was faster and much higher (Figure 6),<br />
indicating that the interaction between UV-B and drought<br />
stresses generated a protective effect on plants. Unlike<br />
the other parameters measured here, the time course<br />
ASA level induced by the stress showed no significant<br />
difference between the two genotypes.<br />
In conclusion, pretreatment of either UV-B or drought<br />
can increase wheat seedling tolerance to the subsequent<br />
stress, and moreover, UV-B induced tolerance against<br />
drought stress was stronger than the drought-induced<br />
defense to UV-B stress evaluated as changes in plant<br />
growth parameters and in amounts of stress markers.<br />
The combined application of drought and UV-B together<br />
brought out strong adverse effects on wheat seedlings of<br />
susceptible variety, but more positive effects on the<br />
tolerant wheat genotype. By scavenging excessive ROS,<br />
antioxidant enzymes and compounds functioned importantly<br />
in the plant responses to UV-B radiation, drought,<br />
and their combined stresses. The expression patterns of<br />
the enzymes and temporary changes of the antioxidants<br />
tested following the treatments displayed differences to<br />
some extent for different stress conditions and genotypes,<br />
leading us to infer that the expression of plant defense<br />
strategy to stress was differently regulated by a<br />
complicate system consisting of plant genetic background,<br />
physiological status, stress factors and their interactions<br />
although the presence of a basic common response in<br />
both genotypes under the stress conditions was observed.<br />
Acknowledgements<br />
This research was supported by the Earmarked Fund for
4056 Afr. J. Biotechnol.<br />
Modern Agro-industry Technology Research System of<br />
China, supported by Shanxi Province Foundation for<br />
Returness, China.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4057-4064, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2012<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
The effects of the regulated deficit irrigation on yield<br />
and some yield components of common bean<br />
(Phaseolus vulgaris L.) under semi-arid conditions<br />
Mehmet Simsek 1 *, Nuray Comlekcioglu 2 and Irfan Ozturk 3<br />
1 Department of Agricultural Structures and Irrigation, Faculty of Agriculture,Harran University, Sanliurfa, Turkey.<br />
2 Department of Horticulture, Faculty of Agriculture, Harran University, Şanliurfa Sanliurfa, Turkey.<br />
3 Department of Biometry Science, Faculty of Agriculture, Harran University, Sanliurfa, Turkey.<br />
Accepted 28 March, 2011<br />
During three consecutive years (2002 to 2004), common bean was grown to investigate the effects of<br />
the regulated deficit irrigation (RDI) on the yield and yield parameters under semi-arid conditions. Field<br />
experiments were conducted on a clay soil. The growing season of common bean was divided into two<br />
phases: (1) Vegetative stage (V); from seed germination to the beginning of flowering and (2)<br />
reproductive stage (R); from the flowering to the last fruit harvesting. The statistical design was a splitplot<br />
with three replications, where the growth stage was the main factor of variation and the irrigation<br />
was the secondary factor. The irrigation treatments consisted of all possible combinations of full<br />
irrigation (T1 and T5 (V100 and R100: 100% of irrigation water (IW)/cumulative pan evaporation (CPE)) or<br />
limited irrigation (T2: V75-R100, T3: V50-R100, T4: V25-R100, T6: V100-R75, T7: V100-R50 and T8: V100-R25) in two<br />
phases. Fresh bean yield and pod weight (PWt), pod length (PL), pod width (PWh) and number of seed<br />
per pod (NSPP) were measured. Yields of T2, T3, T4, T6, T7 and T8 were determined as 27.0, 35.0, 41.0, 4.0,<br />
12.0 and 21.0% lower than the yields obtained from the control (18.36 and 18.40 t ha -1 ) treatments,<br />
respectively. Results demonstrated that, vegetative stage was the more sensitive than the reproductive<br />
stage to the deficit irrigation. The highest irrigation water use efficiency (IWUE) and water use efficiency<br />
(WUE) were found in T6 and T7 treatments as 2.58 and 2.66 kg m -3 , respectively.<br />
Key words: Phaseolus vulgaris L., class A pan, drip irrigation, deficit irrigation.<br />
INTRODUCTION<br />
Common bean is being consumed as a source of<br />
proteins, calories, fibers and minerals in the developing<br />
countries (Ramos et al., 1999; Singh et al., 1999). Also,<br />
since it has an ability to bind atmospheric nitrogen, it<br />
plays a significant role for the crop rotation and sustainable<br />
cropping systems (Donald and Paulsen, 1997).<br />
Common bean is planted on an area of 0.98 million ha<br />
and has a production of 6.82 million t as fresh in the<br />
world. Turkey is ranked second to China with a production<br />
amount of 0.56 million t and a planted area of 0.07<br />
*Corresponding author. E-mail: mehmetsimsek@harran.edu.tr,<br />
simsek0154@gmail.com. Tel: +90 414 3183756. Fax: +90 414<br />
3183682.<br />
Million ha (FAO, 2008). Besides benefits of bean to the<br />
human diet and environment, its production requires a<br />
significant amount of water due to its relatively shallow<br />
root system. Thus, the amount of irrigation applied at the<br />
developmental (vegetative and reproductive) stages,<br />
affect the bean production in that plant growth and yield<br />
are often reduced by periods of water stress (Ramos et<br />
al., 1999).<br />
Efficient use of water in any irrigation systems is vital<br />
especially in arid and semi-arid regions. Drought occurs<br />
in many parts of the world in every year; often with<br />
devastating effects on crop production (Ludlow and<br />
Muchow, 1990; Tonkaz, 2006). As the water supply is<br />
limited worldwide, there is a need of water saving in<br />
irrigation and thus, deficit irrigation should be applied.<br />
Drip irrigation, reduces deep percolation and evaporation
4058 Afr. J. Biotechnol.<br />
and controls water status of the soil more precisely within<br />
the crop root zone (Singandhupe et al., 2003). One<br />
choice for improving irrigation water management is to<br />
alter from furrow or sprinkler irrigation to drip irrigation.<br />
Several alternative irrigation methods have been used<br />
such as surface, drip and sprinkler irrigation systems for<br />
the production of common bean. However, recently using<br />
the drip irrigation has attracted a significant amount of<br />
attention of researchers and farmers instead of using<br />
surface and sprinkler irrigations either to increase the<br />
efficient use of water or the yield (Hanson and May,<br />
2004). Moreover, drip irrigation reduces deep percolation<br />
and evaporation and controls water status of the soil<br />
more precisely within the crop root zone (Singandhupe et<br />
al., 2003). Numerous studies were carried out in the past<br />
for the development and evaluation of irrigation scheduling<br />
techniques under a wide range of irrigation<br />
systems and management techniques, soil, climate and<br />
crop conditions (Kang et al., 2000; Boutraa and Sanders,<br />
2001; Wakrim et al., 2005; Zhang et al., 2006.)<br />
The irrigation scheduling techniques is very critical to<br />
use the drip irrigation most efficiently because while<br />
excessive amount of irrigation causes a decrease in the<br />
yield, inadequate irrigation results in a water stress and a<br />
reduction in production (Sezen et al., 2005; Onder et al.,<br />
2006). Wakrim et al. (2005) reported that, even though<br />
irrigation increases the overall plant growth rate, excess<br />
water might bring about the poor grain yields in common<br />
bean. Also, this statement was in agreement with several<br />
studies which showed that, implementations of limited<br />
and excessive irrigation for common bean result in lower<br />
yield as they create stress during plant development<br />
(Nielsen and Nelson, 1998; Dapaah et al., 1999; Sezen<br />
et al., 2005). The deficit irrigation techniques, including<br />
regulated deficit irrigation (RDI), have been developed to<br />
control excessive vegetative growth or water saving. RDI<br />
is widely used in the horticultural industry since it results<br />
in the more efficient use of irrigation water and often<br />
improves the crop quality (Turner, 2001).<br />
The meteorological-based irrigation scheduling approach,<br />
such as implementations of pan evaporation replenishment,<br />
cumulative pan evaporation (CPE) and ratio between<br />
irrigation water and CPE was employed by many<br />
researchers (Saudan et al., 2000; Ferreia and Carr 2002;<br />
Simsek et al., 2005). This was due to its simplicity, data<br />
availability and high degree of adaptability to the farmer<br />
level (Imtiyaz et al., 2000 and Simsek et al., 2005).<br />
The objective of this study was to determine the effects<br />
of RDI on the yield and yield components of the fresh<br />
common bean in the semi-arid conditions.<br />
MATERIALS AND METHODS<br />
Experimental site and climatic data<br />
This study was conducted on a clay soil at the Agricultural<br />
Experimental Research Field of the Harran University during the<br />
growth periods of 2002, 2003 and 2004. The site is situated on the<br />
37°08' N and 38°46' E and 464 m above sea level. The soil water<br />
contents (w/w; %) of the soil at field capacity were 33.1, 33.2 and<br />
33.7% and at permanent wilting point 21.6, 21.9 and 22.8%, in 0 to<br />
30, 30 to 60 and 60 to 90 cm soil depths, respectively. The<br />
corresponding bulk densities were 1.41, 1.45 and 1.44 Mg m 3 . The<br />
study area was located in a semi-arid climate. The soil water<br />
content was measured gravimetrically in each 0.3 m layer down to<br />
0.9 m depth in two replicates per treatment. These measurements<br />
repeated with intervals of 12 days were done six times for each<br />
year. The lowest level of relative humidity and the highest<br />
temperature and solar radiation occur in summer months. The<br />
recorded maximum and the minimum temperatures were 32.7 and<br />
11.7°C in August and November. The highest and lowest values of<br />
relative humidity were measured as 72.2 and 32.2% for the months<br />
of November and September, respectively (Table 1). The highest<br />
and the lowest solar radiations were measured as 24.0 and 8.8 MJ<br />
m -2 day -1 in August and November.<br />
Crop evapotranspirations under varying irrigation regimes was<br />
calculated using the water balance model (Garrity et al., 1982).<br />
ETc=IW+P–D–R±∆S<br />
Where, ETc is the seasonal crop evapotranspiration (mm); IW is the<br />
total irrigation water applied (mm); P is the precipitation (mm); D is<br />
the drainage (mm); R is the run-off (mm) and ∆S is the variation in<br />
water content (mm) of the soil profile. All terms are expressed in<br />
mm of water in the bean root zone. The effective root depth was<br />
taken as 60 cm. Run-off was taken as to be nil since no run-off was<br />
observed in drip irrigation system.<br />
Total amount of irrigation water applied was calculated by using the<br />
following equation (Doorenbos and Pruitt, 1975):<br />
IW=AxEpan<br />
Where, IW is the amount of irrigation water applied (L); A is the plot<br />
area (m 2 ) and Epan is the amount of cumulative evaporation during<br />
an irrigation interval (mm).<br />
Crop management and irrigation treatments<br />
The experiments were conducted in the field for consecutive 3<br />
years. Common bean cv. Gina was examined in this study. The<br />
seeds were sown in August 05, 11 and 08 in 2002, 2003 and 2004,<br />
respectively. Seeds were spaced with 25 cm in a row and 70 cm<br />
between rows, as used in traditional by the growers of this region.<br />
Each plot was 6 m in length and five rows (21 m 2 ). The plants were<br />
fertilized with 120, 100 and 120 kg ha -1 of N, P2O5, and K2O5,<br />
respectively.<br />
The total crop duration was divided into two phenological stages<br />
in order to distinguish different plant growing stages and irrigation<br />
treatments: Vegetative stage (V); from seed germination to<br />
beginning of flowering and reproductive stage (R); from the first<br />
flowering to the end of harvesting (Tables 2 and 3). The case study<br />
involved four drip irrigation treatments applied, in two growth<br />
stages. Full irrigations (T1 and T5: V100-R100 (IW/CPE ratio 100%))<br />
were applied to the control treatments in the V and R stages, while<br />
25, 50 and 75% limitations, were applied to the other plots and<br />
compared to the control. RDI was studied with treatments T2: V75-<br />
R100, T3: V50-R100, T4: V25-R100, T6: V100-R75, T7: V100-R50, and T8:<br />
V100-R25 (Table 2). The CPE values were obtained by the use of<br />
class A pan evaporometer located in the experimental site. Three<br />
day CPE values were used for irrigation.<br />
The water from water tank was delivered to the field by a submain<br />
32 mm diameter polyethylene (PE), 16 mm diameter laterals<br />
with in-line emitters located 0.4 m apart, surface drip laterals
Table 1. Some climatic data during the experiment.<br />
2002<br />
2003<br />
2004<br />
Long-run data<br />
(1929 to 2004)<br />
Year/month Ta(°C) RH(%) E0(mm d -1 ) Rs(MJ m -2 day -1 ) u2(m s -1 )<br />
August 30.5 43.7 12.2 24.0 3.0<br />
September 26.9 47.8 9.5 19,8 2.4<br />
October 21.8 48.6 4.7 14,2 2.5<br />
November 14.4 62.4 3.2 10.6 1.9<br />
August 32.7 32.2 10.9 22.8 2.6<br />
September 26.4 42.4 9.3 19.7 2.8<br />
October 21.5 51.5 4.0 13.9 1.7<br />
November 12.7 62 2.8 9.8 1.5<br />
August 30.8 40.7 11.8 23.5 2.5<br />
September 27.3 34.8 8.4 19.4 1.9<br />
October 21.7 48.7 4.3 13.1 1.6<br />
November 11.7 72.2 3.0 8.8 1.4<br />
August 31.1 32.1 11.9 21.4 2.7<br />
September 26.6 34.9 9.3 18.2 2.4<br />
October 20.1 44.5 4.5 13.1 1.8<br />
November 12.8 59.0 3.0 8.5 1.6<br />
Simsek et al. 4059<br />
Average of monthly values of air temperature (Ta), relative humidity (RH), evaporation from class “A” pan (ET0), solar radiation<br />
(Rs), and wind speed at 2 m height (u2).<br />
Table 2. Treatments details of irrigation regime in the different<br />
growth stages.<br />
Treatment<br />
Irrigation regime<br />
(CPE ‡ : 100, 75, 50 and 25%)<br />
V R<br />
T1 V100 R100<br />
T2 V75 R100<br />
T3 V50 R100<br />
T4 V25 R100<br />
T5 V100 R100<br />
T6 V100 R75<br />
T7 V100 R50<br />
T8 V100 R25<br />
‡ CPE: Cumulative pan evaporation.<br />
spaced at 0.7 m intervals, one drip lateral each row and operating<br />
at a constant pressure of 152 kPa with 3.0 L h -1 . Irrigation water<br />
was checked by flow water meter in valves.<br />
The harvest was conducted weekly and yield values were<br />
measured for each plot and were calculated for hectare (t ha -1 ). Pod<br />
weight (PWt), pod length (PL) and pod width (PWh) values of the<br />
randomly chosen 50 pods and the values of the number of seed per<br />
pod (NSPP) were determined for each fruit.<br />
Water use efficiency (WUE) and irrigation water use efficiency<br />
(IWUE)<br />
WUE is the ratio between bean yield (t ha -1 ) and seasonal crop<br />
Table 3. Durations of the two growth stages (in days) of<br />
common bean in three years.<br />
Growth stage 2002 2003 2004<br />
V 41 40 42<br />
R 58 58 52<br />
Total duration 99 98 94<br />
evapotranspiration (mm), as can be seen in the equation as follow:<br />
Ya<br />
WUE =<br />
ET<br />
c<br />
If the yield Ya is expressed in kg and the water use ETc is<br />
expressed in m 3 m -2 , then WUE has units of kg m -3 on a unit water<br />
volume basis or g kg -1 when expressed on a unit water mass basis<br />
(Stanhill, 1986; Howell et al., 1990). IWUE is obtained by using the<br />
ratio of the yield per unit IW (mm) (equation).<br />
Ya<br />
IWUE =<br />
IW<br />
Experimental design and statistical analysis<br />
The field experiments were set up with split plot design with three<br />
replicates, where the growth stage was the main factor of variation<br />
and the irrigation was the secondary factor. The data were analyzed
4060 Afr. J. Biotechnol.<br />
using SAS statistical program (SAS Inst 1991). Analysis of variance<br />
(ANOVA) test was conducted and significant differences among<br />
treatments were determined using the TUKEY method. Square root<br />
transformation was performed for number of seed per pod to<br />
analysis of variance.<br />
RESULTS<br />
Effect of RDI on applied water and evapotranspiration<br />
Irrigation treatments were initiated when the soil water<br />
content at the effective root depth decreased to 50% of<br />
the available soil water on August 26 (21 days after<br />
sowing; 21 DAS) in the first, on August 31 (20 DAS) in<br />
the second and on August 30 (22 DAS) in the third year.<br />
The sowing day was considered as 0 day (0 DAS). The<br />
final irrigation treatments were applied November 6,<br />
2002, November 11, 2003 and November 6, 2004. A total<br />
of nineteen irrigations were applied in the first two years,<br />
while eighteen irrigations were applied in the third year.<br />
When the mean of the three years were calculated, the<br />
duration of the vegetative stage was 41 days while the<br />
duration of the reproductive stage was 56 days (Table 3).<br />
Total eight harvests were made in all the experimental<br />
years.<br />
Table 4 shows the values of fresh bean yield, IW and<br />
ETC. The average values of IW and ETc were 721, 672,<br />
623, 575, 721, 682, 642 and 603 mm and 742, 696, 651,<br />
602, 747, 669, 607 and 558 mm for T1, T2, T3, T4, T5, T6,<br />
T7 and T8, respectively. The lowest value was determined<br />
for T8 being applied with 75% water deficit in the reproductive<br />
stage. This was followed by the treatments on<br />
which 50 and 25% water deficit was applied. These<br />
values were higher than the values of IW and ETc<br />
presented in the studies of Onder et al. (2006) and Sezen<br />
et al. (2005). This might be resulted from the decrease in<br />
the relative humidity, the temperature increase and<br />
evaporation ratio increase due to the semi-arid conditions<br />
in this study.<br />
Irrigation water use efficiency and water use<br />
efficiency<br />
IWUE and WUE values were given in Table 4. The<br />
highest IWUE was obtained as 2.58 kg m -3 in T6 whereas<br />
the highest WUE was calculated as 2.66 kg m -3 in T7,<br />
when the mean of the three years was examined. A direct<br />
relation was observed between the depth of the applied<br />
water and evapotranspiration. IWUE and WUE values<br />
were decreased for RDI treatments in the V stage when<br />
the applied and consumed water were decreased. However,<br />
no significant differences in IWUE and WUE values<br />
were observed for the RDI treatments belonging to the R<br />
stage. The lowest mean IWUE and WUE were measured<br />
as 1.88 and 1.80 kg m -3 , respectively on the T4. Wakrim<br />
et al. (2005) determined the WUE values for common<br />
bean between 1.91 and 2.92 kg m -3 which was well<br />
correlated with our data. In addition, values of IWUE and<br />
WUE found by the study of Onder et al. (2006) were in<br />
agreement with our data, in that, values varied from 1.56<br />
to 2.61 kg m -3 for IWUE and from 1.42 to 2.02 kg m -3 for<br />
WUE. The values of IWUE and WUE for the V stage<br />
were determined lower than the ones obtained for the R<br />
stage. The reason was related with the decrease in the<br />
yield in T2, T3 and T4 treatments.<br />
Sezen et al. (2005) reported that, the IWUE and WUE<br />
for Gina variety cultivated by drip irrigation were between<br />
3.80 and 7.29 and 4.14 and 6.16 kg m -3 and the yield was<br />
between 12.20 and 20.60 t ha -1 , respectively. The higher<br />
values of IWUE and WUE had been attributed to the<br />
higher relative humidity, caused by decrease in the water<br />
consumption.<br />
Yield-RDI relationship<br />
As shown in Table 5, the highest mean yield was<br />
observed in control treatment as more than 18 t ha -1 . The<br />
yields of the T2, T3, T4, T6, T7 and T8 treatments were 27,<br />
35, 41, 4, 12 and 21% lower than the yield obtained from<br />
the control treatments, respectively. The lower yield in T2,<br />
T3, and T4, when compared with control treatments, can<br />
be attributed to the use of deficit of water as well as the<br />
high temperature during V stage. The V stage was<br />
defined as the period between the beginning of August<br />
and the middle of September. The mean temperatures<br />
were very high during this period and the relative<br />
humidity was very low, whereas the evaporation ratio was<br />
too high (Table 1). Nielsen and Nelson (1998) reported<br />
that, the water stress lead to the growth of the shortest<br />
plants with the least leaf area during the vegetative<br />
growth stage of black bean. In this study, thin and short<br />
plants and plants with lower number of branches were<br />
observed visually in T2, T3 and T4. Water stress during<br />
the R stage in T6, T7, and T8 can also lead to a significant<br />
decrease in the yield by increasing the number of aborted<br />
flowers and pods per plant. Wakrim et al. (2005) showed<br />
that, the RDI treatments resulted in the mild stress which<br />
affected significantly both plant vegetative and reproductive<br />
growth. Similar decreases in the yield have<br />
already been reported by Singh (1995) in common bean,<br />
Karam et al. (2005) in soybean, Cakir (2004) and Payero<br />
et al.(2006) in corn and Karam et al. (2007) in sunflower.<br />
Application of adequate water during flowering and pod<br />
development is the most significant factor in bean<br />
irrigation. Similar responds were observed in this study<br />
for the common bean that was cultivated in the semi-arid<br />
zone. Similar trend in the yield was seen for the yield<br />
components in all irrigation treatments. Water stress<br />
combined with high temperature during flowering of the<br />
bean brought about a decrease in all yield components.
Table 4. IWUE and WUE for RDI treatments in 2002, 2003 and 2004.<br />
Treatment Fresh bean yield(t ha -1 )<br />
2002<br />
a IW(mm)<br />
Simsek et al. 4061<br />
b ETc(mm) IWUE(kg m -3 ) WUE(kg m -3 )<br />
T1 18.37 786 797 2,34 2,30<br />
T2 13.37 734 751 1,82 1,78<br />
T3 12.17 683 704 1,78 1,73<br />
T4 10.64 631 625 1,69 1,70<br />
T5 18.40 786 811 2,34 2,27<br />
T6 17.64 743 739 2,37 2,39<br />
T7 16.04 700 664 2,29 2,42<br />
T8 14.30 656 608 2,18 2,34<br />
2003<br />
T1 17.77 690 708 2.58 2.51<br />
T2 12.84 643 677 2.00 1.90<br />
T3 11.24 595 622 1.89 1.81<br />
T4 10.57 548 589 1.93 1.79<br />
T5 17.94 690 703 2.60 2.55<br />
T6 17.27 652 623 2.65 2.77<br />
T7 15.80 613 558 2.58 2.83<br />
T8 13.90 575 530 2.42 2.62<br />
2004<br />
T1 18.94 687 721 2.76 2.63<br />
T2 13.97 640 660 2.18 2.12<br />
T3 12.27 593 628 2.07 1.95<br />
T4 11.24 545 591 2.06 1.90<br />
T5 18.80 687 728 2.74 2.58<br />
T6 17.84 651 644 2.74 2.77<br />
T7 16.64 615 600 2.71 2.77<br />
T8 15.40 578 536 2.66 2.87<br />
Average<br />
T1 18.36 721 742 2.55 2.47<br />
T2 13.39 672 696 1.99 1.92<br />
T3 11.89 623 651 1.91 1.83<br />
T4 10.82 575 602 1.88 1.80<br />
T5 18.38 721 747 2.55 2.46<br />
T6 17.58 682 669 2.58 2.63<br />
T7 16.16 642 607 2.52 2.66<br />
T8 14.53 603 558 2.41 2.60<br />
a IW, Irrigation water; b ETc, evapotranspiration.<br />
Yield components<br />
The effects of year, growth stages and irrigation regimes<br />
on considered yield components were significant (P <<br />
0.001), whereas no significant differences were found<br />
between the year for the NSPP and between irrigation<br />
regime for PL (Table 6).<br />
These differences could be attributed to the climate,<br />
since it was considerably cold three weeks before the last<br />
harvest time in the second year. The temperatures<br />
dropped by 10°C, compared with other years. Sudden<br />
climate change caused a drop in the yield and yield<br />
components in 2003. The values obtained in the first and<br />
the third years were similar, whereas the yield for the<br />
second year was lower than the values found in other<br />
years. The interactions between the growth stage and
4062 Afr. J. Biotechnol.<br />
Table 5. The effects of regulated deficit irrigation regimes on common bean yield and its components.<br />
Treatment Year T1 T2 T3 T4 T5 T6 T7 T8<br />
2002 18.37±0.66<br />
Fresh bean yield (t ha-1)<br />
a 13.37±0.20 de 12.17±0.30 ef 10.64±0.34 f 18.40±0.51 a 17.64±0.20 ab 16.04±0.09 bc 14.30±0.21 cd<br />
2003 17.77±0.15 a 12.84±0.32 d 11.24±0.15 e 10.57±0.07 e 17.94±0.26 a 17.27±0.07 a 15.80±0.12 b 13.90±0.06 c<br />
2004 18.94±0.28 a 13.97±0.20 d 12.27±0.45 e 11.24±0.09 e 18.80±0.29 a 17.84±0.20 ab 16.64±0.18 bc 15.40±0.40 cd<br />
Average 18.36±0.27 a 13.39±0.21 e 11.89±0.23 f 10.81±0.15 g 18.38±0.22 a 17.58±0.12 b 16.16±0.14 c 14.53±0.26 d<br />
Pod weight (g)<br />
Pod length (mm)<br />
Pod width (mm)<br />
Number of seed per pod†<br />
2002 9.83±0.30 a 7.97±0.07 bc 7.30±0.17 c 6.90±0.12 c 9.77±0.32 a 9.43±0.09 a 8.87±0.15 ab 8.77±0.24 ab<br />
2003 9.33±0.03 a 6.73±0.22 de 6.17±0.29 e 6.10±0.10 e 9.47±0.12 a 8.70±0.31 ab 8.17±0.13 bc 7.33±0.23 cd<br />
2004 10.10±0.20 a 8.13±0.49 bc 7.40±0.30 c 7.17±0.12 c 9.93±0.26 a 9.70±0.35 a 9.20±0.26 ab 8.97±0.20 ab<br />
Average 9.74±0.15 a 7.61±0.27 d 6.96±0.24 de 6.72±0.17 e 9.72±0.14 a 9.27±0.20 ab 8.74±0.18b c 8.36±0.28 c<br />
2002 115.50±0.98 a 86.37±0.20 de 83.43±0.59 e 77.37±2.09 f 115.10±0.73 a 105.50±0.98 b 98.03±1.18 c 88.57±0.68 d<br />
2003 106.80±1.78 a 84.70±0.75 bc 81.33±1.30 c 75.43±2.75 c 106.80±1.35 a 101.20±2.52 a 91.17±2.03 b 84.67±2.47 bc<br />
2004 118.60±2.11 a 94.57±1.79 bc 90.47±0.29 bc 85.60±0.61 c 117.80±1.68 a 105.10±1.04 ab 104.30±6.81 ab 97.60±1.01 bc<br />
Average 113.63±1.96 a 88.54±1.63 d 85.08±1.44 d 79.47±1.86 e 113.23±1.79 a 103.90±1.07 b 97.84±2.82 c 90.28±2.07 d<br />
2002 13.03±0.58 a 9.80±0.10 b 10.07±0.12 b 9.97±0.23 b 13.47±0.58 a 11.47±0.23 ab 10.80±0.59 b 10.37±0.23 b<br />
2003 11.07±0.69 a 9.80±0.15 ab 9.030±0.07 b 9.30±0.21 ab 11.17±0.48 a 10.30±0.21 ab 9.77±0.52 ab 9.63±0.27 ab<br />
2004 14.33±0.38 a 10.77±0.39 cd 10.27±0.20 d 10.07±0.12 d 14.20±0.32 a 12.70±0.17 ab 12.27±0.43 bc 10.77±0.60 cd<br />
Average 12.81±0.55 a 10.12±0.20 cd 9.79±0.20 d 9.78±0.15 d 12.94±0.51 a 11.49±0.36 b 10.94±0.44 bc 10.26±0.26 cd<br />
2002<br />
2003<br />
2004<br />
2.39±0.013 a<br />
(5.7)<br />
2.32±0.063 a<br />
(5.4)<br />
2.42±0.027 a<br />
(5.8)<br />
Average 2.37±0.024a<br />
(5.6)<br />
2.10±0.023 de<br />
(4.4)<br />
2.08±0.035 cd<br />
(4.3)<br />
2.12±0.023 bc<br />
(4.5)<br />
2.10±0.015 c<br />
(4.4)<br />
2.05±0.023 e<br />
(4.1)<br />
1.99±0.044 cd<br />
(4.0)<br />
2.03±0.021 cd<br />
(4.1)<br />
2.02±0.018 d<br />
(4.1)<br />
2.02±0.030 e<br />
(4.05)<br />
1.95±0.037 d<br />
(3.8)<br />
1.98±0.031 d<br />
(3.9)<br />
1.98±0.019 d<br />
(3.9)<br />
2.39±0.023 a<br />
(5.7)<br />
2.34±0.045 a<br />
(5.5)<br />
2.42±0.041 a<br />
(5.9)<br />
2.38±0.022 a<br />
(5.7)<br />
Number of seed per pod was subjected to square root transformation. Non transformed data are represented by numbers given in brackets.<br />
2.31±0.044 ab<br />
(5.3)<br />
2.27±0.024 ab<br />
(5.2)<br />
2.38±0.007 a<br />
(5.7)<br />
2.32±0.022 ab<br />
(5.4)<br />
2.27±0.030 bc<br />
(5.2)<br />
2.25±0.026 ab<br />
(5.1)<br />
2.33±0.021 a<br />
(5.4)<br />
2.28±0.018 b<br />
(5.2)<br />
2.18±0.043 cd<br />
(4.8)<br />
2.12±0.023 bc<br />
(4.5)<br />
2.18±0.030 b<br />
(4.8)<br />
2.16±0.019 c<br />
(4.7)
Simsek et al. 4063<br />
Table 6. Analysis of variance for bean yield, pod weight, pod length, pod width and number of seed per pod (mean of<br />
2002, 2003 and 2004).<br />
Source of variation<br />
Degree of<br />
freedom<br />
Mean square<br />
FY PWt PL PWh NSPP<br />
Year (Y) 2 5.665*** 7.693*** 630.712*** 22.141*** 0.029 ns<br />
Y X R 6 0.345* 0.105 ns 8.464 ns 0.348 ns 0.006 ns<br />
Growth stage (A) 1 167.445*** 28.880*** 1673.311*** 11.045*** 0.525***<br />
Y X A 2 0.043 ns 0.024 ns 10.125 ns 0.755 ns 0.002 ns<br />
Error 6 0.077 0.106 14.258 0.252 0.006<br />
Irrigation regime (B) 3 107.211*** 16.941*** 2687.815 ns 29.585*** 0.305***<br />
Y X B 6 0.085 ns 0.206 ns 18.782 ns 1.484* 0.001 ns<br />
A X B 3 18.595*** 3.343*** 216.972*** 1.492* 0.054***<br />
Y X A X B 6 0.192 ns 0.121 ns 10.753 ns 0.366 ns 0.001 ns<br />
Error 36 0.248 0.187 12.875 0.465 0.002<br />
Total 71 8.02 1.622 175.301 2.557 0.026<br />
*Significant at P < 0.05; ***significant at P < 0.001; R: replication; RDI, regulated deficit irrigation; FY, fresh yield; PWt, pod<br />
weight; PL, pod length; PWh, pod width; NSPP, number of seed per pod.<br />
RDI were very significant at P < 0.001, for FY, PWt, PL<br />
and NSPP, at P < 0.05 for the PWh (Table 6). Table 5<br />
represents the yield components data of treatments.<br />
Positive correlation was obtained between the yield and<br />
the values of PWt, PL, PWh and NSPP. Similarly, Lyon et<br />
al. (1995) reported that a positive correlation exists<br />
between the seed number, seed weight and the yield.<br />
The reason of the higher yield for the control treatments<br />
was due to mainly increase the vegetative growth parameters.<br />
Similar findings have been reported previously by<br />
Demirtas et al. (2010) in soybean and Onder et al. (2006)<br />
in green bean.<br />
As expected, PWt increase had a significant effect on<br />
the yield. In addition, PWt was significantly influenced by<br />
irrigation level (Onder et al., 2006). The lowest value of<br />
the PWt was found in the T4 on which 75% water stress<br />
was applied in the V stage as 6.72 g, when the pod<br />
weight values were examined. The yields were generally<br />
found to be decreasing, for the treatments on which RDI<br />
was applied on the V stage or R stage, as the PWt was<br />
decreased (Table 5). The PWt values of the control were<br />
observed to be higher for all the three years since water<br />
stress was not applied. RDI regime applied in the V stage<br />
caused a significant decrease in PWt, whereas no<br />
important deviation was observed in the R stage (Table<br />
5).<br />
When the PL was examined for the RDI regime, the<br />
values were decreased with the decreasing of irrigation<br />
water. The longest PL (113.63 and 113.23 mm) was observed<br />
in the T1 and T5, while the lowest PL value was<br />
measured as 79.47 mm for T4.<br />
RDI significantly affected the NSPP and displayed<br />
similar effects on both growing stages. When the mean<br />
values of the years were only considered, it was determined<br />
that the yield increased with increasing the NSPP.<br />
The highest and lowest NSPP were determined as 5.70<br />
for T5 and 3.94 for T4 (Table 5).<br />
Conclusions<br />
The results of this study showed that, more positive<br />
results could be obtained for the RDI regimes in the R<br />
stage than the RDI regimes used in the V stage. It is<br />
found that bean yield is very sensitive to the water stress.<br />
Unless RDI is compulsory, it should not be applied in the<br />
V stage because the sensitivity of the plant is considerably<br />
high at this stage. However, excessive tempe-
4064 Afr. J. Biotechnol.<br />
rature was a dominant factor in the decreasing of the<br />
flowering and fruit set. Therefore, the seeding of the<br />
common bean must be done in the first or second weeks<br />
of the August. Climatic conditions particularly, cumulative<br />
pan evaporation play the most significant role among<br />
others. Therefore, irrigation techniques and programs<br />
considering climatic parameters have become very significant.<br />
The RDI regime with 25% deficit at reproductive<br />
stage could be recommended to growers without a<br />
significant decrease in the yield in semi arid zone.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4065-4071, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2399<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Molecular cloning of a novel GSK3/shaggy-like gene<br />
from Triticum monococcum L. and its expression in<br />
response to salt, drought and other abiotic stresses<br />
Yang Xian-Guang, Fan Jin-Yu and Deng Chuan-Liang*<br />
School of Life Science, Henan Normal University, Xinxiang 453007, China.<br />
Accepted 25 March, 2011<br />
The glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinases are nonreceptor serine/threonine protein<br />
kinases that are involved in a variety of biological processes. Here, a novel GSK-3-like kinase encoding<br />
cDNA was isolated from Triticum monococcum L. seedlings by reverse transcriptase polymerase chain<br />
reaction (RT-PCR). Sequence analysis showed that the full length of cDNA consist of 1,543 bp with an<br />
open reading frame of 1,068 bp, which encodes 355 amino acid residues. The deduced amino acid<br />
sequence showed a high homology with shaggy-like kinases from Triticum aestivum, Zea mays,<br />
Trifolium repens, Nicotine tabacum, Medicago sativa and Arabidopsis thaliana; therefore, the gene was<br />
named TmGSK1 (Triticum monococcum Glycogen Synthase Kinase 1,GenBank Accession No.<br />
DQ443471). Southern blot analysis indicated that there was only one copy of TmGSK1 in the einkorn<br />
wheat genome. Quantitative real-time RT-PCR studies showed that the expression of TmGSK1 in the<br />
einkorn wheat was induced by salt stress, mechanical wounding, ABA hormone, cold and drought.<br />
These results suggest that cells accumulate more TmGSK1 mRNA response to those abiotic stresses.<br />
TmGSK1 was shown to be a positive regulator commonly involved in the tolerance to salt, mechanical<br />
injury, ABA hormone, cold and drought in einkorn wheat.<br />
Key words: TmGSK1, abiotic stress, shaggy-like kinase, signal transduction, Triticum monococcum L.<br />
INTRODUCTION<br />
Abiotic stresses such as salinity, drought, extreme tem-<br />
peratures, chemical toxicity and oxidative stress are<br />
becoming particularly widespread in many regions. It is<br />
estimated that salinity affects at least 20% of the world’s<br />
arable land and more than 40% of irrigated land to various<br />
degrees (Wang et al., 2003). These stresses reduce<br />
average yields for most major crop plants by more than<br />
50%. Further more, drought, salinity, extreme tempe-<br />
ratures and oxidative stress are often interconnected, and<br />
may affect the plants in a similar way (Katerji et al., 2004;<br />
Almansouri et al., 2001). These diverse environmental<br />
stresses often activate similar cell signaling pathways and<br />
cellular responses such as the production of stress<br />
*Corresponding author. E-mail: biol@htu.cn<br />
Abbreviations: GSK-3, Glycogen synthase kinase-3; RT-PCR,<br />
reverse transcriptase polymerase chain reaction; PCR,<br />
polymerase chain reaction.<br />
proteins, up-regulation of anti-oxidants and accumulation<br />
of compatible solutes, leading to plant defense and/or<br />
adjustment to adverse conditions (Bajguz and Hayat,<br />
2009; Zhu, 2002; Knight and Knight, 2001). Various<br />
abiotic stresses lead to the overproduction of reactive<br />
oxygen species (ROS) in plants which are highly reactive<br />
and toxic, and cause damage to proteins, lipids,<br />
carbohydrates and DNA which ultimately results in<br />
oxidative stress (Gill and Tuteja, 2010). Oxidative and<br />
osmotic stress activates several protein kinases including<br />
mitogen-activated protein kinases, which may mediate<br />
osmotic homeostasis and/or detoxification responses. A<br />
number of phospholipid systems are activated by osmotic<br />
stress, generating a diverse array of messenger<br />
molecules, some of which may function upstream of the<br />
osmotic stress-activated protein kinases (Zhu, 2001) and<br />
glycogen synthase kinase3/ shaggy like kinase.<br />
Glycogen synthase kinase-3 (GSK-3) is a ubiquitously<br />
expressed protein kinase. It was originally identified as a<br />
regulator of glycogen synthesis in mammals. Animal<br />
GSK-3 participates in glycogen metabolism and in both the
4066 Afr. J. Biotechnol.<br />
Wnt/β-catenin and PI3K/AKT signaling pathways involved<br />
in the regulation of several physiological processes,<br />
including glycogen metabolism, protein synthesis, trans-<br />
cription factor activity and developmental control.<br />
Whereas, plant GSK-3-like kinases (GSKs) have only<br />
recently entered the scene. In contrast to mammals,<br />
which contain two genes, a multigene family encodes<br />
plant GSKs. GSK-3-like genes have been isolated from<br />
plants such as alfalfa (Pay et al., 1993), arabidopsis<br />
(Rozhon et al., 2010; Tavares et al., 2002; Piao et al.,<br />
1999; Dornelas et al., 1999; Jonak et al., 1995; Bianchi et<br />
al., 1994), Petunia hybrida (Decroocq-Ferrant et al., 1995),<br />
Nicotiana tabacum (Einzenberger et al., 1995), Triticum<br />
aestivum (Chen et al., 2003), Oryza sativa (Koh et al.,<br />
2007) and Zea mays (Zou et al., 2008). Present evidence<br />
indicates that plant GSKs are involved in different<br />
processes such as flower development (Dornelas et al.,<br />
2000), wound responses (Jonak et al., 2000), salt stress<br />
(Piao et al., 2001) and brassinosteroid signaling (Peng et<br />
al., 2010; Li and Nam, 2002). Einkorn wheat is a rare<br />
species that reduces the magnitude of population and the<br />
area of distri- bution under anthropogenic activity. It is<br />
closely related to cultivated wheat and has some<br />
properties, for example, resistance to diseases, and thus<br />
were used in selection to improve wheat varieties. Here,<br />
we isolated a member of GSK family from einkorn wheat<br />
and analyzed its expression patterns in response to<br />
abiotic stresses by real time quantitative polymerase<br />
chain reaction (PCR) approach.<br />
MATERIALS AND METHODS<br />
Plant material and stress treatments<br />
Seed of the einkorn wheat (Triticum monococcum L.) were<br />
surface-sterilized for 5 min in 1% (w/v) sodium hypochlorite and was<br />
then washed in distilled water. The sterilized seeds were soaked in<br />
distilled water at 30°C overnight, for germination. The germinated<br />
seeds were transplanted into a pot containing vermiculite. The<br />
seedings were irrigated with distilled water. Two-week-old plant was<br />
treated with distilled water (as a control for Q-PCR analysis), high<br />
concentration of salt (170 mM NaCl), mechanical injury (shearing),<br />
ABA hormone (100 µM), cold (4°C) and drought (30%PEG6000).<br />
Plant was harvested after stress treatments at intervals of definite<br />
time, then immediately frozen in liquid nitrogen and stored at –80°C<br />
for later use.<br />
RNA isolation and cDNA synthesis<br />
Total RNAs were isolated using TRIzol reagent (Invitrogen) according<br />
to the manufacturer's recommendations. Total RNA was treated with<br />
RNase-free DNaseI (Promega) for 20 min at 37°C, DNase I was<br />
degraded at 65°C for 10 min. The integrity of the RNA was verified<br />
after separation by electrophoresis on a 0.8% agarose gel<br />
containing 0.5% (v/v) ethidium bromide. First-strand cDNA was<br />
synthesized from 500 ng of total RNA using Reverse Transcription<br />
system (Promega) with an oligo-dT15 primer. The reaction solution<br />
was used as templates for reverse transcriptase polymerase chain<br />
reaction (RT-PCR).<br />
TmGSK1 gene cloning and sequence analysis<br />
PCR was performed using the following primers for TmGSK1:<br />
T1-forward (5'- GTTGGTGTGGTGCGTCCTTC-3') and T1-reverse<br />
(5'- TGGGGGCCTCGATCCATGAAC -3') designed from the con-<br />
served sequences of the known plant GSKs. Polymerase chain<br />
reaction (PCR) was initiated with hot start method using the single<br />
strand cDNA template and Ex-Taq polymerase (Takara) on Biometra<br />
T-GRADIENT Themoblock. The PCR reaction was carried out for 35<br />
amplification cycles (94°C for 30 s, 61°C for 45 s and 72°C for 90 s).<br />
The RT-PCR product was cloned into pGEM-T vector (Promega)<br />
and sequenced. Bioinformatic analyses were carried out using<br />
DNASTAR software (DNASTAR, Inc., Madison, WI, USA).<br />
DNA extraction and Southern analysis<br />
Genomic DNA was isolated from green leaves of two-weeks-old<br />
einkorn wheat according to the method described in molecular<br />
cloning laboratory manual (Sambrook and Russell, 2001). For<br />
Southern hybridization, genomic DNA was digested separately with<br />
EcoRΙ and Hind III for 16 h and subjected to electrophoresis in 0.8%<br />
(w/v) agarose for 16 h at 4 V cm -1 (20 µg per lane). After<br />
depurination of DNA within the gel in 0.25 M HCl for 30 min at 25°C,<br />
the DNA was transferred to a Hybond-N+ nylon membrane<br />
(Amersham Bio- sciences, Little Chalfont, UK) in 20×SSC and<br />
hybridized with the probe, which was labeled with [α- 32 P] dCTP by<br />
Random Primer DNA Labeling kit(Takara), for 16 h at 65°C. The<br />
blots were washed once in 0.1% SDS, 2×SSC for 15 min at 65°C<br />
and twice in 0.1% SDS, 0.1×SSC for 15 min at 65°C<br />
(high-stringency conditions), and exposed to X-ray film (Kodak).<br />
Expression analysis by quantitative real-time RT-PCR<br />
The expression of mRNA for TmGSK1 was examined by<br />
quantitative real-time RT-PCR under several different abiotic<br />
stresses on the Rotor-Gene 3000 system (Corbett Robotics).<br />
RT-PCR was performed using the all-in-one qPCR kit<br />
(Genecopoeia), following the manufacturers protocol. Primers were<br />
designed by primer express 2.0 software and synthesized by Beijing<br />
Sunbiotech Co., Ltd. A 189 bp product for TmGSK1 cDNA was<br />
amplified using the following primers: T-forward 5'<br />
GTTTGGTCTGCTGGCTGTGTTCTT 3' and T-reverse 5'<br />
GTGCCATGGGTGAGCTTTGATTT 3'. A 118 bp product of einkorn<br />
wheat housekeeping gene β-actin was amplified as an internal<br />
control using the following primers: ACT-f 5'<br />
TGGCACCCGAGGAGCACCCTG 3' and ACT-r 5' GCGACGTAC<br />
ATGGCAGGAACA 3'. The thermal cycling profile consisted of initial<br />
denaturation at 95°C for 3 min and 40 cycles at 95°C for 15 s, 60°C<br />
for 15 s and 72°C for 30 s. The resultant amplification was purified<br />
and sequenced, and showed 100% homology to target gene. To<br />
confirm amplification specificity, the amplified fragments were<br />
analyzed by 1.5% agarose gel electrophoresis containing ethidium<br />
bromide. The specificity of PCR amplification was determined by<br />
constructing a melting curve after the polymerase chain reaction<br />
amplification, and negative controls containing RNase-free water<br />
instead of sample was run to confirm that the samples were not<br />
cross contaminated. Quantitation of relative expression was<br />
determined by the 2 −∆(∆CT) method (Livak and Schmittgen, 2001).<br />
Each sample was run in triplicate.<br />
RESULTS AND DISCUSSION<br />
cDNA clone and sequence analysis of the TmGSK1<br />
protein<br />
The TmGSK1 cDNA contained 1,543 bp with an open
Xian-Guang et al. 4067<br />
Supplemental material 1. Alignment of sequence of GSKs predicted protein from T. monococcum L., T. aestivum, Z. mays,N. tabacum, M.<br />
sativa and A. thaliana. The black shading indicates identical amino acids. Amino acid sequences were aligned with the MegAlign program<br />
(DNAstar software (DNASTAR, Inc., Madison, WI, USA)) using the CLUSTALW method.<br />
reading frame of 1,068 bp (start codon at 79 bp and stop<br />
codon at 1146 bp) encoding a deduced protein of 355<br />
amino acids (GenBank accession No. DQ443471). The<br />
theoretical isoelectric point (PI) of the putative amino acid<br />
sequence is 8.34 and molecular weight (MW) is 40.3 kDa.<br />
The deduced amino acid sequences of TmGSK1 and<br />
GSKs from T. aestivum (AF525086), Z. mays (AY722707),<br />
Trifolium repens (X99100), N. tabacum (X77763),<br />
Medicago sativa (X68409) and Arabidopsis thaliana<br />
(BT000132) are aligned (Supplemental material 1). The<br />
putative amino acid sequence of TmGSK1 showed high<br />
homology to GSKs from T. aestivum (94.2%), Z. mays<br />
(95.5%), T. repens (90.5%), N. tabacum (83.4%), M.<br />
sativa (84.4%) and A. thaliana (73.2%). Phylogenetic<br />
analysis of TmGSK1 is shown in Supplemental material 2.<br />
Southern analysis of TmGSK1<br />
Southern analysis of genomic DNA digested separately<br />
with two different restriction enzymes, EcoR I and HindIII,<br />
was performed to estimate the copy number of TmGSK1<br />
in the einkorn wheat genome. As shown in Figure 1, the<br />
banding pattern was produced with the TmGSK1<br />
full-length probe (1,543 bp) and a high-stringency wash.<br />
Only one hybridizing band in each digestion was<br />
observed, indicating that the TmGSK1 sequence was<br />
present as a single copy in the einkorn wheat genome.<br />
Quantitative real-time RT-PCR of the TmGSK1 gene in<br />
response to various abiotic stresses<br />
The quantitative real-time RT-PCR analyses of TmGSK1<br />
during various abiotic stresses are shown in Figure 2.<br />
Constitutively expressed β-actin gene was used as an<br />
internal control. TmGSK1 expression was upregulated in<br />
these abiotic stresses. Accumulation of TmGSK1 mRNA<br />
increased step by step under salt stress (Figure 2a). Its<br />
expression was up to 1.5-folds when compared to the con-
4068 Afr. J. Biotechnol.<br />
Supplemental material 2. The phylogenetic tree of predicted protein sequence of GSKs from T. monococcum L.,T. aestivum, Z.<br />
mays, N. tabacum, M. sativa and A. thaliana.<br />
trol when its was stressed 6 h with NaCl and reached its<br />
maximum within 12 h after treatment; under wounding<br />
treatment, the expression pattern of TmGSK1 was in-<br />
duced earlier than that under NaCl treatment (Figure 2b).<br />
Injury inducing its expression increased rapidly, up to<br />
2-folds when it was injured just one hour. For abscissic<br />
acid (ABA) treatment, TmGSK1 transcription was induced<br />
similar to that under wounding. Its expression increased<br />
3-folds after treated with ABA for 1 h (Figure 2c). For<br />
drought treatment, it was 2.0 and 2.6 folds increase after<br />
treated with PEG6000 at 6 and 12 h (Figure 2d); low<br />
temperature (4°C) can induce its high expression too<br />
(Figure 2e). Expression pattern analysis indicates the<br />
possible involvement of TmGSK1 in salt, wounding, ABA,<br />
drought and cold stress signal transduction pathways in<br />
einkorn wheat.<br />
To understand a mechanism of abiotic stress signal<br />
transduction in plant, it is important to identify the<br />
components involved in the pathway. In this study, we<br />
Figure 1. Southern-blot analysis of TmGSK1. Left: Agrose<br />
gel electrophoresis of the genomic DNA which was<br />
completely digested with EcoR I (E) and Hind III (H); Right:<br />
hybridization using the full-length sequence of AbGSK1 as<br />
probe.<br />
isolated the glycogen synthase kinase 3 (GSK3)/<br />
SHAGGY-like kinases from T. monococcum L. Previously,<br />
it has been shown that GSK3 are involved in a variety of<br />
biological processes. Present evidence indicates that<br />
plant GSKs are also involved in different processes such<br />
as hormone signaling, NaCl stress and other abiotic<br />
stresses. In accordance with these previous observations,<br />
we found in the present study that TmGSK1 was also<br />
involved in the response of abiotic stresses such as salt<br />
stress, mechanical injury, abscissic acid hormone, cold<br />
and drought stress. Interestingly, as shown in Figure 2,<br />
the plants accumulate more TmGSK1 mRNA response to<br />
all of these stresses. These means such stresses are<br />
inter- connected and may induce similar cellular signal<br />
transduction pathways. Moreover, these signal pathways<br />
may cross-talk on TmGSK1 or TmGSK1 may integrate all<br />
of these signal pathways in einkorn wheat. Stress signal<br />
activate the glycogen synthase kinase through different<br />
ways and substrates are phosphorylated and activity is
a<br />
Stress time (h)<br />
b<br />
Stress time (h)<br />
c<br />
Stress time (h)<br />
Figure 2. The relative mRNA level of TmGSK1 under salt stress treatment (a);<br />
wounding treatment (b); ABA treatment (c); drought treatment (d); cold treatment<br />
(e). The quantitative real-time RT-PCR analysis of TmGSK1 gene expression in T.<br />
monococcum L in response to salt stress (1% NaCl), mechanical injury, ABA<br />
hormone, drought and cold treatment (4°C) using constitutively expressed β-actin<br />
gene as an internal control.<br />
Xian-Guang et al. 4069
4070 Afr. J. Biotechnol.<br />
Figure 2. Contd.<br />
e<br />
d<br />
Stress time (h)<br />
Stress time (h)<br />
regulated. Then, stress responsive genes express and<br />
start-up stress responsive mechanisms to re-establish<br />
cellular homeostasis and protect and repair damaged<br />
proteins and membranes. Therefore, TmGSK1 was<br />
shown to be a positive regulator commonly involved in the<br />
tolerance to salt, mechanical injury, ABA hormone, cold<br />
and drought in einkorn wheat. However, most<br />
mechanisms in this model are still unknown, and further<br />
investigation should be carried out to indicate the wheat<br />
plant abiotic stress responsive signal transduction in<br />
details.<br />
Acknowledgments<br />
This work was supported by the National Nature Science<br />
Foundation of China (NO.31000165, 31171182), the Key<br />
Project of Chinese Ministry of Education (NO.209076) and<br />
Foundation of science and technology research projects<br />
of Henan Province in China (No. 092300410099,<br />
102300413213).<br />
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Piao HL, Lim JH, Kim SJ, Cheong GW, Hwang I (2001).Constitutive<br />
over-expression of AtGSK1 induces NaCl stress responses in the<br />
absence of NaCl stress and results in enhanced NaCl tolerance in<br />
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Piao HL, Pih KT, Lim JH, Kang SG, Jin JB, Kim SH, Hwang I (1999). An<br />
Arabidopsis GSK3/shaggy-Like gene that complements yeast salt<br />
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Physiol. 119(4): 1527-1534.<br />
Rozhon W, Mayerhofer J, Petutschnig E, Fujioka S, Jonak C (2010).<br />
ASKtheta, a group-III Arabidopsis GSK3, functions in the<br />
brassinosteroid signalling pathway. Plant J. 62(2): 215-223.<br />
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3rd edn. Cold Spring Harbor Laboratory Press, New York.<br />
Tavares R, Vidal J, van Lammeren A, Kreis M (2002). AtSKθ, a plant<br />
homologue of SGG/GSK-3 marks developing tissues in Arabidopsis<br />
thaliana. Plant Mol. Biol. 50(2): 261-271.<br />
Wang wxWX, Vinocur B, Altman A (2003). Plant responses to drought,<br />
salinity and extreme temperatures: towards genetic engineering for<br />
stress tolerance. Planta, 218(1): 1-14.<br />
Zhu JK (2001). Cell signaling under salt, water and cold stresses. Curr.<br />
Opin. Plant Biol. 4(5): 401-406.<br />
Zhu JK (2002). Salt and drought stress signal transduction in plant. Annu.<br />
Ann. Rev. Plant Biol. 53: 247-273.<br />
Zou HW, Wu ZY, Zhang XH, Wang YQ, Yang Q, Cao MQ, Huang CL<br />
(2008). Cloning and characterization of maize ZmASK1, a homologue<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4072-4080, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2419<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Some quality traits and neurotoxin β-N-oxalyl-L-α,βdiaminopropionic<br />
acid (β-ODAP) contents of Lathyrus<br />
sp. cultivated in Turkey<br />
Ugur Basaran 1 *, Ozlem Onal Asci 2 , Hanife Mut 1 , Zeki Acar 3 and Ilknur Ayan 3<br />
1 Bozok University, Faculty of Agriculture, Department of Field Crops, Sivas Street 66200, Yozgat-Turkey.<br />
2 Ordu University, Faculty of Agriculture, Department of Field Crops, Cumhuriyet Street, 52200, Ordu-Turkey.<br />
3 Ondokuz Mayis University, Faculty of Agriculture, Department of Field Crops, 55139, Samsun-Turkey.<br />
Accepted 25 March, 2011<br />
In this study, 52 landraces accessions belonging to the species of Lathyrus sativus and Lathyrus<br />
clymenum collected from different regions of Turkey and one released variety of Lathyrus sativus were<br />
evaluated for some quality traits like seed coat color, 1000 seed weight, crude protein and neurotoxin β-<br />
N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) content. Among the investigated landraces, high<br />
variation was determined for all the investigated traits, which was attributed to both genetic as well as<br />
environmental factors. Protein content was as between 24.07 to 30.90% and β-ODAP content was as<br />
1.35 to 3.86 mg g -1 for seed. Many landraces with low β-ODAP content (< 2.00 mg g -1 ) and high protein<br />
content seem to be promising material for Lathyrus breeding. Furthermore, many investigated<br />
landraces showed lower β-ODAP and higher protein contents compared with the released variety. Also,<br />
the local consumption of L. sativus landraces as a food or feed was determined during the collection<br />
process.<br />
Key words: Lathyrus, landrace, protein, β-ODAP, Turkey.<br />
INTRODUCTION<br />
Lathyrus sativus L. (grass pea, in Turkish “murdumuk,<br />
culban, fasil, feslek”) is a traditional crop use for both<br />
animal consumption as forage and grain and for human<br />
consumption as a pulse. L. sativus has many advantages<br />
including drought and flooding tolerance, disease<br />
resistance, high yield potential and favorable nutritional<br />
composition of seeds. It can easily grow in different soil<br />
types and at high altitudes (Tivari and Campbell, 1996a,<br />
b). L. sativus grows with as little as 250 mm of annual<br />
precipitation and it is typically the last surviving plant in<br />
drought times (Tekele-Haimanot et al., 1990; White et al.,<br />
2002).<br />
Besides the obvious advantages, L. sativus seeds<br />
contain a major anti-nutritional compound namely β-<br />
ODAP (Zhao et al., 1999; Wang et al., 2000). Heavy and<br />
prolonged (3 to 4 month) consumption of L. sativus seeds<br />
*Corresponding author. E-mail: ugurb@omu.edu.tr. Tel: (+90<br />
354) 212 70 01. Fax: (+90 354) 212 27 89.<br />
can cause a disease known as ‘lathyrism’ or ‘neurolathyrism’,<br />
a disease causing paralysis of the limbs<br />
(Spencer et al., 1986; Yan et al., 2006).<br />
Until recently, relatively little effort has been made<br />
towards the improvement of this hardy pulse crop due to<br />
its toxicity. To counter the effects of climate change on<br />
agricultural productivity, L. sativus is gaining interest in<br />
southern Australian Mediterranean-type environments<br />
(Hanbury et al., 1999), North America (Campbell et al.,<br />
1994) and Southern Europe (Crino et al., 2004; Tavoletti<br />
et al., 2005; Polignano et al., 2009). Also, many studies<br />
have been carried out to bring grass pea in areas under<br />
monoculture cereal cultivation in Europe (Vaz Patto et al.,<br />
2006).<br />
In order to reintroduce L. sativus in crop rotations under<br />
marginal environments, breeding programs are in progress<br />
worldwide to improve genotypes combining high<br />
yield, high protein content and low or zeroneurotoxin<br />
content (ODAP) (Bozzini, 1997; Siddique et al., 1998;<br />
Hanbury et al., 2000).<br />
Lathyrus cultivation was not well documented in
Basaran et al. 4073<br />
Figure 1. Distribution of the collection area in Turkey. 02, Adiyaman; 15, Burdur; 16, Bursa; 18, Cankiri; 20, Denizli; 23, Elazig; 43,<br />
Kutahya, 44, Malatya; 48, Mugla; 50, Nevsehir; 55, Samsun; 64, Usak.<br />
Turkey. However, some authors reported that, L. sativus,<br />
Lathyrus cicera, Lathyrus clymenum and Lathyrus ochrus<br />
were cultivated in Turkey (Genc and Sahin, 2001; Cetin,<br />
2006) and L. sativus was widely cultivated in the country<br />
in the past (Davis, 1970). In addition, Lathyrus hirsutus<br />
had been cultivated in the east and inner parts of Turkey<br />
until 1960s (Tosun, 1974). Lathyrus cultivation dramatically<br />
decreased from the 1960s. Today, based on actual<br />
data, only L. sativus is cultivated in 18.000 ha for the<br />
seed in Turkey (TUIK, 2008). This crop is mainly used as<br />
stock-feed; however, it is rarely used for human<br />
cunsumption in some parts of the country (Basaran et al.,<br />
2010).<br />
There is only one released variety of L. sativus,<br />
‘Gürbüz 2001’ in Turkey. This means, approximately all<br />
the cultivated genotypes are landraces; hence, they are<br />
promising materials for breeding programs. Most of these<br />
landraces were not previously screened. However,<br />
Karadag et al. (2010) indicated that, it had a high<br />
variability in β-ODAP levels in winter and spring-sown<br />
grasspea lines. On the other hand, interestingly, there is<br />
no lathyrism case documented for both humans and<br />
animals in Turkey which may result from landraces with<br />
low β-ODAP content. So, the screening of these landraces<br />
regarding their β-ODAP content is very important.<br />
For this purpose, expeditions were carried out to collect<br />
Lathyrus landraces cultivated in Turkey and to evaluate<br />
their quality traits including the protein and β-ODAP<br />
contents. Also, this study provided comprehensive and<br />
additional information about the general status of<br />
Lathyrus cultivation in Turkey.<br />
MATERIALS AND METHODS<br />
In this study, a total of 52 local landrace accessions of Lathyrus (51<br />
L. sativus and 1 L. clymenum) collected from Turkey were<br />
examined. To collect Lathyrus accessions, initially 12 cities in which<br />
Lathyrus are cultivated were determined based on data of the<br />
Turkish Statistical Institute (TUIK, 2007) and Ministry of Agriculture<br />
records. The collection program was carried out in 2007. The<br />
collection area is given in Figure 1.<br />
Lathyrus accessions were taken from different farmers or local<br />
seed markets and their name and altitude of collection cites were<br />
recorded. Three landraces of the collected L. sativus genotypes are<br />
commercial material. In addition, the released variety ‘Gürbüz-2001’<br />
was obtained from Turkish Central Research Institutes for Field<br />
Crops. Seed coat color, 1000 seed weight, protein and β-ODAP<br />
contents were determined for each sample.<br />
Seed coat color was determined by visual observation. Total<br />
nitrogen was determined by the Kjeldahl method and crude protein<br />
content was estimated using a conversion factor of 6.25 (Kacar,<br />
1972). All the data were presented as mean, mimimum, maximum<br />
and standard deviation determined by using SPSS 13.0 statistical<br />
package program.<br />
Determination of β-ODAP<br />
β-ODAP was determined using capillary zone electrophoresis<br />
(CZE) (Zhao et al., 1999) with some modifications. CZE was<br />
carried out using Agilent HP 3D and UV detection at 195 nm. The
4074 Afr. J. Biotechnol.<br />
capillar was 55 cm (44 cm effective length) x 50 µm. The analyses<br />
were performed at a constant voltage of 20 kV at 20°C in an<br />
electrolyte of 75 mM (H3BO3) buffer at pH 7.5. The required pH of<br />
the buffer was adjusted by adding NaOH. All chemicals were of<br />
analytical-reagent grade. The reference standard of β-ODAP was<br />
obtained from Dr. S.L.N.Rao (Lathyrus Technologies, Hyderabad,<br />
India).<br />
Sample extraction<br />
0.5 g powder of L. sativus seeds was soaked in 50 ml ethanol-water<br />
(30:70, v/v) solution and was shaken for 2 h (in ice). After<br />
centrifugation (3500 rpm at 15 min), the upper clear solution was<br />
filtered with 0.45 µm filter paper. Then clear solution was diluted<br />
with ultra distilled water (1:1) and was injected directly into the CZE<br />
system for 40 s at 50 mbar.<br />
RESULTS AND DISCUSSION<br />
There was very limited information about the distribution<br />
and density of Lathyrus cultivation in previous years. But,<br />
according to the old farmer’s statements, Lathyrus cultivation<br />
was more widespread in the past than now and a<br />
dramatically decrease begun in the 1960’s. During the<br />
collection expedition, only landraces of L. sativus and L.<br />
clymenum species were collected. But L. cicera, L.<br />
ochrus and L. hirsutus were not encountered; their<br />
cultivation was reported by different authors (Tosun,<br />
1974; Genc and Sahin, 2001; Cetin, 2006). The absence<br />
of L. cicera and L. ochrus among the encountered species<br />
in the study clearly indicated that, the cultivated<br />
Lathyrus materials had been exposed to genetic erosion<br />
in the last 50 years and a lot of genetic materials<br />
disappeared during the period.<br />
The collection sites and local names of Lathyrus<br />
landraces throughout the country are presented in Table<br />
1. Seed trade for both L. sativus and L. clymenum was<br />
very limited and also, there was only one released variety<br />
named ‘Gürbüz-2001’ in Turkey. So, farmers usually sow<br />
their own seed produced in previous years.<br />
In general, L. sativus is cultivated at high altitudes (600<br />
to 1600 m). The common name of Lathyrus species is<br />
“murdumuk” in Turkey. Moreover, Lathyrus is known by<br />
many local names such as “culban, coluk, fasil feslek”<br />
and L. clymenum is known as “kara murdumuk” (Table<br />
1).<br />
L. sativus is mainly cultivated for its seed and it is used<br />
as feed (Table 2). In some cities, however, its grain has<br />
been rarely consumed by humans as a soup, pilaf or<br />
snack (by mixing with chickpea). L. clymenum is<br />
cultivated only in Mugla city in small field for household<br />
consumption and its seed is used to make soup and it is<br />
added in the traditional food named “stuffed zucchini<br />
flowers”.<br />
According to farmers, L. sativus has higher seed yield<br />
than other legumes especially in dry condition and do not<br />
shatter its pods. So, farmers prefer to harvest the plant<br />
for seed production than for forage. For animal feeding,<br />
seeds are used by soaking in water or grinding and<br />
mixing with cereals in Turkey.<br />
Seed coat colors amongst the landraces of L. sativus<br />
examined in the study were grey, brown, creamy and<br />
mostly mixture of these color. Creamy seed coat color<br />
was observed in few landraces in the mixture. However,<br />
landrace 5501 used only for human consumption had<br />
creamy seed coat color alone. The color variation in a<br />
single landrace can be related to genetic variability. So,<br />
besides inter population variation, intra population genetic<br />
variation was as high as possible, which is a desired<br />
feature in plant breeding. On the other hand, genotypes<br />
with light cream color seed had low neurotoxin content<br />
(Dahiya, 1976). It was reported that, seed coat color<br />
could be a useful visual character to select low toxic lines<br />
(Campbell, 1997). However, Kaul et al. (1986), did not<br />
find any relationship between seed coat color and toxic<br />
contents. In this study, certain relationship between seed<br />
color and β-ODAP contents was not determined. Many<br />
landraces with grey or brown seed coat color had low β-<br />
ODAP than creamy seed’s landraces. Moreover, the<br />
6406 numbered landraces, which had low toxicity, had<br />
grey and brown seed color (Table 2).<br />
1000 seeds weight changed from 72.2 g in landrace<br />
numbered 2303 to 148.0 g in landrace numbered 5501<br />
(Table 2) with mean 100.2 g (Figure 2). According to<br />
farmers’ experience, they had not observed pod shattering<br />
in these landraces. L. sativus showed high<br />
variations (41 to 510 g) regarding 1000 seed weight<br />
(Joshi, 1997; Rybinski et al., 2008). Larger-seeded types<br />
are mostly found around the Mediterranean region, while<br />
small-seeded types are found in Indian subcontinent. In<br />
addition, small-seeded types have a tendency for pod<br />
shattering (Campbell, 1997). So, in general, the cultivated<br />
L. sativus landraces in Turkey showed similar features<br />
with the Mediterranean genotypes.<br />
Crude protein (CP) contents of the landraces varied<br />
from 24.07 to 30.90% for L. sativus and it was 26.33% for<br />
L. clymenum. CP content in the released variety was<br />
25.81% (Table 2.) and mean CP content was 28.23% for<br />
all the landraces (Figure 3). Variation in the seed CP<br />
contents can be attributed more to ecological factors<br />
other than genetic variation due to the ecological differences<br />
of the collection areas. But, Lathyrus is mostly<br />
cultured on poor or marginal lands with no fertilization in<br />
Turkey. So, these hard growing conditions determined<br />
the high CP ratios (28.23% as a mean) which indicated<br />
that many of the collected landraces may display genetic<br />
ability for high protein content. The seed CP ratios<br />
obtained in this study are similar to the results of other<br />
authors (Rotter et al., 1991; Rosa et al., 2000; Urga et al.,<br />
2005; Karadag et al., 2009; Karadag and Yavuz, 2010)<br />
who reported protein content for L. sativus seed between<br />
21.49 and 31.98%. But our results were lower than the<br />
results of Abd El Zaher et al. (2007) who reported that,<br />
seed protein was between 29.17 and 37.33% in L.<br />
sativus and between 36.11 and 36.72% in L. clymenum.
Table 1. Collection sities and local name of accessions of Lathyrus sp.<br />
Basaran et al. 4075<br />
Number Accession** City Village/district Local name Altitude (m)<br />
1<br />
2<br />
LS-0201<br />
LS-0202<br />
Adiyaman<br />
Dardogan/ Merkez<br />
Büklum/ Merkez<br />
Culban/ Coluk<br />
Culban/ Coluk<br />
825<br />
787<br />
3<br />
4<br />
LS-1501<br />
LS-1502<br />
Burdur<br />
Kizilkaya/ Bucak<br />
Harmanli/ Yesilova<br />
Murdumuk<br />
Murdumuk<br />
787<br />
1093<br />
5 LS-1503 Kizilkaya/ Bucak Murdumuk 787<br />
6 LS-1601<br />
Dogancılar/ Harmancık Murdumuk 781<br />
7<br />
8<br />
LS-1602<br />
LS-1603<br />
Bursa<br />
Dogancılar/ Harmancık<br />
Demirciler/ Harmancık<br />
Murdumuk<br />
Murdumuk<br />
781<br />
719<br />
9 LS-1604* Commercial Murdumuk -<br />
10 LS-1801<br />
Yukaripelitozu/ Merkez Murdumuk 768<br />
11 LS-1802 Cankiri Yukaripelitozu/ Merkez Murdumuk 768<br />
12 LS-1803 Elmaci / Eldivan Murdumuk 957<br />
13 LS-2001<br />
Uzunpinar/ Merkez Murdumuk/Fasil 1148<br />
14 LS-2002 Cabar/ Civril Murdumuk/Fasil 1013<br />
15 LS-2003 Ulukent/ Tavas Murdumuk/Fasil 934<br />
16<br />
17<br />
LS-2004<br />
LS-2005<br />
Denizli<br />
Balkica/ Tavas<br />
Eziler/ Guney<br />
Murdumuk/Fasil<br />
Murdumuk/Fasil<br />
1150<br />
841<br />
18 LS-2006 Baklancakirlar/ Cal Murdumuk/Fasil 886<br />
19 LS-2007 Alaaddin/ Acipayam Murdumuk/Fasil 887<br />
20 LS-2008 Karahoyukavsari/ Acipayam Murdumuk/Fasil 900<br />
21 LS-2301<br />
Uzuntarla/ Merkez Culban 995<br />
22 LS-2302 Elazıg Acipayam/ Merkez Culban 987<br />
23 LS-2303* Commercial Culban -<br />
24 LS-4301 Kutahya -/ Domonic Murdumuk 880<br />
25 LS-4401<br />
Yenikoy/ Darende Culban 1600<br />
26<br />
27<br />
LS-4402<br />
LS-4403<br />
Malatya<br />
Yenikoy/ Darende<br />
Basdirek/ Darende<br />
Culban<br />
Culban<br />
1600<br />
1445<br />
28 LS-4404* Commercial Culban -<br />
29 LS-5001<br />
Kalecik/ Kozakli Murdumuk 1120<br />
30 LS-5002 Sadik/ Hacibektas Murdumuk 1159<br />
31 LS-5003 Sahinler/ Gulsehir Murdumuk 880<br />
32 LS-5004 Nevsehir Hacıhalilli/ Gulsehir Murdumuk 1000<br />
33 LS-5005 Cullar/ Acigol Murdumuk 1086<br />
34 LS-5006 Tatlarin/ Aciöl Murdumuk 1113<br />
35 LS-5501 Degirmenci/ Kavak Feslek 600<br />
36 LS-6401<br />
Hacim/ Sivasli Murdumuk/Fasil 938<br />
37 LS-6402 Kasbelen/ Merkez Murdumuk/Fasil 960<br />
38 LS-6403 Kasbelen/ Merkez Murdumuk/Fasil 960<br />
39 LS-6404 Kasbelen/ Merkez Murdumuk/Fasil 960<br />
40 LS-6405 Inay/ Ulubey Murdumuk/Fasil 743<br />
41 LS-6406 Inay/ Ulubey Murdumuk/Fasil 743<br />
42 LS-6407 Inay/ Ulubey Murdumuk/Fasil 743<br />
43<br />
44<br />
LS-6408<br />
LS-6409<br />
Usak<br />
Kisla/ Ulubey<br />
Kisla/ Ulubey<br />
Murdumuk/Fasil<br />
Murdumuk/Fasil<br />
800<br />
800<br />
45 LS-6410 Kisla/ Ulubey Murdumuk/Fasil 800<br />
46 LS-6411 Karacaahmet/ Ulubey Murdumuk/Fasil 780<br />
47 LS-6412 Ilyasli/ Merkez Murdumuk/Fasil 770<br />
48 LS-6413 Ilyasli/ Merkez Murdumuk/Fasil 770<br />
49 LS-6414 Ilyasli/ Merkez Murdumuk/Fasil 770<br />
50 LS-6415 Ilyasli/ Merkez Murdumuk/Fasil 770<br />
51 LS-6416 Ilyasli/ Merkez Murdumuk/Fasil 770<br />
52 LS Released variety ‘Gurbuz-2001’ -<br />
53 LC-4801 Mugla Cumalı- /Datça Kara murdumuk ?<br />
*Commercial material; ** first two numbers are city traffic code and last two numbers are sample number; LS, L. sativus; LC, L. clymenum.
4076 Afr. J. Biotechnol.<br />
Table 2. Local use, morphological and chemical features of accessions of Lathyrus sp.<br />
Accession* Local Use Seed coat color*** 1000 seed weight (g) Crude protein (%) β-ODAP (mg g -1 )<br />
LS-0201 Feed G+B 92.0 30.90 1.90<br />
LS-0202 Feed G+B 84.9 28.03 1.66<br />
LS-1501 Feed+food G 110.6 30.04 2.19<br />
LS-1502 Feed+food G+B 86.4 29.00 1.75<br />
LS-1503 Feed+food G+B 94.4 30.09 3.24<br />
LS-1601 Feed+food G+B+C 102.3 30.57 1.68<br />
LS-1602 Feed+food C+B 134.0 28.00 2.32<br />
LS-1603 Feed+food G+B 107.3 28.61 2.38<br />
LS-1604 Feed G+B 99.6 27.61 2.62<br />
LS-1801 Feed G+B 97.8 27.98 1.83<br />
LS-1802 Feed G+B 122.6 27.50 2.06<br />
LS-1803 Feed B 104.7 28.50 3.86<br />
LS-2001 Feed G+B 76.3 29.34 2.30<br />
LS-2002 Feed G+B 80.6 30.10 2.07<br />
LS-2003 Feed G+B 121.5 26.96 2.02<br />
LS-2004 Feed G+B 115.0 26.67 3.33<br />
LS-2005 Feed G+B 77.2 27.00 3.84<br />
LS-2006 Feed B 73.3 26.23 1.74<br />
LS-2007 Feed G+B 90.5 27.86 2.26<br />
LS-2008 Feed G+B 101.5 27.18 1.71<br />
LS-2301 Feed B 106.3 29.27 1.74<br />
LS-2302 Feed B 72.2 27.34 3.15<br />
LS-2303 Feed G+B 89.94 27.80 2.25<br />
LS-4301 Feed G+B+C 118.6 30.41 1.81<br />
LS-4401 Feed G+B 80.8 30.33 1.41<br />
LS-4402 Feed G+B 101.8 28.62 1.99<br />
LS-4403 Feed B 103.3 28.16 2.02<br />
LS-4404 Feed G 129.0 27.02 2.67<br />
LS-5001 Feed G+B 106.5 28.55 2.18<br />
LS-5002 Feed G+B 102.2 28.69 2.20<br />
LS-5003 Feed G+B 112.2 27.99 2.00<br />
LS-5004 Feed G+B 103.4 27.98 2.07<br />
LS-5005 Feed G+B 140.8 26.23 2.93<br />
LS-5006 Feed B 113.6 28.80 2.06<br />
LS-5501 Food C 148.0 24.07 1.80<br />
LS-6401 Feed G+B 88.3 28.77 1.54<br />
LS-6402 Feed G+B 95.4 28.21 1.42<br />
LS-6403 Feed G+B 107.3 27.91 1.89<br />
LS-6404 Feed G+B 92.2 27.63 1.85<br />
LS-6405 Feed G+B 108.9 29.19 1.72<br />
LS-6406 Feed G+B 75.3 29.78 1.35<br />
LS-6407 Feed G+B 107.5 28.60 2.42<br />
LS-6408 Feed G+B 79.7 28.32 2.59<br />
LS-6409 Feed G+B 76.8 26.60 2.36<br />
LS-6410 Feed G+B 104.0 27.63 2.32<br />
LS-6411 Feed G 85.4 27.78 1.91<br />
LS-6412 Feed G+B 93.4 28.09 1.71<br />
LS-6413 Feed G+B 97.1 27.61 2.38<br />
LS-6414 Feed G+B 85.0 29.70 2.65<br />
LS-6415 Feed G+B 90.3 29.51 1.69<br />
LS-6416 Feed G+B 86.7 29.15 2.90<br />
LS** Feed B 117.8 25.81 2.50<br />
LC-4801 Food B 118.8 26.33 2.68<br />
* LS, L. sativus; LC, L. Clymenum; ** released variety; *** G, grey; B, brown; C, creamy.
Frequency<br />
14<br />
12<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
60.00 80.00 100.00 120.00 140.00 160.00<br />
1000 seed weight (g)<br />
Figure 2. General distribution of Lathyrus landraces for 1000 seed weight.<br />
β-ODAP levels of the samples were between 1.35 and<br />
3.86 mg g -1 (Table 2) with a mean of 2.21 mg g -1 (Figure<br />
4.) The highest toxic level was determined in the landrace<br />
collected from Cankiri numbered 1803, while the lowest<br />
level was obtained in the landrace collected from Usak<br />
numbered 6406 (Table 2). The β-ODAP concentration in<br />
the released variety named ‘Gurbuz- 2001’ was higher<br />
than the mean of all the samples (2.50 mg g -1 ). Low toxic<br />
levels generally, were in landraces belonging to L. sativus<br />
and β-ODAP content was determined as 2.68 mg g -1 in L<br />
clymenum seed (Table 2).<br />
β-ODAP concentration of L. sativus seeds varied between<br />
0.2 to 7.2 mg g -1 in dry matter (Deshpande and<br />
Campbell, 1992). For safe consumption by humans, β-<br />
ODAP content of seeds has to be lower than 2.2 mg g -1<br />
(Abd El Moneim et al., 1999). In the present work, β-<br />
ODAP was lower than 2.00 mg g -1 in 21 L. sativus<br />
landraces (Figure 3), indicating the importance of land-<br />
Mean = 100.2083<br />
Std. Dev. = 17.31015<br />
N = 53<br />
Basaran et al. 4077<br />
races in screening for low toxic content.<br />
There has been no lathyrism case documented in<br />
human or animals up till now in Turkey. Also, farmers did<br />
not know anything about lathyrism or any toxic effect of<br />
Lathyrus seeds on human or animals. In fact, lathyrism<br />
risk is very low in Turkey. This is probablly due to low and<br />
rare consumption, soaking procedure and low β-ODAP<br />
content, because, the grain of Lathyrus sativus is rarely<br />
used in human diet for stock feeding in the country.<br />
Neurotoxic β-ODAP is a water-soluble amino acid that<br />
can be leached from seed by soaking in water (Akalu et<br />
al., 1998). On the other hand, Lahyrism is induced by<br />
heavy and prolonged (more than three months) consumption<br />
of the L. sativus seeds (Mehta et al., 1994).<br />
Genotype is the most important factor determining<br />
ODAP concentration; environment has less influence<br />
(Hanbury et al., 1999). However, environmental factors<br />
such as drought, zinc deficiency, iron oversupply and the
4078 Afr. J. Biotechnol.<br />
Frequency<br />
20<br />
15<br />
10<br />
5<br />
0<br />
24.00<br />
25.00 26.00 27.00 28.00 29.00 30.00 31.00<br />
Protein (%)<br />
Figure 3. General distribution of Lathyrus landraces for seed protein contents.<br />
Frequency<br />
10<br />
8<br />
6<br />
4<br />
2<br />
0<br />
1.00<br />
B-ODAP (mg/g)<br />
Mean = 2.206<br />
Std. Dev. = 0.56494<br />
N = 53<br />
1.50 2.00 2.50 3.00 3.50 4.00<br />
Figure 4. General distribution of Lathyrus landraces for seed β-ODAP contents.
presence of heavy metals in soil can considerably<br />
increase the level of β-ODAP in seeds (Lambein et al.,<br />
2007). So, the development of varieties for a specific<br />
region is one of the effective solutions to avoid lathyrism.<br />
At this point, local landraces will be usefull. Analysis of a<br />
large number of germplasm of L. sativus showed that,<br />
samples originated from Africa, Syria, Turkey and Cyprus<br />
had significantly lower content in the dry seed than in the<br />
samples originated from Bangladesh, Ethiopia, India,<br />
Nepal and Pakistan (Abd El Moneim et al., 2001). Therefore,<br />
the variation in this study on β-ODAP level was<br />
attributed both to genetic factors and environmental<br />
conditions. For this reason, there are needs for new<br />
studies, to determine the β-ODAP levels of these landraces<br />
in different ecological conditions.<br />
L. sativus, which is a drought tolerant legume, is a<br />
promising crop for the diversification of cropping systems<br />
and sustainability of agriculture in marginal and drought<br />
areas. Furthermore, under the risks of climate change<br />
which is supposed to cause serious drought, the plant will<br />
become a more important crop in the world. So, future<br />
studies should concentrate on developing new varieties<br />
with high yield, low β-ODAP and high protein level. The<br />
results of this showed that, L. sativus landraces cultivated<br />
in Turkey, are promising genetic materials especially with<br />
their low β-ODAP contents.<br />
ACKNOWLEDGEMENTS<br />
The authors’ thanks go to Prof. Dr. A. Nur ONAR and Dr.<br />
Meral KARACAN of OMU, Science and Art Faculty,<br />
Chemistry Department, for their help in the CZE analysis.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4081-4085, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2427<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Optimization of a plant regeneration protocol for<br />
broccoli<br />
Ke Huang 1,2,3*,# , Qiuyun Wu 1,2# , Juncheng Lin 3 and Jingui Zheng 3<br />
1 Hunan provincial key laboratory for germplasm innovation and utilization of crop, Changsha, 410128, P. R. China.<br />
2 College of Horticulture and Landscape, Hunan Agricultural University, Changsha, 410128, P. R. China.<br />
3 Institute of Agricultural Product Quality, Fujian Agricultural and Forestry University, Fuzhou, 350002, P. R. China.<br />
Accepted 18 March, 2011<br />
The factors which influence the regeneration of broccoli (Brassica oleracea var. italica) were studied<br />
using an orthogonal design. The results showed that the major factor was the explant type, followed by<br />
naphthylacetic acid (NAA), benzylaminopurine (BAP), sucrose and AgNO3 in turn. The maximum<br />
regeneration was on Murashige and Skoog (MS) medium + NAA 0.107 µM + BAP 17.76 µM + 2% sucrose<br />
+ 0.8% agar. Hypocotyl proved to be the optimum explant source and its regeneration frequency<br />
reached 100%.<br />
Key words: Naphthylacetic acid (NAA), benzylaminopurine (BAP), AgNO3, hypocotyl, broccoli (Brassica<br />
oleracea var. italica).<br />
INTRODUCTION<br />
Broccoli (Brassica oleracea L. var. italica Plenck), which<br />
originated from Italy and is related to the cabbage and<br />
cauliflower, is a very important vegetable crop. It is well<br />
known for its high vitamin, calcium and sulforaphane<br />
content (Zhang et al., 1992; Henzi et al., 2000). Plant<br />
diseases, insects and other stressors can cause enormous<br />
yield reductions during commercial broccoli<br />
cultivation (Yang et al., 2002; Cao and Earle, 2003;<br />
Viswakarma et al., 2004) but, under modern production<br />
systems, genetic engineering can be used to add target<br />
characteristics to broccoli cultivars. Transgenic B. oleracea<br />
plants have been obtained using several methods<br />
involving Agrobacterium-mediated transformation (Boulter<br />
et al., 1990; Toriyama et al., 1991; Berthomieu and<br />
Jouanin, 1992; Christey and Sinclair, 1992; Metz et al.,<br />
1995; Christey et al., 1997; Higgins et al., 2006; Jocelyn<br />
et al., 2007). The efficiency of such Agrobacterium-<br />
*Corresponding author. E-mail: huangke@yahoo.cn Tel.:86-<br />
731-84617028, Fax: 86-731-84617181.<br />
Abbreviations: MS, Murashige and Skoog; BAP,<br />
benzylaminopurine; NAA, naphthylacetic acid.<br />
#Both are Co-authors.<br />
mediated transformation is known to be affected by<br />
multiple factors such as the bacterial strain and<br />
concentration used, the plant genotype, explant type and<br />
co-cultural conditions (van Wordragen and Dons, 1992;<br />
Puddephat et al., 1996; Birch 1997; Cogan et al., 2002;<br />
Chakrabarty et al., 2002; Kim and Botellam, 2002; Nigel<br />
et al., 2002; Suri et al., 2005). Most authors have<br />
reported difficulties with one or more of the stages in the<br />
gene transfer and regeneration process which prevent<br />
any of their protocols from serving as the method of<br />
choice for B. oleracea transformation. Transformation<br />
efficiency can however, be increased by manipulating<br />
many factors, and the important factors are the explant<br />
and the plant growth regulator.<br />
There have been many reports on plant regeneration of<br />
Brassica crops, but they studied the factors that affect<br />
plant regeneration separately, such as explant type (Dai<br />
et al., 2009), plant regulator concentration (Dai et al.,<br />
2009; Ravanfar et al., 2009) and sucrose concentration.<br />
This study reports the optimization of a system for<br />
broccoli regeneration by orthogonal design. The<br />
improved regeneration was achieved by systematically<br />
optimizing the combination of factors that affect plant<br />
regeneration using an orthogonal design (Tang and<br />
Feng, 1997) in Catharanthus Roseus (Sun et al., 2002),<br />
white clover (Zhang et al., 1998) and so on. Together,
4082 Afr. J. Biotechnol.<br />
Table 1. Orthogonal design table.<br />
Medium<br />
no.<br />
Level code NAA (µM) BAP (µM) AgNO3 (mM) Sucrose (%) Explant<br />
1 0 0 0 1 Cotyledon<br />
2 0.054 8.88 0.02 2 Cotyledon plus petiole<br />
3 0.107 17.76 0.04 3 Petiole<br />
4 0.161 26.64 0.06 4 Hypocotyl<br />
Table 2. Broccoli regeneration frequency results*.<br />
NAA<br />
(µM)<br />
BAP<br />
(µM)<br />
AgNO3<br />
(mM)<br />
Sucrose<br />
(%)<br />
Explant<br />
Mean number of<br />
shoots per explant<br />
Regeneration<br />
frequency (%)<br />
1 1(0) 1(0) 1(0) 1(1) 1 (Cotyledon) 1.4±0.54 6±1.58 K<br />
2 1(0) 2(8.88) 2(0.02) 2(2) 2 (Cotyledon plus petiole) 2.0±0.71 22±3.53 F<br />
3 1(0) 3(17.76) 3(0.04) 3(3) 3 (Petiole) 1.6±0.54 9.33±1.78 IJ<br />
4 1(0) 4(26.64) 4(0.06) 4(4) 4 (Hypocotyl) 5.8±1.48 92.22±0.84 B<br />
5 2(0.054) 1(0) 2(0.02) 3(3) 4 (Hypocotyl) 3.6±0.89 72.89±2.28 D<br />
6 2(0.054) 2(8.88) 1(0) 4(4) 3 (Petiole) 2.0±1.22 13.78±1.10 H<br />
7 2(0.054) 3(17.76) 4(0.06) 1(1) 2 (Cotyledon plus petiole) 1.6±0.89 8.22±1.30 JK<br />
8 2(0.054) 4(26.64) 3(0.04) 2(2) 1 (Cotyledon) 0.0±0.00 0±0.00 L<br />
9 3(0.107) 1(0) 3(0.04) 4(4) 2 (Cotyledon plus petiole) 2.0±1.00 9.11±1.22 IJ<br />
10 3(0.107) 2(8.88) 4(0.06) 3(3) 1 (Cotyledon) 2.2±1.30 11.78±1.48 HI<br />
11 3(0.107) 3(17.76) 1(0) 2(2) 4 (Hypocotyl) 6.4±1.14 100±0.00 A<br />
12 3(0.107) 4(26.64) 2(0.02) 1(1) 3 (Petiole) 1.8±1.10 8.67±1.92 JK<br />
13 4(0.161) 1(0) 4(0.06) 2(2) 3 (Petiole) 2.4±0.89 16.89 ±2.59 G<br />
14 4(0.161) 2(8.88) 3(0.04) 1(1) 4 (Hypocotyl) 3.8±0.84 86.67±0.89 C<br />
15 4(0.161) 3(17.76) 2(0.02) 4(4) 1 (cotyledon) 2.4±1.14 8.89±1.64 IJK<br />
16 4(0.161) 4(26.64) 1(0) 3(3) 2 (Cotyledon plus petiole) 2.8±1.30 28.89±1.87 E<br />
*The regeneration frequency was recorded 21 d after inoculated, 450 explants were used for the experiment, A-F expresses differences at the<br />
level ofα=0.01 level.<br />
these modifications resulted in a notable improvement in<br />
the regeneration rate of the species.<br />
MATERIALS AND METHODS<br />
Broccoli (B. oleracea L. var. italica Plenck) cv Xinlv was provided by<br />
the Jiangsu Academy of Agricultural Sciences, Nanjing. Seeds,<br />
surface sterilized in 70% ethanol for 90 s, 0.1% HgCl2 for 12 min,<br />
were germinated at 25°C on solid MS medium (Murashige and<br />
Skoog, 1962) containing 2% sucrose (w/v) (pH 5.8). A 16 h<br />
photoperiod was used.<br />
Medium constituents and culture conditions<br />
All media used in this experiment were of MS type with 0.8% agar<br />
plus different concentrations of benzylaminopurine (BAP: 0, 8.88,<br />
17.76 and 26.64 µM) and naphthylacetic acid (NAA: 0, 0.054, 0.107<br />
and 0.161 µM). The medium with a pH adjusted to 5.8 was<br />
dispensed into 100 ml Erlenmeyer flasks before autoclaving at<br />
121°C and 1.2 ~ 1.3 kgcm -2 for 20 min, and silver nitrate (AgNO3: 0,<br />
0.02, 0.04 and 0.06 mM) was added after being filter sterilized. The<br />
medium was dispensed into 11 cm Petri dishes. The culture<br />
conditions were 25 ± 1°C and a 16 h photoperiod of approximately<br />
28 µEm -2 s -1 . The rooting medium was composed of MS + 0.107 µM<br />
NAA + 2% sucrose + 0.8% agar, at pH 5.8.<br />
Shoot and root induction<br />
Cotyledon, cotyledon with petiole attached, petiole and hypocotyl<br />
explants were isolated from 7 days old germinated seedlings and<br />
cultured on regeneration medium for adventitious bud induction<br />
(Table 1), the orthogonal combination scheme is provided in Table<br />
2. Five replications and 90 explants per replicate was used for each<br />
treatment, and data variance analysis by DPS was used (Tang et<br />
al., 1997).<br />
Explants were inoculated on differentiation regeneration media<br />
for adventitious bud induction. Adventitious buds were then cut and<br />
transplanted onto root induction media. The root induction medium<br />
composed of MS + NAA (0, 0.54 and 1.07 µM) + 3% sucrose +<br />
0.8% agar, and the regenerated plants were transplanted onto<br />
vermiculite medium after 2 weeks of root induction.<br />
RESULTS<br />
By examining the combination of factors that affected<br />
broccoli regeneration using an orthogonal design and by
Huang et al. 4083<br />
Figure 1. The regeneration of broccoli hypocotyle from cv ‘Xinlv’ (B. oleracea var. italica cv ‘Xinlv’). A: Calli formation (7 days); B:<br />
formation of the regenerated bud (21 days); C: further growth of a regenerated bud (42 days); D: root formation in the regenerant<br />
(56 days); E: The transplantation of a regenerated broccoli seedling (90 days); F: flower head formation (150 days).<br />
the use of data variance analysis by DPS (Tang et al.<br />
1997), it was apparent that the most suitable condition for<br />
Xinlv was: MS + NAA 0.107 µM + BAP 17.76 µM + 2%<br />
sucrose + 0.8% agar. Hypocotyls were the best explant<br />
source (Fexplants = 1161.897, F0.01 = 5.29) with a regeneration<br />
frequency of 100% (Table 2 and Figure 1).<br />
The effect of plant growth regulator on broccoli<br />
regeneration<br />
The plant growth regulator kind and concentration have<br />
major effect on the broccoli callus abduction and differentiation.<br />
The results indicated that the different NAA and<br />
BAP level have highly significant difference on broccoli<br />
regeneration (FNAA = 18.8723, FBAP = 8.3232, F0.01 = 5.29),<br />
and 0.107 µM NAA, 17.76 µM BAP is the best for the<br />
broccoli regeneration.<br />
The effect of silver nitrate on broccoli regeneration<br />
Broccoli regeneration is not sensentive on the silver<br />
nitrate, the broccoli adventitious bud regenerated better<br />
while there is no silver nitrate in the medium than the<br />
medium with silver nitrate. So for the broccoli regeneration<br />
medium, the silver nitrate should not be added.<br />
The effect of sucrose concentration on broccoli<br />
regeneration<br />
There is highly significant difference level of different<br />
sucrose concentration on broccoli regeneration (Fsucrose =<br />
6.45344, F0.01 = 5.29), and the 2% sucrose is the best<br />
concentration for broccoli regeneration.<br />
The effect of explant type on broccoli regeneration<br />
We selected the cotyledon plus petiole, hypocotyls,<br />
cotyledon and petiole as the adventitious bud induction<br />
explants. The different explant type has highly significant<br />
difference in the broccoli adventitious bud induction<br />
(Fexplant = 1081.924, F0.01 = 5.29).<br />
Regenerated buds were placed on rooting medium and<br />
that which proved optimum for rooting was MS + 1.07 µM<br />
NAA + 3% sucrose + 0.8% agar with a rooting success<br />
rate of 100%. All roots produced on this media were<br />
rosettes, whilst buds failed to develop on any other media.
4084 Afr. J. Biotechnol.<br />
DISCUSSION<br />
The establishment of a regeneration system is a major<br />
step in the development of transgenic technology for<br />
Brassica vegetables. This paper defined such a system<br />
for B. oleracea L. var. italica Plenck using selection based<br />
on the optimum explants type, plant growth regulators,<br />
sucrose and AgNO3 concentration which proved capable<br />
of achieving 100% regeneration from hypocotyl. The kind<br />
and concentration of plant growth regulators, sucrose<br />
concentration and the explant type all affected the<br />
regeneration of broccoli, while AgNO3 also had some<br />
influence on the successfof regeneration percentage. The<br />
experiment validates the conclusions reached using other<br />
broccoli varieties in which the regeneration frequency<br />
was also 100% and suggests that the regeneration system<br />
is applicable to a range of broccoli varieties (Figure<br />
1). The kind and concentration of plant growth regulators<br />
also have a strong effect on broccoli callus induction and<br />
differentiation (He et al., 1998; Zhang and Gong, 2001;<br />
Zhao et al, 2002; Ravanfar et al., 2009; Dai et al., 2009;<br />
Ravanfar et al., 2009). Significant effects of NAA and<br />
BAP levels on regeneration (FNAA = 23.2379, FBAP =<br />
10.0031, F0.01 = 5.29) indicate that both affect<br />
regeneration frequencies. Ravanfar et al., (2009)<br />
indicated that 96.67% of hypocotyl explant produced<br />
shoot on 3 mg·L - ¹ (13.32 µM) BAP, this study has<br />
presented 100% regeneration at 4 mg·L -1 (17.76 µM),<br />
because there is a combination relationship among the<br />
NAA, BAP, AgNO3 and the explants. We studied the<br />
factors systematically, and the results of regeneration<br />
frequency (100%) and mean number of shoots per<br />
explant (6.4) indicated that this is the best recipe for<br />
broccoli regeneration in this paper.<br />
The results indicated that the medium lacking AgNO3<br />
was optimum for broccoli regeneration (FAgNO3 = 22.1231,<br />
F0.01 = 5.29). It may therefore be appropriate to tailor the<br />
AgNO3 concentration in the medium according to the<br />
plant genotype (Chi et al, 1990). The sucrose<br />
concentration was found to significantly affect the degree<br />
of regeneration (Fsucrose = 8.1219, F0.01 = 5.29). Several<br />
reports have described variety-specific differences in in<br />
vitro organogeny or embryogenic development in<br />
response to contrasting sucrose concentrations (He et<br />
al., 1998; Kantia and Kothari, 2002; Tang et al., 2002).<br />
Sucrose concentration has an effect on adventitious bud<br />
induction and the amount of cluster buds. The<br />
differentiation ability and plant growth regulator level of<br />
explants depend upon the position and timing of excision,<br />
which also affect the regeneration frequency (Murata and<br />
orton, 1987). Hypo-cotyls, cotyledons, petioles and<br />
cotyledons in combi-nation with their petioles have been<br />
used as explants in this study. Some of these explants<br />
have a high regeneration frequency and are easily<br />
infected by Agro-bacterium, making them good transform<br />
explants. Cotyledons plus petioles are usually chosen as<br />
explants during Brassica regeneration because, when cut<br />
from a growth point, they have some primordial meriste-<br />
matic cells and possess higher regeneration frequencies<br />
(Birch, 1997; Kim and Botellam, 2002; Murata and Orton,<br />
1987; Puddephant et al., 1996). It was apparent that<br />
hypocotyls were the best explants for broccoli<br />
regeneration in this study as they exhibited higher<br />
differentiation ability and the plant growth regulator<br />
combination was suitable for bud induction. It has been<br />
proposed that polarity occurs during Brassica hypocotyl<br />
regeneration but, in agreement with Li and Chen (1993;<br />
1994), this was not observed in the present experiment<br />
using B. oleracea L. var. italica Plenck.<br />
Conclusions<br />
The best regeneration recipe for the broccoli is on medium<br />
MS + NAA 0.107 µM + BAP 17.76 µM + 2% sucrose<br />
+ 0.8% agar, and the best explant source is hypocotyl.<br />
Based on this menu, the regeneration frequency of<br />
broccoli reached 100%.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by the Natural Sciences Foundation<br />
of China (30600415). We thank Birt Stevens for<br />
critically reviewing the manuscript.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4086-4092, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2455<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Evaluation of bread wheat genotypes for salinity<br />
tolerance under saline field conditions<br />
Munir Ahmad 1 , Muhammad Munir 1 , Iftikhar Ahmad 2 and Muhammad Yousuf 2<br />
1 Pir Mehar Ali Shah Arid Agriculture University Murree road 46300 Rawalpindi Pakistan.<br />
2 National Agricultural Research Center Park road 45500 Islamabad Pakistan.<br />
Accepted 25 March, 2011<br />
In two consecutive seasons (2007-08 and 2008-09), field experiments were conducted at Soil Salinity<br />
Research Institute, Pindi Bhattian and Biosaline Agricultural Research Station, Pakka Aana, Pakistan.<br />
During 2007-08, 103 wheat landrace genotypes were evaluated for salinity tolerance. During 2008-09, 47<br />
selected genotypes were evaluated at the same locations. Combined analysis of both locations during<br />
both years revealed that genotypes differed significantly for plant height, dry biomass m -2 , fertile tillers<br />
plan -1 , spike length, grain spike -1 , 1000 seed weight and yield m -2 . Grain yield had strong positive<br />
correlation with plant height, dry biomass, spike length, spikelets spike -1 , grain spike -1 and 1000 grain<br />
weight. Due to this positive correlation, yield can be used as a selection criterion under saline field<br />
conditions. Accessions 10807 (Pak), 11299 (Pak), 11917 (Iran) and cultivars Pavon (Pak) performed better<br />
during both years. These genotypes could be effectively used as new sources of salt tolerance.<br />
Key words: Genetic divergence, bread wheat, field evaluation, salinity tolerance.<br />
INTRODUCTION<br />
Salinity is a major abiotic stress limiting crop production<br />
in many agricultural regions of the world (Katerji et al.,<br />
2009). Over 800 million hectares of land are affected by<br />
salt worldwide (Munns, 2005). Some of the most serious<br />
examples of salinity occur in the arid and semiarid<br />
regions. In Pakistan, more than 10 million hectares are<br />
affected by salt (FAO, 2008). A low level of salinity may<br />
not reduce grain yield, although shoot biomass is reduced,<br />
which is reflected in a harvest index. Wheat yield<br />
is substantially reduced as the soil salinity level rises to<br />
100 mM NaCl (Munns et al., 2006).<br />
Screening large population for salinity tolerance in the<br />
field is difficult due to tremendous heterogeneity of saline<br />
soils (Richards, 1983). However, various statistical<br />
*Corresponding author. E-mail: zm_muneer@yahoo.com.<br />
Abbreviations: ECe, Electrical conductivity; SAR, sodium<br />
absorption ratio; SSRI, soil salinity research institute; BARS,<br />
Biosaline Agricultural Research Station; RSCw, residual sodium<br />
carbonate.<br />
techniques have been used to tackle similar problems,<br />
e.g. by blocking, particularly the use of small blocks<br />
(Bartlett, 1978). These have reduced error variations<br />
and increased detection of varietal differences, and can<br />
be adapted to cope with the high heterogeneity of saline<br />
fields. Most of the experiments are carried out under<br />
controlled conditions where plants are not exposed to<br />
those conditions that prevail in salt affected field<br />
conditions such as spatial and temporal heterogeneity of<br />
soil chemical and physical properties (Munns and James,<br />
2003). Therefore, salt tolerance of genotypes under field<br />
conditions needs to be evaluated particularly as a<br />
function of yield that is considered as a foremost target of<br />
the plant breeder (Yamaguchi and Blumwald, 2005).<br />
Kingsbury and Epstein (1984) evaluated 5000<br />
accessions of bread wheat in 50% seawater and identified<br />
29 accessions that produced seed. Jafari-Shabestari<br />
et al. (1995) evaluated 400 Iranian wheat genotypes in<br />
irrigated field conditions in California and identified<br />
numerous accessions that were consistently high for<br />
grain yield in both low and high salinity treatments.<br />
Ahmad et al. (2005) studied six wheat varieties in salt<br />
affected soils and reported that salt tolerant varieties
Table 1 Physico-chemical analysis of the soils sampled from site 1 (BARS) and site 2 (SSRI).<br />
Season 2007-2008 2008-2009<br />
Parameter Unit Site 1 Site 2 Site 1 Site 2<br />
pH – 8.1 8.3 8.2 8.4<br />
ECe dS m -1 16.9 13.4 17.8 14.6<br />
SAR – 1.72 14.32 5.49 16.45<br />
CaCO3 % 1.42 1.23 1.39 1.21<br />
OM % 0.70 0.30 0.69 0.29<br />
N % 0.03 0.04 0.03 0.02<br />
Extractable P mg kg -1 4.12 3.47 3.75 2.10<br />
Extractable K mg kg -1 33.5 54.6 35.4 55.5<br />
Sand % 42.7 12.5 - -<br />
Silt % 37.2 34.2 - -<br />
Clay % 20.1 53.3 - -<br />
Textural class – Sandy loam Clayey - -<br />
Table 2. Analysis of variance of 7 traits studied in 103 wheat genotypes at SSRI and BARS during 2007-08.<br />
Source of variation<br />
Plant height<br />
(cm)<br />
Total bio<br />
mass m -2 (g)<br />
Sum of squares (Percentage of total)<br />
Spike length<br />
(cm)<br />
Spikelets<br />
spike -1<br />
Grains<br />
spike -1<br />
Ahmad et al. 4087<br />
Yield m -2<br />
(g)<br />
1000 seed<br />
weight (g)<br />
Location 22.87** 33.56** 0.17 0.31 10.62* 38.55** 0.38<br />
Replication 0.34 0.21 2.95** 1.22* 1.79** 0.72* 0.73<br />
Block (loc* rep) 9.66** 5.07** 5.33** 5.00 4.61* 4.60** 3.38<br />
Genotypes 35.94** 23.70** 40.89** 33.10** 34.64** 17.88** 39.10**<br />
Loc*genotype 14.48** 17.86** 26.62** 26.38* 25.82** 19.30** 32.54**<br />
Error 16.71 19.60 24.04 33.99 22.52 18.95 23.87<br />
Total sum of squares 141624.11 7974152.40 841.77 2484.76 33713.58 806159.07 1950.14<br />
CV 14.62 53.91 12.52 15.17 22.82 51.62 13.38<br />
*Significant at 5% probability level; **Significant at 1% probability level.<br />
produced greater yield than salt susceptible due to higher<br />
dry weight of shoot and spike and better grain development.<br />
El-Hendawy et al. (2009) evaluated wheat genotypes<br />
and reported that grain weight plant -1 , number of grains<br />
plant -1 and number of fertile spikes plant -1 are good<br />
screening criteria under field conditions.<br />
The objectives of the present study were to evaluate<br />
the genetic diversity in 103 wheat landraces/cultivars for<br />
salinity tolerance under saline field conditions and to<br />
identify the parents for breeding salt tolerant cultivars.<br />
MATERIALS AND METHODS<br />
Seeds of 103 wheat landraces/cultivars (Table 2) including 4 check<br />
cultivars namely: SARC III, SARC IV, SARC V and SARC VII, were<br />
obtained from the gene bank of Plant Genetic Resources Program,<br />
National Agricultural Research Centre, Islamabad; Saline<br />
Agriculture Research Center (SARC), University of Agriculture,<br />
Faisalabad, and Ayyub Agricultural Research Institute, Faisalabad,<br />
Pakistan. The landraces/cultivars included 64 accessions from<br />
Pakistan, 20 from Iran, 7 from Syria, 4 from Egypt and 8 from Italy.<br />
The seeds were sown at Soil Salinity Research Institute (SSRI),<br />
Pindi Bhattian and Biosaline Agricultural Research Station (BARS),<br />
Pakka Aana (Pakistan) during the growing seasons 2007-08.<br />
Incomplete block design was followed with 2 replications keeping<br />
row to row distance of 30 cm and row length of 4 m. The<br />
experiment consisted of 12 small blocks. ECw (electrical conductivity<br />
of water) used for irrigation was 2.0 to 2.4 dSm -1 at SSRI and 3.4 to<br />
3.7 dSm -1 at BARS, while RSCw (residual sodium carbonate) was<br />
1.0 to 1.5 at SSRI and 2.0 to 2.5 meq L -1 at BARS. Soil analysis of<br />
both locations is given in the Table 1. Data were recorded from 10<br />
randomly selected plants from each replication for biomass m -2 ,<br />
plant height, spike length, number of spikelets spike -1 , number of<br />
grains spike -1 , 1000 gain weight and grain yield m -2 . During 2008-<br />
2009, 47 salt tolerant accessions selected from both locations<br />
during 2007-2008 on the basis of yield m -2 and germination and<br />
hydroponic testing at 200 mM NaCl stress (data not given) were<br />
sown at the both earlier mentioned locations following the same<br />
procedure of 2007-2008.<br />
The data were subjected to analysis of variance using the GLM<br />
procedure in SAS (SAS Institute, 2003). The means obtained were<br />
separated by standard error of differences of means. The<br />
correlation coefficients among all the traits were computed following<br />
Kown and Torrie (1964). All the traits showed strong positive cor-
4088 Afr. J. Biotechnol.<br />
Table 3. Salt tolerance categories of 103 wheat genotypes on the basis of average yield of 2 locations.<br />
Salt tolerant<br />
category<br />
Grain yield<br />
(g/m -2 )<br />
Number of<br />
accessions<br />
Accessions (country of collection)<br />
Tolerant 95 (g/m -2 ) and above 5 10795 (Pakistan), 10851 (Pakistan) 11299 (Pakistan), 10807 (Pakistan)<br />
and 11302 (Pakistan)<br />
Moderately<br />
tolerant<br />
Moderately<br />
susceptible<br />
67 - 95 (g/m -2 ) 27 Sakha-92 (Egypt), 11464 (Syria), Maroon (Iran), 13193 (Pakistan),<br />
Roushan (Iran), 11465 (Syria), 11462 (Syria), 11244 (Pakistan), 11186<br />
(Pakistan), 10798 (Pakistan), 10803 (Pakistan), 11386<br />
(Pakistan),11384 (Pakistan), 10862 (Pakistan), 11406 (Pakistan),<br />
11478 (USA), 10834 (Pakistan), Pavon (Pakistan), 10833<br />
(Pakistan),10800 (Pakistan), 11526 (Pakistan), 11193 (Pakistan),<br />
11387 (Pakistan), 11419 (Pakistan), 11272 (Pakistan), SARC-VII<br />
(Pakistan), 11248 (Pakistan)<br />
39 - 67(g/m -2 ) 47 12114 (Pakistan), 11195 (Pakistan), 10783 (Pakistan), Tabasi (Iran),<br />
12119 (Pakistan), Arvand (Iran), ADL (Iran), 10853 (Pakistan), 10849<br />
(Pakistan), SARC IV (Pakistan), 11467 (Syria), 11418 (Pakistan), Giza-<br />
163 (Egypt), Karaj-II (Iran), 11133 (Pakistan), 11414 (Pakistan), 11415<br />
(Pakistan), 11374 (Italy), 12118 (Iran), 11171 (Pakistan), 11457 (Syria),<br />
13184 (Pakistan), Falat (Iran), 10854 (Pakistan), Moghan-1 (Iran),<br />
10809 (Pakistan), 12117 (Pakistan), 11240 (Pakistan), Khazar-1 (Iran),<br />
10777 (Pakistan), 11454 (Syria), 10784 (Pakistan), Chenab (Iran),<br />
11399 (Pakistan), 11407 (Pakistan), Kayeh (Iran), Sardari (Iran), Chods<br />
(Iran), 11458 (Syria), 4098785 (Pakistan), 11409 (Pakistan), 10824<br />
(Pakistan), 11416 (Pakistan), 11373 (Italy), SARC III (Pakistan),<br />
4098795 (Pakistan), 11388 (Pakistan)<br />
Susceptible 39 (g/m -2 ) and below 24 India (Iran), 11378 (Pakistan), 10812 (Pakistan), Karaj-1 (Iran), Darab<br />
(Iran), Omid (Iran), 10788 (Pakistan), 4098815 (Pakistan), Giza-155<br />
(Egypt), 12113 (Pakistan), 11371 (Italy), 11371 (Italy), 10811<br />
(Pakistan), SARC-V (Pakistan), 11372 (Italy), Rasool (Iran), Hirmand<br />
(Iran), Alborz (Iran), 11370 (Italy), 11901 (Italy), 11290 (Pakistan),<br />
11405 (Pakistan), 11792 (Pakistan), 11369 (Italy)<br />
relation with yield m -2 during 2007-2008, therefore, average yield of<br />
both locations was used to classify the genotypes into four groups.<br />
Difference between the range of average yield was divided into four<br />
equal groups (Table 3), namely, salt tolerant, moderately salt<br />
tolerant, moderately salt susceptible and salt susceptible.<br />
RESULTS<br />
Soil analysis of two salt affected soils revealed marked<br />
differences in ECe (electrical conductivity), SAR (sodium<br />
absorption ratio) and soil texture (Table 1). Performance<br />
of wheat genotypes under field conditions is the combined<br />
effect of genotype, environment and genotype ×<br />
environment interaction. Combined analysis of both<br />
locations during 2007-08 and 2008-09 (Tables 2 and 4)<br />
revealed that genotypes differed significantly (P < 0.01)<br />
for all the traits studied. During 2007-08, location effect<br />
was significant (P < 0.05) for plant height, total biomass,<br />
grain spike -1 and yield m -2 . During 2008-09, differences<br />
due to location were significant (P < 0.05) for all the<br />
seven traits studied. This may be due to differences in<br />
ECe and SAR of the two locations (Table 1). Block<br />
(location × replication) effect was significant (P < 0.01),<br />
except spikelets spike -1 and 1000 seed weight during<br />
2007-08, however, during 2008-09, it was significant only<br />
for plant height (P < 0.05). The possible reason for non<br />
significance may the small size of blocks. Location ×<br />
genotype effect was significant (P < 0.01) during the both<br />
years for all the traits, except, spikelets spike -1 where it<br />
was significant at P < 0.05 during 2007-08.<br />
Based on average yield of two locations, 103 wheat<br />
genotypes were classified into four groups (Table 3),<br />
namely: salt tolerant included five genotypes, all from<br />
Pakistan; moderately salt tolerant included 27 genotypes,<br />
mostly from Pakistan, Egypt, Syria and Iran; moderately<br />
salt susceptible included 47 genotypes; and salt susceptible<br />
included 24 genotypes. Most of the susceptible<br />
accessions were from Pakistan, Iran and Egypt.<br />
During 2007-08, moisture stress coupled with high<br />
salinity rise just after sowing created high ideal saline<br />
conditions for crop evaluation. This inhibited the germination<br />
and plant growth at early stage at SSRI. Due to<br />
which most of the accessions at BARS performed better<br />
as compared to SSRI. During 2008-09, a considerable
Table 4. Analysis of variance of 7 traits studied in 47 wheat genotypes at Pindi Bhattian and Pakka Aana during 2008-09.<br />
Trait studied<br />
Plant<br />
height<br />
(cm)<br />
Tiller m -2<br />
Sum of squares (Percentage of total)<br />
Spike length<br />
(cm)<br />
Spikelet<br />
s spike -1<br />
Grains<br />
spike -1<br />
Yield m -2<br />
(g)<br />
Ahmad et al. 4089<br />
1000 seed<br />
weight (g)<br />
Location 58.25** 59.89** 11.46* 28.25** 19.23** 49.26** 21.53**<br />
Replication 0.29 0.08 0.41 0.05 0.01 0.03 0.10<br />
Block (loc × rep) 0.53 2.20* 2.56 1.58 0.34 2.08 1.34<br />
Genotypes 24.36** 20.40** 45.85** 31.60** 60.89** 28.52** 51.09**<br />
Loc × genotype 10.80** 10.10** 25.72** 19.41** 14.74** 13.23** 16.50**<br />
Error 5.77 7.33 14.00 19.11 4.78 6.88 9.43<br />
Total sum of squares 49321.63 2218209.40 256.70 1048.07 12989.14 3294445.11 1050.27<br />
CV 8.53 27.28 9.14 12.29 12.08 39.23 11.74<br />
*Significant at 5% probability level; **Significant at 1% probability level.<br />
Table 5. Correlation coefficient between various traits and yield in wheat genotypes at Pindi Bhattian<br />
(PB) and Pakka Aana (PA) during 2007-08 and 2008-09.<br />
Plant height (cm)<br />
Trait studied Yield (g m -2 ) 2007-08 Yield (g m -2 ) 2008-09<br />
Biomass (g m -2 ) (2007-8)<br />
Fertile tillers plant -1 (2008-09)<br />
Spike length (cm<br />
Spikelets spike -1<br />
Grains spike -1<br />
1000 seed weight (g)<br />
**Significant at 1% probability level.<br />
reduction was manifested in plant height, fertile tillers<br />
plant-1, spike length, spikelets spike-1, grains spike-1,<br />
1000 grain weight and yield m-2 (Table 6). Five accessions<br />
produced above 100 cm tall plants at SSRI but >77<br />
cm at BARS. This showed a significant reduction in plant<br />
height at BARS as compared to SSRI (Table 6).<br />
Total dry biomass production at SSRI was less as<br />
compared to BARS during 2007-08. Accessions 10795,<br />
10807, 10851 and 11299 produced highest total biomass<br />
at both locations. The total biomass production ranged<br />
from 47.6 to 672.2 gm-2 at BARS. It revealed a huge<br />
PB 0.82** PB 0.91**<br />
PA 0.94** PA 0.77**<br />
PB 0.93** -<br />
PA 0.96** -<br />
- PB 0.94**<br />
- PA 0.95**<br />
PB 0.80** PB 0.90**<br />
PA 0.92** PA 0.74**<br />
PB 0.80** PB 0.90**<br />
PA 0.92** PA 0.74**<br />
PB 0.80** PB 0.95**<br />
PA 0.93** PA 0.73**<br />
PB 0.81** PB 0.94**<br />
PA 0.92** PA 0.76**<br />
reduction in biomass in salt susceptible accessions as<br />
compare to salt tolerant. During 2008-09, fertile tillers m-2<br />
showed a considerable reduction at both locations. It<br />
ranged from 125.4 to 409.2 at SSRI and 8 to 259 at<br />
BARS. Accessions 10824, 11287, 11214, 11383 and<br />
Local white were able to maintain maximum number of<br />
fertile tillers at both locations (Table 6). Fertile tillers m-2<br />
had a positive and highly significant correlation with grain<br />
yield m-2 during 2008-09 at both locations (Table 5).<br />
Information on the relationship between yield and its<br />
components and among the components themselves can
4090 Afr. J. Biotechnol.<br />
Table 6. Means of 7 traits studied in 47 wheat genotypes at Pindi Bhattian and Pakka Aana during 2008-09.<br />
Accession<br />
no.<br />
Plant height Fertile Tiller Plant -1 Spike length Spikelets spike -1 Grains spike -1 1000 grain weight Yield m -2<br />
PB PA PB PA PB PA PB PA PB PA PB PA PB PA<br />
Pavon 83.50 58.50 237.60 91.06 9.4 8.00 17.3 12.67 41.8 30.67 40.0 35.20 397.29 90.55<br />
10783 87.00 72.00 232.65 49.24 7.8 8.78 15.0 15.88 29.2 7.78 38.0 32.80 213.89 17.04<br />
10793 88.00 61.00 293.70 18.48 9.4 9.52 16.8 14.67 24.3 21.93 46.0 27.75 68.39 4.20<br />
10800 79.00 53.50 196.35 40.78 9.0 5.42 17.0 9.33 35.5 18.00 38.9 36.40 269.40 6.49<br />
10806 92.50 49.50 325.05 112.50 8.3 7.30 16.0 12.65 23.2 33.83 35.4 27.50 381.13 92.55<br />
10807 83.00 63.50 323.40 61.00 9.8 7.50 15.2 10.50 19.0 21.00 16.5 25.35 105.57 84.25<br />
10812 74.50 49.50 249.15 101.50 8.2 6.83 16.0 10.83 42.7 27.83 38.0 33.60 348.15 22.45<br />
10821 82.00 63.00 140.25 11.22 7.2 9.50 15.0 14.17 11.2 8.00 19.3 25.40 40.66 19.10<br />
10824 99.00 77.00 211.20 102.30 7.4 6.92 14.7 11.50 24.7 21.17 39.0 38.70 211.10 56.00<br />
10828 80.50 56.00 151.80 25.00 7.5 4.83 12.3 7.17 8.3 7.50 30.6 24.40 51.76 45.20<br />
10831 55.50 69.00 125.40 59.00 6.8 7.25 13.8 12.17 23.0 16.55 27.7 24.60 101.15 21.72<br />
11186 99.00 60.50 265.65 29.00 6.4 7.20 10.7 13.50 23.2 24.33 28.0 30.20 296.42 24.94<br />
11214 104.00 67.50 285.45 56.50 7.8 7.25 14.2 14.00 30.7 23.83 32.9 27.80 156.09 18.45<br />
11287 100.50 70.50 364.65 60.50 7.0 7.17 13.8 12.50 25.8 23.33 37.0 25.15 355.59 36.00<br />
11299 106.00 58.50 402.60 259.38 8.5 6.83 15.8 12.50 34.0 17.83 32.1 34.95 385.09 111.24<br />
11383 104.50 66.00 321.75 133.56 9.3 8.62 15.2 11.67 26.3 17.85 31.8 24.75 311.75 67.25<br />
11385 95.00 63.00 199.65 42.44 7.6 7.02 13.7 12.00 28.8 25.00 42.0 26.50 182.18 23.17<br />
11401 94.50 62.00 311.85 57.00 7.4 8.25 15.2 12.83 22.0 21.00 38.7 31.80 391.07 29.70<br />
11409 101.50 59.00 306.90 56.00 7.3 7.75 12.8 12.33 29.5 15.00 34.9 25.25 284.46 24.90<br />
11417 94.50 55.50 379.50 40.62 6.7 7.17 12.3 11.92 22.7 14.83 38.5 21.65 253.54 8.77<br />
11453 79.00 26.50 235.95 24.70 8.2 5.42 15.3 7.50 34.3 17.83 43.3 23.70 264.66 5.24<br />
11454 80.50 57.50 409.20 38.96 7.0 6.92 14.5 12.83 31.8 23.33 36.2 22.65 279.23 6.54<br />
11460 82.50 49.50 211.20 7.62 7.4 5.25 13.0 8.67 22.0 11.33 32.6 18.50 152.28 1.37<br />
11466 94.50 58.50 267.30 37.62 7.3 8.08 15.7 13.00 33.2 19.60 36.0 29.15 205.92 16.12<br />
11478 94.50 53.00 293.70 45.42 6.7 6.25 12.0 9.83 24.5 16.50 37.2 20.70 265.37 9.50<br />
11526 79.50 52.50 292.05 94.00 8.2 6.58 15.3 12.50 34.8 19.33 42.5 28.15 342.54 55.50<br />
11545 81.00 57.50 297.00 117.22 7.3 6.33 12.8 10.83 27.2 19.67 46.2 26.75 385.39 80.02<br />
Sakha 92 72.50 55.00 237.60 168.46 9.5 6.00 18.2 12.00 29.5 24.33 35.0 25.85 242.67 62.17<br />
Karaj II 83.50 57.00 232.65 104.46 7.3 6.85 14.7 12.17 26.0 19.32 29.9 25.75 119.79 34.69<br />
Chenab 80.50 66.50 184.80 49.50 8.6 8.62 14.2 14.67 34.7 26.65 42.3 24.85 217.65 29.10<br />
Maroon 76.50 46.50 343.20 39.78 7.8 5.78 15.0 11.67 31.0 14.92 44.3 32.50 313.14 11.50<br />
Roushan 110.00 61.00 364.65 131.00 8.9 8.32 15.8 13.17 30.8 15.67 39.3 34.75 417.68 62.29<br />
Bayat 71.50 58.50 272.25 29.82 8.0 6.08 16.5 9.67 37.2 17.17 35.3 26.00 260.21 11.23<br />
Omid 79.50 66.00 132.00 16.74 8.1 8.58 14.2 11.50 21.5 14.75 38.8 31.00 26.07 7.35<br />
4098775 89.00 55.00 204.60 50.82 8.6 7.58 15.7 11.83 45.0 22.98 37.1 31.30 391.46 26.00
Table 6. Contd.<br />
4098805 86.50 49.50 313.50 31.16 9.9 8.83 15.8 15.50 39.2 42.50 41.0 28.30 458.40 15.60<br />
Local white 105.50 67.50 260.70 168.30 8.9 7.50 17.5 11.67 30.2 26.67 43.5 30.75 393.57 122.69<br />
Pasban 90 68.00 46.50 255.75 58.36 9.2 7.62 14.3 12.43 33.7 34.97 35.5 27.40 281.16 33.68<br />
SARC IV 68.50 37.00 211.20 151.72 8.8 5.75 14.7 8.67 39.3 20.00 36.2 26.50 255.42 95.00<br />
SARC VII 69.50 59.50 254.10 154.10 8.3 7.3 15.5 15.5 29.5 22.5 37.9 32.50 240.97 90.97<br />
Shorawaki 81.50 70.40 166.65 160.65 9.8 8.1 20.2 16.5 12.7 19.7 16.7 22.40 24.96 86.75<br />
SEdiff x 1.74 1.52 10.31 8.27 0.15 0.19 0.26 0.32 1.24 0.16 1.19 0.72 18.04 5.12<br />
x Standard error of the difference between means; no., number.<br />
be utilized to improve the efficiency of selection in<br />
plant breeding programs. Spike length is an<br />
important yield contributing trait which showed<br />
reduction under saline field conditions. During<br />
2007-08, spike length ranged from 4.85 to<br />
13.97 cm at SSRI and from 6.1 to 11.1 cm<br />
at BARS. During 2008-09, spike length ranged<br />
from 6.4 to 11.3 cm at SSRI and 4.8 to<br />
9.5 cm at BARS (Table 6).<br />
Grains spike -1 also showed reduction at high<br />
salinity level. At SSRI, grains spike -1 ranged from<br />
4.8 to 48.17 during 2007-08, while at<br />
BARS, it was from 16.5 to 46. During 2008-<br />
09, grains spike -1 ranged from 8.3 to 45 at<br />
SSRI, while at BARS, it was from 7.5 to<br />
42.5. Reduction in grain spike-1 also causes<br />
reduction in yield at high salt stress. This<br />
reduc-tion is due to positive association<br />
between grain spike -1 and yield. Most of the<br />
accessions showed reduction in 1000 seed weight<br />
at BARS during both years. The foremost target of<br />
wheat breeder is to improve grain yield. Therefore,<br />
the assessment of final grain yield and<br />
growth parameters determining grain yield is a<br />
significant aspect of breeding programs. Five<br />
accessions: 10795, 10851, 11299, 10807 and<br />
11302 (all from Pakistan) were identified as most<br />
salt tolerant on the basis of average yield performance<br />
at two locations (Table 2). Three of them:<br />
10807, 10851 and 11299 were included in the<br />
second phase of the experiment (2008-09).<br />
Accession 10807 produced 128.71 and 91.0 gm -2<br />
yield during 2007-08 and 105.6 and 84.3 gm -2<br />
during 2008-09 at SSRI and BARS, respectively.<br />
Accession 11299 produced 93.55 and 127.8 gm -2<br />
yield during 2007-08, and 385.1 and 111.2 gm -2<br />
during 2008-09 at SSRI and BARS, respectively.<br />
DISCUSSION<br />
Our results revealed that wheat genotypes responded<br />
differently to salinity stress at the two<br />
locations in terms of yield and yield components.<br />
Similar findings were reported by (Richards et al.,<br />
1987; Slavich et al., 1990). The decline in total dry<br />
biomass in the sensitive accessions was most<br />
likely due to the extra energy utilization for<br />
osmotic accumulation (Wyn Jones and Gorham,<br />
1993). Significant positive correlation between dry<br />
biomass and yield m-2 (Table 5) indicated that<br />
total dry biomass along with yield can be good<br />
selection criteria under salinity stress.<br />
Salts present in the soil solution reduce the<br />
ability of plant to absorb water which slow down<br />
plant growth and ultimately cause reduction in<br />
yield components. This is called osmotic or water<br />
deficit effect of salinity. Salinity stress at different<br />
Ahmad et al. 4091<br />
phenological stages inhibits photosynthetic activities<br />
of the plant because it had a direct inhibitory<br />
effect on the Calvin cycle enzymes (Ottander and<br />
Oquist, 991). Tiller plant -1 is the most salinity<br />
sensitive trait in wheat (El-Hendawy et al., 2005).<br />
Therefore, to increase the yield under<br />
stress condition, it is necessary to maintain<br />
high plant density. Tiller formation included<br />
tiller number and tiller biomass. Salinity reduces<br />
tiller number by delaying and reducing tiller emergence<br />
at the vegetative stage. After tiller emergence,<br />
growth of tillers at all stages is inhibited by<br />
salinity due to its damage on the essential<br />
metabolic reaction in plants, resulting in low tiller<br />
biomass and small tiller size (Mass and Poss,<br />
1989). ECe >7.5 dSm -1<br />
in soil water could eradicate<br />
most of the secondary tillers and greatly<br />
reduce the formation of tertiary and lateral tillers.<br />
The yield potential of wheat is greatly dependent<br />
on the number of tillers plant -1 that is affected in<br />
the early life cycle. Number of tillers regulates<br />
grain yield by its prime influence on the number of<br />
spikes in wheat (Simons and Hunt, 1983). Our<br />
correlation results confirmed these findings.<br />
Spikelets spike -1 along with number of<br />
tillers was identified as the most salt sensitive<br />
yield components in wheat. At the time of spike<br />
emergence, salinity suppresses the reproductive<br />
development, spikelet initiation and ultimately
4092 Afr. J. Biotechnol.<br />
number of spikelets (Mans and Rawson, 2004). Due to<br />
their response to salinity and significant positive<br />
correlation with yield, these two traits could be used as<br />
selection criteria. These traits could be determined at<br />
early growth stages and therefore, may be used to<br />
screen large population. Reduction in grains spike -1 was<br />
due to reduction in spikelets spike -1 as revealed by<br />
positive correlation between them. The 1000 grain weight<br />
was less affected as compare to the other yield<br />
components because it was determined at maturity which<br />
is the least salt sensitive stage in wheat (Frank et al.,<br />
1997).<br />
Grain yield had strong positive correlation with plant<br />
height, dry biomass, spike length, spikelets spike -1 , grain<br />
spike -1 and 1000 grain weight (Table 5). Due to this positive<br />
correlation, yield can be used as a selection criterion<br />
under saline field conditions. Many scientists had classified<br />
crop species on the basis of grain yield under stress<br />
conditions (Sadiq et al., 1994; Jafari-Shabestari et al.,<br />
1995; Anderson et al., 1996). Reduction in yield was due<br />
to the reduction in the yield components and high ECe at<br />
BARS was the main cause of reduction in yield and yield<br />
components. Wheat yield decline 10% when ECe value<br />
goes >10 dSm -1 (Katerji et al., 2009).<br />
Sum of square due to genotype for tillers m -2 , total<br />
biomass m -2 , spikelets spike -1 and yield m -2 revealed<br />
that these traits were adversely affected at both locations.<br />
Our results showed that these traits could be effectively<br />
used as selection criteria for screening wheat under field<br />
conditions. Accessions 10807, 11299, 11917 and cultivars<br />
Pavon performed better during both years. Cultivar Local<br />
white produced better yield at both locations during 2008-<br />
09 but check cultivar Pasban 90 performed better at SSRI<br />
as compare to BARS. The high yield of these genotypes<br />
was actually associated with high total biomass production<br />
and yield components. Accession 11299 (Pak) was found<br />
to be most salt tolerant at germination stage and seedling<br />
stage in hydroponics under 300 and 250 mM NaCl stress,<br />
respectively. Cultivar Pasban 90 and accession 10807<br />
were also found to be salt tolerant at seedling stage in<br />
hydroponics at 250 mM NaCl stress (data not shown).<br />
These genotypes could be used as new sources of salt<br />
tolerance.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4093-4099, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1248<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Antioxidant enzymes activities during secondary<br />
somatic embryogenesis in Persian walnut (Juglans<br />
regia L.)<br />
M. Jariteh 1 *, H. Ebrahimzadeh 1 , V. Niknam 1 , K. Vahdati 2 and R. Amiri 3<br />
1 Plant Physiology Laboratory, Department of Plant Sciences, School of Biology, College of Science, University of<br />
Tehran, P.O.BOX: 14155-6455, Tehran, Iran.<br />
2 Department of Horticulture, College of Abouraihan, University of Tehran, Tehran, Iran.<br />
3 Department of Agronomy and Crop Breeding, College of Abouraihan, University of Tehran, Tehran, Iran.<br />
Accepted 18 March, 2011<br />
Somatic embryogenesis was achieved from immature cotyledon explants of Persian walnut (Juglans<br />
regia L.) cave. "Chandler" on DKW medium. Secondary somatic embryogenesis, the process by which<br />
adventitious embryos are formed from primary somatic embryos, is frequent during somatic<br />
embryogenesis in Persian walnut. It has certain advantages compared with primary somatic<br />
embryogenesis such as high multiplication rate, independence to the explant source and repeatability.<br />
Proteins and activities of antioxidant enzymes including catalase (CAT), peroxidase (POX), superoxide<br />
dismutase (SOD) and polyphenol oxidase (PPO) were evaluated in two types of secondary embryogenic<br />
calli and secondary somatic embryos in Persian walnut. In dark brown calli from which their secondary<br />
somatic embryos were separated, CAT, POX and POX had the highest activities while the activity of<br />
SOD and also protein content showed the lowest level. High SOD activity, protein content and low<br />
activities of CAT, POX and PPO were determined in secondary somatic embryos and light brown calli<br />
containing secondary somatic embryos. According to the obtained results, some proteins and<br />
antioxidant enzymes have been regarded as markers for secondary somatic embryogenesis.<br />
Key words: Persian walnut, secondary somatic embryogenesis, antioxidant enzymes.<br />
INTRODUCTION<br />
Somatic embryogenesis and particularly the secondary<br />
somatic embryogenesis provide an efficient regeneration<br />
system for vegetative propagation and genetic transformation<br />
of walnut (Mc Granahan et al., 1990). Secondary<br />
somatic embryogenesis has been reported in many tree<br />
species and is the basis of embryo cloning (Merkle et al.,<br />
1995; Daigny et al., 1996; Benelli et al., 2001; Agarwal et<br />
al., 2004). Secondary or repetitive somatic embryogenesis,<br />
the process by which adventitious embryos are<br />
*Corresponding author. E-mail: maryam.jariteh@gmail.com.<br />
Fax: (+98) 21 66492992.<br />
Abbreviations: CAT, Catalase; POX, peroxidase; PPO,<br />
polyphenoloxidase; SOD, superoxide dismutase; DBC, dark<br />
brown callus; LBC, light brown callus; SSE, secondary somatic<br />
embryo; DKW, Driver and Kuniyuki Walnut medium.<br />
derived from primary somatic embryos, is frequent during<br />
somatic embryogenesis in walnut. Secondary embryos<br />
are formed on the cotyledons and most frequently at the<br />
root tips of somatic embryos. If these adventitious<br />
embryos removed when their cotyledons began to<br />
expand, they often develop into normal plants. Failure to<br />
remove adventitious embryos at this stage of development<br />
led to the formation of more somatic embryos,<br />
abnormal growth or callus formation. Somatic embryo<br />
tissues often turn into dark brown masses after several<br />
months in culture. These brown tissues frequently<br />
produce globular somatic embryos which could be<br />
removed and used to propagate the embryogenic lines<br />
(Tulecke and Mc Granahan, 1985). Martinelli and<br />
Gribaudo, (2001) called the secondary embryogenesis as<br />
re-initiation of embryogenic calli from somatic embryos.<br />
The secondary somatic embryos of walnut can be<br />
efficiently multiplied by repetitive or recurrent embryo-
4094 Afr. J. Biotechnol.<br />
genesis on hormone-free DKW medium (Deng and<br />
Cornu, 1992).<br />
As an experimental system, secondary somatic embryogenesis<br />
has certain advantages compared with<br />
primary somatic embryogenesis such as high multiplication<br />
rate, independence to the explant source and<br />
repeatability (Karami et al., 2008). Furthermore,<br />
embryogenicity can be maintained for a long period of<br />
time by repeated cycles of secondary embryogenesis<br />
(Raemakers et al., 1995). On the other hand, it is thought<br />
that adventitious embryogenesis including somatic and<br />
anderogenetic ones might be induced by different kinds<br />
of stress treatments (Kamada et al., 1994). There is a<br />
variety of data that link somatic embryogenesis, DNA<br />
methylation and oxidative stress response. Also, an<br />
increasing number of publications link reactive oxygen<br />
species (ROS) and somatic embryogenesis (Zavattieri et<br />
al., 2010).<br />
Some antioxidant enzymes have been introduced as<br />
indicators for somatic embryogenesis. According to<br />
Kairong et al. (1999) the differentiation and development<br />
of embryogenic cells in the somatic embryogenesis of<br />
Lycium barbarum L. are regulated by three antioxidant<br />
enzymes including CAT, POX and SOD. Somatic<br />
embryogenesis from somatic tissues of walnut, rather<br />
than zygotic embryos needs to be further investigated<br />
(Vahdati et al., 2006). Thus, study of antioxidant enzymes<br />
activities during secondary somatic embryogenesis can<br />
help to improve primary somatic embryogenesis and<br />
reduce problems in recalcitrant explants. For this purpose,<br />
two different types of secondary embryogenic calli,<br />
light and dark brown calli containing secondary somatic<br />
embryos at different stages of development and<br />
separated secondary somatic embryos were compared.<br />
Proteins and activities of some antioxidant enzymes<br />
including CAT, POX, SOD and PPO were studied during<br />
secondary somatic embryogenesis in Persian walnut<br />
(Juglans regia L.) cv. "Chandler".<br />
MATERIALS AND METHODS<br />
Plant material<br />
Ten fruits of walnut cultivar "Chandler" were harvested 9 weeks<br />
after pollination and stored at 4°C for 5 days. They were surface<br />
sterilized and dissected immediately. Immature cotyledons were<br />
cultured on DKW solid medium (Driver and Kuniyuki, 1984)<br />
containing 0.01 mg/l IBA, 2 mg/l Kin, 1 mg/l BA and 250 mg/l<br />
glutamine (Tulecke and Mc Granahan, 1985). Cultures were grown<br />
in the dark at room temperature.<br />
The repetitive somatic embryogenic line which initiated from<br />
immature cotyledons of walnut cultivar "Chandler" was used in the<br />
present study. This line has been maintained by subculturing every<br />
two weeks at 25°C in the dark for over 2 years. Somatic embryos<br />
were subcultured on basal DKW medium solidified with 0.21%<br />
Gelrite (w/v). Dark brown calli with secondary somatic embryos at<br />
different developmental stages on their surfaces (DBC+SSE), dark<br />
brown calli without secondary somatic embryo (DBC-SSE),<br />
secondary somatic embryos at different developmental stages<br />
which were separated from the surface of dark brown calli (SSE)<br />
and light brown calli with secondary somatic embryos at different<br />
developmental stages (LBC+SSE) were used for all the analyses.<br />
Protein extraction<br />
For estimation of total protein content and enzymes activity, plant<br />
materials were homogenized at 4°C with a mortar in 1M Tris-HCl<br />
(pH 6.8) and 2.5 % (w/v) polyvinylpolypyrolidone (PVPP) to avoid<br />
phenol oxidative effects. The homogenates were centrifuged at<br />
13000 g for 30 min two times at 4°C using a Heraeus 400R<br />
microfuge. Supernatants were kept at -70°C and used for protein<br />
determination and enzyme assay. An UV-visible spectrophotometer<br />
(UV-160, Shimadzu, Tokyo, Japan) were used for the determination<br />
of the absorbance.<br />
Protein quantification<br />
Protein concentration was measured according to Bradford (1976),<br />
using bovine serum albumin (BSA) as standard. Five milliliters of<br />
the Bradford reagent and 100 µl of the each protein extract were<br />
mixed and then, reaction mixtures were incubated at room<br />
temperature for 20 min. The absorbance values were measured at<br />
595 nm.<br />
Antioxidant enzymes assays<br />
Peroxidase (POX; E.C. 1.11.1.7) activity was measured according<br />
to the method of Abeles and Biles (1991). The assay mixture<br />
consisted of 4 ml of 0.2 M acetate buffer (pH 4.8), 0.4 ml H2O2 (3<br />
%), 0.2 ml 20 mM benzidine and 0.2 ml enzyme extract. The<br />
increase of absorbance was recorded at 530 nm. The POX activity<br />
was defined as 1 µM of benzidine oxidated per min per mg protein<br />
(Unit mg -1 (protein)).<br />
Superoxide dismutase (SOD; E.C. 1.15.1.1) activity was<br />
estimated by measuring its ability to inhibit the photochemical<br />
reduction of nitroblue tetrazolium (NBT) at 560 nm as described by<br />
Giannopolitis and Ries (1977) in a reaction mixture consisted of 0.1<br />
ml enzyme extract, 50 mM sodium phosphate buffer (pH 7.5), 13<br />
mM L-methionine, 75 µM NBT, 0.1 mM EDTA and 75 µM riboflavin.<br />
The reaction mixture was irradiated for 14 min and absorbance was<br />
recorded at 560 nm against the non-irradiated blank. One unit of<br />
SOD activity was defined as the amount of enzyme which caused<br />
50% inhibition in NBT reduction.<br />
Catalase (CAT; E.C. 1.11.1.6) activity was assayed from the rate<br />
of H2O2 decomposition as measured by decrease of absorbance at<br />
240 nm (Aebi, 1974). The reaction mixture contained 0.625 ml of 50<br />
mM sodium phosphate buffer (pH 7.0), 0.075 ml H2O2 (3%) and<br />
0.05 ml enzyme extract.<br />
Polyphenol oxidase (PPO; E.C. 1.14.18.1) activity was estimated<br />
following the method of Raymond et al. (1993) at 40°C. The<br />
reaction mixture contained 2.5 ml of 0.2 M sodium phosphate buffer<br />
(pH 6.8), 0.2 cm 3 pyrogallol 20 mM and 0.02 cm 3 enzyme extract.<br />
The increase in absorbance was recorded at 430 nm. The PPO<br />
activity was defined as 1µM of pyrogallol oxidated per min per mg<br />
protein (Unit mg -1 (protein)).<br />
Gel electrophoresis<br />
For determination of protein patterns, discontinuous sodium<br />
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)<br />
was performed according to Laemmli (1970), using 12%<br />
acrylamide. For detection of proteins, gels were stained with 0.03%<br />
comassie brilliant blue G250. For isoenzyme assay, native PAGE<br />
was carried out according to a modified method of Davis (1964)
with a 12% (SOD) and 10% (POX, CAT and PPO) acrylamide gels<br />
at 4°C.<br />
SOD bands were visualized using the activity staining procedure<br />
described by Wendel and Weeden (1989). Gels were incubated in<br />
0.2 M Tris-HCl (pH 8.0) containing 4% riboflavin, 4% EDTA and<br />
20% NBT for 40 min in the dark at room temperature and then,<br />
exposed to white light until white bands appeared in violet<br />
background. For SOD isoform identification, assays were performed<br />
in the presence of selective inhibitors. Application of KCN (3 mM)<br />
inhibits only Cu/Zn-SOD; H2O2 (5 mM) inhibits both Cu/Zn-SOD and<br />
Fe-SOD. Mn-SOD was not inhibited by KCN or H2O2 (Lee et al.,<br />
2001).<br />
CAT activity was detected using the method of Woodbury et al.<br />
(1971). The gels were incubated in 5 mM H2O2. After 10 min, the<br />
gels were washed with distilled water and stained with a reaction<br />
mixture containing 2% (w/v) ferricyanide (K3Fe(CN)6) and 2% ferric<br />
chloride till yellow bands appeared on dark green background.<br />
Electrophoresis pattern of POX was obtained by staining of the<br />
gels with benzidine according to Van Loon and Geelen, (1971). The<br />
gels were immersed in 0.2 M acetate buffer (pH 4.8) containing 3%<br />
H2O2 and 4% benzidine in 50% methanol in the dark at room<br />
temperature till the brown bands appeared.<br />
PPO bands were visualized using the method of Van Loon and<br />
Geelen, (1971). The gels were incubated in 0.2 M potassium<br />
phosphate buffer (pH 6.8) containing 0.5% 3,4- dihydroxy L-<br />
phenylalanine (L-DOPA) and 3.5% calcium chloride in the dark at<br />
room temperature till brown bands appeared.<br />
Statistical analysis<br />
The data determined in triplicate were analyzed by analysis of<br />
variance (ANOVA) using SAS Software (SAS Institute, Inc., 2002).<br />
Significance of mean differences was determined by the Duncan<br />
multiple range test (DMRT) at P < 0.05.<br />
RESULTS<br />
Tissue culture<br />
Most of the immature cotyledon explants of Chandler formed<br />
callus but only low percentage of them were<br />
embryogenic and the number of somatic embryo per<br />
callus ranged from 1 to 4 (data not shown). The embryos<br />
loosely attached without a vascular connection to the<br />
maternal tissues. They were removed from the<br />
embryogenic calli and cultured on basal medium. When<br />
walnut somatic embryos especially at the primary stages<br />
of development including globular and heart shape were<br />
cultured on basal DKW medium, two distinct responses<br />
were observed; (a) development, maturation and<br />
conversion to whole plant; and (b) secondary callus<br />
induction and repetitive somatic embryogenesis.<br />
In some cases, mature embryos produced new individual<br />
embryos on their cotyledons or root tips directly.<br />
Cotyledon-derived primary embryogenic calli were compact,<br />
hard, yellowish and grow slowly with a few number<br />
of somatic embryos on the surface but secondary calli<br />
were fast growing, very friable and light brown at early<br />
stages of formation and gradually became dark brown<br />
while produced a large number of embryos (Figure 1).<br />
Biochemical assays<br />
Ahmad et al. 4095<br />
The highest and lowest content of proteins was determined<br />
in SSE and DBC-SSE, respectively. Protein<br />
content in LBC+SSE was significantly higher than that in<br />
DBC+SSE. The highest activity of CAT was measured in<br />
DBC-SSE and the lowest activity was detected in<br />
LBC+SSE. There was no significant difference in activity<br />
of CAT between DBC+SSE and SSE. The highest activity<br />
of POX was detected in DBC-SSE and POX activity<br />
decreased in the DBC+SSE, SSE and LBC+SSE,<br />
respectively. PPO showed the highest activity in DBC-<br />
SSE while DBC+SSE had lower activity of PPO. The<br />
lowest and highest activity of PPO was detected in SSE<br />
and LBC+SSE, respectively. The lowest activity of SOD<br />
was detected in DBC-SSE and no significant difference<br />
was determined in SOD activity among other samples<br />
(Table 1). According to the SDS-PAGE protein pattern<br />
(Figure 2), the strongest and weakest protein bands were<br />
detected in SSE and DBC-SSE, respectively. Among the<br />
proteins detected, the most abundant were those with<br />
molecular masses of 83, 53, 45, 40, 36, 30, 29 and 28<br />
kDa, respectively. The 83, 53, 45 and 40-kDa proteins<br />
were present in all samples strongly except DBC-SSE.<br />
Also, the 30, 29 and 28-kDa proteins of light brown<br />
secondary somatic embryos (LBC+SSE) and SSE were<br />
expressed more than the corresponding fraction of dark<br />
brown callus with or without embryos (DBC+SSE and<br />
DBC-SSE).<br />
According to non-denaturing PAGE, two CAT isoforms<br />
were detected in the DBC+SSE. CAT1 which was observed<br />
in SSE and LBC+SSE was stronger than CAT2<br />
and was absent in DBC-SSE. In DBC-SSE only CAT2<br />
was detected and this isoform was absent in SSE and<br />
LBC+SSE (Figure 3a). Thus, CAT 2 could be regarded as<br />
a marker for dark grown calli and embryos.<br />
Five POX isoforms were determined in all samples<br />
examined. According to the activity staining, POX1 band<br />
was stronger than four other bands. This band in<br />
DBC+SSE was very strong compared with LBC+SSE and<br />
SSE. POX2 and POX3 isoforms were similar in all the<br />
samples. POX4 was present only in DBC+SSE. POX5<br />
was weak in DBC-SSE and LBC+SSE in comparison with<br />
DBC+SSE and SSE (Figure 3b). Thus, POX4 could be<br />
regarded as a marker for dark grown calli containing<br />
embryos (DBC+SSE).<br />
Five bands with PPO activity were detected on gels by<br />
native PAGE. PPO1 and PPO3 bands in DBC-SSE were<br />
stronger than the others. PPO2 was present only in<br />
DBC+SSE and DBC-SSE while PPO4 was present only<br />
in SSE and LBC+SSE. PPO5 band was common in all<br />
samples and is very strong in SSE and LBC+SSE in<br />
comparison with other samples (Figure 3c). Thus, PPO4<br />
could be regarded as a specific marker for light grown<br />
calli and secondary somatic embryos.<br />
Six bands of SOD were detected in the samples. The<br />
identities of the major SOD activity bands were tested by
4096 Afr. J. Biotechnol.<br />
Figure 1. Secondary somatic embryogenesis in Persian walnut. (a) Primary embryogenic callus<br />
derived from immature cotyledons of Chandler cultivar with two somatic embryos; 1, arrowed = a<br />
well developed cotyledonary embryo; 2, arrowed = heart shape embryo (bar = 8 mm); (b) direct<br />
secondary somatic embryogenesis; arrowed = two torpedo embryos on the surface of the primary<br />
embryo's cotyledons (bar = 4 mm); (c) light brown secondary embryogenic callus with several<br />
secondary somatic embryos; 1, arrowed = globular secondary embryo, 2, arrowed = precotyledonary<br />
embryo (bar = 4 mm); (d) dark brown secondary embryogenic callus with several secondary somatic<br />
embryos; 1, arrowed = torpedo embryo; 2, arrowed = precotyledonary embryo (bar = 5 mm).<br />
Table 1. Protein content (mg g -1 (F.W.)) and activity of antioxidant enzymes (U mg -1 (protein)) in different stages of<br />
secondary somatic embryogenesis.<br />
Parameter Total protein CAT POX PPO SOD<br />
DBC+SSE 2.52 ± 0.10c 144.16 ± 4.56 b 28.52 ± 1.30b 0.28 ± 0.01b 1.017 ± 0.001a<br />
DBC-SSE 0.55 ± 0.02d 345.26 ± 10.73a 135.51 ± 2.15a 0.85 ± 0.04a 0.980 ± 0.001b<br />
SSE 5.50 ± 0.28a 125.30 ± 4.30b 18.23 ± 1.10c 0.11 ± 0.01c 1.012 ± 0.001a<br />
LBC+SSE 4.26 ± 0.04b 77.26 ± 5.17c 9.24 ± 0.37d 0.13 ± 0.01c 1.015 ± 0.004a<br />
DBC+SSE, Dark brown calli with secondary somatic embryo; DBC-SSE, dark brown calli without secondary somatic embryo;<br />
SSE, secondary somatic embryo; LBC+SSE, light brown calli with secondary somatic embryo. Means ± SE; values marked<br />
with different letters are significantly different according to DMRT at P < 0.05.<br />
preincubating the gels with well-characterized SOD<br />
inhibitors: KCN is an inhibitor of Cu/Zn-SOD, whereas,<br />
H2O2 inhibits both Cu/Zn-SOD and Fe-SOD. The upper<br />
band (Figure 3d), in all samples, represented a<br />
mitochondrial Mn-SOD. Five bands with higher mobility<br />
represented the cytosolic Cu/Zn-SOD isoenzymes. The<br />
band representing a chloroplastic Fe-SOD was not presented.<br />
No significant qualitative differences in the<br />
pattern of SOD isoforms were observed between tissues.<br />
DISCUSSION<br />
In vitro culture conditions represent an unusual combination<br />
of stress factors that plant cells encounter<br />
(oxidative stress as a result of wounding at excision of<br />
the explant tissue, PGRs, low or high salt concentration<br />
in solution, low or high light intensities). The stress not<br />
only promotes dedifferentiation but also induce somatic<br />
embryo formation. The drastic changes in the cellular
Figure 2. SDS-PAGE pattern of proteins during<br />
secondary somatic embryogenesis in Persian walnut;<br />
dark brown calli with secondary somatic embryo<br />
(DBC+SSE) (1), dark brown calli without secondary<br />
somatic embryo (DBC-SSE) (2), secondary somatic<br />
embryos (SSE) (3), light brown calli with secondary<br />
somatic embryo (LBC+SSE) (4) and molecular mass<br />
marker (M). Equal amounts of protein (40 µg) were<br />
loaded in each lane. Arrows indicate the affected and<br />
embryo- specific bands.<br />
environment of the in vitro culture induced by a ‘stressor’<br />
in the culture medium or in the physical environment of<br />
the culture, are responsible for reprogramming of gene<br />
expression (Zavattieri et al., 2010). However, secondary<br />
somatic embryogenesis from a dark brown calli in<br />
absence of any growth regulator might be induced by oxidative<br />
stress. The effect of stress during culture may help<br />
to improve induction of somatic embryogenesis in<br />
recalcitrant somatic tissues of Persian walnut.<br />
Somatic embryos are formed from immature cotyledons<br />
of walnut cultivar and this result is in agreement with<br />
Tulecke and Mc Granahan (1985) and Vahdati et al.<br />
(2006). According to Tulecke and Mc Granahan (1985)<br />
somatic embryo tissues in walnut cultivars often turn into<br />
dark brown masses after several months in culture and<br />
they frequently produce globular somatic embryos which<br />
could be removed and used to propagate the embryogenic<br />
lines. It should be mentioned that, all of the<br />
developmental stages of embryos including globular,<br />
heart shape, torpedo and cotyledonary can be observed<br />
on the surface of secondary embrygenic calli. Although,<br />
all related <strong>articles</strong> have confirmed the secondary<br />
embryogenesis in walnut cultivars but there is no report<br />
Ahmad et al. 4097<br />
on the alterations of antioxidant enzymes and proteins<br />
during somatic embryogenesis.<br />
Protein content in SSE which contained embryos at<br />
different developmental stages was the highest, but DBC-<br />
SSE which lacks any embryos had the lowest level of<br />
protein. The difference in protein content was confirmed<br />
by gel electrophoresis profile. These results are in good<br />
agreement to Junaid et al. (2007), who reported that<br />
protein content in matured and proliferated somatic<br />
embryos were significantly higher than that in secondary<br />
embryogenic calli of Catharanthus roseus L. They<br />
showed that the protein content gradually increased with<br />
advancing stages of somatic embryogenesis. It has been<br />
reported that, the degradation of cell membranes and<br />
browning coincident with the loss of chlorophyll and<br />
browning is associated with cell disorganization<br />
(Laukkanen, et al., 2000). Among the protein bands,<br />
some were stronger than the others. Proteins with molecular<br />
weight of about 83, 53, 45, 40-kDa were expected<br />
to be involved in somatic embryogenesis. Sung and<br />
Okimoto (1983) in the studies on carrot have reported<br />
two somatic embryo-specific proteins (77 and 43 kDa).<br />
Similar studies performed by Chen and Luthe (1987) on<br />
rice revealed the presence of two major proteins of 54<br />
and 24 kDa, which were regarded as somatic embryosspecific.<br />
According to Pakusch et al. (1991) a 24 kDa<br />
band may represent subunits of methyltransferase, an<br />
enzyme associated with lignin synthesis. The production<br />
of lignin compounds in callus of plants species may be as<br />
a result of stress reactions because several stresses are<br />
known to induce the synthesis of phenylpropanoids.<br />
Activities of CAT, POX and PPO in DBC-SSE were<br />
significantly higher than those in the other samples but<br />
the lowest activity of SOD was detected in this tissue.<br />
According to these results, the differentiation and<br />
development of embryogenic tissues during secondary<br />
somatic embryogenesis of Persian walnut could be<br />
regulated by antioxidant enzymes (CAT and POX and<br />
PPO). The study analyses showed that activities of CAT,<br />
POX and PPO rapidly increased along with the seconddary<br />
somatic embryogenesis. According to Kairong et al.<br />
(1999) the acquisition of competence, induction and<br />
development of somatic embryos in L. barbarum L. were<br />
associated with the gradual increase in SOD activity.<br />
POX and CAT activities were high in callus and rapidly<br />
decreased in the early days of the differentiation culture<br />
(Kairong et al., 1999). Laukkanen et al. (1999, 2000)<br />
reported that, tissue browning in Scot pine is associated<br />
with increased POX and PPO activities, as well as cell<br />
disorganization and eventually cell death. From the<br />
zymograms of CAT, it can be concluded that CAT1 was<br />
somatic embryo-specific and CAT2 was present only in<br />
dark brown calli and not in light brown ones. These<br />
differences might be related to tissue browning and thus,<br />
CAT could be an indicator for oxidative stress. Among<br />
five POX isoforms, POX1 band in DBC+SSE was very<br />
strong compared with LBC+SSE and SSE. POX4 band
4098 Afr. J. Biotechnol.<br />
Figure 3. Activity staining for SOD; (a) CAT (b) PPO (c) and SOD (d) during secondary somatic embryogenesis in Persian<br />
walnut: dark brown calli with secondary somatic embryo (DBC+SSE) (1), dark brown calli without secondary somatic embryo<br />
(DBC-SSE) (2), secondary somatic embryos (SSE) (3), and light brown calli with secondary somatic embryo (LBC+SSE) (4).<br />
Equal amounts of protein (40 µg) were loaded in each lane.<br />
was present only in DBC+SSE. Thus, POX4 could be<br />
related to tissue browning in these tissues. PPO1 and<br />
PPO3 bands were stronger in DBC-SSE and PPO2<br />
which was only present in DBC+SSE could be related to<br />
tissue browning, while PPO4 was detected only in SSE<br />
and LBC+SSE and PPO5 could be related to somatic<br />
embryo development (Figure 3c). The contribution of<br />
individual SOD isoforms to total SOD activity was<br />
determined by performing SOD assays directly on protein<br />
extracts separated in native gels. The upper band (Figure<br />
3d), in all samples, represented a mitochondrial Mn-SOD.<br />
Five bands with higher mobility represented the cytosolic<br />
Cu/Zn-SOD isoenzymes. The band representing a<br />
chloroplastic Fe-SOD was not detected in samples. The<br />
levels of superoxide dismutase activity were lower in<br />
browning tissues than that in non-browning tissues in<br />
culture condition in Scot pine (Laukkanen et al., 2000)<br />
and these results are in agreement with the latter<br />
research. In Virginia pine, tissue browning decreases the<br />
efficiency of in vitro regeneration through somatic<br />
embryogenesis (Tang and Newton, 2004). It was reported<br />
that, in non-browning callus cultures, PPO activity<br />
declined while in browning calli, PPO activity continued to<br />
increase. It is believed that the increased PPO activity in<br />
browning calli results from wounding or oxidative<br />
damage. Numerous experiments have recently been<br />
carried out on several plant species with the objective of<br />
explaining the role of oxidative stress in plant<br />
morphogenesis (Gupta and Datta, 2003, 2004; Libik et<br />
al., 2005). It is well known that somatic embryogenesis is<br />
one of the most useful approaches for understanding<br />
plant development. Higher levels of intracellular H2O2<br />
induce and promote embryo-genesis of L. barbarum L.<br />
callus (Kairong et al., 1999). However, the relationship<br />
between the ROS and the callus differentiation and<br />
regeneration has not been well understood till now (Tian<br />
et al., 2003).<br />
In summary, according to the present research, the<br />
differentiation and development of embryogenic tissues<br />
during secondary somatic embryogenesis of Persian<br />
walnut could be regulated by protein and antioxidant<br />
enzymes.
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Enzymatic Analysis 2, <strong>Academic</strong> Press, New York, pp. 673-684.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4100-4105, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2673<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Sugar beet genotype effect on potential of bioethanol<br />
production using Saccharomyces cerevisiae<br />
fermentation<br />
Parviz Mehdikhani 1 *, Hrachya Hovsepyan 2 and Mahmood Rezazadeh Bari 3<br />
1 Agricultural Research Center of West Azerbaijan, Iran.<br />
2 Agricultural Research Center of Yerevan, Armenia.<br />
3 Agricultural Sciences College of Urmia University, Iran.<br />
Accepted 6 April, 2011<br />
In order to study the effect of genotype on sugar beet ethanol production potential, ten beet varieties<br />
including diploid, triploid and tetraploid, multigerm, monogerm sugar beet and fodder beet were planted<br />
in randomized complete block design (RCBD) with three replications in Khoy Agricultural Research<br />
Center for two years (2008 to 2009). Some morphological and physiological traits were recorded. The<br />
fresh root and raw sugar of each treatment were fermented to ethanol using Saccharomyces cerevisiae.<br />
Results showed that, genotype had a remarkable effect on the ethanol production potential. Significant<br />
difference (at the 1% level) in ethanol was observed among the beet varieties in both ethanol production<br />
methods. In addition, more significant differences in most morphological and qualitative traits among<br />
the varieties studied were observed. Variation on ethanol production were intensively related to the<br />
chemical composition of root, especially sugar content, potassium impurity, syrup purity and some<br />
characteristics such as root dry matter and root length. Bioethanol production was enhanced by<br />
increasing the sugar content and root yield in sugar beet. Sugar beet varieties contained less root yield<br />
and more sugar content had higher potential for ethanol production than the fodder beet varieties.<br />
Key words: Bioethanol, Saccharomyces cerevisiae , sugar beet, raw sugar<br />
INTRODUCTION<br />
Oil crises occurrence in the 1970 decade due to consumption<br />
increase over production, and the extraordinary<br />
increase of oil price, persuaded industrial countries to<br />
have different approach on the energy issue. Reduction<br />
of fossil fuel resources, commencement of environmental<br />
concerns and improvement of social living standards<br />
under different conventions and protocols like Kyoto<br />
protocol, led to direct human use of clean fuels<br />
(Miyamoto, 1997). Industry leading manufacturers in the<br />
world and producers attempt to replace petrochemical<br />
materials with agricultural products. Nowadays, bioenergy<br />
forms about 8% of the total annual energy used in<br />
the United States (Anonymous c , 2010). Governments and<br />
*Corresponding author. E-mail: Mehdikhani20@gmail.com. Tel:<br />
+98 461 223 3158 or +98 914 161 3755. Fax: +98 461 223<br />
3159.<br />
industries make use of various initiatives associated with<br />
bio-energy production, increase of production efficiency<br />
and decrease of production cost (National research<br />
council, 2000). Ethanol or ethyl alcohol, unlike past<br />
decades that was only used in few industries, is<br />
considered as one of the important and strategic<br />
commodities in many countries and yet the production<br />
volume of this substance is increasing day by day. More<br />
than 80% of the existing ethanol in the world is used as<br />
fuel consumption and its by-products, and 20% of the<br />
remaining is used for traditional applications in industries<br />
such as medical, health and cosmetics, paint and resin<br />
(Anonymous a, 2010). Brazil and United States are the<br />
world's leading producer of ethanol. In 2008, United<br />
States produced 9 billion U.S. liquid gallons of ethanol<br />
fuel and 10.6 billion U.S. liquid gallons of ethanol fuel in<br />
2009. This represents around 55% of the world's total<br />
ethanol production (Anonymous a , 2010). Ethanol also can<br />
be considered as an agricultural sector stabilizer, for
example, if ethanol conversions factories are enable to<br />
produce ethanol from sugar beet, potato and maize, we<br />
will not be faced with difficulty of production increase of<br />
these products in different periods (Parvin, 2010).<br />
Currently, more than 90% of the existing ethanol in the<br />
world is produced as bio-ethanol and 7% synthetically. In<br />
fact, until the late 1980 a noticeable proportion of ethanol<br />
in the world was artificially produced from petrochemicals,<br />
but after this decade new investment has not<br />
been done in this case. It is obvious that each country<br />
based on the dominant culture and climate conditions can<br />
make use of some agricultural products for producing<br />
ethanol. For an example, more than 20% of the nation’s<br />
total corn supply is consumed for ethanol production in<br />
United States (Anonymous a , 2010), while ethanol is<br />
produced in Iran, countries like South America, Brazil,<br />
India, Pakistan and Thailand and some European<br />
countries from sugar beet and cane molasses. Meanwhile,<br />
countries such as Russia, Canada, China,<br />
Australia and some European countries use wheat for<br />
ethanol production (Venturi and Venturi, 2003).<br />
Sugar beet (Beta Vulgaris L.) is a sucrose-rich product<br />
that has many industrial applications in the today world.<br />
Beets are planted from northern latitudes of 30 to 60° and<br />
from Cairo in Egypt, to Helsinki in Finland and generally,<br />
in Europe, Africa and North America and from the height<br />
of zero to over 2000 meters above sea level (Khajehpour,<br />
2006). Beet has a relatively wide consistency range with<br />
an extensive resistance to water stress, salinity and cold.<br />
Among agricultural products, sugar beet is a plant that<br />
has allocated itself a special place in supplying a part of<br />
energy required for human, in particular, in the third world<br />
countries that do not have access to other energy<br />
sources and it is considered as one of the key of economic,<br />
agricultural and national components (Cook and<br />
Scott, 1998).<br />
Today, sugar beet provides 40% of world sugar trade<br />
within less than 200 years progress (USDA, 2008). Sugar<br />
beet cultivation in 41 countries is estimated as 8.1 million<br />
hectares. In the United States, every year about 0.5<br />
million hectares of lands are planted with sugar beet,<br />
Germany with more than 0.4 million hectares and Italy<br />
and England, each with more than 0.2 million hectares<br />
are placed in next ranks (USDA, 2008). Different researches<br />
have been done on ethanol production from sugar<br />
beet. Theurer et al. (1987) evaluate different varieties of<br />
sugar and fodder beets in different weather conditions<br />
with respect to ethanol production potential. They stated<br />
that the amount of ethanol produced from sugar beet is<br />
more than fodder beet. Gibbons and Westby (1987) did a<br />
study with the aim of optimizing ethanol production from<br />
different sizes of fodder beet slices and achieved 4.83%<br />
of ethanol.<br />
Venturi and Venturi (2003) and Koga (2008) compared<br />
agricultural products of temperate regions and concluded<br />
that, energy consumption efficiency of sugar beet for<br />
producing ethanol is much higher than those of wheat,<br />
Mehdikhani et al. 4101<br />
corn and oil seeds. A research conducted in Turkey<br />
about ethanol production from sugar beet varieties<br />
showed that this country has enough potential to supply<br />
energy required for domestic consumptions and independence<br />
from imports of fossil fuels (Icoz et al., 2009).<br />
With respect to pure carbohydrates, sugar beet has<br />
very high harvest index, because either the aerial part or<br />
underground part of sugar beet can be used to ferment<br />
and produce ethanol. On the other hand, sugar beet<br />
compared with other agricultural products, can be<br />
harvested in a three-month period and this provides a<br />
good chance for ethanol production (Chatin et al, 2004).<br />
In a research conducted in the UK, bio-ethanol values<br />
produced from different crops were studied and it was<br />
announced that 2688 and 5250 L of bio-ethanol were<br />
produced from every hectare of wheat and sugar beet<br />
farms, respectively (Anonymous b , 2010). In another study<br />
in India, 0.38 and 0.126 L of ethanol were produced from<br />
every kg of sugar and fresh sugar beet root, respectively<br />
(Blazek, 2007).<br />
Srivastava et al. (2008) observed significant differences<br />
between different varieties with respect to bio-ethanol<br />
production and announced that the varieties with more<br />
root yield and higher sugar percentage are more<br />
promising for ethanol production. Kosaric et al. (1983)<br />
compared two types of yeast including Saccharomyces<br />
cerevisiae and Saccharomyces diastaticus for bio-ethanol<br />
production from fresh sugar beet root and announced<br />
that the yeast of S. cerevisiae had more efficiency.<br />
Undoubtedly, ethanol production from renewable materials<br />
and biomass mostly depends on physico-chemical<br />
properties of the materials, pre-treatment manner,<br />
efficiency of zymogenic microorganisms and fermentation<br />
conditions such as initial concentration of sugar, PH,<br />
temperature, microbial density and fermentation time<br />
(Camire and Camire, 1994; linko et al., 1983; Zhan et al.,<br />
2003). Increase of fermentable biomass amount is an<br />
issue that must be studied by different varieties with<br />
respect to fermentation efficiency.<br />
Plant breeding strategies can be implemented to<br />
improve biomass yield, biomass quality, biomass<br />
conversion efficiency, resistance to diseases and pests,<br />
sugar content and other characteristics associated with<br />
ethanol production. This study investigated the ethanol<br />
production potential in some beet genotypes and its<br />
relation with some characteristics such as root yield and<br />
sugar content.<br />
MATERIALS AND METHODS<br />
The quality and quantity of the ethanol produced from sugar beet is<br />
strongly dependent on variety. In order to evaluate some characteristics<br />
of sugar beet varieties that affect Bioethanol production,<br />
this experiment was carried out with ten beet varieties in<br />
randomized complete block design (RCBD) with three replications<br />
for two years (2008 to 2009) in Khoy Agricultural Research Station,<br />
Iran. Ten beet varieties including 8 sugar beets and 2 fodder beets<br />
were the most appropriate varieties with respect to plantation
4102 Afr. J. Biotechnol.<br />
season and region. Some traits including green biomass, fresh and<br />
dry weight of root, leaves and petiole, root yield, sugar content,<br />
white sugar content, sugar yield, molasses, nitrogen, sodium and<br />
potassium impurities, syrup purity, leaf area index (LAI), length of<br />
root and green cover percentage were measured and recorded. At<br />
the end of the growth season, all roots and crowns were harvested<br />
and then samples were taken for recording some characteristics in<br />
the laboratory. Fresh sugar beet root and raw sugar were used for<br />
the ethanol production in the laboratory.<br />
Preparing of yeast culture<br />
The special yeast (S. cerevisiae) was maintained on malt agar<br />
medium [contains yeast extract (3 g); malt extract (3 g); peptone (5<br />
g); glucose(10 g); agar (20 g); all dissolved in 1 L of distilled water<br />
and adjusted to pH 5.6] accordingly (Zayed and Foley, 1987).<br />
Inoculum was prepared from 1 ml of yeast with 100 ml of the earlier<br />
stated medium. After maintaining for 30 h in an incubator at 35°C,<br />
the culture contained approximately 5 × 10 8 cells per ml.<br />
Production of ethanol from fresh sugar beet root<br />
After washing the roots of 20 kg of each treatment, using cutting<br />
machine, the roots were cut into slices with a thickness of 1.5 mm<br />
and a length of 5 to 7 cm (Mesbahi, 2003). Then, the slices were<br />
kept in warm water (60°C) for 2 h with agitator timer till their syrup<br />
leaked into the water (Jones et al., 1981). After filtration of the total<br />
solution, the remaining syrup was separated from pulp by pressure.<br />
The sap was boiled for 2 h in a vacuum till its concentration<br />
reached the sufficient limit and also, its microbial contamination was<br />
destroyed. Then the sap was fermented under 35°C and at pH 5.5<br />
for 72 h (Srivastava et al., 2008) using a special strain of S.<br />
cerevisiae Persian type culture collection (PTCC) 5269 obtained<br />
from the Persian type culture collection of yeast cultures, Tehran,<br />
Iran. After fermentation, the obtained solution was filtered and then<br />
purified with a distiller apparatus.<br />
Production of raw sugar from sugar beet<br />
For the production of raw sugar, 20 kg beet root from each<br />
treatment after washing with water was sliced to the dimensions of<br />
1.5 mm in thickness and 5 to 7 cm in length (Mesbahi, 2003). In<br />
whole, the current syrup in the slices after rinsing with pure water of<br />
almost the same volume and temperature of 75°C was exited by a<br />
press machine. In order to refine the obtained solution, lime and<br />
CO2 were added, respectively. In this stage, the pH increased to<br />
about 11 (Clarke, 1988). The solution was maintained in a closed<br />
container for at least an hour with a gentle agitation at 85°C to<br />
some of the impurities deposit. Then the solution was transferred to<br />
a pre-provided apparatus with special tubes passing steam until it<br />
was concentrated up to 70% at 120°C (Mesbahi, 2003). Some<br />
sugar powder was added to the produced syrup in vacuum at<br />
temperature below 100°C and it was kept at the same temperature<br />
till sugar crystals were formed and enlarged. After cooling the<br />
mixture, it was centrifuged at 1800 rpm for 5 min and the produced<br />
sugar was weighed and kept after drying. The remaining syrup was<br />
weighed as molasses (Mesbahi, 2003).<br />
Production of ethanol from raw sugar<br />
The sugar produced from each treatment with 25% concentration<br />
was dissolved in distilled water at 40°C. The obtained solution was<br />
fermented for 72 h under the earlier mentioned temperatures and<br />
pH for all the treatments by using suitable population density of S.<br />
cerevisiae. After filtering the produced solution, it was purified up to<br />
94% by using distiller apparatus (Koga, 2008).<br />
Statistical analysis<br />
Ethanol concentration was determined by specific gravity method<br />
(AOAC, 1990). Ethanol was analyzed using HPLC system with an<br />
Aminex HPX-87H column (Bio-Rad, Hercules, CA) to compare the<br />
results with earlier method. The mobile phase was 5 mm H2SO4<br />
pumped at a flow rate of 0.6 ml/min. Data acquisition and analysis<br />
were performed using the SHIMADZU EZ START 7.1.1 software.<br />
The results reported were based on specific gravity method.<br />
Analysis of variance (ANOVA), least significant difference (LSD)<br />
and comparison of means were done using ASA (SAS, 1995).<br />
RESULTS AND DISCUSSION<br />
In the experiment of the ethanol production from fresh<br />
sugar beet root, the total amount of distilled alcohol was<br />
recorded for each treatment with 94% alcohol. Sugar<br />
beet roots had around 10 to 18% sugar for different<br />
varieties. So the theoretical yield of the produced ethanol<br />
was almost 15% for fresh sugar beet root and around<br />
50% for sugar. An important characteristic of the ethanol<br />
production process is its feedstock quality, which makes<br />
it susceptible to contamination by non-S. cerevisiae<br />
yeasts. The most important aspect of the fuel-ethanol fermentation<br />
is ethanol yield or more generally the industrial<br />
yield. It depends on the fermentative capacity of the yeast<br />
population (Grote and Rogers, 1985) and their resistance<br />
to stress conditions (Bai et al., 2008).<br />
Analysis of variance<br />
Analysis of variance for sugar beet physiological and<br />
quali-tative characteristics for two years showed that,<br />
there was a significant difference among the different<br />
varieties with respect to ethanol production potential from<br />
fresh root and raw sugar. Varieties also showed significant<br />
differences in some recorded traits including root<br />
yield, sugar content, sugar yield, nitrogen, potassium and<br />
sodium impurities, syrup purity and molasses yield. This<br />
difference was completely normal because the various<br />
types of beet including N, E and Z types and/or sugar and<br />
fodder beet and also diploid, triploid and tetraploid were<br />
applied in this study. These differences resulted in<br />
different ethanol yields of the varieties.<br />
Combined analysis of variance for morphological traits<br />
for the two years showed that root length, leaf number,<br />
leaf area index (LAI), dry weight of roots and crowns had<br />
significant differences (p < 0.01).<br />
Comparison of means<br />
Comparison of some traits of beet varieties showed that
Table 1. Comparison of some traits of beet varieties<br />
Mehdikhani et al. 4103<br />
Variety Root Yield Sugar Yield White Sugar Yield Sugar Content K NA N<br />
9597 57.33 c 9.68 bc 8.59 b 16.9 a 3.89 b 1.88 bc 2.23 abc<br />
7233 57.86 c 9.90 bc 8.63 b 17.12 a 4.48 b 2.11 bc 2.34 abc<br />
Shirin 58.14 c 10.37 bc 9.27 b 17.85 a 4.00 b 1.68 c b 2.42 abc<br />
BR1 61.68 bc 10.14 bc 8.65 b 16.43 a 4.81 b 2.21 bc 2.93 ab<br />
7112 71.35 b 11.40 b 9.59 b 16.03 a 4.42 b 3.05 ab 2.76 ab<br />
IR2 87.49 a 14.51 a 12.42 a 16.58 a 4.56 b 2.54 abc 2.60 ab<br />
37RT 65.67 bc 10.78 b 9.23 b 16.45 a 4.42 b 2.62 abc 2.37 abc<br />
19669 68.13 b 10.05 bc 8.85 b 14.75 a 3.78 b 1.69 c 1.90 bc<br />
Fd1 66.60 bc 8.03 cd 5.75 c 11.59 b 7.21 a 2.20 bc 1.17 c<br />
Fd2 69.40 b 6.53 d 4.05 c 9.9 b 8.20 a 3.52 a 3.42 a<br />
Numbers followed by similar letters are not significantly different at 5% level of probability.<br />
Table 2: Comparison of beet varieties regarding ethanol production potential<br />
Variety Ethanol from Root (L Ton–1 ) Ethanol from Root (L ha–1 ) Ethanol from Raw Sugar (L ha–1 )<br />
9597 107.10 a 6194 bc 4725 b<br />
7233 108.40 a 6334 bc 4750 b<br />
Shirin 113.20 a 6637 b 5100 b<br />
BR1 104.80 a 6489 bc 4756 b<br />
7112 102.40 a 7294 b 5275 b<br />
IR2 105.60 a 9285 a 6829 a<br />
37RT 105.70 a 6901 b 5077 b<br />
19669 93.64 a 6436 bc 4868 b<br />
Fd1 74.57 b 4974 cd 3164 c<br />
Fd2 63.25 b 4395 d 2228 c<br />
Numbers followed by similar letters are not significantly different at 5% level of probability.<br />
in the bioethanol production from fresh root of beet,<br />
cultivar of IR2 had the highest root yield with 87.49 ton/h<br />
and the highest ethanol yield with 9285 L/h (Tables 1 and<br />
2). Also, IR2 with 12.42 ton/h of white sugar yield had<br />
higher yield than the rest of the varieties, while fodder<br />
beet varieties (Fd1 and Fd2) with 5.75 and 4.05 tone/h of<br />
white sugar yield, respectively, had the lowest yield and<br />
these varieties had the lowest bioethanol yield (Table 1).<br />
The fodder varieties had impurities amounts of nitrogen,<br />
potassium and sodium. Hence, the amount of molasses<br />
production in these varieties also was more than the rest<br />
(data not shown).<br />
Theurer et al. (1987) evaluated sugar beet and fodder<br />
beet varieties, and declared that sugar beet with 8640 L/h<br />
of ethanol production, compared with 6380 L/h fodder<br />
beet, had more potential for ethanol production.<br />
Ethanol production from raw sugar, IR2 with 6829 L/h<br />
had the highest value, while the fodder beet varieties with<br />
3164 and 2228 L/h had the lowest ethanol yield (Table 2).<br />
According to the qualitative analysis of raw sugar<br />
obtained from the different varieties, it can be stated that,<br />
the quality of sugar in different varieties had no difference<br />
and so ethanol production was related to quantity of the<br />
produced sugar for each variety. The amounts of<br />
potassium and nitrogen impurities of the fodder varieties<br />
were comparable with those of other varieties. Significant<br />
difference between the two years of the tests was not<br />
observed in terms of our studied traits. Regional weather<br />
data also showed that, no large changes in climate<br />
parameters were observed during the years 2008 and<br />
2009 (data not shown).<br />
Correlations of some sugar beet traits with ethanol<br />
production<br />
Correlation between some sugar beet traits with ethanol<br />
production from the fresh root indicated that, ethanol yield<br />
had high positive correlation with the sugar yield, white<br />
sugar yield, sugar content, white sugar content, syrup<br />
purity and dry weight of root. Also, it had significant<br />
negative correlation with molasses sugar (Table 3).<br />
The correlation between some sugar beet traits with<br />
ethanol production from raw sugar is shown in Table 3.<br />
The results indicated that, ethanol yield had high positive<br />
correlation with sugar yield, white sugar yield, sugar
4104 Afr. J. Biotechnol.<br />
Table 3. Correlation of some beet traits with ethanol production from fresh root and raw sugar.<br />
Ethanol from Fresh Root (L/Ton) Ethanol from Raw Sugar (L/H)<br />
Root Yield - 0.575 **<br />
Sugar Yield 0.727 ** 0.969 **<br />
White Sugar Yield 0.838 ** 0.986 **<br />
Sugar Content 0.921 ** 0.828 **<br />
White Sugar Content 0.985 ** 0.830 **<br />
Potassium Impurity 0.266 -0.475 *<br />
Sodium Impurity 0.340 -0.218<br />
Nitrogen Impurity 0.112 0.070<br />
Syrup Purity 0.829 ** 0.655 **<br />
Molasses Sugar -0.628 ** -0.342<br />
Root Length -0.002 0.584 **<br />
Number of leaves -0.128 0.174<br />
Leaf Area Index 0.007 -0.102<br />
Fresh Weight of Crown 0.038 -0.093<br />
Dry Weight of Crown -0.037 0.427 *<br />
Dry Weight of Root 0.369 * 0.582 **<br />
**& * represent correlations with significant levels of 0.01 and 0.05, respectively.<br />
content, white sugar content, root yield, syrup purity, root<br />
length, dry weight of root and crown. Also, it had<br />
significant negative correlation with potassium impurity.<br />
Sodium and nitrogen impurities, number of leaves and<br />
leaf area index (LAI) had no significant correlation with<br />
ethanol production.<br />
In this study, S. cerevisiae was used for the fermentation<br />
and the used sugar type was sucrose that can be<br />
converted to bioethanol. Hence, cellulose tissue in fresh<br />
beet root cannot have great role in ethanol production<br />
(Singh et al., 1995). Therefore, the high correlation<br />
between ethanol yield and white sugar in this study<br />
seems to be reasonable. Variations of 32 to 43% for<br />
ethanol yields were observed among the 10 beet<br />
varieties. The effect of genotype on ethanol production is<br />
related to both the chemical composition and morphological<br />
properties of the beet root samples, with a<br />
stronger effect observed for chemical composition such<br />
as sugar content and impurities. Ethanol production<br />
increased as sugar content increased, whereas the<br />
ethanol production decreased as nitrogen and potassium<br />
impurities increased. Further research is needed to test a<br />
broad number of varieties across a wide range of growing<br />
conditions to further evaluate the effects on ethanol<br />
fermentation yields. Both sugar content and root yield<br />
were strongly correlated with ethanol yield based on<br />
regression analysis using single chemical composition.<br />
The relationship between sugar content and theoretical<br />
percentage of ethanol yield indicated that, sugar content<br />
had significant effect on ethanol yield (Table 3).<br />
Zhan et al. (2003) studied a number of varieties of grain<br />
and fodder sorghum and found that the chemical composition<br />
and physical properties of grain are very important<br />
factors on ethanol production potential.<br />
Conclusions<br />
Bioethanol production from sugar beet via fermentation<br />
technology is a promising fuel alternative. In order to<br />
produce ethanol from sugar beet and by-products via<br />
fermentation, it is important to know the correlations<br />
between some morphological and physiological traits with<br />
ethanol production. The novelty of this study compared<br />
with other investigations is to produce ethanol from fresh<br />
sugar beet root. It was observed that, several varieties<br />
had different ethanol production potentials highly<br />
correlated with root quantitative and qualitative traits. In<br />
fact, this study highlighted breeding of sugar beet<br />
varieties particularly for ethanol production. Based on the<br />
analysis of the experimental data, ethanol production<br />
from fresh root has more efficiency than fermentation of<br />
raw sugar. Among all of the investigated varieties, sugar<br />
beet varieties produced more ethanol per hectare than<br />
fodder beet. Sugar beet varieties had more root yield and<br />
sugar content than fodder beet and these two<br />
characteristics play basic role in ethanol production. The<br />
adapted sugar beet hybrids showed better promise than<br />
fodder beet as a fuel crop in the USA, since sugar beet<br />
produces an equal or greater quantity of fermentable<br />
sugar, it has less bulk to transport and more extractable<br />
sugar per unit mass (Theurer et al., 1987).<br />
The results obtained from this research showed that, a<br />
limited number of beet genotypes had significant effects<br />
on the potential of ethanol production. A significant difference<br />
(p < 0.01) was observed in ethanol yield among<br />
varieties in both methods of ethanol production. Also,<br />
highly significant differences were observed in the<br />
morphological and physiological traits among the varieties.<br />
Genotype variation effects were found on ethanol
yield, that is, highly related to the chemical composition of<br />
roots, especially sugar content, potassium impurity, crude<br />
syrup purity and some morphological characteristics such<br />
as root length and root dry matter. In addition, ethanol<br />
production increased as sugar content and root yield<br />
increased.<br />
When sugar beet was compared with other sugar<br />
crops, its irrigation requirement was less and so, it was<br />
suitable in our agricultural system. It needs more ethanol<br />
from beet genotypes and support from private sugar<br />
industry from all over the world. This subject may create<br />
a new vista.<br />
ACKNOWLEDGMENT<br />
The authors would like to acknowledge all personnel of<br />
biotechnology research institute in Urmia University<br />
especially Dr. Sabzi for preparation of all apparatus and<br />
services. The authors gratefully acknowledge also Dr.<br />
Taleghani and Dr. Mahmoodi, directors of sugar beet<br />
seed institute for the analysis of sugar beet pulps in their<br />
institute lab and Dr. moghaddamnia for kindly reviewing.<br />
REFERENCES<br />
Anonymous a (2010). http://energy-statistics.blogspot.com/2010/11/usethanol-fuel-statistics.html<br />
Anonymous b (2010). http://www.esru.strath.ac.uk/EandE/Web_sites/02-<br />
03/biofuels/quant_bioethanol.htm (Available: 25 May 2010).<br />
Anonymous c (2010)."Renewable Fuel Standard Program (RFS2): Final<br />
Rule". U.S. Environmental Protection Agency. (Available: 9 Feb<br />
2010).AOAC (1990). Official Methods of Analysis, 15th Ed.<br />
Association of Official Analytical Chemists, Arlington, VA.<br />
Bai FW, Anderson WA, Moo-Young M (2008). Ethanol fermentation<br />
technologies from sugar and starch feedstocks. Biotechnol. Adv. 26:<br />
89-105.<br />
Blazek E (2007). http://www.appropedia.org/Ethanol. from organic sugar<br />
beets Categories.<br />
Camire ME, Camire AL (1994). Enzymatic starch hydrolysis of extruded<br />
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Chatin P, Gokhale D, Nilsson S (2004). Sugar beet growing in tropical<br />
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Sugar J. 106: 329-330.<br />
Clarke MA (1988). Chemistry & Processing of Sugar beet & Sugarcane.<br />
Elsevier Science Publishing Co. Inc.<br />
Cook DA, Scott RK (1998). Sugar Beet; science to practice. Translation.<br />
Faculty members sugar beet research institute of Iran. Agric. Sci.<br />
Publication. Tehran.<br />
Gibbons WR, Westby CA (1987). Effect of fodder beet cube size on<br />
ethanol production via diffusion fermentation. Biotechnol. Lett. 9(2):<br />
135-138.<br />
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Grote W, Rogers PL (1985). The susceptibility to contamination of<br />
Zymomonas mobilis process for ethanol production. Ferment<br />
Technol. J. 63: 287-290.<br />
Icoz E, Tuğrul KM, Saral A, Đçöz E (2009). Research on ethanol<br />
production and use from sugar beet in Turkey. Biomass Bioenerg.<br />
33(1): 1-7.<br />
Jones RP, Pamment N, Greenfield PF (1981). Alcohol fermentation by<br />
yeasts-the effect of environmental and other variables. Process<br />
Biochem. 16: 42-49.<br />
Khajehpour MR (2006). Production of industrial plants. Third edition.<br />
Industrial University of Esfahan press. Esfahan. Iran. p. 121.<br />
Koga N (2008). An energy balance under a conventional crop rotation<br />
system in Northern Japan: Perspectives on fuel ethanol production<br />
from sugar beet. Agric. Ecosyst. Environ. 125: 101-110.<br />
Kosaric N, Wieczorek A, Kliza A (1983). Ethanol production from fodder<br />
beet. Biotechnol. Bioeng. 25(7): 1737-1745.<br />
Linko P, Hakulin S, Linko YY (1983). Extrusion cooking of barley starch<br />
for the production of glucose syrup and ethanol. Cereal Sci. J. 1: 275-<br />
284.<br />
Mesbahi GR (2003). Principles of sugar production industries. First<br />
Edition. Agricultural Sciences Publications. Tehran. Iran.<br />
Miyamoto K (1997). Renewable biological systems for alternative<br />
sustainable energy production (FAO Agricultural services Bulletin). p.<br />
128.<br />
National Research Council (2000). Bio-based Industrial Products-<br />
Priorities for Research and Commercialization. National Academy<br />
Press, Washington, DC.<br />
Parvin P (2010). Report of biofuels production and consume in Iran and<br />
the world. Iranian J. Agr. Food Ind. 53: 15.<br />
Renewable Fuel Standard Program (RFS2) (2010): Final Rule. U.S.<br />
Environmental Protection Agency.<br />
SAS (1995). SAS User’s Guide. SAS Institute Inc., Gary, NC.<br />
Singh D, Dahiya JS, Nigam P (1995). Simultaneous raw starch<br />
hydrolysis and ethanol fermentation by glucoamylase from<br />
Rhizoctonia solani and Saccharomyces cerevisiae. J. Basic<br />
Microbiol. 35: 117-121.<br />
Srivastava H, Sharma M, Bhargava VK (2008). Genetic potential of<br />
sugar beet genotypes for ethanol production under different agroclimatic<br />
condition of India. 11th IIRB Congress.<br />
Theurer JC, Doney DL, Smith GA, Lewellen RT, Hogaboam GJ,<br />
Bugbee WM, Gallian JJ (1987). Potential Ethanol Production from<br />
Sugar Beet and Fodder Beet. Crop Sci. Soc. Am. 27: 1034-1040.<br />
USDA (2008). Agricultural Statistics. United States Government Printing<br />
Office, Washington, DC. ARS Publication. Wide Cross. htm. Available<br />
online,<br />
Venturi P, Venturi G (2003). Analysis of energy comparison for crops in<br />
European agricultural systems. Biomass Bioenerg. 25: 235-255.<br />
Zayed G, Foley J (1987). The influence of fermentation conditions on<br />
ethanol yields from sugar beet molasses and fodder beet juice using<br />
Saccharomyces cerevisiae strains. Irish J. Food Sci. Technol. 11: 19-<br />
133.<br />
Zhan X, Wang D, Tuinstra MR, Bean S, Seib PA, Sun XS (2003).<br />
Ethanol and lactic acid production as affected by sorghum genotype<br />
and location. Ind. Crop Prod. 18: 245-255.
African Journal of Biotechnology Vol. 10(20), pp. 4106-4113, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2211<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Modeling and experiment to threshing unit of stripper<br />
combine<br />
Xu Lizhang* and Li Yaoming<br />
Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education and Jiangsu Province, Institute<br />
of Agricultural Engineering, Jiangsu University, Zhenjiang 212013, China.<br />
Accepted 7 April, 2011<br />
The aim of this study was to establish the mathematical models to axial threshing unit of stripper<br />
combine harvester. The exponential distribution model of the un-threshed grain Tu ( z ) , free grain Tf ( z )<br />
and cumulative gain F( z ) along the axial threshing drum z ∈ [0, L]<br />
was obtained. The loss of<br />
separation L f was the free grain which had reached the end of the threshing unit but still not<br />
separated. On the designed testing equipment, experiments of threshing performances were conducted<br />
with the different feed rates and drum rotator speeds for the rice stripped mixtures. Experimental<br />
results showed that the distribution of the cumulative separated gain was in agreement with the<br />
simulation. The results were very useful for practical analysis of grain separation and damage as well<br />
as for threshing unit design and process optimization.<br />
Key words: Stripping, combine harvester, threshing unit, model, experiment.<br />
INTRODUCTION<br />
The stripper combine is combined with a stripper header.<br />
And the basic concept of the stripper header is that a<br />
rearwards rotating rotor fitted in the front of the header is<br />
fitted with 6 to 8 rows of stripping fingers that strip grain<br />
from the crop as the combine moves the head forwards<br />
while it spins backwards (Price,1993; Jiang et al., 2001).<br />
The speed of the rotor can be varied according to crop<br />
conditions. Those stripped by stripping fingers was called<br />
the stripped mixtures, which include the free grain, the<br />
un-threshed grain and some straws etc. For this reason,<br />
characteristic of the stripped mixtures which was fed into<br />
the threshing unit of the stripper combine through the<br />
feeder house was quite different from the material fed into<br />
the threshing unit of conventional combine harvester.<br />
A scientific model which can describe the threshing<br />
process is extremely important to study threshing unit.<br />
Based on the view of mechanics, Trollope (1982) deduced<br />
a set of differential equations which describe the<br />
threshing process, but it is difficult to apply.<br />
*Corresponding author. E-mail: lzxu1979@gmail.com. Tel: +86-<br />
0511-88797214-219. Fax: +86-0511-88780175.<br />
On the assumption of the rule of threshing process, which<br />
meets the qualification of exponential distribution, Huynh<br />
(1982) established a mathematical model, however, it is<br />
too simple and experiential, and it has great differences<br />
with the actual situation. Taking into account the<br />
parameters of threshing process, and properties of gain,<br />
a rather nice probability model of the threshing cylinder<br />
with rasp bar was published (Wan et al., 1990; Zhou et<br />
al., 1998; Yin, 1999).<br />
Miu (2002 a, b) presented a more detailed model of the<br />
axial threshing unit of the conventional combine harvester,<br />
but it is not completely suitable for the threshing unit<br />
of stripper combine. Specific objectives of the work reported<br />
in this study were to: (1) establish a mathematical<br />
model for describing the threshing process; (2) perform<br />
experimental studies on the designed equipment for<br />
threshing performance; (3) simulation by computer on the<br />
mathematical model.<br />
Model development<br />
As shown in Figure 1, a threshing separation unit is in<br />
common use in the stripper combine harvester. The sign
l<br />
rotor<br />
concave<br />
L<br />
straw export<br />
Lizhang and Yaoming 4107<br />
o z<br />
feeding<br />
Figure 1. Schematic diagram of axial threshing unit.<br />
“l ” denotes the breadth of the entrance of the unit and<br />
“L” denotes the length of the threshing platen. Taking the<br />
point of intersection between the axial line of the platen<br />
and the side face of the entrance as the origin, the central<br />
axis of platen is defined as the z axes and the direction of<br />
axial movement of the grain is set for the plus direction of<br />
the z axes.<br />
Assume that the feed rate of the mixture is constant;<br />
the mass of the mixture fed into the threshing unit per<br />
second is 1, then:<br />
z<br />
z ) = ( k 1 + k )<br />
(1)<br />
l<br />
q s ( 2<br />
Where, qs (z ) is the mass of grain fed into the threshing<br />
unit per second over the interval [0,z], z ∈ [ 0,<br />
l]<br />
; k1 is the<br />
percentage of the free grain, which has been threshed<br />
but not separated, in the total mass of the mixture and<br />
k2 is the percentage of the un-threshed grain, which has<br />
not been threshed, in the total mass of the mixture.<br />
Experimental results shows that the mixture in threshing<br />
space includes the free grain (60 to 70%), the unthreshed<br />
grain (15 to 25%) and the straw (about 10 to<br />
20%). Therefore, the threshing process of the rice<br />
stripped mixtures includes two parts. One, the unthreshed<br />
grain are threshed, the other is the free grain<br />
separated form the concave during screwing with the<br />
rotator.<br />
Development of the model is based on the following<br />
assumptions: (1) the probability of grain threshed and<br />
separated during the threshing space is equal in the<br />
plane through the arbitrary point of the z axis; (2) within<br />
the region from z to z+ ∆ z , the probability of threshing for<br />
the un-threshed grain is in direct proportion to the amount<br />
of the un-threshed grain. And the proportion coefficient,<br />
named as 1 u , is related to the parameters, such as the<br />
rotator speed, the cylinder-concave clearance, the structure<br />
of concave and the properties of the grain etc; (3)<br />
within the region z to z+ ∆ z , the probability of separating<br />
form the concave for the free grain is in direct proportion<br />
to the amount of the free grain. And the proportion<br />
coefficient, named as u 2 , is also related to the parameters<br />
such as the rotator speed, the cylinder-concave<br />
clearance, the structure of concave and the properties of<br />
the grain etc.<br />
According to assumptions (1) and (2), if ∆z ->0, then:<br />
[ 1 P(<br />
z)<br />
]<br />
p1( z)<br />
u1<br />
− 1<br />
= (2)<br />
Where, 1( ) z p is the probability of grain threshing at a z<br />
point and 1( ) z P is the mass of the threshed grain within<br />
the region from 0 to z, So:<br />
∫<br />
P ( z)<br />
p ( s)<br />
ds<br />
(3)<br />
1<br />
= z<br />
0<br />
1<br />
It can be obtained as follows:<br />
p ( z)<br />
1<br />
−u1z<br />
= u1e<br />
(4)
4108 Afr. J. Biotechnol.<br />
Similarly, it is known:<br />
p ( z)<br />
2<br />
u<br />
e<br />
−u2<br />
z<br />
= 2<br />
(5)<br />
Where, 2 ( ) z p is the probability of free grain separating<br />
out of the concave at z point; At z ∈[ 0,<br />
l]<br />
is the mass of<br />
the un-threshed grain is equal to subtraction of the<br />
threshed mass from the fed-in mass:<br />
T<br />
u1<br />
( z)<br />
z<br />
z z −u<br />
s z<br />
1<br />
−u1z<br />
= k2<br />
− k2<br />
u1e<br />
ds = k2<br />
e<br />
l l ∫ (6)<br />
l<br />
0<br />
Where, 1( ) z Tu is the mass of the un-threshed grain at<br />
z ∈ [ 0,<br />
l]<br />
.<br />
Similarly, when z ∈ [ l,<br />
L]<br />
, the equation can be expressed<br />
as follows:<br />
T<br />
u2<br />
( z)<br />
z<br />
−u1<br />
( s−l<br />
)<br />
−u1z<br />
= k 2 − Tu1(<br />
l)<br />
∫ u1e<br />
ds = k 2e<br />
(7)<br />
0<br />
Where, 2 ( ) z Tu is the mass of the un-threshed grain;<br />
z ∈ [ l,<br />
L]<br />
.<br />
If z= L, then:<br />
L<br />
t<br />
−u1L<br />
= Tu<br />
2 ( L)<br />
= k2e<br />
(8)<br />
Where, L t is the loss of the un-threshed.<br />
According to the probability theory, the probability of<br />
grain separation is equal to the convolution of the<br />
probability of the un-threshed grain threshing and the<br />
probability of the free grain through which the concave<br />
occurs (Miu, 2002b). As z ∈ [ 0,<br />
l]<br />
:<br />
z z uu z z<br />
f ( z) = k p( z) ∗ p ( z) + k p( z) = k ( e − e ) + k ue<br />
1 2 −u2 z −uz 1 −u2<br />
z<br />
1 2 1 2 1 2 2 1 2<br />
l l u1 −u2<br />
l l<br />
Where, f ( ) is the probability of grain separation;<br />
z ∈ [ 0,<br />
l]<br />
.<br />
Integrate:<br />
F(<br />
z)<br />
1<br />
1 z<br />
k z<br />
[ u1(<br />
1−e<br />
u −u<br />
l<br />
) −u<br />
( 1−e<br />
z<br />
)] + k1<br />
( 1−e<br />
l<br />
)<br />
(9)<br />
2<br />
−u2z<br />
−u1z<br />
−u2z<br />
= (10)<br />
1<br />
2<br />
2<br />
Where, 1( ) z F is the cumulative mass of the separating<br />
grain; z ∈ [ 0,<br />
l]<br />
.<br />
Similarly, when [ l,<br />
L]<br />
z ∈ :<br />
k2<br />
−u2z<br />
−u1<br />
z<br />
−u2z<br />
F2(<br />
z)<br />
= [ u1(<br />
1−e<br />
) −u2(<br />
1−e<br />
)] + k1(<br />
1−e<br />
) (11)<br />
u −u<br />
1<br />
2<br />
Where, 2( ) z F is the cumulative mass of the separated<br />
grain; z ∈ [ l,<br />
L]<br />
.<br />
The grain of the rice stripped mixtures which fed into the<br />
threshing unit finally is divided into three parts: the unthreshed<br />
grain, the free grain and the separated grain.<br />
Hence:<br />
⎧<br />
⎪<br />
⎨<br />
⎪<br />
⎩<br />
z<br />
Tu1(<br />
z)<br />
+ Tf<br />
1(<br />
z)<br />
+ F1(<br />
z)<br />
= ( k1<br />
+ k2)<br />
l<br />
T ( z)<br />
+ T ( z)<br />
+ F ( z)<br />
= k + k<br />
u2<br />
f 2<br />
2<br />
1<br />
2<br />
( z∈[<br />
0,<br />
l])<br />
( z∈[<br />
l,<br />
L])<br />
(12)<br />
Where, T f ( ) , ( ) are the free grain corresponding<br />
1 z<br />
T f 2 z<br />
to different positions z over the rotor length.<br />
Hence:<br />
⎧<br />
⎪T<br />
⎪<br />
⎨<br />
⎪T<br />
⎪⎩<br />
f 1<br />
f 2<br />
z<br />
( z)<br />
= [ k<br />
l<br />
( z)<br />
= k<br />
2<br />
2<br />
u1<br />
u −u<br />
u1<br />
( e<br />
u − u<br />
1<br />
1<br />
2<br />
2<br />
( e<br />
−u2z<br />
−u2z<br />
−e<br />
− e<br />
−u1z<br />
−u1z<br />
) + k e<br />
) + k e<br />
−u2z<br />
1<br />
−u2z<br />
1<br />
]<br />
(13)<br />
The free grain which has reached the end of the<br />
threshing unit but still not separated is called the loss of<br />
separation:<br />
L<br />
u<br />
1 −u2L<br />
−u1L<br />
−u2L<br />
f = Tf<br />
2 ( L)<br />
= k2<br />
( e − e ) + k1e<br />
(14)<br />
u1<br />
−u2<br />
Where, L f is the loss of separation.<br />
MATERIALS AND METHODS<br />
Experimental equipment<br />
The testing equipment of axial threshing unit includes rice stripped<br />
mixtures conveyor, feeding unit, threshing unit, data acquisition and<br />
control system etc., as shown in Figure 2. The rice stripped<br />
mixtures conveyor, feeding unit and threshing unit are driven by<br />
different electromotor whose rotator speed are controllable. The<br />
structural parameters such as the cylinder-concave clearance,<br />
height and screwing angle of the oriented board on the cover
Lizhang and Yaoming 4109<br />
Figure 2. Schematic on testing equipment of axial threshing unit. 1, stripped mixture conveyor; 2, intakes; 3, feeding chain; 4, swath<br />
deflector; 5, threshing drum cover; 6, threshing drum; 7, separation concave; 8, frame; 9, straw outtake; 10, grain box; 11, valve handle;<br />
12 straw boxes.<br />
Figure 3. Distribution of spike tooth on threshing drum.<br />
threshing element are adjustable. The sensor is installed to inspect<br />
the rice stripped mixtures conveyor speed, while sensors of rotate<br />
speed and torque are installed on the axis of feeding chain wheel<br />
and threshing drum. The side boards consist of transparent glass,<br />
so it is convenient to observe movements of grain in the test device<br />
(Xu, 2003).<br />
In order to keep the environment of field, structure and motion<br />
parameters suitable, the testing are similar to the 4LGT-150 stripper<br />
combine harvester in marketing. Variety: late japonica rice; thousand<br />
grain weight: 29.4 g; mass percentage of the free grain in the rice<br />
stripped mixtures: 65%; mass percentage of the un-threshed grain<br />
in the rice stripped mixtures: 20%; mass percentage of the straw in<br />
the rice stripped mixtures: 15%; moisture content of mixture: 37.2%;<br />
moisture content of grain: 24.3%; moisture content of straw: 61.2%;<br />
rotor radius: 297mm; entrance breadth ( l ):480 mm; whole rotor<br />
length ( L ):1580 mm; threshing element: spike tooth (distribution of<br />
spike tooth is shown in Figure 3); number of tooth bar: 6; cylinderconcave<br />
clearance: 8 mm; concave style: grid concave; concave<br />
enveloping angle: 220°; conveyor speed: 1 m/s; feeding chain wheel<br />
rotate speed: 466 r/min.<br />
Experimental methods<br />
The whole length of threshing rotor was divided into 10 testing<br />
zones, the entrance breadth 5 equally uniforms and the rest 5<br />
equally divvied, as shown in Figure 4. The testing arrangements
4110 Afr. J. Biotechnol.<br />
Figure 4. Distribution of the testing zones.<br />
Table 1. Experimental result of the threshing unit.<br />
Serial<br />
number<br />
Feed rate /kg.s -<br />
1<br />
Rotator rotate speed<br />
/r.min -1<br />
Separation loss<br />
rate/%<br />
Un-threshed loss<br />
rate/%<br />
Total loss<br />
rate/%<br />
1 1.5 750 0.154 0.304 0.458<br />
2 1.5 850 0.158 0.226 0.384<br />
3 1.5 950 0.161 0.172 0.333<br />
4 1.8 750 0.206 0.544 0.75<br />
5 1.8 850 0.212 0.376 0.588<br />
6 1.8 950 0.223 0.208 0.431<br />
7 2.0 750 0.328 0.51 0.838<br />
8 2.0 850 0.344 0.448 0.792<br />
9 2.0 950 0.338 0.244 0.582<br />
were carried out on three feed rates and three rotate speeds, as<br />
listed in Table 1. Each group repeats 3 times.<br />
Before the test begin, the rice stripped mixtures of certain mass<br />
are uniformly placed on the conveyer 30 m long. In the process of<br />
test, the rice stripped mixtures were fed into the threshing space, in<br />
which the un-threshed grain is threshed and the free grain is<br />
separated through feeding chain. Grains from the concave grate<br />
drop into the testing zone of the grain box. The ejection from the<br />
threshing unit falls into the straw box with the straw, from which the<br />
un-threshed loss and the separation loss can be obtained.<br />
s<br />
Separation loss rate s 100%<br />
T<br />
L = × (15)<br />
W<br />
Where, T s is the mass of the un- separation grain and W is the<br />
mass of the total grain including un- separation grain and unthreshed<br />
grain.<br />
Un-threshed loss rate:<br />
Tu<br />
L t = × 100%<br />
(16)<br />
W<br />
Where, T u is the mass of the un-threshed grain.<br />
Total loss rate:<br />
Lto = Lt + Ls<br />
(17)<br />
RESULTS AND DISCUSSION<br />
Experimental analysis<br />
The experimental results on the rice stripped mixtures to<br />
the threshing unit are listed in Table 1 with three different<br />
feed rate and three levels of the rotate speeds of the<br />
threshing drum.<br />
It is known from the table that: (1) at the same feed<br />
rate, rotate speed of threshing drum has an obvious<br />
effect to the loss rate of un-threshed and the higher the<br />
rotate speed is, the smaller the un-threshed loss rate. But<br />
it is unobvious to the loss rate of separation; (2) on the<br />
condition that other parameters do not change, the feed
Cumulative mass percent /%<br />
A<br />
B<br />
Figure 5. Axial distribution of grain and straw.<br />
rate of the rice stripped mixtures into the threshing unit<br />
has a remarkable effect on the un-threshed loss rate and<br />
separation loss rate and the larger the feed rate is, the<br />
bigger both the loss rate increase accordingly.<br />
As feed rate is 1.8 kg/s and rotate speed of threshing<br />
drum is 850 r/min, the axial distribution of the grain in<br />
testing zone is shown in Figure 5. It can be deduced from<br />
the figure that: (1) as shown in Figure 5a, the grain<br />
separated from the concave grate increases rapidly in<br />
width of the feeding entrance and reaches maximum at<br />
the center point of the entrance, then reduces fast. The<br />
curve of grain falls slowly in the rest length, which tends<br />
to zero by the end of the threshing drum axis. It means<br />
that the loss of grain is little. The distribution of the straw<br />
separated from the concave grate is average along the<br />
whole length; (2) it is known from Figure 5b that, about<br />
70% of the grain has been separated during width of the<br />
feeding entrance (1/3 whole length). If no measure is<br />
adopted, the load to cleaning must be uneven, so that the<br />
cleaning performances become worse.<br />
Simulation analysis<br />
Lizhang and Yaoming 4111<br />
As feed rate is 1.8 kg/s and rotate speed of threshing<br />
drum is 850 r/min according to the test, it can be<br />
observed that the un-threshed loss rate is 0.376% and<br />
the separation loss rate is 0.212%, then solving equation<br />
(8) and (14), it is obtained as follows:<br />
u 1 =2.5151; 2<br />
u =6.994. (18)<br />
Where, l =480 mm; L =1580 mm; k 1 =65% and<br />
k 2 =20%.<br />
Using equations (1), (6), (7), (10), (11) and (13),<br />
simulations are conducted by computer, as shown in<br />
Figure 6. It is known from the simulation that: (1) the<br />
cumulative mass of the separated grain increased rapidly<br />
as exponential curve during the entrance of the mixture,<br />
after which, curve of the mass went gentle gradually. In
4112 Afr. J. Biotechnol.<br />
Figure 6. Variation of the indices of threshing process.<br />
the whole drum, nearly 70% of the grain has been<br />
separated in the anterior part of one third of the whole<br />
length, which is in agreement with the experimental<br />
results, as shown in Figure 5b; (2) at first, the mass of the<br />
free grain in the threshing space increases continuously<br />
and it reaches maximum at the end of the feeding<br />
entrance, then it decreases gradually. By the end of the<br />
threshing drum, the value of the free grain mass is called<br />
the loss of separation; (3) comparatively, the mass of the<br />
un-threshed grain in the threshing space changes slowly,<br />
it also increases firstly and then reduces, which only<br />
accounts for 20% in the rice stripped mixtures. So, it<br />
seems small comparatively. By the end of the threshing<br />
drum, the value of the un-threshed grain mass is called<br />
the loss of un-threshed.<br />
Conclusion<br />
In this study, a mathematical model of threshing unit with<br />
axial feeding of the stripper combine is established with<br />
the method of probability and simulations are also<br />
conducted. The simulations show the variation of the<br />
indices of threshing process along threshing drum axis,<br />
including un-threshed gain, free gain and cumulative<br />
separated gain. On the self-designed testing equipment,<br />
experiments on threshing performance with different feed<br />
rate and drum rotator speed are conducted for the rice<br />
stripped mixtures. Experimental results show that, the<br />
distribution of the cumulative separated gain is in<br />
agreement with the simulation. It is known from the test<br />
that about 70% of grain is separated in the width of<br />
feeding entrance, so the measures must be taken to<br />
make the cleaning loads even. It is helpful for a better<br />
understanding of threshing processes in axial units and<br />
significant for the design of the threshing unit of the<br />
stripper combine.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by a grant from the National<br />
Natural Science Foundation of China (No. 50475123),<br />
high-tech key laboratory of agricultural equipment &<br />
intelligentization of Jiangsu province (No. BM2009703 )<br />
and A Project Funded by the Priority <strong>Academic</strong> Program<br />
Development of Jiangsu Higher Education Institutions<br />
(PAPD). The authors would like to thank the reviewers for<br />
their helpful suggestions on the manuscript.<br />
REFERENCES<br />
Huynh VM (1982). Threshing and separating process-a mathematical<br />
model. Trans. ASAE, 25(1): 65-73.<br />
Jiang YY, Xu JM, Zhang HY Jiang EC (2001). Rice (Wheat) combine<br />
harvester with cutting and windrowing straw immediately after<br />
stripping. Trans. CSAE, 17(1):64-68.<br />
Miu PI (2002a). Mathematical model of threshing process in an axial<br />
unit with tangential feeding. CSAE, (2): 212-219.<br />
Miu PI (2002b). Kinematics model of material movement through an<br />
axial threshing unit. ASAE meeting, Paper Number: 023052.
Price JS (1993). Evaluation of approach to early separation of grain<br />
thresher by a stripping rotor. Agric. Eng. Resour. (56): 65-79.<br />
Trollope JR (1982). A mathematical model of the threshing Process in a<br />
conventional combine-thresher. J. Agric. Eng. Res. (2): 119-130.<br />
Wan JB, Zhao XD, Zhao JH (1990). Mathematic model and application<br />
of conventional threshing unit. Trans. Chin. Soc. Agric. Machinery, 2:<br />
21-28<br />
Xu LZ (2003). Simulation and test research on threshing unit of stripping<br />
combine. Zhen jiang. Jiangsu University.<br />
Lizhang and Yaoming 4113<br />
Yin HQW (1999). Structure technology analyzing of threshing unit. Agric.<br />
Mechanization Studying, (4): 42-44.<br />
Zhou XC, Zhao HH, Qiao XM (1998). Test research of grain axial<br />
threshing unit. Agric. Food Machinery, (3): 4-6.
African Journal of Biotechnology Vol. 10(20), pp. 4114-4120, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2368<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Yield responses of forage sorghums to salinity and<br />
irrigation frequency<br />
Siti Aishah 1 , H. A. R. Saberi 1 *, R. A. Halim 1 and A. R. Zaharah 2<br />
1 Department of Crop Science, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.<br />
2 Department of Land Management, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.<br />
Accepted 18 March, 2011<br />
Water stress restricts crop yield in both the arid and semi-arid zones of the world. In particular, water<br />
stress is associated with low availability of water, as well as osmotic effects associated with salinity.<br />
The response of forage sorghum [Sorghum bicolor (L.) Moench] varieties to salinity and irrigation<br />
frequency were studied from December 2007 to December 2008. Speedfeed and KFS4 were grown<br />
under salinity levels of 0, 5, 10 and 15 dS m -1 and irrigated when the leaf water potential reached -1.0<br />
(control), -1.5 and -2.0 MPa. The irrigation frequency was found to affect growth and yield of the forage<br />
sorghums. When irrigation was delayed in leaf water potential of -1.0 to -2.0 MPa, the yield and yield<br />
components were found to decrease. The maximum dry forage yields were 45.1, 38.9 and 38.5 g plant -1<br />
for frequent, intermediate and infrequent irrigation regimes, respectively. Increased salinity significantly<br />
reduced forage dry yield from 44.09 g plant -1 in the control to 32.76 g plant -1 at salinity of 15 dS m -1 . For<br />
every one unit increase in salinity, the forage yield decreased by 5.2 units and for every one unit<br />
increase in water stress (irrigation frequency), the forage yield decreased by 3.6 units. The variety<br />
Speedfeed had higher total dry mass than KFS4 under well-watered conditions but KFS4 performed<br />
better than Speedfeed under water stress. For both varieties, infrequent watering reduced dry matter<br />
and biomass accumulation, but increased water use efficiency (WUEs) (6.88).<br />
Key words: Salinity, irrigation frequency, dry matter, water use efficiency (WUEs), forage sorghum.<br />
INTRODUCTION<br />
Salinity is one of the most important environmental<br />
factors limiting crop production of marginal agricultural<br />
soils in many parts of the world (Qureshi et al., 2007).<br />
Salts in soil and water can reduce water availability and<br />
this can lead to stressful conditions. Water stress restricts<br />
crop yields, particularly in the arid and semi-arid zones.<br />
However, cultivation under salinity and water stress<br />
conditions is normally practiced for food supply in developing<br />
countries (Munns, 2002). Sorghum has greater salt<br />
and drought tolerances than other summer forages. Only<br />
warm season annual grasses provide substantial forage<br />
yield in a short period of time. There are a number of<br />
potential forage sorghum varieties which may be appro-<br />
*Corresponding author. E-mail: alireza_sa70@yahoo.com.<br />
Abbreviations: LWP, Leaf water potential; FC, field capacity;<br />
PWP, permanent wilting point.<br />
priate for various salinity levels of seawater, but the levels<br />
of salt tolerance among most of the grown forage sorghum<br />
varieties and cultivars have not been adequately<br />
characterized (Qadir and Oster, 2004). The general effect<br />
of salinity is the reduction in growth (Ghoulam et al.,<br />
2002) which resulted from ion toxicity, osmotic stress,<br />
mineral deficiencies, physiological and biochemical<br />
perturbations, as well as the combinations of these<br />
stresses (Munns, 2002; Hasegawa et al., 2000). Due to<br />
low availability of soil moisture from osmotic effects associated<br />
with salinity, water stress occurs during crop<br />
establishment and development. However, where the<br />
growing season is long, tillering varieties of forage<br />
sorghum are able to recover to a certain extent from<br />
water deficits in the early growth periods by forming additional<br />
head-bearing tillers. The resulting yield reduction<br />
can be partly offset by additional head-bearing tillers<br />
(FAO, 2001). Despite significant contributions, earlier<br />
investigations on sorghum were focused primarily on<br />
grain sorghum. Hence, studies are still needed for forage
sorghum to improve the understanding of the effects of<br />
salt and water stresses for different varieties. Many<br />
factors are still needed to be considered when addressing<br />
the suitability of irrigation water with respect to<br />
salinity. Therefore, the objective of this experiment was to<br />
determine the growth responses of two forage sorghum<br />
varieties to different levels of salinity and irrigation<br />
frequency.<br />
MATERIALS AND METHODS<br />
The factorial experiment was conducted under rain shelter at the<br />
University Putra Malaysia (02°N 59.476´ 101°E 2.867´, 51 m<br />
altitude), from December 2007 to December 2008. The climatic<br />
conditions recorded under the rain shelter were 30°C mean temperature,<br />
90% humidity, 4.5 mm evaporation and 72.5% light at 12<br />
am. Two selected (Fouman et al., 2003) salt tolerant varieties<br />
namely Speedfeed and KFS4, of forage sorghum [Sorghum bicolor<br />
(L.) Moench] were subjected to the salinity levels of 0, 5, 10 and 15<br />
dS m -1 of NaCl concentrations, and irrigated when the leaf water<br />
potential (LWP) reached -1.0 (control), -1.5 and -2.0 MPa. KFS4 is<br />
an open pollinated variety from Iran, while Speedfeed is a hybrid<br />
variety originally from Australia.<br />
The treatments were arranged in a randomized complete block<br />
design with three replications. Polybags (40 × 45 cm) were filled<br />
with a mixture of top soil, peat moss and sand at the ratio of 3:2:1<br />
(v/v), respectively. The soil mixture had a pH of 5.4. During mixing,<br />
60 g of CaCO3, 10 g of complete fertilizer (15% N, P2O5, K2O), 1 g<br />
of triple super phosphate (45% P2O5) and 2.4 g of urea (46% N)<br />
were added to each polybag. Soil field capacity (FC) and<br />
permanent wilting point (PWP) were measured based on pressure<br />
plat and pressure membrane procedures outlined by Richards<br />
(1947), before and after the end of the experiment. Soil moisture<br />
was determined by gravimetric method (Muhammad et al., 2008).<br />
The plants were irrigated with non-saline water for seedling<br />
establishment and with saline water starting from 2 weeks after<br />
germination according to the treatments. Plants were harvested at<br />
pre flowering stage and washed with deionized water. Leaves,<br />
stems and roots were separately weighed and dried at 75°C for 48<br />
h for dry mass determination. Forage weight was calculated based<br />
on cumulative shoot dry weight. Irrigation treatments were applied<br />
based on leaf water potential (-1, -1.5 and -2 MPa) using the<br />
pressure chamber (Santa, Barbara, CA, USA). When leaf water<br />
potential reached the considered potential, the soil samples were<br />
taken. The amount of water required for the respective irrigation<br />
treatment was calculated using the following equation (Muhammad<br />
et al., 2008):<br />
V = SMD × A<br />
Where, V = volume of water to be applied (litre); A = polybag area<br />
= חr 2 .<br />
SMD = ( FC θ<br />
θ<br />
- i ) D Bd /100<br />
θ<br />
SMD = soil moisture deficit; FC = gravimetric soil moisture<br />
content at field capacity (%);<br />
θ i = soil moisture content before irrigation (%); D = rooting depth<br />
(cm); Bd = bulk density (1.5 g cm -3 in this soil).<br />
Water use efficiency (WUE) was determined by dividing the total<br />
dry forage by the volume of irrigation water applied in each<br />
treatment during the experimental period. The data were subjected<br />
Aishah et al. 4115<br />
to analysis of variance (ANOVA) using the Statistical Analysis<br />
System (SAS) procedure. Treatment means were compared using<br />
least significant differences (LSD) at the 5% (P ≤ 0.05) probability<br />
level. Regression analysis was used to determine the relationship<br />
among variables and salinity levels.<br />
RESULTS AND DISCUSSION<br />
The irrigation frequency and salinity affect growth and<br />
yield of forage sorghums. The above ground part which is<br />
important in forage sorghum was highly affected by the<br />
main factor, while the total dry weight was significantly (P<br />
< 0.01) influenced by the interaction of salinity, irrigation<br />
and variety (Table 1).<br />
The total dry mass of both varieties declined as salinity<br />
level increased and water regime decreased (Figure 1).<br />
Salinity may affect the growth by creating an external<br />
osmotic potential that prevents water uptake or due to the<br />
toxic effects of Na + and Cl - ions on the plant, it affects the<br />
uniformity of plant density with negative effect on yield<br />
(Okcu et al., 2005). Our result is also in agreement with<br />
Munns (2002). Between varieties tested, the interaction<br />
effect was more significantly pronounced on the<br />
Speedfeed. This was shown by 41.5% decreased in its<br />
biomass under normal irrigation with increasing salinity to<br />
15 dS m -1 (Figure 1B) as compared to 30.5% for the<br />
KFS4 (Figure 1A). At other levels of irrigation (irrigation at<br />
-1.5 and -2 Mpa), from 0 to 15 dS m -1 , the Speedfeed<br />
biomass decreased by 23.8 and 24.1%, respectively.<br />
Whereas for the KFS4, the greatest biomass differences<br />
between irrigation regimes in respect to salinity, were<br />
found only at the highest salinity level. This response<br />
would indicate that the KFS4 variety is more tolerant to<br />
stress conditions than the Speedfeed. Furthermore,<br />
based on varietal characteristic, their total biomass is not<br />
significantly different from each other (Table 1).<br />
Although irrigation and salinity had highly significant<br />
effects on dry forage yield and root growth, an interaction<br />
effect was only found between irrigation and variety.<br />
Irrespective of variety, the dry forage decreased linearly<br />
with increasing salinity (Figure 2A). A similar result was<br />
reported by Hester et al., (2001) who indicated that the<br />
crop yield performance decreased markedly with the<br />
increase in the concentration of salt. A linear reduction<br />
due to salinity was also found on root mass (Figure 2B).<br />
Based on varietal responses (Table 2), Speedfeed was<br />
more sensitive to water stress as indicated by a<br />
significant reduction in its dry root and dry forage. The<br />
impact on Speedfeed however was greatly shown only<br />
when the irrigation schedule changed from -1 to -1.5 Mpa<br />
and as a result, the forage yield decreased by 22.2% and<br />
roots declined by 29.4%. However, further delay in<br />
irrigation did not show any significant reduction in both<br />
parameters. For KFS4, irrigation schedule did not affect<br />
its forage yield and root growth. This characteristic may<br />
contribute favourably to the persistence of this species in<br />
saline soils under natural condition. In irrigated agriculture,
4116 Afr. J. Biotechnol.<br />
Table 1. Effects of varieties, irrigation and salinity on dry matter yield and root-to-shoot ratio.<br />
Source / Treatment<br />
Dry root<br />
(g plant -1 )<br />
Dry forage<br />
(g plant -1 )<br />
Total dry weight (g<br />
plant -1 )<br />
Root to<br />
shoot ratio<br />
Variety<br />
KFS4 7.47a z 42.25a 49.72a 0.176b<br />
Speedfeed 7.96a 39.41b 47.42a 0.201a<br />
LSD 0.59 2.66 3.23 0.021<br />
Irrigation frequency (MPa)<br />
at LWP -1.0 8.60a 45.12a 53.729a 0.731a<br />
at LWP -1.5 7.41b 38.88b 46.298b 0.611b<br />
at LWP -2.0 7.13b 38.48b 45.624b 0.631b<br />
LSD 0.76 3.42 4.164 0.024<br />
Salinity (dS m -1 )<br />
0 8.93 a 45.73 a 54.669 a 0.657b<br />
5 8.97 a 43.61a 52.592 a 0.736a<br />
10 7.27 b 39.79b 47.073 b 0.653a<br />
15 5.68 c 34.17c 39.867 c 0.591b<br />
LSD 0.91 4.08 4.963 0.029<br />
F value<br />
V*I 5.91** 4.37* 15.66** 75.29**<br />
V*S 0.07 ns 0.03 ns 1.38 ns 17.84**<br />
I*S 0.27 ns 0.29 ns 0.75 ns 15.04**<br />
V*I*S 0.75 ns 0.70 ns 3.08** 22.42**<br />
Error and CV<br />
Error (MS) 1.57 31.43 46.43 0.001<br />
CV (%) 16.25 13.73 14.03 6.08<br />
z Means with same letter within a column are not significantly different at 5% level by LSD. V1 = KFS4, V2 =<br />
Speedfeed; S1, S2, S3 and S4 = salinity 0, 5, 10 and 15 dS m -1 , respectively; I1, I2 and I3 = irrigation<br />
frequency when the LWP reach -1, -1.5 and -2MPa, r<br />
salt would normally be leached from the surface at<br />
sowing, and in dry-land agriculture, the crop is normally<br />
planted after rain (Serraj and Sinclair, 2002).<br />
Unlike the dry forage yield and roots, root to shoot ratio<br />
of both forage varieties responded differently to salinity<br />
and irrigation. The root shoot ratio at normal irrigation<br />
exhibited by KFS4 (Figure 3A) seems to be opposite to<br />
Speedfeed (Figure 3B). The root shoot ratio of KFS4<br />
irrigated at normal frequency was lower than plants under<br />
water stress conditions at all levels of salinity. This<br />
phenomenon is a normal plant growth reaction towards<br />
water stress conditions. On the other hand, the root-toshoot<br />
ratio of well-watered Speedfeed was higher than<br />
that under infrequent irrigation. The higher root-to-shoot<br />
ratio of KSF4 under water stress would probably explain<br />
the better performance of KFS4 under water stress<br />
conditions.<br />
Table 3 shows that infrequent irrigations gave higher<br />
WUE’s when compared to frequently irrigated treatments.<br />
In this study, the WUEs obtained for the infrequently<br />
watered plants were higher than earlier reports (Gulzar et<br />
al., 2003; Mustafa and Abdel Magid, 1982). Although<br />
larger volumes of water were used in this investigation as<br />
compared to the two previous reports, the higher WUEs<br />
obtained here might have been due to the reduction of<br />
irrigation by infrequent watering. Irrespective of variety,<br />
the frequently watered plants accumulated greater dry<br />
matter which eventually had produced high dry forage<br />
yield than other irrigation frequencies (Table 3).<br />
The results derived for the irrigation study showed that<br />
despite the possibility of greater surface evaporation with<br />
the light frequent irrigations, sorghum varieties and other<br />
indicators of plant water stress were found to be<br />
improved with low frequent irrigation. Pearson correlation<br />
matrix reveals that most of the studied characters are<br />
significantly and positively correlated, on the other hand,<br />
root to shoot ratio is negatively correlated with all the<br />
factors. Total dry weight is positively correlated with dry
Figure 1. Effect of salinity and irrigation frequency on total dry weight of (A) KFS4 and (B) Speedfeed<br />
(average of two years).<br />
forage, dry stem, dry leaf and dry root (Table 4). Similar<br />
scenario is observed for the forage dry weight with yield<br />
components.<br />
The regression reveals that the forage yield is<br />
significantly and negatively correlated with salinity and<br />
irrigation frequency (F < 0.05). The relationship between<br />
yield, salinity and irrigation frequency was studied, and<br />
Aishah et al. 4117<br />
the regression equation was: Y=-3.277 -5.207X1-3.586X2,<br />
where R 2 = 0.90 (n = 72). For every one unit increase in<br />
salinity (X1), the forage yield would be decreased by 5.2<br />
units and for every one unit increase in water stress (X2),<br />
the forage yield would be decreased by 3.6 units.<br />
Ultimately, when irrigation was delayed from -1 to -1.5<br />
and -2MPa, the forage yields were decreased significantly,
4118 Afr. J. Biotechnol.<br />
Figure 2. Effects of salinity on forage (A) and root dry weight (B) of forage sorghum.<br />
Table 2. Interaction of variety and irrigation on dry root and dry forage mass.<br />
Irrigation schedule<br />
Dry root (g plant -1 ) Dry forage (g plant -1 V1 V2<br />
)<br />
V1 V2<br />
I1 7.97 a 9.98 a z 43.78 a 46.46 a<br />
I2 7.22 a 7.04 b 41.62 a 36.14 b<br />
I3 7.22 a 6.85 b 41.34 a 35.63 b<br />
LSD (%) 1.231 2.131 4.176 7.961<br />
z Means with same letter within a column are not significantly different at 5% level by LSD; V1 = KFS4, V2 =<br />
Speedfeed; I1, I2 and I3 = irrigation frequency when the LWP reach -1, -1.5 and -2MPa, respectively.<br />
regardless of variety from 45.1 to 38.9 and 38.5 g plant –1<br />
for frequent, intermediate and infrequent irrigation<br />
regimes, respectively. The reduction was found to be<br />
mostly between the frequently and the less and least<br />
frequently watered plants which decreased (Table 3).<br />
This finding suggests that in semi-arid environments<br />
(where water saving is very important), if the aim is to get<br />
high WUE, forage sorghum should be irrigated heavily
Figure 3. Root to shoot ratio of KFS4 (A) and Speedfeed (B) as affected by salinity and<br />
irrigation frequency.<br />
Table 3. Effect of irrigation frequency on yield, total amount of water applied and water use efficiency (WUE).<br />
Irrigation<br />
schedule<br />
Days after treatment Dry forage<br />
4 14 22 31 36 41 48 56<br />
Amount of water applied (liter/plant)<br />
yield<br />
(g plant- 1 )<br />
Total<br />
water<br />
used (liter)<br />
Aishah et al. 4119<br />
WUE (g<br />
plant- 1<br />
liter- 1 )<br />
I1 0.5 1 0.96 0.97 1.14 0.97 0.98 1.13 45.12 az 7.65 a 5.88 b<br />
I2 0.5 1 0 1.19 0 1.42 0 1.98 38.88 b 6.10 b 6.35 ab<br />
I3 0.5 1 0 0 2.02 0 0 2.12 38.48 b 5.64 c 6.88 a<br />
LSD (5%) 3.42 0.61 0.60<br />
I1, I2 and I3 are irrigation frequency when the LWP reaches -1, -1.5 and -2MPa, respectively. z Means with same letter within a column are<br />
not significantly different at 5% level by LSD.
4120 Afr. J. Biotechnol.<br />
Table 4. Correlation coefficients between plant weight parameters.<br />
Parameter DR DL DS DF TDW R:SH<br />
DR 1 0.74** 0.86** 0.87** 0.92** -0.28*<br />
DL 1 0.76** 0.87** 0.86** 0.16 ns<br />
DS 1 0.98** 0.97** -0.48**<br />
DF 1 0.99** -0.32**<br />
TDW 1 -0.32**<br />
R:SH 1<br />
**Correlation is significant at the 0.01 level (2-tailed test); DR = dry root, DL = dry leaf, DS = dry<br />
stem, DF = dry forage, TDW = total dry weight, R:SH = root to shoot ratio.<br />
and infrequently. This contradicted the finding by Saeed<br />
and El-Nadi (2004) who recommended using light<br />
frequency to get high WUEs.<br />
Conclusion<br />
Under salinity and water stress conditions, the KFS4<br />
variety has a better vegetative growth performance as<br />
compared to Speedfeed. Infrequent irrigation had<br />
reduced biomass accumulation; the reduction was higher<br />
when low irrigation frequency was coupled with salinity.<br />
Irrigation may be intensified in saline soils to mitigate the<br />
effect of salinity on plant growth. However, there is a<br />
critical level of salinity after which irrigation cannot<br />
mitigate the effect of salinity. The critical level of salinity<br />
for KFS4 was 15 dS m -1 , while for Speedfeed, it was 10<br />
dS m -1 . WUE of forage sorghum could be increased by<br />
infrequent irrigation, and new fields could be well irrigated<br />
by saving water. The results obtained in this study would<br />
serve as a useful guide for managing forage sorghums in<br />
saline and water stressed field conditions.<br />
REFERENCES<br />
FAO STAT (2001). Land and Water division, FAO AGLw, Water, News,<br />
Contacts Agriculture 21 http://www.fao.arg/ag/agl/. Accessed 10<br />
June 2009.<br />
Fouman A, Majidi Heravan E, Nakano Y (2003). Evaluation forage<br />
sorghum varieties for salt tolerance. Proceedings of 7th International<br />
Conference on Development of Dry Lands. 14-17 September 2003,<br />
Tehran (IRAN).<br />
Ghoulam C, Foursy A, Fares K (2002). Effects of salt stress on growth,<br />
inorganic ions and proline accumulation in relation to osmotic<br />
adjustment in five sugar beet cultivars. Environ. Exp. Bot. 47: 9-50.<br />
Gulzar S, Khan MA, Ungar IA ( 2003). Salt tolerance of a coastal salt<br />
marsh grass. Commun. Soil Sci. Plant Anal. 34: 2595-2605.<br />
Hasegawa PM, Bressan RA, Zhu JK, Bohnert H J (2000). Plant cellular<br />
and molecular response to high salinity. Annu. Rev. Plant Physiol.<br />
51: 463-499.<br />
Hester MW, Mendelssohn IA, Mckee KL (2001). Species and population<br />
variation to salinity stress in Panicum hemitomon, Spartina patens<br />
and Spartina alterniflora: Morphological and physiological<br />
constraints. Environ. Exp. Bot. 46: 277-297.<br />
Muhammad A, Haji Kh, Ahmad H, Muhammad A, Ejaz A, Muhammad A<br />
(2008). Effec of available soil moisture depletion levels and topping<br />
treatments on growth rate total dry biomass in chickpea. J. Agric.<br />
Res. 46(3): 229-243.<br />
Munns R (2002). Comparative physiology of salt and water stress. Plant<br />
Cell Environ. 25: 239-250.<br />
Mustafa MA, Abdel Magid EA (1982). Effects of irrigation interval, urea<br />
and gypsum on N, P and K uptake by forage sorghum on highly<br />
saline-sodic clay. Exp. Agr. 18(2): 177-182.<br />
Okcu G, Kaya MD, Atak M (2005). Effects of salt and drought stresses<br />
on germination and seedling growth of pea (Pisum sativum L.). Turk<br />
J. Agric. For. 29(4): 237-242.<br />
Qadir M, Oster JD (2004). Crop and irrigation management strategies<br />
for saline-sodic soils and waters aimed at environmental sustainable<br />
agriculture (p. 19). Sci. Total Environ. Elsevier Press. 323(1-3) 1-19.<br />
Qureshi AS, Qadir M, Heydari N, Turral H, Javadi A (2007). A review of<br />
management strategies for salt-prone land and water resources in<br />
Iran. International Water Management Institute. 30P (IWMI working<br />
paper 125) Colombo, Sri Lanka.<br />
Richards LA (1947). Pressure-membrane apparatus-construction and<br />
use. Soil Sci. 28: 451-454.<br />
Saeed AM, El-Nadi AH (2004). Forage sorghum yield and water use<br />
efficiency under variable irrigation. Irrig. Sci. 18(2): 67-71.<br />
Serraj R, Sinclair TR (2002). Osmolyte accumulation: Can it really help<br />
increase crop yield under drought conditions? Plant Cell Environ. 25:<br />
333-341.
African Journal of Biotechnology Vol. 10(20), pp. 4121-4126, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2406<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Isolation and characterization of a novel sulfuroxidizing<br />
chemolithoautotroph Halothiobacillus from<br />
Pb polluted paddy soil<br />
Jiyan Shi 1 , Huirong Lin 1, 2 *, Xiaofeng Yuan 3 and Yidong Zhao 4<br />
1 Institute of Environmental Science and Technology, Zhejiang University, Hangzhou, 310029, China.<br />
2 Department of Environmental Science and Engineering, Tan Kah Kee College, Xiamen University, Zhangzhou, 363105,<br />
China.<br />
3 Life Science Department, Zhejiang Chinese Medical University, Hangzhou, 310053, China.<br />
4 Institute of High Energy Physics, Chinese Academy of Science, Beijing Synchrotron Radiation Facility, Beijing, 100049,<br />
China.<br />
Accepted 7 April, 2011<br />
A new mesophilic and chemolithoautotrophic sulfur oxidizing bacterium (SOB) strain HT1 was isolated<br />
from a rice rhizosphere soil polluted by Pb using thiosulfate as electron donor at pH 7.0. The 16S rRNA<br />
gene sequence showed that the new isolate was a sulfur oxidizing obligate chemolithotroph belonging<br />
to Gammaproteobacteria, Halothiobacillus and utilizing different reduced sulfur compounds (sulfide,<br />
elemental sulfur, thiosulfate and sulfite) as chemolithotrophic substrates. Strain HT1 was able to use<br />
CO2 as a carbon source responsible for the reduction of nitrate to nitrite, which represented a halophilic<br />
SOB capable of growth within a broad salinity range of 0 to 3 M NaCl and a heavy-metals-tolerant SOB.<br />
HT1 was Gram negative, motile and was proposed as the type strain of a novel species of sulfur<br />
oxidizing bacteria. The soxB gene could not be detected in strain HT1 during thiosulfate oxidation. The<br />
metabolism pathway of HT1 was ‘S4 intermediate’ (S4I) pathway. Sulfur globules accumulated in HT1<br />
were mainly S8.<br />
Keywords: Halothiobacillus, heavy-metals-tolerant, soxB gene, 16S rRNA gene, sulfur globule.<br />
INTRODUCTION<br />
Sulfur oxidizing bacteria play an important role in mineral<br />
cycling in environments. The species of the genus<br />
Thiobacillus include a wide diversity of Gram negative,<br />
rod sulfur oxidizing bacteria which obtain energy from<br />
oxidation of reduced sulfur. They fall into α, β and γ<br />
subclasses of the Proteobacteria and could be classified<br />
as Acidithiobacillus, Halothiobacillus and Thermithiobacillus<br />
(Kelly and Wood, 2000). Biological reduced sulfur species<br />
such as aqueous (hydro) sulfide as well as insoluble<br />
metal sulfides, polysulfides, elemental sulfur, sulfite, thiosulfate<br />
and polythionates are finally oxidized to sulfate, or<br />
more precisely to sulfuric acid. During these processes,<br />
protons are produced. The oxidation of reduced sulfur to<br />
* Corresponding author. E-mail: linhuirong@yahoo.com.cn. Tel:<br />
+86-571-86971424. Fax: +86-571-86971898.<br />
sulfuric acid is of great importance for biohydrometallurgical<br />
technologies. Contrary to its significant role<br />
in the global sulfur cycle and its biotechnological<br />
importance, the microbial fundamentals of sulfur oxidation<br />
are incompletely understood.<br />
Sulfur oxidizing bacterium (SOB) exhibits a wide range<br />
of metabolic flexibility, particularly with respect to processes<br />
involving respiration and energy transduction.<br />
Previous studies concluded that there were at least two<br />
metabolic pathways of SOB during the thiosulfate<br />
oxidation (Friedrich et al., 2001). Some SOB was<br />
involved in the toxicity of heavy metal ion and possesses<br />
unique metabolic and ecophysiological features with<br />
extraordinary properties. Novel SOB with extraordinary<br />
properties was reported (Sorokin et al., 2006; Ghosh and<br />
Roy, 2007). Wood and Kelly (1991) found the first halophilic<br />
SOB capable of growing at very high salt<br />
concentrations (4 M NaCl). Some SOB that is capable of
4122 Afr. J. Biotechnol.<br />
anaerobic growth with sulfur compounds and nitrogen<br />
oxides as electron acceptors play an important role in<br />
mineral cycle.<br />
Herein, we isolated a novel SOB from a Pb polluted<br />
paddy soil. The nearly complete 16 s rRNA gene sequences<br />
and physiological characteristics of the new isolate<br />
were analyzed. Functional soxB gene was detected in<br />
order to investigate its metabolic pathway. Sulfur K-edge<br />
x-ray absorption near edge structure spectroscopy<br />
(XANES) was used to identify the speciation of sulfur in<br />
the cells of HT1.<br />
MATERIALS AND METHODS<br />
Rhizosphere soil of rice was used as the inoculums for enrichment<br />
cultures. Selected physical and chemical properties of the soil used<br />
are: organic matter, 2.26%; pH, 5.63; Pb concentration, 680.09<br />
mgkg -1 ; Zn concentration, 68.96 mgkg -1 ; Cu concentration, 21.10<br />
mgkg -1 ; S concentration, 247 mgkg -1 . Mineral medium used for<br />
enrichment, isolation and cultivation of SOB contained (per liter of<br />
deionised water, added 15 agar for solid medium): Na2HPO4 1.2 g,<br />
KH2PO4 1.8 g, MgSO4·7H2O 0.1 g, (NH4)2SO4 0.1 g, CaCl2 0.03 g,<br />
FeCl3 0.02 g and MnSO4 0.02 g. Ten grams of Na2S2O3 was added<br />
as model sulfur source (Graff and Stubner, 2003). Enrichment of<br />
SOB was conducted with liquid medium on a rotary shaker at 200<br />
rpm at 28°C. For isolation and purification, solid medium was prepared.<br />
0.2 ml of aliquots of the enrichment cultures were transferred<br />
onto the solid medium. The plates were incubated in an incubator at<br />
28°C. Colonies formed on agar plates of the mineral medium were<br />
transferred at least three times to be pure.<br />
DNA of the new isolate -was obtained for amplification and<br />
sequencing of the 16S rRNA gene. The 16S rRNA gene was<br />
amplified with primers BSF8/20: 5'-AGAGT TTGAT CCTGG<br />
CTCAG-3' and BSR1541/20: 5'-AAGGA GGTGA TCCAG CCGCA-<br />
3'. PCR was run using 50 µl reaction volumes and reactant<br />
concentrations in each 50 µl reaction which were 10 to 15 ng of<br />
DNA template, 25 pM of each primer, 2.5 mM deoxynucleotide<br />
triphosphates (dNTPs, Proega, USA), 10×PCR buffer (applied<br />
Biosystems, USA), 0.1 mM MgCl2 solution (Sigma) and 1 U of Taq<br />
polymerase (Applied Biosystems, USA) in nuclease-free water. The<br />
reaction was carried out as follows: 4 min initial denaturation at<br />
94°C, followed by 30 cycles of denaturation at 94°C for 50 s,<br />
annealing at 57°C for 50 s and DNA extension at 72°C for 40 s.<br />
Cycling was completed by a final elongation step of 72°C for 10<br />
min. The PCR product were purified and sequenced by the<br />
Invitrogen Corporation (USA) in China. The nearly complete 16S<br />
rRNA gene sequences of the new isolate were compared using the<br />
BLAST program. A phylogenetic tree was constructed with sequences<br />
aligned with the CLUSTAL X 1.83 and the Paup v.4.0b.8.a<br />
program.<br />
Physiological characteristics were analyzed as described by<br />
Dong and Cai (2001). Cells were suspended in sterile double<br />
distilled water and adhered on copper mesh. Then, the cells were<br />
dyed with 1% uranygl acetate for about 15 s. Transmission electron<br />
microscope (H-7650, HITACHI) was used to observe the morphology.<br />
Different reduced sulfur compounds (thiosulfate, thiocyanate,<br />
elemental sulfur, sulfide, sulfite and tetrathionate) were used to<br />
determine the utilization of substrates as energy sources. The<br />
sulfite solution was prepared in 50 mM EDTA to prevent<br />
autooxidation (Sievert et al., 2000). Nitrogen (nitrate, nitrite) was<br />
used to test as an electron acceptor in the absence of oxygen with<br />
KNO3 and KNO2. Effects of salinity (NaCl) and heavy metal<br />
tolerance (Pb, Zn and Cu) on bacterial growth were examined in<br />
Luria-Bertani medium containing varying concentrations.<br />
PCR amplifications of soxB gene fragments with extracted DNA<br />
were performed as a two-step PCR using the primer sets and<br />
protocols as described by Petri et al., (2001). Sulfur species in the<br />
cell were analyzed by XANES as described by Prange et al., (1999)<br />
at Beijing synchrotron radiation facility, institute of high energy<br />
physics of China. Spectra were recorded at 4B7A beam line and<br />
scanned in the region between 2420 and 2520 eV.<br />
RESULTS AND DISCUSSION<br />
A new isolate using thiosulfate as sulfur was obtained and<br />
named as strain HT1. Colonies grown on mineral medium<br />
with thiosulfate as sulfur source were smooth and whitelight<br />
yellow (1 to 3 mm). The cells of strain HT1 appeared<br />
singly or in pairs and motile with flagellums (Figure 1).<br />
The 16S rRNA genes were amplified using general<br />
bacterial primers. A phylogenetic tree was obtained as<br />
shown in Figure 2. Comparison of the nearly complete<br />
16S rRNA genes showed that strain HT1 was closely<br />
related to Halothiobacillus and belonged to the γ subclass<br />
of the Proteobacteria. Its lineage is Bacteria,<br />
Proteobacteria, Gammaproteobacteria, Chromatiales,<br />
Halothiobacillaceae and Halothiobacillus. The GenBank<br />
accession number for the nearly complete 16S rRNA<br />
gene sequence of Halothiobacillus HT1 is GU013549.<br />
Table 1 shows the physiological characteristics of HT1<br />
when compared with known Halothiobacillus. Reduced<br />
inorganic sulfur compounds sulfide, polysulfides, elemental<br />
sulfur, sulfite and thiosulfate were oxidized as electron<br />
donors for energy by HT1. No growth was observed on a<br />
mineral medium with thiocyanate as energy source.<br />
Under aerobic conditions, the bacterium was able to<br />
grow with nitrate as electron acceptor. However, no<br />
growth was observed with nitrite. It was concluded that<br />
strain HT1 is a nitrate to nitrite reducer and can serve as<br />
a nitrite provider. The isolate grew autotrophically on<br />
thiosulfate and can use carbon dioxide as carbon source.<br />
HT1 was capable of adapting to high salt (3 M) and could<br />
use CO2 as a carbon source, suggesting that it possessed<br />
carboxysomes that contain enzymes involved in<br />
carbon fixation (Tsai et al., 2007).<br />
HT1 could grow in 621 mg.kg -1 of Pb 2+ , 192 mgkg -1 of<br />
Cu 2+ and 325 mgkg -1 of Zn 2+ , respectively, showing<br />
relatively high tolerance of heavy metals. Possible toxicity<br />
and mechanisms of resistance to Cu have been studied<br />
in Sulfolobus solfataricus and Sulfolobus metallicus<br />
(Ettema et al., 2006; Remonsellez et al., 2006). Microbial<br />
sulfur oxidation is of great importance in influencing the<br />
transformation of heavy metals. This useful application<br />
requires heavy-metals-tolerant SOB which can grow in<br />
heavy metals polluted environment. They may possess<br />
genetic determinants for metal resistance encoding<br />
bacterial metallothioneins and heavy metal-transporting<br />
ATPases to exist in toxic heavy metal ions and contribute<br />
to sulfur oxidation (Auernik et al., 2008).<br />
As a result of the phylogenetic and physiological diversity<br />
of sulfur oxidizing bacteria, several different enzymatic
Figure 1. Morphology of cells of strain HT1 grown in LB medium. (a) In pair; (b) in<br />
pair with flagellums; (c) single.<br />
Shi et al. 4123
4124 Afr. J. Biotechnol.<br />
10<br />
65<br />
Escherichia coli MC4100<br />
100<br />
60<br />
100<br />
Acidithiobacillus thiooxidans FJ172634<br />
Acidithiobacillus ferrooxidans FJ157225<br />
Thiomicrospira thyasirae NR_024854<br />
87<br />
Thiomicrospira halophila DQ390450<br />
Thiomicrospirasp AB301715<br />
Thiofaba tepidiphila AB304258<br />
99<br />
100<br />
97<br />
89<br />
97<br />
63<br />
71<br />
Halothiobacillus sp. EF397577<br />
79<br />
HT1<br />
Halothiobacillus sp. EF397570<br />
Halothiobacillus sp. AY096035<br />
Halothiobacillus neapolitanus AB308268<br />
Thiobacillus halophilus THU58020<br />
Thioalkalibacter halophilus EU124668<br />
Thiobacillus neapolitanus AF173169<br />
Halothiobacillus neapolitanus EU871645<br />
Figure 2. Phylogenetic tree derived from 16S rDNA sequence data of strain selected and other related species.<br />
The tree was rooted with Escherichia coli as an out group.<br />
systems and pathways are involved in the dissimilatory<br />
oxidation of thiosulfate. At least two major pathways have<br />
been proposed for different SOB: (1) the sulfur oxidation<br />
pathway (PSO) and (2) the S4 intermediate pathway<br />
involving polythionates (S4I) (Friedrich et al., 2001). The<br />
S4I pathway includes the formation and oxidation of<br />
polythionate or S and sulfite from thiosulfate. In the PSO<br />
pathway, thiosulfate is oxidized directly to sulfate and<br />
does not accumulate intermediate products such as<br />
polythionate, S and sulfite. Thiosulfate oxidation is carried<br />
out by a thiosulfate-oxidizing multi-enzyme system in<br />
which one enzyme is coded by soxB (Anandham et al.,<br />
2008). SoxB contains a prosthetic manganese cluster in<br />
the reaction center and is essential for thiosulfate
Table 1. Comparative characteristics of strain HT1 and typical Halothiobacillus.<br />
Characteristic HT1 Halothiobacillus *<br />
Gram stain test - -<br />
Oxidase / /<br />
Catalase - /<br />
Indole + /<br />
Methyl red test - /<br />
Amylohydrolysis - -<br />
Oxidative fermentation of glucose Ferment /<br />
Nitrate reduction + +<br />
Nitrite reduction - -<br />
Denitrification - -<br />
Gelatin liquefaction - /<br />
H2S + /<br />
Cu tolerance 621 mgkg -1 /<br />
Pb tolerance 192 mgkg -1 /<br />
Zn tolerance 325 mgkg -1 /<br />
NaCl tolerance 175.5 gkg -1 0-234 g.kg -1<br />
Sulfur sources<br />
sulfide + +<br />
S + +<br />
sulfite + +<br />
thiosulfate + +<br />
tetrathionate + +<br />
thiocyanate - -<br />
“/” Denotes this index does not test or exist; “*” denotes reference control<br />
oxidation. HT1 is a sulfur oxidizing obligate chemolithotroph<br />
capable of using thiosulfate as substrate. In<br />
order to detect the pathway of HT1, different primers<br />
were conducted on it to detect the existence of soxB.<br />
PCR-based analysis showed that no specific bands were<br />
obtained in HT1. Our results were consistent with soxB<br />
gene analysis with Halothiobacillus hydrothermalis conducted<br />
by Petri et al., (2001), suggesting that HT1 oxidize<br />
thosulfate via the S4I path way which is enzymatically<br />
different from the PSO path-way.<br />
It was reported that many SOB took up and stored<br />
sulfur globules in the reduced forms (usually as elemental<br />
sulfur). Different bacterial groups stored various forms of<br />
globules (Prange et al., 1999). Sulfur in the globules of<br />
Beggiatoa alba and Thiomargarita namibiensis consisted<br />
of S8, whereas Acidithiobacillus ferrrooxidans stored<br />
polythionates as globules. Purple and green sulfur<br />
bacteria also consisted mainly of polymeric sulfur chains<br />
while the sulfur chains were found in the globules of<br />
Thermoanaerobacter sulfurigignens and<br />
Thermoanaerobacterium thermosulfurigenes. As an<br />
essential macronutrient for microorganisms, plants and<br />
animals, sulfur (S) exists in soils in a great variety of<br />
organic and inorganic species with oxidation states<br />
ranging from -2 to +6. Reference compounds were used<br />
to explore different sulfur oxidation states and chemical<br />
structures. For analysis, the fitting and plotting package<br />
Shi et al. 4125<br />
WinXAS version 3.1 was used. The spectra were<br />
normalized by fitting first and second order polynomial<br />
functions to the pre- and post-edge regions respectively.<br />
Sulfur K-edge XANES analysis showed that sulfur<br />
globules in HT1 fit well with cyclooctasulfur (S8) which<br />
was consistent with the results of Engel et al., (2007)<br />
(Figure 3). This could be either a biosignature of bacteria<br />
that preferentially take up and oxidize cyclooctasulfur (S8)<br />
or a sign that the polymeric form of sulfur is preferred,<br />
thus leaving cyclooctasulfur to accumulate.<br />
HT1 was a heavy-metals-tolerant SOB which might<br />
play an important role in biogeochemical cycle. Microorganisms<br />
are considered to be important contributors to<br />
the biogeochemical cycle of trace elements through<br />
several microbe mediated processes. Most sulfur transformations<br />
are fundamentally controlled by biosphere<br />
processes, especially by the specialized metabolisms of<br />
microorganisms. Biological oxidation was the principal<br />
pathway for mineralization of organic S. Sulfur oxidation<br />
by metal resistant SOB contributed to the formation of<br />
SO4 2- , leading to the transformation of sulfur.<br />
ACKNOWLEDGEMENTS<br />
This work wassupported by Zhejiang Provincial Natural<br />
Science Foundation of China (R5110031), the National
4126 Afr. J. Biotechnol.<br />
Absorption<br />
2.460 2.465 2.470 2.475 2.480 2.485 2.490<br />
Energy (KeV)<br />
Figure 3. Sulfur K-edge XANES spectra of HT1 cells.<br />
Natural Science Foundation of China (40601086,<br />
20777066), and the Fundamental Research Funds for the<br />
Central Universities (2010QNA6025).<br />
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HG, Steudel R, Dahl C, Hormes J (1999). In situ analysis of sulfur in<br />
the sulfur globules of phototrophic sulfur bacteria by X-ray absorption<br />
near edge spectroscopy. BBA-Gen Subjects, 1428: 446-454.<br />
Remonsellez, F, Orell A, Jerez CA (2006). Copper tolerance of the<br />
thermoacidophilic archaeon Sulfolobus metallicus: possible role of<br />
polyphosphate metabolism. Microbiology, 152: 59-66.<br />
Sievert SM, Heidorn T, Kuever J (2000). Halothiobacillus kellyi sp. nov.,<br />
a mesophilic, obligately chemolithoautotrophic, sulfur-oxidizing<br />
bacterium isolated from a shallow-water hydrothermal vent in the<br />
Aegean Sea, and emended description of the genus Halothiobacillus.<br />
Int. J. Syst. Evol. Micr. 50: 1229-1237.<br />
Sorokin DY, Tourova TP, Kolganova TV, Spiridonova EM, Berg IA,<br />
Muyzer G (2006). Thiomicrospira halophila sp nov., a moderately<br />
halophilic, obligately chemolithoautotrophic, sulfur-oxidizing<br />
bacterium from hypersaline lakes. Int. J. Syst. Evol. Micr. 56: 2375-<br />
2380.<br />
Tsai Y, Sawaya MR, Cannon GC, Cai F, Williams EB, Heinhorst S,<br />
Kerfeld CA, Yeates TO (2007). Structural analysis of CsoS1A and the<br />
protein shell of the Halothiobacillus neapolitanus carboxysome. Plos.<br />
Biol. 5: 1345-1354.<br />
Wood AP, Kelly DP (1991). Isolation and characterisation of Thiobacillus<br />
halophilus sp. nov., a sulphur-oxidising autotrophic eubacterium from<br />
a Western Australian hypersaline lake. Arch. Microbiol. 156: 277-280.<br />
Dong XZ, Cai MY (2001). Identification of common Bacterial Systems<br />
usual Manual, Science Press. Beijing.
African Journal of Biotechnology Vol. 10(20), pp. 4127-4132, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2452<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Biomonitoring of some heavy metal contaminations<br />
from a steel plant by above ground plants tissue<br />
M. Ataabadi, M. Hoodaji* and P. Najafi<br />
Islamic Azad University, Khorasgan Branch, Isfahan, Iran.<br />
Accepted 21 March, 2011<br />
Soil and plants growing in the vicinity of industrial areas display increased concentrations of heavy<br />
metals and give an indication of the environmental quality. The contamination source for aluminum,<br />
iron, nickel and lead in the Botanical garden of Mobarakeh Steel Company was recognized by analyzing<br />
the leaves and topsoil of two evergreen species: Quercus brantii and Ligustrum vulgare. Availability of<br />
the studied elements for plants was investigated by measuring their total and extractable concentration<br />
inthe soils. For ensuring air borne source of these metals, plant tissues were washed with distilled<br />
water. Plant available heavy metals of the soils were few in comparison with the total values which were<br />
related to the high pH value and the CaCO3 percentage in the soils around each plant. On the other<br />
hand, significant differences were obtained between the washed and unwashed leaves in both species<br />
indicating metal contaminations from the atmosphere. Significant differences were detected between<br />
the contaminated and background site samples in both plants for Al, Fe and Ni. In addition, the leaves<br />
of L. vulgare accumulated more elements partially than Quercus brantii. Difference in the accumulation<br />
potentials of the plants were related to the diversity of the physical and chemical properties of the<br />
leaves and the type of elements. The enrichment ratio of plants (Ccontaminated/Cbackground) was calculated<br />
and it indicated that, the Fe in both plants was moderately enhanced by anthropogenic activities.<br />
Key words: Industrial contamination, Quercus brantii, Ligustrum vulgare, enrichment factor.<br />
INTRODUCTION<br />
Human activities cause the slow extermination of plant<br />
and animal species in nature through toxic pollution due<br />
to industrial and technological advancement in recent<br />
decades (Ives and Cardinale, 2004). Many heavy metals<br />
emitted mostly from anthropogenic sources, have now<br />
exceeded or equaled their natural emissions (Biney et al.,<br />
1994) and have been posing a serious threat to the<br />
ecosystems (Wen Kuang et al., 2006).<br />
Soil and plants growing in the nearby zone of industrial<br />
areas display increased concentration of heavy metals,<br />
serving in many cases as biomonitors of pollution loads.<br />
Plants take up large quantities of pollutants and translocate<br />
them into vegetative and generative organs at<br />
various rates (Kovács et al., 1993), which make clear the<br />
quality of the environment. Calzoni et al. (2007) in their<br />
studies on the ability of Rosa rugosa for biomonitoring of<br />
*Corresponding author. E-mail: M_Hoodaji@Khuisf.ac.ir Tel:<br />
0311-5354061, 09131172651. Fax: 0311-5354061.<br />
heavy metal indicated that, leaf accumulation was due to<br />
atmospheric deposition rather than to soil uptake (Calzoni<br />
et al., 2007). Chemical foliar analysis has also been employed<br />
to study the impact and extent of air pollutants<br />
(Djingova et al., 1999; Ericsson et al., 1995; Hüttl and<br />
Fink, 1991) and the pollutant accumulation capacity of<br />
different plants (Bicchiega et al., 1994; Somsak et al.,<br />
2000). Leaves of higher plants have been used for biomonitoring<br />
heavy metals since the 1950s (Al-Shayeb et<br />
al., 1995). Pine needles for the determination of airborne<br />
pollutants are also a suitable technique for monitoring<br />
purposes (Holoubek et al., 2000).<br />
Parts of the deposited p<strong>articles</strong> are not removed by<br />
rainfall and become irreversibly adsorbed or incorporated<br />
into the hydrophobic wax layer of the foliage (Rossini and<br />
Raitio, 2003). Therefore, cleaning has an effect on the<br />
foliar concentrations of Al, As, At, Br, Cd, Cr, Fe, Ni, Pb,<br />
V, T and other heavy metals (Rossini and Raitio, 2003).<br />
Aksoy and Öztürk (1996), for example, used date palm<br />
leaves to monitor the distribution of airborne Pb, Cd, Zn<br />
and Cu in the city of Antalya in Turkey. A comparison of
4128 Afr. J. Biotechnol.<br />
Table 1. Mean values of soil properties.<br />
Site pH O.C (%) CEC (meq/100 g) CaCO3 (%) Texture<br />
Contaminated<br />
Q. brantii 8.1 0.3 11.2 68 Sandy loam<br />
L. vulgare 8.3 0.2 10.5 70 Sandy loam<br />
Background<br />
Q. brantii 8.1 1.3 29.1 26 Clay loam<br />
L. vulgare 8 1.2 28.5 23 Clay loam<br />
O.C, organic carbon; CEC, cation exchange capacity.<br />
washed and unwashed samples showed that, leaf<br />
analyses gave a reasonably reliable measure of the total<br />
aerial fallout of heavy metals in the studied area.<br />
Plants contamination in most cases arises from<br />
atmospheric p<strong>articles</strong> accumulation through their foliage<br />
and leaves and the degree of contamination depend on<br />
be smoothness of leaves, wind speed and on the value of<br />
rainfall. Ward et al. (1977) expressed that plant washing<br />
after sampling, decreased the element contents to about<br />
10 to 30% in comparison with unwashed plants (Ward et<br />
al., 1977).<br />
Hoodaji and Jalalian (2003 a,b) showed that, soil and<br />
plant near the Mobarakeh Steel Company were slightly<br />
contaminated with some heavy metals, such as Fe, Zn<br />
and Mn; for example they also reported that, topsoils (0<br />
to 5 cm) and shoot of rice, wheat and bean were enriched<br />
in Fe (Hoodaji and Jalalian, 2003a, 2003b). The plant<br />
contamination was assessed using the enrichment factor.<br />
The enrichment factor (EF) was based on the<br />
standardization of a tested element against a reference<br />
one (Reinmann et al., 2001). The aim of this study was to<br />
assess the leaves of Quercus brantii and Ligustrum<br />
vulgare as possible biomonitors of heavy metal contamination<br />
in the vicinity of industrial area.<br />
MATERIALS AND METHODS<br />
Site description<br />
Mobarakeh Steel Company (MSC) is located at 65 km south west of<br />
Isfahan, near the city of Mobarakeh, Isfahan Province, Iran. It is<br />
Iran's largest steel maker and one of the largest industrial<br />
complexes operating in Iran and is one of the main contamination<br />
sources in this region. This study was done in the Botanical garden<br />
of Mobarakeh Steel Company located in 32°34'15’N and 51°25' 21,<br />
E southwest of Isfahan, Iran. This region has an arid climate with a<br />
mean annual rainfall of 140 mm and wind direction of SW-NE. The<br />
region’s type of soil, according to information obtained from profile<br />
description was Typic Haplocalcids. The garden was approximately<br />
3 ha in area, with 9 plots and 3 replications. The background site<br />
was located at 50 km away in a village with low traffic in the Bagh<br />
Bahadoran region. Minimum and maximum annual temperatures of<br />
this location were 7.6 and 24.33°C, respectively. Relative humidity<br />
was 40% and mean wind speed was 1.77 km/h.<br />
Two species, Q. brantii and L. vulgare were selected. These<br />
species are widely distributed in landscape and can survive under a<br />
wide temperature range and grow in almost any type of soil. They<br />
have different behaviors to metal contaminants due to various<br />
morphological and physico-chemical characteristics of their leaves.<br />
L. vulgare is a shrub with oval to lance-shaped sub-shiny dark<br />
green leaves, thick cuticle and moderate epicuticular waxy layer,<br />
whilst Q. brantii is a tree with spiny shape of the lightly lobed leaves<br />
and thin cuticle.<br />
Sampling, preparation and analysis of plant samples<br />
The sampling of both species was performed in August 2007 from<br />
the studied and background sites. Leaf sampling was done with<br />
wind direction (SW to NE) and from approximate height of 190 and<br />
140 cm for Quercus and Ligustrum, respectively, along 9 plots in 3<br />
replications. After transferring the samples to a laboratory, leaves<br />
were divided in to two parts. One part was washed with de-ionized<br />
water to clean dust and any deposited substances on the leaves<br />
and another part was not cleaned. At first, all samples were air<br />
dried and then oven dried at 70°C for 48 h to constant mass, milled<br />
and sieved through a 35 mesh screen. 1 g milled powder of each<br />
samples were digested with 10 ml 2N HCl (Chapman and Pratt,<br />
1961).<br />
Sampling, preparation and analysis of soil samples<br />
Soil samples were also collected randomly from a depth of 0 to 30<br />
cm around each plant. Soil samples were air dried, ground and<br />
passed through sieve (2 mm).The main soil chemical properties<br />
were determined by laboratory analysis. Organic carbon was<br />
determined by a modified wet oxidation (Nelson and Sommers,<br />
1982). Soil pH was measured using potentiometric titration of the<br />
soil extract. Cation exchange capacity (CEC) was measured by<br />
ammonium acetate extraction (Rhoads, 1982). Calcium carbonate<br />
was determined by back titration (FAO, 1974). Soil texture was<br />
measured by the Hydrometer method (Gee and Bauder, 1986). The<br />
total metal concentrations (Al, Fe, Ni and Pb) were determined after<br />
digestion with 10 ml HNO3 70% (Pyatt, 1999; Soon and Abbund,<br />
1993). The plant available heavy metals in soils were extracted with<br />
diethylen triamine penta acetic acid (DTPA) solution (0.005 M<br />
DTPA + 0.01 M CaCl2 + 0.1 M TEA at pH = 7.3) (Lindsay and<br />
Norvell, 1978). Finally, heavy metal concentrations in plant and soil<br />
samples were determined by ICP- AES (GBC Integra XL model).<br />
Some chemical and physical properties of soils are given in Table<br />
1.<br />
To identify the possible origin of metals in the plants, an<br />
enrichment factor of plant (EFplant), was calculated with Equation 1<br />
for each metal as follows:
Table 2. Total and plant available mean metal concentration (mg kg -1 ) ± Standard error in topsoil around each plant.<br />
Ataabadi et al. 4129<br />
Location/species<br />
Contaminated site<br />
Al<br />
Q. brantii<br />
Fe Ni Pb Al<br />
L. vulgare<br />
Fe Ni Pb<br />
Total 17651.3 ± 1399.9 22693.3 ± 956.7 40.1 ± 3.3 16.6 ± 0.7 15492 ± 1189.7 20005.3 ± 1364.7 36.7 ± 1.9 18.6 ± 4.1<br />
Plant available 0.4 ± 0.2 9.3 ± 1.1 2 ± 0.2 1.1 ± 0.1 0.3 ± 0.1 12.7 ± 3.3 1.5 ± 0.1 0.8 ± 0.4<br />
Available percent* 0.005 0.04 5.07 6.62 0.002 0.06 3.98 4.31<br />
Background site<br />
Total 19250 ± 1000 30000 ± 680 57.2 ± 1 15.9 ± 1 18750 ± 788 30400 ±1500 44.8 ± 5.5 21.5 ± 2<br />
Plant available 2.3 ± 0.1 18.9 ± 3 1.3 ± 0.09 3.3 ± 0.2 2 ± 0.3 30.3 ± 0.75 1.2 ± 0.1 2.9 ± 0.08<br />
Available percent* 0.01 0.06 2.3 19.8 0.01 0.1 2.71 13.5<br />
*Available percent: (plant available / total) × 100.<br />
M<br />
EF = (1)<br />
plant<br />
plant<br />
M control<br />
Where, Mplant and Mcontrol are the concentrations of the<br />
examined elements of the plant in the contaminated and<br />
background sites, respectively (mg kg -1 ) (Reinmann et al.,<br />
2001).<br />
Data analysis was done by a statistical package SPSS<br />
14.0. To evaluate the effect of washing treatment on<br />
element concentration, the statistical significance (p <<br />
0.05) of the differences between unwashed and washed<br />
samples was determined using t-test.<br />
RESULTS AND DISCUSSION<br />
Relationship between soil properties and<br />
heavy metal uptake by plant<br />
The total and plant available heavy metal values<br />
in the soils are given in Table 2. The total metal<br />
concentrations were high, but only a little fraction<br />
of them were available for the plants. These<br />
results can be explained with the consideration of<br />
the chemical properties of the soils in Table 1.<br />
Total soil heavy metal concentration is commonly<br />
used to indicate the degree of<br />
contamination (Karaca, 2004), although, DTPAextractable<br />
concentration provides a more<br />
suitable chemical evaluation of the amount of<br />
metals available for plant uptake (Lindsay and<br />
Norvell, 1978; Pretuzzelli, 1989; Zufiaurre et al.,<br />
1998). Loading and accumulation of heavy metals<br />
in the soil depend on different factors such as<br />
chemical form of elements, pH, organic matter<br />
content, texture and cation exchange capacity<br />
(CEC) of the soil (Logan and Chaney, 1983). With<br />
increasing pH, organic matter content, CEC and<br />
clay percentage and availability of the metals<br />
reduced. In addition to the existence of carbonate,<br />
sulfate, phosphate and sulfide forms of elements<br />
in soil causes an increase in the metal precipitation<br />
and consequently decrease their availability<br />
for plant (Shuman, 1985 and Forstner,<br />
1985).Therefore, in calcareous soils (CaCO3 ><br />
60%) mobility and uptake of heavy metals were<br />
very low. Kabata-Pendias and Pendias (1999)<br />
also expressed that, uptake and disuptake of<br />
elements depend on plant species, growth stage<br />
and composition of soil solution, especially Ca<br />
(Kabata-Pendias and Pendias, 1999).<br />
Consequently, in the contaminated site, the<br />
existence of more than 60% CaCO3 with pH >8<br />
led to decrease in mobility and availability of these<br />
elements in the soils.<br />
Consideration of the available percents of the<br />
metals also confirmed the results, therefore, it can<br />
be concluded that the source of these elements in<br />
plants cannot be the soil.<br />
Concentration of elements in plants<br />
Leaf samples from contaminated site (MSC) had<br />
significantly higher concentrations of Al, Fe and Ni<br />
than the background site for both species. This<br />
result showed the direct effect of atmospheric<br />
contamination in industrial regions. The effects of<br />
industrial activities on heavy metal pollution have<br />
been reported by many authors (El-Hassan et al.,<br />
2002; Rossini and Mingorance, 2006). Comparison
4130 Afr. J. Biotechnol.<br />
Table 3. The mean values of EFplant.<br />
Plant species Al Fe Ni Pb<br />
Q. brantii 1.5 2.6 1.9 0.9<br />
L. vulgare 1.5 2.9 1.9 1.5<br />
Table 4. Elemental concentrations ± standard error in leaves of Q. brantii compared by t-test.<br />
Element<br />
Site<br />
Al<br />
Unwashed Washed<br />
T-test<br />
Fe<br />
Unwashed Washed<br />
T-test<br />
Ni<br />
Unwashed Washed<br />
T-test<br />
Pb<br />
Unwashed Washed<br />
T-test<br />
Contaminated 367.6 ± 28.7 176.5 ± 27.4 * 1001 ± 157.9 471 ± 41.8 * 85.4 ± 5.2 60.08 ± 9.1 * 3.5 ± 0.7 1.2 ± 0.2 *<br />
Background 250.3 ± 25.3 146.8 ± 16.1 * 380.8 ± 45.25 157.08 ± 28.8 * 45.2 ± 6.85 29.6 ± 5.49 ns 3 ± 0.0.1 1.3 ± 0.5 *<br />
T-test * ns ** * * * ns ns<br />
Significance: *p < 0.05, **p < 0.01. ns: not significant.<br />
Table 5. Elemental concentrations ± Standard error in leaves of L. vulgare compared by t-test.<br />
Site<br />
Al<br />
Unwashed Washed<br />
T-test<br />
Fe<br />
Unwashed Washed<br />
T-test<br />
Ni<br />
Unwashed Washed<br />
T-test<br />
Pb<br />
Unwashed Washed<br />
T-test<br />
Contaminated 415 ± 85.2 266.7 ± 34.6 ns 1024 ± 156.6 486.32 ± 51.7 * 85 ± 10.2 45.1 ± 3.9 * 4.7 ± 0.6 1.1 ± 0.2 *<br />
Background 267.2 ± 35.1 60.9 ± 9.2 * 350.3 ± 55.6 100 ± 9.25 * 43.6 ± 2.80 15.3 ± 1.34 * 3 ± 0.2 05 ± 0.08 *<br />
T-test * * ** * ns * ns ns<br />
*p < 0.05; **p < 0.01; ns, not significant.<br />
of the normal content of Fe in plants (100 to 500<br />
mg kg -1 of dry weight) (Pais and Benton, 1997)<br />
with the detected concentrations, showed that the<br />
contamination of Fe in the study area was<br />
considerable.<br />
Enrichment factor of plant<br />
EFplant showed the different ratio of metal bioaccumulation<br />
in each part of the plant in relation<br />
to the control site. EF > 2 indicates sample<br />
enrichment (Mingorance et al., 2007). 1
Investigation of atmospheric source of elements with<br />
water washing treatment<br />
The ability to distinguish airborne and soil borne<br />
contamination was assessed by washing the leaves.<br />
Washing with water was an effective procedure for the<br />
removal of Pb, Fe, Al, Cr and V from the leaves of<br />
ornamental species (Rossini Oliva and Raitio, 2003)<br />
General comparison between the element concentrations<br />
before and after washing in the contaminated site<br />
indicated significant differences in both species except for<br />
Al in Ligustrum vulgare (Table 4 and 5). In the<br />
contaminated site, after washing, element contents of<br />
leaves decreased about 29-64 and 28-74 percents for<br />
Quercus and Ligustrum, respectively. Therefore, it can be<br />
concluded that metal concentrations in the plants in the<br />
studied area were due to atmospheric dust from industrial<br />
activities. Removal efficiency of the studied elements<br />
after washing with water were much higher in comparison<br />
with Ward et al ΄s results (1977), which reported<br />
reduction in element content about 10-30 percents with<br />
washing (Ward et al., 1977). Reduction in element<br />
content after washing with water is different by physical<br />
and chemical characters of pollutants, plant species and<br />
subsequently properties of leaves (Rea et al., 2000,<br />
(Rossini Oliva and Raitio, 2003)<br />
Conclusions<br />
The findings of this study showed that, both<br />
species were suitable biomonitors for Al, Fe and Ni<br />
contamination in the studied area. L. vulgare acted better<br />
than the Q. brantii in some cases which confirmed that<br />
the accu-mulative ability of plant tissues depend on their<br />
surface properties (Tables 4 and 5). Both species were<br />
mode-rately enriched in Fe due to the EFplant > 2.<br />
Considerable differences between the total and DTPAextractable<br />
heavy metals in soils due to high pH value<br />
and CaCO3 percentage disproved the probability of plant<br />
contamination through the soil and fortified the theory of<br />
atmospheric source of these elements in the industrial<br />
area. On the otherhand, the use of water-washing<br />
procedure which led to significant differences in the<br />
heavy metals content of the leaves in both plants in the<br />
contaminated site, demon-strated the atmospheric source<br />
of the metals.<br />
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Reinmann C, Koller F, Frengstad B, Kashulina G, Niskavaara H,<br />
Englmaier P (2001). Comparison of the element composition in<br />
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Northern Europe. Sci. Total. Environ. 278(1-3): 87-112.<br />
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effects on the chemical composition of foliar samples. Boreal Environ.<br />
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Somsak L, Simurdova B, Liptak J, Kromka M, Antonio J (2000).<br />
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Carter MR (Eds.). Soil sampling and Method of Analysis. pp: 101-<br />
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species in New Zealand. Environ. Sci. Technol. 11(9): 917-920.<br />
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in bark of Pinus massoniana (Lamb.) as an indicator of atmospheric<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4133-4137, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1388<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Isolation, purification and identification of bacteria from<br />
the shoes worn by children<br />
Hui Li, Changqing Zhao, Jin Zhou, Huanhuan Shao and Wuyong Chen*<br />
National Engineering Laboratory for Clean Technology of Leather Manufacture, Institute of Life Sciences, Sichuan<br />
University, Chengdu 610065, China.<br />
Accepted 4 April, 2011<br />
13 strains of bacteria were isolated from 12 shoes that were worn by children aged 6 to 12 for more than<br />
half a year. Through morphological observation, physiological and biochemical measurements, as well<br />
as 16SrRNA sequence analysis, the bacteria were identified as follows: Bacillus licheniformis, Bacillus<br />
subtilis (5 subspecies), Bacillus spore (3 subspecies), Bacillus anthracis, Staphylococcus aureus,<br />
Bacillus amyloliquefaciens and Bacillus thuringiensis. The results may contribute to the selection of<br />
efficient antimicrobial agents for children's shoes and insole.<br />
Key words: Children’s shoes, bacteria, isolation, identification.<br />
INTRODUCTION<br />
Shoes are necessary for everyone, especially as they are<br />
worn by all in almost 2/3 of humans’ life time. They are<br />
vulnerable to contamination by microorganisms when<br />
worn. Some published results show that a great amount<br />
of bacteria and fungi attach themselves to the feet and<br />
socks of humans, with the density of 5,000 to 100,000<br />
colonies/cm 2 , and more severely with a density of<br />
10,000,000 to 1,000,000,000 colonies/cm 2 (Xu, 2006). It<br />
is these microorganisms that react with the sweat of<br />
humans and cause an undesired odor. However, various<br />
microorganisms could grow and breed largely in the<br />
micro-environment of inner shoes and would rightly<br />
cause incomparable beriberi by reacting with human’s<br />
sweat. On the other hand, there was little information<br />
about these bacteria and fungi in shoes, although the<br />
pathogens which these microorganisms belong to have<br />
not been reported.<br />
Since children are usually engaged in many physical<br />
activities, they could secrete more sweat than adults for<br />
their vigorous metabolism. As a result, more bacteria<br />
would breed and lead to more serious foot diseases (Cai,<br />
2005). In the long-term result, a series of diseases could<br />
be induced, which would severely do harm to children's<br />
physical and mental health ultimately (Dorman et al., 2000).<br />
*Corresponding author. E-mail: wuyong.chen@163.com.<br />
Tel/Fax: + (86)28 8540 4462.<br />
Shamez had successfully isolated a strain of<br />
Staphylococcus from children’s shoes (Shamez and<br />
Mehdi, 2005), but up till now, no report has been made<br />
on the isolation of other bacteria from children’s shoes. If<br />
the specific pathogenic microorganisms could be isolated<br />
and identified from the shoes, some effective antimicrobial<br />
agents for the different species of bacteria<br />
could be selected for materials used in making shoes to<br />
protect children's feet from being infected by the microorganisms.<br />
For this purpose, the bacteria from children’s<br />
shoes were isolated and identified with morphological<br />
observation, physiological and biochemical tests, extraction<br />
of genomic DNA, PCR amplification and analysis of<br />
16SrRNA sequence.<br />
MATERIALS AND METHODS<br />
12 pairs of shoes that were worn by children aged 6 to 12 for more<br />
than half a year, were selected as the shoes’ sample. Beef extract<br />
peptone medium was used for the isolation of bacteria (Ausubel et<br />
al., 2005). LB medium, TE buffer (pH 8.0), CTAB/NaCl solution (pH<br />
8.0) and dNTPs mixture were selected for 16SrRNA sequence<br />
analysis (Joseph et al., 2001).<br />
Isolation of bacteria<br />
The leather insole, plastic insole and leather lining from the 12 pairs<br />
of shoes were cut into pieces. Then, the pieces were put in a sterile<br />
conical flask with glass beads and 100 ml sterile water was added<br />
under sterile conditions. After the pieces were soaked in sterile
4134 Afr. J. Biotechnol.<br />
water for 1 h, the conical flask was shook in a vibrator (CHZ-82,<br />
Jintan Fuhua Instrument Co., China) at 200 rpm for 3 h to disperse<br />
the cells of germs. When the cells were singly dispersed, 1 ml<br />
sample of the cell suspension and 9 ml sterile water were added<br />
together into a test tube and mixed fully. Then 1 ml mixture suspension<br />
was taken into 9 ml sterile water to obtain the 10 -2 dilution.<br />
According to the same procedure, different concentrations of<br />
bacteria (10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 ) were obtained (Lu et al.,<br />
2006). For isolation of the aerobic bacteria, 0.05 mg/ml nystatin was<br />
added to sterile beef extract peptone medium to inhibit the growth<br />
of molds and yeasts (Papamanoli et al., 2003). When the nystatin<br />
and medium were mixed uniformly, 15 ml of the mixture was poured<br />
into culture plates. After the medium was solidified by cooling, 0.2<br />
ml diluents of 10 -5 , 10 -6 and 10 -7 for each concentration were drawn<br />
into plates by a sterile pipette. Each concentration of germ<br />
suspension was inoculated for 3 plates, and then the plates were<br />
coated by a sterile glass rod uniformly. Lastly, the culture plates<br />
were stored in a biochemical incubator (SPX-80BS-II, Shanghai<br />
Medical Equipment Manufacture Co., China) at 37°C for 2 days.<br />
For isolation of the anaerobic bacteria, 15 ml of beef extract<br />
peptone medium was poured into culture plates. After the medium<br />
was solidified by cooling, 0.2 ml diluents of 10 -4 , 10 -5 and 10 -6 for<br />
each concentration were drawn into plates by a sterile pipette. Each<br />
concentration of germ suspension was inoculated for 3 plates and<br />
then plates were coated by a sterile glass rod uniformly. Lastly, the<br />
culture plates were sealed by sterile Vaseline and placed in a<br />
vacuum dryer. After been vacuumed, the plates were stored in a<br />
biochemical incubator at 37°C for 2 days to enable the anaerobic<br />
bacteria grow largely on the plates.<br />
Purification of the isolates<br />
The morphology of colonies was observed with optical microscope<br />
(XZE-H, Chongqing Optical Instrument Co., China). According to<br />
different morphology of the colonies, the single colony of<br />
predominant bacteria was picked and inoculated to the medium<br />
slant, and in total, there were 13 strains of bacteria identified<br />
preliminarily. Then purified colonies were obtained by repeated<br />
streaking of the single colony on fresh agar plates and their<br />
morphology was recorded as the basis for classification in detail<br />
(Christine, 2002). In the experiment, there were no strict anaerobe<br />
bacteria on the vacuum dryer flat.<br />
Morphological identification<br />
After it was cultured at 37°C for 24 h, the strains were picked from<br />
the slants and were stained with gram as well as spore (Kandler et<br />
al., 1986). Then, the cell and spore of the strains were observed<br />
with optical microscope. At the same time, the common standard<br />
strains of Escherichia coli and Bacillus were selected as references<br />
(Kim et al., 2000).<br />
Physiological and biochemical appraisal<br />
There were a total of 10 trials based on the identification of a<br />
common bacterial system for appraising physiological and<br />
biochemical characteristics of the bacteria (Joseph et al., 2001).<br />
The common standard strains such as E. coli and Bacillus were<br />
also used as references.<br />
Extraction of genomic DNA, amplification and analysis of<br />
16SrRNA<br />
According to the manufacturer’s instructions, the extraction of the<br />
total genomic DNA was performed using DNA extraction kit<br />
(Promega, USA) and PCR amplification was performed. A portion of<br />
the bacterial 16SrRNA gene was amplified using the primers, 27F<br />
(5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCT<br />
ACCTTGTTACGACTT-3') (Mora et al., 1998). The reaction mixture<br />
was set up on ice and it included: 10 × Taq buffer (with Mg 2 + ) (5 μl),<br />
dNTP (3.5 μl), 1.5 μl each for forward and reverse primer, Taq DNA<br />
polymerase (0.5 μl), template DNA (2 μl) and an addition of ddH2O<br />
up to 36 μl as the final volume. The PCR program was denaturized<br />
at 94°C for 5 min, and at 94°C for 30 s, while it was annealed at<br />
57°C for 40 s and extended at 72°C for 90 s. These steps cycled a<br />
total of 32, while the program was finally extended at 72°C for 10<br />
min (Mora et al., 1998). Subsequently, the PCR product was<br />
separated by 1% agar-gel electrophoresis, and the band of expected<br />
size was cut-off and purified with a purification kit (Promega,<br />
USA). The expected bands were sequenced by Huada Genomic<br />
Company (Beijing, China).<br />
Sequence identification was initially estimated using the BLAST<br />
facility of the National Center for Biotechnology Information. All<br />
available subsets of 16SrRNA gene sequences were selected,<br />
analyzed and aligned with CLUSTALX 1.8 (Thompson et al., 1997).<br />
RESULTS AND DISCUSSION<br />
Morphology, physiological and biochemical<br />
identification<br />
According to the different morphology of the colony<br />
(Figure 1), there were 13 strains of bacteria distinctly<br />
labeled from 1 to 13#, respectively. The morphological<br />
characteristics of the bacteria are shown in Table 1, and<br />
the physiological and biochemical characteristics of the<br />
bacteria are shown in Table 2. The colony characteristics<br />
of the isolates could thus be helpful for their genetics<br />
identification.<br />
Identification of the sequences of 16SrRNA<br />
According to Table 3, the similarities of the 13 strains of<br />
16SrRNA sequence reached up to 98% when compared<br />
with the standard strains in the GenBank (Kolbert and<br />
Persing, 1999). Combining it with the morphological and<br />
physiological characteristics (Buchanan et al., 1984;<br />
Breed et al., 1994), these strains were identified as:<br />
Bacillus licheniformis CICC 10037 (1#); Bacillus subtilis<br />
64-3 (2#); Bacillus spore CO64 (3#); B. subtilis SRS-15<br />
(4#); B. subtilis W-9 (5#); B. subtilis I527 (6#); B. subtilis<br />
I3 (7#); Bacillus anthracis (8#); Staphylococcus aureus<br />
(9#); 1Bacillus amyloliquefaciens SDG-3 (10#); B. spore<br />
DC3158 (11#); B. spore LY (12#) and Bacillus<br />
thuringiensis 61436 (13#).<br />
Among the strains isolated from the children's shoes, a<br />
strain of S. aureus accounted for 7.70%, while 12 strains<br />
of Bacillus accounted for 92.30%. Published researches<br />
show that most Bacillus can secrete amylase, lipase and<br />
protease to strongly decompose carbon pollutants, complex<br />
polysaccharides and proteins, and they also play an<br />
important role in decomposing water-soluble organic<br />
matter such as sweat, leading to foot odor (Wan, 2001;
Li et al. 4135<br />
Figure 1. Morphology of the bacteria strains. The morphology of the 13 strains of the bacteria were observed with optical<br />
microscope (labeled from 1 to 13#), respectively; the size of bacteria observed with optical microscope were magnified by<br />
1000 times.<br />
Table 1. Morphological characteristics of bacteria.<br />
S/N Gram Shape Spore Colonies color Morphology Transparency Edge<br />
1 G + Short rod Had a spore Yellowish Round Opaque Irregular<br />
2 G + Short rod Had a spore White Irregular Opaque Irregular<br />
3 G + Short rod Had a spore White Round Sub transparent Regular<br />
4 G + rod Had a spore Yellowish Round Opaque Irregular<br />
5 G + Long rod Had a spore White Round Opaque Irregular<br />
6 G + Short rod Had a spore Yellowish Round Opaque Regular<br />
7 G + Long rod Had a spore Yellowish Round Opaque Regular<br />
8 G + Long rod Had a spore Yellowish Irregular Opaque Irregular<br />
9 G + Spherical No spore Yellowish Round Opaque Regular<br />
10 G + Rod Had a spore Yellowish Round Opaque Irregular<br />
11 G + Rod Had a spore White Round Transparent Regular<br />
12 G + Rod Had a spore White Round Opaque Irregular<br />
13 G + Rod Had a spore Yellowish Round Opaque Irregular<br />
Bruce et al., 1988). B. anthracis could spread widely in<br />
dust pollution and easily cause skin anthrax (Quan et al.,<br />
2005). Also, S, aureus is the most common infection<br />
pathogen of the purulent, and it can produce toxin and<br />
cause serious harm, as aggressive bacteria, to the skin of<br />
people’s feet (Proctor et al., 1995). In this study, 13<br />
strains of bacteria from children’s shoes were identified.<br />
The results of the identification will be helpful in screening<br />
efficient antibacterial agents for shoes and insole to<br />
protect children's feet effectively. Also, the results of the<br />
strain’s identification will provide more useful information<br />
for the medical care of children’s feet.<br />
CONCLUSION<br />
13 strains of bacteria were isolated from the children’s<br />
shoes and were identified as B. licheniformis, Bacillus<br />
subtilis (5 subspecies), B. spore (3 subspecies), B.<br />
amyloliquefaciens, S. aureus, Bacillus amyloliquefaciens<br />
and B. thuringiensis. This is the first report on the<br />
bacteria isolated and identified from children’s shoes in<br />
this area. Based on the results, some efficient antibacterial<br />
agents could be chosen against the pathogenic<br />
microorganisms for children's shoes and insole.<br />
ACKNOWLEDGEMENTS<br />
The authors wish to thank the Ministry of Science and<br />
Technology, China for co-operating in the Science and<br />
Technology project between Romania and the People’s<br />
Republic of China (Item No. 2009DFA42850). Also, the<br />
authors would also like to thank the Agency of Science and
4136 Afr. J. Biotechnol.<br />
Table 2. Characteristics of the physiology and biochemistry of bacteria.<br />
S/N 1 2 3 4 5 6 7 8 9 10 11 12 13<br />
Amylolysis + + + + + + + + − + + + +<br />
Gelatinolytic + + − + + + + + + + + − +<br />
Methyl red test − − − − − + + − + − − − +<br />
Indole test − − − − − − − + − − − − +<br />
Vp + + − + + + + + + − − − +<br />
Urea test − − + − − − − − + − + + −<br />
Sugar fermentation + + + + + + + + + + + + +<br />
Glucose fermentation + + + + + + + + + + + + +<br />
Mannitol fermentation + + + + + + + + + + + + +<br />
Catalase test + + + + + + + + + + + + +<br />
+ Indicates positive reaction results; - indicates negative reaction results.<br />
Table 3. Identification of the sequences of 16S Rrna.<br />
S/N Sequence number Strain number Species number Similarity (%)<br />
1 AY871102.1 CICC10037 B. licheniformis 99.29<br />
2 EF472462.1 64-3 B. subtilis 99.14<br />
3 DQ643066.1 CO64 B. spore 99.89<br />
4 GU056808.1 SRS-15 B. subtilis 99.92<br />
5 EU815066.1 W-9 B. subtilis 99.86<br />
6 GQ199597.1 I527 B. subtilis 98.21<br />
7 GQ199595.1 I3 B. subtilis 100<br />
8 AY138383.1 2000031664 B. anthracis 99.47<br />
9 AB114634.1 A9784 S. aureus 99.31<br />
10 FG436406.1 SDG-31 B. amyloliquefaciens 99.98<br />
11 GU121479.1 DC3158 B. spore 99.49<br />
12 AY787805.1 LY B. spore 98.54<br />
13 FJ932761.1 61436 B. thuringiensis 99.48<br />
Technology of Sichuan Province for financially supporting<br />
this study (Item No. 2009HH0004).<br />
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Genotypic Characterization of Thermophilic Bacilli: a study on new<br />
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Papamanoli E, Tzanetakis N, Litopoulou-Tzanetaki E, Kotzekidou P<br />
(2003). Characterization of lactic acid bacteria isolated from a Greek<br />
dry-fermented sausage in respect of their technological and probiotic<br />
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(1995). Persistent and relapsing infections associated with small<br />
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the company Promega in USA.<br />
Quan CS, Liu Q, Tian WJ, Kikuchi J, Fan SD (2005). Biodegradation of<br />
an endocrine disrupting chemical,di-2-ethylhexyl phthalate,by Bacillus<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4138-4146, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1410<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Analyzing the two-dimensional plot of the interannual<br />
climate variability for detection of the climate change in<br />
the Large Karoun River Basin, Iran<br />
Narges Zohrabi 1 *, Ali Reza Massah Bavani 2 , Hossein Sedghi 3 and Abdol RasolTelvari 4<br />
1 Department of irrigation, Science and Research Branch, Islamic Azad University, Khouzestan -Iran.<br />
2 Department of Irrigation and Drainage Engineering, College of Abouraihan, University of Tehran, Iran.<br />
3 Department of Water Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran.<br />
4 Department of Civil Engineering, Islamic Azad University, Ahwaz, Iran.<br />
Accepted 20 January, 2011<br />
In most studies on climate change, the first problem often faced by the researcher is detecting the<br />
climate change of the study area during the past periods and attributing it to the greenhouse gases. In<br />
this study, an attempt has been made to introduce a method for detecting the climate change during the<br />
past periods in regional scale and attributing it to greenhouse gases with regard to climate processes<br />
in a region. For this purpose, at first it is necessary to calculate the interannual variability range of the<br />
region climatic variables, resulting from the interaction between the climate systems of the ‘earth’<br />
(atmosphere, biosphere, etc.). Hence, long-term statistics (1000 years) of the temperature and<br />
precipitation, resulting from control run (fix greenhouse gases) of AOGCM models (HadCM3 and<br />
CGCM3), were used for Large Karoun River Basin. Then, based on the two-variant normal distribution,<br />
the interannual climate variability range of the study area was plotted as two-dimensional temperatureprecipitation<br />
graphs. Next, the annual temperature and precipitation anomaly values of the observation<br />
stations in different regions of Large Karoun River Basin were compared with the region interannual<br />
variability range for detecting the climate change of the study area during the past and attributing it to<br />
greenhouse gases. The results show increase in temperature and decrease in precipitation trends,<br />
denoting the fact that the temperature variable has been influenced by the climate change. So, in all<br />
regions of the Large Karoun River Basin, the final years of the period (1971 to 2009) have almost been<br />
located outside the interannual climate variability range, indicating the effect of climate change on the<br />
climatic variables of the said years.<br />
Key words: Interannual climate variability, detecting the climate change, AOGCM models, Large Karoun River<br />
Basin.<br />
INTRODUCTION<br />
Different factors can unbalance the stationary time series<br />
of a region’s climatic variables, and a recognition of this<br />
fact can greatly contribute to the climate assessment of<br />
that specific region in future periods. Part of these factors<br />
relate to the interactions between the components of the<br />
Earth’s climate system (atmosphere, hydrosphere,<br />
cryosphere, land surface and biosphere), such as Atlantic<br />
Multidecadal Oscillation (AMO), Pacific Decadal<br />
*Corresponding author. E-mail:<br />
n.zohrabi@khouzestan.srbiau.ac.ir.<br />
Oscillation (PDO), El Niño/Southern Oscillation (ENSO)<br />
and El Nino which can cause internal variability in time<br />
series of climatic variables (Wang and Schimel, 2003;<br />
Hegerl et al., 2007). Various studies have been<br />
accomplished so far in order to detect such variability.<br />
Kazadi and Kaoru (1996) in their research on interannual<br />
variability, during long term periods of the climate (30<br />
years from 1960 to 1992) in Zaire river basin (Africa),<br />
more attentively dealt with understanding the ENSO<br />
phenomenon, its relationship with it and its effect on<br />
internal climate variability and showed that solar annual<br />
cycles determine the seasonal changes of climatic
variables over a region. Timilsena et al. (2009) investtigated<br />
the influence of interannual/interdecadal climate<br />
variability on the Colorado river basin. In this research,<br />
the relationship between individual impact of ENSO, PDO<br />
and AMO and its combined effect on stream-flow was<br />
determined using the non parametric ‘rank sum’ test. The<br />
results indicated an increase in stream-flow during<br />
ElNiNo and a decrease during LaNiNo phenomena,<br />
respectively in the Colorado river basin. Peel and<br />
McMahon (2006) investigated the recent changes in<br />
internal variability of temperature and precipitation on a<br />
global scale. For this purpose, they used empirical mode<br />
decomposition (EMD) (Huang et al. 1998) for quantifying<br />
the proportion of variation in the annual temperatureprecipitation<br />
variability. They reported that the annual<br />
variability of temperature and precipitation have marginally<br />
decreased in the USA, Canada, Europe, Central<br />
Asia, China, Japan and Australia since 1970.<br />
Another part of the factors causing non-stationary<br />
status in climatic variables in a region may be external<br />
factors related to the changes in solar radiation and<br />
changes in the earth orbital and volcanic activity.<br />
Although changes in solar radiation or in the earth orbital<br />
may occur in a several thousand years scale, the<br />
temperature of the region tends to get cold for a few<br />
months by volcanic eruption. Overall changes due to<br />
natural external factors and internal climatic variability<br />
within the system are called natural climate variability.<br />
Another important factor affecting the stationary status of<br />
the climatic variables in a region and thereby resulting in<br />
different trends in such variables is increasing the<br />
greenhouse gases volume in the atmosphere due to<br />
anthropogenic activity (Wang and Schimel, 2003; Hegerl<br />
et al., 2007; Baede et al., 2001). Many scientists believe<br />
that the primary cause for higher earth's surface temperature<br />
in recent decades has been the increased<br />
concentration of the greenhouse gases (Pagano and<br />
Garen, 2005; Garfin et al., 2008; Milly et al., 2008). To<br />
investigate the effect of the said factors on climatic<br />
variables of a region, the trend tests are usually used for<br />
examining the observation records. Khliq et al. (2009)<br />
documented guidelines of different statistical trend tests<br />
in time series for detecting the hydrological trends on<br />
local scales. Moreover, studies by Chen and Grasby<br />
(2009) demonstrated the impacts of the natural<br />
fluctuations of the quasi-cyclic components on the Mann-<br />
Kendall and Thiel-Sen tests, which are the most common<br />
methods used in data analysis and detection of trend in<br />
time series. Although, from the results of this study and<br />
other similar previous researches, this study provides a<br />
better insight for appropriate temporal trend analyses of<br />
the hydroclimate and associate climate data time series,<br />
inference can be drawn that the main method used in<br />
researches for detecting the climate change during the<br />
past, has been analyzing the trends in climatic variables<br />
using non-parametric tests and one-dimensional test in<br />
most cases. The reason for the fame of such tests, proba-<br />
Zohrabi et al. 4139<br />
bly lie in the fact that for the application of nonparametric<br />
methods, no assumptions on parent<br />
distribution of the time series are necessary (Khliq et al.,<br />
2009). It should be noted that although an increase in<br />
greenhouse gases in the atmosphere can create a trend<br />
(especially in temperature), the opposite case is not true,<br />
that is, the existence of a trend in the climate data of a<br />
region can not necessarily be attributed to the increase in<br />
greenhouse gases. In other words, after proving the<br />
existence of a trend in the past climate data of a region,<br />
the relationship between the trend and the increase in<br />
greenhouse gases should be proved. In this case, it can<br />
be said that the climatic variables of the region in the past<br />
have been affected by the climate change. Therefore, in<br />
the climate impact assessment studies, the system<br />
behavior which is a result of alteration by natural<br />
fluctuations or human-induced climate change needs to<br />
be separately investigated (Sridhar and Nayak, 2010).<br />
Braganza et al. (2004) showed the influence of the<br />
external natural forces (solar radiation and volcanic activities)<br />
and the forces resulting from anthropogenic<br />
activities on the climate change observed throughout the<br />
twentieth century, in the course of considering 5 simple<br />
indices of the surface temperature including: the globalmean<br />
temperature (GM), mean land-ocean temperature<br />
contrast (LO), mean magnitude of the annual cycle in<br />
temperature over land (AC), the meridional temperature<br />
gradient in the northern hemisphere mid-latitudes (MTG)<br />
and the northern/southern hemisphere temperature<br />
contrast (NS). For attributing the changes that occurred in<br />
the temperature indices, they used simple and multiple<br />
linear regression between observation records of the<br />
temperature indices and the simulated data derived from<br />
the control run (under the influence of natural forces and<br />
fix greenhouse gases) of 5 models coupled in the<br />
atmosphere-ocean. The linear trends observed in all the<br />
indices except for the hemispheric temperature contrast<br />
(NS) are significantly larger than the values that resulted<br />
from the simulation of such models.<br />
In this research, the detection and attribution of internal<br />
variability to the greenhouse gases and surveying the uncertainty<br />
of different models has been perfectly performed,<br />
but only in one dimension of “temperature”, while<br />
nothing has been done about the “precipitation” variable.<br />
More detailed researches in the field of detection and<br />
attribution of variability which occurred in greenhouse<br />
gases can be found in the paper presented by the<br />
“International ad hoc detection and attribution group”<br />
(Barnett et al. 2005). The mentioned paper, while<br />
reviewing the best part of the studies regarding the<br />
detection of internal variability and attributing them to the<br />
greenhouse gases, shows that the main goal of detection<br />
and attribution studies during the past several years has<br />
been a comparison of the observed changes in climate,<br />
primarily during the past century, with the data simulations<br />
by the Coupled General Circulation Model<br />
(CGCM) that have been forced by estimates of historical
4140 Afr. J. Biotechnol.<br />
Figure 1. Location of the Large Karoun River Basin in Iran and some of its important<br />
installations.<br />
changes in anthropogenic and (natural external forces)<br />
most frequently in “temperature” on a global-scale. Also,<br />
it is indicated that according to the results of the studies<br />
carried out in different parts of the globe, the temperature<br />
variable has been influenced by the climate change.<br />
Accordingly, although in a study conducted in the UK<br />
by Hulme and Brown (1998), the internal climate<br />
variability range and its attribution to greenhouse gases is<br />
well recognized, but the uncertainty of the simulation<br />
models in internal climate variability calculations has<br />
been ignored. This is why in climate change studies there<br />
are various uncertainty resources in different stages<br />
required by simulation of the climatic variables by<br />
Atmosphere-Ocean General Circulation Mode (AOGCM)<br />
model on regional scales (Jones and Page, 2001)<br />
It can be seen therefore in most previous researches<br />
that first, the natural and internal climate variability have<br />
been investigated often in one-dimensional state using<br />
trend tests in order to detect the climate change that is<br />
not attributed to the greenhouse gases. Secondly, the<br />
uncertainty of the simulation models of the internal<br />
climate variability has not been considered. Thus, the<br />
present study intends to propose a new method for twodimensional<br />
detection of the climate change during the<br />
past periods, considering the uncertainty and the way it is<br />
attributed to greenhouse gases. Meanwhile, considering<br />
the fact that this is contrary to numerous studies in Iran,<br />
concerning the trend detection in climate variables time<br />
series, no works have been carried out regarding the<br />
occurred variability’s attribution to the greenhouse gases<br />
and uncertainty analysis.<br />
Description of the study area<br />
The Large Karoun River Basin is located in the southwestern<br />
part of Iran, and due to the fact that it<br />
encompasses the country's largest water potential, this<br />
basin houses the most and the greatest water projects in<br />
Iran. The basin area of 67,257 km 2 encompasses about<br />
5% of the total area of Iran. 67% of the basin area is<br />
mountainous and the remaining 33% is made of high<br />
plains. Its two main tributaries, including Karoun and Dez<br />
rivers, flow over Khouzestan Plain after journeying separately<br />
through anfractuous routes in mountains and join<br />
together at Band Ghir located 50 km off north of Ahwaz<br />
city. As such, the Karoun River eventually terminates at<br />
the Persian Golf (Figure 1).<br />
A brief description of the study area is thus presented<br />
and details can be found in Jamab (1999). Climatic
Mean Monthly precipitation(m m /m onth)<br />
200<br />
150<br />
100<br />
50<br />
0<br />
Jan Feb Mar Apr Mar Jun Jul Aug Sep Oct Nov Dec<br />
P Khoram abad P Shahrekord P Ahwaz P Pol shalo<br />
P Talezang P Gotvand T Khoramabad T Shahrekord<br />
T Ahwaz T Talezang T T Gotvand<br />
Gotvang Gotvan<br />
T Pol Shalo<br />
Figure 2. Mean monthly precipitation (P) and mean monthly temperature in different regions of the Large<br />
Karoun basin.<br />
variation in Large Karoun River Basin is numerous and<br />
the types of dry desert, semi dry, semi wet, wet, very wet<br />
Mediterranean and cold humid climates are found in it.<br />
Precipitation regime in the basin is Mediterranean, that is,<br />
the dry season exactly matches the summer and the<br />
rainfall season coincides with winter. About 96% of<br />
rainfall occurs between October and May which varies<br />
from 1800 mm /year in the heights of 150 mm/year in<br />
plain areas (Jamab, 1999).<br />
Fundamentally, Figures 2 and 3 show the precipitation<br />
and temperature conditions in different regions of the<br />
Large Karoun River Basin. As it is clear from the figures,<br />
a wide range of precipitation data have been recorded in<br />
basin stations, indicating various climates dominating the<br />
Large Karoun River Basin. This is due to the large extent<br />
and considerable heights of the basin which makes the<br />
investigation of the precipitation variable and climate<br />
conditions analysis dependant on the division of the basin<br />
into its constituting micro-climates. The classification of<br />
the micro-climates has been performed based on the<br />
precipitation pattern in relation with the region<br />
temperature, vegetation and height of different regions<br />
and lesser attention has been paid to the precipitation<br />
volume. This means that in classification of microclimates,<br />
some parameters like altitude, temperature and<br />
vegetation of land are more influential than precipitation<br />
volume. For example, as it can be seen from Figure 3,<br />
Zohrabi et al. 4141<br />
despite the identical volume of the rainfall in pluviometery<br />
stations of Ahwaz and Shahrkurd, these two regions have<br />
different climates due to their different height and<br />
temperature. Also, Sepid-dasht and Yasouj despite their<br />
identical volume of precipitation are located in two<br />
regions with different climates. So, the Large Karoun<br />
River Basin has been divided into four distinct climatic<br />
regions (Figure 1 and Table 1).<br />
Each region will be discussed as a separate uniform subbasin<br />
(Table 1) as follows:<br />
(1) Mountainous region of Karoun River Basin.<br />
(2) Mountainous region of Dez River Basin.<br />
(3) Middle part of the Large Karoun River Basin (from<br />
installed stations at an input of Karoun1 and Dez dams<br />
reservoirs to Ahwaz).<br />
(4) Southern regions of the Large Karoun River Basin<br />
(from Ahwaz to the Large Karoun estuary (Arab, 2009).<br />
MATERIALS AND METHODS<br />
Data<br />
Considering the climatic classification, data verification and<br />
suitability of the statistical period, precipitation data of more than 50<br />
years (1956 to 2009) and temperature data of more than 30 years<br />
(1971 to 2009), four main stations were selected in this basin<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
-5<br />
Mean Monthly T em perature( o C/m onth)
Row<br />
4142 Afr. J. Biotechnol.<br />
Mean annual precipitation (mm)<br />
1000<br />
900<br />
800<br />
700<br />
600<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0<br />
Shahrekord<br />
Figure 3. Mean annual precipitation (P) during 1956 to 2009 in different regions of the Large Karoun basin.<br />
Table 1. Geographical characteristics of the selected river stations.<br />
Different regions of Large<br />
Karoun River Basin<br />
Station<br />
name<br />
Long Lat<br />
Elevation<br />
masl<br />
Drainage<br />
area (km 2 )<br />
Time period<br />
P T<br />
1 Mountainous region of Karoun River Basin Pol Shalo 50.08 31.45 700 23400 1956- 2008 1971-2009<br />
2 Mountainous region of Dez River Basin Tale Zang 48.46 32.49 480 16213 1956- 2008 1971-2009<br />
3 Middle basin of Large Karoun River Basin Gotvand 48.49 32.15 75 32425 1956- 2008 1958-2009<br />
4 Southern region of Large Karoun River Basin Ahwaz 48.41 31.2 20 60737 1957- 2008 1957-2009<br />
(Table 1). The aforementioned data were acquired from “Iran<br />
Meteorological Organization” and “Ministry of Energy” [Iran Water<br />
Resource Management Company and Khouzestan Water and<br />
Power Authority (KWPA)].<br />
According to this classification, Pol-e Shaloo (located at Karoun3<br />
dam), Tale Zang (located upstream of Dez dam), Gotvand and<br />
Ahwaz stations are considered as the representative of the<br />
mountainous region of Karoun river basin, the mountainous region<br />
of Dez river basin, the middle part of the Large Karoun River Basin<br />
and the southern region of the Large Karoun River Basin,<br />
respectively (Figure 1 and Table 1).<br />
Simulation of climatic variables during the past periods using<br />
HadCm3 and CGCM3 models<br />
One of the methods for simulating internal climate variability within<br />
a region is to explore climatic variables time series in long-term<br />
records (that is, more than several hundred years). Due to the lack<br />
of long-term observation records around the world, the alternative<br />
method will be the use of a three-dimensional coupled ‘atmosphereocean<br />
general circulation model’ or AOGCM (Wilby and Harris,<br />
2006; Mitchell, 2003). These models are based on the laws of<br />
physics represented by mathematical relationships which are<br />
resolved in a three-dimensional network on the earth surface. In<br />
order to simulate the earth’s climate, the main climatic processes<br />
including atmosphere, cryosphere, biosphere and hydrosphere,<br />
were simulated in independent sub-models. So far, different general<br />
Ahwaz<br />
Pol Shalo<br />
Talezang<br />
Gotvand<br />
Station<br />
Sepiddasht<br />
Barz<br />
Armand<br />
circulation models have been developed by different research<br />
institutes including UKMO-HadCM3, CCCma-CGCM3, GFDL_CM2,<br />
CM2.1, NCAR_PCM, CCSM3 and CSIRO-MK3 (cited in IPCC-AR4,<br />
2007). In this research, to consider the uncertainty of the model, the<br />
outputs of two CGCM3 and HadCM3 models with spatial resolution<br />
of roughly 3.75 degrees lat/lon and 2.5/3.75 lat/lon, respectively,<br />
were used. In a specific type of simulation by this model, the<br />
amount of greenhouse gases over simulation time is kept at an<br />
observed level in the year 1850. In this type of simulation, known as<br />
the control run, the climatic variables are often simulated over a<br />
1000-year period. Obviously, due to the fixed amount of<br />
greenhouse gases in such simulations, the time series of the<br />
variable under study is only affected by internal forces of the<br />
climate system and thus determines the internal variability of the<br />
climate system. If it is assumed that no changes will occur in the<br />
external factors including solar radiations and volcanic activities<br />
over future periods (that is, the next one hundred years), one can<br />
conclude that such simulation reflects natural climate variability<br />
(Oldenborgh et al., 2005; Lambert et al., 2005).<br />
Calculation of interannual climate variability<br />
In this study, in order to separate climate changes caused by<br />
greenhouse gases from interannual variability, the variability range<br />
of the temperature and precipitation are first examined. On the<br />
other hand, the recorded statistical periods’ length available at<br />
gauging stations is not adequate for calculating the interannual<br />
Marghak<br />
Yasoj
climate variability of the study area. Also, the observation records of<br />
the recent decades have been probably affected by radiation force<br />
resulting from increasing greenhouse gases, thereby making<br />
separation of interannual variability from climate changes quite<br />
difficult. Thus, in order to show the interannual variability across a<br />
region, long-term records of 1000 years generated by the control<br />
run of AOGCM models are used (Ruosteenoja et al., 2002). To<br />
analyze the interannual variability range of two “temperature” and<br />
“precipitation” variables of the study area, first, their annual<br />
anomaly time series with respect to the average base period is<br />
calculated. [By definition, temperature anomaly is the temperature<br />
difference from a base temperature, while precipitation anomaly is<br />
the precipitation ratio difference from a base precipitation]. The<br />
world meteorological organization (WMO) has suggested that in<br />
order to harmonize the selection of the 30 years base period in<br />
different climate change studies and the possibility of comparing<br />
them, the 1961 to 1990 base period should be considered. On the<br />
other hand, as the organization recommends, where data recorded<br />
on the station of the study area for this period is not available, the<br />
period of 1971 to 2000 will be replaced (IPCC-TGCIA, 1999).<br />
So, because the statistics of the temperature variable of the Large<br />
Karoun River Basin upstream do not cover the 1961 to 1990 period,<br />
the period of 1971 to 2000 is considered as the base period. Then,<br />
it is assumed that the time series of these two variables follow twovariant<br />
normal distribution. With this assumption, the relationship<br />
governing the two-variant normal distribution of the temperature<br />
and precipitation anomaly will be realized as follows (Von Storch<br />
and Zwiers, 2002):<br />
1<br />
1<br />
2<br />
2<br />
⎧ ′ ′ ′ ′<br />
T<br />
⎨<br />
⎩<br />
T<br />
− 2ρ<br />
σ<br />
R<br />
σ<br />
2 2<br />
2<br />
− ρ σ T<br />
T R σ R<br />
+<br />
R<br />
⎫ 2<br />
⎬ = χ<br />
⎭<br />
Where, T ′ and R′ are temperature and precipitation anomalies,<br />
σ T σ and R are standard deviation of temperature and<br />
precipitation and ρ �represents the correlation between the<br />
2<br />
χ temperature and precipitation anomalies. The amount of is<br />
2<br />
χ determined by square distribution of with 2 degrees of freedom<br />
which is extracted from the respective tables at 95% confidence<br />
2<br />
χ level (df = 2, 95% → = 5.99). By plotting Equation (1) for 95%<br />
confidence level, elliptic circuits whose internal area indicates the<br />
interannual climate variability range for temperature and<br />
precipitation are obtained and its external area represents the<br />
variability related to other factors (like greenhouse gases).<br />
RESULTS<br />
Approximation of interannual climate variability in<br />
the study area<br />
Figure 4 shows the interannual climate variability range<br />
with regard to the calculated 95% confidence level in the<br />
study area. In this figure, the horizontal axis represents<br />
temperature anomaly, while the vertical axis shows<br />
precipitation anomaly with respect to the base period<br />
average of 1971 to 2000. As it can be seen from this<br />
figure, most of the points showing annual anomalies are<br />
depicted inside the ellipse or too close to its perimeter. It<br />
means that the interannual climate variability range of the<br />
(1)<br />
Zohrabi et al. 4143<br />
region has been plotted quite well by the ellipses. Overall,<br />
due to low skewness, the depicted figure indicates the<br />
assumed two-variant normal distribution of temperature<br />
and precipitation as acceptable for plotting the<br />
interannual climate variability range of the region under<br />
study. On the other hand, the excellent overlap obtained<br />
from each of the two models shows low uncertainty of the<br />
model selection. Therefore, it can be concluded with 95%<br />
probability that the combined interannual temperatureprecipitation<br />
variability anomaly values of the Large<br />
Karoun River Basin are less than 1.5°C and 75%,<br />
respectively.<br />
After the determination of the interannual climate variability<br />
range within the study area, for evaluating the<br />
significance of climate change in the past half century (its<br />
attribution to greenhouse gases), the values of these<br />
variability were compared with the observed annual<br />
anomalies of temperature and precipitation for each<br />
region of the Large Karoun River Basin. Figure 5 shows<br />
the two-variant trend of temperature-precipitation anomalies<br />
during the past half century with respect to<br />
average (1971 to 2000) and the interannual climate<br />
variability range in different regions of the Large Karoun<br />
River Basin.<br />
Since the inside area of each ellipse represents the<br />
combined interannual temperature-precipitation variability<br />
range within the study area, a point outside this range is<br />
indicative of the temperature and precipitation changes<br />
being dependant on factors other than interannual<br />
climate variability. Frequency time evolution of annual<br />
climate in the Large Karoun River Basin over the last<br />
years (1971 to 2009) can be traced from A to Z. As the<br />
points, A and Z, are considered as the start and ending<br />
years of the recent century, respectively, the results of<br />
the two-dimensional trend of temperature-precipitation<br />
anomalies for the past half-century with respect to the<br />
average (1971 to 2000 period) of four regions specified in<br />
Figure 5 shows an increase in temperature and a<br />
decrease in precipitation. Approximately, in all regions of<br />
the Large Karoun River Basin, especially the most<br />
southern part of it, the “temperature” found throughout<br />
the ending years of this period lies outside the interannual<br />
climate variability range, indicating the significance<br />
of climate change (that is, its relationship with the<br />
increase of greenhouse gases) in these years. The<br />
droughts that occurred in the ending years of the period<br />
confirm these results. Meanwhile, water years of 1999 to<br />
2000 and 2000 to 2001 and also 2007 to 2008 and 2008<br />
to 2009 have been defined as ‘dry years’. A review of<br />
annual precipitation changes in the representative<br />
northern stations of Karoun and Dez river basins, namely<br />
Pol-e Shallo and Taleh Zang stations shows more than<br />
50% decrease in precipitation than under normal<br />
conditions. As such, higher precipitation in these regions<br />
of the basin compared with that of downstream regions,<br />
considerably reduces the basin yield, especially on<br />
upstream Dez and Karoun 3 dams at Pol-e Shallo and<br />
Taleh Zang stations (Arab, 2009). The recent trend towards
4144 Afr. J. Biotechnol.<br />
Figure 4. Interannual temperature-precipitation variability, with respect to the average period of 1971<br />
to 2000 for Large Karoun River Basin.<br />
Figure 5. Two-dimensional trend of annual temperature–precipitation anomalies for the past half<br />
century with respect to the average period of 1971 to 2000 for the Large Karoun River Basin,<br />
together with interannual climate variability range. a: Mountainous region of Karoun river (Pole<br />
Shalo station); b: Mountainous region of Dez river (Talezang station); c: Middle basin of Large<br />
Karoun River Basin (Gotvand station); d: Southern region of the Large Karoun River Basin<br />
(Ahwaz station). Black points: observed data Grey points: Ten last years.
Figure 5. Continued<br />
warming and drying, evidenced in Figure 5 is reinforced<br />
and, in the context of the past half century of Large<br />
Karoun River Basin climate variability, it becomes more<br />
significant. Therefore, detecting the range of inter-annual<br />
climatic variability of a region helps in identifying the<br />
years when extreme events such as drought and flood<br />
occur.<br />
DISCUSSION<br />
In this study, an attempt has been made to present a<br />
method of detecting past climate change in a regional<br />
scale and attributing it to greenhouse gases with regard<br />
to the climate process in a region. For this purpose,<br />
initially the variability range of climate variables related to<br />
the regional interannual variability was determined using<br />
control run data of two AOGCM models. The two models<br />
Zohrabi et al. 4145<br />
data were used to study the uncertainty of two AOGCM<br />
models in interannual variability of the study area. As<br />
such, it was assumed that the probability function of the<br />
temperature and precipitation was a two-variant normal<br />
distribution. With this assumption, the interannual climate<br />
variability range of the region was determined. The<br />
results of this study showed that: Firstly, the assumption<br />
of normality of temperature-precipitation probability function<br />
was acceptable due to low skewness of the plotted<br />
range. Secondly, the uncertainty between AOGCM<br />
models in estimating the natural climate variability range<br />
is low.<br />
The comparison between the two-dimensional observed<br />
temperature-precipitation trend of the region and the<br />
interannual variability range showed that the climatic<br />
variable trend in the past half century in Large<br />
Karoun river basin had led to increased temperature and<br />
decreased precipitation, denoting the fact that the
4146 Afr. J. Biotechnol.<br />
temperature variable has been influenced by the climate<br />
change. Khordadi et al. (2008) studies also show that<br />
regional climate in Iran is becoming dryer and warmer,<br />
and probably this will continue in the future (Montazeri<br />
and Fahmi, 2003). Also, the occurrence of low-flow and<br />
drought crisis in the second half of the century has been<br />
more frequent, incurring considerable damages because<br />
of decreased quality water resources on regional and<br />
national scales. Meanwhile, the impacts of greenhouse<br />
gases are clearly noticeable in the ending years of the<br />
observation period. In general, the proposed method in<br />
this paper can be used as an alternative to conventional<br />
stochastic methods in which only the data trend tests<br />
(such as Mann-Kendall test) for detecting and attributing<br />
climate change in the past periods are used.<br />
ACKNOWLEDGEMENTS<br />
The authors wish to thank organizations and individuals<br />
who supported this work. Also, they wish to thank, in a<br />
special way, the Ministry of Energy, Khouzestan Water<br />
and Power Authority (KWPA) and Iran Water Resource<br />
Management Company for the provision of necessary<br />
data sets and for their valuable support during this study.<br />
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KHozestan Water and Power Authority Ministry of Energy, Iran (In<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4147-4156, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1905<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Screening and optimization of extracellular lipases by<br />
Acinetobacter species isolated from oil-contaminated<br />
soil in South Korea<br />
Periasamy Anbu # *, Myoung-Ju Noh # , Da-Hye Kim, Jun-Seok Seo, Byung-Ki Hur* and Kyeong<br />
Ho Min<br />
Department of Biological Engineering, College of Engineering, Inha University, Incheon 402-751, Republic of Korea.<br />
Accepted 6 December, 2010<br />
A total of 53 strains of bacteria were isolated from oil contaminated soil collected in South Korea. The<br />
isolated bacteria were screened using spirit blue agar and Rhodamine-B agar media. Two of the<br />
isolated strains exhibited a greater clear zone than the others, indicating higher lipase activity.<br />
Therefore, these two strains (BK43 and BK44) were selected and identified based on their<br />
morphological and physiological characteristics. The 16S rRNA gene sequencing was also<br />
implemented. Phylogenetic analyses based on the results of 16S rRNA gene sequencing revealed that<br />
BK43 and BK44 were close in identity to Acinetobacter junii. The optimum pH and temperature for<br />
lipase production by BK43 were found to be 6.0 at 30°C, after 24 h of incubation, while BK44 were found<br />
to be 6.0 at 25°C, after 12 h of incubation. In addition, increased enzymatic production was obtained<br />
when the organisms were cultured in medium supplemented with 1% sucrose as the carbon source.<br />
Among the different lipase inducers tested, both strains utilized Tween 80 and produced a great level of<br />
extracellular lipase. Overall, the results of the present study demonstrate that the genus Acinetobacter<br />
is good for extracellular lipase production under acidic conditions.<br />
Key words: Acinetobacter, lipase, optimization, screening.<br />
INTRODUCTION<br />
Lipases are triacylglycerol acylhydrolases (EC 3.1.1.3)<br />
that catalyze the hydrolysis of triacylglycerol to glycerol<br />
and fatty acids (Sharma et al., 2001). Lipases are ubiquitous<br />
in nature and produced by animals, plants and<br />
microorganisms. Currently, microbial lipases are receiving<br />
a great deal of attention due to their potential for use<br />
in industrial processes. Most commercial extracellular<br />
products are obtained from microorganisms and have the<br />
ability to catalyze a wide variety of reactions in aqueous<br />
and non-aqueous phases (Saxena et al., 2003). Microbial<br />
lipases have also received more attention due to their<br />
*Corresponding author. E-mail: anbu25@yahoo.com,<br />
biosys@inha.ac.kr. Tel: +82-32-860-7512. Fax: +82-32-872-<br />
4046.<br />
#These authors contributed equally to this work.<br />
selectivity, stability and substrate specificity (Treichel et<br />
al., 2010). Particularly, extracellular bacterial lipases are<br />
of commercial importance in the food, detergent, cosmetic<br />
and pharmaceutical industries, as well as<br />
inprocesses involving organic synthesis and fat/oil degradation<br />
(Jaeger et al., 1994).<br />
Extracellular lipase production by bacteria are influenced<br />
by the composition of the growth medium, cultivation<br />
conditions and many physico-chemical (pH and temperature)<br />
and nutritional factors (carbon, nitrogen and lipid<br />
sources) (Jaeger et al., 1994). Most bacterial lipases are<br />
generally induced in medium that contains the proper<br />
fatty acids and oils (Joseph et al., 2006; Immanuel et al.,<br />
2008; Kiran et al., 2008). However, a few investigators<br />
have reported that the activity of lipase produced by<br />
Pseudomonas aeruginosa EF2 and Acinetobacter<br />
calcoaceticus was repressed by the presence of a lipid<br />
source in the medium (Gilbert et al., 1991; Mahler et al.,<br />
2000). In addition, Lin et al. (2006) have found that
4148 Afr. J. Biotechnol.<br />
vitamins influenced lipase production by the edible Basidiomycetes,<br />
Antrodia cinnamomea.<br />
A variety of extracellular lipases of bacterial origin with<br />
different properties and specificities have been described<br />
and characterized. Extracellular lipase was isolated from<br />
many different bacterial species, including Bacillus<br />
(Ertugrul et al., 2007) and Pseudomonas (Kiran et al.,<br />
2008; Wang et al., 2009). Additionally, many studies have<br />
been con-ducted to evaluate lipase production under<br />
alkaline condi-tions (Chen et al., 1998; Kasana et al.,<br />
2008; Kiran et al., 2008; Wang et al., 2009) but few<br />
studies have focused on acidic lipases (Bradoo et al.,<br />
1999; Liu et al., 2007).<br />
Lipase production by Acinetobacter radioresistens<br />
under alkaline conditions in the presence of n-hexadecane<br />
was evaluated (Chen et al., 1998). In recent<br />
years, most studies conducted to improve lipase production<br />
have focused on their production by one important<br />
genus, Acinetobacter (Chen et al., 1998; Dharmsthiti et<br />
al., 1998). Acinetobacter strains have been isolated from<br />
a variety of sources, including soil (Bompensieri et al.,<br />
1996) and water (Blaise and Armstrong, 1973). However,<br />
industries are still seeking strains of bacteria that produce<br />
a high yield of potent lipase with excellent properties<br />
using cost-effective methods. Therefore, the present<br />
study was conducted to isolate novel lipase producing<br />
bacteria, after having the isolated strains identified and<br />
the culture conditions for optimal production of extracellular<br />
lipase determined.<br />
MATERIALS AND METHODS<br />
Materials<br />
Gum arabic, p-nitrophenyl palmitate, sodium deoxycholate and<br />
tributyrin were purchased from Sigma (USA). Tween 80 was<br />
obtained from Duchefa, Biochemica (The Netherlands). All other<br />
chemicals used in this study were of analytical grade.<br />
Isolation and screening of lipase-producing microorganisms<br />
Fifty three strains of bacteria were isolated from oil contaminated<br />
soil collected in South Korea using serial dilution. The isolates were<br />
maintained on LB agar plates at 4°C and stored at -80°C in glycerol.<br />
The isolated bacteria were screened for the production of lipase<br />
using spirit blue agar containing lipase reagent as Marshall (1992)<br />
method. The lipolytic activities of all isolates were then compared by<br />
measuring the width of the areas of clearing or areas of deep blue<br />
color around the colonies. In addition, the same strains were<br />
screened using the Rhodamine-B method (Kouker and Jaeger,<br />
1987). The bacterial strains were inoculated on the agar plates,<br />
after which the lipolytic activity was determined by the formation of<br />
an orange fluorescent zone around the fungal colonies that was<br />
visible upon UV irradiation (350 nm).<br />
The liquid culture medium (Tryptic soy broth) used for lipase<br />
production contained the following (g/L): Pancreatic casein, 17;<br />
enzymatic digest soybean, 3; NaCl, 5; dipotassium phosphate, 2.5;<br />
glucose, 2.5; pH 7.5. To produce the lipase, an Erlenmeyer flask<br />
(250 ml) containing 50 ml of medium was inoculated with an aliquot<br />
of approximately 1% of the preculture prepared in LB broth (g/L):<br />
Tryptone, 10; yeast extract, 5; NaCl, 10; pH 7.0. The inoculated<br />
flasks were then incubated at room temperature with constant<br />
shaking at 180 rpm. Then, the cell-free supernatant was recovered<br />
by centrifugation (10,000 rpm, 10 min at 4°C) and the clear supernatant<br />
was used to determine the lipase activity. The growth of the<br />
microorganisms was then determined by measuring the absorption<br />
at 600 nm.<br />
Identification of the microorganisms<br />
The lipase producing bacteria was identified by morphological and<br />
biochemical characterizations. A gram (positive/negative) stain<br />
reaction was conducted using the Biomerieux system according to<br />
the manufacturer’s instructions. The biochemical tests were conducted<br />
using the API 20NE system according to the manufacturer’s<br />
protocols (Biomerieux). The identification was further confirmed by<br />
the 16S rRNA gene sequencing method. Briefly, genomic DNA was<br />
extracted using a genomic purification kit (Promega, USA). The<br />
DNA was then amplified by PCR using the following universal 16S<br />
rRNA gene primers, 8-27F: 5’-AGAGTTTGATCCTGGCTCAG-3’<br />
and 1472R: 5’-TACGGYTACCTTGTTACGACTT-3’. PCR was<br />
conducted by subjecting a reaction mixture to initial denaturation at<br />
94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 55°C for 1<br />
min, 72°C for 1 min and a final extension step at 72°C for 10 min.<br />
Phylogenetic analysis<br />
The 16S rRNA gene sequence was compared with sequences<br />
available in the nucleotide database using the BLAST algorithm at<br />
the NCBI. Phylogenetic tree was constructed using the neighbor<br />
joining method (http://www.phylogeny.fr).<br />
Fatty acid analysis<br />
To analyze total cellular fatty acid content, the bacterial cells were<br />
cultured at 30°C for 24 h, after which the fatty acid profiles were<br />
determined based on the method described by Lepage and Roy<br />
(1984) using a gas chromatograph (Hewlett Packard 6890, USA)<br />
equipped with a flame-ionized detector (FID) and a DB23 (30 m ×<br />
0.25 mm × 0.26 µm, Agilent Technologies, USA) capillary column.<br />
Lipase assay<br />
Lipase activity was assayed quantitatively using 4-nitrophenyl<br />
palmitate as the substrate according to the method described by<br />
Winkler and Stuckmann (1979). Briefly, 10 ml of isopropanol<br />
containing 30 mg of 4-nitrophenyl palmitate (pNPP) was mixed with<br />
90 ml of 0.05 M phosphate buffer (pH 8.0) containing 207 mg of<br />
sodium deoxycholate and 100 mg of gum arabic. A total of 2.4 ml of<br />
freshly prepared substrate solution was then pre-warmed at 37°C<br />
and mixed with 0.1 ml of enzyme solution. After incubation at 37°C<br />
for 15 min, the absorbance was measured at 410 nm against an<br />
enzyme free control. One enzyme unit was defined as the amount<br />
of enzyme that liberated 1 µmol of 4-nitrophenol per minute under<br />
the assay conditions.<br />
Optimization of lipase production<br />
Initially, the lipase production was determined after culture in tryptic<br />
soy broth from 6 to 48 h at 28°C. The lipase production was then<br />
evaluated at different pHs ranging from 5.0 to 10.0 and at tempera-
Table 1. Screening isolated bacterial strains for extracellular lipase.<br />
Isolates No.<br />
Sprit blue<br />
agar (mm)<br />
Rhodamine-B agar<br />
(mm)<br />
Isolate No.<br />
Sprit blue<br />
agar (mm)<br />
Anbu et al. 4149<br />
Rhodamine-B agar<br />
(mm)<br />
BK01 - ++ BK28 - -<br />
BK02 ++ - BK29 + -<br />
BK03 ++ + BK30 ++ +<br />
BK04 - - BK31 + +++<br />
BK05 - ++ BK32 ++ +<br />
BK06 + ++ BK33 - +<br />
BK07 ++ ++ BK34 - ++<br />
BK08 - + BK35 - -<br />
BK09 - ++ BK36 ++ +++<br />
BK10 + + BK37 - -<br />
BK11 - - BK38 ++ +<br />
BK12 - - BK39 - ++<br />
BK13 - - BK40 - -<br />
BK14 - + BK41 ++ +<br />
BK15 - - BK42 + -<br />
BK16 - - BK43 +++ +++<br />
BK17 - + BK44 +++ +++<br />
BK18 ++ + BK45 - -<br />
BK19 - - BK46 - -<br />
BK 20 ++ - BK47 - -<br />
BK21 - - BK48 + -<br />
BK22 + - BK49 ++ +<br />
BK23 + - BK50 - -<br />
BK24 + - BK51 + -<br />
BK25 ++ + BK52 ++ ++<br />
BK26 - ++ BK53 - +<br />
BK27 ++ ++<br />
+++, High activity (above 50 mm); ++, moderate activity (above 25 to below 50 mm); +, low activity (below 25 mm); -, no activity.<br />
tures of 20, 25, 30, 37 and 45°C. The pH of the medium was<br />
adjusted prior to autoclaving. Further, the changes in lipase<br />
production in response to the following carbon sources (1%) were<br />
evaluated: glucose (control), fructose, xylose, maltose, lactose,<br />
sucrose, mannitol and starch. The carbon sources were sterilized<br />
separately and then aseptically added to the autoclaved medium.<br />
The following substances were used as lipase inducers (1%): olive<br />
oil, sesame oil, soybean oil, tributyrin and Tween 80. Medium that<br />
contained no lipase inducer was used as a control.<br />
RESULTS AND DISCUSSION<br />
Isolation, screening and identification of lipase<br />
producing bacteria<br />
A total of 53 distinct morphological bacterial strains were<br />
isolated from oil-contaminated soil in South Korea. The<br />
isolated strains were screened for extracellular lipase<br />
using spirit blue agar and Rhodamine-B agar media. Two<br />
of the isolates produced a larger clear zone than the<br />
others, indicating higher lipase activity. These two strains<br />
that produced blue color around bacterial colonies, were<br />
grown on spirit blue agar medium due to the hydrolysis of<br />
tributyrin and Tween 80. Furthermore, a fluorescent zone<br />
on Rhodamine-B agar medium was observed under UV<br />
irradiation (350 nm), indicating that the two strains were<br />
able to hydrolyze olive oil (Table 1). The earlier results<br />
confirmed that the two strains were potent to produce<br />
lipase and also indicate that lipolytic bacteria are widespread<br />
in the oil-contaminated environments. The selected<br />
strains (BK43 and BK44) were then identified based<br />
on morphological, physiological and biochemical characterizations.<br />
The physiological and biochemical characteristics<br />
are presented in Table 2. The results showed that<br />
both strains are gram negative, aerobic and coccoid rod<br />
shape. A biochemical test was conducted using an API<br />
identification kit. Both strains were oxidase negative and<br />
capable of assimilating caprate and malate (Table 2),<br />
which indicates that strains BK43 and BK44 were closely<br />
related to Acinetobacter junii. Taken together, these<br />
characteristics indicated that both strains belong to the<br />
genus Acinetobacter.
4150 Afr. J. Biotechnol.<br />
Table 2. The morphological, biochemical and physiological characteristics of strains BK43 and<br />
BK44.<br />
Tests Reactions BK43 BK44<br />
Gram staining - Negative Negative<br />
Shape - Coccoid rod Coccoid rod<br />
NO3 Nitrate/nitrite reduction - -<br />
TRP indole production - -<br />
GLU Acid production from glucose - -<br />
ADH arginine dihydrolase - -<br />
URE Urease - -<br />
ESC β-glucosidase - -<br />
GEL Protease (gelatin hydrolysis) - -<br />
PNPG β-galactosidase - -<br />
GLU assimilation of glucose - -<br />
ARA assimilation of arabinose - -<br />
MNE assimilation of mannose - -<br />
MAN assimilation of mannitol - -<br />
NAG assimilation of N-acetyl-glucosamine - -<br />
MAL assimilation of maltose - -<br />
GNT assimilation of gluconate - -<br />
CAP assimilation of caprate + +<br />
ADI assimilation of adipate - -<br />
MLT assimilation of malate + +<br />
CIT assimilation of citrate - -<br />
PAC assimilation of phenyl-acetate - -<br />
OX cytochrome oxidase - -<br />
+; Positive result, -; negative result.<br />
Sequencing and phylogenetic analysis<br />
The identities of the bacteria were further confirmed by<br />
16S rRNA sequencing. Approximately 1381 and 1383 bp<br />
sequences were obtained from BK43 and BK44,<br />
respectively, and then aligned with other 16S rRNA sequences<br />
available in the GenBank database. A phylogenetic<br />
tree was then constructed using the neighbor-<br />
joining method by only culturable Acinetobacter species<br />
(Figure 1). The phylogenetic analysis indicated that the<br />
sequences of strain BK43 and BK44 were highly homologous<br />
(99 and 100%, respectively) with the sequence of<br />
A. junii (NCBI accession No. AM184300) followed by<br />
Acinetobacter sp. (NCBI accession No. AM412159). The<br />
16S rRNA sequences of strains BK43 and BK44 were<br />
deposited in GenBank under accession numbers<br />
GQ202270 and GQ202271, respectively. Based on these<br />
results, BK43 and BK44 are Acinetobacter sp.<br />
Fatty acid analysis<br />
The fatty acid profiles of the Acinetobacter species<br />
isolated in this study are presented in Table 3. Although<br />
the patterns of fatty acid compositions of both strains<br />
were similar, the fatty acid contents differed. The fatty<br />
acid profiles were more similar to A. junii (Yoon et al.,<br />
2004) than to other Acinetobacter species such as<br />
Acinetobacter antiviralis (Lee et al., 2009) and A.<br />
radioresistens (Nishimura et al., 1988). The major fatty<br />
acids produced by the strains were C16:0, C16:1 (n-7)<br />
and C18:1 (n-9). However, the C18:1 (n-9) accumulated<br />
more than 50% in both strains. These results further<br />
suggest that the isolated strains BK43 and BK44 are<br />
Acinetobacter sp. The identified bacterial strains BK43<br />
and BK44 were used for further studies to optimize their<br />
extracellular lipase production.<br />
Production of extracellular lipase<br />
Most extracellular bacterial lipases are influenced by<br />
nutritional and physiological factors such as pH, temperature<br />
and carbon sources (Immanuel et al., 2008; Kiran<br />
et al., 2008; Wang et al., 2009). The lipase activity of both<br />
isolates was evaluated from 6 to 48 h. The maximum<br />
lipase production was observed after 12 h of incubation<br />
for BK44 and after 24 h for BK43 (Figure 2). Furthermore,<br />
the enzyme activity was gradually decreased after 12 and<br />
24 h, respectively. Therefore, the optimum incubation
Figure 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the<br />
relationships between strains BK43, BK44 and related strains. Bar, 0.005 changes per nucleotide<br />
position.<br />
periods for each strain were maintained throughout the<br />
studies. The results of the present study are similar to<br />
those of several other studies in which the optimum incubation<br />
period for lipase production was found to be 12 to<br />
24 h (Chen et al., 1998; Dharmsthiti et al., 1998).<br />
However, some bacterial species required more than 2<br />
days for the maximum lipase activity to occur (Joseph et<br />
al., 2006; Kiran et al., 2008; Wang et al., 2009).<br />
Anbu et al. 4151<br />
Theresults of the present study indicate that the<br />
organisms isolated here can be used to produce large<br />
quantities of lipase within a short period of time.<br />
Both strains were able to produce a high level of lipase<br />
at pH 6.0. However, the enzyme activity decreased<br />
rapidly at alkaline pHs. Figure 3A shows that both strains<br />
prefer an acidic pH. These findings differ from the results<br />
of other studies in that the optimum lipase production
4152 Afr. J. Biotechnol.<br />
Lipase activity (U/m l)<br />
1.6<br />
1.4<br />
1.2<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0<br />
Table 3. Fatty acid profiles of Acinetobacter species.<br />
Fatty acids Contents in BK43 (%) Contents in BK44 (%)<br />
C10:0 1.504 1.864<br />
C12:0 1.602 1.497<br />
C14:0 trace trace<br />
C15:1 3.6 3.09<br />
C16:0 19.2 20.81<br />
C16:1 (n-9) trace 1.506<br />
C16:1 (n-7) 16.31 16.86<br />
C17:0 trace trace<br />
C17:1 trace trace<br />
C18:0 trace trace<br />
C18:1(n-9) 54.2 51.524<br />
Trace: fatty acids present in amounts less than 1%.<br />
BK43<br />
BK44<br />
6 12 24 36 48<br />
Incubation periods (h)<br />
Figure 2. Effect of different incubation periods on lipase production. Each point represents the<br />
mean ± SEM of three independent experiments.<br />
occurred at neutral pH (Joseph et al., 2006) or alkaline<br />
pH (Chen et al., 1998; Kiran et al., 2008; Wang et al.,<br />
2009), but similar at pH 6.0 (Ertugrul et al., 2007). Most<br />
bacterial species are able to produce greater amounts of<br />
lipase at pH 6.5 to 7.0 (Dharmsthiti et al., 1998; Gao et al.,<br />
2004; Joseph et al., 2006). Most reports, available regarding<br />
the production of lipases by fungi, involved studies<br />
conducted under acidic conditions (Cihangir and Sarikaya,<br />
2004; Mhetras et al., 2009).<br />
Among different temperatures tested (20 to 37°C), the<br />
highest production by BK43 was obtained at 30°C, while<br />
the highest production by BK44 was obtained at 25°C<br />
(Figure 3B). In the case of BK44, approximately 25% of<br />
the enzyme activity was lost when the temperature increased<br />
from 25 to 30°C. In addition, the enzyme<br />
production by both strains has decreased dramatically at<br />
37°C, which indicates that these strains are unable to<br />
grow and produce lipase at higher temperatures. Many<br />
species of Acinetobacter have been reported to be<br />
psychrophilic and psychrotrophic (Kasana et al., 2008;
Figure 3. Effect of different pHs (A) and temperatures (B) on lipase production. Each point<br />
represents the mean ± SEM of three independent experiments.<br />
Park et al., 2009), however, the strains isolated here<br />
failed to grow and produce lipase at low temperatures<br />
even after 1 week of incubation. The optimum temperature<br />
for lipase production was 25 to 30°C, which agrees<br />
with the production of lipase by many microorganisms,<br />
including A. radioresistens and A. calcoaceticus LP009<br />
(Chen et al., 1998; Dharmsthiti et al., 1998; Kiran et al.,<br />
2008).<br />
A<br />
B<br />
Anbu et al. 4153<br />
Several studies have shown that lipase production was<br />
influenced by different carbon sources present in media<br />
(Mahler et al., 2000; Immanuel et al., 2008). In general,<br />
the required specific carbon source and its concentration<br />
differ among organisms. Therefore, in this study, various<br />
carbon sources (1%) were added to the culture medium<br />
and their effects on lipase production, by both strains,<br />
were evaluated. The greatest increase in lipase production
4154 Afr. J. Biotechnol.<br />
B<br />
A<br />
Figure 4. Effect of various carbon sources (A) and lipase inducers (B) on lipase production.<br />
Each point represents the mean ± SEM of three independent experiments.<br />
by both strains was observed in response to supplementation<br />
of the culture medium with sucrose as the<br />
carbon source, followed by lactose in the case of BK43<br />
and xylose in the case of BK44 (Figure 4A) compared<br />
with glucose (control). Sucrose has been reported to<br />
have similar effects on lipase production by Bacillus<br />
pumilus SG2 (Sangeetha et al., 2008). The other carbon<br />
sources evaluated in this study, such as fructose, starch
and mannitol, led to a decrease in lipase production by<br />
BK44 of more than 50%. These results suggest that the<br />
above carbon sources act as inhibitors of lipase production<br />
and indicate that the carbon source has the potential<br />
to increase the lipase production significantly.<br />
Lipase inducers were evaluated as shown in Figure 4B,<br />
lipase production by both strains increased dramatically<br />
(2-fold) when 1% Tween 80 was added to the media.<br />
However, a moderate level of lipase production by BK43<br />
and BK44 was obtained when soybean and sesame oil,<br />
respectively, were used as the lipase inducers (Figure<br />
4B). These findings agree with the results of other studies<br />
in which other lipids induced lipase production (Gilbert et<br />
al., 1991; Joseph et al., 2006; Immanuel et al., 2008;<br />
Kiran et al., 2008). Similarly, Dharmsthiti et al. (1998) and<br />
Gao et al. (2004) reported that lipase production by A.<br />
calcoaceticus LP009 and Serratia marcescens ECU1010<br />
was induced by 1 and 0.5% Tween 80, respectively.<br />
Conversely, Joseph et al. (2006) reported that Tween 20<br />
and 80 induced a poor level of lipase production by<br />
Staphylococcus epidermis. In the present study, other<br />
lipase inducer such as tributyrin resulted in very low<br />
enzyme production by both strains.<br />
The results of the present study provide useful information<br />
for the optimization of culture conditions such as<br />
carbon sources and lipase inducers, and physicochemical<br />
properties such as pH and temperature to<br />
provide the best lipase production by Acinetobacter<br />
species. These results clearly demonstrated that lipaseproducing<br />
bacteria are widespread in oil contaminated<br />
soil. The optimized growth conditions developed in this<br />
study can be used for a large scale in industrial purposes.<br />
Furthermore, the majority of studies conducted to date<br />
have evaluated lipase production under alkaline conditions;<br />
therefore, the results of the present study will be<br />
useful for development of methods of lipase production<br />
under acidic conditions. To our knowledge, this is the first<br />
report on the highest lipase production in Acinetobacter<br />
species under acidic conditions. The final optimized medium<br />
resulted to about 5.9 fold (BK43) and 5.7 fold<br />
(BK44) more lipase production when compared with<br />
lipase obtained in the original medium. More studies will<br />
be conducted to purify and characterize the acidic lipase<br />
produced by the two strains isolated here under optimized<br />
culture and physico-chemical conditions.<br />
ACKNOWLEDGEMENT<br />
This work was supported by Inha University Grant, Inha<br />
University, Republic of Korea.<br />
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Purification and characterization of extracellular lipases from<br />
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other polysaccharides greatly enhance the formation of exolipase by<br />
Serratia marcescens. J. Bacteriol. 138: 663-670.<br />
Yoon MY, Shin PK, Han YS, Lee SH, Park JK, Cheong CS (2004).<br />
Isolation of an Acinetobacter junii SY-01 strain producing an<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4157-4165, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2085<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> Journal<br />
Full Length Research Paper<br />
Antifungal activity of selected plant leaves crude<br />
extracts against a pepper anthracnose fungus,<br />
Colletotrichum capsici (Sydow) butler and bisby<br />
(Ascomycota: Phyllachorales)<br />
Lucy Johnny*, Umi Kalsom Yusuf and R. Nulit<br />
Department of Biology, Faculty of Science, University Putra Malaysia, 43400 UPM<br />
Serdang, Selangor Darul Ehsan, Malaysia.<br />
Accepted 8 April, 2011<br />
The antifungal activities of the leaves extract of 15 selected medicinal plants; Alpinia galanga (L.) Willd.,<br />
Alstonia spatulata Blume., Annona muricata L., Blechnum orientale L., Blumea balsamifera L., Centella<br />
asiatica L., Dicranopteris linearis (Burm. f.) Underw., Dillenia suffruticosa (Griff ex Hook.f. and<br />
Thomson) Martelli, Litsea garciae Vidal., Melastoma malabathricum L., Momordica charantia L.,<br />
Nephrolepis biserrata (Sw.), Pangium edule Reinw., Piper betle L. and Polygonum minus Huds., were<br />
evaluated on the plant pathogenic fungus, Colletotrichum capsici which was isolated from chilli. The<br />
antifungal assay was carried out in potato dextrose media in five different treatments, which were<br />
distilled water as the negative control, crude extract of leaves in methanol, chloroform, acetone and<br />
Kocide 101 as the positive control. They were carried out in three replicates. The two-way analysis of<br />
variance (ANOVA) was carried out on all the data to justify the difference between critical difference<br />
(CD) of mean (P = 0.05) and coefficient of variation (CV %) in terms of mean percent reduction in colony<br />
diameter, sporulation and minimum inhibitory concentration (MICs) of C. capsici to take statistical<br />
decisions. Crude extract of P. betle in all the solvents was found to be the most effective and exhibited<br />
the highest antifungal activities. Crude extract of P. betle in methanol inhibited 85.25% of radial growth<br />
of C. capsici followed by 78.53% leaves crude extract in chloroform and 73.58% leaves crude extract in<br />
acetone at the concentration of 10 µg/ml (p < 0.05). The exact concentrations that had definite potential<br />
to fully restrict the growth (100% inhibition) of C. capsici (MIC) by P. betle was 12.50 µg/ml in methanol,<br />
17.50 µg/ml in chloroform and 15.00 mg/ml in acetone. The sporulation assay also revealed that, P. betle<br />
leaves crude extracts showed the highest inhibition of spore germination rate of C. capsici overall at<br />
the concentration of 10 µg/ml; with 80.93% inhibition by leaves crude extracts in methanol, 74.09% by<br />
leaves crude extracts in chloroform and 72.91% by leaves crude extracts in acetone. Concentration of<br />
plant leaves crude extracts that inhibited 50% or more of the radial growth and sporulation was<br />
considered as effective (LC ≥ 50). As a conclusion, the leaf crude extracts that exhibited effectiveness<br />
by showing more than 50% inhibition against C. capsici should be considered for further evaluation. P.<br />
betle leaf crude extracts was the most effective in inhibiting the fungus respectively and thus, exhibited<br />
the highest potential as a new leading biofungicide in the agriculture field.<br />
Key words: Plant leaves crude extracts, antifungal activities, Colletotrichum capsici.<br />
INTRODUCTION<br />
Pepper fruit anthracnose which is caused by Colleto-<br />
*Corresponding author. E-mail: lucyjohnny13784@yahoo.com.<br />
Tel: +6017-3434717. Fax: +603-86567454.<br />
trichum species is one of the most serious disease which<br />
leads to serious yield loss and quality deterioration in<br />
many Asian countries and in tropical areas (Oanh et al.,<br />
2004; Sang et al., 2007). The most destructive disease of<br />
pepper anthracnose is caused by Colletotrichum capsici<br />
(Sydow) Butler and Bisby (1931) (Ascomycota:
4158 Afr. J. Biotechnol.<br />
Phyllachorales) (Amusa et al., 2004). It is well known for<br />
infection on leaves, stems, mummification of unripe green<br />
pepper fruits, pre-mature fruit drop and fruit rot (Agrios,<br />
1988; Marvel, 2003). It also has been reported that, pre-<br />
and post-harvest fruit losses of up to 50% was caused by<br />
this fungi (Boali, 1991). Generally, Colletotrichum<br />
diseases can be controlled by a wide range of chemicals<br />
such as copper compounds, dithiocarbamates, benzimidazole<br />
and trizole compounds; other fungicides such as<br />
chlorothalonil, imazalil and prochloraz are also effective<br />
against Colletotrichum (Waller, 1992). Although, the use<br />
of systemic fungicides simplifies the management strategy,<br />
not many systemic fungicides are practically in use on<br />
chilli. This limits the choice of systemic fungicides on<br />
chilli, thus, there is a strong need to find alternative<br />
systemic fungicides to the existing chemical carbendazim,<br />
which is the only systemic fungicide currently<br />
used in chilli fields (Gopinath et al., 2006). Furthermore,<br />
there is also the raise of concerns for problems of<br />
fungicide insensitivity, residues on edible produce and for<br />
tree crops; efficiency of spraying has increased in<br />
importance (Bailey and Jeger, 1992). One approach<br />
might be the testing of plants traditionally used for their<br />
antifungal activities as potential sources for drug development.<br />
Hence, this study provided broader options in agriculture<br />
by evaluating the antifungal activity of plant’s<br />
leaves crude extracts from selected medicinal plants as<br />
Alpinia galanga (L.) Willd., Alstonia spatulata Blume.,<br />
Annona muricata L., Blechnum orientale L., Blumea<br />
balsamifera L., Centella asiatica L., Dicranopteris linearis<br />
(Burm. f.) Underw., Dillenia suffruticosa (Griff ex Hook.f.<br />
and Thomson) Martelli, Litsea garciae Vidal., Melastoma<br />
malabathricum L., Momordica charantia L., Nephrolepis<br />
biserrata (Sw.)., Pangium edule Reinw., Piper betle L.,<br />
and Polygonum minus Huds., in order to test their<br />
antifungal potential against phytopathogenic fungi C.<br />
capsici. These plants were selected for extraction due to<br />
their well-known medicinal properties in traditional uses<br />
by local ethnics in Malaysia. The collected plant samples<br />
were identified at Herbarium, Department of Biology,<br />
Universiti Putra Malaysia. Leave samples were extracted<br />
in methanol, chloroform and acetone. Then, C. capsici<br />
was treated with different concentration of plant crude<br />
extract and antifungal activities were determined by<br />
measuring the percentage inhibition of radial growth,<br />
sporulation rate and minimum inhibition concentration<br />
(MIC).<br />
MATERIALS AND METHODS<br />
Plant collection and extraction<br />
The leaves of 15 plants (A. galanga, A. spatulata, A. muricata, B.<br />
orientale, B. balsamifera., C. asiatica, D. linearis, D. suffruticosa, L.<br />
garciae, M. malabathricum, M. charantia, N. biserrata, P. edule, P.<br />
betle, and P. minus) were collected locally from the nearby areas of<br />
Sarikei, Sarawak (Table 1).<br />
The leaf samples were air dried and weighed. Leaves samples<br />
were then, ground using mortar and pestle into coarse powder.<br />
Leaves of the plants were extracted in polar solvent (methanol),<br />
semi-polar solvent (chloroform), and non-polar solvent (acetone) by<br />
following cold percolation method (Valsaraj et al., 1997). Leaves<br />
sample were then, soaked in three different solvents; methanol,<br />
chloroform and acetone, at room temperature for 72 h. The<br />
obtained extract was then filtered through Whatman (no.1) filter<br />
paper extracts solution and was transferred into 250 ml round<br />
bottom flasks which were previously weighed. Then, the extracts<br />
solution was evaporated using Buchi Rotavapor R-210, Switzerland<br />
to concentrate the extracts. Concentrated extracts were allowed to<br />
dry in fume cupboard, weighed again and were kept in 4°C for<br />
bioassays evaluation. Their volume was made up to obtain respective<br />
concentrations.<br />
Source of isolate<br />
The culture of C. capsici from Capsicum annuum L. was obtained<br />
from the Faculty of Agriculture, Universiti Putra Malaysia. Pure<br />
cultures were maintained on potato dextrose agar (PDA) and slants<br />
(Gupta, 2004).<br />
Antifungal assays<br />
Agar-dilution assay<br />
The agar dilution assay was carried out according to Alam (2004)<br />
with a slight modification. 39 g of potato dextrose agar (PDA)<br />
powder was boiled until the agar completely dissolved in 1 L of<br />
distilled water. The solution was then transferred to the blue cap<br />
bottle and sterilized using autoclave at 121°C for 15 min. 19 ml of<br />
the sterilized PDA and 1 ml of plant extract were mixed and plated<br />
on the sterilized Petri dishes (8.5 mm in diameter). 10 mm diameter<br />
of mycelia discs were inoculated at the centre of the medium. The<br />
antifungal assay was divided into five different treatments as crude<br />
extract of leaves in methanol, chloroform and acetone, commercial<br />
fungicide Kocide 101 as positive control and a negative control.<br />
Colony growth was determined on the basis of linear dimensions.<br />
Assay was carried out in three replicates. The percent reduction<br />
(Rr) or stimulation (Rs) of colony diameter by each extract was<br />
determined using the following formula (Nduagu et al., 2008):<br />
Rr =<br />
Rr =<br />
(R1 – R2) x 100<br />
R1<br />
(R2 – R1) x 100<br />
R2<br />
Where, Rr = percent reduction in colony diameter; Rs = percent<br />
stimulation in colony diameter; R1 = colony diameter on the<br />
untreated medium (control); and R2 = colony diameter on the<br />
treated medium.<br />
Minimum inhibitory concentration (MIC) assay<br />
Minimum inhibitory concentration (MIC) was determined using agardilution<br />
method (Yazdani et al., 2007). 1 ml of various crude<br />
extracts concentrations (0.001, 0.005. 0.050, 0.500, 5.0, 12.5, 15.0,<br />
17.5 and 20.0 µg/ml) were prepared in the laboratory universal<br />
bottle containing 9 ml of PDA and were sterilized. The mixture of<br />
PDA and extracts were poured into Petri dish under sterile<br />
condition. Then, 2 µl of adjusted spore suspension was added to
Table 1. Details about plants used, their family, English and local names.<br />
Plant used Family English name Local name<br />
A. galanga (L.) Willd. Zingiberaceae Greater galangal or blue<br />
ginger<br />
A. spatulata Blume. Apocynaceae Hard milkwood or siamese<br />
balsa<br />
A.muricata L. Annonaceae Brazilian pawpaw, soursop,<br />
prickly custard apple<br />
Johnny et al. 4159<br />
Lengkuas (Malay), Engkuas/Lankwas/Puar<br />
(Iban)<br />
Pulai puteh/Rejang (Iban), Pulai basong (Malay)<br />
Hampun kapal (Kadazan), Durin mekah (Iban),<br />
Durian belanda (Malay)<br />
B. orientale L. Blechnaceae Oriental blechnum, Paku ikan/Paku Lipan/Paku ular/Paku ulat<br />
centipede fern<br />
(Malay)<br />
B.balsamifera L. Asteraceae Sambong Susuoh (Bidayuh), Urok bung (Kayan), Dun<br />
supiro (Kiput), Keymabo (Selakau), Sembong<br />
(Malay)<br />
C.asiatica L. Mackinlayaceae Indian pennywort Pegaga (Malay)<br />
D. linearis (Burm. f.)<br />
Underw.<br />
D. suffruticosa (Griff ex<br />
Hook.f. and Thomson)<br />
Martelli<br />
Gleicheniaceae Uluhe, staghorn fern, false<br />
staghorn, resam<br />
Bengkawang (Iban), Resam (Malay)<br />
Dilleniaceae Simpoh ayer Buan (Iban), Abuan (Semai), Bu’ua (Bidayuh),<br />
Simpoh air (Malay)<br />
L. garciae Vidal. Lauraceae Bagnolo, wuru lilin Madang enkala/Pedar (Iban), Pong Labon<br />
(Sabah), Ta’ang (Bidayuh)<br />
M.malabathricum L. Melastomaceae Malabar melastome,<br />
Singapore rhododendron,<br />
senduduk<br />
Sekenduduk/kenduduk/seduduk (Iban),<br />
Senduduk (Malay)<br />
M. charantia L. Cucurbitaceae Bitter melon Peria (Malay)<br />
N. bisserrata (Sw.) Polypodiaceae Giant sword fern Paku larat (Iban), Paku uban (Malay)<br />
P. edule Reinw. Salicaceae football fruit, kepayang,<br />
kluwak<br />
Kepayang (Iban, Malay)<br />
P. betle L. Piperaceae Betel leaf Sireh (Malay, Iban)<br />
P.minus Huds. Polygonaceae Pygmy smartweed Kesum (Malay)<br />
each Petri dish plate. The agar without any plant crude extract<br />
served as the control. Assay was carried out in three replicates. The<br />
minimum inhibitory concentration (MIC) was regarded as the lowest<br />
concentration of the extract that did not show any visible growth<br />
(100% inhibition) after 14 days of incubation (compared with<br />
control). The minimum inhibitory concentration (MIC) was expressed<br />
in µg/ml.<br />
Sporulation assay<br />
The sporulation assay was carried out according to Nduagu et al.<br />
(2008) with a slight modification. The rate of sporulation was<br />
determined by adding 10 ml sterile distilled water to each seven<br />
days old plate that were obtained from agar dilution assay and<br />
gently scraping with a sterile glass rod to dislodge the spores. The<br />
spore suspensions obtained were filtered through sterile cheese<br />
cloth into a sterile 50 ml glass beaker and homogenized by manual<br />
shaking. The spores were then counted using a haemocytometer.<br />
Assay was carried out in three replicates. The percent sporulation<br />
reduction (Sr) or stimulation (Ss) by each extract was<br />
determined using the following formula (Nduagu et al., 2008):<br />
Sr =<br />
(R1 – R2) x 100<br />
R1<br />
(R2 – R1) x 100<br />
Ss =<br />
R2<br />
Where, Sr = percent reduction in sporulation; Ss = percent<br />
stimulation in sporulation; S1 = sporulation on the untreated<br />
medium (control); and S2 = sporulation on the treated medium.<br />
Statistical analysis<br />
The two-way analysis of variance (ANOVA) was carried out on all<br />
the data to justify the difference between critical difference (CD) of<br />
mean (P = 0.05) and coefficient of variation (CV %) in terms of<br />
mean percent reduction in colony diameter, sporulation and MICs of<br />
C. capsici to take statistical decisions (Snedecor and Cochran,<br />
1989). Results with p < 0.05 were considered to be statistically<br />
significant. If the result was significant, CD test was adapted to find<br />
which of the concentrations were same or different in their percent<br />
reduction in colony diameter, sporulation and MICs. For the<br />
calculation of CV, standard deviation was converted into a relative<br />
measure of dispersion for the purpose of comparison. If CV was<br />
greater, it was said that, the treatment was more variable and less<br />
stable in terms of action and vice versa. CD and CV were<br />
calculated using the following formula: CD = Standard error<br />
difference X table value for error degrees of freedom at 5% level<br />
and CV = Standard deviation / mean X 100.
4160 Afr. J. Biotechnol.<br />
RESULTS<br />
Inhibition of radial growth of C. capsici by plant crude<br />
extracts<br />
Table 1 shows that only 5 out of the15 plants screened<br />
showed more than 50% inhibition of radial growth. These<br />
plants were P. betle, A. galanga, C. asiatica, M. charantia<br />
and P. minus. Crude extracts of P. betle in all the<br />
solvents exhibited significant reduction in colony radial<br />
growth against C. capsici in all the concentrations (Table<br />
2). Furthermore, these studies revealed that, the percent<br />
inhibition of radial growth against C. Capsici increased as<br />
the concentration of plant crude extract increased.<br />
Methanol crude extract of P. betle exhibited the highest<br />
antifungal activity with 72.30 to 85.18% inhibition against<br />
C. capsici. This was followed by methanol crude extract<br />
of A. galangal and C. asiatica which exhibited 68.77 to<br />
74.60% and 57.60 to 71.87% inhibition, respectively.<br />
Chloroform crude extracts of P. betle also showed the<br />
highest inhibition (72.36 to 78.53%) against radial growth<br />
of C. capsici followed by A. galangal (50.72 to 63.57%),<br />
M. charantia (57.46 to 61.31%) and C. asiatica (56.58 to<br />
50.61%) (Table 2). While in B. balsamifera and P. edule<br />
less than 50% antifungal activity was exhibited (46.61to<br />
49.82% inhibition), respectively.<br />
Acetone crude extracts of P. betle, A. galanga, M.<br />
charantia, C. asiatica, B. balsamifera and P. edule were<br />
found to be effective against C. capsici which showed<br />
more than 50% inhibition of radial growth with different<br />
concentration (Table 2). P. betle exhibited 75.02%<br />
inhibition in 10.00 µg/ml, 75.38% inhibition in 1.00 µg/ml,<br />
73.73% inhibition in 0.10 µg/ml and 70.36% inhibition in<br />
0.01 µg/ml against C. capsici. This was followed by<br />
acetone crude extract of B. balsamifera which exhibited<br />
74.56% inhibition in 10.00 µg/ml, 74.56% inhibition in<br />
1.00 µg/ml, 70.82% inhibition in 0.10 µg/ml and 65.80%<br />
inhibition in 0.01 µg/ml against C. capsici. Acetone crude<br />
extract of M. charantia exhibited 57.24% inhibition in<br />
10.00 µg/ml, 54.94% inhibition in 1.00 µg/ml, 51.44%<br />
inhibition in 0.10 µg/ml and 51.53% inhibition in 0.01<br />
µg/ml against C. capsici. Acetone crude extract of D.<br />
suffruticosa exhibited effective antifungal activities only in<br />
10.00 µg/ml with 51.33%.<br />
MIC of C. capsici by plant crude extracts<br />
Plant crude solvent extracts with the lowest concentration<br />
that did not show any visible growth (100% inhibition) of<br />
C. capsici after 14 days of incubation (compare to<br />
control) were determined as MIC. Among the plants<br />
screened, P. betle exhibited the lowest MIC value against<br />
C. capsici in 12.5 µg/ml in methanol crude extract. This<br />
was followed by methanol crude extract of A. galanga<br />
(15.00 µg/ml), C. asiatica (17.5 µg/ml) and finally, by M.<br />
charantia and B. balsamifera (20 µg/ml), respectively<br />
(Table 3). In chloroform crude extracts, both P. betle and<br />
A. galanga exhibited the lowest MIC value against C.<br />
capsici in 17.5 µg/ml, followed by C. asiatica and M.<br />
charantia (20.00 µg/ml). The lowest MIC value was<br />
observed in P. betle (15.00 µg/ml), B. balsamifera (17.50<br />
µg/ml) and M. charantia and P. minus (20.00 µg/ml) in<br />
acetone crude extracts (Table 3).<br />
Inhibition/stimulation of sporulation of C. capsici by<br />
plant crude extracts<br />
Inhibition of sporulation of C. capsici by leaf crude<br />
extracts in methanol, chloroform and acetone of the 15<br />
medicinal plants species are summarized in Table 4.<br />
Plant crude solvent extracts with concentration that<br />
inhibited more than 50% of the normal sporulation were<br />
considered as effective (Begum et al., 2007). Among the<br />
plants screened, five species showed 50% and more of<br />
antifungal activity against; C. capsici A. galanga, C.<br />
asiatica, B. balsamifera, M. charantia, P. betle and P.<br />
minus. P. betle exhibited the highest antifungal activity in<br />
all the chosen four treatments of solvent extracts. P. betle<br />
leaf crude extract of 10.00 µg/ml exhibited the highest<br />
percent of inhibition against C. capsici between 72.91<br />
and 80.93%, respectively (Table 4).<br />
DISCUSSION<br />
The findings from this study revealed that, leaf crude<br />
extracts of P.betle exhibited the highest antifungal<br />
activities overall in inhibiting the mycelial growth of C.<br />
capsici among the 15 medicinal plants. The methanol<br />
crude extract of P. betle in 10.00 µg/ml exhibited the<br />
highest inhibition overall with 85.25% inhibition (P <<br />
0.05). Compared with the positive control, commercial C.<br />
capsici fungicide (Kocide 101), the percentage of<br />
inhibition by methanol crude extract of P. betle in 10.00<br />
µg/ml exhibited almost the same percentage of Kocide<br />
101 (87.24%) of the respective concentration. At the<br />
lowest concentration of acetone crude extract which was<br />
0.01 µg/ml, P. betle still effectively inhibited the mycelial<br />
growth with 70.36% inhibition. This showed that the<br />
inhibitory action of the P. betle crude extracts was<br />
recorded even at very low dose, which is a clear indication<br />
that the crude extract contained active components<br />
that have antifungal properties.<br />
The methanol crude extract of P. betle in 10.00 µg/ml<br />
exhibited the highest inhibition in sporulation of C. capsici<br />
overall with 80.93% inhibition (P < 0.05). The positive<br />
control, commercial C. capsici fungicide (Kocide 101),<br />
exhibited 91.73% inhibition of the respective concentration.<br />
At the lowest concentration of acetone crude<br />
extract which was 0.01 µg/ml, P.betle still effectively<br />
inhibited the sporulation with 68.96% inhibition. This<br />
showed that the inhibitory action of the P. betle crude
Johnny et al. 4161<br />
Table 2. Mean ± S.E of inhibiton of radial growth (mm) of C. capsici by leaf extracts in methanol, chloroform and acetone with<br />
various concentrations.<br />
Leaf extract in methanol<br />
Mean ± S.E of inhibiton of radial growth (mm)<br />
0.01 (µg/ml) 0.10 (µg/ml) 1.00 (µg/ml) 10.00 (µg/ml)<br />
A. galanga L. 55.22 ± 1.08* 56.99 ± 0.59* 59.31 ± 1.28* 62.78 ± 1.51*<br />
A. spatulata Blume. 19.13 ± 0.92 19.89 ± 1.04 22.06 ± 0.56 23.64 ± 0.91<br />
A. muricata L. 30.25 ± 1.23 31.86 ± 1.21 35.60 ± 1.20 39.96 ± 1.54<br />
B. orientale L. NI NI 2.68 ± 1.07 4.02 ± 1.13<br />
B. balsamifera L. 25.86 ± 0.73 27.24 ± 1.19 30.37 ±1.22 34.33 ±1.46<br />
C. asiatica L. 54.78 ± 1.48* 46.90 ± 1.80* 55.26 ± 1.58* 59.07 ± 1.44*<br />
D. linearis 4.44 ± 1.35 5.84 ± 0.77 7.85 ± 0.90 9.78 ± 0.85<br />
D. suffruticosa 30.57 ± 0.59 31.08 ± 0.77 33.77 ± 0.78 36.71 ± 0.76<br />
L.garciae Vidal. 13.59 ± 0.84 15.83 ± 1.22 29.34 ± 0.66 31.02 ± 1.41<br />
M. malabathricum L. 30.71 ± 2.52 30.27 ± 1.24 33.41 ± 1.08 34.51 ± 2.16<br />
M.charantia L. 42.17 ± 1.32* 42.42 ± 2.81* 46.25 ± 1.59* 48.32 ± 1.57*<br />
N.bisserrata (Sw.) NI NI 2.89 ± 0.11 3.67 ± 1.13<br />
P.edule Reinw. NI NI 1.68 ± 0.43 4.26 ± 1.17<br />
P. betle L. 61.22 ± 0.53* 65.50 ± 1.10* 66.59 ± 0.89* 71.87 ± 0.78*<br />
P. minus Huds. 39.10 ± 1.72 40.20 ± 1.42 40.37 ± 2.03 46.92 ± 1.11*<br />
Leaf extracts in chloroform<br />
A. galanga (L.) Willd. 42.64 ± 1.30* 46.40 ± 0.72* 48.97 ± 1.36* 53.38 ± 1.05*<br />
A. spatulata Blume. 24.96 ± 1.13 24.74 ± 1.12 27.44 ± 0.72 27.40 ± 0.73<br />
A. muricata L. 8.39 ± 0.84 10.61 ± 1.16 12.65 ± 1.19 18.01 ± 0.92<br />
B. orientale L. NI NI 1.77 ± 1.01 5.33 ± 1.13<br />
B. balsamifera L. 35.50 ± 0.43 39.17 ± 1.35 44.87 ± 1.10* 46.26 ± 1.34*<br />
C. asiatica L. 38.69 ± 1.87 40.82 ± 1.54 42.61 ± 1.01* 47.41 ±1.82*<br />
D. linearis (Burm. f.) Underw. 32.05 ± 1.02 34.23 ± 0.62 36.19 ± 1.13 37.89 ± 0.62<br />
D. suffruticosa 21.41 ± 0.50 22.10 ± 0.93 23.98 ± 1.26 29.90 ± 2.20<br />
L. garciae Vidal. 1.94 ± 1.70 5.84 ± 1.26 8.85 ± 1.99 9.68 ± 1.53<br />
M. malabathricum L. 25.58 ± 1.55 23.56 ± 0.69 27.27 ± 1.13 41.12 ± 1.55<br />
M. charantia L. 48.12 ± 1.53* 50.24 ± 2.12* 51.85 ± 1.94* 50.09 ± 1.56*<br />
N. bisserrata (Sw.) NI NI 3.27 ± 0.15 5.60 ± 0.62<br />
P. edule Reinw. 36.93 ± 1.43 37.73 ± 0.70 41.24 ± 1.17 44.01 ± 1.40*<br />
P.betle L. 61.27 ± 0.75* 63.90 ± 1.72* 65.37 ± 0.90* 66.20 ± 1.15*<br />
P. minus Huds. 20.23 ± 2.27 25.62 ± 1.51 29.43 ± 0.52 31.57 ± 1.38<br />
Leaf extracts in acetone<br />
A. galanga L. 19.70 ± 0.95 21.68 ± 0.66 23.17 ± 1.55 26.94 ± 1.26<br />
A. spatulata Blume. 28.87 ± 0.97 31.06 ± 0.84 34.65 ± 0.43 37.27 ± 1.06<br />
A. muricata L. 16.00 ± 0.85 17.56 ± 1.04 28.75 ± 1.01 33.55 ± 1.25<br />
B. orientale L. NI 3.77 ± 1.32 2.83 ± 1.15 4.70 ± 1.47<br />
B. balsamifera L. 55.86 ± 0.36* 60.06 ± 1.00* 62.55 ± 1.15* 62.44 ± 1.13*<br />
C. asiatica L. 14.47 ± 0.84 24.16 ± 1.24 28.48 ± 1.23 35.28 ± 1.59<br />
D. linearis 21.74 ± 0.99 23.28 ± 1.26 28.25 ± 1.21 34.80 ± 0.91<br />
D. suffruticosa 39.14 ± 0.49 39.14 ± 1.42 38.78 ± 1.38 43.23 ± 1.50*<br />
L. garciae Vidal. NI NI 24.48 ± 1.38 25.48 ± 1.26<br />
M. malabathricum L. 22.74 ± 1.25 31.07 ± 1.63 28.98 ± 1.89 37.72 ± 0.84<br />
M. charantia L. 43.16 ± 0.72* 43.23 ± 1.25* 46.45 ± 0.94* 47.78 ± 1.28*<br />
N. bisserrata (Sw.) NI NI 2.05 ± 0.92 3.18 ± 1.00<br />
P. edule Reinw. 8.12 ± 1.28 36.91 ± 2.03 36.92 ± 1.27 35.36 ± 0.75<br />
P. betle L. 59.57 ± 1.39* 62.17 ± 1.18* 63.77 ± 0.55* 63.56 ± 0.77*<br />
P. minus Huds. 25.34 ± 1.88 27.65 ± 1.73 28.08 ± 1.30 42.64 ± 0.78<br />
Positive control (Kocide 101) 61.60 ± 0.64 66.08 ± 0.90 72.55 ± 0.71 73.54 ± 1.06<br />
Negative control (distiiled water) 84.66 ± 0.24 84.34 ± 0.57 85.00 ± 0.00 84.30 ± 0.65<br />
Each value represented the mean (3 replicates) ± standard error; NI = no inhibition; * represent crude extracts that effectively inhibited growth<br />
(P < 0.05).
4162 Afr. J. Biotechnol.<br />
Table 3. Mean ± S.E of inhibiton of radial growth (mm) of C. capsici<br />
by leaf extracts in methanol, chloroform, and acetone with various<br />
concentrations.<br />
Leaf extracts in methanol Minimum inhibition<br />
concentration (µg/ml)<br />
A. galanga L. 15.00<br />
A. spatulata Blume. >20.00<br />
A. muricata L. >20.00<br />
B. orientale L. >20.00<br />
B.balsamifera L. 20.00<br />
C. asiatica L. 17.50<br />
D. linearis >20.00<br />
D.suffruticosa >20.00<br />
L. garciae Vidal. >20.00<br />
M. malabathricum L. >20.00<br />
M. charantia L. 20.00<br />
N.. bisserrata (Sw.) >20.00<br />
P.edule Reinw. >20.00<br />
P.betle L. 12.50<br />
P.minus Huds. >20.00<br />
Leaf extracts in chloroform<br />
A.galanga L. 17.50<br />
A.spatulata Blume. >20.00<br />
A. muricata L. >20.00<br />
B.orientale L. >20.00<br />
B. balsamifera L. 20.00<br />
C. asiatica L. 20.00<br />
D.linearis >20.00<br />
D. suffruticosa >20.00<br />
L.garciae Vidal. >20.00<br />
M. malabathricum L. >20.00<br />
M.charantia L. >20.00<br />
N.bisserrata (Sw.) >20.00<br />
P. edule Reinw. >20.00<br />
P.betle L. 17.50<br />
P.minus Huds. >20.00<br />
Leaf extracts in acetone<br />
A.galanga L. 20.00<br />
A. spatulata Blume. >20.00<br />
A.muricata L. >20.00<br />
B.orientale L. >20.00<br />
B.balsamifera L. 17.50<br />
C. asiatica L. 20.00<br />
D.linearis >20.00<br />
D. suffruticosa >20.00<br />
L.garciae Vidal. >20.00<br />
M.malabathricum L. >20.00<br />
M.charantia L. >20.00<br />
N. bisserrata (Sw.) >20.00<br />
P. edule Reinw. >20.00<br />
P.betle L. 15.00
Table 3. continued<br />
P.minus Huds. >20.00<br />
Positive control (Kocide 101) 12.50<br />
Negative control (distilled water) NI<br />
NI = No Inhibition<br />
Johnny et al. 4163<br />
Table 4. Mean ± S.E of inhibiton of sporulation (x10 5 ) of C. capsici by leaf extracts in methanol, chloroform, and acetone with<br />
various concentrations.<br />
Leaf extracts in methanol<br />
Mean ± S.E of inhibition/stimulation of sporulation (x10 5 )<br />
0.01 (µg/ml) 0.10 (µg/ml) 1.00 (µg/ml) 10.00 (µg/ml)<br />
A.galanga (L.) Willd. 2.40 ± 0.07* 2.62 ± 0.06* 2.57 ± 0.12* 2.73 ± 0.10*<br />
A. spatulata Blume. 0.84 ± 0.02 0.86 ± 0.03 1.11 ± 0.02 1.09 ± 0.03<br />
A. muricata L. 0.94 ± 0.04 0.91 ± 0.03 1.35 ± 0.06 1.59 ± 0.06<br />
B.orientale L. NI NI NI NI<br />
B.balsamifera L. 1.29 ± 0.08 1.28 ± 0.10 1.43 ± 0.04 1.62 ± 0.08<br />
C.asiatica L. 1.49 ± 0.13 1.57 ± 0.14 2.03 ± 0.10* 2.41 ± 0.09*<br />
D.linearis (Burm. F.) Underw. 0.22 ± 0.04 0.17 ± 0.03 0.32 ± 0.05 0.48 ± 0.04<br />
D. suffruticosa 1.08 ± 0.08 1.30 ± 0.03 1.58 ± 0.13 1.61 ± 0.06<br />
L.garciae Vidal. 0.40 ± 0.02 0.54 ± 0.02 0.93 ± 0.03 0.95 ± 0.04<br />
M. malabathricum L. 1.46 ± 0.03 1.39 ± 0.05 1.59 ± 0.11 1.54 ± 0.12<br />
M.charantia L. 1.65 ± 0.11 1.70 ± 0.14 2.00 ± 0.10* 2.01 ± 0.04*<br />
N.bisserrata (Sw.) NI NI NI NI<br />
P.edule Reinw. NI NI NI NI<br />
P.betle L. 2.78 ± 0.09* 2.92 ± 0.06* 2.95 ± 0.03* 3.20 ± 0.07*<br />
P. minus Huds. 1.81 ± 0.08 1.73 ± 0.03 1.77 ± 0.07 2.06 ± 0.05*<br />
Leaf extracts in chloroform<br />
A. galanga (L.) Willd. 1.91 ± 0.05 1.96 ± 0.15* 2.25 ± 0.09* 2.49 ± 0.06*<br />
A. spatulata Blume. 1.23 ± 0.02 1.17 ± 0.03 1.19 ± 0.03 1.29 ± 0.03<br />
A.muricata L. 0.12 ± 0.03 0.11 ± 0.04 0.45 ± 0.04 0.55 ± 0.04<br />
B. orientale L. NI NI NI NI<br />
B. balsamifera L. 1.53 ± 0.06 1.63 ± 0.13 1.92 ± 0.08 2.13 ± 0.07*<br />
C.asiatica L. 1.50 ± 0.09 1.52 ± 0.15 1.82 ± 0.10 2.00 ± 0.06*<br />
D. linearis (Burm. f.) Underw. 1.16 ± 0.02 1.60 ± 0.03 1.58 ± 0.04 1.57 ± 0.04<br />
D. suffruticosa 1.00 ± 0.04 1.06 ± 0.04 1.07 ± 0.03 1.11 ± 0.03<br />
L. garciae Vidal. 0.06 ± 0.02 0.07 ± 0.02 0.21 ± 0.03 0.30 ± 0.02<br />
M. malabathricum L. 1.09 ± 0.07 1.05 ± 0.11 1.26 ± 0.08 1.76 ± 0.08<br />
M. charantia L. 1.74 ± 0.12 1.88 ± 0.05 2.02 ± 0.05* 2.11 ± 0.04*<br />
N.bisserrata (Sw.) NI NI 0.20 ± 0.03 0.20 ± 0.03<br />
P.edule Reinw. 1.39 ± 0.06 1.58 ± 0.05 1.58 ± 0.04 1.83 ± 0.04<br />
P.betle L. 2.71 ± 0.06* 2.84 ± 0.04* 2.86 ± 0.13* 2.93 ± 0.04*<br />
P.minus Huds. 1.03 ± 0.09 1.25 ± 0.04 1.30 ± 0.09 1.43 ± 0.05<br />
Leaf extracts in acetone<br />
A.galanga (L.) Willd. 0.98 ± 0.08 1.01 ± 0.04 1.01 ± 0.07 1.21 ± 0.10<br />
A. spatulata Blume. 1.31 ± 0.03 1.33 ± 0.03 1.49 ± 0.03 1.47 ± 0.03<br />
A. muricata L. 0.60 ± 0.05 0.53 ± 0.04 1.06 ± 0.04 1.09 ± 0.04<br />
B. orientale L. NI NI NI 0.10 ± 0.03<br />
B. balsamifera L. 1.91 ± 0.07 2.48 ± 0.15* 2.73 ± 0.10* 2.80 ± 0.10*<br />
C. asiatica L. 0.66 ± 0.10 0.89 ± 0.08 1.43 ± 0.06 1.81 ± 0.09<br />
D. linearis (Burm. f.) Underw. 1.05 ± 0.05 1.08 ± 0.03 1.47 ± 0.04 1.56 ± 0.04
4164 Afr. J. Biotechnol.<br />
Table 4. Continued<br />
D. suffruticosa 1.70 ± 0.04 1.67 ± 0.12 1.64 ± 0.03 1.77 ± 0.09<br />
L. garciae Vidal. NI NI 0.83 ± 0.04 0.89 ± 0.04<br />
M. malabathricum L. 1.06 ± 0.04 1.40 ± 0.05 1.41 ± 0.05 1.62 ± 0.10<br />
M.charantia L. 1.66 ± 0.13 1.85 ± 0.06 1.93 ± 0.04* 1.99 ± 0.06*<br />
N. bisserrata (Sw.) NI NI NI 0.07 ± 0.01<br />
P. edule Reinw. NI 1.17 ± 0.04 1.38 ± 0.05 1.42 ± 0.03<br />
P.betle L. 2.71 ± 0.07* 2.82 ± 0.02* 2.83 ± 0.12* 2.88 ± 0.07*<br />
P. minus Huds. 1.14 ± 0.06 1.26 ± 0.13 1.33 ± 0.03 2.05 ± 0.04*<br />
Positive control (Kocide 101) 0.76 ± 0.09 0.79 ± 0.05 0.55 ± 0.04 0.33 ± 0.02<br />
Negative control (distilled water) 3.93 ± 0.02 3.95 ± 0.01 3.97 ± 0.01 3.95 ± 0.02<br />
Each value represented the mean (3 replicates) ± standard error; NI = No Inhibition; * represent crude extracts that effectively inhibited<br />
growth (P < 0.05).<br />
extracts was recorded even at very low dose, which is a<br />
clear indication that the crude extract contained active<br />
components that had antifungal properties. Phangthip et<br />
al. (2005) found that Piper betle leaves have antimicrobial<br />
activity due to the essential oils of the plant which<br />
contained phenolic compounds such as cavicol,<br />
cavibetol, carvacrol, eugenol and allilpyrocatechol. These<br />
compounds are assumed could inhibit bacteria and fungi.<br />
Begum et al. (2007) had found the extract of P.betle to<br />
have a wide spectrum of antifungal activity. It has also<br />
been reported that the leaves of P. betle possess various<br />
medicinal properties such as antioxidant, antibacterial,<br />
digestive, stimulant, antifungal and nematocidal properties<br />
(Phangthip et al., 2006).<br />
Johann et al. (2007) stated that the chemistry of Piper<br />
species has been widely investigated and phytochemical<br />
investigations from all parts of the world have led to the<br />
isolation of a number of physiologically active compounds<br />
such as alkaloids/amides, propenylphenols, lignans,<br />
neolignans, terpenes, steroids, kawapyrones, piperolides,<br />
chalcones, di-hydrochalcones, flavones and flavanones<br />
which exhibited high antimicrobial and antifungal<br />
properties. According to Lee et al. (2004) most Piper<br />
chemistry has been conducted to find potential pharmaceuticals<br />
or pesticides and over 90% of the literature<br />
focuses on compounds that are cytotoxic, antifungal,<br />
antitumor, fragrant or otherwise useful to humans.<br />
The antimicrobial properties of different species of the<br />
genus Piper have also been studied (Johann et al.,<br />
2007). In a screening for medicinal plants with<br />
antimicrobial activity in Colombia, the methanolic extract<br />
of the leaf of Piper lanceafolium showed activity against<br />
Candida albicans, Klebsiella pneumoniae, Enterococcus<br />
faecalis, Mycobacterium phlei, Bacillus subtilis and<br />
Staphylococcus aureus. Piper nigrum (black pepper) is<br />
known to have antifungal activity due to lactones,<br />
terpenoids, alkaloids and saponins. 4,5-Dimethoxy-2,3-<br />
(methylenedioxy)-l-allylbenzene, a natural isolate of Piper<br />
hispidum and Piper aduncum, also has strong<br />
antimicrobial activity. This natural product and three other<br />
related compounds, [4-(5’-hydroxy-5’-nonanyl)-1,2<br />
(methylenedioxy) benzene, 4-(5’-non-4’-enyl)-1,2-<br />
(methylenedioxy) benzene and 6-methoxy-2,3-<br />
(methylenedioxy)-4-allylphenol], were synthesized from<br />
piperonal and screened for their biological activity. These<br />
four compounds showed high levels of antifungal and<br />
antibacterial activity against several fungi and bacteria.<br />
In conclusion, the leaves crude extracts that exhibited<br />
good potential and showed effectiveness as fungicides of<br />
C. capsici should be drawn with an in-depth study of<br />
testing the phytoextracts for their potentiality under in vivo<br />
condition.<br />
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clinically relevant fungal pathogens. Brazilian J. Microbiol. 38: 632-<br />
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(M.S.). Virginia Polytechnic Inst. State University,<br />
Nduagu C, Ekefan EJ, Nwankiti AO (2008). Effect of some crude plant<br />
extracts on growth of Colletotrichum capsici (Synd.) Butler & Bisby,<br />
causal agent of pepper anthracnose. J. Appl. Biosci. 6(2): 184-190.<br />
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of anthracnose fungus, Colletotrichum capsici on various Thai chilli<br />
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Phangthip P, Thaewnon-ngiw B, Kanchanarach W, Sangdee A,<br />
Rittideth S (2005). Antifungal efficacy of Piper betle Linn. extract<br />
against the growth of mat fungi. 31st Congress on Science and<br />
Technology of Thailand at Suranaree University of Technology.<br />
Sang HK, Jae BY, Hyo GP (2007). Inheritance of anthracnose<br />
resistance in a new genetic resource, Capsicum baccatum PI594137.<br />
J. Crop Sci. Biotech. 11(1): 13-16.<br />
Snedecor GW, Cochran WG (1989). Statistical methods, 8th edn. Iowa<br />
State University Press, Ames, Iowa.<br />
Waller JM (1992). Colletotrichum diseases of perennial and other cash<br />
crops. Bailey, J.A. and Jeger, M. J., eds. In: Colletotrichum: Biology,<br />
Pathology and Control. CAB. Int. Wallingford, UK. pp. 167-186.<br />
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Valsaraj R, Pushpangadan P, Smitt UW (1997). Antimicrobial screening<br />
of selected medicinal plants from India. J. Ethnopharmacol. 58: 75-<br />
83.<br />
Yazdani D, Rezazadeh SH, Amin GH, Zainal AMA, Shahnazi S,<br />
Jamalifar H (2009). Antifungal activity of dried extracts of anise<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4166-4174, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2250<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Isolation, identification and application in lignin<br />
degradation of an ascomycete GHJ-4<br />
Huiju Gao, Yanwen Wang, Wenting Zhang, Weile Wang and Zhimei Mu*<br />
College of Forestry, Shandong Agricultural University, Shandong, China.<br />
Accepted 24 March, 2011<br />
This study was undertaken to isolate an ascomycete producing ligninolytic enzyme and characterize its<br />
lignin degradation capability. Among 20 isolates, GHJ-4 was isolated from decayed wood of Salix<br />
matsudana Koidz in Mount Tai, China, by different indicator compounds assay. The taxonomy of the<br />
fungi was Paraconiothyrium variabile Damm, Verkley and Crous, which had been confirmed by both<br />
morphological and 5.8S rDNA/ITS analyses. The capability of utilizing several lignin model compounds<br />
and decoloration of aromatic dyes by GHJ-4 strain revealed its ligninolytic potentiality. After incubation<br />
for 40 days, the weight loss of the wood was 20.91% and lignin loss was 22.99%, which indicated that,<br />
GHJ-4 strain had higher degradation ability for lignin. To our best of knowledge, this study represented<br />
the first report that P. variabile could produce ligninolytic enzyme and degrade lignin.<br />
Key words: Ligninolytic enzyme, ascomycete, identification, lignin degradation.<br />
INTRODUCTION<br />
Biofuel plays an essential role in replacing petroleumbased<br />
fuels in current worldwide energy situation (Yuan<br />
et al., 2008). Among all the feedstocks, lignocellulosic<br />
biomass, which could be acquired from biomass crop and<br />
agricultural residuals, might be the best choice in the long<br />
term (Smeets and Faaij, 2007). However, the key<br />
obstacle for transitioning lignocellulosic feedstock was<br />
the complicated structure of the plant cell wall,<br />
particularly the presence of lignin. It was highlighted that,<br />
microbial degradation of lignin had potential advantages<br />
over the prevailing chemical degradation, due to energy<br />
and environmental concerns (Keller et al., 2003). However,<br />
degrading lignocellulosic biomass by current<br />
available microorganism is still far to meet the industrial<br />
*Corresponding author. E-mail: ghj@sdau.edu.cn. Tel: (86)538-<br />
8249561. Fax: (86)538-8249164.<br />
Abbreviations: LiP, Lignin peroxidise; MnP, manganese<br />
peroxidase; PDA, potato dextrose agar; SNA, synthetic nutrient<br />
agar medium; OA, oatmeal; CMA, cornmeal; MEA, malt extract;<br />
PDA, potato dextrose; ITS, internal transcribed spacer; PCR,<br />
polymerase chain reaction; BLAST, Basic Local Alignment<br />
Search Tool; NJ, Neighbors joining; RBBR, Remazol Brilliant<br />
Blue R; LiP, lignin peroxidise; MnP, manganese peroxidise.<br />
demands (Shary et al., 2007; Afrida et al., 2009). Thereby,<br />
isolation of new strains for degradation of lignin is still<br />
essential.<br />
Extracellular oxidoreductases, including lignin peroxidase<br />
(LiP), manganese peroxidase (MnP) and laccase<br />
produced by wood-decomposing microorganism, are<br />
directly involved in the degradation of lignin in their<br />
natural lignocellulosic substrates and various xenobiotic<br />
compounds including dyes (Abrahão et al., 2008;<br />
Thurston, 1994; Call and Mücke, 1997; Leonowicz et al.,<br />
2001). Some wood-degrading fungi contain all three<br />
classes of lignin-modifying enzymes, while the others<br />
contain only one or two of these enzymes (Hatakka,<br />
1994; Dhouib et al., 2005). The production of ligninolytic<br />
enzymes is observed as a colourless halo around<br />
microbial growth (Dhouib et al., 2005). So, the lignin<br />
degradation capability of microbes is initially screened<br />
indirectly on solid media containing different indicator<br />
compounds.<br />
Although, wood decay fungi are primarily basidiomycetes,<br />
other microorganisms are also involved in the<br />
lignocellulosic decaying processes (Nilsson et al., 1989).<br />
Wood biodegradation by certain ascomycetes was first<br />
described in detail and designated as “soft rot” by Savory<br />
(1954). Nilsson et al. (1989) demonstrated that, some<br />
higher ascomycetes, particularly Daldinia concentrica,
degraded Aspen wood with the same intensity as<br />
Trametes versicolor, a basidiomycete typically classified<br />
as white-rot fungus. The ascomycete, Chrysonilia sitophila<br />
could degrade rice hull and Pinus radiata bark products<br />
and also produce ligninolytic and cellulolytic enzymes<br />
(Ferraz et al., 1991). Whereafter, different Penicillium<br />
strains was described as potential degraders or was able<br />
to degrade compounds with related lignin structures<br />
(Rodriguez et al., 1994). The family Xylariaceae had<br />
been reported to cause a special type of extensive woodrot,<br />
which could produce laccase and low amounts of<br />
peroxidase as potential ligninolytic enzymes along with<br />
different polysaccharide-cleaving hydrolases as well as<br />
esterase (Liers et al., 2006).<br />
Ascomycetous fungi with coniothyrium-like anamorphs<br />
are common colonizers of woody host plants (Damm et<br />
al., 2008). Nilsson (1973) found that, Coniothyrium<br />
cerealis could degrade wood. A new woody plants hosted<br />
genus Paraconiothyrium was established by Verkley et<br />
al. (2004), which included the old Coniothyrium minitans<br />
and Coniothyrium sporulosum. Later species were frequently<br />
isolated from wood and leaves of Prunus,<br />
Actinidia, Laurus and Dendrobium. Recently, two<br />
additional new species, Paraconiothyrium africanum and<br />
Paraconiothyrium variabile, were identified based on their<br />
DNA sequence data and unique morphological characteristics<br />
(Damm et al., 2008).<br />
In this study, an ascomycete producing ligninolytic<br />
enzyme, forming coniothyrium-like anamorphs, was<br />
isolated from decayed wood of Salix matsudana Koidz in<br />
Mount Tai. It was a new strain with lignin-transformation<br />
capability. The fungus was morphologically and phylogenetically<br />
identified and its ability for lignin degradation<br />
was characterized.<br />
MATERIALS AND METHODS<br />
Isolation of ligninolytic enzyme producing fungi GHJ-4 strain<br />
Different indicator compounds were added to potato dextrose agar<br />
(PDA) in order to detect ligninolytic enzyme production. These<br />
compounds included 0.04% Remazol Brilliant Blue R (RBBR),<br />
0.04% guaiacol and 0.02% tannic acid (Dhouib et al., 2005). The<br />
plates were incubated at 28°C for at least a week under dark.<br />
Positive strains were subcultured when clear positive reactions<br />
were visible.<br />
Morphology<br />
To enhance sporulation, autoclaved wood sawdust was placed onto<br />
the surface of synthetic nutrient agar (SNA) medium (Kwaśna and<br />
Bateman, 2007) and incubated at 25°C for 1 to 4 weeks under<br />
natural light. Measurements, photographs of characteristic structures<br />
and vertical sections through conidiomata were made<br />
according to Damm et al. (2007). Cultural characteristics and radial<br />
growth rates were determined on oatmeal (OA), cornmeal (CMA),<br />
3% malt extract (MEA) and PDA agars (Damm et al., 2008). Plates<br />
were incubated in a growth chamber at 25°C under natural light.<br />
Growth characteristics were studied on MEA plates incubated in the<br />
Gao et al. 4167<br />
dark at temperatures ranging from 5 to 35°C, with 5°C intervals.<br />
DNA isolation, amplification and analyses<br />
Genomic DNA of GHJ-4 strain was extracted from fungal mycelium<br />
grown on PDA plates following previously described protocols<br />
(Pryor and Gilbertson, 2000). The internal transcribed spacer (ITS)<br />
regions of 5.8S rDNA gene was amplified and sequenced using<br />
primer ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-<br />
TCCTCCGCTTATTGATATGC-3'). Polymerase chain reaction (PCR)<br />
was performed by denaturation for 5 min at 95°C followed by 35<br />
cycles of 1 min at 94°C for 1 min at 56°C and 1 min at 72°C, with a<br />
final extension of 10 min at 72°C. The PCR products with the<br />
expected size (about 600 bp) were cut from gel and purified by DNA<br />
gel extraction kit (TaKaRa, Japan), cloned into pEASY-T3 vector<br />
(TransGen, China) and transformed into Escherichia coli competent<br />
cells DH5α. Sequences were performed with an AB13730XL DNA<br />
autosequencer (Applied Biosystems). Sequence homolog analysis<br />
was performed by Basic Local Alignment Search Tool (BLAST)<br />
(http://www.ncbi.nlm.nih.gov). Fungi identification was based on<br />
5.8S rDNA gene sequence similarity to BLAST hits. Neighbors<br />
joining (NJ) phylogenetic trees were generated based on partially<br />
sequenced 5.8S rDNA and closely related sequences. In the NJ<br />
analyses, the Kimura distance calculation was used. A bootstrap<br />
analysis (1000 replicates using NJ option) was performed to<br />
determine the confidence levels of the nodes.<br />
Cultivations in liquid media<br />
GHJ-4 strain was cultivated on a basic media (Media V) for<br />
production of laccase on 200 rpm at 28°C. Media V contained 15.0<br />
g sucrose and 200.0 g potato extract per liter. Subsequently, it was<br />
cultivated in four types of rich media to improve laccase production.<br />
Media I contained (g/l): 3.0 soluble starch, 2.0 yeast extract, 0.17<br />
KH2PO4, 0.44 MgSO4, 0.37 CaCl2, 0.5 Tween-80 ; media II contained<br />
(g/l): 200.0 potato extract, 25.0 glucose, 3.0 yeast extract,<br />
3.0 KH2PO4, 2.5 MgSO4, 0.1 vitamin B1, pH 6.0; Media III contained<br />
(g/l): 24.0 soluble starch, 24.0 glucose, 10 bran, 3.2 KH2PO4, 0.2<br />
MgSO4, 0.006 CuSO4, 3.2 NH4Cl, pH6.5; Media IV contained (g/l):<br />
30 bran, 2.0 peptone, 3.0 beef extract, 3.0 KH2PO4, 0.5 MgSO4,<br />
0.02 Vitamin B1, 0.01 CaCl2, 0.1 NaCl.<br />
LiP and MnP production by GHJ-4 strain was followed for 14<br />
days. The fungus was inoculated into 250 ml Erlenmeyer flasks<br />
containing 50 ml medium which involve 30.0 g bran, 58.0 g soluble<br />
starch, 3.5 g beef extract, 1.0 g NaNO3, 3.0 g KH2PO4 and 0.1 g<br />
MnSO4.<br />
Laccase activity assay<br />
Laccase activity was measured spectrophotometrically with guaiacol<br />
as substrate. A modified method of Chakraborty et al. (2000)<br />
was used. An aliquot of enzyme solution was incubated in 4.0 ml of<br />
50 mM sodium acetate buffer (pH 4.5) containing 1.0 mM guaiacol<br />
at 30°C. The changes in absorbance due to oxidation of guaiacol in<br />
the reaction mixture was monitored at 465 nm (ε465=12,100 M -1 cm -<br />
1 ). One unit of enzyme activity was defined as the amount of<br />
enzyme that oxidized 1 µmol guaiacol per minute under the<br />
aforementioned condition.<br />
Lignin peroxidase activity assay<br />
The lignin peroxidase activity was evaluated by UV spectrometry of<br />
the veratraldehyde produced ( TM 310 =9,300 M −1 cm −1 ) during veratryl<br />
alcohol oxidation (Moldes et al., 2003). The reactive mixture was
4168 Afr. J. Biotechnol.<br />
composed of 2.5 ml sodium tartrate buffer (100 mM, pH 3.0), 1.0 ml<br />
veratryl alcohol (10 mM), 100 µl hydrogen peroxide (10 mM) and<br />
400 µl enzyme extract. The reaction was started by adding<br />
hydrogen peroxide and the veratraldehyde was determined at 310<br />
nm. One enzyme unit was defined as 1 µmol product formed per<br />
minute under the assay conditions.<br />
Manganese peroxidase activity assay<br />
Manganese peroxidase activity was assayed following the method<br />
of Moldes et al. (2003). The reaction mixture contained 3.4 ml<br />
sodium malonate (50 mM pH 4.5), 0.1 ml MnSO4 (15 mM) and 400<br />
µl enzyme extract. The reaction was started by adding 0.1 ml H2O2<br />
(10 mM) and the produced Mn 3+ complexes was determined at 240<br />
nm (ε240=6,500 M -1 cm -1 ). One activity unit was defined as the<br />
amount of enzyme that oxidized 1 µmol MnSO4 per minute and the<br />
activities were expressed in U ml -1 .<br />
Characterization of ligninolytic potential<br />
GHJ-4 strain was incubated in 250 ml Erlenmeyer flasks containing<br />
25 ml minimal salts medium, which involves 2.0 g NH4Cl, 0.5 g<br />
MgSO4, 1.0 g KH2PO4, 0.007 g CuSO4, 0.2 g Na2HPO4, 0.035 g<br />
MnSO4, 0.007 g FeSO4 per liter and lignin (Sigma-Aldrich) and<br />
guaiacol as sole carbon source. The flasks were inoculated with 2<br />
pieces (1.0 cm diameter) of 7-day precultured agar plates and the<br />
cultures maintained under stationary at 28°C for 15 days. Growth of<br />
the fungus and indication of the lignin degradation capability was<br />
measured based on the dry weight of the mycelia after filtration,<br />
followed by drying at 80°C to constant weight.<br />
Detection of decolourization ability<br />
For dye decolourization studies, GHJ-4 strain was incubated on<br />
PDA plate supplemented with 0.02% RBBR, aniline blue and<br />
phenol red at 28°C (Rodriguez et al., 1996). Decolourization was<br />
observed after 15 days.<br />
Wood biodegradation by GHJ-4 strain<br />
Wood chips (2.0×2.0×0.1 cm) were obtained from Populus bonatii<br />
Levl (10-year old). Wood biodegradation was performed under<br />
solid-state fermentation at 28°C for 40 days. Inoculum was<br />
prepared as follows: 3 pieces (1.0 cm diameter) of 7-day precultured<br />
agar plates were inoculated into a 250 ml Erlenmeyer flask<br />
containing 5% Wood chips, 15% bran and 80% nutrient solution<br />
that was composed of 2.0 g NH4Cl, 0.5 g MgSO4, 1.0 g KH2PO4,<br />
0.007 g CuSO4, 0.2 g Na2HPO4, 0.035 g MnSO4, 0.007 g FeSO4<br />
and 1,000 ml of water. After biodegradation, bio-treated wood chips<br />
were washed to remove the mycelium grown on wood surfaces and<br />
then dried to constant weight at 105°C. Initial and final dry weights<br />
were used to determine weight losses. A set of uninoculated<br />
sterilized wood chips served as a control wood sample.<br />
Decayed and non-decayed wood samples were milled in a knife<br />
mill to pass through a 0.5 mm screen. Klason insoluble and soluble<br />
lignins in benzene/ethanol extracted milled wood samples were<br />
determined by acid hydrolysis as described by Ferraz et al. (2000).<br />
RESULTS<br />
Isolation of GHJ-4 strain<br />
Reactions with three different indicators, guaiacol,<br />
Remazol Brilliant Blue R (RBBR) and tannic acid, were<br />
tested with collected samples. The positive reactions with<br />
guaiacol, RBBR and tannic acid correlated well with each<br />
other. Altogether, 20 fungal strains showing positive<br />
reactions on indicator plates were isolated from decayed<br />
wood of S. matsudana Koidz in Mount Tai. Fungal strain<br />
GHJ-4 was selected for further studies, as the oxidative<br />
polymerization of guaiacol to form reddish brown zones in<br />
the medium was higher than other isolated strains. Its<br />
lignin degradation capability was further confirmed by<br />
other indicators (Figure 1).<br />
Phylogenetic analysis<br />
An expected 589 bp DNA fragment of the 5.8S rDNA/ITS<br />
region was obtained from the GHJ-4 strain by PCR amplification.<br />
The sequence was deposited in the GenBank<br />
database (Accession no. GQ331986). BLAST analysis of<br />
the amplificons indicated that, it had the highest<br />
similarities (over 99%) sequences generated from<br />
Paraconiothyrium or Microdiplodia strains. To clarify the<br />
phylogenetic position of GHJ-4, a phylogenetic tree was<br />
constructed based on the 5.8S rDNA sequences (Figure<br />
2). The result revealed that GHJ-4 strain, P. variabile and<br />
Paraconiothyrium brasiliense formed one cluster, which<br />
implied they were closely related.<br />
Taxonomy<br />
The strain GHJ-4, obtained from wood of S. matsudana<br />
Koidz, could be assigned to P. variabile based on the<br />
DNA sequence data and its morphology (Damm et al.,<br />
2008). Its conidiomata were pycnidia and the shape of<br />
the conidiogenous cells was more variable. Conidia were<br />
smaller than those of most other Paraconiothyrium<br />
species and grew more slowly than P. brasiliense.<br />
Conidiomata pycnidial, produced on wood sawdust on<br />
SNA in 1 to 4 weeks was solitary, subglobose, 1 to 3<br />
ostioles, black, superficial or semi-immersed, 200 to 600<br />
µm diameter, wall consisting of 6 to 8 cell-layers (30 to 45<br />
µm) of thick-walled dark brown textura angularis, becoming<br />
hyaline and thin-walled towards the inside of the<br />
pycnidium, that was surrounded by brown hyphal<br />
appendages. Conidiophore, lining the inner conidiomatal<br />
cavity, was hyaline, branched with conspicuous umbilici,<br />
3 to 10 × 2 to 5 µm. Conidiogenous cells varied in shape,<br />
conical to subulate or subcylindrical, broadly or elongated<br />
ampulliform, phialidic with periclinical wall thickening or<br />
with one or more percurrent proliferations near the apex.<br />
Conidia was pale brown or light green, subcylindrical to<br />
ellipsoidal, both ends obtuse, 1-celled, smooth walled, 3<br />
to 4.5 × 1.5 to 2.5 µm. Vegetative hyphae were 2 to 5 µm<br />
wide, hyaline to pale brown, septate and smooth. On<br />
PDA medium, parts of the hyaline vegetative hyphae was<br />
transformed to very dark-walled hyphal pieces, which<br />
could become locally swollen or accumulate amorphous
Figure 1. Reactions of GHJ-4 strain with different indicators on PDA plates. A, Reddish brown zones of guaiacol; B,<br />
decolourize of RBBR; C, yellow zones of tannic acid; D, decolourize of aniline blue; E, decolourize of phenol red.<br />
Figure 2. 5.8S rDNA/ITS region phylogenetic tree of GHJ-4 strain obtained by the<br />
neighbor-joining method based on the two-parameter Kimura correction of<br />
evolutionary distances. Numbers at the nodes are the bootstrap confidence values<br />
obtained after 1000 replicates. Camarosporium leucadendri was used as the outgroup.<br />
Gao et al. 4169
4170 Afr. J. Biotechnol.<br />
5 µm D<br />
A B C<br />
E F<br />
Figure 3. Morphology of GHJ-4 strain. A, Dark walled hyphae; B, conidia oozing from pycnidia; C, longitudinal section through a<br />
pycnidium; D, pycnidial wall with conidiophorese; E, conidiophores; F, conidia.<br />
brown material on the outer wall surface (Figure 3).<br />
Cultural characteristics<br />
Colonies on OA reached 53.2 mm after 7 days and 85<br />
mm after 14 days (25°C, in diffuse daylight). It was flat,<br />
with an even to slightly ruffled colourless and glabrous<br />
margin, mycelium whitish, aerial mycelium absent or<br />
consisting of sparse, scattered white to grayish tufts.<br />
Colonies on MEA reached 42.6 mm diameter in 7 days<br />
and 68.5 mm in 14 days, with an even to slightly ruffled,<br />
glabrous and colourless margin, colony surface almost<br />
entirely covered by a dense mat of woolly aerial<br />
mycelium, which was pale olivaceous-grey, in the centre<br />
olivaceous-black and near the margin paler to almost<br />
pure white. Colonies on PDA reached 54.7 mm diameter<br />
in 7 days, 82.8 mm diameter in 14 days. It was flat, with<br />
an even, whitish margin, most of the colony surface<br />
covered by felty floccose aerial mycelium, in the centre<br />
becoming olivaceous-grayish. Colonies on CMA reached<br />
85 mm diameter in 14 days (46 mm in 7 days), as on<br />
PDA. It was flat, white, in the centre becoming olivaceousgrayish<br />
(Figure 4).<br />
Growth characteristics<br />
The growth temperature range of GHJ-4 was 5 to 35°C<br />
and the optimum growth temperature was approximately<br />
25°C.<br />
Production of ligninolytic enzyme by GHJ-4 strain<br />
Laccase production by GHJ-4 strain was studied in liquid<br />
cultures and proved to be highly dependent on the<br />
medium. The production level of laccase in liquid cultures<br />
was quite low in basic media. Laccase production was<br />
enhanced by the addition of bran. Among the five media<br />
tested in this study, media IV had the highest laccase<br />
production (Figure 5). The production of laccase activity<br />
occurred on day 2 and reached its maximum (484.72<br />
U/ml) on day 10 and then declined gradually.<br />
Furthermore, GHJ-4 also produced LiP and MnP. LiP<br />
activity first appeared on the 2 nd day (0.31 U/ml) and<br />
reached its highest level of activity (7.28 U/ml) on the 12 th<br />
day, after which the activity started to decrease (Figure<br />
6). As regards MnP activity, it began on the 4 th day (0.73<br />
U/ml) and then it increased peaked on the 8 th day of
Figure 4. Cultural characteristics of GHJ-4 strain incubated on CMA (A), PDA (B), OA (C)<br />
and MEA (D) (25°C, natural light).<br />
-1)<br />
Enzyme activity (U ml -1 )<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0<br />
0 2 4 6 8 10 12<br />
Time(day)<br />
MediaⅠ Media Ⅱ Media Ⅲ<br />
Media Ⅳ Media Ⅴ<br />
Figure 5. Time course of extracellular laccase activity of GHJ-4 on different media.<br />
Gao et al. 4171
4172 Afr. J. Biotechnol.<br />
Enzyme activity(U ml<br />
-1 )<br />
cultivation (3.81 U/ml) (Figure 7).<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
0 2 4 6 8 10 12 14<br />
Time(day)<br />
Figure 6. Time course for LiP activity produced by GHJ-4.<br />
Lignin model compounds utilization capability<br />
The ability of GHJ-4 strain to utilize guaiacol or lignin as<br />
the sole carbon and energy source is shown in Table 1.<br />
Results showed that lignin and guaiacol, particularly<br />
lignin, could be utilized by GHJ-4 strain. So, GHJ-4 was<br />
able to utilize some lignin model substrates.<br />
Dye decolourization ability of GHJ-4<br />
The polymeric dye RBBR, an anthracene derivative, was<br />
used as a starting material in dye production and represented<br />
an important class of often toxic and recalcitrant<br />
organopollutants. Results showed that, GHJ-4 could<br />
readily decolorize RBBR (Figure 1). We had also found<br />
the decolourization of another two polymeric dyes, aniline<br />
blue and phenol red, by GHJ-4 in solid culture (Figure 1).<br />
Biodegradation capacity for wood<br />
The decay capacity of GHJ-4 strain for wood blocks was<br />
examined. Results showed that, GHJ-4 had 20.91%<br />
losses of wood weight and approximately 22.99% lignin<br />
loss after 40 days incubation period. So, GHJ-4 strain<br />
could be considered as an efficient wood degrader.<br />
DISCUSSION<br />
P. variabile was frequently isolated from different plant<br />
species worldwide. Riccioni et al. (2007) isolated it from<br />
wood of Actinidia chinensis and Actinidia deliciosa in<br />
association with trunk or vine disorders and from necrotic<br />
wood under pruning cut surfaces. There was also a<br />
report on the isolation of this species from leaves of<br />
Laurus nobilis (Göre and Bucak, 2007) and branches of<br />
Prunus persica and Prunus salicina with dieback symptoms<br />
or pruning debris (Damm et al., 2008). However,<br />
there is no isolation of P. variabile having been reported<br />
in China. Our present study represents the first report of<br />
isolation of this species. This indicated its broad host<br />
spectrum, including several distantly related host plants<br />
and a wide geographical distribution.<br />
The basidiomycetous white-rot fungi and related litterdecomposing<br />
fungi are the most efficient lignin-degrading<br />
organisms in nature (Fackler et al., 2006; Ohkuma et al.,<br />
2001). However, the degradation of lignocellulose by<br />
ascomycetes is an important route for carbon cycling in<br />
plant litter and soils (Nilsson and Daniel, 1989; Liers et
Enzyme activity(U ml<br />
-1 )<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
0 2 4 6 8 10 12 14<br />
Time(day)<br />
Figure 7. Time course for MnP activity produced by GHJ-4.<br />
Table 1. Growth of GHJ-4 on different medium.<br />
Carbon source<br />
3 days<br />
Dry weight of mycelial (mg)<br />
6 days 9 days 12 days 15 days<br />
Lignin 2.2 4.9 10.0 16.9 25.5<br />
Guaiacol 0.5 1.2 6.4 8.8 12.3<br />
al., 2006). Our results showed that, the newly isolated<br />
ascomycete GHJ-4 could produce extracellular oxidoreductases<br />
including laccase, lignin peroxidase (LiP) and<br />
manganese peroxidase (MnP), which is the first record of<br />
P. variabile producing ligninolytic enzyme. Furthermore,<br />
laccase production by GHJ-4 was largely affected by<br />
culture conditions, particularly bran, which was in<br />
agreement with the reported results (Lee et al., 1999;<br />
Schlosser et al., 1997; Dong et al., 2005). Chen et al.<br />
(1982) had clearly demonstrated that, part of lignin<br />
degradation proceeds via low-molecular-weight aromatic<br />
acids and the dye serves as a substrate for fungal enzymes<br />
possibly related to lignin degradation (Glenn and<br />
Gold, 1983). Our results showed that, the newly isolated<br />
P. variabile GHJ-4 strain was capable of utilizing several<br />
lignin model compounds and decoloration of aromatic<br />
dyes, which provided additional evidence of degrading<br />
lignin of GHJ-4. In addition, this finding proved that GHJ-<br />
4 was able to degrade natural lignin.<br />
In conclusion, our results showed that this new isolated<br />
P. variabile GHJ-4 had a certain lignin degradation<br />
capacity and provided a new microorganism resource for<br />
Gao et al. 4173<br />
lignin degradation. The ability of this fungi strain could be<br />
of interest for lignocellulosic biomass degradation as well<br />
as treatments of pulp and paper mill effluent. However, it<br />
remains to be studied concerning the role of every<br />
ligninolytic enzyme of GHJ-4 strain on lignin biodegradation<br />
and how lignin is degraded by it. Furthermore,<br />
it would be more informative to compare the ligninolytic<br />
capabilities of this fungus with other lignin-degrading<br />
ascomycetes and basidiomycetes and further research<br />
concerning the role of ascomycetes on lignin biodegradation<br />
should be carried out using P. variabile GHJ-4<br />
and other microorganisms of this class.<br />
ACKNOWLEDGEMENT<br />
This work was funded by Natural Science Foundation of<br />
Shandong province, China (Grant No. Q2008D08).<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4175-4182, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2285<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Improved taxol production in Nodulisporium sylviforme<br />
derived from inactivated protoplast fusion<br />
Kai Zhao 1,2 , Lixin Sun 3 , Xi Ma 1 , Xiuliang Li 1 , Xin Wang 1 , Wenxiang Ping 1 and Dongpo Zhou 1 *<br />
1 Key Laboratory of Microbiology, College of Life Science, Heilongjiang University, 74 Xuefu Road, Harbin 150080, China.<br />
2 Biotechnology Research Center, Heilongjiang Academy of Agricultural Sciences, 368 Xuefu Road, Harbin 150086,<br />
China.<br />
3 Hospital of Heilongjiang University, Harbin 15008.<br />
Accepted 6 April, 2011<br />
Inactivated protoplast fusion by UV irradiation and UV+LiCl mutation was conducted using<br />
Nodulisporium sylviforme strain UV40-19 and UL50-6 to breed a high taxol-producing fungus. Qualitative<br />
and quantitative analysis of taxol production was confirmed using thin-layer chromatography, high<br />
performance liquid chromatography and mass spectrometry. The protoplasts of UV40-19 and UL50-6 were<br />
fully inactivated by heating at 54°C for 5 min and by UV irradiation (30 w UV light and vertical distance 30<br />
cm) for 85 s. The highest fusion rate (14.31 ± 1.13%) between UV40-19 and UL50-6 was obtained under the<br />
conditions of 35% PEG, 90 s fusion time and the addition of 0.01 mol/l CaCl2. One high taxol<br />
production strain HDF-68 was obtained. The taxol production was up to 468.62 ± 37.49 �g/l, which was<br />
increased by 24.51 and 19.35% compared with the parental strain UV40-19 and UL50-6, respectively. This<br />
study provided a good basis for the application of this technique to the breeding of the strains<br />
with high taxol output.<br />
Key words: Taxol, endophytic fungi, protoplast preparation, protoplast fusion.<br />
INTRODUCTION<br />
Taxol is a diterpenoid with anticancer activities. It was first<br />
isolated from the bark of Taxus brevifolia Nutt by Wani et<br />
al. (1971) and is still mainly extracted from the bark of<br />
yews. The action of taxol is to inhibit the depolymerization<br />
of microtubulin and disturb the function of microtubes thus,<br />
affect the formulation of spindle and inhibit tumor cell<br />
mitosis (Zhou et al., 2003). Recently, great effort has been<br />
made to develop alternative means of taxol production,<br />
including complete chemical synthesis (Holton et al.,<br />
1994a, b; Nicolaou et al., 1994), semi-synthesis<br />
(Comnercon et al., 1995; Holton et al., 1995) and the<br />
Taxus spp. plant cell culture (Christem et al., 1991; Arteca<br />
and Wickremesinhe, 1993; Ketchum et al., 1995;<br />
Furmanowa and Syklowska-Baranek, 2000; Wang at al.,<br />
2001).<br />
Using microbe fermentation to produce taxol is a<br />
*Corresponding author. E-mail: zhoudp2003@yahoo.<br />
com.cn. Tel: +86 0451 86609016. Fax: +86 0451<br />
86609016.<br />
very prospective method for obtaining a large<br />
amount of taxol. Several endophytic fungi that produce<br />
taxol have been isolated (Stierle et al., 1993; Strobel et al.,<br />
1996; Zhou et al., 2009). Since 1993, the authors have<br />
isolated five endophytic fungal species that can produce<br />
taxol by screening samples from the inner bark<br />
(phloem-cambium) and xylem of Taxus cuspidata Sieb. et<br />
Zucc. These fungi are Nodulisporium sylviforme (Zhou et<br />
al., 2001), Pleurocytospora taxi (Sun et al., 2003),<br />
Alternaria taxi (Ge et al., 2004), Botrytis (Zhao et al.,<br />
2008a) and Aspergillus niger var. taxi (Zhao et al., 2009).<br />
However, the method is still at the experimental<br />
stage due to the low yield of the isolated and bred<br />
strain, which makes industrial production difficult.<br />
Therefore, the bottleneck lies in the breeding of high<br />
yield strain (Zhou et al., 2003). Two high taxol-<br />
production mutants UV40-19 and UL50-6, were screened<br />
from N. sylviforme HQD33 by UV irradiation and UV+LiCl<br />
mutating in our lab to induce mutagenesis. The<br />
common mutagenesis may have little effect on taxol<br />
production. Alternatively, protoplast fusion can be used<br />
to produce high yield stable strain and is widely applied to
4176 Afr. J. Biotechnol.<br />
fungal breeding (Gokhale, 1992; Muralidhar and Panda,<br />
2000). Up to date, no report on the breeding of high<br />
taxol-production strain by inactivated protoplast fusion<br />
is available. In this study, we report the production<br />
of a high taxol-producing strain by inactivated<br />
protoplast fusion of two parental strains.<br />
MATERIALS AND METHODS<br />
Strains<br />
The spores from N. sylviforme HQD33 (a taxol-producing endo-<br />
phytic fungus isolated from T. cuspidata in China, CCTCC M 202049)<br />
with a taxol output of 51.06 to 125.70 µg/l were subjected to<br />
a series of mutagenesis screening (UV, EMS, 60 Co and NTG).<br />
A mutagenesis-derived strain NCEU-1 with a taxol output of<br />
314.07 μg/l was used as the primary starting strain. UV 40-19<br />
with taxol output of 376.38 µg/l and UL50-6 with taxol output<br />
of 392.63 µg/l were the mutants from strain NCEU-1<br />
protoplasts which underwent UV mutagenesis and the<br />
combined mutagenesis by UV and LiCl, respectively (Zhao<br />
and Zhou, 2004). UV40-19 and UL50-6 were used as parental strains in<br />
this study for inactivated protoplast fusion.<br />
Media<br />
Potato dextrose agar (PDA) medium and potato dextrose liquid<br />
medium (Shen et al., 1999) were used as the growing medium. For<br />
the regeneration of protoplasts, solid regeneration medium (PDA<br />
medium containing 0.7 mol/l NaCl) and semi-solid regeneration<br />
medium (reducing the content of agar in solid regeneration medium<br />
to 6 to 8 g/l) were used as hypertonic media. S-7 medium (Stierle<br />
et al., 1993) with the addition of tyrosine, linoltic acid and<br />
phenylalanine at a final concentration of 1.5 to 5.0 mg/l was<br />
used as the fermentation medium.<br />
Lytic enzyme solution<br />
Lytic enzyme solution was prepared by dissolving 30 mg/ml<br />
lywallzyme (Institute of Microbiology of Guangdong, Guangzhou,<br />
China), 20 mg/ml snailase (Beijing BioTech Co., China) and 10<br />
mg/ml lysozyme (Institute of Biochemistry, Academy of China) in 0.7<br />
mol/l NaCl, followed by clarifying the solution by centrifuged at 4000<br />
r/min at 4°C for 15 min. The supernatants were collected by filtration<br />
and the pH was adjusted to 5.5 to 6.0.<br />
Osmotic stabilizer and fusogen<br />
NaCl at 0.7 mol/l was used as the osmotic stabilizer. Polyethylene<br />
glycol (PEG, MW 6000) was dissolved in the osmotic stabilizer to<br />
yield final concentration of 25, 30, 35and 40% (w/v), respectively.<br />
The PEG solutions containing 0.01 mol/l CaCl2 and 0.05 mol/l<br />
glycine were used as fusogen.<br />
Preparation and regeneration of protoplasts<br />
Preparation and regeneration of protoplasts were carried out<br />
as previously described (Zhao and Zhou, 2004).<br />
Inactivation and fusion of protoplasts<br />
Protoplasts of strain UV40-19 were inactivated by heating at 54°C for<br />
5 min. Protoplasts of strain UL50-6 were inactivated by UV irradiation<br />
(30 w UV light and vertical distance 30 cm) for 85 s. Two<br />
inactivated protoplast suspensions were selected<br />
randomly with a concentration of 1.0×10 6 protoplasts/ml. 1<br />
ml of each was mixed and centrifuged at 3000 r/min for 10<br />
min. The protoplasts were collected and suspended in 0.2 ml<br />
osmotic stabilizer, then 1.8 ml different concentrations of<br />
PEG preheated at 30°C was added to the suspensions. The<br />
mixed liquid was treated at a 30°C water bath for different<br />
periods, then 5 ml osmotic stabilizer at 4°C were added to<br />
stop the fusion, followed by washing and centrifugation to<br />
discard the fusion agent. The washed protoplasts were<br />
resuspended into osmotic stabilizers and regenerated by<br />
double-layer culture for 3 to 5 days at 28°C (Zhao et al., 2008b).<br />
The fusion rate was calculated based on the regeneration of<br />
syzygies and inactivated parents.<br />
The fusion time was set at 60 s with PEG concentrations at<br />
25, 30, 35 and 40%. Then, the fusion time was set at 30, 60,<br />
90 and 120 s under the optimal PEG concentration. In<br />
addition, the effect of Ca 2+ (0.01 mol/l CaCl2) in the fusion<br />
reagents on the fusion rate of the protoplasts was estimated.<br />
The fusion rate of the protoplasts was calculated as follows<br />
(Zhao et al., 2008b):<br />
a= ((b-c)/d) ×100%<br />
Where, a, refers to the fusion rate of the protoplasts; b refers<br />
to the number of colonies on the regeneration plate; c refers<br />
to the number of colonies on the inactivated parent<br />
regeneration plate; d refers to the number of parent colonies<br />
on regeneration plate.<br />
Screening of fusants with high taxol yield<br />
The obtained fusants with good mycelia growth and spore-<br />
production abilities were successively transferred and cultured.<br />
They were transferred onto plates containing 135 μg/ml Nystatin and<br />
cultured at 28°C for 3 days (Zhao et al., 2005). Single colony<br />
growing fast on the plates with large diameter was selected as<br />
resistant strains through primary screening which were used for the<br />
screening of high-output strains. Other colonies were discarded.<br />
The fusants obtained by primary screening were activated on PDA<br />
slope culture at 28°C, then transferred into 50 ml PDA liquid medium<br />
in 250 ml flask and cultured at 28°C for 3 days. The products were<br />
inoculated into modified S-7 culture at the concentration of 3% (v/v)<br />
and fermented at 28°C and 150 r/min for 12 days. At the end of<br />
fermentation, the filtrate and mycelium were collected. The filtrate<br />
was extracted twice using acetic ether, one hour each time and<br />
supernatants were collected. The mycelium was whetted fully and<br />
extracted using 30 ml acetic ether for 1 h. The organic phase was<br />
combined and then, distilled to remove the organic solvent.<br />
Purification by column chromatography<br />
The silica gel used for 60 to 100 um column chromatography<br />
was dipped in CHCl3 overnight and packed (15 × 260 mm) by<br />
gravity settling. The impurities were washed out from silica<br />
gel thoroughly using CHCl 3. The sample was dissolved into<br />
the CHCl3 and loaded. The unabsorbed impurities were<br />
washed out using CHCl3. Thereafter, methanol: CHCl3 (3:97,<br />
v/v) was used for elution and the peaks were collected. All<br />
the process was carried out at room temperature.<br />
Thin-layer chromatography (TLC) analysis<br />
The chloroform-methanol (7:1, v/v) was used as developer
Regeneration rate of protoplasts (%)<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
1 2 3 4 5 6 7 8 9 10 11 12<br />
Heat inactivation time (min)<br />
Figure 1. Effect of heat inactivation time and temperature on<br />
regeneration rate of strain UV40-19 protoplasts.<br />
and taxol standard (Sigma Ltd) was used as the control. 1%<br />
vanillin-concentrated sulfuric acid was used as<br />
chromatography agent. After TLC developing,<br />
chromategenic agent was sprayed and dried at 90 to 105°C<br />
for chromatogenic reaction.<br />
High performance liquid chromatography (HPLC) analysis<br />
Using taxol standard as control, the corresponding TLC area of 1<br />
cm 2 of the sample was scratched and dissolved using 1 ml methanol<br />
and eluted using ultra sonication. The eluate was filtered. A Waters<br />
Millennium32 HPLC workstation equipped with a photodiode array<br />
detector was used for quantitative analysis. An aliquot of taxol<br />
extract (10 μl) was lyophilized and dissolved in 1% methanol and<br />
was injected onto a 250 × 4.6 mm Taxsil-3 C18 reverse phase<br />
column. The mobile phase was a mixture of methanol-water (60:40)<br />
and the flow-rate was set at 1.0 ml/min. Taxol in the eluent was<br />
detected by measuring the absorbance at 227 nm where taxol has<br />
the maximum absorbance. Qualification was achieved using the<br />
standard curve generated from the taxol standard over a<br />
concentration range of 0.05 to 1.00 mg/ml at which the peak area<br />
showed linear relationships with the absorbance (r = 0.9988).<br />
Mass spectrometry analysis<br />
The HPLC peak with the same retention time as the standard<br />
was collected and dried. The structure of the extracted taxol was<br />
confirmed with a waters triple quadrupole tandem LC-MS system<br />
(Waters, MA, USA). The HPLC portion was run isocratically with<br />
acetonitrile: water (49:51) as mobile the phase. The sample was<br />
loaded onto a 250 × 4.6 mm Taxsil-3 C18 reverse phase column<br />
(Metachem, Co. Ltd) and separated at a flow rate of 0.8 ml/min with<br />
the column temperature at 35°C. The MS scanning ranged from 100<br />
to 1000 m/z and the shell gas (N2) and assistant gas (N2) were 65<br />
international units (IU) and 20 IU, respectively. The discharge<br />
current was 5 µA. The evaporator and capillary temperatures were<br />
465 and 180°C, respectively.<br />
Measurement of hereditary stability of fusants<br />
Fusants with high taxol output were cultured for ten gene-<br />
Zhao et al. 4177<br />
44°C<br />
52°C<br />
54°C<br />
rations. Strains with good hereditary stability and high yield<br />
of taxol were stored.<br />
RESULTS<br />
Preparation, inactivation and fusion of protoplasts<br />
The yield of N. sylviforme UV40-19 and UL50-6 protoplasts<br />
was up to 1.26 × 10 and 1.82 × 10 7 /ml, respectively.<br />
Regeneration tests of inactivated protoplasts of both<br />
strains showed that no colony grew on the regeneration<br />
medium (Figures 1 and 2), demonstrating that the<br />
inactivation process was successful.<br />
The protoplast fusion rate at different PEG<br />
concentration and different fusion time was shown in<br />
Tables 1 and 2, respectively. It can be seen that both PEG<br />
concentration and fusion time had great effects on the<br />
protoplast fusion. The optimal PEG concentration was<br />
35% and the optimal fusion time was 90 s.<br />
The addition of Ca 2+ into the fusion agent can<br />
improve the fusion rate of the protoplasts signifi-<br />
cantly. Figure 3 showed that under conditions of 90<br />
s and 35% PEG, the addition of 0.01 mol/l CaCl2<br />
increased the fusion rate of the protoplasts, from<br />
10.92 ± 0.76 to 14.31 ± 1.13%.<br />
Screening of fusants with high taxol output<br />
Isolated fusants were transferred onto the PDA medium<br />
and incubated at 28°C for 2 to 3 days. There were great<br />
differences on the morphological characters and growth<br />
rate among fusants. The fusants growing faster were<br />
selected out and employed to fermentation and hereditary<br />
stability tests. From thirty five fusants, one high yield<br />
fusant HDF-68 was obtained. Morphological comparisons<br />
between parents and HDF-68 were shown in Table 3.
4178 Afr. J. Biotechnol.<br />
Regeneration rate of protoplasts (%)<br />
45<br />
40<br />
35<br />
30<br />
25<br />
20<br />
15<br />
10<br />
5<br />
0<br />
30 40 50 60 70 80 90 100<br />
UV irradiation time<br />
Figure 2. Effect of UV irradiation time on regeneration rate of strain UL50-6 protoplasts.<br />
Table 1. Effect of PEG concentration on fusion rate.<br />
PFG concentration (%, w/v) Fusion rate (×10 -2 )*<br />
25 1.53 ± 0.11<br />
30 4.92 ± 0.21<br />
35 9.61 ± 0.52<br />
40 6.44 ± 0.48<br />
*Data were shown as Means ± SD and mean values were based<br />
on five replicates.<br />
Clearly, the morphological characters of fusant HDF-68<br />
were not identical to either of the parents. It displayed<br />
mixed characters of both parents’ strains.<br />
Taxol production in the fusant HDF-68<br />
TLC analysis showed that the fermentation extractions<br />
from the fusant HDF-68 appeared one blue dot located at<br />
the same place as the standard taxol Rf. This test<br />
indicated that the fusant HDF-68 can produce taxol or<br />
taxol-like compounds. Extracted from fermented fungal<br />
culture, the taxol sample showed the characteristic peaks<br />
as the standard taxol in the HPLC chromatogram (Figure<br />
4a and b). After the addition of the taxol standard into the<br />
samples, the peaks from the samples increased,<br />
supporting that the purified fermentation product from the<br />
fusant HDF-68 is taxol. Based on taxol standard curve,<br />
the taxol content produced in the sample was 468.62 ±<br />
37.49 �g/l. The taxol output of the fusant HDF-68 was<br />
higher than their parent strains N. sylviforme UV40-19 and<br />
UL50-6 by 24.51 and 19.35%, respectively (p < 0.01).<br />
Waters LC-MS analysis confirmed that purified product<br />
Table 2. Effect of fusion time on fusion rate.<br />
Fusion time (s) Fusion rate (×10 -2 )*<br />
30 4.08 ± 0.26<br />
60 6.18 ± 0.28<br />
90 10.92 ± 0.76<br />
120 8.27 ± 0.41<br />
*Data were shown as Means ± SD and mean values<br />
were based on five replicates.<br />
is taxol with the expected molecular ion mass (M+H) + (m/z<br />
854.92) (Figure 5).<br />
Stability of strain HDF-68<br />
After ten generations of successive subculturing, the<br />
colony morphological characteristics of strain HDF-68<br />
were identical to those of the original fusant strain and the<br />
taxol output of successive strain of the fusant HDF-68<br />
was almost the same as the original fusant strain (Table<br />
4). The results showed that strain HDF-68 remained<br />
stable after being cultured for ten generations.<br />
DISCUSSION<br />
Effects of PEG concentration and fusion time on<br />
fusion rate<br />
PEG is the most commonly used fusogen and its con-<br />
centration has a significant effect on the rate of protoplast<br />
fusion (Gokhale, 1992). Previous studies showed that, in
Fusion rate (%)<br />
16<br />
15<br />
14<br />
13<br />
12<br />
11<br />
10<br />
9<br />
8<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
(%)<br />
A B<br />
Figure 3. Effect of Ca 2+ on fusion rate. (A) With 0.01 mol/l Ca 2+ ; (B) without Ca 2+ .<br />
Table 3. Morphological characters of parents and fusant HDF-68.<br />
Strain UV40-19 UL50-6 HDF-68<br />
Colony morph<br />
Colony diameter<br />
(PDA, 5 days)<br />
Colonies loose, hoary,<br />
reverse light yellow, margin<br />
irregular, mycelium effuse<br />
Colonies dense, hoary,<br />
reverse light brown, margin<br />
regular, mycelium effuse<br />
5.8-6.7 cm 5.4-6.2 cm 6.1-6.2 cm<br />
Zhao et al. 4179<br />
Colonies dense, hoary,<br />
reverse light yellow,<br />
margin irregular,<br />
mycelium effuse<br />
Spore volume* 33.68 ± 7.58 �m 3 29.45 ± 12.77 �m 3 36.95 ± 6.36 �m 3<br />
*Spore volume was calculated by the equation: V = 4/3·π·a/2·(b/2) 2 . a: length of long axis; b: length of minor axis. Data were<br />
shown as Means ± SD and mean values were based on ten replicates.<br />
case of fungi, 25 to 40% PEG had the optimal effect on<br />
improving fusion rate (Zhou and Ping, 1990). In this study,<br />
35% PEG was the optimal concentration for protoplast<br />
fusion. Compared with the concentration of fusogen,<br />
fusion time has less influence on the fusion rate<br />
(Wesseling, 1982; Zhou and Ping, 1990). Only short<br />
period (about 1 min) of exposure to PEG was enough to<br />
induce protoplast fusion and with the prolonged fusion<br />
time, the fusion frequency did not increase accordingly<br />
(Hopwood and Wright, 1979; Baltz and Matsushima,<br />
1981). The results of this study showed that, over the<br />
range of 30 to 90 s, with the increasing of fusion time, the<br />
fusion rate increased. The optimal fusion time for N.<br />
sylviforme was 90 s. Further prolonging the fusion time<br />
will reduce the fusion rate. The optimal fusion time varies<br />
with the diffe- rent species.<br />
Production of HDF-68 using inactivated protoplast<br />
fusion<br />
In this study, the inactivation rates of both parents were<br />
100%, so all the colonies growing on the regeneration<br />
medium should be fusants, which made the selection<br />
procedure of fusants easier. The regeneration rate in this<br />
study was up to 14.31 ± 1.13%. This is because heat
4180 Afr. J. Biotechnol.<br />
Figure 4. HPLC chromatograms of taxol extracted from strain HDF-68. Arrows indicate the taxol-specific peaks. A, Taxol<br />
sample extracted from strain HDF-68; B, taxol molecule standard.<br />
treatment and UV-irradiation affected the different<br />
processes of the protoplasts. For example, UV-irradiation<br />
could enhance crossing over between the two genomes,<br />
leading to high complementation ability (Gokhale, 1992).<br />
Although, a lot of fusants were obtained, many were<br />
unstable. This may be because the protoplasts derived<br />
from mycelium of N. sylviforme contain several nuclei,<br />
which usually leads to multinucleate protoplasts (Gokhale,<br />
1992).<br />
Through protoplast fusion with inactivated parents, we<br />
have succeeded in obtaining a high yield fusant HDF-68<br />
that could produce 468.62 �g/l taxol and the fusant HDF-<br />
68 exhibited characteristics of both parents and genetic<br />
stability after subculturing. These results suggest that<br />
intraspecific chromosomal recombination between N.<br />
sylviforme UV40-19 and UL50-6 might have occurred since<br />
the parents were inactivated.<br />
Up to date, there is no report on using inactivated<br />
protoplast fusion to produce a strain with high taxol<br />
output. This is the first successful application of the<br />
methods in improving taxol output of endophytic fungi.<br />
Although, the yield of taxol obtained in this study is<br />
insufficient for industrial production, we proved that the<br />
inactivated protoplast fusion is an effective method in<br />
fungus breeding and provided a good basis for the<br />
application of this technique to the breeding of the<br />
strains with high taxol output. Further improvement of<br />
strain HDF-68 and the optimization of fermentation<br />
conditions and culture medium might lead to higher<br />
production of taxol.<br />
ACKNOWLEDGEMENTS<br />
This study was supported by National Natural<br />
Science Foundation of China (30970090), China<br />
Postdoctoral Science Foundation (20090450136),<br />
Innovation Foundation of Harbin (2010RFQXS043),<br />
Program for New Century Excellent Talents in<br />
Heilongjiang Provincial University, Research<br />
Program Heilongjiang Education Bureau (11551377),<br />
High-level Talents (innovation team) Project of
Figure 5. Mass spectrum of taxol extracted from strain HDF-68. Arrow indicates the molecular ion of taxol at m/z 855(M+H) +<br />
Table 4. Properties of HDF-68 after 10 generations.<br />
Heilongjiang University (Hdtd2010-17) and<br />
Outstanding Youth Science Fund of Heilongjiang<br />
University.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4183-4189, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2286<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Production of ethanol from mango (Mangifera indica L.)<br />
peel by Saccharomyces cerevisiae CFTRI101<br />
Lebaka Veeranjaneya Reddy 1 *, Obulam Vijaya Sarathi Reddy 2 and Young-Jung Wee 3 *<br />
1 Department of Microbiology, Yogi Vemana University, Kadapa (A.P.) 516 003, India.<br />
2 Department of Biochemistry, Sri Venkateswara University, Tirupati 517 502, India.<br />
3 Department of Food Science and Technology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Korea.<br />
Accepted 4 April, 2011<br />
Mango fruit processing industries generate two types of waste, including solid waste (peel and stones)<br />
and liquid waste (juice and wash water). Utilization of this waste is both a necessity and challenge. This<br />
work was aimed to investigate the suitability of dried mango peel for ethanol production. The mango peel<br />
contained good amount of reducing sugars up to 40% (w/v). Direct fermentation of mango peel extract<br />
gave only 5.13% (w/v) of ethanol. The rate of the fermentation was very slow. Nutrients such as yeast<br />
extract, peptone and wheat bran extract were tested for the supplementation of mango peel medium and<br />
it was observed that the nutrient supplementation increased the ethanol production significantly up to<br />
7.14% (w/v). The suitability of wheat bran extract (WBE) based medium, which is cheap and abundantly<br />
available for mango peel fermentation was also discussed.<br />
Key words: Mango peel, ethanol fermentation, nutrient supplementation, wheat bran extract.<br />
INTRODUCTION<br />
The excessive consumption of fossil fuels, particularly in<br />
large urban areas, has greatly contributed to generation<br />
of high levels of pollution. There is a need for environmentally<br />
sustainable energy sources to find a viable and<br />
long-term substitute for liquid petroleum. As a step to<br />
solve this problem, the use or addition of biofuels to<br />
gasoline, which reduces emission of carbon monoxide<br />
and unburned hydrocarbons that form smog, has widely<br />
been enforced in recent years (Wyman, 1994). In this<br />
regard, India reforms are taken by blending 10 to 15%<br />
ethanol in its gasoline usages. Converting a renewable<br />
non-fossil carbon, such as organic wastes and biomass<br />
consisting of all growing organic matter (plants, grasses,<br />
fruit wastes and algae) to fuel would assure a continual<br />
energy supply (Wyman, 1996).<br />
The economics of ethanol production by fermentation<br />
*Corresponding author. E-mail: lvereddy@yahoo.com;<br />
yjwee@ynu.ac.kr. Tel: +91 92909 75611; +82 53 810 2951.<br />
Fax: +82 53 810 4662.<br />
are significantly influenced by the cost of the raw materials,<br />
which accounts for more than half of the production costs<br />
(Classen et al., 1999). To achieve a lower production<br />
cost, the supply of cheap raw material is thus a necessity.<br />
Production of value added products from agro-industrial<br />
and food processing wastes is now a focusing area, as it<br />
reduces pollution in the environment in addition to energy<br />
generation. The annual availability of these wastes<br />
amounts to 1.05 billion tons (Anonymous, 2004). The<br />
major part of this is mostly discarded and it is the main<br />
source for increasing the pollution in environment on<br />
occasions and also, the discarding process become a<br />
very expensive step due to high transportation costs.<br />
Majority of fruit and vegetable wastes available from their<br />
processing industries are seasonal and they do not<br />
decompose rapidly. The mechanical drying of these<br />
wastes (mango peel, citrus peel, pineapple peel and<br />
tomato processing wastes) gave opportunity to store the<br />
substrate all over the year. The yeast Saccharomyces<br />
cerevisiae and facultative bacterium Zymomonas mobilis<br />
are better candidates for industrial alcohol production. Z.<br />
mobilis possesses advantages over S. cerevisiae with
4184 Afr. J. Biotechnol.<br />
respect to enthanol productivity and tolerance. However,<br />
ethanol is produced commercially by yeast because it<br />
ferments glucose to ethanol as a virtually sole product<br />
and it is known for its high ethanol tolerance, rapid<br />
fermentation rates and insensitivity to temperature and<br />
substrate concentration (Linden and Hahn-Hägerdal,<br />
1989).<br />
Mango is processed to a maximum extent, thereby producing<br />
high quality of solid and liquid wastes. Solid<br />
wastes, stones, stalks, trimmings and fibrous materials<br />
are obtained during the preparation of raw material. This<br />
contributes about 40 to 50% of total fruit waste out of<br />
which, 5 to 10% is pulp waste and 15 to 20% is kernel<br />
(Anonymous, 2004; Madhukara et al., 1993; Maini et al.,<br />
2000; Pandey et al., 2000). Liquid waste is the waste<br />
material that comes out of a factory after washing of<br />
fruits, packaging, blanching, cooling and plant and machinery<br />
clean up and so on. Utilization of this mango waste<br />
is both a necessity and a challenge. If a factory is<br />
processing five tons of Totapuri mangoes per hour, about<br />
six tons of peel would be available as waste per day of 8<br />
h work. Approximately, 0.4 to 0.6 million tons of mango<br />
peel is generated annually in India (Anonymous, 2004).<br />
This waste is either used as cattle feed or dumped in<br />
open areas, where it adds to environmental pollution. The<br />
use of mango peel as a source of pectin and fibre<br />
production also has been reported (Pandia et al., 2004).<br />
Grohmann et al. (1994; 1995; 1996; 1998) previously<br />
reported ethanol production from orange peel. Ethanol<br />
production from banana (Manikandan et al., 2008) and<br />
pineapple peels (Ban-koffi and Han, 1990) were also<br />
investigated. Mango peel is difficult to decompose, as it<br />
takes a very long time, because of its complex composition.<br />
Suitability of mango peel for biogas production was<br />
investigated by Madhukara et al. (1993). However,<br />
ethanol fermentation from fruit and vegetable wastes like<br />
mango peel appears to give better returns. The presence<br />
of high amount of reducing sugars in dried and fresh<br />
mango peel prompted us to make an attempt to utilize it<br />
as a raw material for ethanol production and development<br />
of cheap medium. As far as we know, this is the first<br />
report of its kind on ethanol production from mango peel.<br />
MATERIALS AND METHODS<br />
Strain and medium<br />
Non-amylolytic and ethanol-producing yeast strain S. cerevisiae<br />
CFTRI 101 was used throughout the experiments; was obtained<br />
from CFTRI, Mysore, India. The culture was maintained on MPYD<br />
(malt extract 0.3%, peptone 0.5%, yeast extract 0.3% and dextrose<br />
2%) agar (1.5%) slants at 4°C. The inoculum was prepared by<br />
inoculating the slant culture into 25 ml of the sterile MPYD liquid<br />
medium in 100 ml conical flask and growing it on a rotary shaker<br />
(100 rpm) for 48 h. 10% (v/v) inoculum (3 × 10 4 cells ml -1 ) was<br />
inoculated into 100 ml sterile mango peel extract broth in 250 ml<br />
conical flask and was incubated up to 5 days under stationary<br />
conditions. All the stated experiments were conducted at pH 5.0<br />
and 30°C.<br />
Mango peel<br />
Mango peel was procured from local mango pulp industry (Vinsari<br />
Fruit Pulp Industries Ltd., Renigunta, Tirupati, India). It was dried<br />
and milled to a particle size of 40 BS (British Standard) mesh in an<br />
apex mill.<br />
Extraction of sugars from mango peel<br />
Mango powder (100 g) was mixed with water (1:3) and left overnight.<br />
The liquid containing sugars was extracted with the help of<br />
cheesecloth by squeezing. This acted as control. In the case of<br />
enzymatic digestion with 1% (v/v) pectinase, Trizyme 50 (Triton<br />
Chemicals, Mysore, India) was used for improved results. The<br />
extraction medium pH was 5.0 and the temperature was 37°C. The<br />
extract was suitably diluted to obtain the desired concentration of<br />
sugars (15-17%, w/v), and was supplemented with various nutrients<br />
(yeast extract 1%, peptone 1.5%, ammonium phosphate 2% and<br />
wheat bran 3%) in order to study the effect of nutrients on fermentation.<br />
The unsupplemented medium acted as the control. In the<br />
case of wheat bran extract supplementation experiments, mango<br />
peel sugars were extracted into the wheat bran extract solution<br />
instead of water.<br />
Preparations of wheat bran extract (WBE)<br />
Wheat bran obtained from the local market was used in preparation<br />
of the wheat bran extract. Wheat bran (30 g) was boiled with 500 ml<br />
of water for 10 min. After cooling, it was filtered and equal volume<br />
(500 ml) of the extract was collected by washing the residue and<br />
made to 1 L (Shamala and Sreekantiah, 1988).<br />
Analytical methods<br />
Mango peel analysis for the determination of moisture, non-reducing<br />
sugars, protein, total soluble solids, cellulose and lignin was<br />
carried out according to the methods of Ranganna (1986).<br />
Reducing sugar concentration was estimated by Shaffer and<br />
Somogyi (1933) method. Ethanol and other metabolites were determined<br />
by gas chromatography coupled with flame ionization detector<br />
(Antony, 1984). Final cell biomass was estimated by weighing the<br />
dried yeast cells after fermentation. All data are shown as the<br />
average values and standard deviations from three independent<br />
experiments, unless otherwise stated.<br />
RESULTS<br />
Extraction of sugars from dried mango peel<br />
From the aqueous extraction, low amount of sugars were<br />
obtained; only 20% (w/v). Mango peel treated with crude<br />
pectinase yielded higher levels of solubilisation and<br />
reducing sugars (30 ± 5%, w/v) (Tables 1 and 2). The<br />
optimum incubation period for solubilisation of the maximum
Table 1. Composition of fresh and dried mango peel used in this<br />
study.<br />
Content Fresh mango<br />
peel<br />
sugars was found to be 24 h (Figure 1). The results also<br />
indicated a relatively low inhibition of hydrolytic enzymes<br />
(amylases and cellulases) by the sugars released from<br />
Dried mango<br />
peel<br />
Moisture 70 ± 5 10 ± 1.2<br />
Total solids 25.6 ± 4.6 70.5 ± 2.7<br />
Reducing sugars 7.0 ± 1.8 30 ± 2.5<br />
Non-reducing sugars 5.9 ± 0.4 4.3 ± 0.5<br />
Protein 3.5 ± 0.5 4.0 ± 0.8<br />
Cellulose and lignin 25.2 ± 2.0 23 ± 1.2<br />
Table 2. Effect of pectinase enzyme (1%, v/v) on sugar extraction<br />
from dried mango peel.<br />
Time (h)<br />
Reducing sugar (%, w/v)<br />
Pectinase non-treated Pectinase treated<br />
5 5.8 ± 0.5 10 ± 0.63<br />
10 10 ± 0.65 15.6 ± 2.3<br />
15 15.5 ± 1.5 21 ± 1.6<br />
20 18 ± 1.3 24.3 ± 1.3<br />
25 20 ± 1.5 30 ± 5.5<br />
Figure 1. Total percentage of water soluble solids and sugars in the<br />
dried mango peel extract after pectinase (1%, v/v) treatment. –●–,<br />
total water soluble solids; –■–, soluble sugars.<br />
Reddy et al. 4185<br />
the mango peel. Another significant observation made<br />
during this study was decrease in the initial pH from 5 to<br />
4.5 at the end.
4186 Afr. J. Biotechnol.<br />
Effect of pH, temperature and nutrients on the dried<br />
mango peel fermentation<br />
The levels of reducing sugars were adjusted to 15% (w/v)<br />
with the dilution and the required nutrients were supplemented<br />
for fermentation. The direct fermentation of<br />
mango peel extract gave 5.14% (w/v) ethanol. The<br />
results of optimizing the culture conditions such as pH<br />
and temperature indicated that, the changes in pH and<br />
temperature could affect the final ethanol concentration.<br />
The final ethanol concentration at different pH and temperature<br />
experiments (Figure 2a, b) showed that 30°C<br />
and pH 4.5 were optimum for ethanol production from<br />
mango peel extract.<br />
The supplementation of nutrients significantly increased<br />
the ethanol concentration and fermentation rate. Yeast<br />
extract alone and combination with peptone supple-<br />
Figure 2. Effect of (a) pH and (b) temperature on ethanol<br />
fermentation from the dried mango peel extract.<br />
mentation attributed to the ethanol formation very rapidly<br />
and formed 7.0 and 7.14% (w/v) ethanol, respectively,<br />
instead of 5.14% (w/v) from unsupplemented media at<br />
the end of the fermentation. The data on fermentation of<br />
mango peel extract with nutrient supplementation are<br />
presented in Table 3. The mango peel extract with wheat<br />
bran extract medium significantly increased the yeast<br />
growth and the ethanol formation when compared with<br />
the peel extract alone (Table 3). The wheat bran extract<br />
increased the ethanol concentration from 51.4 to 67.5 g l -<br />
1 (Figure 3). We also tried the fresh mango peel, which<br />
contained low levels of reducing sugars (7 to 10%, w/v)<br />
because of its high amount of water (90%, v/v). Experiments<br />
with the fresh mango peel extract without supplementation<br />
of nutrients was also done, but it gave low<br />
ethanol productivity (3%, w/v).<br />
For cell viability when compared with the control medium,
Table 3. Periodical analysis of ethanol and final cell mass during fermentation of mango peel extract supplemented with various nutrients a .<br />
Supplement<br />
Ethanol<br />
concentration<br />
(g L -1 )<br />
24 h 48 h 72 h<br />
Theoretical<br />
ethanol<br />
yield (%)<br />
Volumetric<br />
productivity<br />
(g L -1 h -1 )<br />
Ethanol<br />
concentration<br />
(g L -1 )<br />
Theoretical<br />
ethanol<br />
yield (%)<br />
Volumetric<br />
productivity<br />
(g L -1 h -1 )<br />
Ethanol<br />
concentration<br />
(g L -1 )<br />
Theoretical<br />
ethanol yield<br />
(%)<br />
Reddy et al. 4187<br />
Volumetric<br />
productivity<br />
(g L -1 h -1 )<br />
Control 23 30.7 0.95 45.6 60.0 0.93 51.3 68.4 0.71 5.2<br />
Peptone 30 40 1.25 55.4 73.3 1.15 68.8 91.7 0.95 6.3<br />
Yeast extract 33.2 44.3 1.38 60.5 80.7 1.26 70.0 93.7 0.92 6.6<br />
Ammonium<br />
phosphate<br />
25.4 33.6 1.05 46.7 62.3 0.97 53.2 70.9 0.73 5.1<br />
Peptone +<br />
yeast extract<br />
34.5 46.0 1.43 62.2 82.9 1.29 71.4 95.2 0.99 6.8<br />
Wheat bran<br />
extract<br />
30.8 41.1 1.28 53.9 71.9 1.12 67.5 90 0.93 6.2<br />
a The values presented in the table are mean values of three independent experiments.<br />
the supplemented medium had high cell count<br />
at the end of the fermentation (data not shown).<br />
The stated results were supported by the increase<br />
of final biomass in the nutrient supplemented<br />
mango peel extract medium (Table 2).<br />
DISCUSSION<br />
The dried mango peel contained high amount of<br />
reducing sugars (up to 45%, w/v) and the results<br />
are in accordance with those of previous reports<br />
(Anonymous, 2004; Madhukara et al., 1993). In<br />
the case of aqueous extraction, the sugar content<br />
was very low. The reason for the low content<br />
could be due to the presence of pectin, which held<br />
the sugar molecules and could not be released<br />
with simple water extraction. The presence of<br />
other enzymes like amylase and cellulase in the<br />
crude pectinase, may aid to increase sugar concentration<br />
by hydrolyzing the respectable substances<br />
(Grohmann et al., 1995). The significant<br />
pH drop during the enzymatic hydrolysis of mango<br />
peel is undoubtedly caused by the release of Dgalacturonic<br />
acid from pectin. The pKa value of Dgalacturonic<br />
acid is 3.51 (Filippov et al., 1978) and<br />
the pH values of peeled hydrolyzates appeared be<br />
to stabilized in the range of 3.3 and 3.5. The low<br />
yields of ethanol from dried mango peel increase<br />
the cost of production. Increase in the ethanol<br />
production up to 7 to 7.5% (w/v) as in general<br />
industrial output from molasses can economize<br />
the process. The development of cheap medium<br />
for fruit waste fermentation to ethanol also<br />
required low-cost ethanol production.<br />
To improve the concentration of ethanol, the fermentation<br />
medium was supplemented with variety<br />
of nutrients like yeast extract, peptone and<br />
Biomass<br />
(g L -1 )<br />
ammonium phosphate to overcome the nutritional<br />
deficiency. The result presented in Table 3 clearly<br />
indicates that, in the case of supplementation of<br />
nutrients, not only the rate of ethanol synthesis<br />
but also the final ethanol concentration increased<br />
significantly. The combination of yeast extract and<br />
peptone gave the maximum improvement in rate<br />
of ethanol synthesis as well as final concentration<br />
in the medium. The ethanol production as well as<br />
viable cell count was significantly increased up to<br />
50 and 20%, respectively, in the mango peel<br />
extract with yeast extract and peptone supplementation.<br />
However, the ammonium phosphate<br />
supplemented one did not stimulate the ethanol<br />
production. Similar results were also obtained in<br />
the case of ethanol fermentation from orange peel<br />
by genetically modified Escherichia coli, which<br />
can utilize the glucose, galactose and galcturonic<br />
acid for ethanol product (Grohmann et al., 1994,
4188 Afr. J. Biotechnol.<br />
Figure 3. Effect of wheat bran extract on the conversion of mango peel to<br />
ethanol fermentation. –▲–, mango peel extract medium supplemented with<br />
wheat bran extract; –■–, control (unsupplemented medium).<br />
1996). It is possible to produce high ethanol<br />
concentrations by extending exponential growth phase of<br />
yeast to longer periods and soluble sugar concen-tration<br />
as in the case of beer production (Kirsop, 1978; Casey<br />
and Magnus, 1984). It is expected that the nutrient<br />
supplementation would overcome nutritional deficiencies<br />
of yeast and allow them to stay longer in growth phase. In<br />
order to verify whether the amount of nutrients and their<br />
mode of feeding influenced the alcoholic fermentation by<br />
S. cerevisiae, experiments were conducted in batch fermentation<br />
with various amounts of nutrients and different<br />
feeding strategies.<br />
Compared with initial total supplementation, exponential<br />
feeding strategy improved the performance of the<br />
fermentation process and the ethanol tolerance of the<br />
yeast. In a recent study reported by Reddy and Reddy<br />
(2005), the nutrients and polyphenols rich horse gram<br />
flour improved the ethanol formation in very high gravity<br />
fermentation. The suitability of wheat bran extract as a<br />
medium for ethanol production from mango peel investigations<br />
successfully replaced the costly medium components<br />
and developed a novel wheat bran extract (WBE)<br />
medium which could provide a cheap source of amino<br />
acids and other nutrients (Shamala and Sreekantiah,<br />
1988). Reddy and Basappa (1996) also successfully<br />
replaced the nutrients with wheat bran extract in the<br />
direct fermentation of starch to ethanol by Endomycopsis<br />
fibuligera and Z. mobilis. However, supplementation with<br />
yeast extract and peptone was superior to that of wheat<br />
bran extract. This showed that wheat bran extract has<br />
limited amount of nutrients when compared with yeast<br />
extract or peptone. Further optimization studies on the<br />
supplementation of WBE are to be made to make the<br />
process economically viable. The initial fermentability of<br />
the unsupplemented peel extracts by S. cerevisiae was<br />
extremely poor because of insufficient growth nutrients in<br />
the peel medium. The fermentation of mango peel extract<br />
was stimulated by supplementation with low amounts of<br />
yeast extract and peptone. The wheat bran extract<br />
though stimulated not only the rate of fermentation and<br />
also the final concentration of ethanol, but it was not as<br />
good as the yeast extract and peptone. However, higher<br />
concentrations of WBE is needed to be supplemented to<br />
make the mango peel extract fermentation process more<br />
economical since the supplementation with yeast extract<br />
and peptone is obviously more expensive. Given the<br />
promise of the proposed WBE based medium for ethanol<br />
fermentation, it should be tested beyond the bench scale.<br />
Generally, the production concentration in commercial<br />
ethanol production plants is between 7.5 and 10% (w/v).<br />
Based on previous reports, the ethanol production<br />
concentration was 4.02% (w/v) in citrus peel waste, 3.5%<br />
(w/v) in grape fruit peel and 4.2% (w/v) in pineapple peel<br />
(Ban-koffi and Han, 1990; Nishio et al., 1980; Wilkins et<br />
al., 2007a, b).<br />
In this study, mango peel was proved as one of the<br />
novel and potential raw material for ethanol production.<br />
Ethanol production from mango peel requires supplementation<br />
of nutrients because of its low nutrient availability.<br />
Supplementation of yeast extract, peptone and<br />
optimization of fermentation conditions enhanced the<br />
fermentation rate and final ethanol concentration. WBE<br />
supplementation showed comparable improvement in all<br />
terms with the very expensive, yeast extract and peptone.<br />
Further optimization studies on peel hydrolysis using<br />
commercial enzymes and optimization of WBE supple-
mentation will make the process economically viable and<br />
it should be tested beyond the bench scale.<br />
ACKNOWLEDGMENTS<br />
We profusely thank Dr T.N Bhavanishankar, plant manager,<br />
Bacardi-Martine India Limited (Nanjangud, Karnataka<br />
State) for his support in the GC–FID analysis. Special<br />
thanks to Dr S. C. Basappa, former deputy director and<br />
scientist, Central Food Technological Research Institute<br />
(CFTRI), Mysore, for his encouragement and critical<br />
comments on the manuscript.<br />
REFERENCES<br />
Anonymous (2004). By-products and waste utilization (value addition of<br />
mango processing waste). Food Digest. 27: 14-17.<br />
Antony JC (1984). Malt beverages and malt brewing materials: gas<br />
chromatographic determination of ethanol in beer. J. Assoc. Anal.<br />
Chem. 67: 192-193.<br />
Ban-koffi L, Han YW (1990). Alcohol production from pineapple waste.<br />
World J. Microbiol. Biotechnol. 6: 281-284.<br />
Casey GP, Magnus CA (1984). High gravity brewing: effects of nutrition<br />
on yeast composition, fermentative ability and alcohol production.<br />
Appl. Environ. Microbiol. 48: 639-646.<br />
Classen PAM, Sijistsma L, Stams AJM, Deveries SS, Westhuls RA<br />
(1999). Utilization of the biomass for the supply of energy carries.<br />
Appl. Microbiol. Biotechnol. 52: 741-755.<br />
Filippov MP, Shkolenko GA, Kohn R (1978). Determination of the<br />
esterification degree of the pectin of different origin and composition<br />
by the method of infrared spectroscopy. Chem. Zvesti. 32: 218-222.<br />
Grohmann K, Baldwin EA, Buslig SB (1994). Production of ethanol from<br />
enzymatically hydrolyzed orange peel by yeast Saccharomyces<br />
cerevisiae. Appl. Biochem. Biotechnol. 45/46: 383-388.<br />
Grohmann K, Cameron GR, Buslig SB (1995). Fermentation of sugars<br />
in orange peel hydrolisates to ethanol by recombinant E. coli KO11.<br />
Appl. Biochem. Biotechnol. 51/52: 383-388.<br />
Grohmann K, Cameron GR, Buslig SB (1996). Fermentation of orange<br />
peel hydrolysates by ethanogenic E. coli: effects of nutrient<br />
supplements. Appl. Biochem. Biotechnol. 57/58: 383-388.<br />
Grohmann K, Manthey JA, Cameron RG, Buslig BS (1998).<br />
Fermentation of galacturonic acid and pectin-rich materials to ethanol<br />
by genetically modified strains of Erwinia. Biotechnol. Lett. 20: 195-<br />
200.<br />
Reddy et al. 4189<br />
Kirsop BH (1978). European Brewer’s Convention Monograph. V:<br />
Fermentation and Storage Symposium. Zoeterwoude, Elsevier,<br />
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Linden T, Hahn-Hägerdal B (1989). Fermentation of lignocellulosic<br />
hydrolysates with yeast and xylose isomerase. Enzyme Microb.<br />
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Madhukara K, Krishnanad N, Srilatha HR (1993). Ensilage of mango<br />
peel for methane generation. Process Biochem., 28: 119-123.<br />
Manikandan K, Sreenivasan V, Viruthagiri T (2008). Kinetics studies on<br />
ethanol production from banana peel waste using mutant strain S.<br />
cerevisiae. Ind. J. Biotechnol. 7: 83-88.<br />
Pandey A, Soccol CR, Nigam P, Soccol VT (2000). Biotechnological<br />
potential of agro-industrial residues. I: Sugarcane bagasse. Bioresour.<br />
Technol. 74: 69-80.<br />
Pandia B, Stephen K, Louise W (2004). Texture and distribution of<br />
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esterase and calcium. J. Sci. Food Agric. 8: 1493-1499.<br />
Ranganna S (1986). Handbook of Analysis and Quality Control of Fruit<br />
and Vegetable Products. Tata McGraw-Hill publishing Company,<br />
New Delhi, India.<br />
Reddy LVA, Reddy OVS (2005). Improvement of ethanol production in<br />
very high gravity fermentation by horse gram (Dolichos biflorus) flour<br />
supplementation. Lett. Appl. Microbiol. 41: 440-444.<br />
Reddy OVS, Basappa SC (1996). Direct fermentation of starch to<br />
ethanol by Endomycopsis fibuligera and Zymomonas mobilis:<br />
synergism and limitations. Biotechnol. Lett. 18: 1315-1318.<br />
Shaffer PA, Somogyi M (1933). Copper-iodometric reagents for sugar<br />
determination. J. Biol. Chem. 100: 695-713.<br />
Shamala TR, Sreekantiah KR (1988). Use of wheat bran as a nutritive<br />
supplement for the production of ethanol by Zymomonas mobilis., J.<br />
Appl. Bacteriol. 65: 433-436.<br />
Wilkins MR, Widmer WW, Grohmann K (2007a). Simultaneous<br />
saccharification and fermentation of citrus peel waste by<br />
Saccharomyces cerevisiae to produce ethanol. Process Biochem. 42:<br />
1614-1619.<br />
Wilkins MR, Widmer WW, Grohmann K, Cameron RG (2007b).<br />
Hydrolysis of grapefruit peel waste with cellulase and pectinase<br />
enzymes. Bioresour. Technol. 98: 1596-1601.<br />
Wyman CE (1994). Ethanol from lignocellulosic biomass: technology,<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4190-4196, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2298<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Secretory expression of Rhizopus oryzae α-amylase in<br />
Kluyveromyces lactis<br />
Song Li, Wei Shen, Xianzhong Chen, Guiyang Shi and Zhengxiang Wang*<br />
Research Center of Bioresource and Bioenergy, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi<br />
214122, China.<br />
Accepted 17 March, 2011<br />
Kluyveromyces lactis is a non-conventional yeast species extensively used in the expression of<br />
heterologous genes. In this study, a genetically modified K. lactis with high-level expression of αamylase<br />
from Rhizopus oryzae was obtained, which could successfully hydrolyze and use starch for<br />
growth very well. Shake flask fermentation indicated that, the recombinant yeast was able to produce<br />
the α-amylase at considerable secretion levels using a variety of carbon sources. The highest level of<br />
amylase expression was 22.4 U/ml when cultivated at 30°C and pH 7.0 in the presence of galactose.<br />
Moreover, it was shown that the recombinant yeast, which could efficiently degrade starch, yielded a<br />
final biomass of 12.25 g/l with enzyme activity of 11 U/ml in the culture medium using 20 g soluble<br />
starch/l as the sole carbon source.<br />
Key words: Kluyveromyces lactis, α-amylase, secretion, starch, fermentation.<br />
INTRODUCTION<br />
Yeasts are eukaryotic micro-organisms that have been<br />
used for centuries in traditional fermented food (Romanos<br />
et al., 1992). They have recently become attractive host<br />
organisms for the production of foreign proteins because<br />
of developments in recombinant DNA technology (Romanos<br />
et al., 1992; Dominguez et al., 1998). Compared with the<br />
extensively used Escherichia coli expression system, the<br />
yeast expression system has important advantages in the<br />
expression of proteins from eukaryotic micro-organisms;<br />
these advantages stem from its effective posttranslational<br />
modification system and ability to produce<br />
foreign proteins in soluble and correctly folded form (van<br />
Ooyen et al., 2006).<br />
Kluyveromyces lactis is a Crabtree-negative, nonconventional<br />
yeast that is able to grow solely on lactose<br />
as the carbon source (Dominguez et al., 1998). For decades,<br />
K. lactis has been used in the production of lowlactose<br />
milk because of its lactase-producing ability,<br />
which is necessary for lactose degradation (Freitas et al.,<br />
2008). Its GRAS (generally regarded as safe) status, efficient<br />
secretion capacity and low catabolite repression<br />
*Corresponding author. E-mail: zxwang@jiangnan.edu.cn.<br />
Tel/Fax: +86-510-85918121.<br />
makes it an attractive alternative expression system to<br />
traditional baker’s yeast in the production of certain<br />
proteins (Dominguez et al., 1998; Schaffrath and Breunig,<br />
2000). As an expression host, K. lactis is best known for<br />
its use in the production of bovine chymosin on an<br />
industrial scale, widely recognised as a major biotechnological<br />
achievement (van Ooyen et al., 2006). The yeast<br />
was successfully used in the production of β-galactosidase<br />
in the food industry (Dagbagli and Goksungur,<br />
2008) and was able to achieve high-level expression of<br />
human serum albumin (Lodi et al., 2005) and human<br />
interleukin-1β (Fleer et al., 1991) in pharmaceutical<br />
research. In addition, a large variety of proteins from<br />
different sources such as bacteria, fungi, plants and<br />
mammals (van Ooyen et al., 2006), have been produced<br />
by K. lactis, including α-amylase from Bacillus<br />
amyloliquefaciens (Bartkeviciute and Sasnauskas, 2003),<br />
mouse (Tokunaga et al., 1997) and wheat (Russell et al.,<br />
1993).<br />
The aim of this study was to enhance the expression<br />
and secretion of the Rhizopus oryzae α-amylase using K.<br />
lactis. The amylase gene was integrated into the K. lactis<br />
genome and expressed under the control of the K. lactis<br />
lactase (LAC4; β-galactosidase) promoter and<br />
Saccharomyces cerevisiae α-mating factor pre-pro<br />
sequence. Different cultivation conditions, including tem-
perature, pH and carbon sources were investigated to<br />
study their effects on the production of the R. oryzae αamylase<br />
in K. lactis. Specifically, the starch fermentation<br />
characteristics of the recombinant K. lactis strain were<br />
also observed.<br />
MATERIALS AND METHODS<br />
Strains, plasmids and culture media<br />
E. coli JM109 was used for the propagation of plasmids. The host<br />
strain K. lactis and the expression plasmids pKLAC1 were<br />
purchased from New England BioLabs Company. Recombinant<br />
plasmid pMD-RoAmy containing the R. oryzae α-amylase gene<br />
coding sequence was constructed in previous work (Li et al., 2011)<br />
LB medium (5 g yeast extract, 10 g peptone, 10 g NaCl per litre)<br />
was used for cultivation of E. coli. YPD medium (10 g yeast extract,<br />
20 g peptone, 20 g dextrose per litre) and YCBA agar plate (10 g<br />
yeast carbon base, 15 g agar powder per litre, 5 mM acetamide)<br />
were prepared for the pre-cultivation of K. lactis and selection of<br />
yeast recombinants, respectively. YPDS agar plate (YPD medium<br />
supplemented with 5 g soluble starch, 15 g agar powder per litre)<br />
was used for halo assays of α-amylase activity. YPX medium (10 g<br />
yeast extract, 20 g peptone, 20 g carbon source per litre) was used<br />
for the expression of α-amylase.<br />
Construction and transformation of expression plasmid<br />
The mature RoAmy coding sequence (RA) was amplified by the<br />
polymerase chain reaction (PCR) from pMD-RoAmy with primers:<br />
5'-ATTGTCGACGTGCCTGTCATCAA-3' and 5'-GTAGCGGCCGCG<br />
ATAAGCTTGCACAAACGAAC-3'. The amplification yielded a copy<br />
of the gene flanked by SalI and NotI sites provided by the two<br />
primers (underlined), respectively. The PCR product was gelpurified,<br />
digested with the two indicated enzymes and inserted at<br />
the corresponding site of pKLAC1 vector; this procedure yielded a<br />
recombinant expression plasmid pKLRA, in which the α-amylase<br />
gene was under the control of the K. lactis lactase (LAC4; βgalactosidase)<br />
promoter and the S. cerevisiae α-mating factor prepro<br />
sequence.<br />
The secretion cassette pKLRA was linearized with SacII and<br />
introduced into K. lactis cells by electroporation transformation<br />
method (Delorme, 1989). The electroporated cells were spread on<br />
YCBA agar plates. After incubation, transformants appeared on the<br />
plates at 30°C for 3 to 4 days and were selected for further study.<br />
α-Amylase halo assay<br />
For halo assay, the transformed yeast cells were patched onto<br />
YPDS agar plates and incubated for 48 h at 30°C. The halo-forming<br />
ability of transformants was detected by staining with weak iodine<br />
solution (20 g KI and 2 g I2 per litre).<br />
Expression of α-amylase<br />
The selected K. lactis transformants with different halo-forming abilities<br />
were cultured in shake flasks to screen the RoAmy expression<br />
levels. The recombinant strains were inoculated into 10 ml YPD<br />
medium and grown in a shaking incubator with 200 rpm at 30°C<br />
overnight; 0.2 ml of the overnight culture was transferred into 50 ml<br />
fresh medium and cultivated to an OD600=6.0 to 10.0. Then, the precultures<br />
were inoculated into 50 ml YPX medium to a final optical<br />
Li et al. 4191<br />
density (600 nm) of 0.2. In all the shake flask cultivation processes,<br />
250 ml baffled flasks were used.<br />
Analytical techniques<br />
The α-amylase activity was determined as follows: 1 ml 1% (w/v)<br />
soluble starch was mixed with 0.25 ml citric acid–Na2HPO4 buffer<br />
(0.2 M, pH 5.0) and incubated at 55°C for 5 min, followed by<br />
addition of 0.1 ml enzyme solution. It was further incubated for 5<br />
min. The reducing sugar formed was determined according to the<br />
method described previously (Miller, 1959), using maltose as a<br />
standard. One unit of α-amylase activity was defined as the amount<br />
of enzyme that released 1 mg reducing sugar per minute under the<br />
mentioned conditions.<br />
The yeast growth was measured in terms of dry weights, which<br />
were determined by drying 3 ml samples to constant weight at<br />
65°C. The starch concentration was quantified according to the method<br />
described by Xiao et al. (2006).<br />
RESULTS<br />
Expression of α-amylase in K. lactis<br />
More than one hundred transformants appeared on YCBA<br />
agar plates. Eleven colonies were randomly selected and<br />
replicated to YCBA agar plates for further purification.<br />
Then, the purified clones were patched onto YPDS agar<br />
plates and grown at 30°C for 3 to 4 days. After staining<br />
with weak iodine solution, clear zones were formed<br />
around clones no. 1, 2, 5 and 11, as shown in Figure 1. It<br />
was seen that clones no. 1 and 11 shown larger zones on<br />
the plate assay, but the α-amylase activities observed in<br />
shake flask fermentation were almost the same (data not<br />
shown).<br />
Effects of culture conditions on α-amylase<br />
expression in K. lactis<br />
To investigate the effects of initial pH on α-amylase<br />
expression, this study tested the YPD media with initial<br />
pH values ranging from 4.0 to 8.0. The highest obtained<br />
level of amylase expression, calculated from the triplicate<br />
fermentations, ranged from 1.4 to 11.2 U/ml, as shown in<br />
Figure 2a. To study the effects of temperature on αamylase<br />
expression, the temperature of the shake flask<br />
incubator was set from 20 to 35°C and the initial pH value<br />
of the medium was adjusted to 7.0. Among the cultivation<br />
temperatures observed, the highest amylase expression<br />
level (11.8 U/ml) was achieved at 30°C (Figure 2b).<br />
α-Amylase expression with different carbon sources<br />
The carbon source for amylase expression in the shake<br />
flask culture was screened and the initial amount of<br />
different carbon source was kept at 20 g/l. Figure 3<br />
shows that, amylase was successfully secreted in all the
4192 Afr. J. Biotechnol.<br />
Figure 1. Test of the amylase activity of selected clones of the<br />
K. lactis transformats. The clones’ labelled no. 3, 4, 6 to 10<br />
and the colony labelled “C” of non-transformed control host K.<br />
lactis cells show no clearance zones indicating the absence of<br />
amylase activity.<br />
tested culture media, of which the galactose-containing<br />
culture provided the highest level of amylase secretion at<br />
22.4 U/ml- approximately two times higher than that<br />
obtained in other carbon source-containing cultures.<br />
However, the highest biomass (15.75 g/l) was achieved<br />
using glycerol as the carbon source. Interestingly, at the<br />
end of the shake flask fermentation, the starch- and<br />
maltose-containing cultures yielded similar biomass,<br />
which was slightly higher than that observed in the<br />
glucose-containing culture. Samples from the galactosecontaining<br />
culture media of recombinant K. lactis were<br />
analyzed by SDS-PAGE for the presence of the αamylase,<br />
as shown in Figure 4, both glycosylated and<br />
non-glycosylated α-amylases were secreted into the<br />
culture medium.<br />
Starch fermentation characteristics of the<br />
recombinant K. lactis strain<br />
The characterisation of the growth of the recombinant K.<br />
lactis strain and the K. lactis host strain as well as their<br />
starch hydrolysis and fermentation, were observed using<br />
soluble starch as the sole carbon source. Figure 5a<br />
shows that, the native K. lactis strain could not attain<br />
continuous growth in starch, whereas the recombinant<br />
strain, which has the α-amylase secretion cassette,<br />
displayed a high growth rate in starch. The effects of<br />
initial starch concentrations ranging from 10 to 40 g/l<br />
were examined to study the growth of recombinant yeast.<br />
Because of the α-amylase secreted by the recombinant<br />
yeast, the starch in the cultures was efficiently and com-<br />
Figure 2. (A) Effects of pH and (B) temperatures on αamylase<br />
production in YPD culture medium. Samples<br />
were harvested from 48 h fermentation cultures. All data<br />
are mean values of three independent experiments; error<br />
bars indicate standard deviation.<br />
pletely degraded during fermentation from 12 to 24 h<br />
(Figure 5b). The maximum cell density increased with<br />
rising initial starch concentrations from 10 to 30 g/l;<br />
although, the 40 g starch/l in the medium was completely<br />
degraded in the first 24 h of fermentation, the maximum<br />
cell density observed at 84 h was slightly lower than that<br />
obtained at 20 and 30 g starch/l concentrations. In<br />
addition, the maximum yeast growth rate during the first<br />
12 h was achieved with 10 g starch/l and the yeast exhibited<br />
better growth with 20 g starch/l during the 24 to 60 h<br />
cultivation time. Further details are given in Table 1.<br />
DISCUSSION<br />
It is difficult to accurately compare the expression levels<br />
of various α-amylases in K. lactis, because of variations<br />
in the starch origin and α-amylase activity definition, as<br />
well as in the details of the enzyme assays. However, the
Figure 4. SDS-PAGE analyses of α-amylase<br />
produced in galactose-containing culture medium by<br />
recombinant K. lactis. Proteins were concentrated 5fold<br />
by 10% (w/v) trichloroacetic acid precipitation,<br />
before loading (15 µl). Protein bands were stained<br />
with Coomassie brilliant blue R-250. Lane 1 to 5,<br />
samples from 0, 6, 12, 24 and 48 h fermentation<br />
cultures, respectively; lane M, molecular mass<br />
marker.<br />
highest yield of the R. oryzae α-amylase obtained in the<br />
galactose-containing culture medium by shake-flask<br />
fermentation was 22.4 U/ml (approximately 20 mg/l),<br />
Figure 3. Effects of carbon sources on α-amylase secretion and<br />
biomass production. Samples were harvested from 48 h fermentation<br />
cultures. All data are mean values of three independent experiments;<br />
error bars indicate standard deviation.<br />
Li et al. 4193<br />
which was much higher than the expression level of<br />
mouse α-amylase (0.527 U/ml) (Tokunaga et al., 1997) or<br />
Schwanniomyces occidentalis α-amylase (30 mU/ml)<br />
(Strasser et al., 1989) obtained in K. lactis. To the best of<br />
this study’s knowledge, among various α-amylases expressed<br />
in K. lactis, the expression level of the R. oryzae<br />
α-amylase obtained in this study is one of the highest<br />
reported? In the previous work of these authors, the R.<br />
oryzae α-amylase expression levels observed in E. coli<br />
and S. cerevisiae were only approximately 0.11 and 1.3<br />
U/ml, respectively. In addition, the α-amylase expression<br />
level obtained by recombinant Pichia pastoris was up to<br />
45 U/ml in shake-flask fermentation, however, the<br />
application of recombinant α-amylase in food industry<br />
could be limited due to the characteristics of the host<br />
stain (not a GRAS status strain) and slight residual<br />
methanol in the final fermentation broth. Thus, it was<br />
conclude that K. lactis could be more promising for the<br />
expression of the R. oryzae α-amylase at food status.<br />
Among physical parameters, cultivation temperature<br />
and pH of growth media are important for organism<br />
growth and enzyme secretion (Gupta et al., 2003). Similar<br />
to the temperatures used in many studies (Rocha et<br />
al., 1996; Mincheva et al., 2002), 28 to 30°C yielded the<br />
highest α-amylase activity and biomass. Attempts to<br />
express amylase at an initial pH of 4.0 resulted in an<br />
absence of enzyme activity and the amylase activity<br />
produced at pH 8.0 was only half of the highest amylase<br />
expression level at pH 7.0 (Figure 2). This result indicates<br />
that pH control is crucial for R. oryzae α-amylase<br />
expression in K. lactis.<br />
The expression and secretion of R. oryzae α-amylase
4194 Afr. J. Biotechnol.<br />
Figure 5. Time courses in the fermentation of starch using<br />
recombinant K. lactis and host strain. Biomass produced<br />
(A) and starch hydrolyzed (B) by recombinant K. lactis with<br />
initial starch concentrations of 10 g/l (●), 20 g/l (▲), 30 g/l<br />
(▼) and 40 g/l ( ) and host strain with 20 g starch/l (■). All<br />
data are mean values of three independent experiments;<br />
error bars indicate standard deviation.<br />
in K. lactis was under the control of the LAC4 promoter,<br />
which is one of the most frequently used promoters for<br />
high-level heterologous gene expression in K. lactis and<br />
induced by the presence of lactose or galactose in the<br />
growth medium, but not fully repressed in the absence of<br />
the inducer (van Ooyen et al., 2006). A number of carbon<br />
sources were tested for the growth and α-amylase<br />
expression of recombinant K. lactis. Galactose generated<br />
the highest α-amylase expression probably because the<br />
LAC4 promoter was induced by galactose present in the<br />
culture medium (Rubio-Texeira, 2006). By contrast, αamylase<br />
expressed in lactose-containing cultures were<br />
almost the same as that observed in other carbon source<br />
cultures, indicating that lactose failed to induce the LAC4<br />
promoter for the enhancement of α-amylase expression.<br />
From another point of view, the LAC4 promoter was not<br />
repressed by the presence of a variety of studied carbon<br />
sources. However, considering the price of galactose,<br />
other carbon sources, such as glucose, glycerol, lactose<br />
and starch, are more suitable for α-amylase production.<br />
Most of the wild-type yeast strains cannot directly utilise<br />
starches because of their inability to produce starchdecomposing<br />
enzymes (Jamai et al., 2007). Numerous<br />
studies have demonstrated that, both α-amylase and<br />
glucoamylase are required for efficient starch hydrolysis<br />
in industrial ethanol production using S. cerevisiae strains<br />
(Knox et al., 2003; Kosugi et al., 2009). In previous study,<br />
R. oryzae α-amylase was secreted through an S.<br />
cerevisiae strain. However, the recombinant strain in the<br />
starch-containing culture medium showed extremely low<br />
α-amylase activity (approximately 0.06 U/ml), a level insufficient<br />
for efficient starch degradation without exogenous<br />
glucoamylase. In this study, the host K. lactis strain<br />
used could not grow in the medium with starch as the<br />
sole carbon source, whereas the recombinant K. lactis<br />
strain obtained was able to hydrolyse soluble starch<br />
efficiently because of its ability to express α-amylase at<br />
high concentrations in the starch-containing culture (11.2<br />
U/ml), as shown in Figure 5 and Table 1.<br />
The maltose uptake system of S. cerevisiae has been<br />
extensively studied and is a highly specific strain-dependent.<br />
For example, Weusthuis et al. (1993) observed a<br />
linear decrease in cell yield with increasing amounts of<br />
maltose in the medium feed for S. cerevisiae CBS 8066<br />
strain, whereas Batistote et al. (2006) reported that, S.<br />
cerevisiae VIN7 wine strain yielded a higher final biomass<br />
in the presence of maltose rather than glucose under<br />
certain conditions. Similar results were found for both S.<br />
cerevisiae 70 and 254 strains, as described by Zastrow et<br />
al. (2000). These findings are similar with those observed<br />
in the recombinant K. lactis strain obtained in this study.<br />
Specifically, the recombinant K. lactis produced higher<br />
final cell density in the presence of soluble starch rather<br />
than glucose, probably because of its ability to produce<br />
R. oryzae α-amylase. Unlike bacterial α-amylase, fungal<br />
α-amylase can be used to produce high-maltose syrup<br />
(Doyle et al., 1989), in which the maltose concentration is<br />
up to 50% (w/w) and the glucose concentration is usually<br />
around 10% (w/w). In addition, considerable amounts of<br />
maltotriose, which also yielded higher final biomass than<br />
in the presence of glucose (Zastrow et al., 2000), can be<br />
released during starch hydrolysis (Doyle et al., 1989). In<br />
previous study, soluble starch hydrolysed with R. oryzae<br />
α-amylase yielded a final concentration of glucose and<br />
maltose at 12 and 67% (w/w), respectively and a large<br />
amount of maltotriose was still released at the initial<br />
hydrolysis stage of the gelatinised starch (data not<br />
shown). Overall, it can be conclude that the ability of R.<br />
oryzae α-amylase to produce high levels of maltose and<br />
maltotriose accounts for the higher cell density obtained<br />
by the recombinant K. lactis in the presence of soluble
starch.<br />
Conclusion<br />
Table 1. Soluble starch fermentation characteristics of recombinant K. lactis strain.<br />
Parameter<br />
Biomass (g l -1 h -1 ) a<br />
Starch (g l -1 h -1 ) b<br />
Starch concentration (g/l)<br />
Time (h) 10 20 30 40<br />
0-12 0.064 0.064 0.063 0.601<br />
12-24 0.289 0.343 0.261 0.262<br />
24-36 0.031 0.333 0.292 0.281<br />
36-84 0.146 0.271 0.563 0.261<br />
0-6 0.717 0.852 0.683 0.883<br />
6-12 - c 1.667 1.517 1.602<br />
12-15 - 4.001 2.933<br />
15-18 - 3.967<br />
18-21 -<br />
a Biomass production rate, gram of dry weight per litre per hour; b starch degradation rate,<br />
gram of soluble starch per litre per hour; c the calculation of starch degradation rate in its<br />
running out time is not accurate and the data are not shown. All data are mean values of<br />
three independent experiments.<br />
In conclusion, because of the high-level expression and<br />
secretion of R. oryzae α-amylase in the culture medium,<br />
the recombinant K. lactis was able to efficiently grow<br />
using soluble starch as the sole carbon source and<br />
attained a relatively high cell density probably because of<br />
the considerable amounts of maltose and maltotriose<br />
released by R. oryzae α-amylase. These findings indicate<br />
that, the production of heterologous proteins or other<br />
specific products from K. lactis using starchy materials as<br />
the sole carbon source may be achieved through the coexpression<br />
of the genes of interest and R. oryzae αamylase<br />
gene.<br />
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Jamai L, Ettayebi K, Yamani JE, Ettayebi M (2007). Production of<br />
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Knox AM, du Preez JC, Kilian SG (2003). Starch fermentation<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4197-4206, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2139<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Volatile compounds of maari, a fermented product from<br />
baobab (Adansonia digitata L.) seeds<br />
Charles Parkouda 1 *, Brehima Diawara 1 , Samuel Lowor 2 , Charles Diako 3 , Firibu Kwesi Saalia 4 ,<br />
Nana T Annan 3 , Jan S Jensen 5 , Kwaku Tano-Debrah 4 and Mogens Jakobsen 6 .<br />
1 Département Technologie Alimentaire/IRSAT/CNRST 03 BP 7047 Ouagadougou 03, Burkina Faso.<br />
2 Cocoa Research Institute of Ghana, Physiology/Biochemistry Division P.O. Box 8, Akim-Tafo, Ghana.<br />
3 Council for Scientific and Industrial Research, Food Research Institute, P.O. Box M-20, Accra, Ghana.<br />
4 Department of Nutrition and Food Science, University of Ghana, Legon, Accra, Ghana.<br />
5 University of Copenhagen, Faculty of Life Science, Forest and Landscape, DK-2970, Hørsholm, Denmark.<br />
6 University of Copenhagen, Faculty of Life Science, Centre for Advance Food Studies, Department of Food Science,<br />
Rolighedsvej 30 DK 1958 Frederiksberg C, Denmark.<br />
Accepted 1 April, 2011<br />
The volatile compounds associated with baobab seeds fermentation for Maari production were<br />
extracted and analysed by Likens-Nickerson simultaneous steam distillation-extraction method and gas<br />
chromatography-mass spectrometry (GC-MS), respectively. Furthermore, the titratable acidity, tannin<br />
content and proximate composition were evaluated. A total of 96 compounds were identified with<br />
esters, acids, alcohols and ketones being quantitatively the major groups. Fermentation led to an<br />
increase in the concentration of total volatile compounds from 121.6 in unfermented cooked seeds to<br />
809.1 mg kg -1 in the fermented product. Drying resulted in a significant loss of up to 80.7% of the total<br />
volatiles.<br />
Key words: Volatile compounds, maari, baobab, fermentation.<br />
INTRODUCTION<br />
Several fermented food condiments are commonly found<br />
in several African and Asian countries (Parkouda et al.,<br />
2009). In these countries, they are extensively used due<br />
to their pleasant taste and particular odour as well as<br />
their important nutritional qualities (Azokpota et al., 2008;<br />
Ouoba et al., 2005; Owens et al., 1997). Volatile compounds<br />
are one of the most valuable traits contributing to<br />
their quality and sensorial attributes (Beaumont, 2002).<br />
Maari is one of these fermented food condiments<br />
obtained by processing and spontaneous fermentation of<br />
seeds from the baobab tree (Adansonia digitata L.) in<br />
Burkina Faso. The process is laborious, energy- and<br />
time-consuming and procedures are based on traditional<br />
knowledge and experience of each processor. Contrary<br />
to similar fermented condiments produced from African<br />
locust beans (Soumbala, afitin, iru, sonru and netetu), the<br />
*Corresponding author. E-mail: cparkouda@yahoo.fr.<br />
baobab cooked seeds were not dehulled before the<br />
fermentation; this can lead to a difference in the chemical<br />
content and the profile of volatile compounds. Maari is<br />
especially used as flavouring agent for sauces, soups<br />
and other dishes in some African countries (Chadare et<br />
al., 2008; Parkouda et al., 2010). Traditional maari is<br />
characterized by a specific strong odour which is the<br />
main criterion used by consumers to appreciate the<br />
quality of the condiment. Similar products like afitin,<br />
chungkuk-jang, iru, natto, sonru, soumbala and thua-nao<br />
have been extensively studied and a diversity of volatile<br />
compounds reported (Azokpota et al., 2008;<br />
Leejeerajumnean et al., 2001; Ouoba et al., 2005;<br />
Tanaka et al., 1998). The flavour of these condiments<br />
has mainly been attributed to various volatile compounds<br />
produced through the metabolic activities of microorganisms<br />
during fermentation or the processing conditions<br />
(Azokpota et al., 2008; Leejeerajumnean et al.,<br />
2001; Ouoba et al., 2005). Differences in the traditional<br />
processes for fermented condiments occur among ethnic
4198 Afr. J. Biotechnol.<br />
tribes and these differences presumably influence the<br />
quality of the final products. The identification of volatile<br />
compounds is one of the steps which could give information<br />
for a future selection of starter culture to be used<br />
in controlled fermentation (Azokpota et al., 2010). In<br />
afitin, iru and sonru, compounds belonging to pyrazines,<br />
aldehydes, ketones, esters, alcohols, acids, alkanes,<br />
alkenes, benzenes, phenols, sulphurs and furans groups<br />
were identified (Azokpota et al., 2008). In addition to<br />
these compounds, Ouoba et al. (2005) found amines and<br />
pyridines groups in soumbala. However, volatile compounds<br />
that characterize maari have not yet been<br />
reported. The aim of the present study was to investigate<br />
the volatile compounds associated with spontaneous<br />
fermentation of baobab seeds for maari production using<br />
Likens-Nickerson simultaneous distillation-extraction<br />
method and gas chromatography mass spectrometry<br />
(GC-MS).<br />
MATERIALS AND METHODS<br />
Maari processing and sampling<br />
Maari samples were obtained by spontaneous fermentation of<br />
baobab seeds by three producers (V, C and B) from Pousghuin (a<br />
village in Zorgho, Burkina Faso) according to their own procedure.<br />
Briefly, maari production was done as follows: The baobab seeds<br />
were cleaned and boiled for about 36 h. After the 24 h of boiling, an<br />
ash lye solution was added as softening agent. After the boiling<br />
period, the seeds were drained and transferred into a basket and<br />
left to ferment spontaneously (first fermentation) for 72 h at room<br />
temperature (30 to 35°C). The fermenting mash was pounded and<br />
moulded with further addition of the alkaline ash lye solution. It was<br />
left to undergo a second spontaneous fermentation for about 24 h<br />
at room temperature before drying, including an initial steam<br />
cooking step. The final product obtained was characterized by dark<br />
brownish colour, stickiness and strong smell. Maari is sometime<br />
slightly roasted to give a specific trait to the product. For each of the<br />
three producers, duplicate unfermented boiled seeds, fermenting<br />
mash just before steam cooking and the final dried maari were<br />
collected from the producers and stored at -20°C before analysis.<br />
Additional commercial dried maari ready for consumption was also<br />
collected at Toulfé (a village in Burkina Faso) for comparison. The<br />
analyses were performed in triplicate.<br />
Proximate analysis<br />
Moisture, protein, lipid and ash analyses were determined according<br />
to AOAC official methods (AOAC, 2005). The tannins content<br />
of the samples were estimated by the vanillin-HCl colorimetric<br />
method as described by Addy et al. (1995). The tannins were<br />
extracted with methanol (Merck, Darmstadt, Germany) and made to<br />
react with the vanillin reagent (Merck, Darmstadt, Germany), to<br />
produce a colour reaction, which was measured spectrophotometrically<br />
at 500 nm. The calibration curve was established with<br />
(+)-catechin (Merck, Darmstadt, Germany). For titratable acidity<br />
(TA), 5 g of sample was mixed with 50 ml of distilled water and<br />
mixed in a stomacher (Stomacher 400 Lab Blender, London,<br />
England) at normal speed for 4 min. After filtration through a<br />
Whatman paper filter, the TA was evaluated by titrating the filtrates<br />
with 0.1 N sodium hydroxide (Merck, Darmstadt, Germany) using<br />
phenolphthalein as the indicator (Jideani and Okeke, 1991).<br />
Extraction and identification of the volatile compounds<br />
Extraction of volatile compounds<br />
The simultaneous distillation and extraction method described by<br />
Nickerson and Likens (1966) was used for the investigation of the<br />
volatile compounds of the different types of maari. The volatile<br />
compounds of the samples were extracted using a microscale<br />
steam distillation low density solvent extraction device (micro-SDE;<br />
Chrompack, Middelburg, the Netherlands) as described by Annan<br />
et al. (2003) and modified as follows: a known quantity of pounded<br />
samples (according to their moisture content) was mixed in 400 ml<br />
distilled water to obtain 2.5% slurries (on dry weight basis) of<br />
samples (w/v). A 1 ml internal standard solution (2-methyl-1propanol)<br />
was added to the sample slurry in a 1000 ml Erlenmeyer<br />
flask. About 6 ml of a mixture of pentane and diethyl ether (1:1) was<br />
placed in a 9 ml pear-shaped solvent flask. Both flasks were<br />
connected to the distillation apparatus and the solutions were<br />
brought to boil. Extraction of volatile compounds was carried out for<br />
30 min, from the beginning of condensation of vapours on the walls<br />
of the condenser. The organic solvent phase was collected and<br />
stored at -20°C to freeze out any water present. The solvent extract<br />
was poured off, dried over 2 g of anhydrous Na2SO4 and concentrated<br />
to 100 mg by gently blowing N2 gas over the surface. The<br />
concentrated extract was analyzed for volatile compounds using<br />
gas chromatography and mass spectrometry (GC-MS). Extractions<br />
were performed in triplicate.<br />
Separation and identification of the volatile compounds by GC-<br />
MS<br />
Separation and identification of volatile compounds in the<br />
concentrated extract was performed on a Varian CP 3900 GC/MS<br />
equipped with Varian CP-8400 Autosampler (GC-MS; Varian, NA,<br />
USA). Two microlitres of extract were injected (split ratio, 1:20) into<br />
Varian factor four capillary column (VF-5ms) (30 m x 0.25 µm x<br />
0.39 mm film thickness) using the temperature program: 10 min at<br />
40°C, raised at 6°C min -1 to 240°C and then, maintained for 30 min<br />
at 240°C. The carrier gas was helium with a constant flow rate of 1<br />
ml min -1 .<br />
The MS detector conditions were as follows; transfer line<br />
temperature, 250°C; manifold temperature, 80°C; ion trap<br />
temperature, 20°C; ionisation mode, EI auto; mass range, 25 to 550<br />
m/z; multiplier delay, 1.77 min.<br />
Identification of volatile compounds was determined in the total<br />
ion mode scanning a mass to charge ratio (m/z) range between 25<br />
and 550. Further identification was obtained by probability-based<br />
matching with mass spectra in the NIST 05 mass spectral library<br />
(Varian). Only compounds showing high quality index (degree of<br />
agreement between mass spectrum of sample and mass spectrum<br />
in database ≥ 90) were retained. Concentrations of volatile compounds<br />
were estimated by comparing the relative peak areas of the<br />
compounds with that of the 2-methyl-1-propanol internal standard<br />
and reported in mg kg -1 based on the concentration of the internal<br />
standard.<br />
Statistical analysis<br />
Differences between mean values were determined by analysis of<br />
variance and Student-Newman-Keuls test using SAS statistical<br />
software package (SAS, release, 8.1, Cary, N.C., USA).
RESULTS<br />
As seen from Table 1, during fermentation, protein content<br />
decreased from 156 ± 4.1 g kg -1 to values ranging<br />
from 147 ± 0.7 to 134 ± 0.5 g kg -1 dry weight basis, while<br />
lipid content increased from 122 ± 5.3 g kg -1 to values<br />
ranging from 151 ± 3.8 to 181 ± 1.5 g kg -1 . Ash content<br />
increased from 45 ± 0.4 up to 88 ± 3.6 g kg -1 . The<br />
titratable acidity of unfermented baobab seeds had a<br />
value of 0.17% OAE and increased to 1.87% in the<br />
fermented product. The tannins content decreased from<br />
0.29 mg g -1 CE to values between 0.25 and 0.09 mg g- 1<br />
after fermentation. Changes resulting from the<br />
fermentation were statistically significant (P < 0.05).<br />
The results in Table 2 show that, a variety of volatile<br />
compounds characterized the studied samples and a<br />
total of 96 volatile compounds were found. They belonged<br />
to 10 groups of compounds including acids (6),<br />
alcohols (13), aldehydes (6), alkanes (2), alkenes (2),<br />
esters (23), ketones (22), pyrazines (7), sulphur compounds<br />
(4) and others (11). Esters, ketones, alcohols<br />
groups constituted the groups with the largest number of<br />
volatile compounds.<br />
No volatile compounds belonging to the aldehydes,<br />
alkenes and pyrazines group were found in the unfermented<br />
cooked seeds (UCS). Fermentation led to an<br />
increase in the concentration of total volatile compounds<br />
from 121.6 mg kg -1 in an unfermented cooked seeds<br />
(UCS) up to 809.1 mg kg -1 in the fermented product<br />
(ZV1). In the fermented samples, the total volatile compounds<br />
were 809.1, 582.9 and 636.8 mg kg -1 ,<br />
respectively, for ZV1, ZC1 and ZB1; after the drying<br />
process, they were 291.3, 204.03 and 123 mg kg -1 for<br />
ZV1, ZC1 and ZB1, respectively. Drying process resulted<br />
in an important loss up to 80.7% of the total volatiles<br />
including principally esters, alcohols and acids groups.<br />
The amount of the volatile compounds in the dried commercials<br />
maari sample (DTA) was 352.9 mg kg -1 .<br />
Qualitatively, only few volatile compounds including 2nonadecanone,<br />
γ-dodecalactone, 2-decanone, 3-ethyl-2pentanone,<br />
14-methyl pentadecanoate, di-tert-Butyl<br />
dicarbonate, ethyl acetate, Z,Z,Z-4,6,9-nonadecatriene,<br />
nonadecane, benzeneacetaldehyde and 2,4-dimethyl-3pentanol<br />
were common in all dried fermented samples<br />
from the three producers, showing a specificity of each<br />
sample. Table 3 shows that, the relative amounts of the<br />
compound groups varied widely among samples and<br />
ranged from 0.0 to 65.4%. The major compounds group<br />
found in unfermented cooked seeds was sulphur compounds<br />
(26% of its total amount of volatiles). No<br />
aldehydes, alkynes, furans, phenols and pyrazines compounds<br />
were found in the unfermented cooked seeds. In<br />
the fermented products, the major compounds found<br />
generally were alcohols, aldehydes, alkanes, esters and<br />
ketones groups. The sample ZB1 showed the highest<br />
relative amount of esters (65.4%), ZV1 for ketones<br />
(37.3%) and ZC1 for alcohols (26%), which were the<br />
Parkouda et al. 4199<br />
common components groups among all the samples. The<br />
commercial samples DTA stands out significantly as the<br />
sample richest in the content in pyrazines group (20%).<br />
DISCUSSION<br />
Production of maari resulted in a decrease of carbohydrates.<br />
Decrease in carbohydrates has been reported<br />
in previous studies on similar products like bikalga and<br />
daddawa (Parkouda et al., 2008; Ibrahim and Antai,<br />
1986). The decrease is likely to be explained by cooking<br />
and loss of carbohydrate in the cooking water and by the<br />
fermentation where carbohydrates are metabolised by<br />
the microorganisms responsible for fermentation<br />
(Parkouda et al., 2008). The formation of some volatile<br />
aroma compounds belonging to ketones and aldehydes<br />
groups which are generally formed via Maillard reactions<br />
between saccharides and peptides may also play a role<br />
(Ledauphin et al., 2003). The increase of the titratable<br />
acidity observed is likely due to the accumulation of acid<br />
from the hydrolytic activities of the amylolytic enzymes<br />
(Schaffner and Beuchat, 1986) as well as the role of<br />
microbial metabolism such as lactic acid bacteria and<br />
Bacillus spp. during fermentation. The observed decrease<br />
in tannins during fermentation is in line with previous<br />
studies on baobab seeds (Addy et al., 1995; Nnam and<br />
Obiakor, 2003). Hydrolysis of the polyphenolic compounds<br />
to simpler substances by the enzyme polyphenol<br />
oxidase or the breakdown of the tannin complexes for<br />
example, tannin-protein, tannic acid-starch and tanniniron<br />
complexes to release free nutrients could explain this<br />
decrease (Obizoba and Atti, 1991). Chemical content,<br />
including titratable acidity and tannins is expected to<br />
contribute to the sensorial quality and volatile compounds<br />
profiles of the different samples. For instance, free fatty<br />
acids are known to contribute positively to the production<br />
of characteristic flavours in food but high levels of fatty<br />
acids may easily cause rancidity (Nawar, 1985).<br />
In maari, as other fermented condiments, volatile aroma<br />
compounds are important for quality and sensorial<br />
attributes. A number of volatile compounds are present in<br />
the unfermented baobab cooked seeds; others are<br />
produced during fermentation. The volatile compounds<br />
found in the unfermented cooked seeds can be explained<br />
by a complex sequence of chemical reactions, for example,<br />
strecker degradation, taking place as a function of<br />
moisture, cooking temperature and duration (Rodríguez-<br />
Bernaldo De Quirós et al., 2000). Esters group found in<br />
the unfermented cooked seeds were also reported<br />
previously in unfermented cooked Africa locust bean<br />
(Azokpota et al., 2008; Ouoba et al., 2005). In contrast,<br />
no ester was reported in unfermented cooked Africa<br />
locust bean during controlled production (Azokpota et al.,<br />
2010). The latter result can be explained by the severe<br />
heat treatments applied to the locust beans, leading to<br />
complete loss of esters due to thermal degradation
4200 Afr. J. Biotechnol.<br />
Table 1. Chemical composition † of Unfermented and Fermented baobab seeds*.<br />
Sample<br />
Compound<br />
Moisture (g kg -1 ) Protein (g kg -1 ) Lipid (g kg -1 ) Ash (g kg -1 ) Carbohydrate (g kg -1 ) Titratable acidity (% OAE) Tannin (mg g -1 CE)<br />
Seeds 59 ± 0.5 d 156 ± 4.1 a 122 ± 5.3 c 45 ± 0.4 d 619 0.17 ± 0.03 d 0.29 ± 0.09 a<br />
ZB2 134 ± 0.5 a 147 ± 0.7 b 181 ± 1.5 a 73 ± 3.1 b 465 1.47 ± 0.07 b 0.10 ± 0.06 b<br />
ZC2 103 ± 0.6 c 142 ± 4.1 c 177 ± 3 a 68 ± 1.3 c 511 1.87 ± 0.08 a 0.25 ± 0.08 a<br />
ZV2 131 ± 0.2 b 134 ± 0.5 d 151 ± 3.8 b 88 ± 3.6 a 491 0.77 ± 0.01 c 0.09 ± 0.01 b<br />
Results are expressed on dry weight basis. *Data are expressed as mean ± standard deviation (n = 3). Means values in a column with the same letter are not significantly different at 95% confidence.<br />
ZC2, ZV2 and ZB2, Dried maari samples ready for consumption collected respectively from producer C, V and B; CE, catechin equivalents; OAE, oleic acid equivalent.<br />
Table 2. Volatile compounds of cooked unfermented baobab seeds (UCS) and various samples of maari.<br />
RT<br />
Mean concentration (mg kg -1 Acid (6)<br />
Volatile compound UCS<br />
of product dry weight basis)<br />
ZV 1 ZV2 ZC1 ZC 2 ZB 1 ZB 2 DTA<br />
12.14 2,2-dimethyl propanoic acid 1.6 b - - - - 7.5 a - -<br />
14.67 2-methyl-Propanoic acid - - - - 13 a 0.7 b - -<br />
19.99 2-(aminooxy) pentanoic acid 1.3 b - - - - 4 a - -<br />
22.7 Benzoic acid - - - - - 1.0 a 0.7 a -<br />
38.78 Palmitic acid 2.9 f - 8.1 d 30.6 a 5.3 e 18.5 b - 14.7 c<br />
41.58 Oleic acid - 44.3 a 2.7 e 6.5 d - 14.4 b 0.9 f 8.7 c<br />
Total acids 5.83 44.34 10.78 37 18.35 46.18 1.6 23.39<br />
Alcohol (13)<br />
13.88 1-isopropoxy-2-methyl-2-Propanol 1 d 5.5 a - 4.4 b - 2.5 c - -<br />
14.91 3,3-Dimethyl-2-pentanol 0.2 b - - - - 6.8 a 6.4 a -<br />
18 2-methyl-3-hexanol 0.8 d 9.2 a 4.5 b - 1.5 c - - 1.1 c,d<br />
22.2 2,4-dimethyl-3-pentanol 3.6 f 86 a 19 c 50.3 b 14.3 d 4 f 4.3 f 11.3 e<br />
22.22 2,4,4-trimethyl-1-pentanol 0.5 d 75 a 11.5 c - 19.7 b - - -<br />
22.37 1,5-hexadien-3-ol 12 c - - 61.2 a - 48.1 b 13.7 c -<br />
22.43 5-methyl-4-octanol - 12.5 a - 3.9 b - - - -<br />
23.53 2-dodecanol 0.2 b - - - - 0.6 a - -<br />
25.07 2-pentadecyn-1-ol - - - 31.6 a 1.1 b - - -<br />
26.42 2,5-dimethyl-1,5-hexadiene-3,4-diol - - - - - 9.3 a 3.5 b -<br />
26.43 2-(hydroxymethyl)-2-methyl-1,3-propanediol 1.8 a - - - - 1.7 a - -<br />
30.93 1-hexadecanol - - - - - 1.2 a 1.0 b -
Table 2 cont.<br />
Parkouda et al. 4201<br />
45.11 2-hexyl-1-octanol 1.0 c 7.2 a - - - 1.5 b 0.8 c -<br />
Total alcohols 21.04 195.19 34.88 151.28 36.53 75.67 29.62 12.45<br />
Aldehyde (6)<br />
6.69 3-Bromopropionaldehyde Ethylene acetal - - - - - 30.6 b 15.6 c 44.1 a<br />
13.26 Heptanal - - - 4.8 a - - - 1.3 b<br />
17.58 Octanal - - 1.6 d 21.3 a - 3.7 c 0.2 e 4.3 b<br />
19.01 Benzeneacetaldehyde - - 22.6 a - 8.3 c 2.8 d 1.8 e 9.7 b<br />
26.41 (E,E)-2,4-nonadienal - - 2.9 b 56.5 a 3.6 b - - -<br />
26.42 2,4-Decadienal - - - 15.4 a - 1.2 c - 2.9 b<br />
Total aldehydes 0 0 27.1 97.95 11.86 38.3 17.61 62.35<br />
Alkane (2)<br />
45.52 Nonadecane - - 1.1 c 16.1 a 1.4 b 1 c,d 1 d -<br />
47.68 Tetratetracontane 0.9 d - - 8.3 a 0.8 e 1.7 b 0.6 f 1.3 c<br />
Total alkanes 0.9 0 1.11 24.35 2.22 2.69 1.51 1.3<br />
Alkenes(2)<br />
20.85 Z-1,9-hexadecadiene - - - 5.3 a 4.2 b - 1.8 c 5.1 a<br />
33.47 Z,Z,Z-4,6,9-Nonadecatriene - - 9.7 a 4.8 b 0.9 d - 1.9 c 1.5 c,d<br />
Total alkenes 0 0 9.7 10.08 5.06 0 3.7 6.55<br />
Ester (23)<br />
7.7 Methoxyacetic acid, pentyl ester 1.2 b - - - - - - 5.2 a<br />
13.96 Isobutyl isobutyrate 2.1 c 10.3 a - 9.4 b 2.4 9.2 a,b 2.6 c 2.1 c<br />
14.29 2-butanol, 2-nitrosoacetate 1.3 a - - - 0.8 b - - -<br />
14.74 Ethyl acetate 2.1 c - 2.6 c 10.3 a 3.5 b 3.4 b 3.4 b 2.4 c<br />
18.79 Vinyl butyrate 5.9 b - - 11.8 a 2.2 d 2.7 c 2.3 c 2.1 c,d<br />
24.87 2-phenylethyl acetate - - 1.1 c 15.2 a 1.3 b - - -<br />
24.98 di-tert-butyl dicarbonate 5 c 37.8 a 5 c 28 b 1.5 d 5.4 c 1 e 1.1 d,e<br />
38.11 14-methyl pentadecanoate - - 4.7 c 14.3 b 17.1 a - 2.2 d -<br />
38.12 Methyl hexadecanoate 0.3 d 4.7 c - - - 12 b - 13.3 a<br />
38.89 L-(+)-ascorbic acid 2,6-dihexadecanoate - 116.9 a - - - 64.1 b - -<br />
39.22 Ethyl hexadecanoate 0.9 f 26 b 14.6 d 9.7 d 7.9 e 109.5 a - 15.3 c<br />
40.36 Methyl 7,10-octadecadienoate - - - - - 4 a - 1.3 b<br />
40.72 Propyl hexadecanoate - - 1.5 b - - 7.8 a - -<br />
40.78 Methyl 9,12-octadecadienoate - 7 c 1.9 f 5.2 d 2.5 e 18.1 a - 7.7 b<br />
40.9 Methyl (Z)-9-octadecenoate 10.6 b 3 f 6.9 d 3.6 e 20.5 a 8.3 c
4202 Afr. J. Biotechnol.<br />
Table 2. cont.<br />
41.36 9,12-octadecadienoate - - - - - 52.5 - -<br />
41.83 Ethyl (Z,Z) 9,12-octadecadienoate - - 5.3 c - 2.3 d 33.1 a - 6.4 b<br />
41.84 Ethyl linoleate 0.5 b 17.8 a - - - 0.2 c - -<br />
41.87 Ethyl (E)-9-octadecenoate - - - 6.7 b - - - 11.1 a<br />
41.9 Ethyl oleate 0.6 f 8 c 8.2 b 4.6 d 3 e 68.4 a - 8 b,c<br />
42.21 Butyl hexadecanoate - 7.8 a - - - 0.7 b - -<br />
44.06 Isopropyl linoleate - 8.9 a - - - 2.5 b - -<br />
44.39 Decyl oleate - 11.8 a - - - 2.3 b - -<br />
Total esters 20.47 267.46 47.79 121.98 48.13 416.31 11.51 84.19<br />
Ketone (22)<br />
8.23 3,4-dimethyl-2-pentanone 9.1 b - - - - 10.5 b 10.3 b 15.8 a<br />
8.24 3-methyl-2,4-pentanedione - - 16.9 a - 15.3 a - - -<br />
8.96 3-ethyl-2-pentanone 3 d - 4.7 b 18 a 4.1 c 1 e 0.9 e 4.2 b,c<br />
9.77 4-methyl-2-hexanone 0.4 e - 4.2 b 26.1 a 3.5 c 2 d - 3.5 c<br />
14.31 3-methyl-2-hexanone 2.2 b 5.3 a - - 1.2 c 2.1 b - -<br />
14.81 3-hydroxy-2-butanone 2.8 a - - - - - 2.6 b 3 a<br />
15.94 2-chloro-3-methyl-1-phenyl-1-butanone - 26.7 a - - 7.8 b - - -<br />
15.95 2-chloro-acetophenone - - 17.9 b - 0.6 c - 16.5 b 21.1 a<br />
16.01 2,2-dihydroxy-1-phenyl-ethanone - - - - 1.7 b - - 7.4 a<br />
18.78 3-Hydroxybutanone - 74.1 a - - - - - 12.3 b<br />
20.48 2,5-dimethyl-4-hydroxy-3-hexanone 1.3 d 19 a - 8.2 b 4 c 1.2 d - -<br />
23.24 2-decanone - 27.9 a 5.5 c 14.6 b 1.9 e 2.6 e 1 f 4.1 d<br />
25.46 4,4,6-trimethyl-2-Cyclohexen-1-one - - - - 0.8 b - - 3.4 a<br />
25.75 2-undecanone - - 3.3 a - - - 2.7 b -<br />
26.88 3,5-dimethyl-2-octanone - 78.9 a 16.5 b 9.4 c 1 e 1.5 d - -<br />
26.89 3-methyl-2-heptanone - - - - 2.4 a - 0.9 b -<br />
28.05 2-dodecanone - 16.9 a - 2.6 b - 0.6 c - -<br />
29.12 1-cyclododecyl-ethanone - 14.9 a - - - 0.4 c 1 b -<br />
29.57 2,5-cyclohexadiene-1,4-dione 1.3 e 21.8 a 2.1 d 11.5 b - 7.3 c 0.9 f -<br />
30.21 2-tridecanone - - 6.7 a - - 1.7 b 1.4 b -<br />
33.91 Gamma. dodecalactone - 16.7 a 8.3 b - 1.6 c 0.8 d 0.6 d -<br />
34.16 2-nonadecanone - - 5.6 b 11.1 a 1.2 d 1 d 0.9 d 4.1 c<br />
Total ketones 20.12 302.1 91.55 101.49 46.91 32.65 39.67 78.91<br />
Pyrazine (7)<br />
17.65 Trimethyl-pyrazine - - - - - - - 2.4
Table 2 cont.<br />
Parkouda et al. 4203<br />
19.38 1-(2-tetrahydrofurylmethyl) pyrazine - - - - - 1.7 - -<br />
20.32 Tetramethyl-pyrazine - - - - - - 56.9<br />
21.79 2-isobutyl-3-methylpyrazine - - - - - 0.4 - -<br />
22.32 2,3,5-trimethyl-6-ethylpyrazine - - - - - - - 9.9<br />
25.36 2-(2-methylpropyl)-3,5,6-trimethylpyrazine - - - - - - - 1.4<br />
27.59 2,6-Bis(2-methylpropyl)pyrazine - - - - - 2.2 a 2 a -<br />
Total pyrazines 0 0 0 0 0 4.27 1.98 70.61<br />
Sulphur compounds (4)<br />
2.66 Thioacetic acid 29.3 b 52.3 a - - - - -<br />
4.52 Disulfide, dimethyl - - - - 3.6 b 4.1 a - -<br />
12.93 Diacetyl sulphide 0.8 b - - - - 4.3 a 0.5 c -<br />
16.08 Dimethyl trisulfide 1.5 d - - 7.4 b 7.8 a 3.1 c 3.9 c 1.7 d<br />
Others (11)<br />
Total sulphur compounds 31.63 0 52.28 7.44 11.48 11.56 4.44 1.71<br />
4.02 Tetrahydro-thiazole 20.3 a - - - - - 9.4 b -<br />
11.79 d-Threo-O-ethylthreonine 1.1 b - - 5.5 a - - 0.8 b 1.6 b<br />
16.92 Phenol - - 7.6 b - 9.3 a - - -<br />
17 2-pentyl-furan - - - 4.5 a - 1.2 b - -<br />
17.13 O-(3-methylbutyl)-hydroxylamine - - - - - 1.2 a - 1.4 a<br />
18.19 1-undecyne - - 5.5 b 21.3 a 5.1 b 0.1 c - -<br />
25.91 Indole - - 1.8 b - 9.1 a - - 1.5 c<br />
26.25 2-methoxy-4-vinylphenol - - - - - 1.2 a - 1.5 a<br />
35.73 Benzamide - - 1.2 b - - 1.9 a 1.2 a,b 1.6 a,b<br />
35.93 O-decyl-hydroxylamine 0.2 b - - - - - - 1.5 a<br />
37.88<br />
7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-<br />
Diene-2,8-dione<br />
- - - - - 3.6 a - 2.3 a<br />
Total others 21.64 0 16.14 31.32 23.49 9.15 11.35 11.41<br />
Total all compounds (96) 121.63 809.09 291.33 582.89 204.03 636.78 122.99 352.87<br />
Only compounds showing high quality index were retained. Values are means of triplicate determinations. Means with the same letters in the same compound in a row are not significantly different (P <<br />
0.05). –Compound not detected. UCS, Unfermented cooked beans; ZV1 and ZV2, maari samples from the producer V, collected respectively at the end of the fermentation (before the drying process)<br />
and after drying process; ZC1 and ZC2, maari samples from the producer C, collected respectively at the end of the fermentation (before the drying process) and after drying process; ZB1 and ZB2,<br />
maari samples from the producer B, collected respectively at the end of the fermentation (before the drying process) and after drying process; DTA, commercial sample collected at Toulfé in Burkina<br />
Faso; RT, retention time.
4204 Afr. J. Biotechnol.<br />
Table 3. Relative amounts of volatile compound groups identified in baobab unfermented cooked seeds and maari samples.<br />
Aroma compound<br />
group<br />
Relative amount of the compound groups (%) a<br />
UCS b ZV1 ZV2 ZC1 ZC2 ZB1 ZB2 DTA<br />
Acids 4.8 5.5 3.7 6.3 9.0 7.3 1.3 6.6<br />
Alcohols 17.3 24.1 12.0 26.0 17.9 11.9 24.1 3.5<br />
Aldehydes 0.0 0.0 9.3 16.8 5.8 6.0 14.3 17.7<br />
Alkanes 0.7 0.0 0.4 4.2 1.1 0.4 1.2 0.4<br />
Alkenes 0.0 0.0 3.3 1.7 2.5 0.0 3.0 1.9<br />
Esters 16.8 33.1 16.4 20.9 23.6 65.4 9.4 23.9<br />
Ketones 16.5 37.3 31.4 17.4 23.0 5.1 32.3 22.4<br />
Pyrazines 0.0 0.0 0.0 0.0 0.0 0.7 1.6 20.0<br />
Sulfur Compounds 26.0 0.0 17.9 1.3 5.6 1.8 3.6 0.5<br />
Others 17.8 0.0 5.5 5.4 11.5 1.4 9.2 3.2<br />
a % Concentration of volatile compounds relative to the total concentration of volatile compounds identified; b unfermented cooked<br />
baobab seeds– group not detected; ZV1 and ZV2, maari samples from the producer V, collected respectively at the end of the<br />
fermentation (before the drying process) and after drying process; ZC1 and ZC2, maari samples from the producer C, collected<br />
respectively at the end of the fermentation (before the drying process) and after drying process; ZB1 and ZB2, maari samples from<br />
the producer B, collected respectively at the end of the fermentation (before the drying process) and after drying process; DTA,<br />
commercial sample collected at Toulfé in Burkina Faso.<br />
(Rodríguez-Bernaldo De Quirós et al., 2000).<br />
This study showed that, the fermentation of the baobab<br />
seeds for maari production led to an increase of volatile<br />
compounds. Increase of volatile compounds in other<br />
fermented condiments during fermentation was also<br />
reported in previous studies (Azokpota et al., 2008;<br />
Ouoba et al., 2005; Owens et al., 1997). The formation of<br />
volatile compounds during fermentation observed is<br />
obviously due to the microorganisms involved in<br />
fermentation as well as the processing factors such as<br />
boiling or roasting (Azokpota et al., 2010; Dakwa et al.,<br />
2005; Ouoba et al., 2005; Yuliana and Garcia, 2009).<br />
Indeed, difference between the volatile compounds<br />
profiles of soumbala samples was reported to rely on the<br />
variable ability of the species of microorganism to<br />
degrade the proteins, lipids and carbohydrates of African<br />
locust beans (Ouoba et al., 2005). Comparing volatile<br />
compounds produced during spontaneous and controlled<br />
fermentation of afitin, iru and sonru, a very pronounced<br />
increase of the concentration of total volatile compounds<br />
was reported during the use of Bacillus subtilis as starter<br />
culture (Azokpota et al., 2010). This increase was<br />
explained by the use of pure culture (Azokpota et al.,<br />
2010; Leejeerajumnean et al., 2001). Investigating on<br />
processing factors, Dakwa et al. (2005) found pretreatment<br />
of soybeans by either boiling or roasting before<br />
fermentation to generate different volatile compounds<br />
profiles of soy-dawadawa.<br />
The amount of the total volatile compounds decreased<br />
during the drying process resulting in a loss of up to<br />
80.7% of the total aroma volatile compounds. A previous<br />
study on Bacillus fermented soybeans also reported a<br />
loss of aroma compounds during drying<br />
(Leejeerajumnean et al., 2001). This loss is attributable to<br />
most of the compounds simply evaporating during the<br />
open-air drying process. On the other hand, other volatile<br />
components like 2-undecanone, 2-chloro-acetophenone<br />
and 3-methyl-2-heptanone appeared in the dried maari<br />
and a few volatile compounds like dimethyl trisulfide were<br />
higher in the dried maari than in the fermented non-dried<br />
maari. These changes could probably be related to<br />
variable factors such as enzymatic activities, Maillard<br />
reaction, strecker degradation or microbial metabolism<br />
that could continue to occur during the drying process.<br />
Overall, the volatile compounds reported in this study<br />
are highly variable and confirmed the non-standard<br />
production practices among producers. The present<br />
results support earlier studies that show the diversity of<br />
aroma volatile compounds in fermented food condiments<br />
such as Beninese afitin, iru and sonru, Burkinabe soumbala,<br />
Ghanaian soy-dawadawa, Japanese natto, Korean<br />
chungkuk-jang and Thai thua-nao (Azokpota et al., 2010;<br />
Azokpota et al., 2008; Dakwa et al., 2005;<br />
Leejeerajumnean et al., 2001; Ouoba et al., 2005;<br />
Tanaka et al., 1998). For soumbala, Ouoba et al. (2005)<br />
revealed the presence of pyrazines, aldehydes, ketones,<br />
esters, alcohols, acids, alkanes, alkenes, benzenes, phenols,<br />
sulphurs, furans pyridines and amines. Amines and<br />
pyridines were not found in afitin, iru and sonru (Azokpota<br />
et al., 2008). In addition to the chemical groups found in<br />
soumbala, (excluding pyridines) in this study, the<br />
presence of alkynes compounds was reported. In natto<br />
and thua-nao, ketones, acids and pyrazines were reported<br />
to be the major volatile compounds (Owens et al.,<br />
1997). Ester, mainly formed by esterification of carboxylic<br />
acids and alcohols was reported to determine the<br />
characteristic pleasant aromatic notes (Klesk and Qian,<br />
2003; Rodríguez-Bernaldo De Quirós et al., 2000).<br />
According to this study, maari shows a low level of<br />
pyrazines contrary to what has been reported for the
African locust bean fermented condiments such as afitin,<br />
iru, sonru and soumbala (Azokpota et al., 2008; Ouoba et<br />
al., 2005). Similar to our results, low presence of<br />
pyrazines was also recorded in soy-dawadawa obtained<br />
by boiled soybeans compared with roasted soybeans<br />
(Dakwa et al., 2005). The formation of pyrazines is<br />
generally associated with heating (Owens et al., 1997).<br />
Shu (1998) revealed that, pyrazines are characterized by<br />
their extremely high volatility and reactivity. Due to the<br />
physical proprieties of baobab seeds, the seeds are<br />
intensively cooked for about 36 h prior to the fermentation;<br />
this may be explained as the difference observed<br />
in the pyrazines content in the present study. The<br />
presence of arginine was also reported to decrease the<br />
yield of the pyrazines production (Hwang et al., 1995). In<br />
others studies, arginine content was recorded to be<br />
higher in baobab seeds (2.21 mg g -1 ) than in African<br />
locust bean (0 mg g -1 ), (Konlani et al., 1999; Glew et al.,<br />
1997) which could also explain the low content of<br />
pyrazines in fermented baobab seeds.<br />
Diversity of the volatile components from each typical<br />
sample was observed. This difference may be explained<br />
by the specificity of each typical product giving specific<br />
compounds which are presumably more critical for the<br />
sensory characteristics as perceived (and probably<br />
expected) by consumers in tasting the product. In<br />
previous studies, the use of pure culture of microorganisms<br />
led to uniform final fermented product<br />
preferred by a panel of consumers (Ouoba et al., 2005;<br />
Azokpota et al., 2010). This study is one of the steps<br />
which could give information for a future selection of<br />
starter culture to be used in controlled fermentation.<br />
ACKNOWLEDGEMENTS<br />
This work was supported by the Danida (Danish<br />
International Development Agency) funded project titled<br />
“Improving food potential in West African parkland trees”.<br />
Special thanks to the women who helped us with the<br />
maari processing during the study.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4207-4211, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2476<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Antioxidant activities of Rosmarinus officinalis L.<br />
essential oil obtained by hydro-distillation and solvent<br />
free microwave extraction<br />
Okoh O. O. 1 , Sadimenko A. P. 1 and Afolayan A. J. 2 *<br />
1 Department of Chemistry, University of Fort Hare Alice, 5700, South Africa.<br />
2 Department of Botany, University of Fort Hare Alice, 5700, South Africa.<br />
Accepted March 3, 2011<br />
The essential oils of Rosmarinus officinalis L. growing in a rural area within the Nkonkobe Municipality<br />
of the Eastern Cape, South Africa, were extracted using the solvent free microwave extractor (SFME)<br />
and hydro-distillation (HD) methods. The antioxidant and free radical scavenging activity of the<br />
obtained oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH + ) assay and βcarotene<br />
bleaching test. In the DPPH + assay, while the free radical scavenging activity of the oil<br />
obtained by SFME method showed percentage inhibitions of 48.80, 61.60 and 67.00%, the HD oil<br />
showed inhibitions of 52.20, 55.00 and 65.30% at 0.33, 0.5 and 1.00 mg/ml, respectively. In the βcarotene<br />
bleaching assay, the percentage inhibition increased with increasing concentration of both<br />
oils, with a high antioxidant activity of the oil obtained through the SFME than through the HD method.<br />
The significance of this observation is discussed with respect to the properties of essential oils<br />
obtained using different methods.<br />
Key words: Essential oil, antioxidant, hydrodistillation, solvent free microwave extraction; Rosmarinus<br />
officinalis.<br />
INTRODUCTION<br />
Plants, especially herbs and spices, have many phytochemicals<br />
which are potential sources of natural antioxidants.<br />
These include phenolic diterpenes, flavonoids,<br />
tannins and phenolic acids (Dawidowicz et al., 2006).<br />
Some antioxidants have been widely used as food<br />
additives to protect food against oxidative degradation by<br />
free radicals. Spices which are used in different types of<br />
food to improve flavors are well known for their<br />
antioxidant properties (Madsen and Bertelsen, 1995).<br />
Recently, there has been increasing interest in the use of<br />
natural antioxidants such as tocopherols, flavonoids and<br />
extracts from rosemary (Rosmarinus officinalis L.) for<br />
food preservation (Hras et al., 2000; Bruni et al., 2004;<br />
Williams et al., 2004; Fruitos and Hernandez-Herrero,<br />
2005). According to these authors, these natural<br />
*Corresponding author. E-mail: aafolayan@ufh.ac.za. Fax:<br />
+27866282295.<br />
antioxidants do not present health problems that may<br />
arise from the use of synthetic antioxidants such as<br />
butylated hydroxy anisole (BHA), butylated hydroxy<br />
toluene (BHT) and propyl gallate (PG) which have side<br />
effects (Amarowicz et al., 2000; Aruoma et al., 1992).<br />
Antioxidants are compounds that neutralize chemically<br />
active products of metabolism, such as free radicals<br />
which can damage the body. It has been documented<br />
that plant phenols, with their potential to act as<br />
antioxidants, play a major role in the prevention of<br />
cancer, cardiovascular and neurogenerative diseases<br />
which are believed to be caused by oxidative stress<br />
(Losso et al., 2007).<br />
R. officinalis L. is a perennial herb with ever-green<br />
needle like leaves that belongs to the Lamiaceae family.<br />
The Lamiaceae is a large family, rich in aromatic species<br />
that are used as culinary herbs, in folk medicines and<br />
fragrances, and many members of this family possess<br />
essential oils that are secreted by glandular trichomes<br />
(Marin et al., 2006). Previous studies have shown that rose-
4208 Afr. J. Biotechnol.<br />
mary essential oil have antimicrobial, antioxidant, anticarcinogenic,<br />
cognition-improving and certain glucose<br />
level lowering properties which make it useful as a<br />
natural animal feed additive (Fahim et al., 1999;<br />
Debersac et al., 2001; Fu et al., 2007).<br />
Whilst the general antioxidant potential of R. officinalis<br />
essential oil have been reported before (Gachkar et al.,<br />
2007), there is no information on the possible effect of the<br />
method of extraction of the essential oils on the<br />
antioxidant property of this herb. The biological and<br />
phytochemical properties of essential oils extracted<br />
through different methods have been found to depend on<br />
the extraction method (Okoh et al., 2010). In this paper,<br />
we report the effects of solvent free microwave extraction<br />
and hydro-distillation extraction methods on the<br />
antioxidative properties of the essential oils of R.<br />
officinalis. The effects of these two extraction methods on<br />
the chemical compositions and antimicrobial activities of<br />
the oils have been previously reported in our laboratory<br />
(Okoh et al., 2010).<br />
MATERIALS AND METHODS<br />
All solvents and reagents used in these experiments such as<br />
methanol, chloroform and ethanol were of analytical grade and<br />
together with 2,2-diphenyl-2-picylhydrazyl hydrate (DPPH), 3,5-ditert-butyl-4-hydroxytoluene<br />
(BHT) -tocopherol, gallic acid (GA),<br />
rutin, β-carotene, linoleic acid and Tween 40, were all purchased<br />
from Sigma Aldrich (Pty) Ltd- Johannesburg, South Africa.<br />
Collection of plant material and extraction of the essential oil<br />
Collection, processing of the plant materials and extraction of the<br />
essential oils using SFME and HD methods were in accordance<br />
with our previous report (Okoh et al., 2010). Briefly, two samples of<br />
fresh leaves (250 g each) of R. officinalis were collected in<br />
February, 2009 from the vicinity of the University of Fort Hare, Alice<br />
campus in the Eastern Cape Province of South Africa (latitudes<br />
30°00-34°15S and longitudes 22°45-30°15E). The plant was<br />
identified by Prof Don Grierson of the Botany Department,<br />
University of Fort Hare, Alice and a voucher specimen (Okoh/10)<br />
was deposited at the University herbarium.<br />
SFME was carried out with a Milestone DryDIST (2004)<br />
apparatus. The multimode reactor has a twin magnetron (2 × 800<br />
W, 2450 MHz) with a maximum delivered power of 500 W in 5 W<br />
increments. A rotating microwave diffuser ensures homogeneous<br />
microwave distribution throughout the plasma coated PTFE cavity.<br />
The temperature was monitored by an external IR sensor. Constant<br />
conditions of temperature and water were guaranteed by the reflux<br />
of condensed water, which was achieved by a circulating cooling<br />
system at 5°C. Two hundred and fifty grams (250 g) of the plant<br />
leaves were placed into the reactor without addition of water or any<br />
solvent. The exhaustive extraction of the essential oil was obtained<br />
in 40 min.<br />
For the HD, 250 g of the plant leaves were hydrodistilled for 3 h<br />
in an all-glass Clevenger apparatus in accordance with the<br />
description of the British Pharmacopoeia (1980). Heat was supplied<br />
to the heating mantle (50°C) and the essential oil was extracted<br />
with 4 L of water for 3 h (until no more essential oil was released).<br />
The oils collected from both extraction methods were analyzed<br />
immediately after each collection using GC-MS. The compositions<br />
of these oils have been previously reported (Okoh et al., 2010).<br />
Antioxidant activity<br />
DPPH radical scavenging activity<br />
The method of Liyana-Pathiranan and Shahidi (2005) was used for<br />
the determination of scavenging activity of DPPH free radical in the<br />
essential oils. A solution of 0.135 mM DPPH in methanol was<br />
prepared and 1.0 ml of this solution was mixed with 1.0 ml of oil<br />
prepared in methanol containing 0.025 to 0.5 mg of the essential oil<br />
and standard drugs (BHT and rutin). The reaction mixture was<br />
vortexed thoroughly and left in the dark at room temperature for 30<br />
min. The absorbance of the mixture was measured spectro-photometrically<br />
at 517 nm. The ability of the plant essential oils to<br />
scavenge DPPH radical was calculated by the following equation:<br />
DPPH radical scavenging activity = {(Abscontrol – Abssample)} / (Abs<br />
control} × 100<br />
where, Abscontrol is the absorbance of DPPH radical + methanol,<br />
Abssample is the absorbance of DPPH radical + sample oil /standard.<br />
β- carotene bleaching assay<br />
This method evaluates the capacity of the oil to reduce the<br />
oxidative loss of β-carotene in a β-carotene linoleic acid emulsion<br />
(Taga et al., 1984). β-Carotene (10 mg) was dissolved in 10 ml of<br />
chloroform (CHCl3). An aliquot (0.2 ml) of this solution was added<br />
into a boiling flask containing 20 mg of linoleic acid and 200 mg of<br />
Tween 40. The chloroform was removed using a rotary evaporator<br />
at 40°C for 5 min. Distilled water (50 ml) was slowly added to the<br />
residue with vigorous agitation, to form an emulsion. The emulsion<br />
was added to a tube containing 0.2 ml of essential oil. The<br />
absorbance was immediately measured at 470 nm and the test<br />
emulsion was incubated in a water bath at 50°C for 5 min, after<br />
which the absorbance was measured again. BHT was used as the<br />
positive control. In the negative control, the essential oils were<br />
substituted with an equal volume of ethanol. The antioxidant activity<br />
(%) of the oil was evaluated in terms of the bleaching of the βcarotene<br />
using the following formula:<br />
% inhibition = {(At-Ct) / (CO-Ct)} × 100<br />
where, At and Ct are the absorbances measured for the oil and<br />
control, respectively, after incubation for 5 min, and CO is the<br />
absorbance values for the control measured at zero time during the<br />
incubation.<br />
The oil concentration providing 50% antioxidant activity (EC50)<br />
was calculated by plotting antioxidant percentage against oil<br />
concentration.<br />
RESULTS AND DISCUSSION<br />
The principle of antioxidant activity is based on the<br />
availability of electrons to neutralize free radicals. In this<br />
study, the antioxidant activity of R. officinalis oil was<br />
evaluated by two complementary tests: scavenging of<br />
DPPH + free radicals and the β-carotene bleaching test.<br />
The results are as shown in Figures 1 and 2, respectively.<br />
The free radical scavenging activity of essential oil of R.<br />
officinalis obtained by SFME revealed percentage<br />
inhibitions of 48.80 , 61.60 and 67.00%, while that of the<br />
HD oil showed percentage inhibitions of 52.20, 55.00 and
Inhibition of beta carotene (%)<br />
Inhibition of DPPH + (%)<br />
65.30% both at concentrations of 0.33 , 0.5 and 1.0<br />
mg/ml, respectively. These results show that activity<br />
increases as the concentrations of the oils were<br />
increased, at least within the limit of the test concentrations<br />
of the oils (Figure 1). The SFME essential oil<br />
showed a slightly higher DPPH-radical scavenging<br />
activity (IC50 = 0.34 mg/ml) than the hydrodistilled<br />
essential oil (IC50 = 0.46 mg/ml), whereas for BHT, IC50<br />
Figure 1. Free radical-scavenging activity of R. officinalis<br />
essential oil evaluated by the 1,1-diphenyl-2-picrylhydrazyl<br />
(DPPH).<br />
Figure 2. Antioxidant activity of R. officinalis L. essential oil<br />
determined by β-carotene bleaching test.<br />
Okoh et al. 4209<br />
was 0.22 mg/ml at concentration of 0.33 mg/ml.<br />
The result of lipid peroxidation inhibitory activity of the<br />
essentials oils, assessed by the β-carotene bleaching test<br />
are shown in Figure 2. β-Carotene usually undergoes<br />
rapid discoloration in the absence of an antioxidant. This<br />
is because the oxidation of β-carotene and linoleic acid<br />
generates free radicals (Jayaprakasha et al., 2001). The<br />
linoleic acid free radical formed upon the abstraction of a
4210 Afr. J. Biotechnol.<br />
hydrogen atom from one of its diallylic methylene groups<br />
attacks the highly unsaturated β-carotene molecule,<br />
hence β-carotene is oxidized, losing its orange color<br />
which is then monitored spectrophotometrically (Shon et<br />
al., 2003). The results obtained from this assay are<br />
similar to the data obtained from DPPH test. The<br />
percentage inhibitions were 48.62, 68.06 and 86.79% for<br />
hydrodistilled oil; and 35.87, 76.29 and 91.19% for<br />
solvent free microwave extracted oil at concentrations of<br />
0.33, 0.5 and 1.0 mg/ml, respectively. Although, both oils<br />
prevented the bleaching of β-carotene, the SFME<br />
extracted oil had a slightly higher activity than the HD<br />
extracted oil. These results are also consistent with the<br />
results obtained from DPPH test. The concentrations<br />
providing 50% inhibition were 0.338, 0.282, and 0.22<br />
mg/ml for HD, SFME and BHT, respectively. It was noted<br />
that the antioxidant activities of the tested samples were<br />
dependent on their concentrations. The IC50 of SFME was<br />
however lower than that of HD essential oil.<br />
In plant essential oils, oxygenated monoterpenes and<br />
monoterpene hydrocarbons are mainly responsible for<br />
the antioxidant potential (Ruberto and Baratta, 2000), and<br />
in our previous findings (Okoh et al., 2010) on the<br />
chemical constituents component of this study, we<br />
reported that oxygenated monoterpenes and monoterpene<br />
hydrocarbons were the main components of R.<br />
officinalis essential oil.<br />
Many reports on the investigations of the activity of R.<br />
officinalis have shown that there are biologically active<br />
compounds in R. officinalis essential oil that exhibit<br />
cytotoxic, antioxidant, anti-carcinogenic and cognitionenhancing<br />
properties (Okamura et al., 1994; Frankel et<br />
al., 1996; Thorsen and Hildebrandt, 2003). These<br />
compounds that have the potential to influence glucose<br />
level in diabetic patients, modify rumen microbial<br />
fermentation and enhance bone desorption, but do not<br />
enhance immune response (Oluwatuyi et al. 2004; Wang<br />
et al., 2008). Essential oils, despite their wide uses and<br />
fragrances, constitute effective alternatives to synthetic<br />
compounds produced by chemical industry without<br />
showing the same side effects as the latter (Faixova and<br />
Faix, 2008). In this study, both SFME and HD extracted<br />
oil exhibited remarkable antioxidant activities. In general,<br />
the essential oil obtained from SFME showed greater<br />
activity than the one obtained from HD.<br />
It is very difficult to attribute the antioxidant effect of a<br />
total essential oil to one or a few active principles<br />
because an essential oil always contains a mixture of<br />
different chemical compounds. In addition to the major<br />
compounds, minor molecules may make significant<br />
contributions to the oil activity. Therefore, the antioxidant<br />
property of the oils from this herb might be the combined<br />
activities of the various major and minor components of<br />
the oils.<br />
In conclusion, the results of this study showed some<br />
differences in the antioxidant activities and chemical<br />
composition of the essential oils obtained by SFME and<br />
HD extraction methods. The SFME extracted oil showed<br />
a higher activity than that obtained by HD. This is probably<br />
due to the higher proportions of oxygenated compounds<br />
in SFME extracted oils. According to Okoh et al. (2010),<br />
higher amounts of oxygenated compounds with lower<br />
amounts of monoterpene hydrocarbons were present in<br />
the essential oils of this plant isolated by SFME in<br />
comparison with the oil obtained by traditional hydrodistillation.<br />
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and prooxidant properties of active rosemary constintuents: Carnosol<br />
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Debersac P, Heydel JM, Amiot MJ, Goudonnet H, Artur Y, Suschetet M,<br />
Siess MH (2001). Induction of cytochrome P 450 and/or detoxication<br />
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patterns. Fd. Chem. Toxicol. 39: 907-918.<br />
DryDIST (2004). Milestone srl, Via Fratebenefratelli, 1/5, I-24010<br />
Sorisole, Bergamo, Italy. Available from www.milestonesrl.com.<br />
Fahim FA, Esmat AY, Fadel HM, Hassan KF (1999). Allied studies on<br />
the effect of Rosmarinus officinalis L. on experimental hepatotoxicity<br />
and mutagenesis. Intl. J. Food. Sci. Nutri. 50: 413-427.<br />
Faixova Z, Faix S, S (2008). Biological effects of rosemary (Rosmarinus<br />
officinalis L. essential oil (A Review) .University of Veterinary<br />
Medicine, Komenskho 73, 041 81 Kosice lnstitute of Animal<br />
Physiology Slovak Academy of Sciences, Soltesovej 4, 040 0l,<br />
Kosice, The Slovak Republic.<br />
Frankel EN, Huang SW, Aeschbach R, Prior E (1996). Antioxidant<br />
activity of a rosemary extract and its constituents, carnosic acid,<br />
carnosol and rosmarinic acid in bulk oil and oil in water emulsion. J.<br />
Agri. Food. Chem. 44: 131-135.<br />
Fruitos MJ, Hernandez–Herrero JA (2005). Effects of rosemary extract<br />
(Rosmarinus officinalis) on the stability of bread with an oil, garlic and<br />
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651-655.<br />
Fu Y, Zn Y, Chen L, Shi X, Wang Z, Sun S, Efferth T (2007).<br />
Antimicrobial activity of clove and rosemary essential oils alone and<br />
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Gachkar L, Yadegari D, Rezaei MB, Taghizadeh M, Alipoor AS, Rasooli<br />
I (2007). Chemical and biological characteristics of Cuminum<br />
cyminum and Rosmarinus officinalis essential oils. Food. Chem. 102:<br />
898-904.<br />
Hras AR, Hadolin M, Knez Z, Bauman D (2000). Comparison of<br />
antioxidative and synergistic effects of rosemary extract with<br />
tocopherol, ascorbyl palmitate and citric acid in sunflower oil. Food.<br />
Chem. 71: 229-233.<br />
Jayaprakasha GK, Singh RP, Sakariah, KK (2001). Antioxidant activity<br />
of grape seed (Vitis vinifera) extracts on peroxidation models in vitro.<br />
Food. Chem. 73: 285–290.<br />
Liyana- Pathiranan CM, Shahidi F (2005). Antioxidant activity of<br />
commercial soft and hard wheat (Triticum aestivum L) as affected by<br />
gastric conditions. J. Agric. Food . Chem.. 53: 2433-2440.<br />
Losso JN, Shahidi F, Bagchi D (2007). Anti-angiogenic functional and<br />
medicinal foods. Boca Raton, FL: Taylor & Francis.<br />
Madsen HL, Bertelsen G (1995). Spices as antioxidants. Trds. Food<br />
Sci. Tech. 6: 271-277.<br />
Marin M, Koko V, Duletic-Lausevic S, Marin PD, Rancic D, Dajic–<br />
Stevanovic Z (2006). Glandular trichomes on the leaves of<br />
Rosmarinus officinalis: Morphology, stereology and histochemistry.<br />
South Afr. J. Bot. 72: 378-382.<br />
Okamura N, Fujimoto Y, Kuwabara S, Yagi A (1994). High performance<br />
liquid chromatographic determination of carnosic acid and carnosic
acid and carnosol in Rosmarinus officinalis and salvia officinalis. J.<br />
Chrom. A, 679: 381-386.<br />
Okoh OO, Sadimenko AP, Afolayan AJ (2010). Comparative evaluation<br />
of the antibacterial activities of the essential oils of Rosmarinus<br />
officinalis L. obtained by hydrodistillation and solvent free microwave<br />
extraction methods. Food Chem. 120: 308-312.<br />
Oluwatuyi M, Kaatz GW, Gibbons S (2004). Antibacterial and resistance<br />
modifying activity of Rosmarinus officinalis. Phytochem. 65(24):<br />
3249-3254.<br />
Ruberto G, Baratta MT (2000). Antioxidant activity of selected essential<br />
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838-849.
African Journal of Biotechnology Vol. 10(20), pp. 4212-4216, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2656<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Effects of bagging on sugar metabolism and the activity<br />
of sugar metabolism related enzymes during fruit<br />
development of Qingzhong loquat<br />
Zhaojun Ni 1 , Zhen Zhang 1 , Zhihong Gao 1 *, Linping Gu 2 and Leifang Huang 2<br />
1 College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, People’s Republic of China.<br />
2 Service Centre of Agriculture and Forestry, Suzhou, Jiangsu, People’s Republic of China.<br />
Accepted 17 March, 2011<br />
To investigate the effects of bagging on sugar metabolism and the activity of sugar metabolism related<br />
enzymes in Qingzhong loquat fruit development, the contents of sucrose, glucose and soluble solids as<br />
well as the activities of sugar metabolism related enzymes were evaluated. The content of sucrose,<br />
glucose and soluble solids increased, while the content of fructose, sorbitol and titratable acidity<br />
decreased in ripe fruit in response to bagging. In addition, the activities of acid invertase (AI) and<br />
neutral invertase (NI) in the bagged fruit were lower than that in the non-bagged fruit, and the activities<br />
of sucrose synthase (SS) and sucrose-phosphate synthase (SPS) in the bagged fruit were higher than<br />
that in the non-bagged fruit. The activities of SDH (sorbitol dehydrogenase) and SOX (sorbitol oxidase)<br />
in the bagged fruit were lower than those in the non-bagged fruit, but there was no significant<br />
difference, whereas the activities of SS and SPS in the bagged fruit were significantly higher than that<br />
in the non-bagged fruit, suggesting that bagging mainly increased the products of photosynthesis by<br />
enhancing the activities of SS and SPS.<br />
Key words: Loquat, bagging, sugar metabolism.<br />
INTRODUCTION<br />
Bagging is an effective method of improving fruit quality<br />
in fruit production and has been widely used to improve<br />
fruit appearance, decrease pesticide residues and<br />
increase commercial value. Moreover, bagged fruit is<br />
preferred by the consumers. Yang et al. (2009) proved<br />
that bagging could modify the microenvironment during<br />
fruit development, decreasing the rate of fruit drop and<br />
reducing the content of organic acid in longan fruit. Kim et<br />
al. (2008) reported that the appearance of bagged<br />
peaches improved, while the content of soluble solids,<br />
anthocyanin and chlorophyll increased in white coloured<br />
*Corresponding author. E-mail: gaozhihong@njau.edu.cn. Tel:<br />
+86 2584395724. Fax: +86 2584395266.<br />
Abbreviations: AI, Acid invertase; NI, neutral invertase; SS,<br />
sucrose synthase; SPS, sucrose-phosphate synthase; SDH,<br />
sorbitol dehydrogenase; SOX, sorbitol oxidase.<br />
bags. Lin et al. (2008) demonstrated that bagged pear<br />
fruits were more attractive with less russet and visible<br />
dots than non-bagged fruit, while the residues of<br />
chlorpyrifos carbendazim and cyhalothrin were also greatly<br />
reduced. Consumers also showed preference for bagged<br />
fruit.<br />
Loquat (Eriobotrya japonica Lindl.) originated in China,<br />
is distributed in Zhejiang, Fujian and Jiangsu Province<br />
(Qiu and Zhang, 1996). ‘Qingzhong’ loquat is a local<br />
cultivar from Suzhou, Jiangsu Province. The fruit has<br />
good flavour and storage ability. Xu et al. (2008, 2010)<br />
and Feng et al. (2009) studied the effects of bagging on<br />
‘Baiyu’ loquat and ‘Ninghaibai’ loquat fruit quality and<br />
antioxidative capacity. However, the effects of bagging on<br />
sugar metabolism during fruit development were not<br />
explored. The purpose of this study was to determine the<br />
effects of bagging on sugar metabolism and the activities<br />
of related enzymes during loquat fruit development, and<br />
establish a basis for the improvement of this cultivation<br />
technique.
MATERIALS AND METHODS<br />
Materials<br />
Thirty-year-old ‘Qingzhong’ loquat trees grown at Suzhou, Jiangsu<br />
Province were used in this project. The fruits were bagged (white,<br />
single-layer sulphuric acid paper) about 40 days after initial fruit set,<br />
with bagging 80 fruits per plant. Ten fruits were sampled every 15<br />
days until harvest. Sampling was repeated three times. Flesh was<br />
separated from the fresh fruit for each treatment and frozen at -<br />
70°C for further study.<br />
Sugar content<br />
Dried fruit (0.1 to 0.2 g) was powdered in a chilled mortar with 5 ml<br />
of distilled water. The homogenate was centrifuged at 5000 ×g for<br />
10 min. The supernatant was poured into a Sephadex G-25 column<br />
and re-extracted once. The supernatants were combined and the<br />
volume was increased to 25 ml. The contents of sucrose, glucose<br />
and fructose were determined by the anthrone colorimetric method.<br />
Sampling was repeated three times.<br />
Frozen fruit was powdered in a mortar chilled with liquid nitrogen.<br />
Two grams of fruit were added to 7 ml 70% ethanol and mixed into<br />
a homogenate for about 20 min in 35°C water. Then, the<br />
homogenate was centrifuged at 10, 000 ×g for 5 min. The<br />
supernatant was poured into a Sephadex G-25 and re-extracted<br />
once. The supernatants were combined and the volume increased<br />
to 25 ml. A 2 ml sample of supernatant was evaporated to dryness<br />
and dissolved with 1 ml of distilled water. The content of sorbitol<br />
was quantified by HPLC (high performance liquid chromatography,<br />
Agilent 1100). A 15 µl aliquot was passed through a CA column (10<br />
µm diameter). Flowing water was used for the mobile phase. The<br />
temperature of the column was 85°C. Sampling was repeated three<br />
times.<br />
Enzyme activity<br />
Extraction and determination of acid invertase (AI), neutral<br />
invertase (NI), sucrose synthase (SS) and sucrose-phosphate<br />
synthase (SPS) were determined by the method described by<br />
Nielsen et al. (1991). Frozen fruit was powdered in a mortar chilled<br />
with liquid nitrogen. Fruit (1 g) was added to 5 ml of pre-cooled<br />
extraction buffer (0.2 M Tris-Hcl buffer, pH = 8.8) containing 25 mM<br />
MgCl2 and 15 mM NaN3, and made into a homogenate in an icebath.<br />
Then, the homogenate was centrifuged at 10,000 ×g for 15<br />
min. The supernatant was poured into a Sephadex G-10 and reextracted<br />
once. The supernatants were combined and the volume<br />
was increased to 10 ml, which was used as the enzyme extract.<br />
Sampling was repeated three times.<br />
The activity of sorbitol dehydrogenase (SDH) was determined by<br />
the following method: Enzyme solution (150 l) and 1.05 ml of 25<br />
mM NAD + were combined, to which 1.05 ml of 150 mM D-sorbitol<br />
was added. The absorbance was determined at 340 nm. In the<br />
enzyme-free reaction system, 150 l of 0.2 M Tris-HCl (pH = 8.8)<br />
was used as the control instead of enzyme solution. The treatment<br />
was repeated three times. The units (OD • mL –1 • min –1 ) of SDH<br />
activity was the change of absorbance of 1 ml reaction solution per<br />
minute. The activity of SOX (sorbitol oxidase) was determined by<br />
the method of Yamaki (1980) and Yamaki and Ishikawa (1986).<br />
Statistical analysis<br />
Statistical analysis was carried out using analysis of variance and<br />
the significance was determined using SPSS 16.0 (Statistical<br />
package for the social science) with LSD (least significant<br />
difference) values at P < 0.05.<br />
RESULTS<br />
Effects of bagging on the content of sugar<br />
Ni et al. 4213<br />
The glucose content in ripe fruit increased in response to<br />
bagging (P < 0.05), while the levels of sorbitol and titratable<br />
acid decreased (Table 1). Soluble solid to acidity ratio<br />
increased in bagged fruit. The contents of sucrose,<br />
fructose and glucose in the bagged and non-bagged fruit<br />
increased with fruit development (Figure 1 A, B and C).<br />
The content of fructose in the bagged fruit was slightly<br />
lower than that in the non-bagged fruit before 70 days<br />
after initial fruit set and decreased by 2.01 mg·g -1 in ripe<br />
fruit when compared with control. The amount of sucrose<br />
and glucose in the bagged fruit were slightly lower than<br />
those in the non-bagged fruit before 85 days after initial<br />
fruit set, after which the amounts were higher for bagged<br />
than non-bagged fruit. The contents of sucrose and<br />
glucose were higher in bagged than in non-bagged fruits<br />
by 1.30 mg·g -1 and 2.75 mg·g -1 in ripe fruit, respectively.<br />
The accumulation of sorbitol in the bagged fruit was<br />
very similar to that in non-bagged fruit during fruit development,<br />
decreasing at first and then increased during<br />
development. The content of sorbitol in the bagged fruit<br />
was lower than that found in the non-bagged fruit (Figure<br />
1D).<br />
Effects of bagging on the activities of sucrose<br />
metabolism related enzymes<br />
Invertase belongs to an enzyme class in which the<br />
regulation of sucrose conversion into fructose and<br />
glucose in the plant is mediated by NI and AI (Ni et al.,<br />
2009). The activities of NI and AI in the bagged fruit were<br />
quite similar during fruit development (Figure 2A and B).<br />
The activities of NI and AI in the non-bagged fruit initially<br />
increased faster and then more slowly until the fruit<br />
ripened. The activity of AI was slightly higher in the<br />
bagged fruit than in the non-bagged fruit when fruit<br />
ripening approached.<br />
SS is an enzyme that plays a key role in sucrose<br />
decomposition in the course of sucrose metabolism and<br />
SPS is a key enzyme that regulates sucrose synthesis in<br />
the plant (Zheng et al., 2006). The activity of SS in the<br />
bagged fruit increased during fruit development and was<br />
higher than that in the non-bagged fruit (Figure 2C). The<br />
change in SS and SPS activities in the bagged and nonbagged<br />
fruit was almost the same before 85 days after<br />
the initial fruit set. After this date, the activities of SS and<br />
SPS in the bagged fruit increased more rapidly (Figure<br />
2D).<br />
Effects of bagging on the activities of sorbitol<br />
metabolism related enzymes<br />
SDH is an enzyme that catalyses the conversion of sorbitol
4214 Afr. J. Biotechnol.<br />
Table 1. Effects of bagging on the contents of sugar, sucrose, fructose, glucose, sorbitol, soluble solids and titratable acidity in fruit of<br />
Qingzhong loquat.<br />
Treatment<br />
Content<br />
of sugar<br />
(mg·g – 1 )<br />
Content of<br />
sucrose<br />
(mg·g – 1 )<br />
Content of<br />
fructose<br />
(mg·g – 1 )<br />
Content of<br />
glucose<br />
(mg·g – 1 )<br />
Content of<br />
sorbitol<br />
(mg·g – 1 )<br />
Soluble<br />
solid<br />
(%)<br />
Titratable<br />
acidity<br />
(%)<br />
Solid/acid<br />
ratio<br />
Control 60.04 b 21.62 a 24.46 a 7.55 b 6.41 a 14.35 a 0.45 a 31.89<br />
Bagged 57.95 a 22.92 a 22.45 a 10.3 a 2.28 b 15.13 a 0.35 b 43.23<br />
The different letters indicate significant difference at 0.05 level; sugar = sucrose + fructose + glucose + sorbitol. Solid/acid ratio represents the<br />
ratio of soluble solid to titratable acidity.<br />
Content of sucrose(mg.g-1)<br />
Content of glucose(mg.g-1)<br />
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Content of fructose(mg.g-1)<br />
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Figure 1. Effects of bagging on the content of (A) sucrose, (B) fructose, (C) glucose and (D) sorbitol in fruit<br />
of Qingzhong loquat.<br />
to fructose and SOX is an enzyme that catalyses the<br />
conversion of sorbitol to glucose (Liang et al., 2004). The<br />
activities of SDH and SOX in the bagged and nonbagged<br />
fruit were quite similar during fruit development<br />
and decreased before 85 days after initial fruit set before<br />
increasing slowly until the fruit ripened (Figure 3A and B).<br />
The correlation between sugar accumulation and<br />
activities of sucrose and sorbitol-metabolizing<br />
enzymes during fruit development<br />
Sucrose content displayed a very significant positive<br />
correlation with the activities of SS and SPS during<br />
bagged fruit development, while glucose content also<br />
positively correlated very significantly with the activity of<br />
sucrose-metabolizing enzymes. Sorbitol content also<br />
exhibited a significant positive correlation with the activity<br />
of SDH. The content of sucrose, fructose, glucose and<br />
the activity of sucrose-metabolizing enzymes during nonbagged<br />
fruit development showed a very significant<br />
positive correlation, suggesting that bagging affects the<br />
accumulation of fructose and the activity of sucrosemetabolizing<br />
enzymes (Table 2).<br />
DISCUSSION<br />
The results demonstrated that average fruit weight in the<br />
bagged fruit was lower than that in non-bagged fruit. The
Activity of neutral invertase<br />
(mg.g-1FW.h-1)<br />
Activity of sucrose synthase<br />
(mg.g-1 FW.h-1)<br />
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control � bagged fruit<br />
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Activity of acid invertase<br />
(mg.g-1FW.h-1)<br />
Acticity of sucrose phosphate<br />
synthase<br />
(mg/g.h FW)<br />
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Figure 2. Effects of bagging on the activities of (A) neutral invertase, (B) acid invertase, (C) sucrose synthase and (D)<br />
sucrose-phosphate synthase in fruit of Qingzhong loquat.<br />
Activity of sorbitol dehydrogenase<br />
(OD.ml-1.min-1FW)<br />
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Activity of sorbitol oxidase<br />
(mg.g-1FW.h-1)<br />
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Ni et al. 4215<br />
Figure 3. Effects of bagging on the activities of (A) sorbitol dehydrogenase and (B) sorbitol oxidase in fruit of Qingzhong loquat.<br />
trend in sucrose, fructose, glucose and sorbitol accumulation<br />
were quite similar but the rates of accumulation<br />
were not synchronous in the bagged and non-bagged<br />
fruit during fruit development. The accumulation of<br />
sucrose and glucose in the bagged fruit increased, while<br />
the accumulation of fructose and sorbitol decreased. The<br />
content of soluble solids in the bagged fruit increased and<br />
the content of titratable acidity decreased when compared<br />
with the non-bagged fruit. These results coincide with<br />
what Xu et al. (2008, 2010) report on the ‘Baiyu’ loquat<br />
treated with a white single-layer paper bag.<br />
This study found that the content of sorbitol in<br />
‘Qingzhong’ loquat mature leaf was 24.52 mg·g -1 , 3.83<br />
times that of the mature fruit (data was not shown in the<br />
results), which revealed that the content of sorbitol in the<br />
mature fruit was much lower than that in the mature leaf.
4216 Afr. J. Biotechnol.<br />
Table 2. The correlation between sugar accumulation and activities of sucrose-metabolizing enzyme and<br />
sorbitol-metabolizing enzyme during fruit development.<br />
Treatment Sugar content NI AI SS SPS SDH SOX<br />
Sucrose 0.996** 0.990** 0.991** 0.986** — —<br />
Control<br />
Fructose<br />
Glucose<br />
0.988**<br />
0.994**<br />
0.978**<br />
0.989**<br />
0.977**<br />
0.983**<br />
0.970**<br />
0.981**<br />
—<br />
—<br />
—<br />
—<br />
Sorbitol — — — — 0.836 0.76<br />
Bagging<br />
Sucrose 0.981* 0.984* 0.994** 0.999** — —<br />
Fructose 0.957* 0.958* 0.972* 0.984* — —<br />
Glucose 0.993** 0.993** 0.997** 0.994** — —<br />
Sorbitol — — — — 0.968* 0.558<br />
**Correlation is significant at the 0.01 level (2-tailed); *correlation is significant at the 0.05 level (2-tailed); — correlation is<br />
negative. AI, acid invertase; NI, neutral invertase; SS, sucrose synthase; SPS, sucrose-phosphate synthase; SDH,<br />
sorbitol dehydrogenase; SOX, sorbitol oxidase.<br />
However, the accumulation of sorbitol in bagged fruit was<br />
lower than that of the non-bagged fruit. Ruan et al. (1997)<br />
reported that the difference in sugar content in fruit was<br />
independent of export from a source leaf but the transport<br />
capacity of sugar was different in different kinds of fruit<br />
trees, which suggested that the content of sorbitol<br />
entering the fruit did not depend on the output capacity of<br />
the leaf but on the gathering ability of the fruit. A possible<br />
reason why the content of sorbitol in the bagged fruit was<br />
lower than that in the non-bagged fruit is that bagging<br />
might affect the ability to transport sorbitol from the<br />
leaves to the fruits. Bagging influenced the activities of<br />
sucrose and sorbitol metabolism related enzymes. The<br />
activities of NI and AI were lower in bagged fruit than<br />
those in the non-bagged fruit and were significantly<br />
different before 85 days after initial fruit set. The activity<br />
of NI was similar in the bagged and non-bagged fruit,<br />
whereas the activity of AI was higher in the bagged than<br />
in the non-bagged fruit until the fruit ripened. The<br />
activities of SS and SPS were higher in the bagged than<br />
in the non-bagged fruit, which suggested that bagging<br />
mainly increased the activities of sucrose metabolism<br />
related enzymes.<br />
Sugar introduced into the fruit from the leaf depends on<br />
the sink strength of the fruit to a large extent. An important<br />
physiological marker that determines sink strength is<br />
the activities of sucrose metabolism related enzymes<br />
(Vizzotto et al., 1996). As photosynthesis in fruit decreased<br />
after bagging (Xu et al., 2010), the activities of sugar<br />
related enzymes may be required to increase the sink<br />
strength of the fruit to obtain more products of photosynthesis.<br />
This study suggests that bagging mainly<br />
offsets the loss induced by lack of sunlight by increasing<br />
the activities of SS and SPS.<br />
REFERENCES<br />
Feng JJ, Chen JW, Xu HX, Zhang YC, Xie M, Hu YC (2009). Effects of<br />
different light transmittance paper bags on fruit quality and<br />
antioxidant capacity in Ninghaibai loquat cultivar. J. Fruit Sci. 26: 66–<br />
70.<br />
Kim YH, Kim HH, Youn CK, Kweon SJ, Jung HJ, Lee CH (2008).<br />
Effects of bagging material on fruit coloration and quality of<br />
‘Janghowon Hwangdo’ peach. Acta Hort. 772: 81-86.<br />
Liang D, Ma FW, Guan QM, Zhang JK, Xu LF (2004). Advances in the<br />
research of enzymes related to sorbitol metabolism in plants of<br />
Rosaceae. Acta Bot. Bor.-Occi. Sinica. 24: 1362–1366.<br />
Lin J, Chang YH, Yan ZHM, Li XG (2008). Effects of bagging on the<br />
quality of pear fruit and pesticide residues. Acta Hort. 772: 315-318.<br />
Ni ZJ, Shen DT, Gu LP, Zhang Z, Huang LF, Gao ZH (2009). Sugar<br />
accumulation and related enzyme activity during the loquat fruit<br />
development. Acta Bot. Bor.-Occi. Sinica. 29: 478–493.<br />
Nielsen TH, Skjærbæk HC, Karlsen P (1991). Carbohydrate metabolism<br />
during fruit development in sweet pepper(Capsicum annuum) plants.<br />
Physiol. Plantarum. 82: 311–319.<br />
Qiu WL, Zhang HZ (1996). China Fruit Records - Logan and Loquat.<br />
China forestry press, Beijing, China.<br />
Ruan YL, Patrick JW, Bardy C (1997). Protoplast hexose carrier activity<br />
is a determinate of genotypic different in hexose storage in tomato<br />
fruit. Plant Cell Environ. 20: 341–349.<br />
Vizzotto G, Pinton R, Varanini Z, Costa G (1996). Sucrose accumulation<br />
in developing peach fruit. Physiol. Plant. 96: 225-230.<br />
Xu HX, Chen JW, Xie M (2010). Effect of different light transmittance<br />
paper bags on fruit quality and antioxidant capacity in loquat. J. Sci.<br />
Food Agri. 90: 1783-1788.<br />
Xu HX, Chen JW, Zhang YC, Xie M, Zhou LM, Qian JL, Tang QM, Xu<br />
CM (2008). Effects of bagging on fruit quality and antioxidant capacity<br />
in ‘Baiyu’ loquat. Acta. Hort. Sinica. 35: 1193-1198.<br />
Yamaki S (1980). A sorbitol oxidase that converts sorbitol to glucose in<br />
apple leaf. Plant Physiol. 21: 591-599.<br />
Yamaki S, Ishikawa K (1986). Roles of four sorbitol related enzymes<br />
and invertase in the seasonal alteration of sugar metabolism in apple<br />
tissue. J. Am. Soc. Hort. Sci. 111: 134–137.<br />
Yang WH, Zhu XC, Bu JH, Hu GB, Wang HC, Huang XM (2009).<br />
Effects of bagging on fruit development and quality in cross-winter<br />
off-season longan. Sci. Hort. 120: 194-200.<br />
Zheng GQ, Song YX, Guo SH, Ma HA, Niu DL (2006). Soluble sugar<br />
accumulation and the activities of sugar metabolism related enzymes<br />
in cistanche deserticola and its host haloxylon ammodendron. Acta<br />
Botanica Boreali-Occidentalia Sinica. 26: 1175-1182.
African Journal of Biotechnology Vol. 10(20), pp. 4217-4225, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2181<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Anti-inflammatory evaluation of immature fruit and seed<br />
aqueous extracts from several populations of Tunisian<br />
Citrullus colocynthis Schrad<br />
Belsem Marzouk 1¥ *, Zohra Marzouk 2¥ , Ehsen Haloui 2 , Manel Turki 3 , Abderrahman Bouraoui 3 ,<br />
Mahjoub Aouni 1 and Nadia Fenina 2<br />
1 Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives, Faculté de Pharmacie de Monastir,<br />
Rue AVICENNE 5000 Monastir, Tunisie.<br />
2 Unité de Pharmaco-économie et Développement des Médicaments, Laboratoires de Biologie Végétale et Laboratoire<br />
de Pharmacologie, Faculté de Pharmacie de Monastir, Tunisie.<br />
3 Unité de recherche URSAM, Laboratoire de Pharmacologie, Faculté de Pharmacie, Monastir, Tunisie.<br />
Accepted 21 February, 2011<br />
Plant extracts are some of the most attractive sources of new drugs and have shown promising results<br />
for the treatment of inflammation and immune-related diseases, including rheumatoid arthritis. Citrullus<br />
colocynthis Schrad. (Cucurbitaceae) endemic in Tunisia, is widely used in folk medicine to treat many<br />
inflammation disorders. The aim of this study is to quantify the alkaloid and the flavonoid levels of<br />
different populations of C. colocynthis fruit and seed aqueous extracts at immature state. After acute<br />
toxicity assay, these extracts were screened for anti-inflammatory activity using the carrageenaninduced<br />
paw edema assay in rats. Alkaloid and flavonoid levels vary among the population. The best<br />
anti-inflammatory activities were obtained with immature fruits from south Tunisia. Therefore, C.<br />
colocynthis Schrad. could be a potential useful product suitable for further evaluation for inflammatory<br />
diseases.<br />
Key words: Citrullus colocynthis Schrad., alkaloids, flavonoids, toxicity, anti-inflammatory, Tunisia.<br />
INTRODUCTION<br />
Since ancient times, several diseases have been treated<br />
by administration of plant extracts. Interest in ethnopharmacy<br />
as a source of active compounds has increased<br />
worldwide, particularly for the development of antiinflammatory<br />
drugs. Plants with analgesic and antiinflammatory<br />
activities have become more interesting<br />
because some of these plants are part of the arsenal of<br />
*Corresponding author. E-mail: belsemmarzouk@yahoo.fr. Tel:<br />
216 73 450 389.<br />
Abbreviations: TOF, Total oligomeric flavonoids; TLC, thin<br />
layer chromatography; ASL, acetyl salicylate of lysine.<br />
¥ These authors contributed equally.<br />
modern medicine and many people are aware of<br />
problems associated with the over-prescription and<br />
misuse of usual drugs.<br />
Citrullus colocynthis Schrad. (Cucurbitaceae), growing<br />
in Tunisia (Pottier-Alapetite, 1981), is widely used in<br />
Tunisian folk medicine for treating many diseases such<br />
as various contagious diseases, hypertension and<br />
rheumatism (Le Flock, 1983; Boukef, 1986). Anti-inflammatory<br />
traditional healers seem not to pay attention to the<br />
fruit’s degree of maturity and literature rarely mention if<br />
seeds are present in preparations involving ground<br />
fruit/pulp. Common preparations use juice, fresh or dried<br />
(often ground) fruit material. Extracts (maceration or<br />
boiling) are prepared either in water or in aqueous<br />
mixtures (honey, milk and water/olive oil at various ratios).<br />
Methods of administration are by ingestion or massage<br />
(Le Flock, 1983; Boukef, 1986; Bellakhdar, 1999; El-
4218 Afr. J. Biotechnol.<br />
Ghadi and Bshana, 1988).<br />
Some studies have demonstrated the medicinal effect<br />
of C. colocynthis Schrad. as anti-tumour (Tannin-Spitz et<br />
al., 2007), immunostimulant (Bendjeddou et al., 2003),<br />
anti-microbial (Marzouk et al., 2009, 2010a) and antioxidant<br />
(Marzouk et al., 2010b), and against hepatic diseases<br />
(Gebhardt, 2003), hyperglycaemia (Al-Gaithi et al., 2004)<br />
and hair loss (Roy et al., 2007).<br />
However, plants in the environment are exposed to a<br />
range of abiotic stresses like osmotic, salinity, temperature<br />
and heavy metal toxicity which affects their growth<br />
and other metabolic processes such as alkaloid and<br />
flavonoid productions. These secondary metabolites were<br />
synthesized by plants to defend themselves against the<br />
harmful action of external agents (Heller et al., 1993;<br />
Waller and Nowacki, 1978). Thus, alkaloid and flavonoid<br />
contents depend on the geographical distribution.<br />
In a previously published paper, C. colocynthis<br />
immature fruits and seeds were demonstrated as the<br />
most efficient analgesic and anti-inflammatory parts<br />
(Marzouk et al., 2010c). The current study measured in<br />
vivo toxicity and anti-inflammatory activity of seven<br />
populations of C. colocynthis Schrad. using mice and rat<br />
as models. Preparation and tests were carried out on the<br />
reconstituted lyophilized aqueous extracts.<br />
MATERIALS AND METHODS<br />
Plant materials<br />
C. colocynthis Schrad. plants were collected in August (2007) from<br />
seven stations (Table 1 and Figure 1). The identification was<br />
performed according to the flora of Tunisia (Pottier-Alapetite, 1981)<br />
and voucher specimens were deposited in the biological laboratory<br />
of the Faculty of Pharmacy of Monastir.<br />
Extraction protocol<br />
The extraction was performed on fresh immature fruits or seeds.<br />
Each plant part was ground with a mixer and added to distilled<br />
water. The mixture was allowed to reflux for 30 min, after which the<br />
solution was allowed to cool (4 h at 4°C). The mixture was then<br />
filtered using filter paper (Whatman no.1) under the vacuum of a<br />
water pump. The filtrate obtained was lyophilized, yielding the<br />
lyophilized aqueous extract. Yields are given in Table 2.<br />
Quantitative alkaloid screening<br />
Alkaloids were quantified according to the volumetric procedure of<br />
the Official Method of European Pharmacopoeia. Briefly, to 3 g of<br />
each dried plant parts, ammonium hydroxide (5 ml), ethanol (10 ml)<br />
and diethyl ether (30 ml) were successively added. After 4 h of<br />
maceration, the mixture was lixiviated by 150 ml of ether and 50 ml<br />
of chloroform. The obtained solution was reduced to 50 ml by a<br />
rotavapory (Heidolph) and then treated with sulphuric acid (0.5 N, 3<br />
x 20 ml). Bases were removed from the extract through precipitation<br />
with an excess of ammonium hydroxide (3 ml). The supernatant<br />
was then extracted with chloroform (3 x 10 ml). The precipitate<br />
containing alkaloids was separated by means of filtration and<br />
treated again with chloroform which was evaporated under low<br />
pressure. The presence of alkaloids was verified by Dragendorff’s<br />
reagent confirmed with Bouchardat’s and Meyer’s reagent (Treas<br />
and Evans, 1984). To the residue, 20 ml of sulphuric acid was<br />
added. The excess sulphuric acid was titrated with sodium<br />
hydroxide (0.02 N) using the phenolphthalein indicator. The<br />
percentage of total alkaloids was then calculated according to the<br />
following formula:<br />
Alkaloid level (%) = ((VH2SO4-V H2SO4ex) x 5.788 / W) x 100<br />
Where, VH2SO4 = 20 ml, V H2SO4ex = volume of excess sulphuric acid,<br />
W = 3 g and 1 ml of sulphuric acid correspond to 5.788 mg of<br />
alkaloids.<br />
Quantitative oligomeric flavonoid screening<br />
In order to obtain total oligomeric flavonoids (TOF), the powdered<br />
seeds were macerated separately for 24 h in a mixture of<br />
acetone/water (2:1). Each extract was then filtered and the acetone<br />
was evaporated under low pressure. Tannins were removed from<br />
the aqueous phase through precipitation with NaCl for 24 h at 5°C<br />
and the supernatant was then extracted with ethyl acetate,<br />
concentrated and precipitated with excess chloroform. The<br />
precipitate containing TOF was separated by means of filtration<br />
(Paris and Moyse, 1976). The presence of flavonoids was verified<br />
by the reaction of ‘cyanidine’ in the presence of hydrochloric acid<br />
and magnesium. After release of hydrogen, an orange colour with<br />
red purple indicates the presence of the flavonoids. The<br />
appearance of the spots corresponding to the flavonoids on the<br />
plates of thin layer chromatography (TLC) is done by pulverization<br />
using a solution of 2% AlCl3 in methanol. The plates of TLC are<br />
then observed under UV with 366 nm; the spots corresponding to<br />
the flavonoids have a yellow fluorescence at intense yellow<br />
fluorescence (Paris and Moyse, 1976). The percentage of total<br />
oligomer flavonoids was then calculated according to the following<br />
formula:<br />
Total oligomer flavonoids level (%) = (weight of total oligomer<br />
flavonoids / powdered material weight) x 100<br />
Animals<br />
Male adult Wistar rats weighing 160 to 180 g and Swiss albino mice<br />
(weighing 18 to 25 g) of both sex were obtained from Pasteur<br />
institute (Tunis, Tunisia). They were housed in polypropylene cages<br />
and were left for 2 days for acclimatization to animal room<br />
maintained under controlled condition (a 12 h light – dark cycle at<br />
22 ± 2°C) on standard pellet diet and water ad libitum. Before the<br />
day of assay, only the Wistar rats were fasted overnight with free<br />
access to water. Housing conditions and in vivo experiments<br />
approved according to the guidelines established by the European<br />
Union on Animal Care (CFE Council (86/609)) were used. The rats<br />
were used for the anti-inflammatory evaluation of the aqueous<br />
extracts, while the mice were used for the analgesic investigation<br />
and for the acute toxicity testing. Animals were divided into drugtreated<br />
‘test’ and saline-treated ‘control’ groups of six or eight<br />
animals per group.<br />
Acute toxicity<br />
For acute toxicity, mice were divided into groups of eight animals<br />
each. One group served as a control and received 0.9% NaCl alone<br />
(10 ml/kg) given intraperitoneally (i.p.), while the remaining groups<br />
were treated with increasing doses of the aqueous extract; 50, 100,
Table 1. Sites and voucher specimens of C. colocynthis Schrad. populations.<br />
Population Site Voucher specimens<br />
Hammamet Hammamet Sud (36°25 N, 10°34 W) C.C-07.07<br />
Mahdia Chorban (35°20 N, 10°32 W) C.C-06.07<br />
Kasserine Mozgam (35°11 N, 8°46 W) C.C-05.07<br />
Sbeitla Sbitla (35°14 N, 9°07 W) C.C-04.07<br />
Sidi-Bouzid Jelma (35°16 N, 9°25 W) C.C-03.07<br />
Sfax Skhira (34°10 N, 10°04 W) C.C-02.07<br />
Medenine Sidi Makhlouf (33°33 N, 10°27 W) C.C-01.07<br />
N: North; W: west.<br />
Figure 1. Geographical map of Tunisia.<br />
250, 500, 750, 1000, 1500, 2000, 3000 and 4000 mg/kg (i.p.),<br />
respectively.<br />
The mortality rate within a 48 h period was determined and the<br />
LD50 was estimated according to the method described by Miller<br />
and Tainter (1944). According to the results of acute toxicity test,<br />
doses were chosen for pharmacological evaluations. After the last<br />
observation, the mice were killed and the liver, lungs, heart, spleen<br />
and kidneys were withdrawn, weighed and stored for next<br />
evaluations.<br />
Anti-inflammatory activity<br />
The anti-inflammatory activity was assessed on the basis of<br />
Marzouk et al. 4219<br />
inhibition of paw edema induced by the injection of carrageenan (an<br />
edematogenic agent) into the subplantar region of the right hind<br />
paw of the rat (Winter et al., 1962). Male Wistar rats were divided<br />
into different groups of eight animals. The control group received<br />
2.5 ml/kg of saline, the standard group received the reference drug<br />
(acetyl salicylate of lysine (ASL), 300 mg/Kg) and the test groups<br />
received different population extracts of C. colocynthis at a dose of<br />
1 and 4 mg/kg. Thirteen minutes after intraperitoneal administration<br />
of different substances, 0.05 ml of 1% of carrageenan suspension<br />
was injected into all animals in the right hind paw.<br />
The paw volume, up to tibiotarsal articulation, was measured<br />
using a plethysmometer. The measures were determined at 0 h (V0:<br />
before edematogenic agent injection) and 1, 2, 3, 4, 5, 6 and 24 h<br />
intervals later (VT). The difference between VT (1, 2, 3, 4, 5, 6 and
4220 Afr. J. Biotechnol.<br />
Table 2. Extraction yields (w/w %), alkaloid and oligomeric flavonoid levels (%) and LD50 (mg/Kg) of different populations of C. colocynthis Schrad. seeds and fruits.<br />
Population of Citrullus colocynthis Schrad.<br />
Parameter Hammamet Mahdia Kasserine Sbeitla Sidi-Bouzid Sfax Medenine<br />
Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits<br />
Extraction yields 1.68 0.93 1.90 1.70 2.88 2.94 1.93 2.68 1.76 2.44 2.15 2.81 2.94 2.76<br />
Alkaloid levels 2.01 1.57 3.10 2.08 2.74 1.61 2.48 1.55 2.90 1.71 3.90 2.86 3.87 2.90<br />
Flavonoid levels 0.24 - 0.18 - 0.18 - 0.20 - 0.17 - 0.13 - 0.10 -<br />
LD50 2043.84 795.45 818.61 749.97 653.51 750.03 1535.21 799.64 2298.48 795.49 438.62 385.54 744.15 553.73<br />
24 h) and V0 was taken as the edema value. The<br />
percentage of inhibition was calculated according to the<br />
following formula:<br />
Percentage inhibition = ((VT-V0) control – (VT-V0) treated group) x<br />
100 / (VT-V0) control<br />
Statistical analysis<br />
Data obtained from animal experiments were expressed as<br />
mean ± S.E.M. and as percentage. Results were statistically<br />
evaluated by ANOVA and using student’s t-test.<br />
P ≤ 0.05 were considered significant.<br />
RESULTS<br />
Extraction yields<br />
After all population (Table 1 and Figure 1)<br />
extractions, experimental results (Table 2) reveal<br />
that seeds and fruits from Kasserine, Sfax and<br />
Medenine have significantly more higher extraction<br />
yields than the other population plant parts.<br />
Hammamet population showed the lowest yield.<br />
Quantitative alkaloid and oligomeric flavonoid<br />
screening<br />
Alkaloid presence was reported in all C.<br />
colocynthis organs (immature seeds and fruits<br />
whose alkaloids are quantified in this study)<br />
except the roots (Marzouk et al., 2009). Results<br />
(Table 2) were dependent upon the population.<br />
Sfax (3.90 and 2.86%, respectively, for seeds and<br />
fruits) and Medenine (3.87 and 2.90% correspondingly,<br />
for seeds and fruits) populations<br />
contained more alkaloids, but Hammamet organs<br />
presented the lowest amounts of it. Differences<br />
were also noted between plant parts: seeds<br />
showed better percentage (from 2.01 to 3.90%)<br />
than fruits (from 1.55 to 2.90%). Flavonoids which<br />
are only detected in seeds (Marzouk et al., 2009)<br />
were present just at low levels (from 0.10 to<br />
0.24%, Table 2). Hammamet was the richest<br />
population in these components.<br />
Toxicity studies<br />
Swiss-albino mice were observed for 48 h and<br />
morbidity and/or mortality were recorded, for each<br />
group at the end of observation period. Due to<br />
death index, the LD50 of all extracts were<br />
determined (Table 2). This value is in relation with<br />
plant parts and also with origin states. The LD50 of<br />
seeds ranged from 438.62 mg/kg (Sfax<br />
population) to 2298.48 mg/kg (Sidi-Bouzid<br />
population). For the C. colocynthis fruits, Sfax<br />
population had the lowest LD50 (385.54 mg/kg)<br />
and that of Sbeitla was evaluated as the less toxic<br />
(LD50 = 799.64 mg/kg). Except Kasserine<br />
population, immature seeds were less toxic than<br />
immature fruits. C. colocynthis seeds and fruits<br />
from Sfax seem to be the most toxic plant parts of<br />
all. Regarding the LD50, the quantitative alkaloid<br />
and oligomeric flavonoid screening, this toxicity<br />
could be, probably, attributed to the alkaloid<br />
levels.<br />
Anti-inflammatory effect<br />
In carrageenan-induced rat paw edema, all extracts<br />
produced a significant reduction of the edema<br />
throughout the entire period of observation<br />
(Tables 3 and 4). The intraperitoneal administration<br />
of the aqueous extracts of seeds and fruits<br />
significantly reduced the paw edema induced by<br />
the noxious agent. This inhibition differs with the<br />
plant part aqueous extract and its origin state. For<br />
this second assessment of the anti-inflammatory<br />
activity, the obtained results demonstrated that<br />
the reduction of the paw edema vary in a dose<br />
dependent fashion with its maximum at 4 mg/kg.<br />
On the contrary, at 1 mg/kg, only a weak to<br />
moderate activities were noted. In terms of plant<br />
parts, immature fruits showed better activity than<br />
immature seeds. Experimental results, at 4 mg/kg,<br />
reveal that the anti-inflammatory effect seems to<br />
remain moderate with Mahdia seeds and<br />
Hammamet fruits which showed a very weak<br />
property during the first phase and increase
Table 3. Effects of different populations of C. colocynthis Schrad. seed aqueous extracts and reference drug on carrageenan-induced paw edema.<br />
Seed population Dose (mg/Kg)<br />
Mean swelling thickness (10-2) ± S.E.M. (% inhibition)<br />
1 h 2 h 3 h 4 h 5 h 6 h 24 h<br />
Marzouk et al. 4221<br />
C1 - 15.00±1.76 34.50±4.33 57.00±6.22 61.50±6.42 67.00±7.37 71.00±6.39 40.50±5.84<br />
C2 - 33.00±3.66 48.00±4.77 61.50±7.20 76.25±7.37 95.00±9.20 106.25±9.85 77.75±6.66<br />
C3 - 15.00±1.76 24.00±2.66 49.00±4.77 57.50±5.56 59.50±5.25 64.50±8.20 32.50±3.51<br />
Hammamet 1<br />
(C1)<br />
4<br />
(C2)<br />
Mahdia 1<br />
(C2)<br />
4<br />
(C2)<br />
Kasserine 1<br />
(C2)<br />
4<br />
(C2)<br />
Sbeitla 1<br />
(C1)<br />
4<br />
(C2)<br />
Sidi-Bouzid 1<br />
(C3)<br />
4<br />
(C2)<br />
Sfax 1<br />
(C1)<br />
4<br />
(C2)<br />
14.00±1.41 ns<br />
(6.67)<br />
15.75±0.96***<br />
(52.27)<br />
33.00±0.82 ns<br />
(0.00)<br />
27.00±1.15***<br />
(18.18)<br />
23.75±1.71***<br />
(28.03)<br />
12.75±0.96***<br />
(61.36)<br />
20.00±1.63***<br />
(39.39)<br />
18.00±0.82***<br />
(45.45)<br />
14.00±1.41 ns<br />
(6.67)<br />
14.50±1.29***<br />
(56.06)<br />
15.00±2.45 ns<br />
(0.00)<br />
14.75±1.50***<br />
(55.30)<br />
30.50±1.91*<br />
(11.59)<br />
22.50±1.29***<br />
(53.13)<br />
40.25±1.26***<br />
(16.15)<br />
35.25±0.96***<br />
(26.56)<br />
31.00±1.41***<br />
(35.42)<br />
18.50±1.29***<br />
(61.45)<br />
25.00±1.15***<br />
(47.91)<br />
22.00±2.16***<br />
(54.17)<br />
16.75±2.52***<br />
(30.21)<br />
20.00±2.16***<br />
(58.33)<br />
31.50±2.38 ns<br />
(8.69)<br />
19.75±0.96***<br />
(58.85)<br />
44.25±2.75***<br />
(22.37)<br />
20.50±0.58***<br />
(66.66)<br />
33.00±0.82***<br />
(46.34)<br />
32.00±1.41***<br />
(47.97)<br />
29.00±2.16***<br />
(52.85)<br />
23.50±1.29***<br />
(61.79)<br />
28.50±1.29***<br />
(53.66)<br />
25.75±1.71***<br />
(58.13)<br />
33.25±2.16***<br />
(32.14)<br />
24.50±1.29***<br />
(60.16)<br />
44.25±2.75***<br />
(22.37)<br />
23.50±2.52***<br />
(61.79)<br />
46.75±3.10***<br />
(23.98)<br />
19.00±1.41***<br />
(75.08)<br />
31.00±2.31***<br />
(59.34)<br />
26.75±1.26***<br />
(64.92)<br />
32.50±2.52***<br />
(57.38)<br />
24.00±0.82***<br />
(68.52)<br />
35..00±0.82***<br />
(54.10)<br />
26.75±1.71***<br />
(64.92)<br />
31.75±1.29***<br />
(44.78)<br />
28.00±0.82***<br />
(63.28)<br />
47.00±3.16***<br />
(23.58)<br />
28.00±0.82***<br />
(63.28)<br />
36.00±2.16***<br />
(46.27)<br />
22.75±0.96***<br />
(76.05)<br />
30.50±0.58***<br />
(67.89)<br />
29.75±0.96***<br />
(68.68)<br />
34.50±1.91***<br />
(63.68)<br />
29.00±0.82***<br />
(69.47)<br />
30.50±1.73***<br />
(67.89)<br />
25.75±1.71***<br />
(72.89)<br />
34.50±1.29***<br />
(43.69)<br />
24.25±2.06***<br />
(74.47)<br />
53.00±1.83***<br />
(20.89)<br />
28.75±0.96***<br />
(69.74)<br />
32.00±2.16***<br />
(54.93)<br />
21.75±0.96***<br />
(79.53)<br />
27.00±0.82***<br />
(74.59)<br />
25.75±0.96***<br />
(75.76)<br />
28.25±2.06***<br />
(73.41)<br />
19.00±1.41***<br />
(82.12)<br />
30.00±1.63***<br />
(71.76)<br />
18.25±0.96***<br />
(82.82)<br />
29.50±3.41***<br />
(54.26)<br />
23.00±2.16***<br />
(78.35)<br />
49.50±1.91***<br />
(25.00)<br />
20.50±1.29***<br />
(80.71)<br />
18.25±1.71***<br />
(54.94)<br />
14.00±0.82***<br />
(81.99)<br />
20.50±0.58***<br />
(73.63)<br />
17.75±0.96***<br />
(77.17)<br />
24.50±1.29***<br />
(68.49)<br />
13.75±0.50***<br />
(82.31)<br />
24.50±1.29***<br />
(68.49)<br />
12.75±0.96***<br />
(83.60)<br />
14.50±1.29***<br />
(55.38)<br />
16.00±0.82***<br />
(79.42)<br />
28.00±0.82***<br />
(30.86)<br />
8.75±1.71***<br />
(88.75)
4222 Afr. J. Biotechnol.<br />
Table 3. Contd.<br />
Seed population Dose (mg/Kg)<br />
Medenine<br />
1<br />
(C3)<br />
4<br />
(C3)<br />
ASL 300<br />
(C1)<br />
Mean swelling thickness (10-2) ± S.E.M. (% inhibition)<br />
1 h 2 h 3 h 4 h 5 h 6 h 24 h<br />
14.50±2.89 ns<br />
19.25±2.21** 33.25±2.21*** 32.50±2.38*** 39.00±3.91*** 29.25±3.86*** 14.25±0.96***<br />
(3.33)<br />
(19.79) (32.14) (43.48) (34.45) (54.65) (56.15)<br />
1.75±0.96*** 2.00±0.82*** 3.50±0.58*** 1.50±1.29*** 2.50±1.29*** 1.25±0.50*** 0.75±0.95***<br />
(88.33) (91.66) (92.86) (97.40) (95.80) (98.06) (97.69)<br />
7.25±0.96***<br />
(51.66)<br />
14.25±1.71***<br />
(58.69)<br />
17.50±2.38***<br />
(69.30)<br />
15.75±2.22***<br />
(74.40)<br />
19.50±2.08***<br />
(70.89)<br />
19.50±1.29***<br />
(72.53)<br />
23.75±1.71***<br />
(41.36)<br />
C1,: Control 1; C2, control 2; C3, control 3; 1 (C1) and 4 (C1), in comparison with control 1; 1 (C2) and 4 (C2), in comparison with control 2; 1 (C3) and 4 (C3), in comparison with control 3.<br />
Values are expressed as mean ± M.S.E. (n = 8); *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 significant from the control; ns, not significant from the control; ASL, acetyl salicylate of lysine.<br />
Table 4. Effects of different populations of C. colocynthis Schrad. fruit aqueous extracts and reference drug on carrageenan-induced paw edema.<br />
Fruit population Dose (mg/kg)<br />
Mean swelling thickness (10-2) ± S.E.M. (% inhibition)<br />
1 h 2 h 3 h 4 h 5 h 6 h 24 h<br />
C1 - 15.00±1.76 34.50±4.33 57.00±6.22 61.50±6.42 67.00±7.37 71.00±6.39 40.50±5.84<br />
C2 - 33.00±3.66 48.00±4.77 61.50±7.20 76.25±7.37 95.00±9.20 106.25±9.85 77.75±6.66<br />
C3 - 15.00±1.76 24.00±2.66 49.00±4.77 57.50±5.56 59.50±5.25 64.50±8.20 32.50±3.51<br />
Hammamet 1<br />
15.00±2.45<br />
(C1)<br />
ns<br />
31.75±1.26* 46.50±2.08*** 47.00±3.16*** 53.00±1.83*** 53.25±3.20*** 18.00±0.82***<br />
(0.00) (7.97) (18.42) (23.58) (20.90) (30.28) (30.86)<br />
4<br />
(C2)<br />
Mahdia 1<br />
(C2)<br />
4<br />
(C2)<br />
Kasserine 1<br />
(C1)<br />
4<br />
(C2)<br />
Sbeitla 1<br />
(C2)<br />
4<br />
(C2)<br />
24.00±0.82***<br />
(27.27)<br />
18.75±0.96 ***<br />
(43.18)<br />
17.00±0.82***<br />
(48.48)<br />
10.50±1.71***<br />
(30.00)<br />
17.75±0.96***<br />
(46.21)<br />
23.50±1.73***<br />
(28.79)<br />
19.25±0.96***<br />
(41.67)<br />
27.00±0.82***<br />
(43.75)<br />
24.75±1.26***<br />
(48.44)<br />
22.75±1.71***<br />
(52.60)<br />
23.25±1.41***<br />
(32.61)<br />
24.25±0.96***<br />
(49.48)<br />
26.50±1.29***<br />
(44.79)<br />
22.50±1.29***<br />
(53.13)<br />
31.00±0.58***<br />
(49.59)<br />
31.25±0.96***<br />
(49.19)<br />
27.5±0.58***<br />
(55.28)<br />
33.25±2.08***<br />
(41.67)<br />
29.75±1.71***<br />
(51.63)<br />
33.25±2.21***<br />
(45.93)<br />
22.75±2.06***<br />
(63.01)<br />
23.00±0.82***<br />
(69.84)<br />
30.50±1.29***<br />
(60.00)<br />
27.75±1.71***<br />
(63.61)<br />
27.00±0.82***<br />
(56.10)<br />
24.00±1.41***<br />
(68.52)<br />
32.50±2.38***<br />
(57.38)<br />
25.25±1.71***<br />
(66.89)<br />
23.75±0.96***<br />
(75.00)<br />
32.50±2.08***<br />
(65.79)<br />
22.50±0.58***<br />
(76.32)<br />
36.00±2.16***<br />
(46.27)<br />
23.00±1.86***<br />
(75.79)<br />
30.50±1.73***<br />
(67.89)<br />
22.00±2.16***<br />
(76.84)<br />
15.75±0.96***<br />
(85.18)<br />
25.50±2.08***<br />
(76.00)<br />
20.50±0.58***<br />
(80.71)<br />
32.00±2.06***<br />
(54.93)<br />
17.50±1.29***<br />
(83.53)<br />
30.00±1.63***<br />
(71.76)<br />
16.00±0.82***<br />
(84.94)<br />
11.25±0.96***<br />
(85.83)<br />
17.25±0.96***<br />
(77.81)<br />
14.25±0.96***<br />
(81.67)<br />
13.00±1.15***<br />
(67.90)<br />
12.75±0.50***<br />
(83.60)<br />
24.50±1.29***<br />
(68.49)<br />
11.25±0.96***<br />
(85.53)
Table 4. Contd.<br />
Sidi-Bouzid 1<br />
(C3)<br />
4<br />
(C2)<br />
Sfax 1<br />
(C2)<br />
4<br />
(C2)<br />
Medenine 1<br />
(C3)<br />
4<br />
(C3)<br />
ASL 300<br />
(C1)<br />
14.50±2.89 ns<br />
(3.34)<br />
18.25±1.71***<br />
(44.96)<br />
33.00±2.08 ns<br />
(0.00)<br />
14.50±3.11***<br />
(56.06)<br />
13.25±2.22<br />
(11.67)<br />
1.50±0.57***<br />
(90.00)<br />
7.25±0.96***<br />
(51.66)<br />
21.75±1.50***<br />
(9.38)<br />
22.50±1.29***<br />
(53.13)<br />
40.25±1.26***<br />
(16.14)<br />
16.75±0.96***<br />
(65.10)<br />
16.75±3.30**<br />
(30.21)<br />
1.75±0.96***<br />
(92.71)<br />
14.25±1.71***<br />
(58.69)<br />
42.75±2.16***<br />
(12.76)<br />
26.00±0.82***<br />
(57.72)<br />
43.75±1.26***<br />
(43.50)<br />
20.75±1.71***<br />
(66.26)<br />
29.00±2.16***<br />
(40.82)<br />
2.75±0.50***<br />
(94.39)<br />
17.50±2.38***<br />
(69.30)<br />
37.50±2.38***<br />
(34.78)<br />
25.75±1.71***<br />
(66.23)<br />
32.50±2.52***<br />
(57.38)<br />
15.75±1.50***<br />
(79.34)<br />
31.75±3.86***<br />
(44.78)<br />
1.75±0.96***<br />
(97.65)<br />
15.75±2.22***<br />
(74.40)<br />
39.00±1.29***<br />
(34.45)<br />
21.50±2.08***<br />
(77.37)<br />
34.50±1.92***<br />
(63.68)<br />
17.50±0.58***<br />
(81.58)<br />
34.50±1.29***<br />
(42.02)<br />
2.75±0.96***<br />
(95.38)<br />
19.50±2.08***<br />
(70.89)<br />
29.25±2.08***<br />
(54.65)<br />
21.00±2.54***<br />
(80.24)<br />
27.00±0.82***<br />
(74.59)<br />
14.75±1.26***<br />
(86.12)<br />
21.75±3.95***<br />
(66.28)<br />
0.75±0.96***<br />
(98.84)<br />
19.50±1.29***<br />
(72.53)<br />
Marzouk et al. 4223<br />
17.75±1.71***<br />
(45.38)<br />
5.75±0.25***<br />
(92.60)<br />
24.50±1.29***<br />
(68.49)<br />
5.50±0.50***<br />
(92.93)<br />
16.75±2.22***<br />
(48.46)<br />
0.50±0.58***<br />
(98.46)<br />
23.75±1.71***<br />
(41.36)<br />
C1: Control 1; C2: Control 2; C3: Control 3; 1 (C1) and 4 (C1): in comparison to control 1; 1 (C2) and 4 (C2): in comparison to control 2; 1 (C3) and 4 (C3): in comparison to control 3; Values are<br />
expressed as mean ± M.S.E. (N=8); *p ≤ 0.05, **≤0.01, ***≤0.001 significant from the control; ns: not significant from the control; ASL: Acetyl Salicylate of Lysine.<br />
significantly at the second one, the other populations<br />
throughout the experiment, showed an<br />
important effect. This difference may be correlated<br />
to the process of synergism or antagonism<br />
between all extract components (Marzouk et al.,<br />
2009). With all extracts, 3 h after carrageenan<br />
injection, the anti-inflammatory activity instigated<br />
unambiguous increase to attain the maximum<br />
level at 6 and 24 h. The inhibition percentages range<br />
at 6 and 24 h respectively, are from 75.76 to<br />
98.06% and from 77.17 to 97.69% for seeds and<br />
from 80.24 to 98.84% and 81.67 to 98.46% for<br />
fruits. South Tunisia aqueous extracts (at 4<br />
mg/kg) were strongly more active than the other<br />
population (1 to 6 h) and seem to have close<br />
values after 24 h after the carrageenan injection.<br />
The highest activity was found for immature fruit<br />
extract from Medenine. Standard drug decreased<br />
paw edema by a maximum of 74.39% after 4 h.<br />
DISCUSSION<br />
This is the first study evaluating the in vivo acute<br />
toxicity and the anti-inflammatory activities of<br />
extracts from different population of Tunisian C.<br />
colocynthis immature fruits and seeds.<br />
The study clarified complexes of biodiversity<br />
pictures in terms of plant parts and plant geographical<br />
distribution. Results previously obtained<br />
(Marzouk et al., 2010c) showed the importance of<br />
immature seeds and fruits. Fruits and seeds<br />
differed, as made obvious by their extraction<br />
yields, analgesic and anti-inflammatory activeties.<br />
The present investigation showed, in addition, that<br />
for a given plant part, the intensity of the pharmacological<br />
activity can change from a population to<br />
another. The trend for all the populations to have<br />
higher activity for immature fruit aqueous extracts<br />
and immature seeds points towards various<br />
active, potential or antagonistic compounds present<br />
in various concentrations according to the plant
4224 Afr. J. Biotechnol.<br />
population, especially flavonoids and alkaloids. These<br />
two phytochemical key families are quantified and the<br />
results indicated that the geographical distribution of C.<br />
colocynthis Schrad. influenced second metabolite levels<br />
and subsequently their toxicity and biological activities.<br />
Based on the LD50 calculation, the acute administration<br />
doses of all population aqueous extracts were estimated<br />
(1 and 4 mg/kg). Carrageenan has been widely used as a<br />
noxious agent which is able to induce experimental<br />
inflammation for the screening of compounds possessing<br />
anti-inflammatory activity. This phlogistic agent, when<br />
injected locally into the rat paw, produced a severe<br />
inflammatory reaction, which was discernible within 30<br />
min (John and Nodine, 1999). The development of<br />
edema induced by carrageenan is a biphasic event: The<br />
early phase (90 to 180 min) of the inflammation is due to<br />
the release of histamine, serotonin and similar substances.<br />
The later phase (270 to 360 min) is associated<br />
with the activation of kinin-like substances and the<br />
release of prostaglandins, proteases and lysosome<br />
(Olajide et al., 1999). All population extracts inhibited hind<br />
paw edema and showed a dose-dependent anti-inflammatory<br />
activity but the results were different for each<br />
population plant parts depending on the early/later<br />
phases. All tested extracts inhibited both the phases of<br />
the carrageenan-induced edema by reducing the release<br />
of histamine and serotonin and also the kinin-like substances<br />
and prostaglandins. Pharmacological properties<br />
of all C. colocynthis Schrad. populations may be attributed<br />
to a possible molecular mechanism by effectively<br />
decreasing the production of the pro-inflammatory<br />
cytokines like IL-6 and IL-1β and the expression of COX-<br />
2, and simultaneously elevating the level of anti-inflammatory<br />
cytokine IL-4 in the carrageenan-injected rat paw<br />
tissues (Moulin and Coquerel, 2002).<br />
Differences in the LD50 and the anti-inflammatory<br />
efficacy are related to the plant population compositions.<br />
Alkaloids and flavonoids found in C. colocynthis plant part<br />
were dependent upon the population. At any rate, these<br />
results indicate that the anti-inflammatory activities could<br />
not be imputed to one family of phytochemicals only (or<br />
its absence). Alkaloids are commonly found to have antiinflammatory<br />
properties (Moulin and Coquerel, 2002).<br />
Flavonoids (detected in all the seed extracts) are known<br />
to have the same activities (Borgi et al., 2008). However,<br />
alkaloids and flavonoids cannot be only responsible for<br />
the pharmacological effect. Steroids and iridoids which<br />
are present in this plant (Marzouk et al., 2009) may<br />
contribute to better performance as an anti-inflammatory<br />
agent (Bames and Adcock, 2009). Differences found<br />
between the seven populations tested, in the alkaloid and<br />
flavonoid levels, in the toxicity and in the pharmacological<br />
activities, may be attributed to the climatic conditions and<br />
soils.<br />
But, despite the complexity of the chemistry of the<br />
inorganic and organic nature, it is possible to find some<br />
general trends like genetics and breeders which are more<br />
and more interested in the link between genetic factors<br />
and phenotypic variation of qualitative and quantitative<br />
composition.<br />
With these anti-inflammatory property, C. colocynthis<br />
Schrad., the Tunisian population, can be considered an<br />
effective agent to treat inflammation diseases. Immature<br />
seeds and fruits demonstrated a high activity at very low<br />
aqueous extract doses (1 and 4 mg/kg). The study<br />
corroborated the pharmacological effects of this species,<br />
justified and supported scientifically, the use of C.<br />
colocynthis as an anti-inflammatory agent to treat pain<br />
and rheumatoid arthritis. Additional studies are ongoing<br />
to confirm this C. colocynthis Schrad. properties with<br />
other models. Further, attempts are made to isolate and<br />
define the active anti-inflammatory fractions and its<br />
components.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4226-4233, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB07.500<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Effects of artemether on the plasma and urine<br />
concentrations of some electrolytes in rats<br />
R.O. Akomolafe 1 *, I.O. Adeoshun 1 , J.B. Fakunle 2 , E.O. Iwalewa 3 , A.O. Ayoka 1 , O.E. Ajayi 1 , O.M.<br />
Odeleye 4 and B.O. Akanji 5<br />
1 Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria.<br />
2 Department of Chemical Pathology, Obafemi Awolowo University, Ile-Ife, Nigeria.<br />
3 Department of Pharmacology, Obafemi Awolowo University, Ile-Ife, Nigeria.<br />
4 Department of Pharmacognosy, Obafemi Awolowo University, Ile-Ife, Nigeria.<br />
5 Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Nigeria.<br />
Accepted 24 March, 2011<br />
This study was carried out to determine the changes in the urine levels of sodium (Na + ), potassium (K + ),<br />
and calcium (Ca 2+ ) of rats during a week of intramuscular administration of artemether (12.5 to 50.0<br />
mg/kg/day), another one week thereafter and their concentrations in the plasma at the end of the study.<br />
At 12.5 and 25.0 mg/kg of artemether, urine Na + concentration was significantly increased throughout<br />
the study (p < 0.05), except on Day 7 (at 12.5 mg/kg) and Day 11 (at 25.0 mg/kg), when it was not<br />
significantly different from the control. At 12.5 mg/kg of the drug, urine K + concentration was<br />
significantly increased throughout the study (p < 0.05). Artemether caused no significant changes in<br />
urine Ca 2+ concentration in the control rats as well as those that received 12.5 and 25.0 mg/kg of<br />
artemether. Progressive and significant reductions in the urine concentrations of all the electrolytes at<br />
50.0 mg/kg of artemether were observed. Their concentrations in the plasma were also significantly<br />
reduced at this dose of the drug. A dose-dependent degeneration of the renal tissue of all the<br />
experimental rats was also observed. We concluded that high doses of artemether caused progressive<br />
degeneration of the renal tissue of rats, inability of the damaged kidneys to concentrate urine, which<br />
manifested as excessive water loss and electrolyte depletion.<br />
Key words: Artemether, electrolytes in plasma, urine concentrations, rats.<br />
INTRODUCTION<br />
Artemether, one of the derivatives of artemisinin, is<br />
reputed for its efficacy in the treatment of malaria,<br />
including those resulting from infection by chloroquineresistant<br />
strains of plasmodium (Qinghaosu antimalarial<br />
coordinating research group, 1979; China cooperative<br />
research group on Qinghaosu and its derivatives as antimalarials,<br />
1982). Though in combination with other antimalarials,<br />
it is gaining ground as one of the first lines of<br />
treatment of cerebral malaria caused by chloroquineresistant<br />
plasmodium (Van Vugt et al., 1999; Nosten et<br />
al., 2000). It has been reported that intramuscular administration<br />
of multiple doses of the drug to dogs, rats<br />
and rhesus monkeys produced neurotoxic effects such as<br />
*Corresponding author. E-mail: rufakom@yahoo.co.uk.<br />
gait disturbances, loss of spinal and pain reflexes (Petras<br />
et al., 1997; Sumalee et al., 1997; Nontprasert et al.,<br />
1998; 2000; Xiao et al., 2002). High doses of artemether<br />
were also reported to have caused neuronal necrosis in<br />
the region of the brainstem of rats (Raymond et al., 1998;<br />
Xiao et al., 2002). Anorexia and a dose-dependent<br />
reduction in body weight have also been reported at<br />
these high doses (Qigui et al., 1998). We have studied<br />
the changes in some of the visceral functions of Wistar<br />
rats following one week of intramuscular administration of<br />
12.5 to 50.0 mg/kg of artemether (Akomolafe et al.,<br />
2006). We reported a pattern of anorexia which<br />
manifested as a significant reduction in the food and<br />
water intake of all the treated rats. This was accompanied<br />
by significant increases in their urine output. These<br />
effects persisted until even one week after the stoppage<br />
of drug administration in those rats that received 50.0
mg/kg of the drug, whereas those that received lower<br />
doses had only their food intake restored during this<br />
period (Akomolafe et al., 2006). We concluded that the<br />
significant increase in urine output without a corresponding<br />
increase in the water intake of the rats could<br />
exacerbate dehydration and lead to a deleterious effect<br />
on the ionic balance of the body fluid of the rats<br />
(Akomolafe et al., 2006). We also postulated that high<br />
doses of artemether could cause impaired renal function<br />
of the treated rats and that the significant increase in<br />
urine output could be due to other effects of the drug on<br />
thirst, anti-diuretic hormone output and the osmotic<br />
pressure of their blood (Akomolafe et al., 2006). The<br />
plasma levels of some electrolytes namely sodium (Na + ),<br />
potassium (K + ), and calcium (Ca 2+ ) are very important for<br />
the proper functioning of the neuromuscular and<br />
cardiovascular systems (Guyton and Hall, 2001; Ganong,<br />
2003). Excessive loss of these ions form the body<br />
through urine stool or sweat could have serious<br />
deleterious effects on these two systems likewise their<br />
excessive retention (Guyton and Hall, 2001; Ganong,<br />
2003). There is a dearth of information on the influence of<br />
artemether on the plasma and urine levels of these<br />
important electrolytes of the body fluid in laboratory<br />
animals, especially rats. This study was carried out to<br />
determine the effects of artemether on the plasma and<br />
urine levels of Na + , K + , and Ca 2+ of rats with a view to<br />
shedding more light on its toxicity in their body fluid and<br />
renal tissue.<br />
MATERIALS AND METHODS<br />
Experimental animals<br />
Eighty adult Wistar rats (200 to 250 g) were used for this study. The<br />
rats were obtained from the Animal Holding of the Department of<br />
Physiological Sciences, Obafemi Awolowo University, Ile-Ife,<br />
Nigeria. They were kept in the laboratory under natural light/dark<br />
cycle and were fed on normal mouse cubes (Ladokun feeds,<br />
Ibadan, Nigeria) and water. The rats were divided into four groups<br />
labeled I, II, III and IV. Each of the groups consisted of twenty rats,<br />
ten males and ten females. Each of the rats was housed in a<br />
separate metabolic cage (Ohaus R Model; Ohaus, Pine Brook, NJ,<br />
USA) with access to food and water ad libitum. The rats were<br />
acclimatized for 2 weeks before the commencement of the<br />
experiments.<br />
Drug administration<br />
Injectable form of artemether (80 mg/ml) manufactured by Kunming<br />
Pharmaceutical Factory, Kunming, People’s Republic of China) was<br />
dispensed in 1 ml ampoules for intramuscular injection.<br />
Dose regimens<br />
Each rat in Group I that weighed 250 g was given 0.16 ml of normal<br />
saline (equivalent to the volume of the drug that was administered<br />
to each 250 g rat that received 50.0 mg/kg/day) for 1 week. This<br />
group served as the control. Each of the rats in Groups II, III and IV<br />
Akomolafe et al. 4227<br />
received 12.5, 25.0 and 50.0 mg/kg/day of artemether respectively<br />
via the intramuscular route for 1 week.<br />
Urine samples were collected into clean specimen bottles for 24<br />
h on the day before the commencement of drug administration and<br />
this was taken as the Day 0 urine for each of the rats. This<br />
procedure was repeated for Days 3, 7, 11 and 14 of the study, that<br />
is, one week of drug administration and another one week later.<br />
The concentrations of Na + , K + and Ca 2+ in the samples were<br />
measured. The urine concentrations of Na + and K + were determined<br />
by Flame Photometry using Coming 410C Flame Photometer. The<br />
Ca 2+ level was measured using Cresolphthalein Complexone<br />
method (Burtis and Ashwood, 2001).<br />
On Day 14, the rats were sacrificed under chloroform anaesthesia.<br />
A midline incision was made with a surgical blade to expose<br />
the abdominal organs. Blood was collected from the hearts of the<br />
rats by cardiac puncture and delivered into lithium heparinized<br />
specimen bottle. A new syringe was used for the collection of blood<br />
from each rat; the blood was immediately centrifuged at 3000<br />
revolutions per minute for 20 min. The plasma was thereafter<br />
separated into a specimen bottle in readiness for analysis. The<br />
concentrations of Na + , K + , and Ca 2+- in the samples were<br />
determined using the same methods that were used in the analysis<br />
of urine.<br />
The rats kidneys were dissected out and kept inside 10%<br />
formalin until their sections were cut and stained with eosin and<br />
hematoxylin for histological studies. Photomicrographs of the<br />
tissues were taken using Lect3 Dialux Microscope (Bright Field) at<br />
×40 magnification.<br />
Statistical analysis<br />
The results were expressed as mean ± SEM and subjected to oneway<br />
analysis of variance (ANOVA). Significant differences were<br />
further tested by the Duncan’s Multiple Range and Student Neuman<br />
Keuls tests. Student’s t-test was used to compare the urine<br />
concentration of the electrolyte for each day with the Day 0 value<br />
for each group. Differences with probability values of p < 0.05 were<br />
considered significant.<br />
RESULTS<br />
Effect of artemether on urine concentration of<br />
electrolytes<br />
Sodium (Na + ): Throughout the study, there was no<br />
significant change in the concentration of Na + in the urine<br />
of the control rats (Figure 1). At 12.5 and 25.0 mg/kg,<br />
urine Na + concentration was significantly higher than the<br />
Day 0 value and the control rats throughout the study (p <<br />
0.05), except on Day 7 (at 12.5 mg/kg) and Day 11 (at<br />
25.0 mg/kg), when it was not significantly different from<br />
these values. At 50.0 mg/kg, the urine Na + concentration<br />
decreased gradually and significantly throughout the<br />
study (p < 0.05).<br />
Potassium (K + ): The K + concentration in the urine of the<br />
control rats did not vary significantly throughout the study<br />
(Figure 2). At 12.5 mg/kg, K + concentration in the urine<br />
was significantly higher than that of the control rats and<br />
that of the Day 0 value throughout the study (p < 0.05).<br />
At 25.0 mg/kg, there was no significant change in K +
4228 Afr. J. Biotechnol.<br />
Urinary potassium concentration (mmol/L)<br />
Urinary concentration of sodium (mmol/L)<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
*<br />
*<br />
NS<br />
12.5 mg/Kg<br />
25.0 mg/Kg<br />
50.0 mg/Kg<br />
a<br />
*<br />
a * a<br />
a<br />
* *<br />
0<br />
0 3 7 11 14<br />
Figure 1. Variation in the urine concentration of sodium due to a week of intramuscular administration of<br />
artemether to rats. Each point is mean ± SEM (n = 20). *, Significantly different from the Day 0 value (p <<br />
0.05); a, significantly different from control (NS) (p < 0.05).<br />
600<br />
500<br />
400<br />
300<br />
200<br />
100<br />
0<br />
(mmol/L)<br />
*<br />
*<br />
a<br />
a<br />
a<br />
Day<br />
0 3 7 11 14<br />
Day<br />
a<br />
NS<br />
12.5 mg/Kg<br />
25.0 mg/Kg<br />
50.0 mg/Kg<br />
Figure 2. Variation in the urine concentration of potassium due to a week of intramuscular administration of<br />
artemether to rats. Each point is mean ± SEM. (n = 20). *, Significantly different from the Day 0 value (p
DISCUSSION<br />
Urinary concentration of calcium (mmol/L)<br />
1.6<br />
1.4<br />
1.2<br />
1<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
(mmol/L)<br />
*<br />
a a<br />
0<br />
0 3 7 11 14<br />
*<br />
Day<br />
*<br />
NS<br />
Akomolafe et al. 4229<br />
12.5 mg/Kg<br />
25.0 mg/Kg<br />
50.0 mg/Kg<br />
Figure 3. Variation in the urine concentration of calcium due to a week of intramuscular administration of<br />
artemether to rats. Each point is mean ± SE (n = 20). *, Significantly different from the Day 0 value (p <<br />
0.05). a, significantly different from control (NS) (p < 0.05)<br />
This study showed a dose-dependent degeneration of the<br />
renal tissue of the rats under the influence of high doses<br />
of artemether (Plates 1 to 4). Chemical compounds which<br />
furnish oxygen based radicals are reputed for their cell<br />
membrane lipid peroxidation properties. Lipid<br />
peroxidation refers to the oxidative degradation of lipids.<br />
It is the process whereby free radicals take electrons<br />
away from lipids in cell membranes, resulting in cell<br />
damage. The endoperoxide bridge in artemether molecule<br />
produces free radicals which are responsible for the lipid<br />
peroxidation properties of the drug (Goodman and<br />
Gilman, 1985; Meshnick, et al., 1991, 1993; Maeno et al.,<br />
1993). The degenerative changes observed in the renal<br />
tissue of the treated rats could be attributed to the<br />
destruction of the membrane of the renal tubular cells of<br />
the rats by the free radicals produced by the drug.<br />
Analysis of the urine of the rats for electrolytes revealed<br />
a progressive and significant reduction in the concentration<br />
of the electrolytes at 50.0 mg/kg of artemether.<br />
This indicated that the rats that received this dose of<br />
artemether produced more dilute urine with each passing<br />
day of the study, despite the anorexia induced by the<br />
drug. Inability of the kidney to concentrate or dilute the<br />
urine is one of the features of damaged nephrons (Leaf<br />
and Cotran, 1980; Neinman, and Lorenz, 1989; Guyton<br />
and Hall, 2001; Dunn, 2003; Ganong, 2003). Rapid<br />
tubular flow occurs in the remaining nephrons of the<br />
diseased kidneys. The renal tubules lose their ability to<br />
concentrate or dilute the urine (Guyton and Hall, 2001).<br />
This study indicated that high doses of artemether are<br />
toxic to the renal tissue of the rats. We have earlier<br />
reported a significant increase in the volume of the urine<br />
produced by rats that received 50.0 mg/kg of artemether,<br />
during treatment and even one week post-treatment,<br />
without a corresponding increase in their food and water<br />
intake for the same period (Akomolafe et. al., 2006). The<br />
excessive fluid loss of rats that received this dose of<br />
artemether could lead to dehydration and a severe<br />
depletion of the major electrolytes of their body fluid. The<br />
photomicrograph of the kidney of rats that were given this<br />
dose of artemether showed a very high degree of tissue<br />
degeneration. The continuous significant decreases in<br />
the urine concentration of the electrolytes could not have<br />
been due to increased reabsorption of the electrolytes by<br />
the renal tubules, but rather to the artemether-induced<br />
anorexia and the excessive water loss that usually<br />
accompanies increased electrolyte loss in urine (osmotic<br />
diuresis).<br />
Throughout the study, there was no significant change<br />
in the concentration of Na + in the urine of the control rats<br />
(Figure 1). This is an indication that the kidney function of<br />
the rats was not impaired, as evidenced by the photomicrograph<br />
of their kidneys (Plate 1). At 12.5 and 25.0<br />
mg/kg, urine Na + concentration was significantly higher<br />
than the Day 0 and control values throughout the study (p<br />
< 0.05), except on Day 7 (at 12.5 mg/kg) and Day 11 (at<br />
25.0 mg/kg), when it was not significantly different from<br />
these values. Our earlier report indicated that rats that<br />
*<br />
a
4230 Afr. J. Biotechnol.<br />
M<br />
S<br />
G<br />
B<br />
Plate 1: Photomicrograph of the kidney of rats that received normal saline i.m. for 7 days (Control 1 rats). Mag x 400.<br />
The kidney tissue shows no pathological changes, as reflected by the Bowman’s capsule (B), the glomerulus (G), renal<br />
tubules (T) and other cells of the renal tissue. The glomerulus (G) and the capsular space (S) are distinct and intact.<br />
The tubules retain their normal shapes and the tubular cells have well defined boundaries, and nuclei. The mesangial<br />
cells (M) are normal in size shape and number.<br />
.<br />
T<br />
C<br />
Plate 2. Photomicrograph of the kidney of rats that received 12.5 mg/kg of artemether i.m. for 7 days.<br />
Mag×400. The glomerulus (G) has expanded to the extent of almost closing the capsular space(S). The tubular<br />
cells (C) are densely stained. The tubules (T) have started losing their integrity as reflected in the distortion of<br />
their shape.<br />
received 25.0 mg/kg of artemether had significantly<br />
increased urine output which lasted for even a week after<br />
treatment, without a corresponding increase in food and<br />
water intake, most especially during treatment (Akomolafe<br />
et al., 2006).<br />
The significant increase in urine Na + concentration at<br />
25.0 mg/kg of artemether is an evidence of dehydration<br />
induced by the drug. The integrity of the nephrons of the<br />
rats had been severely compromised as revealed by<br />
Plate 3. The significant increase in urine Na +<br />
concentration at this dose of the drug could also be a<br />
reflection of excessive sodium loss by the damaged<br />
T<br />
G<br />
kidneys of the rats.<br />
The K + concentration in the urine of the control rats did<br />
not vary significantly throughout the study (Figure 2). At<br />
12.5 mg/kg, K + concentration in the urine was significantly<br />
higher than the control and Day 0 values throughout the<br />
study (p < 0.05). At 25.0 mg/kg, there was no significant<br />
change in K + concentration throughout the study.<br />
Potassium, once absorbed by the intestinal tract, is<br />
partially removed from the plasma by glomerular filtration<br />
and is then nearly completely reabsorbed in the proximal<br />
tubule. Unlike sodium, it is effectively re-excreted by the<br />
distal tubules There is no threshold level for K + ( Guyton<br />
S
.<br />
Plate 3. Photomicrograph of the kidney of rats that received 25.0 mg/kg of artemether, i.m. for 7 days.<br />
Mag×400. The Bowman’s capsule (B) started breaking down. The glomerulus (G) has undergone<br />
degeneration. The capsular space (C) has been completely obliterated. The tubular cells (S) are still<br />
densely stained. The tubules (T) have lost much of their integrity as their boundaries are no more well<br />
defined. The intertubular space (P) are almost completely closed due to inflammation of the tubules.<br />
T<br />
C<br />
B<br />
T<br />
C<br />
G<br />
B<br />
Akomolafe et al. 4231<br />
Plate 4.Photomicrograph of the kidney of rats that received 50.0 mg/kg of artemether i.m. for 7 days. Mag×400. The<br />
Bowman capsule (B) is almost non-existent due to degeneration. The densely stained tubular cells (C) are still<br />
observable, though the tubular intergrity (T) has been grossly distorted as a result of inflammation and degenerative<br />
changes.<br />
and 25.0mg/kg of artemether in this study could be<br />
attributed to the differences in the renal handling of the<br />
two electrolytes. Potassium is mainly an intracellular<br />
electrolyte. The damaged renal tissue could also be a<br />
source for additional K + in the body fluid of the rats.<br />
Artemether caused no significant change in urine Ca 2+<br />
concentration in the control rats as well as those that<br />
received 12.5 and 25.0 mg/kg of artemether, throughout<br />
the study (Figure 3). It could be due to the fact that<br />
artemether does not have any appreciable effects on<br />
renal Ca2 + excretion at these doses of the drug.<br />
However, this needs further verification.<br />
The plasma Na + concentration was significantly increased<br />
at 25.0 mg/kg of artemether only (p < 0.05) (Figure<br />
4). This is in conformity with the observed significant<br />
elevation of the urine concentration of this electrolyte in<br />
rats that received this dose of artemether. The<br />
significantly increased Na + concentration in the plasma of<br />
rats that received 25.0 mg/kg of the drug could be due to<br />
the same reasons given for the elevated urine level of the<br />
electrolyte. The reductions observed at 12.5 and 50.0<br />
mg/kg of the drug were not significant. The plasma K +<br />
concentration increased significantly at all the doses of<br />
artemether studied, except at 50.0 mg/kg where it was<br />
significantly reduced (Figure 5). The significant increase<br />
in the plasma concentrations of this electrolyte at lower<br />
doses of artemether could be as a result of the druginduced<br />
renal tissue damage at these doses, while the
4232 Afr. J. Biotechnol.<br />
Potassium (mmol/L)<br />
7<br />
6<br />
5<br />
4<br />
3<br />
2<br />
1<br />
0<br />
Sodium (mmol/L)<br />
(mmol/L)<br />
142<br />
140<br />
138<br />
136<br />
134<br />
132<br />
130<br />
128<br />
(mmol/L)<br />
NS 12.5 25.0 50.0<br />
Dose (mg/kg)<br />
Figure 4. Effect of artemether on the plasma sodium concentration of rats. *Significantly different<br />
from control (p < 0.05). The plasma Na + concentration was significantly increased at 25.0 mg/kg of<br />
artemether only (p < 0.05). The reduction observed at 12.5 and 50.0 mg/kg of the drug was not<br />
significant.<br />
*<br />
NS 12.5 25.0 50.0<br />
Dose (mg/kg)<br />
Figure 5. Effect of artemether on the plasma potassium concentration of rats. *Significantly different from control (p <<br />
0.05). The plasma K + concentration increased significantly at all the doses of artemether studied, except at 50.0 mg/kg<br />
where it was significantly reduced.<br />
significant decrease at 50.0 mg/kg could be attributed to<br />
the earlier reported sustained significant reduction in food<br />
consumption, increased urine output, as well as much<br />
degeneration of the nephrons induced by artemether.<br />
There was no significant difference in the plasma Ca 2+<br />
level of the control rats and those that received 12.5 and<br />
25.0 mg/kg of artemether (Figure 6). This is an indication<br />
that, at these doses of artemether, calcium excretion was<br />
not adversely affected by the renal tubular damage<br />
induced by the drug. However, a significant decrease in<br />
*<br />
*<br />
the plasma level of this electrolyte was observed at 50.0<br />
mg/kg of the drug.<br />
Conclusion<br />
High doses of artemether caused progressive degeneration<br />
of the renal tissue, inability of the nephrons to<br />
concentrate urine, excessive water loss and electrolyte<br />
depletion in rats.<br />
*
Calcium (mmol/L)<br />
2.5<br />
2<br />
1.5<br />
1<br />
0.5<br />
0<br />
NS 12.5 25.0 50.0<br />
Dose (mg/ ) kg)<br />
Akomolafe et al. 4233<br />
Figure 6. Effect of artemether on the plasma calcium concentration of rats. *Significantly different from control (p < 0.05).<br />
There was no significant difference in the plasma Ca 2+ level of the control rats and those that received 12.5 and 25.0 mg/kg<br />
of artemether. At 50.0 mg/kg, plasma Ca 2+ decreased significantly.<br />
ACKNOWLEDGMENTS<br />
We are grateful to Obafemi Awolowo University, Ile-Ife for<br />
financing this study and to Mr. J.A. Ibeh, of the<br />
Department of Anatomy and Cell Biology, and Messrs T.<br />
A. Ogundoyin, F. A. Abidoye, C. O. Ola, R. T. Olatoye,<br />
and A. E. Adebiyi of the Department of Physiological<br />
Sciences for their technical assistance in the process of<br />
carrying out this research work<br />
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Petras JM, Kyle DE, Gettayacamin M, Young GD, Bauman RA,<br />
Webster HK, Corcoran KD, Peggins JD, Vane MA, Brewer TG<br />
(1997). Arteether; Risk of two-week administration of Macaca<br />
mulatta. Am. J. Trop. Med. Hyg. pp. 390-396<br />
Qigui L, Thomas GB, James OP (1998). Anorexic toxicity of<br />
dihydroartemisinin, artemether, and arteether in rats following<br />
multiple intramuscular doses. Int. J. Toxicol. 17: 663-676<br />
Qinghaosu Antimalarial Coordinating Research Group (1979).<br />
antimalarial studieson qinghaosu. Chin. Med. J. 92: 811-816<br />
Qinghaosu Antimalarial Coordinating Research Group, Haiman Island<br />
(1979). Observations on the clinical effect of qinghaosu in the<br />
treatment of chloroquine-resistant malaria. J. Nat. Drug. 9: 12-16.<br />
Nosten F, Van Vugt M, Prince R, Luxemburger C, Thyway KL,<br />
Brockman A, McGready R Kuile F, Looareesuuvan S, White NJ<br />
(2000). Effects of artesimate-mefloquine combination on incidence of<br />
plasmodium falciparum malaria and mefloquine resistance in<br />
Western Thailand; A prospective study. Lancet. 356: 297-302.<br />
Raymond GG, Donald BN, Qigui Li JO, Thomas JB (1998). Dosedependent<br />
brainstem neuropathology following repeated arteether<br />
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Sumalee K, Paul M, Paul H, Herman Z, Steven RM (1997). Artemisinin<br />
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Van Vugt M, Wilariratana P, Gemperti B, Gathman I, Phaipum L,<br />
Brockman A, Luxemburger C, White NJ, Nosten F, Looareesuwan S<br />
(1999). Efficacy of six doses of artemether-lumefantrine (Benflumetol)<br />
in multidrug-resistant plasmodium falciparum malaria. Am. J. Trop.<br />
Med. Hyg. 60(6): 736-942.<br />
Xiao S, Yang Y, You Q, Utzinger J, Guo H, Peiying J, Mei J, Guo J,<br />
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*
African Journal of Biotechnology Vol. 10(20), pp. 4234-4241, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2174<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Expression of biological active VHH camelid single<br />
domain antibody in transgenic tobacco<br />
Behnaz Korouzhdehy 1 , Mehdi Dadmehr 1 *, Issa Piri 1 , Fatemeh Rahbarizadeh 2 and Mahmood<br />
Solouki 3<br />
1 Department of Agriculture, University of Payame Noor, Tehran, Iran.<br />
2 Department of Medical Biotechnology, University of Tarbiat Modares, Tehran, Iran.<br />
3 Department of Agriculture, University of Zabol, Zabol, Iran.<br />
Accepted 1 April, 2011<br />
Functional VHH single domain antibody lacking light chains occur naturally in Camelidae. The single<br />
domain nature of VHH gives rise to several unique features when compared to antigen-binding<br />
derivatives of conventional antibodies. The level of expression in Escherichia coli was found to be too<br />
low for therapeutic purposes. Therefore, there is a need to examine other production systems such as<br />
plants. Several plants are the facile and economic bioreactor for large-scale production of industrial<br />
and pharmaceutical agents like proteins and antibodies. Here, we have selected tobacco as the host<br />
plant because of large scale production capability and many other advantages such as greater safety<br />
and lower production costs when compared to animal-based systems. In this study, we have subcloned<br />
VHH gene into pBI121 using phasmid pCANTAB5E. The new construct was used to transform the<br />
Agrobacterium strains C58GV3101 and LBA4404. Agrobacterium strain C58GV3101 showed a higher<br />
virulence on leaf disks of Nicotiana tabacum (NC25). Transgenic tobacco plants were then developed by<br />
introducing VHH gene under the control of CaMV 35S promoter. The presence of the VHH antibody gene<br />
in the plant genome was verified by PCR analysis and Southern hybridization experiments. Northern<br />
blot analysis showed that the genes coding for the VHH could be expressed in tobacco plants. Three<br />
lines of transgenic plant that expresses high levels of mRNA were screened in a further analysis. The<br />
expression of VHH was then observed in transgenic plants by ELISA using the specific antibody<br />
developed, the results showed three to five folds higher than non-transgenic tobaccos.<br />
Key words: VHH antibody fragment, subcloning, antibodies, transgenic tobacco, bioreactor.<br />
INTRODUCTION<br />
Antibodies have been used as diagnostic or therapeutic<br />
agents in vivo as well as ex vivo for the last two decades.<br />
Their clinical applications are evident in the treatment of<br />
diseases such as cancer, transplantation, autoimmunity<br />
and cardiovascular disorders. They have been specifically<br />
used in cancer therapy because of their high<br />
specificity for tumour antigens and low cross-reactivity<br />
with normal cells. VHHs are heavy chain variable domains<br />
derived from immunoglobulins (IgG) naturally devoid of<br />
light chains such as those derived from Camelidae in pre-<br />
*Corresponding author. E-mail: mehdidadmehr@yahoo.com.<br />
vious studies (Hamers-Casterman et al., l993) Furthermore,<br />
their H chain is devoid of the CH1 domain due to<br />
an unconventional splicing event during mRNA maturation.<br />
Therefore, the antigen-binding fragment of the heavychain<br />
antibody consists of a single domain referred to as<br />
VHH that replaces a four-domain Fab fragment in the<br />
immunoglobulin structure (Muyldermans et al., 1994).<br />
VHH molecules are about 10 times smaller than IgG<br />
molecules. They are single polypeptides and very stable,<br />
resisting extreme pH and temperature conditions.<br />
Moreover, they are resistant to the action of proteases<br />
which is not the case for conventional antibodies.<br />
Furthermore, in vitro expression of VHH’s produces high<br />
yield, properly folded functional VHHs. In addition,<br />
antibodies generated in Camelids will recognize epitopes
than those recognised by antibodies generated in vitro<br />
through the use of antibody libraries or through immunization<br />
of mammals other than Camelids (Ghahroudi et<br />
al., 1997). As such, anti-MUC1 VHHs may interact more<br />
efficiently with its target than conventional antibodies,<br />
thereby blocking its interaction with the target ligands<br />
more efficiently. Since VHH’s are known to bind into<br />
unusual epitopes such as cavities or grooves (Spinelli et<br />
al., 2000), the affinity of such VHH's may be more<br />
suitable for therapeutic treatment. Besides the<br />
advantages of easy cloning (single gene) and selection<br />
from in vivo matured library, it has other technological,<br />
physiochemical and functional advantages, such as close<br />
homology to human VH fragments, high expression yield,<br />
highly soluble and the generation of antigen-specific high<br />
affinity binders (Nguyen et al., 2001). Recently, Camelid<br />
single domain antibodies have been used to target MUC1<br />
antigens in breast cancer (Taylor-Papadimitriou et al.,<br />
2002). These fragments have been expressed in both<br />
Escherichia coli and P. pastoris (Rahbarizadeh et al.,<br />
2006). However, these expression systems have several<br />
limitations that hinder maximum output of biologically<br />
active and safe therapeutic agents. Some of these<br />
limitations that have been reported are: (1) formation of<br />
inclusion bodies in bacteria, (2) formation of non-native<br />
proteins having different biological activities in yeast, (3)<br />
low transgene expression levels, (4) transgene induced<br />
instability of certain cell lines in mammalian cell cultures<br />
and (5) contamination of animal-based products with<br />
human pathogens (Koprowski et al., 2005). Such<br />
shortcomings invite alternative methods of production to<br />
ensure the safety and economical benefits of recombinant<br />
therapeutic proteins (Carmer et al., 1999). Plantbased<br />
systems are increasingly used for the production<br />
of recombinant proteins including antibodies (During et<br />
al., 1990; Peeters et al., 2001; Torres et al., 1999;<br />
Schillberg et al., 2005). Plant-based systems have<br />
several advantages over the other production systems,<br />
such as the ability to carry out necessary posttranslational<br />
modifications not available in bacterial<br />
systems, as well as greater safety and lower production<br />
costs when compared to animal-based systems. Plantbased<br />
technology has been recently reviewed, with full<br />
description of commonly used plants (Hellwig et al.,<br />
2004). Processing of transgenic crops would require<br />
relatively little capital investment, making the commercial<br />
production of biopharmaceuticals an exciting prospect. It<br />
has been estimated that the cost of producing<br />
recombinant proteins in plants could be 10 to 50 fold<br />
lower than that for producing the same protein in E. coli<br />
or mammalian cells (Stoger et al., 2000, 2002). Several<br />
proteins, enzymes and antibodies have been produced in<br />
plants and used in clinical trials, with a prospect of<br />
commercial exploitation (Ma et al., 2003). In this report,<br />
which is the first of its kind, we described the cloning and<br />
expression of VHH single domain antibody in tobacco<br />
plants as a first step in the development of a plant based<br />
Korouzhdehy et al. 4235<br />
vaccine that could produce VHH and a low-cost biomass<br />
scale-up was performed with this bioreactor.<br />
MATERIALS AND METHODS<br />
Construction of the expression vector pBI-VHH<br />
The phasmid vector pCANTAB5E containing the VHH coding<br />
sequence that was partially optimized for expression in competent<br />
E. coli TG1 was obtained from the Department of Medical<br />
Biotechnology, Tarbiat Modares University (AY544575). The VHH<br />
encoding region was amplified from pCANTAB5E phasmid DNA<br />
using the primers back: 5´-<br />
GGAAATTCGAGCTCTTAGTGAGATGGTGAC-3´ and forward:<br />
5´TCTAGAGGA TCCTAAACAATGGTCCTGCTACAGTCA-3´. Each<br />
PCR mixture was prepared in a final volume of 50 µl containing 50<br />
ng pCANTAB5E phasmid DNA template, 50 pM forward primer, 50<br />
pM of the corresponding backward primer, 40 pM dNTPs, 25 mM<br />
MgCl2, 1.25 U Taq DNA polymerase (Cinnagen) and 5 µl 10 × PCR<br />
buffer II (Cinnagen). Hot start PCR was performed at 95°C for 5<br />
min, followed by 35 cycles of denaturation at 94°C for 30 s,<br />
annealing at 52°C for 45 s and elongation at 72°C for 1 min. The<br />
reaction was completed by a final extension time at 72°C for 10<br />
min. The amplified variable regions were purified by electrophoresis<br />
on a 1.5% agarose gel and subsequently extracted with an<br />
AccuePrep Gel Extraction Kit (Bioneer) according to the<br />
manufacturer’s instructions.<br />
Amplified DNA was cloned into T/A vector (pTZ57R) using the<br />
InsT/A clone PCR product cloning kit from Fermentas. pTZ57R<br />
plasmid was cut with BamH I and Sac I (Roche Applied Science).<br />
After another electrophoresis on a 1.5% agarose gel, the variable<br />
regions were excised and extracted with the AccuPrep Gel<br />
Extraction Kit (Bioneer). The fragment of interest was ligated with<br />
the corresponding cut vector pBI 121 in a 10 µl volume containing<br />
50 ng vector DNA, threefold molar excess of the PCR product and<br />
5 U of T4 DNA ligase (Fermentas) for 16 h at 14°C. E. coli TG1<br />
cells (50 µl) were transformed with 2 µl of the ligation product via<br />
CaCl2 method. One milliliter of prewarmed SOC medium<br />
(Sambrook et al., 2001) was immediately added, and the cells were<br />
grown at 37°C for 1 h shaking with 250 rpm. Cells were plated on<br />
LB agar medium containing kanamycin (25 µg/ml) and incubated<br />
overnight at 30°C.<br />
Colonies were picked for plasmid extraction using the Accuprep<br />
Plasmid Extraction Kit (Bioneer). Agrobacterium tumefaciens strains<br />
C58GV3101 and LBA4404 have been previously described<br />
(Hoekema et al., 1983; Koncz et al., 1986). The engineered pBI 121<br />
plasmids were used to transform the both earlier mentioned<br />
Agrobacteriums using a freeze and thawing standard protocol<br />
(Sambrook et al., 2001).<br />
Transformation<br />
Axenic plants of tobacco (Nicotiana tabaccum var.NC25) were<br />
maintained under in vitro growth conditions at 26°C and 16 h<br />
photoperiod in the laboratory. For tobacco transformation,<br />
Agrobacterium cells were used for transformation. Leaf explants of<br />
in vitro-grown were inoculated with Agrobacterium in a YEP liquid<br />
medium for 10 min. For effective transformation, the explants were<br />
placed in a co-culture medium containing 2.0 mg/l BAP and 0.01<br />
mg/l NAA. Two days later, the explants were transferred to the<br />
regeneration medium (MS medium, supplemented with 2.0 mg/l<br />
BAP, 0.01 mg/l NAA, 200 mg/l cefatoxim and 100 mg/l kanamycin).<br />
The explants were transferred to the fresh medium at 2 weeks<br />
intervals. As a control, non-inoculated explants were cultured in the<br />
same medium without hormones and antibiotics. The induced
4236 Afr. J. Biotechnol.<br />
shoots were then dissected from the explants and transferred to<br />
free hormone MS rooting medium containing cefatoxim (200 mg/l)<br />
for Agrobacterium elimination and kanamycin (100 mg) as a<br />
selective antibiotic. Also, specific modified MS media were<br />
developed and used for co-culture, regeneration and rooting steps<br />
by Dadmehr (Unpublished data). When the roots were induced on<br />
the regenerated shoots, plantlets were transplanted to moist<br />
vermiculite for acclimatization. When the shoots had began to grow,<br />
the plantlets were transplanted to moist soil and the plants were<br />
grown to maturity.<br />
PCR analysis and Southern blot hybridization<br />
Genomic DNA from 200 mg each of non-transgenic plants as<br />
negative control and all putative kanamycin resistant plants was<br />
extracted from transgenic plants using the CTAB method (Rogers et<br />
al., 1994). Transformed and control plant genomic DNA was used<br />
as a template to detect the VHH gene by polymerase chain reaction<br />
(PCR) under the conditions that were described before and with<br />
specific primers. The 327 bp amplified DNA fragments were<br />
analyzed by electrophoresis on a 1.5% agarose gel stained with<br />
EtBr and observed under UV illumination. The genomic DNA also<br />
was digested with SacI to confirm the copy number of inserted T-<br />
DNAs for the VHH derived transformants. The digested DNA was<br />
electrophoresed on a 1.0% agarose gel and then transferred to<br />
positively charged nylon membrane (Roche Co., Germany) using a<br />
Turboblotter system (Schleicher and Schuell). Dig-labeled probes<br />
were generated by PCR Dig Labeling Mix (Roche Co., Germany)<br />
with the specific primer set for the VHH gene. DNA gel blots were<br />
hybridized at 50°C in a DIG easy hybridization buffer. After<br />
hybridization overnight, the membrane was washed in an SSC<br />
buffer series and then detected using the Dig Detection Kit following<br />
the manufacturer’s instructions (Roche Co., Germany).<br />
Northern blot analysis<br />
Total RNA from the leaves of transformed plants was isolated using<br />
the RNAgents ® Total RNA isolation system (Promega Co., USA)<br />
according to the manufacturer’s instructions. Samples of 30 µg of<br />
total RNA were denatured with formaldehyde and formamide and<br />
fractionated in a 3.0% agarose gel using a 3-(N-morpholino)<br />
propane sulfonic acid (MOPS) buffer. The RNA was blotted to a<br />
positively charged nylon membrane, and fixed by ultraviolet<br />
irradiation. The membranes were hybridized with Dig-labeled<br />
probes using a template containing most of the coding region for<br />
VHH. After hybridization for 10 h at 68°C, the blots were washed in<br />
an SSC buffer series and then detected by means of the Dig<br />
Detection Kit according to manufacturer’s instructions (Roche Co.,<br />
Germany).<br />
Quantification of VHH levels in transgenic tobaccos<br />
Antibodies from leaves were obtained by homogenization in a<br />
blender with liquid nitrogen, and the resulting powder was<br />
resuspended in cold extraction buffer containing PBS, pH 7.2, 10<br />
mM EDTA, 0.03% Triton X-100, 5 mM mercaptoethanol and 1 mM<br />
PMSF (1 g of fresh weight/ml). The extract was filtered and<br />
centrifuged for 20 min at 12,000 ×g and the resulting supernatant<br />
was used for the VHH protein expression analysis. ELISA plates<br />
were coated with 100 µl of coating buffer containing the sample<br />
proteins from the transgenic plants overnight at 4°C. After standing<br />
overnight, the plates were washed and blocked 2 h at RT with 5%<br />
skim milk in PBS-Tween 20. The plates were then washed three<br />
times with 0.05% Tween 20 in PBS and 100 µl of MUC1 synthetic<br />
peptide TSA-P1-24 as the primary antibody diluted at 1:1000 in<br />
PBS-Tween 20% was added to each well and allowed to react for 2<br />
h at RT. The plates were again washed three times, and treated<br />
with 100 µl of anti-(camel) rabbit antibodies conjugated with<br />
horseradish peroxidase as the second-antibody, diluted at 1:2000 in<br />
PBS-Tween 20 for 2 h at RT. Finally, the plates were again washed<br />
four times, and 200 µl of a freshly prepared solution of<br />
ophenylenediamine dihydrochloride and H2O2 was added.<br />
Reactions were stopped by adding 2.5 M H2SO4. The absorbance<br />
was measured in a Multiskan ELISA Reader (Labsystems, Helsinki,<br />
Finland) using a 492-nm filter.<br />
RESULTS<br />
Construction and transformation of vectors<br />
Single domain antibody gene of the pCANTAB5E was<br />
subcloned into the expression vector pBI121. The<br />
transfer of this gene is schematically shown in Figure 1.<br />
The isolation and PCR amplification of the gene encoding<br />
the VHH antibody was performed using pCANTAB5E<br />
phasmid DNA as template. Targeting the restriction sites<br />
BamH1 and Sac1 in pTZ57R plasmid facilitated<br />
subcloning. New construct (plasmid) was transformed<br />
into TG1 E. coli and the colonies were appeared on the<br />
kanamycin containing plate. After the plasmid extraction,<br />
both pTZ57R and pBI121 plasmids were cut by BamH1<br />
and Sac1 restriction enzymes. Electrophoresis showed<br />
327 bp bands from pTZ57R and 12000 and 1900 bp GUS<br />
removed from pBI121. The amplified 327 and 12000 bp<br />
bands were gel-purified from agarose. During the ligation,<br />
VHH gene was inserted into pBI121 plasmid and was<br />
called PBI-VHH (Figure 1). PBI-VHH plasmid was<br />
transferred to Agrobacterium strains C58GV3101 and<br />
LBA4404.<br />
Effects of two strains of Agrobacterium on the<br />
efficiency of transformation in tobacco<br />
To improve the efficiency of transformation, this study<br />
examined the effects of two Agrobacterium strains on<br />
shoot formation from leaf disks. 168 and 162 leaf disks<br />
were infected by Agrobacterium strains C58GV3101 and<br />
LBA4404, respectively with a copy of pBI-VHH. All of<br />
these were transferred to plates with media containing<br />
cefatoxim. The frequency of regenerated shoots from leaf<br />
disks that were infected with C58GV3101 was higher.<br />
Fourteen (14) transformants were obtained from leaf<br />
disks infected with the Agrobacterium strain LBA4404<br />
after 3 months in culture. In this experiment, the<br />
transformation efficiency was at least 8.3%, a ratio of<br />
about 7.8-fold of C58GV3101 (Table 1). These results<br />
indicated that kind of Agrobacterium strains affects the<br />
efficiency of transformation in N. tabacum. When the leaf<br />
explants were inoculated with Agrobacterium immediately<br />
after excision, shoots on the cut edges of the explants<br />
were observed in the presence of 100 mg/l kanamycin<br />
and 200 mg/l cefatoxim after two weeks. The putatively
Figure 1. Schematic representation of pBI-VHH construction. VHH gene inserted<br />
between CaMV 35S promoter and NOS terminator.<br />
Table 1. Effect of two Agrobacterium strains on transformation frequency.<br />
Number of experiment Strain<br />
EX1<br />
EX2<br />
EX3<br />
Total<br />
transformed shoots were excised when they were about<br />
1 cm tall and transferred to a shoot elongation medium<br />
containing cefatoxim and kanamycin.<br />
Consequently, shoots were transferred to rooting media<br />
and after hardenization to the greenhouse. The genes<br />
that had been stably integrated into the plant genome<br />
were transcripted under the control of CaMV35S,<br />
resulting in the expression of the VHH gene.<br />
Number of leaf<br />
disk<br />
Number of<br />
regeneration shoot<br />
Korouzhdehy et al. 4237<br />
Number of<br />
transgenic shoot<br />
C58GV3101 68 26 7<br />
LBA4404 52 10 3<br />
C58GV3101 24 3 1<br />
LBA4404 30 0 0<br />
C58GV3101 76 28 6<br />
LBA4404 80 14 0<br />
C58GV3101 168 57 14 (8.3%)<br />
LBA4404 162 24 3 (1.85%)<br />
Expression of VHH antibody fragment in plants and<br />
molecular analysis<br />
More than 160 kanamycin-resistant putative transformants<br />
were obtained (Table 1) and carried out further<br />
analysis of VHH gene expressions on selected transgenic<br />
plants. Untransformed tobacco plants that had been<br />
regenerated from leaf discs without kanamycin selection,<br />
were used as negative controls. PCR analysis was
4238 Afr. J. Biotechnol.<br />
carried out as the first method to confirm the transgenic<br />
nature of the regenerated plants. The presence of VHH<br />
DNA in the genomic DNA isolated from regenerated<br />
tobacco was confirmed. Transformants were detected by<br />
PCR amplification of inserted VHH with specific primers.<br />
The expected 327 bp VHH bands were found in the<br />
transformants (Figure 2). No DNA product was detected<br />
in untransformed control plant DNA. The same fragment<br />
was amplified using plasmid pBI 121 as our positive<br />
control. Southern blot analysis of PCR positive transgenic<br />
lines (T1) was done to verify site-specific integration and<br />
to establish copy number. In order to determine the T-<br />
DNA copy number in each transformant, the DNA was<br />
digested with SacI, which cuts the T-DNA between VHH<br />
Figure 2. Agarose gel electrophoresis of VHH amplification<br />
products from transgenic plants. Lane M: 100 bp ladder; lane WT:<br />
wild type tobacco; lanes 1 to 7: transgenic tobacco plants lines 2,<br />
3, 6, 7, 12, 18 and 19.<br />
Figure 3. Southern blot analysis of transgenic tobacco lines 1, 9 and<br />
22 for the presence of VHH gene. Lane WT: genomic DNA from nontransgenic<br />
tobacco; Lanes 1 to 5: genomic DNA from transgenic<br />
tobacco lines 2, 6, 7, 9, 12 and 18.<br />
Figure 4. Expression of VHH mRNA extracted from leaf tissue.<br />
Lane WT: non-transgenic tobacco; Lanes 1 to 4: transgenic<br />
tobacco lines 2, 6, 7 and 12.<br />
and the nos terminator. The probe then hybridizes to one<br />
T-DNA end fragment from each single-copy insertion,<br />
thus permitting the copy number of the VHH gene to be<br />
established. The transgene copy number as estimated by<br />
Southern hybridization ranged from one to two copies<br />
(Figure 3).<br />
Detection of the VHH transcripts<br />
The transformants were analyzed by hybridizing RNA<br />
samples with a labeled probe encompassing the coding<br />
region of the VHH gene. Figure 4 shows the results of an<br />
experiment, where selected transformants harboring the
Figure 5. VHH expression levels in transgenic tobacco lines. Production of VHH in tobaccos<br />
of four independent transgenic plants (T2, T6, T7 and T12) and one wild type plant (WT) was<br />
analyzed by ELISA. Mean values and standard deviations of three independent analysis are<br />
represented. A reaction buffer (B) was used as a blank control.<br />
pBI 121constructs and a non-transgenic control were<br />
probed. A VHH transcript was found in the four lines of<br />
transgenic tobacco (nos. 2, 6, 7 and 12), and the mRNA<br />
levels in individual transgenic lines were all quite different<br />
(Figure 4). A high level of a VHH transcript was found in<br />
the 6 and 7 transgenic lines. The signal was variable<br />
among different transformants, due to random integration<br />
of the foreign gene into the host tobacco genome. The<br />
size of VHH specific transcript was confirmed by RNA<br />
Century TM Size Markers (Ambion, Inc., Austin, TX USA)<br />
and supplied manuals. Five micrograms of RNA Century<br />
Markers was electrophored along with RNA transcripts<br />
from transformants and cutted for staining with EtBr. The<br />
transcripts from the pBI 121 transformants were ≈ 327 bp<br />
in length by comparison with stained RNA Century<br />
Markers, which is consistent with the expected size.<br />
Detection of VHH protein expression in transgenic<br />
plants<br />
Based on northern analysis, the transformed plants were<br />
further checked for recombinant protein expression by<br />
ELISA analysis using a monoclonal specific antibody. As<br />
a result, the expression of recombinant VHH protein was<br />
confirmed by an ELISA assay. The results demonstrated<br />
that concentrated extracts from all selected plants were<br />
positive by ELISA (Figure 5). Expression level of VHH<br />
varied significantly among the four transformed plants.<br />
Plant derived protein in the transformed plants ranged<br />
from 0.9 to 2.5 ng/50 µg of total soluble protein.<br />
Transgenic plant line 6 showed the highest level of<br />
Korouzhdehy et al. 4239<br />
expression, accounting for 0.005% of the total soluble<br />
protein.<br />
DISCUSSION<br />
Expression of the first recombinant antibody in tobacco<br />
plant opened the field of the expression of recombinant<br />
antibodies as a real alternative to eliminate many<br />
constraints of monoclonal antibody production in<br />
bioreactors (Hiatt et al., 1989). Expression of antibodies<br />
in plants is being studied for their potential uses in<br />
biotechnology. Camelidae are known to produce<br />
immunoglobulin (Igs) devoid of light chains and constant<br />
heavy-chain domains (CH1). Antigen-specific fragments<br />
of these heavy-chains IgGs (VHH) are of great interest in<br />
biotechnology applications (Muyldermans et al., 2001).<br />
VHH single domain antibody fragments against MUC1<br />
antigen have been produced, but exclusively in bacteria<br />
and yeast. We have described here for the first time, the<br />
expression of a MUC1 specific VHH antibody in plants.<br />
Selecting the most appropriate vector for use in plant<br />
transformation can be a complex task and various factors<br />
need to be considered. Here, we have used pBI-VHH<br />
plasmid which carries origins of replication from spa and<br />
colE1 to facilitate maintenance in Agrobacterium and E.<br />
coli, respectively. The presence of unique multiple<br />
cloning sites (MCSs) for introducing target genes into the<br />
T-DNA region and a bacterially expressed marker gene<br />
(kanamaycin), permits selection and maintenance of the<br />
vector in E. coli and Agrobacterium. To facilitate immunohistochemical,<br />
biochemical and bioassay investigation,
4240 Afr. J. Biotechnol.<br />
large amount of VHH is required; therefore, we used the<br />
CaMV 35S promoter which is a very strong constitutive<br />
promoter, causing high levels of gene expression in dicot<br />
plants. Prior to that, we used the T/A cloning vector<br />
(PTZ57R) to take advantage of inserted MCS (multi<br />
cloning site) which is beneficial for cloning. M13 primers<br />
around the MCS that facilitate the gene sequencing were<br />
also used. The resulting PCR product ligated into a linear<br />
vector with a 3´ terminal 'T' or 'U' at both ends. Tobacco<br />
as a laboratory bench model was our target plant<br />
because of its large scale production and as compared to<br />
other plants represents a potentially safer production<br />
bioreactor for human proteins from the standpoint of<br />
containment.<br />
This study established an efficient protocol for plant<br />
regeneration and the Agrobacterium mediated<br />
transformation of tobacco. The effects of different<br />
Agrobacterium strains C58GV3101 and LBA 4404 on<br />
transformation rate of tobacco was investigated. The<br />
results indicate that the presence of pBI-VHH plasmid<br />
with kanamycin resistance in C58GV3101 increased the<br />
level of T-DNA transfer into the tobacco cells. This may<br />
be due to the higher sensitivity of C58GV3101 when<br />
compared to LBA4404. Therefore, using this<br />
Agrobacterium strain may provide a better opportunity to<br />
use tobacco for genetic engineering. In an effort to study<br />
the safety and reproducibility of VHH single domain<br />
antibody production in plant-based systems, a transgenic<br />
tobacco plant expressing this antibody was grown as a<br />
leaf disk culture on selective medium. Two weeks later,<br />
the plant cell cultures were initiated in selective medium<br />
and examined for VHH antibody production. Expression<br />
of the VHH appeared to remain constant throughout the<br />
growth periods, with no effect on the growth rate.<br />
Transient and stable plant system used here to express<br />
VHH gene, has the same advantages and limitations that<br />
have been already described in literature for similar<br />
cases. Various methods have been recently applied to<br />
produce and select functional antibodies. The transgene<br />
copy number as estimated by Southern hybridization<br />
ranged from one to two copies (Figure 3). According to<br />
the same experiment (Olhoft et al., 2001), we can<br />
conclude that multiple integrations of the T-DNA are due<br />
to the nature of the binary plasmid. Usually, the<br />
correlation between transcript level and T-DNA copy<br />
number is known in the transgenic plant. However, we<br />
guess that differences in the amount of specific transcript<br />
for foreign gene could be due to the promoter used or to<br />
a position effect, rather than to the number of transgene<br />
copies. One of the most commonly used methods to<br />
estimate the expression level of the recombinant antibody<br />
produced in transgenic plants is the enzyme-linked<br />
immunosorbent assay. In this work, we reported the<br />
validation of an enzyme (alkaline phosphatase)-linked<br />
immunosorbent assay to determine a plant-derived<br />
antibody directed against the MUC1 concentrations up to<br />
2.5 ng//50 µg of total protein. The binding between MUC1<br />
synthetic peptide and the plant-derived antibody in this<br />
assay was not affected by the impurities of the samples,<br />
demonstrating that our assay is free from interference in<br />
the range of concentrations. This VHH appears to be an<br />
intrinsically stable molecule, able to accumulate in the<br />
plant cell cytosol and to maintain its functionality. At<br />
present, the expression levels of a foreign antibody in<br />
transformed plants have been shown in relatively small<br />
amounts (0.01 to 0.1% of the total soluble protein). The<br />
highest expression level for the cholera toxin B subunit in<br />
transgenic tobacco chloroplasts, up to 4.1% of the total<br />
soluble protein has been reported (Daniell et al., 2001).<br />
The use of different promoters (Arakawa et al., 2001;<br />
Chong et al., 1997), the use of plant-derived leader<br />
sequences and signal peptides (Kong et al., 2001) and<br />
mainly the modification of codon usage of the foreign<br />
protein (Mason et al., 1980) could improve expression<br />
levels in plants. More studies are required to use the<br />
different subcloning procedures by different plant vectors,<br />
and optimize the expression and production of VHH gene<br />
in plants and the extraction/purification procedures that<br />
have substantial impacts on the final outcome<br />
ACKNOWLEDGEMENTS<br />
The authors thanks Dr. Reza Sheikhnejad (ProNAi<br />
Therapeutics Inc., Kalamazoo, Michigan, USA) for his<br />
valuable advice and support in this study.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4242-4248, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2343<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Aloe arborescens aqueous gel extract alters the<br />
activities of key hepatic enzymes and blood<br />
concentration of triglycerides, glucose and insulin in<br />
alloxan-induced diabetic rats<br />
Motetelo Alfred Mogale 1 , Sogolo Lucky Lebelo 2 , Leshweni Jeremia Shai 3 * and Jacobus<br />
Nicholaas Eloff 4<br />
1 Department of Physiology, Faculty of Medicine, University of Limpopo, P. O. Box 130, Medunsa 0204, South Africa.<br />
2 Department of Life and Consumer Sciences, University of South Africa, Private Bar X6, Florida 1710, South Africa.<br />
3 Department of Biomedical Sciences, Tshwane University of Technology, Acardia campus, Pretoria, South Africa.<br />
4 Phytomedicine Programme, University of Pretoria, Onderstepoort, South Africa.<br />
Accepted 22 February, 2011<br />
The present study investigated the antidiabetic activity and the possible mechanisms of action of<br />
aqueous extract of Aloe arborescens leaf gel (AALGEt) on normal and alloxan-induced diabetic rats.<br />
Diabetes was induced in 12 h fasted rats by intraperitoneal injection of 140 mg/kg body weight of<br />
alloxan. Blood glucose levels, body weight and water intake were determined on day 7, 14 and 21 of<br />
AALGEt treatment. Plasma insulin and triglycerides levels, as well as activities of hepatic glucokinase<br />
and glucose 6-phosphatase (G6Pase) were determined at the end of the study. Blood glucose levels,<br />
plasma triglyceride and insulin levels, as well as the activity of hepatic G6Pase were significantly<br />
increased in diabetic rats. With the exception of hepatic glucokinase activity, daily oral administration<br />
of AALGEt to diabetic rats significantly reversed the effects induced by alloxan. The activities of<br />
glucokinase and glucose-6-phosphatase as well as plasma insulin levels in AALGEt-treated normal rats<br />
were comparable with those observed in untreated normal rats. The results suggest that AALGEt<br />
ameliorates physiological parameters altered by the diabetic state. These effects may be mediated in<br />
part, through the protection of pancreatic beta cells from further damage by alloxan.<br />
Keywords: Aloe arborescens, alloxan, diabetic rats, antidiabetic activity, hepatic enzymes.<br />
INTRODUCTION<br />
Ethnomedical reports suggest that more than 1200 plants<br />
are used to control diabetes mellitus in traditional medicinal<br />
systems of different cultures, worldwide (Marles and<br />
Farnsworth, 1995; Grover et al., 2002). The hypoglycemic<br />
*Corresponding author. E-mail: shailj@tut.ac.za. Tel: +2712 382<br />
6342. Fax: +2712 382 6342.<br />
Abbreviations: AALGEt, Aqueous Aloe arborescens leaf gel<br />
extract; FFAs, Free fatty acids; G6Pase, glucose 6phosphatase;<br />
HDL, high density lipoprotein; HSL, hormone<br />
sensitive lipase; LPL, lipoprotein lipase; SANBI, South African<br />
National Biodiversity Institute.<br />
effects of a large number of these plants have been<br />
evaluated and confirmed in animal models of diabetes<br />
(Day and Bailey, 2006; Frode and Medeiros, 2006) as<br />
well as in clinical studies (Jayawardena et al., 2005; Day<br />
and Bailey, 2006). However, many of these plants still<br />
need to be validated and characterized in terms of<br />
toxicity, active principles and hypoglycemic mechanism of<br />
action.<br />
Aloe arborescens Miller., also known as Krantz aloe, or<br />
Kidachi aloe, is a multi-stemmed succulent shrub with a<br />
height of about 3 m. It belongs to the Asphodelaceae<br />
family (van Wyk and Smith, 2004; Smith et al., 2008). It is<br />
indigenous to southern Africa but it is reported to be<br />
cultivated all over the world (Smith et al., 2008). Some<br />
black communities in the Northwest Province (Odi district)
of South Africa use the leaf components (gel and juice) of<br />
A. arborescens as antidiabetic remedies (Mogale,<br />
personal communication). The hypoglycemic activity of A.<br />
arborescens leaf components as evaluated and confirmed<br />
in experimental animal models of diabetes (Beppu et al.,<br />
1993; 2006) are attributed to the presence of the<br />
polysaccharides, arboran A and B in the leaves of this<br />
plant (Hikino et al., 1986; Beppu et al., 2006). Katerere<br />
and Eloff (2005) also discussed the use of Aloes in<br />
diabetes. However, the mechanism whereby A.<br />
arborescens leaf components exert their hypoglycaemic<br />
effect remains unknown.<br />
An antidiabetic agent may be effective in reducing<br />
blood glucose concentration through: 1) stimulating<br />
insulin secretion from pancreatic beta-cells; 2) enhancing<br />
glucose uptake by fat and muscle cells; 3) altering the<br />
activity of some enzymes (e.g. α-glucosidase, α-amylase,<br />
glucokinase and glucose-6-phosphatase) that are<br />
involved in glucose metabolism and 4) slowing down the<br />
absorption of sugars from the gut (Tanira, 1994; Klover<br />
and Mooney, 2004; Cheng and Fantus, 2005). The<br />
present study was undertaken to investigate the effects of<br />
aqueous A. arborescens leaf gel extract on fasting blood<br />
glucose levels, insulin secretion and activities of selected<br />
hepatic enzymes in normal and alloxan-induced diabetic<br />
rats.<br />
MATERIALS AND METHODS<br />
Plant material and preparation of AALGEt<br />
Leaves of A. arborescens were collected from a private garden in<br />
the North West province of the Republic of South Africa and<br />
confirmed as those of A. arborescens (Miller) by the South African<br />
National Biodiversity Institute (SANBI) (Genspec no. 2206- 12).<br />
Leaf gels were dried and milled into a fine powder after which<br />
AALGEt was prepared by homogenizing 25 g of the powder in 250<br />
ml of water, followed by centrifugation at 3000 xg for 20 min. The<br />
resulting supernatants were pooled and stored at 4°C until use.<br />
Animals and induction of diabetes<br />
Male albino Wistar rats weighing 220 to 280 g were obtained from<br />
the animal facility of the University of Cape Town, South Africa. All<br />
animals were kept in individual cages in an environmentally<br />
controlled room with a 12 h light/12 h dark cycle. The animals had<br />
free access to water and standard rat diet. The study was approved<br />
by the Ethics Committee of the University of Limpopo, South Africa<br />
(Animal Ethics Approval number AEC09/06). Diabetes mellitus was<br />
induced in 12 h fasted animals by intraperitoneal injection of alloxan<br />
monohydrate (Sigma, St. Louis, MO., USA) dissolved in sterile<br />
normal saline at a dose of 140 mg/kg body weight. Since alloxan is<br />
capable of producing fatal hypoglycaemia as a result of massive<br />
pancreatic insulin release (Szudelski, 2001; Dhandapani et al.,<br />
2002), rats were treated with 20% glucose solution intraperitoneally,<br />
8 h after alloxan treatment. The rats were then kept for the next 24<br />
h in their cages with 5% glucose in dispenser bottles to prevent<br />
hypoglycaemia (Dhandapani et al., 2002). Diabetes was confirmed<br />
in rats by measuring fasting blood glucose, 72 h post alloxan<br />
treatment. Rats with marked hyperglycaemia (blood glucose level<br />
above 11.0 mM) were selected for use in the study. The highest<br />
Mogale et al. 4243<br />
blood glucose concentration measured in a diabetic rat was 25 mM.<br />
Experimental procedure<br />
The experiment was carried out using four groups of eight rats<br />
each: Group I (untreated normal rats), group II (treated normal rats),<br />
group III (untreated diabetic rats) and group IV (treated diabetic<br />
rats). Experimental animals (groups II and IV) received a daily dose<br />
of 300 mg/kg body weight of AALGEt incorporated in their drinking<br />
water for 3 weeks. Control animals received water without AALGEt<br />
during the same period. Fasting blood glucose levels were<br />
determined by means of MediSense ’s Optimum Xceed Diabetes<br />
Monitoring system and blood glucose test strips before treatment<br />
(day 0) and on day 1 (4 h), day 7, day 14 and day 21 after initiation<br />
of treatment. Body weights and water intake of all groups of rats<br />
were assessed on the same days that blood glucose levels were<br />
measured.<br />
On day 22, blood was withdrawn from the heart of each rat under<br />
general anaesthesia. The collected blood was heparinised and<br />
centrifuged for 20 min at 3000 xg and used for the mea-surement of<br />
plasma glucose, plasma triglycerides and plasma insulin. Following<br />
blood collection, rats were euthanized using sodium thiopentone<br />
(200 mg/kg). Liver tissue was removed from each animal, washed<br />
with saline and stored at -70°C until used for the assay of<br />
glucokinase and glucose-6-phosphatase activities.<br />
Determination of plasma glucose, triglycerides and insulin<br />
levels<br />
Plasma glucose and plasma triglyceride levels were measured<br />
using commercially available kits based, respectively on the glucose<br />
oxidase and the glycerol blanked methods. Both methods<br />
were adapted to the Beckman Coulter®’s UniCell DXC 800<br />
Synchron® Clinical System. Plasma insulin levels were determined<br />
by the enzyme linked immunosorbant assay (ELISA) adapted to the<br />
Beckman Coulter® Ireland Inc’s UniCell DXI 800 Access®<br />
Immunoassay System.<br />
Measurement of hepatic glucokinase activity<br />
About 500 mg liver tissues from each rat was homogenized in 5 ml<br />
ice-cold homogenization buffer containing 100 mM KCl and 20 mM<br />
HEPES (pH 7.9). The homogenates were centrifuged at 3000 xg for<br />
20 min at 4°C. Glucokinase activity in the supernatants was<br />
measured by the pH sensitive colorimetric assay published by<br />
Chapman and Wong (2002) in a reaction mixture containing 20 mM<br />
HEPES pH 7.9, 5 mM ATP, 5 mM MgCl2, 5 mM D-glucose and 0.2<br />
mM Cresol Red. Absorbance measurements were taken at 570 nm<br />
after incubation at 37°C for 30 min. The glucokinase activity in the<br />
supernatants was expressed as units/mg protein. One unit of<br />
glucokinase is defined as the amount of protons liberated/minute at<br />
37°C under the specified assay conditions. Protein concentration in<br />
supernatants was assayed by the method of Lowry et al. (1951)<br />
using bovine serum albumin as a standard.<br />
Assay of hepatic glucose-6-phosphatase activity<br />
Liver microsomal fractions were prepared according to the ultracentrifugation<br />
method published by Pari and Satheesh (2006).<br />
Briefly, rat liver (1.0 g) was homogenized in 5 ml ice-cold 5 mM<br />
HEPES (pH 7.4) containing 0.25 M sucrose. The homogenate was<br />
centrifuged at 10 000 ×g for 20 min, at 4°C after which the<br />
supernatant was centrifuged again at 105 000 ×g for 60 min. The<br />
microsomal pellets obtained were re-suspended in same buffer and
4244 Afr. J. Biotechnol.<br />
Table 1. Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on blood glucose<br />
levels of normal and alloxan-induced diabetic rats.<br />
Rat groups<br />
Average blood glucose (mmol/L)<br />
Day 0 Day 7 Day 14 Day 21<br />
Normal control rats 4.1 ±0.5 5.9 ±0.6 4.7±0.9 4.8 ±0.7<br />
Normal + AALGEt 4.3 ±1.3 5.2 ±0.8 4.6 ±0.5 5.1 ±0.7<br />
Diabetic control rats 1.5 ±1.3 2.2 ±8.2 >27.8** >27.8**<br />
Diabetics + AALGEt 20.1 ±4.2 1.2 ±5.6 ¥ 9.4 ±4.3 ¥¥ 7.2 ±6.8 ¥¥<br />
Each value represents ± SD, n = 8. Mean values were significantly different from those of normal control<br />
rats. *p < 0.01, **p < 0.001. Mean values were significantly different as compared to diabetic control<br />
rats, ¥ p < 0.01, ¥¥ p < 0.001.<br />
their glucose-6-phosphatase enzyme activity was assayed according<br />
to the method developed by Baginski et al. (1974). In this<br />
method, glucose-6-phosphate is converted by glucose-6-phosphatase<br />
(G6Pase) in liver microsomal fractions into glucose and<br />
inorganic phosphate. The inorganic phosphate liberated is allowed<br />
to react with ammonium molybdate in the presence of ascorbic acid<br />
(Fiske and Subbarow, 1925). The amount of phosphate liberated<br />
per unit time determined as the blue phosphomolybdenum complex<br />
at 700 nm is a measure of G6Pase. G6Pase activity was expressed<br />
as units/mg of protein. One unit of glucose-6-phosphatase activity<br />
was defined as the amount of Pi liberated/min at 37°C under the<br />
specified assay conditions.<br />
Water consumption and body weight determination<br />
Daily water consumption was determined by calculating the difference<br />
between amount of water (200 ml) supplied and the volume<br />
of water remaining after 24 h. Body weight was determined<br />
gravimetrically.<br />
Statistical analysis<br />
Data expressed as mean ± SD were analyzed using the Sigma Stat<br />
statistical program (version 8.0). Comparisons were made between<br />
normal and alloxan-induced diabetic rats as well as between<br />
treated and untreated alloxan-induced diabetic rats by means of<br />
unpaired Student’s t-test and their significance were established by<br />
analysis of variance analysis (ANOVA). Differences of p < 0.05<br />
were considered statistically significant.<br />
RESULTS<br />
There was no significant difference in the fasting blood<br />
glucose levels of normal rats treated with AALGEt when<br />
compared with that of normal untreated rats (Table 1).<br />
However, a steady increase in the blood glucose levels of<br />
diabetic control rats was observed throughout the entire<br />
experiment. The increase in the blood glucose levels of<br />
untreated diabetic rats was significantly higher on day 7<br />
(p < 0.01), day 14 (p < 0.001) and day 21 (p < 0.001)<br />
when compared with normal control rats. Treatment of<br />
diabetic rats with AALGEt for 3 weeks significantly<br />
reduced their blood glucose levels when compared with<br />
those of diabetic control rats on day 7 (p < 0.01), day 14<br />
(p < 0.001) and day 21 (p < 0.001).<br />
At the end of the experimental period, plasma glucose,<br />
triglyceride and insulin were measured. Plasma glucose<br />
and triglyceride levels in untreated diabetic animals were<br />
significantly higher (p < 0.001 and p < 0.05, respectively)<br />
(Figure 1A and B).<br />
Treatment of diabetic rats with AALGEt for 3 weeks<br />
significantly reduced plasma glucose (p < 0.01) by 66.7%<br />
and plasma triglycerides by 25% when compared with<br />
untreated diabetic rats. Oral administration of AALGEt to<br />
diabetic rats for 3 weeks significantly increased plasma<br />
insulin levels when compared to diabetic controls (Figure<br />
1C).<br />
Changes in body weight and water intake of normal<br />
control, treated normal rats, diabetic controls and<br />
AALGEt treated diabetic rats on days 1, 14 and 21 of<br />
treatment are shown in Figure 2A and B. The changes in<br />
the activities of hepatic glucokinase (GK) and G6Pase in<br />
normal and diabetic control are shown in Table 2. Alloxan<br />
treatment caused a decrease in the hepatic GK activity<br />
and a significant increase (p < 0.05) in the activity of<br />
hepatic G6Pase of diabetic rats when compared to<br />
normal control rats (Table 2). Furthermore, oral administration<br />
of AALGEt to normal and diabetic rats did not<br />
cause any significant difference in the activity of hepatic<br />
GK. On the other hand, oral administration of AALGEt for<br />
3 weeks resulted in a significant decrease (p < 0.05) in<br />
hepatic G6Pase activity.<br />
A significant decrease in body weight was observed in<br />
untreated diabetic rats when compared with normal<br />
control rats on day 14 (p < 0.01) and day 21 (p < 0.001)<br />
(Figure 2A). There was a significant increase in the water<br />
intake of diabetic rats than in normal control rats on days<br />
7, 14 and 21 (p < 0.05, p < 0.01 and p < 0.001, respecttively)<br />
(Figure 2B). Treatment of diabetic rats with<br />
AALGEt did not significantly attenuate the observed<br />
weight loss. However, treatment of diabetic rats with<br />
AALGEt significantly reduced the water intake to lower<br />
levels than untreated diabetic rats (p < 0.01 on day 14<br />
and p < 0.001 on day 21).
Plasma glucose (mM/L)<br />
50Normal controls<br />
AALGEt treated normals<br />
2.0<br />
Diabetic controls<br />
AALGEt treated diabetics 1.8 B<br />
40<br />
30<br />
20<br />
10<br />
0<br />
A<br />
Rat groups<br />
**<br />
¥¥<br />
Plasma triglyceride (mmol/L)<br />
1.6<br />
1.4<br />
1.2<br />
1.0<br />
0.8<br />
0.6<br />
0.4<br />
0.2<br />
0.0<br />
B<br />
Rat groups<br />
Plasma insulin (umol/L)<br />
250<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
Mogale et al. 4245<br />
Rat groups<br />
Figure 1. Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on plasma glucose (A), plasma triglyceride (B) and<br />
plasma insulin (C) levels of normal and alloxan-induced diabetic rats. Mean values were significantly different as compared to normal<br />
Figure 1: Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on plasma glucose (A)<br />
plasma triglyceride (B) and plasma insulin (C) levels of normal and alloxan-induced diabetic rats. Mean<br />
values were significantly different compared to normal control rats, * P < 0.05, ** P < 0.01. Mean values<br />
were significantly different compared to diabetic control rats, ¥ P < 0.05, ¥¥ control rats, *p < 0.05, **p < 0.01. Mean values were significantly different as compared to diabetic control rats,<br />
P < 0.01.<br />
¥ P < 0.05, ¥¥ p < 0.01.<br />
DISCUSSION<br />
mice.<br />
Alloxan induces diabetes by damaging the insulin secreting<br />
cells of the pancreas leading to hypoinsulinemia and<br />
hyperglycaemia (Szudelski, 2001). In agreement with<br />
these known effects of alloxan, the blood glucose levels<br />
of untreated alloxan-induced diabetic rats were significantly<br />
increased throughout the current study when<br />
compared to those of normal control rats. Continuous<br />
treatment of alloxan-induced diabetic rats with AALGEt<br />
(300 mg/kg body weight) for a period of 3 weeks resulted<br />
in a significant decrease in the blood glucose levels of<br />
treated diabetic rats when compared to diabetic controls<br />
but no such effect was observed in normal treated rats.<br />
Thus, unlike the use of insulin or sulfonylurea drugs,<br />
which cause severe hypoglycaemia when taken in excessive<br />
doses (Bastaki, 2005; Cheng and Fantus, 2005),<br />
continuous use of AALGEt or its accidental overdose will<br />
not result in hypoglycaemic shock. Furthermore, results<br />
of the current study suggest that the blood glucose<br />
lowering effect of AALGEt can last for a longer period of<br />
time. These observations concur with the findings of<br />
Beppu et al. (1993, 2006) in alloxan-induced diabetic<br />
*<br />
¥<br />
Continuous treatment of alloxan-induced diabetic rats<br />
with AALGEt (300 mg/kg body weight) for 3 weeks also<br />
significantly increased (but did not normalize) the plasma<br />
insulin levels of diabetic rats as compared to diabetic<br />
controls. However, AALGEt did not significantly alter<br />
plasma insulin levels of normal rats. Thus, like the aloe<br />
vera leaf gel extract (Reynolds and Dweck,1999),<br />
AALGEt might exert its blood glucose lowering effects<br />
through stimulation of insulin secretion from pancreatic<br />
beta cells. However, the fact that AALGEt did not<br />
significantly alter the plasma insulin levels of normal rats<br />
but significantly increased plasma insulin levels of alloxan<br />
induced-diabetic rats towards the normal value, suggest<br />
that, AALGEt may either regenerate damaged pancreatic<br />
beta cells or protect them from further damage by alloxan.<br />
Since alloxan is known to exert its diabetogenic<br />
effect through generation of oxygen free radicals, this<br />
hypothesis is in agreement with the findings of Beppu et<br />
al. (2003) and Chacko et al. (2008), who reported that A.<br />
arborescens components possess free radical scavenging<br />
effects that may prevent pancreatic islet beta-cell<br />
destruction by free radicals generated by alloxan. Thus,<br />
C<br />
**<br />
C<br />
¥
4246 Afr. J. Biotechnol.<br />
Water intake (ml/rat/day)<br />
Figure 2. Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on body weight (A) and water intake (B) of<br />
normal and alloxan-induced diabetic rats. Mean values were significantly different compared to normal control rats, * p <<br />
0.05, *** p < 0.001. Mean values were significantly different compared to diabetic control rats, ¥ p < 0.05, ¥¥ p < 0.01, ¥¥¥ p <<br />
0.001.<br />
Table 2. Effects of treatment with AALGEt (300 mg/kg body weight) on the activities of hepatic glucokinase<br />
and glucose-6-phosphatase<br />
Rat groups Glucokinase (U/g protein) Glucose 6- phosphatase (U/mg protein)<br />
Normal control rats 298.01 ± 11.4 0.198 ± 0.05<br />
Normal + AALGEt 304.56 ± 8.34 0.213 ± 0.06<br />
Diabetic control rats 282.82 ± 6.18 0.521 ± 0.02*<br />
Diabetics + AALGEt 288.13 ± 9.81 0.282 ± 0.06 ¥<br />
Each value represents ± SD, n = 8. Mean value was significantly different from those of normal control rats. *p <<br />
0.05. Mean value was significantly different as compared to diabetic control rats, ¥ p < 0.05.<br />
in this respect, AALGEt probably act like plant ex-tracts of<br />
Gymnema sylverstre (Chattopadhyay, 1998) and Ipomea<br />
batatas L. (Kusano and Abe, 2000) which are reported to<br />
repair pancreatic islet cells damaged by either alloxan or<br />
streptozotocin (Bnouham et al., 2006).<br />
Uncontrolled diabetes mellitus is often associated with<br />
abnormal plasma lipid levels, in particular, elevated<br />
plasma triglyceride levels and reduced plasma HDLcholesterol<br />
levels (Sniderman et al., 2002; Mooradian,<br />
2009). Hypertriglyceridemia represents an independent<br />
risk factor for the development of coronary heart disease<br />
250<br />
200<br />
150<br />
100<br />
50<br />
0<br />
in people with type 2 diabetes (Ooi and Ooi, 1998;<br />
Sniderman et al., 2002). We have observed in this study,<br />
that continuous treatment of alloxan-induced diabetic rats<br />
with AALGEt (300 mg/kg body weight) for a period of 3<br />
weeks significantly reduced their plasma triglyceride<br />
levels when compared with diabetic control rats. Under<br />
normal circumstances, insulin inhibits the enzyme hormone<br />
sensitive lipase (HSL), which mobilizes free fatty<br />
acids (FFAs) from adipose tissues (Kwiterovich, 2000;<br />
Smith et al., 2005). FFAs released by the action of HSL<br />
are the major substrates for the hepatic triglyceride
synthesis (Kwiterovich, 2000). Insulin also activates the<br />
enzyme lipoprotein lipase (LPL), which clears triglyceride<br />
rich proteins from blood plasma (Kwiterovich, 2000;<br />
Smith et al., 2005). However, in a diabetic state, HSL is<br />
not inhibited and LPL is not activated due to insulin<br />
deficiency (Kwiterovich, 2000; Mlinar et al., 2007). Thus,<br />
the effect of AALGEt on plasma triglyceride levels could<br />
be attributed to its effects on plasma insulin levels.<br />
Hepatic glycogenolysis and gluconeogenesis are major<br />
causes of fasting hyperglycemia seen in both type 1 and<br />
type 2 diabetes mellitus (Smith et al., 2005). Inhibition of<br />
enzymes involved in gluconeogenesis and/or glycolgenolysis<br />
therefore constitutes an alternative approach to<br />
suppress hepatic glucose production and lower fasting<br />
plasma glucose (Agius, 2007). G6Pase catalyses the<br />
final reaction in hepatic glucose production by both gluconeogenesis<br />
and glycogenolysis, and has been proposed<br />
as a potential target for antihyperglycaemic drugs for<br />
type-2 diabetes (Pari and Satheesh, 2006; Agius, 2007).<br />
A significant increase in the activity of the hepatic<br />
G6Pase of untreated alloxan-induced diabetic rats as<br />
compared to normal rats was observed in the current<br />
study. Treatment of diabetic rats with AALGEt (300 mg/kg<br />
body weight) for 3 weeks resulted in a significant<br />
reduction of GPAase activity when compared to diabetic<br />
controls. Thus, AALGEt could contain substances that act<br />
like the oral hypoglycaemic agent, metformin (Bastaki,<br />
2005; Cheng and Fantus, 2005) and plant extracts of<br />
Boerhaavia diffusa L. (Pari and Satheesh, 2004) and<br />
Pterocarpus marssupium (Pari and Satheesh, 2006)<br />
which have been shown to lower blood sugar levels by<br />
inhibiting hepatic production of glucose.<br />
Glucokinase is a rate limiting enzyme involved in the<br />
hepatic storage and utilization of glucose (Smith et al.,<br />
2005). Following a carbohydrate rich meal, hepatic GK<br />
clears a significant amount of glucose from the blood<br />
circulation and facilitates its conversion into glycogen and<br />
fatty acids (Smith et al., 2005; Agius, 2007). Thus,<br />
hepatic GK play a significant role in the prevention of<br />
postprandial hyperglycemia. The activity of hepatic GK is<br />
reported to be reduced in alloxan-induced experimental<br />
diabetes (Zhang et al., 2007) and some antidiabetic plant<br />
extracts are reported to exert their hypoglycemic activity,<br />
in part by increasing the activity of this enzyme (Pari and<br />
Satheesh, 2006). In agreement with the known effects of<br />
alloxan on the activity of GK (Zhang et al., 2007), we<br />
have observed in this study that the hepatic GK activity<br />
was significantly reduced when compared to normal<br />
controls. However, oral administration of AALGEt (300<br />
mg/kg body weight) to both normal and alloxan-induced<br />
diabetic rats for 3 weeks did not significantly alter the<br />
activity of hepatic GK in these animals.<br />
Uncontrolled diabetes mellitus is also associated with<br />
body weight loss, polyurea, polydypsia and polyphagia<br />
(Bastaki, 2005). Weight loss in diabetes mellitus results<br />
from a combination of dehydration (caused by frequent<br />
urination), increased breakdown of muscle proteins (for<br />
Mogale et al. 4247<br />
provision of gluconeogenic amino acids) and enhanced<br />
mobilization of fat stores (provision of FFAs to be used as<br />
fuel) (Bastaki, 2005; Smith et al., 2005). These events<br />
are directly or indirectly related to insulin deficiency or<br />
lack of insulin actions (Smith et al., 2005). We have<br />
observed a significant decrease in the body weight of<br />
untreated alloxan-induced diabetic rats as compared to<br />
normal control rats. The loss of weight in alloxan-induced<br />
diabetic rats was accompanied by increased water intake<br />
(polydypsia) by these rats, suggesting that dehydration<br />
was a contributing factor towards weight loss. Continuous<br />
treatment of alloxan-induced diabetic rats with AALGEt<br />
for 3 weeks significantly prevented body weight loss.<br />
However, the mean body weight of AALGEt treated rats<br />
was still significantly reduced when compared to that of<br />
the normal control rats. The marked improvement in body<br />
weight loss of treated diabetic rats as compared to<br />
untreated alloxan induced diabetic rats could also be<br />
attributed to the effect of AALGEt on plasma insulin levels<br />
and hence on improved glucose metabolism in these<br />
rats.<br />
The findings of this study suggest that oral administration<br />
of an aqueous extract of the leaf gel of A.<br />
arborescens (AALGEt) (300 mg/kg body weight) for 3<br />
weeks brings about significant beneficial effects in<br />
various physiological/biochemical parameters altered by<br />
the diabetic state. Although, the exact chemical compound(s)<br />
responsible for the antidiabetic activity of<br />
AALGEt are currently unknown, AALGEt appears to exert<br />
its blood glucose lowering effect in alloxan-induced<br />
diabetic rats by protecting insulin secreting pancreatic<br />
beta cell from further damage by alloxan. Further studies,<br />
in particular, histological studies of pancreatic beta cells<br />
of AALGEt treated alloxan-induced diabetic rats are<br />
needed in order to confirm this hypothesis. Furthermore,<br />
the isolation and identification of active compounds<br />
AALGEt will go a long way in promoting the use and<br />
acceptance of A. arborescens leaf as an antidiabetic<br />
remedy. Although, lower doses may also have led to a<br />
positive effect, the dosage that was used in these experiments<br />
(equivalent to 2.4 g/day per 80 kg human) were<br />
probably higher than the dosage used by traditional<br />
healers. In general, these results strongly support the<br />
traditional use and preparation of AALGEt for treating<br />
diabetes in humans.<br />
ACKNOWLEDGEMENTS<br />
The authors would like to thank Mrs A. Freitas for her<br />
assistance in handling and maintaining the animals. The<br />
study was funded by the Department of Physiology,<br />
University of Limpopo, South Africa. The authors would<br />
also like to thank Prof Leon Hay, Head of the Department,<br />
for making funds available for this study. L.J. Shai<br />
is a recipient of NRF funding under the Thutuka-REDIBA<br />
programme.
4248 Afr. J. Biotechnol.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4249-4255, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2158<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Increased vascular endothelial growth factor (VEGF)<br />
expression in rats with spinal cord injury by<br />
transplantation of bone marrow stromal cells<br />
Deshui Yu 1,3 , Libo Liu 2 , Xiaodong Zhi 3 , Yang Cao 3 and Gang lv 1 *<br />
1 Department of Orthopaedics, the First Affiliated Hospital, China Medical University, Shenyang 110001, People’s<br />
Republic of China.<br />
2 Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001, People’s<br />
Republic of China.<br />
3 Department of Orthopaedics, The First Affiliated Hospital, Liaoning Medical University, Jinzhou 121001, People’s<br />
Republic of China.<br />
Accepted 24 March, 2011<br />
Vascular endothelial growth factor (VEGF), a well known angiogenic factor, has been shown to have<br />
direct and/or indirect influence on spinal cord injury (SCI). The purpose of this study is to observe<br />
VEGF expression changes in rats with SCI by bone marrow stromal cells (BMSCs) treatment. The mRNA<br />
expression of VEGF in rats was analyzed by semi-quantitative RT- PCR and the protein expression level<br />
was quantified by means of western blot and immunohistochemistry technology. It was found that<br />
VEGF was significantly up-regulated in BMSCs treatment group in comparison to the sham group and<br />
DMEM group on mRNA level. On protein level, VEGF was also highly increased in BMSCs treatment<br />
group, which reached the highest level after 14 days treatment. The present study suggests a potential<br />
local role for VEGF as mediators of SCI which might provide a certain reasonable clue to clarify<br />
molecular mechanisms of BMSCs treatment in SCI.<br />
Key words: Vascular endothelial growth factor (VEGF), spinal cord injury, bone marrow stromal cells, reverse<br />
transcription-polymerase chain reaction (RT-PCR), western blot, immunohistochemistry.<br />
INTRODUCTION<br />
Each year, many people worldwide suffer from spinal<br />
cord injury (SCI). These injuries cause death of neural<br />
cells, severance and demyelination of descending and<br />
ascending axons, and consequently, loss of motor and<br />
sensory function. Endogenous repair efforts fail to repair<br />
*Corresponding author. Email: ganglv514@yahoo.com.cn. Tel:<br />
+86-0416-467-3886. Fax: +86-0416-467-3528.<br />
Abbreviations: SCI, Spinal cord injury; BMSCs, bone marrow<br />
stromal cells; DMEM, Dulbecco's modified Eagle's medium;<br />
FBS, fetal bovine serum; RT, room temperature; DEPC,<br />
diethylpyrocarbonate; VEGF, vascular endothelial growth factor;<br />
PCR, polymerase chain reaction; IDV, integrated densities<br />
value; EDTA, ethylenediaminetetraacetic acid; DAB,<br />
diaminobenzidine; COX-2, cyclooxygenase-2.<br />
the spinal cord and, as a result, the functional impairments<br />
are permanent (Wrigley et al., 2009; Wollaars et al.,<br />
2007). Potential treatments for SCI are being tested in<br />
the clinic but so far, none of these have emerged as one<br />
that reverses the devastating functional consequences of<br />
SCI, however, several studied demonstrated that bone<br />
marrow stromal cells (BMSCs) may also have therapeutic<br />
promise for SCI (Shields et al., 2010; Bunge., 2008).<br />
Survival, migration and differentiation of BMSCs after<br />
transplantation have been widely researched in recent<br />
years (Shields et al., 2010; Bunge, 2008; Someya et al.,<br />
2008). The underlying molecular mechanisms of repairing<br />
injured spinal cord and improving functional recovery with<br />
BMSCs have not, however, been largely understood.<br />
Better understanding of the molecular mechanism of<br />
BMSCs treatment for SCI may contribute significantly to<br />
the development of BMSCs reagents as well as help in
4250 Afr. J. Biotechnol.<br />
SCI control. Consequently, it is of vital importance to<br />
define the molecular mechanisms of BMSCs treatment<br />
for successful treatment of SCI.<br />
Angiogenesis plays an important role in increasing<br />
blood flow at the lesioned site, where hypoxia occurs<br />
after SCI and is an essential component of nerve regeneration<br />
across a gap of injured spinal cord (Carmeliet et<br />
al., 2002). Similarly, neo-angiogenesis is also essential<br />
for trauma repair, which requires nutrients and oxygen to<br />
overcome hypoxia (Carmeliet et al., 2002). It has been<br />
reported that SCI may induce angiogenesis (Casella et<br />
al., 2002), however, under normal circumstances after<br />
SCI, the angiogenesis of the injured spinal cord is<br />
insufficient to support the spinal cord plasticity required<br />
for functional recovery. Recent data from the medical<br />
literature have also shown a positive correlation between<br />
the density of blood vessels and functional improvement<br />
following SCI (Blight., 1991; Widenfalk et al., 2003).<br />
These findings suggest that stimulating angiogenesis<br />
may improve recovery of function after SCI. BMSCs have<br />
the ability to stimulate angiogenesis, which had been<br />
reported in recent medical literature (Tang et al., 2006;<br />
Chen et al., 2003; Kayo et al., 2005). BMSC may<br />
promote angiogenesis through VEGF expression in the<br />
spinal cord, according to the results of the present study,<br />
which may contributes to shed more light on the<br />
molecular mechanism of BMSCs treatment in SCI.<br />
In this study, we investigated whether BMSCs transplantation<br />
could induce the expression of VEGF in the<br />
injured spinal cord and provided a certain reasonable<br />
clue that shed more light on the molecular mechanisms<br />
of BMSCs treatment in SCI.<br />
MATERIALS AND METHODS<br />
The adult male Wistar rats (12 weeks, 250 to 300 g) were<br />
purchased from the Center for Experimental Animals of China<br />
Medical University. All animal experiments were carried out in<br />
accordance with the National Institute of Health Guide for the Care<br />
and Use of Laboratory Animals.<br />
Culture and identification of MSCs<br />
BMSCs were isolated from rat bone marrow and cultured as<br />
described by previous literature (Yano et al., 2005). Briefly, fresh<br />
whole bone marrow was harvested aseptically from tibias and<br />
femurs by 5 ml syringe needle. The marrow was extruded with 5 ml<br />
of Dulbecco's modified Eagle's medium (DMEM)-low glucose<br />
medium. Bone marrow was mechanically dissociated to obtain a<br />
homogeneous cell suspension. The cells were placed in a 25 cm 2<br />
plastic flask for cell culture with 5 ml DMEM-low glucose medium<br />
containing 10% fetal bovine serum (FBS). The cells were incubated<br />
at 37°C in 95% humidity and 5% CO2 for 24 h. At this time, nonadherent<br />
cells were removed by replacing the medium. The culture<br />
medium was replaced three times a week. After the cultures have<br />
reached 80 ~ 90% confluency, they were lifted by incubation in a<br />
solution containing 0.25% trypsin for 5 min at 37°C for passage.<br />
Cultured BMSCs were indentified with antibodies against CD34 and<br />
CD44 according to previous literature (Yano et al., 2005).<br />
Spinal cord injury model preparation and intramedullary spinal<br />
cord injection<br />
The adult male Wistar rats (12 weeks, 250 to 300 g) were<br />
anesthetized with pentobarbital and laminectomies were performed<br />
at the T8 to T10 level. Spinal cords were injured using the weight<br />
drop technique according to Allen’s method (Allen, 1914) with a<br />
slight modification. A plastic impounder (2-mm diameter) was<br />
placed gently on the exposed dura and a 10-giron weight was<br />
dropped from a height of 10 cm onto the impounder. The weight<br />
and impounder were immediately removed after impact and<br />
paravertebral muscle and skin were closed. Rats were randomly<br />
divided into three groups: Sham group, DMEM group and BMSCs<br />
group.<br />
Thirty minutes after injury, rats received four injections of a<br />
suspension of BMSCs (passage 3 ~ 4 cells, 75000 in 3µl of DMEM<br />
each) into peri-lesion area of the exposed spinal cord (BMSCs<br />
group). Two injections were applied 1 mm rostral and two injections<br />
1 mm caudal to the lesion epicenter, and one injection at each side<br />
0.5 mm lateral to the cord midline. The microneedler was carefully<br />
inserted from the dorsal surface 1.75, 1.25 and 0.75 mm deep into<br />
the spinal cord. Each rat received a total of 3.0 × 10 5 BMSCs. In<br />
DMEM group, the rats received four injections of an equal volume<br />
of DMEM alone at the same position and depth. Sham controls<br />
underwent the same operations but without spinal cord insult. Rats<br />
used in this study were treated strictly according to the NIH Guide<br />
for Care and Use of Laboratory Animals.<br />
RNA preparation and semi-quantitative RT-PCR<br />
Total RNA was isolated from frozen spinal tissue using TRIzol<br />
reagent (Invitrogen Corp., Carlsbad, CA) according to the<br />
manufacturer’s protocol and as described in the online supplement.<br />
In short, the spinal tissue was ground with mortar and pestle,<br />
cooled by liquid nitrogen of the ground tissue, and 100 mg was<br />
incubated with 1 ml TRIzol for 5 min at room temperature (RT). Cell<br />
debris was removed by centrifugation (12,000 ×g at 4°C for 10 min)<br />
and 0.4 ml chloroform was added. After vortexing, the mix was<br />
incubated for 5 min at RT. The phases were separated by<br />
centrifugation (12,000 ×g at 4°C for 15 min) and the aqueous phase<br />
was transferred to a new tube. 0.6× volume of isopropyl alcohol and<br />
a 0.1× volume of 3 M sodium acetate were added to this aqueous<br />
phase and incubated for 10 min at 4°C. The precipitated RNA was<br />
pelleted by centrifugation (12,000 ×g at 4°C for 15 min) and after<br />
the removal of the supernatant, the RNA pellet was washed twice<br />
with 70% ethanol. After drying, the RNA was resuspended in 30 µl<br />
diethylpyrocarbonate (DEPC)-treated water. The quality and<br />
quantity of the RNA was verified by the presence of two discrete<br />
electropherogram peaks corresponding to the 28S and 18S rRNA<br />
at a ratio approaching 2:1. Using mRNA as template, singlestranded<br />
cDNAs were generated by Superscript II reverse<br />
transcriptase (Invitrogen) according to the manufacturer’s<br />
directions. The vascular endothelial growth factor (VEGF) primer<br />
sequences were as follows: Sense prime: 5′-GAGTATATCT<br />
TCAAGCCGTCCTGT-3′; anti-sense prime: 5′- ATCTGCATAG<br />
TGACGTTGCTCTC -3′. GAPDH (Applied Biosystems) served as<br />
the internal control. The polymerase chain reaction (PCR)<br />
conditions were 94°C for 3 min, followed by 30 cycles of DNA<br />
amplification (45 s at 94°C, 1 min at 61°C and 1 min 30 s at 72°C)<br />
and 8 min incubation at 72°C. PCR products were separated by<br />
electrophoresis at a constant voltage (2 V/cm) in a 1.2% (w/v)<br />
agarose gel. Images were captured using a Gel Print 2000i/VGA<br />
(Bio Image), and the integrated densities value (IDV) was analyzed<br />
with computerized image analysis system (Motic Images Advanced<br />
3.2). All DNA manipulations were performed as described by<br />
Sambrook and Russell, (2001).
Western blot<br />
Yu et al. 4251<br />
Figure 1. Bone marrow stromal cells, culture and detect. A, Third passage of bone marrow mesenchymal stem cells was<br />
observed by inverted phase contrast microscope; B, the expression of CD44 in bone marrow stromal cells was observed<br />
by phase contrast the inverted microscope<br />
In order to detect protein expression level of VEGF, western blot<br />
was performed as described in the online supplement. The spinal<br />
cord tissues were obtained from the peri-lesion region at days 3, 7<br />
and 14 post-surgery. Protein homogenates of spinal cord samples<br />
were prepared by rapid homogenization in 10 volumes of lysis<br />
buffer (2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM<br />
ethylene glycol tetraacetic acid (EGTA), 0.4% NaF, 20 mM Tris-<br />
HCl, pH7.5). Tissue homogenate were centrifuged at 17,000 xg for<br />
1 h at 4°C and the protein concentration in the supernatant was<br />
determined by the Coomassie (G250) binding method. Equal<br />
amounts of protein (20 µg) from each sample were loaded and<br />
separated into a 4 to 7.5% gradient sodium dodecyl sulfate<br />
polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing<br />
conditions. Electroblotting proteins were transferred onto nitrocellulose<br />
membranes (Santa Cruz Biotechnology, Inc). After<br />
blocking with 5% nonfat dry milk overnight at 4°C, membranes were<br />
incubated for 2 h at room temperature in agitation with the following<br />
antibodies: rabbit polyclonal anti-VEGF (dilution 1:400; Santa Cruz<br />
Biotechnology, Inc), and rabbit polyclonal anti-β-actin (dilution<br />
1:400; Santa Cruz Biotechnology, Inc). Secondary horseradish<br />
peroxidase conjugated rabbit anti-goat/goat anti-rabbit antibodies<br />
(Santa Cruz Biotechnology, Inc) were used at 1:5000 dilution for 2 h<br />
at room temperature in agitation. Immunoreactive bands were<br />
visualized using the enhanced chemiluminescence (ECL kit, Santa<br />
Cruz Biotechnology, Inc) and scanned using Chemi Imager 5500<br />
V2.03 software. The integrated densities value (IDV) was analyzed<br />
with computerized image analysis system (Fluor Chen 2.0) and<br />
normalized with that of β-actin.<br />
Immunohistochemistry<br />
To detect expression and localization of VEGF in spinal cord tissue,<br />
immunohistochemistry was performed. The cryostat sections were<br />
thawed, air-dried and then blocked with goat serum for 30 min at<br />
room temperature, and then incubated with rabbit polyclonal anti-<br />
VEGF antibody (dilution 1:150, Santa Cruz Biotechnology, USA) at<br />
4°C over night. Following reaction with primary antibodies, the<br />
sections were stained using the ABC Kit (Santa Cruz Biotechnology,<br />
USA), and the color was developed with diaminobenzidine<br />
(DAB). Negative controls were conducted by exchange of primary<br />
antibody for phosphate buffered saline (PBS).<br />
Statistics analysis<br />
To calculate the statistical differences among the sham, DMEM and<br />
BMSCs groups, the statistical package SPSS13.0 (SPSS Incorporated,<br />
USA) was used for all analysis. One-way analysis of<br />
variance (ANOVA) followed by Bonferroni’s post hoc test were<br />
utilized to determine the significant difference among multiple<br />
groups. Student’s t test was used to determine the significance of<br />
differences between the groups. All values were expressed as<br />
mean ±SD. In general, p values less than 0.05 were considered<br />
statistically significant.<br />
RESULTS<br />
BMSC cell culture and detection<br />
Most cells suspended in culture medium, non-adherent<br />
cells were removed by replacing the medium. Adherent<br />
cells began to proliferate and form cell colony at about 72<br />
h later. Cells surrounded the cell colony center to<br />
distribute toward circumference. Cell morphology was<br />
homogeneous, which appeared spindle-shaped with<br />
serial sub-cultivation (Figure 1A). Most cultured adherent<br />
passage 3 cells expressed CD44 (Figure 1B). In contrast,<br />
a majority of adherent passage 3 cells were negative for<br />
CD34.<br />
Semi-quantitative RT-PCR analysis of VEGF<br />
expression<br />
In order to detect the mRNA expression of VEGF in rat<br />
with SCI by BMSC treatment, reverse transcription PCR<br />
was conducted. As shown in Figure 2, VEGF mRNA<br />
levels in BMSCs group were significantly increased at 1,
4252 Afr. J. Biotechnol.<br />
1 3 5<br />
Day<br />
Figure 2. The mRNA expression of VEGF at different time points in rats from each group<br />
after transplantation. A, The expression of VEGF in different group by RT-PCR; M: marker; 1:<br />
sham operation group; 2, 4, 6: DMEM group at transplantation after 1, 3, 5 days,<br />
respectively; 3, 5, 7: BMSCs group at transplantation after 1, 3, 5 days, respectively. B: The<br />
integrated density value of vascular endothelial growth factor mRNA at different time points in<br />
rats from different group after transplantation. Different mark represent the significant<br />
difference at p < 0.05<br />
3 and 5 days after transplantation of BMSC as compared<br />
to DMEM group (P < 0.05), moreover, DMEM group were<br />
significantly higher than sham group (P < 0.05) in<br />
different time. These results showed that transplantation<br />
of BMSC could up-regulate VEGF mRNA expression.<br />
Western blot analysis of VEGF expression<br />
The western blot was performed to detect protein expression<br />
of VEGF. The result showed that VEGF expression<br />
was at a low level in the normal group rats. The<br />
expression level of VEGF protein in the DMEM group rats<br />
increased and peaked at 3 days (P < 0.01), then decreased<br />
quickly with the extension of time, approaching the<br />
expression levels of the sham group rats at 7 and 14<br />
days (P < 0.05) after spinal cord injury. In the BMSCs<br />
group rats, the up-regulation of VEGF protein expression<br />
was observed at 7 and 14 days after SCI as compared<br />
with that in DMEM group rats (P < 0.05), which were<br />
time-dependent and reached the highest level at 14 days<br />
after spinal cord injury (Figure 3).<br />
Immunohistochemistry analysis of VEGF expression<br />
In order to further confirm VEGF protein expression,<br />
immunohistochemistry was conducted. The results<br />
showed that there were no obvious differences (P < 0.05)<br />
in VEGF expression between BMSCs and DMEM groups<br />
rats at 3 day after transplantation (Table 1). The expression<br />
level of VEGF in BMSCs group rats were increased
3 7 14<br />
Day<br />
Figure 3. The expression of VEGF at different time points in rats from each group after<br />
transplantation in protein level. A: The expression of VEGF in different group by western blot; 1:<br />
sham operation group; 2, 4, 6: DMEM group at transplantation after 3, 7, 14 days, respectively; 3,<br />
5, 7: BMSCs group at transplantation after 3, 7, 14 days, respectively. B: The integrated density<br />
value of vascular endothelial growth factor protein at different time points in rats from different<br />
group after transplantation. Different mark represent the significant difference at p < 0.05.<br />
Table 1. The mean optical density of VEGF at different time points in rat in all groups after the transplantation by<br />
immunohistochemical staining (n = 6,x ± s). Different mark represent the significant difference at p < 0.05.<br />
Time point after the<br />
transplantation (day)<br />
3 7 14<br />
Sham group 0.1800±0.0061 0.1811±0.0089 0.1823±0.0063<br />
DMEM group 0.3153±0.0098 ##<br />
0.2381±0.0095 ## 0.1954±0.0038<br />
BMSCs group 0.3150±0.0097 0.2520±0.0052* 0.2281±0.0079**<br />
significantly at 7 and 14 days (P < 0.05) after<br />
transplantation when compared with that in DMEM group<br />
(Figure 4 and Table 1).<br />
DISCUSSION<br />
VEGF is essential for developmental angiogenesis and<br />
Yu et al. 4253<br />
plays important roles in adult animals to control vascular<br />
permeability and homeostasis, blood pressure, and<br />
pathological angiogenesis associated with wound healing<br />
(Ku et al., 1993; Shibuya, 2006; Lee et al., 2007). Based<br />
on its major role as an angiogenic factor and autocrine<br />
growth factor for many diseases, therapeutic angiogenesis<br />
drugs have been developed for clinical use by<br />
improvement of VEGF expression. Previous studies
4254 Afr. J. Biotechnol.<br />
Figure 4. The expression of VEGF by immunohistochemical staining at 7 days after transplantation. A, Sham operation group; B,<br />
DMEM group; C, BMSCs group.<br />
had shown that administration of VEGF could significantly<br />
increase the microvascular density in central nervous<br />
system (Blight., 1991; Widenfalk et al., 2003). In this<br />
study, we found that BMSCs treatment not only increased<br />
the level of VEGF expression, but also extended the time<br />
of VEGF expression in SCI, which would strengthen the<br />
biological effects of VEGF in promoting angiogenesis of<br />
SCI, and this seems to be compatible with the reported<br />
results that BMSCs implantation could induce<br />
angiogenesis in a rat model of chronic hind limb ischemia<br />
(Chen et al., 2003), acute myocardial infarction and<br />
stroke (Rubén et al., 2006). The present study suggests<br />
that VEGF probably plays a pivotal role in the treatment<br />
of SCI by BMSC.<br />
BMSCs are mesenchymal elements normally providing<br />
structural and functional support for hemopoiesis<br />
(Pittenger et al., 1999), which have the potential to differentiate<br />
into other kinds of cells such as osteoblasts,<br />
adipocytes, and chondrocytes (Pittenger et al., 1999;<br />
Prockop, 1997), and produce a variety of neurotrophic<br />
factors, cytokines, cell adhesion molecules, and growth<br />
factor such as VEGF, thereby providing the pathway for<br />
regenerating axons. BMSCs transplanted into a rat model<br />
of chronic hind limb ischemia (Al-Khaldi et al., 2003),<br />
acute myocardial infarction (Tang et al., 2006) and<br />
ischemic brain (Chen et al., 2003) have provided their<br />
beneficial effects by enhancing angiogenesis. The<br />
angiogenesis stimulated by the BMSCs transplantation is<br />
considered to at least in part, result from angiogenic<br />
factors secreted by BMSCs and/or stimulating endogenous<br />
parenchymal cells (Chen et al., 2003). VEGF is<br />
one of the most potent angiogenic factors, capable of promoting<br />
proliferation, migration and survival of endothelial<br />
cells and plays the most important role in the initiation of<br />
new blood vessel formation (Kayo et al., 2005; Breen.,<br />
2007; Leung et al., 1989), therefore, VEGF may play<br />
important role in the treatment of SCI by BMSC.<br />
In conclusion, we have demonstrated that VEGF<br />
expression was significantly up-regulated in BMSC group<br />
in comparison to the normal group and DMEM group. The<br />
current study provides a new approach for studying the<br />
molecular mechanism underlying BMSCs treatment in<br />
SCI. However, this molecular mechanism is largely<br />
unknown; elucidation of these questions will depend on<br />
further experiments. The next proposal of our study will<br />
be to analyze whether VEGF expression could be accompanied<br />
by cyclooxygenase-2 (COX-2), a protein relevant<br />
in regulating pro-inflammatory processes, which have<br />
been demonstrated to play a relevant role in VEGF<br />
regulation (Siner et al., 2007). These data have to be<br />
confirmed in future experiments, which would provide<br />
further valuable clues to clarify molecular mechanisms of<br />
BMSCs treatment in SCI.<br />
ACKNOWLEDGEMENTS<br />
We thank Dr. Yanfeng Wang for his assistance with<br />
histological preparations. This work was supported by<br />
Special Fund for Scientific Research of Doctor-degree<br />
Subjects in Colleges and Universities of Ministry of Education<br />
(20060159019) and Natural Science Foundation of<br />
Liaoning Province (20052096).<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4256-4258, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2225<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Short Communication<br />
In-ovo evaluation of the antiviral activity of methanolic<br />
root-bark extract of the African Baobab (Adansonia<br />
digitata Lin)<br />
Sulaiman, Lanre K. 1,2 , Oladele, Omolade A. 2 *, Shittu, Ismaila A. 1 , Emikpe, Benjamin O. 3 ,<br />
Oladokun, Agnes T. 1 and Meseko, Clement A. 1<br />
1 Viral Research Department, National Veterinary Research Institute, Vom, Plateau State, Nigeria.<br />
2 Department of Veterinary Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria.<br />
3 Department of Veterinary Pathology, University of Ibadan, Ibadan, Oyo State, Nigeria.<br />
Accepted 18 March, 2011<br />
Application of ethnoveterinary medicine in the control of poultry diseases is being embraced in many<br />
parts of the world for more profitable production. This study investigated the antiviral property of the<br />
root-bark extract of the African Baobab tree (Adansonia digitata Lin) against Newcastle disease virus.<br />
One hundred and seventy five specific antibody negative embryonated chicken eggs and a field strain<br />
of Newcastle disease virus were used to test for the antiviral activity of the methanolic root bark extract<br />
of the tree. Following a 2-h exposure of the virus to eight graded concentrations of the extract, it was<br />
incubated at 37°C and observed 12-hourly for mortality. Dead embryos were spot tested for<br />
haemagglutinating activity. The 100EID50 concentration of the virus and the highest concentration of the<br />
extract were inoculated as positive and negative controls, respectively. All eggs inoculated with the<br />
virus alone as well as 5 and 2 mg/ml extract/virus suspensions, died by 72 h post inoculation, while no<br />
mortality was observed amongst those inoculated with 250 and 200 mg/ml virus/extract suspensions as<br />
well as those inoculated with the pure extract. Mortalities of 40, 40 and 20%, due to viral activity were<br />
recorded for 25, 50 and 100 mg/ml suspensions, respectively. This study showed that methanolic rootbark<br />
extract of A. digitata has antiviral activity against Newcastle disease virus in ovo, particularly when<br />
used at dose rates of 200 and 250 mg/ml.<br />
Key words: Ethnoveterinary, African Baobab, antiviral activity, Newcastle disease virus.<br />
INTRODUCTION<br />
A major constraint to profitable broiler production is<br />
disease outbreaks. The tropical environment provides<br />
optimum conditions like high environmental temperature<br />
and relative humidity as well as poor biosecurity for<br />
disease agents to thrive. These are major challenges<br />
being faced by the poultry industry in Nigeria. In the face<br />
of persistent disease outbreaks and in spite of the use of<br />
*Corresponding author. E-mail: laprecieux@yahoo.com or<br />
lade.oladele@gmail.com. Tel: 234-806-113-8531.<br />
Abbreviation: DMSO, Dimethylsulfoxide; EID50, embryo<br />
infective dose 50; HA, haemagglutination; ND, Newcastle<br />
disease; NVRI, Nigerian Veterinary Research Institute; SAN,<br />
specific antibody negative.<br />
conventional chemicals as disinfectants and drugs, it has<br />
become imperative to seek means of achieving effective<br />
biosecurity and control measures which ethnoveterinary<br />
preparations could offer (Gueye, 1999; Musa et al.,<br />
2008).<br />
The Baobab tree (Adansonia digitata Lin) is indigenous<br />
in many African countries (Wickens, 1982; Sidibe and<br />
Williams, 2002). Many parts of the plant, especially<br />
leaves, fruit pulp, seeds and bark fibers, have been used<br />
traditionally for medicinal and nutritional purposes (Sidibe<br />
and Williams, 2002; Chadare et al., 2009) and some<br />
commercial enterprises produce standardized preparations<br />
derived from its parts. The medicinal applications<br />
include treatment of intestinal and skin disorders and<br />
various uses as anti-inflammatory, anti-pyretic and<br />
analgesic agents (Ramadan et al., 1994; Palombo, 2006;
Ajose, 2007; Karumi et al., 2008). In addition, antibacterial,<br />
antiviral and anti-trypanosomal activities of the<br />
plant extracts have been reported (Anani et al., 2000;<br />
Hudson et al., 2000; Atawodi et al., 2003; Vimalanathan<br />
and Hudson, 2009). Most of the reports were on the<br />
efficacy of the leaves, fruit pulp, seeds and bark fibers of<br />
this plant with very little information on the antiviral<br />
properties associated with the root-bark extracts.<br />
However, the root bark is commonly used in traditional<br />
African Medicine in the treatment of fever (SCUC, 2006;<br />
Wickens, 2008). This study was therefore carried out to<br />
evaluate the antiviral property of the methanolic root bark<br />
extracts of the African Baobab tree (A. digitata Lin)<br />
against Newcastle disease (ND) virus.<br />
MATERIALS AND METHODS<br />
Extract preparation<br />
Root-bark of A. digitata was harvested, cut into small pieces and<br />
air-dried on a clean surface for 10 days. Two kilograms of the rootbark<br />
was soaked (completely immersed) in 5 L of methanol in a<br />
covered container for 3 days (Wickens, 2008; Egunyomi et al.,<br />
2010). The methanol was decanted and fresh methanol was added<br />
to the root-bark. The procedure was repeated twice and the extract<br />
was poured into a small glass container and refrigerated.<br />
Antiviral activity test<br />
Preparation of virus: Extract inoculum<br />
The virus used was a field strain of ND virus from the Virology<br />
Research Laboratory of the National Veterinary Research Institute<br />
(NVRI), Vom, Nigeria. The virus was propagated and titrated in 9<br />
day-old specific antibody negative (SAN) embryonated chicken<br />
eggs and the 100EID50 was calculated. The extract was diluted to<br />
the desired concentrations of 250, 200, 100, 50, 25, 10, 5 and 2<br />
mg/ml. These extract concentrations were substituted for the virus<br />
buffer diluent at the end point dilution of the virus to achieve the<br />
100EID50. The virus/extract suspensions were kept at 4°C for 2 h to<br />
allow reaction to occur.<br />
Egg inoculation<br />
The embryonated chicken eggs were labeled according to the<br />
extract concentrations used. A set of plastic egg trays were<br />
thoroughly cleaned with Virkon ®, the eggs were swabbed with<br />
70% alcohol in cotton wool and transferred into the cleaned trays.<br />
The swabbed eggs were placed in the micro-safety cabinet where<br />
they were punched and immediately inoculated with the<br />
extract/virus suspension via the allantoic route. Each egg was<br />
inoculated with 0.1 ml of the inoculum and 5 eggs were inoculated<br />
with each con-centration of the extract/virus suspension, while<br />
inoculating the 250 mg/ml extract only (without virus) and the<br />
100EID50 concentration of the virus (without extract- virus control)<br />
as negative and positive controls, respectively. The eggs were<br />
sealed with molten wax and incubated at 37°C. The inoculated<br />
eggs were monitored for embryonic death 12-hourly for up to 96 h<br />
post inoculation. Dead embryos were removed and chilled in the<br />
refrigerator for 3 h after which they were opened to check for<br />
haemagglutinating (HA) activity by spot testing.<br />
Spot haemagglutination test<br />
Sulaiman et al. 4257<br />
Dead embryos that had been chilled were brought out of the<br />
refrigerator and kept at room temperature for about 30 min. The<br />
eggs were swabbed and placed in the biosafety cabinet. The shell<br />
of each egg was opened to reveal the air space and a pipette was<br />
used to dispense a drop of 1% washed chicken red blood cells on a<br />
white tile. A wire loop was thoroughly flamed and used to pick a<br />
drop of the allantoic fluid which was mixed with the drop of blood.<br />
The tile was gently rocked and observed for visible agglutination,<br />
indicating viral activity (Thayer and Beard, 1998; Murakawa et al.,<br />
2003). This was done for every egg and the observations were<br />
recorded.<br />
RESULTS AND DISCUSSION<br />
All eggs inoculated with the virus control, 5 and 2 mg/ml<br />
of the extract/virus suspension died between 48 and 72 h<br />
of inoculation and showed positive haemagglutination by<br />
spot-testing. However, no mortality was observed amongst<br />
the embryonated eggs inoculated with the extract only,<br />
250 and 200 mg/ml of extract/virus suspension up till 96 h<br />
post inoculation. There were varying percentages of<br />
embryo mortality amongst the 100, 50, 25 and 10 mg/ml<br />
extract/virus suspension groups (Table 1). The haemagglutination<br />
test was carried out to associate embryo<br />
mortality with viral activity. The results of mortalities and<br />
HA spot-testing are shown in Table 1. Only the group of<br />
eggs inoculated with 5 mg/ml extract/virus suspension<br />
had a dead embryo at 24 h post inoculation.<br />
This study has evaluated the potentials of the methanolic<br />
root-bark extract of A. digitata Lin as an antiviral<br />
agent against ND virus using multiplication of the ND<br />
virus in embryonated egg as an indicator for antiviral<br />
property. The results of the virus propagation showed<br />
that 250 and 200 mg/ml concentrations of the extract<br />
completely inhibited the growth of ND virus in<br />
embryonated chicken eggs, indicating that the methanolic<br />
root bark extract of A. digitata at these doses are<br />
effective against the virus when exposed in-ovo.<br />
The results of this study corroborrates the findings of<br />
Vimalanathan and Hudson (2009) that the methanolic,<br />
water and dimethylsulfoxide (DMSO) extract of the leaf,<br />
stem and pulp of A. digitata has both antiviral effect,<br />
especially against influenza virus and anti-inflammatory<br />
effects. The antiviral property observed might be ascribed<br />
to the presence of various potentially bioactive ingredients<br />
(Chadare et al., 2009), including triterpenoids,<br />
flavonoids and phenolic compounds, but at present, the<br />
antiviral activity observed in this study could not be<br />
ascribed to specific compounds.<br />
At extract concentrations of 100, 50 and 25 mg/ml,<br />
there were 20, 40 and 40% mortalities due to viral activity<br />
when compared with 0% at concentrations of 250 and<br />
200 mg/ml. This indicates inhibitory rather than virucidal<br />
effect of the extract on the ND virus at these doses.<br />
Whereas, at concentrations of 2 to 10 mg/ml, as well as<br />
at 0 mg/ml (virus control), the study recorded total or near
4258 Afr. J. Biotechnol.<br />
Table 1. Embryo mortality and haemagglutination (HA) test result of embryonated chicken eggs inoculated with graded doses<br />
of A. digitata extract/ND virus suspension.<br />
Virus<br />
Mortality HA Test Mortality due to<br />
(mg/ml) 24 h pi 48 h pi 72 h pi 96 h pi +ve -ve viral activity (%)<br />
250 0/5 0/5 0/5 0/5 0 5 0<br />
200 0/5 0/5 0/5 0/5 0 5 0<br />
100 0/5 0/5 1/5 0/4 1 4 20<br />
50 0/5 1/5 3/4 1/1 2 3 40<br />
25 0/5 0/5 2/5 0/3 2 3 40<br />
10 0/5 0/5 3/5 2/2 5 0 100<br />
5 1/5 0/4 4/4 - 4 1 80<br />
2 0/5 0/5 5/5 - 5 0 100<br />
Vc 0/5 0/5 5/5 - 5 0 100<br />
Uic 0/5 0/5 0/5 0/5 0 5 0<br />
Ec 0/5 0/5 0/5 0/5 0 5 0<br />
Vc = Virus control; Uic = uninoculated control; Ec = egg control; pi = post inoculation.<br />
total mortalities due to viral activity, indicating the ineffectiveness<br />
of the extract at these doses. The dead<br />
embryos in the 50 and 5 mg/ml extract concentrations<br />
that were negative for haemagglutinating activity must be<br />
a result of nonspecific deaths from manipulations during<br />
inoculation (Villegas, 1998).<br />
In conclusion, this work has shown that the methanolic<br />
root-bark extract of A. digitata Lin has direct antiviral<br />
activity against ND virus and could therefore be useful in<br />
the control of the disease in poultry birds.<br />
REFERENCES<br />
Ajose FOA (2007). Some Nigerian plants of dermatologic importance.<br />
Int. J. Derm. 46(suppl 1): 48-55.<br />
Anani K, Hudson JB, de Souza C, Akpagana K, Towers GHN, Arnason<br />
JT, Gbeassor M (2000). Investigation of Medicinal Plants of Togo for<br />
Antiviral and Antimicrobial Activities. Pharm. Biol. 38: 40-45.<br />
Atawodi SE, Bulus T, Ibrahim S, Ameh DA, Nok AJ, Mamman N,<br />
Galadima M (2003). In Vitro trypanocidal effect of methanolic extract<br />
of some Nigerian savannah plants Afr. J. Biotechnol. 2: 317-321.<br />
Chadare FJ, Linnemann AR, Hounhouigan JD, Nout, MJR, Van Boekel<br />
MAJ (2009). Baobab Food Products: A Review on their Composition<br />
and Nutritional Value. Crit. Rev. Food Sci. Nutr. 49: 254-274.<br />
Egunyomi A, Gbadamosi IT, Osiname KO (2010). Comparative<br />
effectiveness of ethnobotanical mosquito repellents used in Ibadan,<br />
Nigeria. J. Appl. Biosci. 36: 2382-2388.<br />
Gueye EF (1999). Ethnoveterinary medicine against poultry diseases in<br />
African villages. World Poult. Sci. J. 55: 187-198.<br />
Hudson JB, Anani K, Lee MK, De Souza C, Arnason JT, Gbeassor M<br />
(2000). Further Investigations on the Antiviral Properties of Medicinal<br />
Plants of Togo. Pharm. Biol. 38: 46-50.<br />
Karumi Y, Augustine AI, Umar IA (2008). Gastroprotective effects of<br />
aqueous extract of Adansonia digitata leaf on ethanol-induced<br />
ulceration in rats. J. Biol. Sci. 8: 225-228.<br />
Murakawa Y, Sakaguchi K, Soejima K, Eriguchi S, Takase K, Sueyoshi<br />
M, Nagatomo H, Ito T, Otuski K (2003). Heamagglutinating activity of<br />
the lentogenic Newcastle disease virus strain MET95. Avian Pathol.<br />
32(1): 39-45.<br />
Musa U, Abdu PA, Dafwang II, Katsayal UA, Edache JA, Karsin PD<br />
(2008). Ethnoveterinary remedies used for the management of<br />
Newcastle disease in some selected local government areas of<br />
Plateau state, Nigeria. Nig. J. Pharm. Sci. 7(1): 126-130.<br />
Palombo EA (2006). Phytochemicals from traditional medicinal plants<br />
used in the treatment of diarrhoea: Modes of action and effects on<br />
intestinal function. Phytother. Res. 20: 717-724.<br />
Ramadan A, Harraz FM, El-Mougy SA (1994). Anti-inflammatory,<br />
analgesic and antipyretic effects of the fruit pulp of Adansonia<br />
digitata. Fitoterapia, 65: 418-422.<br />
SCUC (2006). Baobab manual, Field manual for extension workers and<br />
farmers. University of Southampton, Southampton, UK.<br />
Sidibe M, Williams JT (2002). Baobab. Adansonia digitata. International<br />
Centre for Underutilized Crops. University of Southampton p. 105<br />
(ISBN 0854327649).<br />
Thayer SG, Beard CW (1998). Serologic procedures. In: Swayne E,<br />
Glisson JR, Jackwood MW, Pearson JE, Reed WM (eds). A<br />
laboratory manual for the isolation and identification of avian<br />
pathogens. American Association of Avian Pathologists. Kennett<br />
Square, PA. USA pp. 255-266<br />
Villegas P (1998). Titration of biological suspensions. In: In: Swayne E,<br />
Glisson JR, Jackwood MW, Pearson JE, Reed WM (eds). A<br />
laboratory manual for the isolation and identification of avian<br />
pathogens. American Association of Avian Pathologists. Kennett<br />
Square, PA. USA, pp. 248-254.<br />
Vimalanathan S, Hudson JB (2009). Multiple inflammatory and antiviral<br />
activities in Adansonia digitata (Baobab) leaves, fruits and seeds. J.<br />
Med. Plan. Res. 3(8): 576-582.<br />
Wickens GE (1982). The baobab-Africa’s upside-down tree. Kew<br />
Bulletin. 37: 173-209.<br />
Wickens GE (2008). The Baobabs: Pachycauls of Africa, Madagascar<br />
and Australia. Springer.
African Journal of Biotechnology Vol. 10(20), pp. 4259-4264, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2064<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Effect of gamma radiation on different stages of Indian<br />
meal moth Plodia interpunctella Hübner (Lepidoptera:<br />
Pyralidae)<br />
Hosseinzadeh Abbas 1 , Shayesteh Nouraddin 2 , Zolfagharieh Hamid Reza 3 , Bernousi Iraj 5 ,<br />
Babaei Mohammad 3 , Zareshahi Hasan 4 , Ahari Mostafavi Hossein 3 and Fatollahi Hadi 3<br />
1 Department of Entomology, Agricultural Faculty, Urmia University, Urmia, Iran.<br />
2 Department of Plant Medicine, Mahabad Branch, Islamic Azad University, Mahabad, Iran.<br />
3 Agricultural, Medical and Industrial Research School, Karaj, Iran.<br />
4 Radiation Application Research School, Yazd, Iran.<br />
5 Department of Agronomy and Plant Breeding, Agricultural faculty, Urmia University, Urmia-Iran.<br />
Accepted 4 March, 2011<br />
Indian meal moth Plodia interpunctella Hübner is one of the most important stored products pests in<br />
the world. In this research, the effect of gamma irradiation was studied on different developmental<br />
stages of this pest and the doses required to prevent each of these developmental stages was<br />
investigated. From the results, required dose to prevent larval emergence from irradiated 1 to 24 h eggs<br />
was 400 Gray (Gy), and 400 Gy was required to prevent pupae from 15 days old larvae. Also, the dose of<br />
radiation required to prevent adult emergence from irradiated 5 days old pupa was 650 Gy. According to<br />
the results, dose of 650 Gy is adequate to control all immature stages of this pest. In addition, the effect<br />
of gamma ray was studied on developmental stage period of each irradiated existence stage till adult<br />
eclosion. The results revealed that there was a dose-dependent increase in the developmental periods,<br />
and the growth index of the adults was significantly decreased with increasing dose of radiation<br />
administered to the eggs, larvae and pupae too. It is concluded that irradiation can be used as a safe<br />
method to control stored pests.<br />
Key words: Gamma irradiation, prevention dose, developmental period, growth index, Plodia interpunctella.<br />
INTRODUCTION<br />
Iran is the largest producer of pistachios and dates in the<br />
world and according to the Food and Agriculture Organization<br />
(FAO), approximately 10% of stored products in<br />
Iran are lost due to insect infestation (Zolfagharieh,<br />
2002). The Indian meal moth Plodia interpunctella<br />
Hübner is a cosmopolitan pest whose larvae can infest a<br />
variety of stored grain, nuts, pulses, meals, dried fruits<br />
and processed foods (Simmons and Nelson, 1975).<br />
Methyl bromide (MB) is a widely used fumigant for the<br />
control of insect infestation in many agricultural commo-<br />
*Corresponding author. E-mail: abas1354@yahoo.com or<br />
abmelis@gmail.com.<br />
Abbreviations: MB, Methyl bromide; PTM, potato tuber moth.<br />
dities. As methyl bromide has the potential to destroy the<br />
ozone layer in the environment, alternative methods had<br />
to be sought to replace its use in insect control<br />
(Gochangco et al., 2002). It has been reported that<br />
irradiation could offer an important and attractive alternative<br />
for the export commodity fumigation uses of MB<br />
and for eliminating the problem of chemical residues<br />
(Ahmed, 1991; Marcotte, 1993). Brower and Tilton (1983)<br />
reported that irradiation is an approved method of direct<br />
control for stored product insect in wheat and wheat flour<br />
in many countries, and indications were that it would<br />
soon be approved for all grain, grain products and other<br />
dry food commodities. Commodities are exposed to<br />
gamma rays from the radioactive isotopes cesium 137 or<br />
cobalt 60, electron beams, or X rays converted from an<br />
electron beam generator. The mode of action of ionizing<br />
irradiation as a quarantine treatment involves breaking
4260 Afr. J. Biotechnol.<br />
chemical bonds. When this happens to DNA, normal<br />
development or reproduction of the organism may be<br />
prevented. Decades of research conducted worldwide on<br />
radiation disinfestation of food and agricultural products<br />
have shown that this method is effective. The advantages<br />
of irradiation processing include no undesirable residues<br />
in the foods treated, no resistance developed by pest<br />
insects and few significant changes in the physicchemical<br />
properties or the nutritive value of the treated<br />
products (Ahmed, 2001; Lapidot et al., 1991; Boshra and<br />
Mikhaiel, 2006). Unlike other disinfestations techniques,<br />
irradiation does not need to kill the pest immediately to<br />
provide quarantine security; therefore, live (but nonviable<br />
or sterile) insects may occur with the exported commodity<br />
(Follett and Griffin, 2006).<br />
In the present work, we have tested gamma radiation<br />
sensitivity of different life stages of P. interpunctella<br />
Hübner. The study was also designed to assess the<br />
effect of gamma irradiation on different stages of P.<br />
interpunctella.<br />
MATERIALS AND METHODS<br />
Insect culture<br />
P. interpunctella Hübner used in all experiments were derived from<br />
a laboratory culture initially established for larvae collection from<br />
infested pistachios in Urmia Province, Northwest Iran. The larvae<br />
were reared on artificial diet containing bran (800 g), dry yeast<br />
powder (160 g), honey (300 g) and glycerol (200 ml / 1000 g) dry<br />
ingredient. The diet was decontaminated at 60°C for 6 h.<br />
Throughout the experiments, insect cultures were maintained under<br />
controlled laboratory conditions of 28 ± 1°C and 65% ± 5% relative<br />
humidity (RH) with a 14:10 h light : dark photoperiod. Larvae were<br />
placed in 500 ml glass containers half full of rearing medium.<br />
Fifteen virgin pairs of males and females (0 to24 h old) were<br />
selected for the experiments. These adults were placed in funnels<br />
capped with screen lids. Funnels were placed over radiology film<br />
for 24 h and oviposited eggs on the film were collected every 12 h.<br />
Irradiation of eggs<br />
Twenty eggs (1 to 2 days old) were placed in glass Petri dishes and<br />
irradiated in a calibrated 60 Co irradiator (Issledovatel type PX-30)<br />
with the activity of 4.5 kCi and dose rate of 0.65 Gy/s at the Iran<br />
Nuclear Research Center for Agriculture and Medicine. Ferric<br />
dosimetry system was used for the facility.<br />
Radiation was applied at nine dose levels ranging from 0 to 400<br />
Gy to the eggs. Controls were subjected to the same conditions as<br />
the irradiated eggs in that, they were transferred to the irradiator<br />
and subsequently removed without exposure to radiation. After<br />
irradiation, eggs were returned immediately to the laboratory and<br />
then placed in Petri dishes that had a central well made by ringing a<br />
central area (2 cm diameter) with glue and allowing the glue to dry.<br />
Rearing medium was placed in the Petri dish around the central<br />
well to provide food for any hatched larvae, and the apparatus was<br />
placed at 28 ± 1°C and 65 ± 5% RH. The Petri dishes were<br />
examined under a binocular microscope, and the number of<br />
hatched eggs was counted daily until no further hatched egg was<br />
observed. The rearing medium containing the small larvae was then<br />
transferred to Petri dishes containing food. Pupation and adult<br />
emergence of these larvae were recorded. Four replicate batches<br />
of eggs were used for each dose level and the control.<br />
Irradiation of 15 days old larvae<br />
Fifteen days old larvae were selected for uniformity in size before<br />
irradiation. Larvae were irradiated in the same irradiator and dose<br />
ranges from 0 to 400 Gy. Immediately after treatment, irradiated<br />
and control larvae were transferred to Petri dishes containing<br />
rearing medium. The Petri dishes were subsequently transferred to<br />
the rearing conditions. Pupation, adult emergence and mortality of<br />
these larvae were recorded every 2 days. Mortality of larvae was<br />
determined by the brown color with no observable movement. The<br />
adults that developed from irradiated larvae were removed and<br />
counted daily. Four replicate (20 larvae for each replicate) were<br />
used for each dose level and control.<br />
Developmental periods and longevity of the adults derived<br />
from irradiated eggs, larvae and pupae<br />
The developmental periods, from egg to adult of P. interpunctella<br />
Hübner that developed from irradiated eggs, larvae and pupae were<br />
recorded. The longevity of the adults was also determined. The<br />
growth index (percentage adult eclosion/total developmental period)<br />
was calculated for the irradiated eggs, larvae and pupae.<br />
Irradiation of pupae<br />
Five-day old pupae were placed in Petri dishes and irradiated at six<br />
dose levels of 0, 150, 250, 350, 450, 550 and 650 Gy. The number<br />
of adults that emerged from irradiated pupae was recorded. Four<br />
replicates (20 pupae in each) were used for each dose level.<br />
Statistical analysis<br />
Data from the experiments were subjected to analysis of variance<br />
(ANOVA) using the Statistical Package for the Social Sciences<br />
(SPSS) for windows 11.5. Data were transformed using arcsine √x<br />
before ANOVA. Means were separated at the 5% significance level<br />
by the Tukey test.<br />
RESULTS AND DISCUSSION<br />
Effects of gamma radiation on eggs<br />
The data obtained from experiments dealing with the irradiation<br />
of eggs are summarized in Table 1. A significant<br />
reduction in hatched eggs was observed and it was<br />
correlated with the radiation doses (F = 596.522; df = 8; P<br />
< 0.05). Percentage larval emergence from irradiated<br />
eggs was 93.75% in the untreated controls but was<br />
reduced to 2.81% at a dose of 350 Gy, and all irradiated<br />
eggs were completely sterile at 400 Gy. The percentage<br />
of emergent larvae that survived to the pupa stage also<br />
decreased with increasing radiation doses (F = 769.212;<br />
df = 8; P < 0.05). At doses of 250 and 300 Gy, pupal<br />
development was 10.31 and 4.37%, respectively.<br />
The percentage of pupation that survived to the adult<br />
stage was also decreased with increasing radiation doses<br />
(F = 300.892; df = 8; P < 0.05). Percentage adult
Table 1. Egg hatch, pupation, and adult emergence of the P. interpunctella irradiated as eggs (1 to 2 days old).<br />
Dose (Gy) Egg hatch (%) ± SE Pupation (%) ± SE Adult emergence (%) ±SE<br />
0 93.75±1.61 a 75.00±1.02 a 61.25±0.72 a<br />
50 78.12±1.20 b 65.00±1.02 b 60.00±1.02 a<br />
100 62.5±1.02 c 52.50±1.44 c 48.75±1.25 b<br />
150 43.13±1.20 d 35.00±1.02 d 31.87±1.20 c<br />
200 31.87±1.20 e 20.62±1.20 e 16.25±0.72 d<br />
250 21.25±0.72 f 10.31±0.79 f 8.75±.72 e<br />
300<br />
350<br />
400<br />
11.87±1.20 g<br />
2.81±0.79 h<br />
0 i<br />
4.37±1.20 g<br />
Means within each column followed by the same latter are not significantly different (P > 0.05).<br />
0 h<br />
0 h<br />
1.87±1.20 f<br />
Table 2. Pupation, adult emergence and mortality (%) of the P. interpunctella irradiated as<br />
larvae (15 days old).<br />
Dose (Gy) Pupation(%)±SE Adult emergence (%) ±SE Mortality(%) ±SE<br />
0 85.62±1.57 a 60.00±1.77 a 11.87±1.20 g<br />
150 76.25±0.72 b 45.94±0.60 b 17.81±1.07 f<br />
200 51.87±1.20 c 21.25±0.72 c 35.31±1.07 e<br />
250 37.50±1.77 d 8.12±1.20 d 51.87±1.20 d<br />
300 20.31±1.02 e 0 e 61.87±1.20 c<br />
350 5.00±1.02 f 0 e 81.87±1.20 b<br />
400 0 g 0 e 100 a<br />
Means within each column followed by the same latter are not significantly different (P > 0.05).<br />
emergence from irradiated eggs was 61.25% in the<br />
untreated controls but was reduced to 1.87% at a dose of<br />
300 Gy and no adult emerged at doses of 350 Gy and<br />
above.<br />
Effects of gamma radiation on 15 days old larvae<br />
The effects of gamma radiation on irradiated larvae are<br />
shown in Table 2. The data showed that the percentage<br />
of irradiated larvae that survived to the pupal stage were<br />
decreased with increasing radiation doses (F = 1077.006;<br />
df = 6; P < 0.05). The percentages of pupae that developed<br />
from irradiated larvae were 5% at 350 Gy when<br />
compared with 85.62% in the control. The percentage of<br />
pupation that survived to the adult stage were also<br />
decreased with increasing radiation doses (F = 998.931;<br />
df = 6; P < 0.05). Statistical analysis of data indicated that<br />
irradiation significantly affected mortality percentages of<br />
larvae (F = 1038.098; df = 6; P < 0.05). Mortality of<br />
irradiated larvae began 4 days after the treatment and<br />
continued through the time of observation. Irradiation at<br />
400 Gy caused 100% mortality.<br />
0 f<br />
0 f<br />
Abbas et al. 4261<br />
Effects of gamma radiation on the developmental<br />
period and longevity of the adults derived from<br />
irradiated eggs, larvae and pupae<br />
There was a dose-dependent increase in the<br />
developmental time of P. interpunctella Hübner adults<br />
developed from irradiated eggs (F = 21.876; df = 7; P <<br />
0.05), larvae (F = 42.931; df = 7; P < 0.01) and pupae (F<br />
= 21.650; df = 7; P < 0.05) (Table 3). The mean developmental<br />
time from egg to adult was 44.20 days in the<br />
controls, which rose to 52.50 days for insects derived<br />
from eggs irradiated with a dose of 350 Gy. The<br />
development of the irradiated larvae was also extended<br />
from 23.40 days in the controls to 36.40 days for insects<br />
exposed to a dose of 350 Gy. The mean developmental<br />
time from pupa to adult was 3.70 days in the controls,<br />
which rose to 6.90 days at a dose of 350 Gy. The growth<br />
index of the adults was significantly decreased with<br />
increasing doses of radiation administered to the eggs (F
4262 Afr. J. Biotechnol.<br />
Table 3. Mean (± SE) developmental period of P. interpunctella adults irradiated as eggs, larvae and pupae a .<br />
Dose<br />
(Gy)<br />
Developmental period from eggs to adults (days) Growth index b<br />
Eggs treated Larvae treated Pupae treated Eggs treated Larvae treated Pupae treated<br />
0 44.20±0.63 e 23.40±0.65 c 3.70±0.15 d 1.38 a 2.56 a 25.00 a<br />
50 45.20±0.44 de 24.20±0.74 c 3.80±0.20 d 1.32 a 2.12 b 23.87 b<br />
100 45.60±0.70 de 25.80±0.49 bc 4.50±0.17 cd 1.05 b 1.86 c 19.46 c<br />
150 46.40±0.65 cde 27.30±0.67 b 5.00±0.30 c 0.67 c 1.68 c 17.59 d<br />
200 47.40±0.65 bcd 30.50±0.72 a 5.50±0.28 bc 0.33 d 0.70 d 11.68 e<br />
250 48.20±0.61 bc 32.00±0.80 a 6.10±0.28 ab 0.17 e 0.25 e 7.77 f<br />
300 49.40±0.37 b - 6.90±0.23 a 0.03 f - 5.37 g<br />
350 52.50±0.40 a - - - - 5.16 g<br />
a Means followed by the same letter in a column within each dose are not significantly different (P > 0.05); b growth index: percentage adult<br />
eclosion/total developmental period.<br />
Adult emergence (%)<br />
100<br />
90<br />
80<br />
70<br />
60<br />
50<br />
40<br />
30<br />
20<br />
10<br />
0<br />
92.5a<br />
66.87b<br />
35c<br />
23.75d<br />
12.5e<br />
Control 150 250 350 450 550 650<br />
Dose (Gy)<br />
Figure 1. Adult emergence of the P. interpunctella irradiated as pupae (5 days old).<br />
= 1141.967; df = 7; P < 0.05), larvae (F = 456.928; df = 7;<br />
P
obtain complete sterility for Ephestia cautella (Walker)<br />
and P. interpunctella eggs respectively (Ozyardimci et al.,<br />
2006). In our study, a dose of 350 Gy completely<br />
prevented adult emergence from irradiated eggs. Ayvaz<br />
and Tuncbilek (2006) found that no Ephestia kuehniella<br />
Zeller late-instar larvae received an irradiation dose of<br />
250 Gy as emerged adults.<br />
When the effects of six gamma radiation doses ranging<br />
from 50 to 1000 Gy were investigated against all life<br />
stages of E. cautella, the development of adults from<br />
treated eggs and larvae was prevented by 300 and 200<br />
Gy, respectively (Cogburn et al., 1973). In the present<br />
study, a dose of 400 Gy completely prevented larval<br />
development and the larvae did not reach pupation. The<br />
minimum dose required to prevent adult emergence from<br />
P. iterpunctella exposed as larvae was 300 Gy, while<br />
doses required to cause death were 450 Gy and above<br />
(Azelmat et al., 2005). A dose of 250 Gy was sufficient to<br />
give complete mortality for last-instar larvae of E.<br />
kuehniella Zeller (Ayvaz and Tuncbilek, 2006). Our<br />
results showed that, when the prevention of adult<br />
emergence is used as a standard for measuring the<br />
effectiveness of radiation against P. interpunctella larvae,<br />
a dose of 300 Gy is required. It was also found that the<br />
ultimate development of adults from 11-days-old P.<br />
interpunctella larvae was completely prevented when<br />
these larvae were exposed to 149 Gy (Johnson and Vail,<br />
1988). Ayvaz et al. (2006) reported that younger larvae<br />
were more sensitive to radiation than the older ones, and<br />
that the dose required to prevent adult emergence was<br />
lower (200 Gy) than for mature larvae (250 Gy). Cogburn<br />
et al. (1973) reported that from the larvae of Cadra<br />
cautella Walker treated with 20 krad (200 Gy), no adults<br />
were produced. Mansour (2002) also reported that<br />
200 Gy doses were enough to prevent adult emergence<br />
from irradiated mature larvae of codling moth Cydia<br />
pomonella (L.). Ayvaz and Tunçbilek (2006) found that a<br />
dose of 200 Gy applied to the young larvae of E.<br />
kuehniella completely prevented female adult emergence<br />
(all emerged moths were males).<br />
There was a dose-dependent delay in the developmental<br />
period of the P. iterpunctella eggs, larvae and<br />
pupae. The longevity of the treated larvae increased<br />
when compared with controls that continued development<br />
to adult emergence. Hallman (2000) noted that insects<br />
may live for considerable periods of time after irradiation.<br />
Makee and Saour (2003) assumed that unbalanced<br />
hormonal system led to prolonged developmental time in<br />
the F1 larvae of the potato tuber moth (PTM),<br />
Phthorimaea operculella Zeller. Hosseinzadeh et al.<br />
(2010) found that there was a dose-dependent increase<br />
in the developmental time of Oryzaephilus surinamensis<br />
L. adults developed from irradiated eggs, larvae and<br />
pupae. Increasing post-treatment longevity for irradiated<br />
P. interpunctella, the false codling moth, Cryptophlebia<br />
leucotreta Meyrick, the Mediterranean flour moth, E.<br />
kuehniella, and the red flour beetle Tribolium castaneum<br />
Abbas et al. 4263<br />
Herbst have been reported previously (Azelmat et al.,<br />
2005; Johnson and Vail, 1988; Bloem et al., 2003; Mehta<br />
et al., 1990; Ayvaz and Tuncbilek, 2006). Typically, irradiated<br />
nymphs or larvae will have a prolonged nymphal<br />
or larval stage and may live longer than non-irradiated<br />
control insects (Hasan and Khan, 1998).<br />
When the pupae of P. interpunctella were irradiated,<br />
the percentage of adult emergence was decreased by<br />
doses up to 650 Gy. When male and female pupae of<br />
Ephestia calidella (Guenee) were irradiated with doses of<br />
200 to 800 Gy, the percentage adult emergence was<br />
decreased in accordance with increasing gamma<br />
radiation doses (Boshra and Mikhaiel, 2006). This work<br />
also showed that a 1000 Gy dose prevented the emergence<br />
of both sexes.<br />
Hallman (2000) reported that some stored-product<br />
moths, especially the Angoumois grain moth, Sitotraga<br />
cerealella (Oliver) and the Indian meal moth P.<br />
interpunctella (Hübner), were among the most tolerant<br />
arthropods to radiation. In comparison with dipteran<br />
species, Lepidoptera require much higher levels of<br />
ionizing radiation to obtain full sterility (LaChance and<br />
Graham, 1984). The main cause of this difference is<br />
thought to be as a result of the different kinetic organization<br />
of chromosomes in these two groups of insects.<br />
The Diptera possess typical monocentric chromosomes<br />
with kinetic activity restricted to the centromere, whereas<br />
lepidopteran chromosomes are holokinetic (Gassner and<br />
Klemetson, 1974).<br />
The results of this study support the assumption that a<br />
dose of 400 Gy is required in order to prevent hatching of<br />
eggs and 300 Gy is required to prevent the larvae from<br />
reaching adulthood. It is recommended that dose of 650<br />
Gy should be used to control population growth of P.<br />
interpunctella when targeting pupae are within stored<br />
products. Delayed developmental periods were observed<br />
after irradiation of the eggs, larvae and pupae. Irradiation<br />
could be used as a disinfestation treatment in stored<br />
products as an alternative to chemical fumigants. Phostoxin<br />
and methyl bromide are the most common fumigants<br />
for insect disinfestation in storage. Fumigants leave<br />
residues in treated products, and due to the ozonedepleting<br />
properties, methyl bromide was banned in<br />
many countries. Therefore, irradiation is a safe alternative<br />
to fumigation.<br />
Acknowledgments<br />
The authors wish to express their deep appreciation to<br />
Urmia University and Karaj Nuclear Research Center for<br />
Agriculture and Medicine. The authors also thank Dr.<br />
Aramideh for assistances analyzing data.<br />
REFERENCES<br />
Ahmed M (2001). Disinfestation of stored grain, pulses, dried fruits and
4264 Afr. J. Biotechnol.<br />
nuts, and other dried foods. In: Molins R (ed.) Food Irradiation<br />
Principles and Applications, Wiley, New York. pp. 77-112.<br />
Ahmed M (1991). Irradiation disinfestation and packaging of dates.<br />
Insect disinfestation of food and agricultural products by irradiation.<br />
IAEA, Vienna, pp. 7-26.<br />
Ayvaz A, Albayrak S, Karaborklu S (2008). Gamma radiation sensitivity<br />
of eggs, larvae and pupae of Indian meal moth Plodia interpunctella<br />
(Hubner) (Lepidoptera: Pyralidae). Pest Manage. Sci. 64: 505-512.<br />
Ayvaz A, Tuncbilek AS (2006). Effects of gamma radiation on life stages<br />
of the Mediterranean flour moth Ephestia kuhniella Zeller<br />
(Lepidoptera: Pyralidae). J. Pesticide Sci. 79: 215-222.<br />
Azelmat K, Sayah F, Mouhib M, Ghailani N, Elgarrouj D (2005). Effects<br />
of gamma irradiation on forth-instar Plodia interpunctella (Hubner)<br />
(Lepidoptera: Pyralidae). Journal of Stored Product Research, 41:<br />
423-431.<br />
Bloem S, Carpenter JE, Hofmeyr JH (2003). Radiation biology and<br />
inherited sterility in false codling moth (Lep- idoptera: Tortricidae). J.<br />
Econ. Entomol. 96: 1724-1731.<br />
Boshra SA, Mikhaiel AA (2006). Effect of gamma radiation on pupal<br />
stage of Ephestia calidella (Guenee). J. Stored Prod. Res. 42: 457-<br />
467.<br />
Brower J.H, Tilton EW (1983). The potential of irradiation as<br />
commodities. In: Moy JH (ed) Proceedings: Radiation disinfestation<br />
of food an agricultural products conference. Hawaii Institute of<br />
Tropical Agricultural and Human Research, University of Hawaii, pp.<br />
75-86.<br />
Brower JH (1974). Age as a factor in determining radio sensitivity of<br />
eggs of Plodia interpunctella (Hubner). Environ. Entomol. 3: 945-946.<br />
Cogburn RR, Tilton EW, Brower JH (1973). Almond moth: gamma<br />
radiation effect of the life stages. J. Econ. Entomol. 66: 745-751<br />
Follett PA, Griffin R (2006). Irradiation as a phytosanitary treatment for<br />
fresh horticultural commodities: research and regulations. In:<br />
Sommers CH, Fan X (eds). Food Irradiation and Research and<br />
Technology, Blackwell Publishing, Ames, Iowa. pp. 143-168.<br />
Gassner G, Klemetson DJ (1974) A transmission electron microscope<br />
examination of hemipteran and lepidopteran gonial centromeres.<br />
Can. J. Genet. Cytol. 16: 457-464<br />
Gochangco MU, Sanjuan EM, Lster AO (2002). Irradiation as an<br />
alternative treatment to methyl bromide for disinfestations of<br />
Tribolium castaneum in stored cacao. Proceeding of a final research<br />
coordination meeting organized by the joint FAO/IAEA Division of<br />
Nuclear Techniques in Food and Agriculture. Vienna, Austria, pp.<br />
111-123.<br />
Hallman GJ (2000). Expanding radiation quarantine treatments beyond<br />
fruit flies. Agric. Entomol. 2: 85-95.<br />
Hasan M, Khan AR (1998). Control of stored-product pests by<br />
irradiation. Integrated Pest Manage. Rev. 3: 15-29.<br />
Hosseinzadeh A, Shayesteh N, Zolfagharieh HR, Babaei M, Zareshahi<br />
H, Ahari H, Mostafavi H, Fatollahi H (2010). Gamma radiation<br />
sensitivity of different stages of Saw-toothed grain beetle<br />
Oryzaephilus surinamensis(L.) (Coleoptera: Silvanidae). J. Plant Prot.<br />
Res. 50(3): 250-255.<br />
Johnson JA, Vail PV (1988). Post treatment survival development and<br />
feeding of irradiated Indian meal moth and navel orangeworm larvae<br />
(Lepidoptera: Pyralidae). J. Econ. Entomol. 81: 376-380.<br />
LaChance LE, Graham CK (1984). Insect radiosensitivity: dose curves<br />
and dose-fractionation studies of dominant lethal mutations in the<br />
mature sperm of 4 insect species. Mutat. Res. 127: 49-59<br />
Lapidot M, Saveanu S, Padova R, Ross I (1991). Insect disinfestation<br />
by irradiation. Insect Disinfestation of food and Agricultural Products<br />
by Irradiation. Proceedings IAEA, Vienna, p. 103.<br />
Marcotte M (1993). United Nations Environment programme Methyl<br />
Bromide Technical Options Committee. Food Irrad. Newslett. 17: 27-<br />
32.<br />
Makee H, Saour G (2003). Noninherited sterility in irradiated<br />
Phthorimaea operculella females. J. Appl. Entomol. 127:489-493<br />
Mansour M (2002). Gamma irradiation as a quarantine treatment for<br />
apples infested by codling moth (Lep. Tortricidae). J. Appl. Entomol.<br />
127: 137-141.<br />
Mehta VK, Sethi GR, Garg AK (1990). Effect of gamma radiation on the<br />
development of Tribolium castaneum after larval irradiation. J.<br />
Nuclear Agric. Biol. 19(2): 124-127<br />
Ozyardimci B, Cetinkaya N, Denli E, Ic E, Alabay M (2006). Inhibition of<br />
egg and larval development of the Indian meal moth Plodia<br />
interpunctella (Hubner) and almond moth Ephestia cautella (Walker)<br />
by gamma radiation in decorticated hazelnuts. J. Stored Prod. Res.<br />
42: 183-196.<br />
Simmons P, Nelson HD (1975). Insects on Dried Fruits. USDA Agric.<br />
Handbook 464, ARS, Washington, DC, p. 25.<br />
Zolfagharieh HR (2002). Irradiation to control Plodia interpunctella and<br />
Oryzaphillus surinamensis in in Pistacios and Dates. In: Proceeding<br />
of a final research coordination meeting organized by the joint<br />
FAO/IAEA Division of Nuclear Techniques in Food and Agriculture.<br />
Vienna, Austria, pp. 101-109.
African Journal of Biotechnology Vol. 10(20), pp. 4265-4268, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1903<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Body composition of freshwater Wallago attu in relation<br />
to body size, condition factor and sex from southern<br />
Punjab, Pakistan<br />
Muhammad Yousaf*, Abdus Salam and Muhammad Naeem<br />
Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan 60800, Pakistan.<br />
Accepted 3 February, 2011<br />
Wallago attu is one of the large freshwater catfish found in Pakistan. The rapid growth and high<br />
nutritional quality encouraged investigation into the aquaculture potential of this excellent food fish. It<br />
was observed that body size had a positive influence on percent ash, percent fat and percent protein<br />
contents (wet weight) but there was no significant effect on percent water content. The condition factor<br />
had a positive correlation with fat, ash and protein contents (%wet weight) but no influence on percent<br />
water content. There was no significant influence of sex on body composition of W. attu. If it is<br />
impossible to find out the water content, then the body constituents can be estimated from the wet<br />
body weight, total length and condition factor of this species. As the variations in body composition are<br />
related to these variables, so the equations of each constituent were estimated. The predictive<br />
equations can be used to estimate the body composition with a fair level of accuracy.<br />
Key words: Wallago attu, body size, condition factor, body composition, sex.<br />
INTRODUCTION<br />
Wallago attu is one of the large freshwater catfish found<br />
in Pakistan, India, Sri Lanka, Nepal, Bangladesh, Burma,<br />
Thailand, Vietnam, Kampuchea, Malay Peninsula,<br />
Afghanistan, Sumatra and Java (Talwar and Jhingran,<br />
1991; Giri et al., 2002). The rapid growth (Goswami and<br />
Devraj, 1992) and high nutritional quality of its flesh<br />
(Lilabati and Viswanath, 1996) encourage investigation<br />
into the aquaculture potential of this excellent food fish.<br />
Taking into consideration the various health risks, fish’s<br />
mineral and body composition and their health status<br />
were assessed in order to establish the safety level of the<br />
table sized species prior consumption (Fawole et al.,<br />
2007). Body composition illustrates the nutritional quality<br />
of food because analysis of biochemical composition<br />
including protein, fat and ash is very important in<br />
assessing food value (Kamal et al., 2007). So, biochemical<br />
evaluation is necessary to ensure the nutritional<br />
value as well as eating quality fish (Azam et al., 2004).<br />
However, the value of these body constituents vary<br />
*Corresponding author: E-mail: mahyousaf@hotmail.com.<br />
significantly from one species and one individual fish to<br />
another depending on age, sex, feeding season,<br />
sampling time, activity and environmental condition<br />
(Weatherley and Gill, 1987; Jobling, 1994; Tang et al.,<br />
2009). The aim of the present study is to examine<br />
changes in the proximate composition in relation to body<br />
weight, length, condition factor and sex. Predictive<br />
equations are developed to describe these relationships<br />
in wild W. attu.<br />
MATERIALS AND METHODS<br />
Seventy-eight, wild W. attu of different body sizes, ranging from<br />
16.7-50.2 cm total length and 14.54-648.82 g body weight, were<br />
obtained from different localities of Indus River Southern Punjab<br />
using a cast net and were transported live to the Institute of Pure<br />
and Applied Biology in plastic containers. On arrival at the<br />
laboratory, fresh fish were washed with tap water several times to<br />
remove adhering blood and slime. They were anaesthetized with<br />
Tricaine Methanesulfonate (MS 222), weighed to nearest 0.01 g on<br />
an electronic digital balance (MP-3000 Chyo, Japan) and their<br />
length measured to the nearest 0.1 cm on wooden measuring tray.<br />
These fish were placed in a pre-weighed aluminum foil tray in an<br />
electric oven (Memmert ® 8540) at 70°C until a constant weight
4266 Afr. J. Biotechnol.<br />
Table 1. Wet body weight (g) versus body constituents of Wallago attu.<br />
Body constituents (%) r a b S.E. (b) t value when b = 0<br />
Water contents 0.010 76.112 -0.0001 0.002 -0.05<br />
Ash contents (wet weight) 0.120 3.224 0.001 0.0003 1.667<br />
Fat contents (wet weight) 0.448*** 3.570 0.003 0.001 4.143<br />
Protein contents (wet weight) 0.265** 15.035 0.003 0.001 2.462<br />
Statistical parameters of various relationships, correlation coefficient (r), intercept (a), regression coefficient (b),<br />
standard error of b (S.E.) and n = 78 in each case.<br />
Significance level: **P
Table 3.Condition factor (K) versus body constituents of Wallago attu.<br />
Body constituent (%) r a b S.E. (b) t value when b = 0<br />
Water contents 0.009 74.601 1.189 15.424 0.077<br />
Ash contents (wet weight) 0.108 3.027 0.679 0.720 0.943<br />
Fat contents (wet weight) 0.434*** 1.146 6.743 1.605 4.203<br />
Protein contents (wet weight) 0.117 14.166 3.410 3.324 1.026<br />
Yousaf et al. 4267<br />
Statistical parameters of various relationships, correlation coefficient (r), intercept (a), regression coefficient (b), standard<br />
error of b (S.E.) and n = 78 in each case.<br />
Significance level: ***P
4268 Afr. J. Biotechnol.<br />
375.<br />
Kamal D, Khan AN, Rahman MA, Ahamed F (2007). Biochemical<br />
composition of some small indigenous fresh water fishes from the<br />
river Mouri, Khulna, Bangladesh. Pak. J. Biol. Sci. 10(9): 1559-1561.<br />
LeCren ED (1951). The length-weight relationship and seasonal cycle in<br />
gonad weight and condition in the perch (Perca fluviatilis). J. Anim.<br />
Ecol. 20: 201-219.<br />
Lilabati H, Viswanath W (1996). Nutritional quality of freshwater catfish<br />
(Wallago attu) available in India. Food Chem. 57: 197-199.<br />
Love RM (1980). The chemical biology of fishes. Vol II. <strong>Academic</strong><br />
press, London.<br />
McComish TS, Anderson RO, Goff FG (1974). Estimation of Bluegill<br />
Lepomis macrochirus proximate composition with regression models.<br />
J. Fish. Res. Bd. Can. 31: 1250-1254.<br />
Memid D, Celikkale MS, Ercan E (2006). Effects of different diets on<br />
growth performance and body composition of Russian sturgeon<br />
(Acipenser gueldenstaedtii, Brandt & Ratzenburg, 1833). J. Appl.<br />
Ichthyol. 22(1): 287-290.<br />
Salam A, Davies PMC (1994). Body composition of northern pike (Esox<br />
lucius L) in relation to body size and condition factor. Fish. Res. 19:<br />
193-204.<br />
Salam A, Ali M, Anas M (2001). Body composition of Oreochromis<br />
nilotica in relation to body size and condition factor. Pak. J. Res. Sci.<br />
12(1): 19-23.<br />
Talwar PK, Jhingran AG (1991). Inland fishes of India and adjacent<br />
countries. Volume 2. A.A. Balkema, Rotterdam.<br />
Tang H, Chen L, Xiao C, Wu T (2009). Fatty acid profiles of muscle<br />
from large yellow croaker (Pseudosciaena crocea R.) of different age.<br />
J. Zhejiang Univ. Sci. B. 10(2): 154-158.<br />
Weatherley AH, Gill HS (1987). The biology of fish growth. <strong>Academic</strong><br />
Press. London. pp. 1-443.<br />
Wootton RJ (1990). Ecology of Teleost Fishes. Chapman and Hall,<br />
London<br />
Wootton RJ (1998). Ecology of Teleosts fishes 2 nd Dordrecht: Kluwer.<br />
Zar JH (1996). Biostatistical Analysis. Prentice-Hall. New Jersey.
African Journal of Biotechnology Vol. 10(20), pp. 4269-4275, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB09.786<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Rhizopus stolonifer exhibits dimorphism<br />
C. O. Omoifo<br />
Department of Crop Science, Ambrose Alli University, Ekpoma, Edo State, Nigeria. E-mail: coomoifo@yahoo.com.<br />
Accepted 4 March, 2011<br />
This study showed that multiple morphologies could be induced from sporangiospores of Rhizopus<br />
stolonifer in minimal medium. These included moniliform hyphae, septate hyphae and terminal budding<br />
yeast cells. When considered along the normal coenocytic filamentous growth form, the study showed<br />
that R. stolonifer could be polymorphic.<br />
Key words: Rhizopus stolonifer, polymorphic, multiple morphologies.<br />
INTRODUCTION<br />
Rhizopus stolonifer, a Zygomycete has a filamentous<br />
growth habit. Its filaments are coenocytic, that is, they are<br />
non-septate. It is the only fungus yet known to produce<br />
rhizoids which penetrate the substratum in order to obtain<br />
nutrients. The rhizoids also serve as support. Opposite<br />
the rhizoids, a sporangiophore juts into the atmosphere<br />
and this terminates in a club-shaped collumelum<br />
enclosed within the sporangial wall. Between the collumelum<br />
and wall are numerous asexual reproductive<br />
structures known as sporangiospores. This organism is<br />
also characterized by the presence of stolons, which<br />
connect rhizoid joints.<br />
Other members of the Zygomycetes, especially species<br />
of the genus Mucor, have been shown to undergo fungal<br />
dimorphism (Bartnicki-Garcia and Nickerson, 1962a, b, c,<br />
d; Friedenthal et al., 1974; Schulz et al., 1974; Ruiz-<br />
Hereira, 1985; McIntyre et al., 2002; Lubberhusen et al.,<br />
2003). In such phase transitions, sporangiospores convert<br />
to yeast like cells, with a central globose mother cell<br />
which multilaterally produces daughter buds by blastic<br />
action. This occurs under CO2 tension or high hexose<br />
concentration.<br />
However, terminal budding yeast cells have been<br />
induced from sporangiospores of Mucor circinelloides<br />
Tieghem in minimal medium (Omoifo, 2005). When the<br />
external medium was subjected to K + variation, the 1.0 g/l<br />
K + supplementation was found to be critical for protoplast<br />
formation, a transient morphology, but Na + was required<br />
for yeast induction. Thus, a treatment of 1.0 g/l K + , 0.10<br />
g/l Na + gave optimum yeast induction; even though thallic<br />
growth forms, including holoblastic-, holothallic- and<br />
enterothallic conidia were also induced, uracil supplementation<br />
drastically diminished the presence of these<br />
thallic forms (Omoifo, 2006a). This suggested that these<br />
ions impacted on the structural modification led to the<br />
induction of yeast cells from sporangiospores. A follow-up<br />
study showed that the incorporation of myoinositol and<br />
zinc into the minimal medium of growth of the<br />
microorganism enhanced yeast induction by more than<br />
63%. These studies further demonstrated that environmental<br />
factors influence mould-yeast interconversion.<br />
In the study of Bartnicki-Garcia and Nickerson (1962b),<br />
several members of the Mucurales were tested for their<br />
ability to undergo mould-yeast interconversion, but R.<br />
stolonifer was found to be incapable of it. Their study was<br />
conducted in solid cultures of complex nature (yeast<br />
extract-peptone-glucose, YPG) and incubated under 30%<br />
CO2 pressure. This claim in minimal medial composition<br />
that is known to induce terminal budding yeast cells from<br />
M. circinelloides Tieghem was validated or otherwise<br />
disproved (Omoifo, 2005, 2006a, b).<br />
It was also found that Mucor species could only convert<br />
to multipolar budding yeastlike cells under CO2 atmosphere<br />
when there is fermentable carbon substrate like<br />
galactose, fructose, mannose, glucose and disaccharide<br />
like sucrose; when it was grown with xylose as substrate,<br />
intact sporangiospores remained in the broth after 72 h<br />
(Bartnicki-Garcia and Nickerson, 1962b).<br />
On the other hand, McIntyre et al. (2002) cultivated M.<br />
circinelloides in xylose substrate-Vogel’s minimal medium<br />
which was incubated in CO2 atmosphere and the<br />
morphological expression was multipolar budding yeast<br />
like cells. Lubberhussen et al. (2003) used similar<br />
medium (Vogel’s) with xylose or glucose as substrate for<br />
the cultivation of M. circinelloides and obtained filamenttous<br />
growth in cultures sparge under the atmosphere.<br />
Since preliminary study showed that R. stolonifer could<br />
utilize glucose for growth, this study examined the effect
4270 Afr. J. Biotechnol.<br />
of the pentose sugar, xylose, on its habit.<br />
Reported here is the induction of several morphological<br />
forms of R. stolonifer in minimal medium.<br />
MATERIALS AND METHODS<br />
Fungal strain<br />
The strain of R. stolonifer used in this study was a gift from Miss<br />
Elsa Thomas, the Project Assistant to Dr. G. S. Prasad of Microbial<br />
Type Culture Collection 1, Institute of Microbial Technology, Sector<br />
39A, Chandigarh, India. It was purified through three different<br />
inoculations and subculturing and maintained as malt extract (MEA,<br />
31.2 g/L) solid cultures which exhibited filamentous growth habit. A<br />
fresh culture was prepared after seven days.<br />
Inoculum preparation for growth<br />
Inoculum was obtained by pouring sterile deionized distilled water<br />
over aerobic growth and a sterile glass rod was gently passed over<br />
the surface so as to dislodge the spores. The suspension was not<br />
subjected to extreme preparation as was done in the method of<br />
Omoifo (1996, 2003, 2006a).<br />
This was to ensure that some mycelia fragments were retained in<br />
the inoculum. This showed to be so on examination under the<br />
microscope (x400 magnification). But generally, spore count which<br />
was taken with Neubauer Haemocytometer. (BSS No. 784<br />
Hawksley, London Vol. 1/4000) was adjusted to 10 x 10 5 spores per<br />
ml in sterile deionized distilled water, with the aid of tally counter.<br />
Reagent and culture media<br />
This has been described (Omoifo, 1996, 2006a) and subsequently<br />
modified. Briefly, media were prepared per litre: Xylose (Hymedia),<br />
10 g; (NH4)2SO4 (Merck), 5 g; K2HPO4 (Qualigens), 5 g; KH2PO4<br />
(Merck), 5 g; MgSO4 (Qualigens), 2 g; FeS04 (Hymedia), 0.1 g;<br />
NaCl (Qualigens), 0.1 g; CuSO4 (Analytical Rasayan), 0.06 g;<br />
MnSO4 (Qualigens), 0.065 g; ZnSO4 (BDH), 0.06 g; CaCl2 (Sigma)<br />
uracil (Hymedia), 100 mg; myoinositol (Sigma), 0.25 g.<br />
The media where prepared in 2000 ml beaker. Weights of buffer<br />
components 0.2 M Na2HPO4: 0.1 M citrate were obtained using<br />
H54Ar meter balance and added to the beaker. The pH was<br />
adjusted to 4.5 with 2 N NaOH or 1 N HCl using a control dynamics<br />
pH meter model APX 175 E/C in the 2000 ml beaker before<br />
dispensing 80 ml of broth in each duplicate of 250 ml Erlenmeyer<br />
flasks for the test. The solution in each flask was made up to 100 ml<br />
with glass distilled deionized water and sterilized at 121°C per 15<br />
min.<br />
Inoculation, growth conditions and sample collection<br />
A 1 ml spore suspension was drawn and inoculated into each broth<br />
flask using Eppendorf micropipette. Each culture flask was then<br />
shaken for 30 s and thereafter incubated at 28 ± 1°C (laboratory<br />
temperature). Culture flasks were examined after 72 and 168 h of<br />
growth.<br />
The culture flasks were returned for further incubation. Slides<br />
were prepared for viewing by putting one or two drops of<br />
lactophenol on a glass slide with the aid of wire loop, one drop from<br />
the culture flask properly shaken was then added and covered with<br />
a glass cover slip and viewed under a binocular microscope at x<br />
1000 magnification using the microscope Olympus BX51 attached<br />
with camera model No. C-3250. The observed morphologies were<br />
recorded.<br />
RESULTS<br />
Visual examination showed the emergence of mycelia on<br />
the surface of growth medium. This appeared white to<br />
grey on broth surface but aerial mycelia were grey.<br />
The culture was cloudy and sediments were formed by<br />
day 3. After one week, aerial mycelia became profuse<br />
and thick sediment was formed at the bottom of the<br />
culture. Microscopic examination revealed that, aerial<br />
mycelia originated from broth level mycelia made up of<br />
rhizoids and stolons. Figure 1a showed a section of a<br />
long sporangiophore, which is vertically opposite the<br />
rhizoids that penetrate into the broth as well as a side<br />
(horizontal) branch, which is a section of the stolon<br />
interconnecting two rhizoid joints. Shown in Figure 1b is<br />
globose sporangium enclosing numerous sporangiospores;<br />
within it also is the collumelum which is the terminal<br />
end of the characteristically long sporangiophore.<br />
Figure 1c showed collapsed sporangia that had released<br />
their content of spores. Sporangiospores are grey to dark<br />
brown in colour, have striated walls and vary in shape<br />
(Figure 1d).<br />
Three different types of hyphae were formed within the<br />
broth. These were tan to light brown coenocytic hyphae<br />
(Figure 2c), dark brown septate hyphae (Figure 2s) and<br />
moniliform hyphae which has narrow diameter and simple<br />
septa-like partitions which have no cell wall material<br />
deposition (Figure 2m).<br />
On the other hand, yeast cells which were profusely<br />
produced, were terminal budding and assumed varying<br />
shapes. They could be globose, subglobose, variations of<br />
cylindrical and allantoidal cells (Figure 2a and y). Yeast<br />
formation also occurred by moniliform budding whereby<br />
the growing tip enlarges; a constriction occurs behind the<br />
expanded apex which assumes the yeast shape. It could<br />
be globose, subglobose or ellipsoidal. This eventually<br />
detaches from the hyphae into the medium where it<br />
continues to proliferate by terminal budding (Figure 3a<br />
and b).<br />
DISCUSSION<br />
Several growth forms were observed in submerged<br />
cultures in this study. These included coenocytic hyphae,<br />
septate hyphae, moniliform hyphae and terminal budding<br />
yeast cells, which was preponderant. Therefore, R.<br />
stolonifer is polymorphic in minimal medium.<br />
The study of Bartnicki-Garcia and Nickerson (1962b)<br />
showed that R. stolonifer could not convert to yeast-like<br />
morphology under carbon dioxide pressure, unlike the<br />
Mucor rouxii used in their study. In the aforementioned<br />
study, it was shown that M. rouxii proliferate by multipolar<br />
budding, whereby a yeast mother cell gave rise to<br />
numerous multipolar daughter buds by blastic action.
Figure 1. Substrate-level and aerial mycelia of Rhizopus stolonifer showing (a) part of<br />
stolon; (b) part of sporangiophore; (c) rhizoids; (d) sporangium; (e) collapsed sporangia<br />
and (f) striated sporangiospores.<br />
This is in contrast with the results obtained in the<br />
present study whereby R. stolonifer exhibited different<br />
growth forms in minimal medium. A weight proportion for<br />
each morphological type in submerged culture was not<br />
determined. Subjective assessment however showed that<br />
the terminal budding yeast morphology was the dominant<br />
form.<br />
Although, nuclei were not visualized in this study, it<br />
should be pointed out that dimorphic M. rouxii produces<br />
yeast like cells which are multipolar budding and the<br />
mother cell and daughter buds, each contain numerous<br />
nuclei, as revealed by Giemsa staining (Bartnicki-Garcia<br />
and Nickerson, 1962b) or 6-diamidino 2-phenylidole<br />
(DAPI) staining (McIntyre et al., 2002; Lubberhussen et<br />
Omoifo 4271<br />
al., 2003), whereas terminal budding yeast cell like that of<br />
Saccharomyces cerevisiae, harbours one nucleus per<br />
cell. Thus, after conversion to the yeast cell from sporangiospore<br />
or after intermediary stage as in moniliform<br />
hypha, proliferation of R. stolonifer was in the manner<br />
similar to the budding pattern of yeast cells of S.<br />
cerevisiae.<br />
It does not appear that formation of septa by<br />
Zygomycetous species in minimal medium has been<br />
previously reported. In the study of McIntyre et al. (2002),<br />
the use of calcofluor white revealed the presence of<br />
septa in filaments formed by M. circinelloides. But in that<br />
case, it was due to the formation of arthrospores that<br />
occurred after the growth and ceased (Lubberhussen et
4272 Afr. J. Biotechnol.<br />
Figure 2. Submerged mycelia of R. stolonifer showing (c) coenocytic hyphae; (m) moniliform<br />
hyphae; (s) septate hypha; (c) terminal budding yeast cells and (a) subglobose, ellipsoidal and<br />
allantoid yeast cells, which may be 1 to 3 celled.<br />
al., 2003). In the present study, arthrospores were not<br />
observed. Septation is a characteristic of the higher fungi<br />
where it compartmentalizes cellular components. Since<br />
septate hyphae were observed in submerged culture in<br />
this study, it thus means that R. stolonifer could exhibit<br />
characteristics of the higher fungi. As this study showed,<br />
it also applies to the formation of terminal budding yeast<br />
cells.<br />
Formation of terminal budding yeast cell took two<br />
different lines. These include induction from (a)<br />
sporangiospore and (b) moniliform hypha. The process of<br />
induction of terminal budding yeast cells from<br />
sporangiospores has been previously observed (Omoifo,<br />
2003, 2005).<br />
Swollen and then lysed sporangiospore, released<br />
nucleates into medium of growth. These gave rise to<br />
protoplasts which subsequently became yeast cells,<br />
thereafter, proliferation by terminal bud formation<br />
occurred. Omoifo (2006a, b) demonstrated the effect of<br />
myoinositol and zinc on the induction and subsequent<br />
proliferation of terminal budding yeast cells from sporangiospores<br />
of M. circinelloides Tieghem in a system thought<br />
to be of high ionic strength that possibly led to the<br />
establishment of a pH-transmembrane gradient; such<br />
would stimulate second messengers generation and<br />
signals transduction within the living cell (Dawes, 1986;<br />
Mitchell, 1978).<br />
It was hypothesized that, activity at the molecular level<br />
activated mitogenic factors that led to yeast form<br />
induction (Omoifo, 2005). Thereafter, it could assume<br />
that terminal yeast budding observed here is in the<br />
manner of proliferation of S. cerevisiae, that is, daughter<br />
bud production by blastic action whereby the inner wall of<br />
the mother cell extends into the new bud, and this entails<br />
the extension of the existing cell wall (Marchant and<br />
Smith, 1968; Sentandreu and Northcote, 1969).<br />
As shown in this study, formation of an intermediary<br />
structure, similar to that elaborated for the initial stages of<br />
formation of sporogenous hyphae was involved in<br />
moniliform yeast induction (Omoifo, 2005). It was shown<br />
that sporogenous hyphae emerges from sporangiospore<br />
as coenocytic thin walled vegetative filament which<br />
possesses thin lumen that becomes compartmentalized;<br />
with the centripetal growth of the plasma membrane thus<br />
forming septa-like partition (without the involvement of<br />
cell wall). A morphologically distinct conidium is produced<br />
at the distal end of this filament, and this holoblastically<br />
produced catenate conidia (Omoifo, 2005). In the present<br />
study, the growing tip of moniliform filament formed yeast<br />
mother cell instead, and this on detachment becomes<br />
terminal budding. This method of primary bud formation<br />
by R. stolonifer contrasts with that described for
Figure 3. Yeast cells (y) and moniliform hyphae (m) of R. stolonifer budding off yeast cells; (a) young<br />
bud; (b) mature bud near detachment. The moniliform hypha may have simple septa-like partition, (i).<br />
Phialophora dermatitidis whereby there is bud eruption<br />
from hyphal wall; bud wall is continuous with inner hyphal<br />
wall, whereas the outer wall forms a collar (Grove et al.,<br />
1973). In contrast too, is the study of Hardcastle and<br />
Szaniszlo (1974) that showed that moniliform hyphae<br />
emerged from induced yeast cells of Cladosporium<br />
Omoifo 4273<br />
werneckii but these produced lateral conidia, which on<br />
release did not exhibit any form of budding.<br />
In the present study, terminal budding yeast cells were<br />
induced from sporangiospores in two different ways, viz<br />
(a) sporangiospore to nucleates to protoplasts to yeast<br />
cells; (b) sporangiospore to moniliform hypha to yeast
4274 Afr. J. Biotechnol.<br />
cell. This study confirmed earlier reports which showed<br />
that terminal budding yeast cells can be induced from<br />
sporangiospores (Omoifo, 1996, 1997, 2003, 2006a, b;<br />
Omoifo et al., 2006). For biochemical and molecular<br />
studies to be conducted successfully, pure cultures of the<br />
morphotypes or anamorphs of any particular fungus is<br />
required. The achievement of this objective has been<br />
demonstrated in several studies (Omoifo, 2003, 2005,<br />
2006a, b). This is further confirmed in the foregoing<br />
report.<br />
ACKNOWLEDGEMENTS<br />
This work was done at the MTCC-1, Institute of Microbial<br />
Technology, Chandigarh, India during the Postdoctoral<br />
fellowship programme supported by the Third World<br />
Academy of Sciences, Triestie, Italy and the Council for<br />
Scientific and Industrial Research, India. To both<br />
institutions, I express my sincere gratitude.<br />
REFERENCES<br />
Bartnicki-Garcia S, Nickerson WJ (1962a). Induction of yeast-like<br />
development in Mucor by carbon dioxide. J. Bacteriol. 84: 829- 840.<br />
Bartnicki-Garcia S, Nickerson WJ (1962b). Nutrition, growth and<br />
morphogenesis of Mucor rouxii. J. Bacteriol. 84: 841-858.<br />
Bartnicki-Garcia S, Nickerson WJ (1962c). Isolation, composition and<br />
structure of cell walls of filamentous and yeast-like forms of Mucor<br />
rouxii. Biochim. Biophys. Acta. 58: 102-119.<br />
Bartnicki-Garcia S, Nickerson WJ (1962d). Assimilation of carbon<br />
dioxide and morphogenesis of Mucor rouxii. 64: 584-551.<br />
Dawes EA (1986). Microbial Energetics: Tertiary Level Biology<br />
Chapman and Hall, New York, p. 189.<br />
Friedenthal JW Epstein A, Passon S (1974). Effect of potassium<br />
cyanide, glucose and anaerobiosis on morphogenesis of Mucor<br />
rouxii. J. Gen. Microbiol. 82: 15-24.<br />
Grove SN, Oujezdsky KB, Szaniszlo PJ (1973). Budding in the<br />
Dimorphic Fungus Phialophora dermatitidis. J. Bacteriol. 115(1): 323-<br />
329.<br />
Hardcastle RV, Szaniszlo PJ (1974). Characterization of dimorphism in<br />
Cladosporium werneckii. J. Bacteriol. 119: 294-302.<br />
Lubberhusen TL, Nielsen T, McIntyre M (2003). Characterization of the<br />
Mucor circinelloides life cycle by on-line image analysis. J. Appl.<br />
Microbiol. 95: 1152-1160.<br />
Marchant R, Smith DG (1968). Bud formation in Saccharomyces<br />
cerevisiae and a comparison with the mechanism of cell division in<br />
other yeasts. J. Gen. Microbiol. 53: 163-169.<br />
McIntyre M, Breum J, Arnau J, Nielsen J (2002). Growth physiology and<br />
dimorphism of Mucor circinelloides (synracemosus) during<br />
submerged batch cultivation. Appl. Microbiol. Biotechnol. 58: 495-<br />
502.<br />
Mitchell P (1978). Translocation through natural membranes. Adv.<br />
Enzymol. 29: 33-87.<br />
Omoifo CO (1996). Modeling sporangiospore-yeast transformation of<br />
Dimorphonyces strain. Hind. Antibiot. Bull. 38: 12-31.<br />
Omoifo CO (1997). Auxotrophic requirement for sproangiospore -yeast<br />
transformation of Dimorphomyces diastaticus Strain C12. Hind.<br />
Antibiot. Bull. 39: 11-15.<br />
Omoifo CO (2003). Sequential sporangiosopore-yeast transformation<br />
hypothesis. Afr. Scientist. 4: 191-273.<br />
Omoifo CO (2005). Development of Yeast Cells from Sporangiospores.<br />
Idehuan Publishing Company, Nigeria, p. 402.<br />
Omoifo CO (2006a). Effect of K+, Na+ and uracil on sporangiosporeyeast<br />
transformation of Mucor circinelloides Tieghe. Afr. J.<br />
Biotechnol. 5(9): 715-722.<br />
Omoifo CO (2006b). Effect of myoinositol and zinc on sporangiosporeyeast<br />
transformation of Mucor circinelloides Tieghe Afr. J. Biotechnol.<br />
5(9): 723-730.<br />
Omoifo CO, Aruna MB, Omamor IB (2006). Effect of myoinositol on<br />
sporangiospore-yeast transformation of Mucor circinelloides Tieghem<br />
cultivated in synthetic broth. Hind. Antibiot. Bull. 47-48: 24-30.<br />
Ruiz-Hereira J (1985). Dimorphism in Mucor species with emphasis on<br />
M. Rouxii and M. Baciliformis. In: Szaniszlo PJ (ed). Fungal<br />
Dimorphism With Emphasis on Fungi Pathogenic for Humans, New<br />
York. pp. 361-385.<br />
Schulz BE, Kraepelin G, Hinkelmann W (1974). Factors affecting<br />
dimorphism in Mycophyta (Mucorales): a correlation with the<br />
fermentation/respiration equilibrium. J. Gen. Microbiol. 83: 1-13.<br />
Sentandreu R, Northcote DH (1969). The formation of buds in yeast. J.<br />
Gen. Microbiol. 55: 393-398.
African Journal of Biotechnology Vol. 10(20), pp. 4276-4288, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB09.787<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Effect of extracellular calcium chloride on<br />
sporangiospore-yeast transformation of Rhizopus<br />
stolonifer<br />
C. O. Omoifo<br />
Department of Crop Science, Ambrose Alli University, Ekpoma, Nigeria. E-mail: coomoifo@yahoo.com.<br />
Accepted 4 March, 2011<br />
Previous studies showed that zinc and myoinositol, which are intracellular, serve as components of<br />
signalling elements in many eukaryotes, and participate in stimulating the induction and proliferation of<br />
yeast cells from sporangiospores of fungi. It was thought that a transmembrane-pH-gradient, similar to<br />
what is obtained in bacterial mitochondrion or proton-substrate symport in yeasts, permitted<br />
influx/efflux of materials into the cell that triggered the induction and subsequent proliferation of yeast<br />
cells. To examine this model further, this study evaluated the ability of sporangiospores of Rhizopus<br />
stolonifer to undergo morphogenetic transformation in the presence of different levels of extracellular<br />
calcium (0.0, 0.20, 0.25, 0.50, 1.0, 1.5 and 1.8 mM). It was found that calcium supported yeast induction<br />
and proliferation to varying extent. Ca 2+ at 0.50 and1.8 mM, which was outstanding, was compared with<br />
broth treatment at 0.25 mM ZnSO4: pH 4.5 which gave optimum biomass in a previous study. Medium<br />
pH was further varied: pH 4.2, 4.5 and 5.0. The results showed that there was interaction between the<br />
level of divalent cations and pH. At all levels tested, Ca 2+ was better stimulatory than Zn 2+ . Direct<br />
induced yeast cell count, which eliminated possible impact of protoplasts and calcium phosphate<br />
precipitate on optical density measurements, made these results more valid. Treatment at 0.50 mM Ca 2+ :<br />
pH 5.0 gave the most profound result. This was therefore chosen for further biochemical work.<br />
Key words: Rhizopus stolonifer, synthetic broth, dimorphism, extracellular Ca 2+ , sporangiospores, induced<br />
yeast cells.<br />
INTRODUCTION<br />
Dimorphism is the conversion of a microorganism from<br />
one growth habit to another, an effect caused by changes<br />
in environmental conditions (Romano, 1966). In a report<br />
presented over a decade ago, it was shown that<br />
sporangiospores of a mucoraceous strain is transformed<br />
to terminal budding yeast cells (Omoifo, 1996). This was<br />
opposite the multipolar budding yeast-like morphology<br />
that has been reported for many mucoraceous species<br />
including Mucor rouxii (Bartnicki-Garcia, 1968; Bartnicki-<br />
Garcia and Nickerson, 1962a, b, c, d; Lubberhusen et al.,<br />
2003; McIntyre et al., 2002), Mycotypha (Hall and<br />
Kolankaya, 1974; Schulz et al., 1974; Price et al., 1973).<br />
The preceding report showed that several distinct<br />
morphologies can be induced from the fungus, Rhizopus<br />
stolonifer. These include yeast cells, coenocytic hyphae,<br />
moniliform hyphae and septate hyphae. This is in<br />
contrast to the multipolar budding yeast-like cells that<br />
have been reported for other members of the<br />
Zygomycetes. It is also in sharp contrast to the study of<br />
Bartnicki-Garcia and Nickerson (1962b) whereby it was<br />
shown that R. stolonifer was incapable of morphogenetic<br />
interconversion.<br />
Conversion of sporangiospores to terminal budding<br />
yeast cells require structural modifications that involved<br />
transient morphologies, including growth spheres and<br />
cytosolic nucleates that are released when cell wall is<br />
lysed, and protoplasts till the yeast is formed. This<br />
becomes the stable form, which is proliferated by terminal<br />
budding. These various forms have been shown in<br />
several publications (Omoifo, 1996, 1997, 2003; Omoifo<br />
and Omamor, 2005; Omoifo et al., 2006) including the<br />
research monograph “development of yeast cells from<br />
sporangiospores” (Omoifo, 2005).<br />
In an attempt to explain the cryptic biophysical changes<br />
that occurred leading to the formation of yeast, a system<br />
model known as the sequential sporangiospore-yeast
transformation (SSYT) hypothesis was formulated<br />
(Omoifo, 2003). This was based on electrochemical<br />
gradient which permits transport processes and energy<br />
metabolism in mitochondria or proton-substrate symport<br />
through plasma membranes (Alderman and Hofer, 1981;<br />
Albert et al., 1994; Dawes, 1986; Mitchell, 1967; Slayman<br />
and Slayman, 1974; Voet and Voet, 1995; West and<br />
Mitchell, 1972; 1973). It holds that the multiple conversions<br />
are possible because the individual entities are<br />
bathed in a milieu whereby a transmembrane-pHgradient<br />
is set up and this encourages influx/efflux of<br />
materials with consequent fundamental biochemical and<br />
biophysical changes in cytosolic components and cell<br />
wall integrity. It was shown that monovalent elements like<br />
H + , K + and Na + , play important roles in the induction of<br />
terminal budding yeast cells from sporangiospores of<br />
muco-raceous strains. H + was involved in the<br />
maintenance of the transmembrane-pH-gradient; K + was<br />
absolutely necessary for the formation of protoplasts from<br />
spora-ngiospores of Mucor circinelloides Tieghem<br />
(Omoifo, 2005). On the other hand, accumulation of Na + ,<br />
which is antiported with K + (Tonomura, 1986) was<br />
necessary for the protoplasts to convert to the yeast form<br />
(Omoifo, 2005). The effect of K + was confirmed in another<br />
study where protoplast formation peaked at a high level<br />
of K + 1.0 g/l, and yeast induction was optimal at 0.10 g/l<br />
Na + . In a combinational manner under various<br />
treatments, the interaction between 1.0 g/l K+ and 0.1 g/l<br />
Na + had the greatest impact on yeast induction,<br />
notwithstanding the occurrence of other anamorphic<br />
subtypes including enterothallic and holothallic conidia,<br />
which were scanty. However, these anamorphs were<br />
eliminated when 100 mM uracil was incorporated into the<br />
medium of growth (Omoifo, 2006a).<br />
Subsequent study showed the effect of intracellular<br />
signalling precursors on yeast induction from sporrangiospores.<br />
Although, myoinositol and zinc were not the<br />
primary initiators, they individually enhanced yeast<br />
induction from sporangiospores (Omoifo and Aruna,<br />
2006; Omoifo et al., 2006). When the cyclic hexitol and<br />
divalent ion were used in combination, induced yeast<br />
sigmoid growth pattern ensued and biomass was upped<br />
by 63 to 65% in comparison with control cultures<br />
(Omoifo, 2006b). Calcium is a primary element in signal<br />
transduction in eukaryotic cells where it’s binding to<br />
proteins triggers changes in conformation as well as<br />
altering local electrostatic fields, thereby controlling<br />
cellular processes (Kretsinger, 1979; Albert et al., 1994;<br />
Berridge, 1987; Berridge et al., 2000; Voet and Voet,<br />
1995; Clapham, 2007). Since zinc which plays key role in<br />
catalytic and structural component of many proteins<br />
(Eide, 2006) has been shown to enhance yeast induction<br />
from sporangiospores, perhaps calcium, which functions<br />
as an intracellular messenger (Albert et al., 1994) and<br />
further involved in cytosolic Ca 2+ homeostasis (Forster<br />
and Kane, 2000; Ton and Rao, 2004), would also<br />
promote yeast induction from sporangiospores.<br />
Omoifo 4277<br />
The objective of this study was to evaluate the effect of<br />
CaCl2 on the ability of R. stolonifer to convert to terminal<br />
budding yeast cells. Since exogenous culture pH<br />
influence Ca 2+ uptake through Ca 2+ channels in plasma<br />
membrane (Youatt, 1990), the effect of pH on the<br />
stimulatory ability was also evaluated.<br />
MATERIALS AND METHODS<br />
Chemicals<br />
Reagent grade chemicals were obtained from BDH Laboratory<br />
supplies (Poole, UK) except Bacto peptone (Sigma), myoinositol<br />
(Sigma) and yeast extract (Oxoid).<br />
Fungal strain<br />
R. stolonifer used in this study was isolated at the Microbial Type<br />
Culture Collection Laboratory 1, Institute of Microbial Technology,<br />
Chandigarh India as reported in the preceding report. It was<br />
maintained on glucose-yeast extract-peptone agar (GYPA: 10-0.3-<br />
5.0-15g/l) slants. Fresh culture was prepared on Petri dish unto<br />
which 20 ml of agar was poured and used after 7days of growth.<br />
Inoculum preparation for growth studies<br />
A 7 day old culture of R. stolonifer was flushed with sterile deionized<br />
distilled water and a sterile glass rod was gently passed over<br />
the fungal growth so as to dislodge the spores. The suspension,<br />
poured into centrifuge bottles, was washed with 3 changes of sterile<br />
deionized distilled water in an MSE 18 centrifuge at 5000 rpm for 7<br />
min at 25°C. Spore count was taken with a Neubauer hemocytometer<br />
and was made up to 10 6 /ml in sterile deionized distilled<br />
water.<br />
Reagents and culture media<br />
Procedure for preparing the base medium has been described<br />
(Omoifo, 1996) and has been used in subsequent studies (Omoifo,<br />
2003, 2006a, b; Omoifo and Aruna, 2005; Omoifo et al., 2006). It<br />
contained per litre: 10 g glucose, 5 g (NH4)2SO4, 5 g K2HPO4, 5 g<br />
KH2PO4, 0.10 g NaCl, 0.10 g FeSO4, 2.0 g MgSO4, 0.060 g CuSO4,<br />
0.060 g ZnSO4, 0.065 g MnCl2, 100 mg uracil and 2 Mm<br />
myoinositol. Where the effect of Zn 2+ was to be tested, zinc<br />
sulphate was not added to the basal preparation. The initial culture<br />
was buffered at Ph 4.5 with 0.2 M Na2HPO4: 0.1 M citrate. When Ph<br />
level was the test factor, culture was then buffered with equivalent<br />
weights of the buffer species. Since levels of calcium were to be<br />
tested, these were prepared separately. Each of the duplicate<br />
flasks of the synthetic broth was incorporated with the various<br />
concentrations of calcium chloride at the following levels: 0.0, 0.20,<br />
0.25, 0.50, 1.0, 1.5 and 1.8 Mm. The Ph was adjusted with 2 N<br />
NaOH or 1 N HCl using a Cole-Parmer Ph Tester model 59000.<br />
The solution in each flask was made up to 100 ml with glass<br />
distilled deionized water and sterilized at 121°C for 15 min.<br />
Inoculation, growth conditions and sample collection<br />
A 1 ml spore suspension was inoculated into each broth flask using<br />
Eppendorf micropipette in an operation done in a laminar flow<br />
chamber model CRC HSB-60-180. Before each inoculation, the
4278 Afr. J. Biotechnol.<br />
Table 1. Treatments and biomass estimates with their respective standard errors, and form of growth<br />
during the cultivation of R. stolonifer in synthetic broth for 120 h at Ph 4.5 and temperature 28°C.<br />
Treatment: Calcium chloride (Mm) Mean biomass at 620 nm Form of growth<br />
Control 0.1399 ± 0.0075 Yeast cell<br />
0.2 0.1361 ± 0.0062 Yeast cell<br />
0.25 0.1462 ± 0.0107 Yeast cell<br />
0.5 0.1666 ± 0.0095 Yeast cell<br />
1.0 0.1381 ± 0.0153 Yeast cell<br />
1.5 0.1360 ± 0.0086 Yeast cell<br />
1.8 0.1854 ± 0.0418, turbidity Yeast cell<br />
spores were kept in suspension by shaking for 30 s and the broths<br />
were similarly shaken after each inoculation before transferring to a<br />
laboratory side bench for incubation at 28 ± 1°C. At 24 h intervals,<br />
the culture flasks were brought to the chamber and 10 ml were<br />
withdrawn from each culture flask with sterile pipettes, one for each<br />
flask, into factory-sterilized plastic sample tubes. The culture flasks<br />
were thereafter returned for further incubation. The samples were<br />
kept at -18°C until further analysis. Slides were prepared for<br />
viewing by putting one or two drops of lactophenol-in-cotton blue on<br />
a glass slide and with the aid of wire loop, one drop from the culture<br />
flask properly shaken was then added and covered with a glass<br />
cover slip and viewed under a binocular microscope at x1000<br />
magnification using the microscope Olympus CHB14. The observed<br />
morphologies were recorded.<br />
Biomass determination<br />
Culture samples were thawed up to room temperature before<br />
biomass determination. The absorbance was obtained at 620 nm,<br />
using a Camspec M105 spectrophotometer (Cambridge, UK).<br />
Statistical analysis<br />
Results were subjected to a 2-way analysis of variance (ANOVA)<br />
test for the single factor, or a split-plot format for combined factors<br />
and considered significant if p < 0.05 and comparison between<br />
means was performed using the Genstat 5 package.<br />
RESULTS AND DISCUSSION<br />
The effect of various levels of Ca 2+ on morphological<br />
expression of R. stolonifer cultivated in still cultures is<br />
shown in Table 1. Yeast cell was the predominant<br />
morphology. In other dimorphic fungi, Ceratocystyis ulmi<br />
(Paranjape et al., 1990) and Ophiostoma ulmi (Gadd and<br />
Brunton, 1992), exogenous Ca 2+ stimulated germ tube<br />
formation in media designated for yeast production, while<br />
it led to a reduction of mycelia production in<br />
Aureobasidium pullulans (Madi et al., 1997). Madi and<br />
colleagues attributed this difference to the use of<br />
‘restrictive medium’ in the former, as compared to their<br />
use of what they termed ‘general medium’ for the growth<br />
of A. pullulans. The preceding report illustrates the fact<br />
that multiple morphologies could be induced from<br />
sporangiospores in buffered synthetic broth, but this<br />
could be streamlined to obtain specific morphology,<br />
depending on the environmental factor incorporated into<br />
the medium of growth. For example, multipolar budding<br />
yeast like cell was obtained in complex nitrogen sourced<br />
medium incubated at elevated temperature (Omoifo,<br />
1996, 2005) and terminal budding yeast cells from simple<br />
nitrogen sourced-medium (Omoifo, 1996), or myoinositolincorporated<br />
medium (Omoifo, 1997, 2003, 2005; Omoifo<br />
et al., 2006).<br />
In the present study, proliferation of terminal budding<br />
yeast cells induced from sporangiospores of R. stolonifer<br />
occurred at all concentration levels tested. Growth profile<br />
did not describe a sigmoid pattern (Figure 1). Mean<br />
biomass showed differences in growth between cultures<br />
where control broth apparently had higher values than<br />
0.2, 1.0 and 1.5 mM Ca 2+ (Table 1). Analysis of variance<br />
of O.D. readings showed that only time had significant<br />
impact on yeast induction and growth (Table 2). However,<br />
on separation of means, LSD 0.04889 p < 0.05, the<br />
aforementioned concentration levels were not distinct<br />
except for 0.5 and 1.8 mM Ca 2+ , which were in separate<br />
subsets (Table 3).<br />
In this study, control culture or cultures with low level<br />
Ca 2+ made had no difference with yeast proliferation,<br />
except when there was high level challenge, 0.5 or 1.8<br />
mM Ca 2+ . But the magnitude of yeast cells formed in the<br />
1.5 mM Ca 2+ supplemented broth compares with that of<br />
control although protoplasts were part of entities in the<br />
latter culture. This study decided to subject the Ca 2+<br />
levels with the higher O.D. values to different pH<br />
treatments.<br />
The effect of extracellular pH levels on sporangiosporeyeast<br />
transformation had earlier been shown (Omoifo,<br />
1996). In synthetic broth, pH 4.5 was found to be most<br />
ideal for conversion of sporangiospores to yeast cells<br />
(Omoifo, 1996). A subsequent report showed that<br />
buffered synthetic broth pH 4.5, supplemented with 0.25<br />
mM ZnSO4 further enhanced conversion of sporangiospore<br />
of M. circinelloides to yeast (Omoifo, 2006a).<br />
In the present study, Zn 2+ was contrasted with Ca 2+ under<br />
the optimum conditions previously determined but also<br />
varied this with a ≤0.5 unit pH differences in either<br />
direction. Cumulative mean values showed that the<br />
highest biomass was obtained at 0.5 mM Ca 2+
Figure 1. Biomass profiles of R. stolonifer cultivated in buffered synthetic broth supplemented with different levels<br />
of calcium chloride for 120 h at pH 4.5 and temperature of 20°C, ambient.<br />
Omoifo 4279
4280 Afr. J. Biotechnol.<br />
Table 2. Analysis of variance of optical density readings (620 nm) of induced yeast<br />
cells of R. stolonifer.<br />
Source of variation DF SS MS F-pr.<br />
Concentration 6 0.043517 0.007253 0.316<br />
Time 4 0.068225 0.017056 0.029*<br />
Concentration x time 24 0.187666 0.007819 0.192<br />
Residual 105 0.638335 0.006079<br />
Total 139 0.937743<br />
*Significant at p < 0.05 probability level; DF, degree of freedom; SS, sum of square, MS,<br />
mean sum; F-pr, frequency of probability.<br />
Table 3. Homogenous subsets of mean biomass estimates<br />
obtained during sporangiospore-yeast transformation of R.<br />
stolonifer cultivated in synthetic broth for 120 h with pH 4.5<br />
at 28°C, ambient.<br />
Treatment : Calcium chloride (mM) Mean<br />
Subset 1<br />
1.8 0.1854<br />
Subset 2<br />
0.5 0.1676<br />
Subset 3<br />
Control 0.1399<br />
0.2 0.1361<br />
0.25 0.1462<br />
1.0 0.1381<br />
1.5 0.1360<br />
Means separation based on L.S.D. 0.04889 at p < 0.05.<br />
supplementation but was the least with the 0.25 mM Zn 2+<br />
challenge which was barely lower than 1.8 mM Ca 2+<br />
insult (Table 4). This indicated that Ca 2+ ion at 0.5 mM<br />
concentration was more effective in promoting yeast<br />
induction/proliferation.<br />
To know the contribution of the various factors<br />
incorporated into the growth medium, a split-plot analysis<br />
of variance of growth data was done. It was shown that<br />
the main factors including ionic concentration, pH and<br />
time, as well as their interactions made significant impact<br />
(p < 0.1) on growth of R. stolonifer as yeast cells (Table<br />
5). The specific contribution of pH levels to yeast proliferation<br />
is shown in Figure 2. The pH influenced yeast<br />
proliferation. At 0.5 mM Ca 2+ concentration where<br />
optimum growth occurred, maximum growth was at pH<br />
4.5, whereas it was at pH 5.0 when the 1.8 mM Ca 2+<br />
concentration (which had lower optimum values than the<br />
0.5 mM Ca 2+ concentration) was used. While the least<br />
biomass was recorded at 0.25 mM Zn 2+ concentration,<br />
maximum growth with this ion also occurred at pH 5.0.<br />
But at pH4.2 where the least growth occurred for all the<br />
ionic concentrations, the 0.25 mM Zn 2+ had higher growth<br />
value than the 1.8 mM Ca 2+ concentration. While growth<br />
was compromised at pH 4.2 in the 1.8 mM Ca 2+ culture,<br />
the pressure was relieved as the external pH rise. This<br />
indicated that at the cytosolic level, there was interaction<br />
between the type of ion and pH on the one hand, and the<br />
ionic concentration and pH level on the other. A<br />
comparison of biomass of the Ca 2+ challenge at all the pH<br />
levels tested suggested that yeast induction and proliferation<br />
is more sensitive to higher level Ca 2+ insult.<br />
Since there is interest in conditions where determinable<br />
growth phases (lag, log and stationary) occurred, a timecourse<br />
pattern should illustrate this. Induced proliferating<br />
yeast cells of R. stolonifer assumed sigmoid curve with<br />
0.25 mM Zn 2+ supplementation at the lower pH values,<br />
whereas at pH 5.0 where the biomass was higher (Figure<br />
2), there appeared to be a resurgence of growth after the<br />
initial rapid log growth and wind-down 96 h from<br />
commencement of experiment (Figure 3a). The occurrence<br />
of sigmoid growth of yeast cells induced from<br />
sporangiospores of M. circinelloides cultivated in 0.25<br />
mM Zn 2+ supplemented pH 4.5 minimal cultures has been<br />
previously reported (Omoifo, 2006a). In that study, other
Optical density at 620<br />
Table 4. Mean biomass of induced yeast cells from sporangiospores of R.<br />
stolonifer cultivated in buffered synthetic broth at different levels of<br />
exogenously applied Ca 2+ and Zn 2+ ions.<br />
Parameter<br />
Optical density at 620 nm<br />
0.25 mM ZnSO4 0.50 mM CaCl2 1.80 mM CaCl2<br />
Mean 0.3795 0.8252 0.3941<br />
S.E. 0.003 0.010 0.006<br />
Fold-increment 2.2 2.1<br />
Table 5. Analysis of variance of growth data of R. stolonifer cultivated in buffered synthetic<br />
broth at different pH levels and exogenously applied ions.<br />
Source of variation d.f. Sum of square F-value<br />
Concentration 2 7.693008
4282 Afr. J. Biotechnol.<br />
Figure 3. Time-course pattern in the growth of induced yeast<br />
cells of R. stolonifer at different combinations of ions and pH<br />
levels.<br />
overcame inherent repression and assumed proliferation,<br />
and hence the resurgence of growth prior to termination<br />
of experiments. In contrast, in the 0.5 mM Ca 2+ challenge,<br />
a sigmoid growth pattern was described in the buffered<br />
pH 5.0 culture. However, a resurgence of growth occurred<br />
after sigmoid pattern when the cultures were buffered<br />
at the lower levels of pH (Figure 3b). Perhaps similar<br />
explanation for this phenomenon stated earlier is here<br />
forth attributable. Growth profiles at the 1.8 mM Ca 2+<br />
concentration showed greater variability from the sigmoid<br />
pattern (Figure 3c). Growth had extended lag: 96 h at pH<br />
4.2 and 72 h at pH 4.5; but it was very short at pH 5.0
Table 6. Overall yield of yeast cells induced from sporangiospores of R. stolonifer<br />
cultivated in buffered synthetic broth at different levels of exogenously applied Ca 2+<br />
and Zn 2+ ions.<br />
Parameter<br />
Unicellular count (x10 6 )<br />
0.25 mM ZnSO4 0.50 mM CaCl2 1.80 mM CaCl2<br />
Mean 8.99 87.31 53.56<br />
Standard error 0.149 1.629 1.64<br />
Fold-increment 9.71 1.63<br />
Comparing tables 4 and 6, the difference that exists between 0.25 mM ZnSO4 and 0.5<br />
mM CaCl2 on one hand and 0.5 mM CaCl2 and 1.8 mM CaCl2 on the other are also<br />
reflected in the direct cell count data, although the magnitude vary. As the CaCl2 level<br />
increases, growth of induced yeast cells is suppressed.<br />
where a resurgence of growth also occurred after 96 h.<br />
The higher profile description at pH 5.0 is a reflection of<br />
the magnitude of growth shown in Figure 2 and shows<br />
similarity with the pattern in the 0.25 mM Zn 2+ supplementation<br />
(Figure 3a) although its magnitude was greater<br />
(Figure 2). Microscopic examination revealed that<br />
morphological expression in the pH 5.0 buffered cultures<br />
was protoplast and yeast cell, except when the ionic<br />
concentration was 0.5 mM Ca 2+ , which was of mainly<br />
terminal budding yeast cell.<br />
In all experiments, except with control and 0.25 mM<br />
Zn 2+ supplementation, turbidity appeared to occur probably<br />
resulting from precipitation of calcium phosphate<br />
and this appeared to increase with Ca 2+ levels. Similar<br />
observation was made in the study which showed the<br />
effect of exogenous Ca 2+ on the growth of Aureobasidium<br />
pullulans (Madi et al., 1997). In order to validate the<br />
findings of this study, especially where sigmoid curves<br />
were obtained for optical density readings, direct cell<br />
count was do. This would eliminate any discrepancy in<br />
O.D. readings that could arise from calcium phosphate<br />
precipitation.<br />
Unicellular population size in the 0.5 mM Ca 2+ culture<br />
was 9.7-fold that of the 0.25 mM Zn 2+ , while it was 1.6fold<br />
the magnitude of the 1.8 mM Ca 2+ broth (Table 6). In<br />
contrast, when biomass estimation was done through<br />
measurement of O.D., the value of 0.5 mM Ca 2+ broth<br />
was 2.2-fold that of 0.25 mM Zn 2+ and 2.1-fold the<br />
magnitude in 1.8 mM Ca 2+ culture. Earlier experiment had<br />
shown that protoplast, cytosolic units that is, nucleates in<br />
earlier studies, are released when cell wall lysed, and<br />
thallic subtypes and conidia were induced in Zn 2+<br />
supplemented broths (Omoifo, 2006a). However, thalli<br />
were absent in the presence of 100 mM uracil (Omoifo,<br />
2006b). In the present study, protoplasts and yeast cells<br />
contributed to the O.D. readings in the Zn 2+ supplemented<br />
medium, whereas only yeast cells were herein<br />
counted. Calcium phosphate precipitated only in Ca 2+<br />
supplemented media. Thus, the contributors to the O.D.<br />
readings included the induced cellular entities and<br />
calcium phosphate precipitate. Since the value differential<br />
between the 1.8 and 0.5 mM Ca 2+ estimate was larger, as<br />
Omoifo 4283<br />
reflected in the greater than 2-fold increase when O.D.<br />
was used in biomass assessment in contrast to the use<br />
of direct unicellular count (less than 2-fold increase), it<br />
was difficult to assume that calcium phosphate precipitation<br />
was the sole cause of the reduction in the biomass<br />
estimate in the 1.8 mM Ca 2+ supplemented medium.<br />
Perhaps, the high Ca 2+ concentration suppressed yeast<br />
cell induction and proliferation.<br />
This was probably confirmed at pH 5.0 where yeast<br />
count in the 1.8 mM Ca 2+ supplementation was significantly<br />
less than that of 0.5 mM Ca 2+ (Figure 4).<br />
Furthermore, the 1.8 mM Ca 2+ medium contained terminal<br />
budding yeast cells, unbudded yeast cells as well as<br />
protoplasts, whereas 0.5 mM Ca 2+ broth contained<br />
terminal budding yeast cells only. Apparently, excess<br />
Ca 2+ influenced the ability of protoplasts to convert to<br />
yeast form on the one hand, and of unicellular yeast form<br />
to undergo mitogenesis and or proliferation on the other.<br />
Similar trend, higher values for 0.5 mM Ca 2+ as against<br />
1.8 mM Ca 2+ , occurred at pH 4.2 or 4.5 and further<br />
supports the results obtained when optical density was<br />
used for biomass estimation (Figure 2). These results<br />
illustrate the effect of interaction between pH level and<br />
exogenous Ca 2+ supply.<br />
To further test all such interactions, an analysis of<br />
variance of induced yeast count data was done (Table 7).<br />
There was significant (p < 0.001) difference for the<br />
induction and proliferation because of treatments, indicating<br />
the existence of variability in the transformation<br />
process.<br />
The existence of variation in the treatments is in agreement<br />
with such findings when optical density was the<br />
measure of biomass, as shown earlier. This also agrees<br />
with the finding of Omoifo (2006a) when M. circinelloides<br />
Tieghem was the test microorganism and Zn 2+ and<br />
myoinositol were incorporated into the growth medium. In<br />
the present study, the mean performances of treatments<br />
are presented in Table 8.<br />
Of the treatments, the least induced yeast cell count<br />
was obtained in the 0.25 mM ZnSO4: pH 4.2 medium and<br />
progressively increased with pH until the optimum for this<br />
ion in 0.25 mM ZnSO4: pH 5.0 broth. The 0.5 mM Ca 2+
4284 Afr. J. Biotechnol.<br />
Ind uced yeast cell count (1 x 10 6 )<br />
0.25 mM ZnSO4<br />
0.5 mM CaCl2<br />
1.8 mM CaCl2<br />
Figure 4. Microscopic cell count of induced yeast cells of R. stolonifer in Ca 2+ and Zn 2+ incorporated buffered<br />
synthetic broth as affected by different levels of pH.<br />
Table 7. Treatment and induced cell estimates with their respective standard errors and form<br />
of growth during the cultivation of R. stolonifer in synthetic broth for 120 h at temp28°C.<br />
Treatment Mean cell count (x10 6 ) Form of growth*<br />
0.25 mM ZnSO4: pH4.2 4.94±2.04 Y,P<br />
0.25 mM ZnSO4: pH4.5 9.68±4.47 Y,P<br />
0.25 mM ZnSO4: pH5.0 12.42±4.83 Y,P<br />
0.50 mM CaCl2: pH4.2 56.60±29.87 Y, P<br />
0.50 mM CaCl2: pH4.5 71.02±33.99 Y<br />
0.50 mM CaCl2: pH5.0 134.08±62.67 Y<br />
1.80 mM CaCl2: pH4.2 24.24±14.21 Y, P<br />
1.80 mM CaCl2: pH4.5 41.38±19.61 Y, P<br />
1.80 mM CaCl2: pH5.0 80.15±35.84 Y, P<br />
*Y, yeast cell; P, protoplast.<br />
concentration on average had higher growth value. The<br />
buffered pH 4.2 culture had mean count of 56.60 ± 29.87<br />
and this increased as the medium pH until the maximum<br />
at 134.08 ± 62.27 at pH 5.0. Comparatively, 0.5 mM<br />
CaCl2 supported higher level of growth than 1.8 mM<br />
CaCl2. The increase in growth value also followed the<br />
same trend as those at the other levels. Therefore, the<br />
results of treatment means confirmed the influence of<br />
extracellular pH on the induction from, and subsequent<br />
proliferation of yeasts from sporangios pores of R.<br />
stolonifer.<br />
Figure 5 shows yeast proliferation profiles in the different
Table 8. Analysis of variance of induced yeast cell count of R. stolonifer cultivated in<br />
buffered synthetic broth with different pH levels and exogenously applied ions.<br />
Source of variation Degree of freedom Sum of square F-value<br />
Concentration 2 184113.5
4286 Afr. J. Biotechnol.<br />
Figure 5. Time-course pattern in induced yeast cell count of R.<br />
stolonifer at different combinations of ions and pH levels. Growth<br />
pattern was sigmoid in the buffered pH 4.5 synthetic broths<br />
when Ca2+ was exogenously supplied, and at pH 4.2and 5.0,<br />
growth was still at the logarithmic phase when the experiments<br />
were terminated. Growth magnitude was reduced at pH 4.2.<br />
Table 9. Paired t-test on total unicellular count in media incorporated with Ca 2+ or<br />
Zn 2+<br />
Ionic concentration Mean Variance N<br />
0.5 mM Ca 2+ 9.31567E7 1.05997E16 60<br />
0.25 mM Zn 2+ 8.10667E6 6.52549E13 60<br />
t = 6.87839, p = 4.34432E-9. At the 0.05 level, the two means are significantly different
Log10 (number of induced yeast cells)<br />
Log10 (number of induced yeast cells)<br />
Time (h)<br />
Time (h)<br />
0.5 mM CaCl2 pH 4.2<br />
0.5 mM CaCl2 pH 4.5<br />
0.5 mM CaCl2 pH 5.0<br />
1.8 mM CaCl2 pH 4.2<br />
1.8 mM CaCl2 pH 4.5<br />
1.8 mM CaCl2 pH 5.0<br />
Figure 6. Log transformed induced yeast cell count of R. stolonifer in cultures where sigmoid growth was exhibited<br />
Table 10. Lag phase and specific growth rate in cultures where sigmoid growth pattern was<br />
exhibited.<br />
Extracellular concentration 0.5 mM Ca 2+ 1.8 mM Ca 2+<br />
Extracellular pH 4.2 4.5 5.0 4.2 4.5 5.0<br />
Lag phase (h) 24 24 24 48 48 48<br />
µ 0.025 0.026 0.029 0.037 0.039 0.031<br />
The longer lag possibly means that growth was repressed at that concentration/pH level resulting in lack<br />
of cell number increase. Conceivably, the eventual increase in cell number may then have been due to<br />
a minority of cells in the medium not initially suppressed, which could continue to grow and divide. In<br />
this situation, the lag would represent the time required for the subpopulation of cells not repressed to<br />
overgrow the arrested population. Alternatively, the cells could overcome the repressive effect of Ca 2+,<br />
by genetic adaptation and subsequently proliferate, thus achieving higher specific growth rate.<br />
Omoifo 4287
4288 Afr. J. Biotechnol.<br />
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Energy-transducing ATPase-structure and kinetics, Cambridge<br />
University Press, London, pp. 240-281.<br />
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and Sons Inc. p. 1390<br />
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in non-metabolizing Escherichia coli. J. Bioenerget. 3: 445-453<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4289-4295, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1198<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Genetic variation and bottleneck in Japanese quail<br />
(Coturnix japonica) strains using twelve microsatellite<br />
markers<br />
Hossein Emrani 1 *, Cyrus Amirinia 1 and Mohammad Ali Radjaee Arbabe 2<br />
1 Department of Biotechnology, Animal Science Research Institution of Iran (ASRI), Karaj, Iran.<br />
2 Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.<br />
Accepted 28 January, 2011<br />
The genetic structure of four strains of Japanese quail (Pharach, Panda, Tuxedo and Golden) was<br />
investigated by 12 microsatellite markers in Iran. Whole blood samples were collected from 200<br />
individuals belonging to four strains and genomic DNA was extracted by salting out procedure. The 12<br />
microsatellite markers were amplified through polymerase chain reaction (PCR). The results indicated<br />
that the average heterozygosity between strains ranged from 0.4343 to 0.7902. The Chi-square and<br />
likelihood ratio test performed to examine strains for Hardy-Weinberg equilibrium showed some highly<br />
significant deviations from deficiency. FIS value, which indicates the degree of departure from random<br />
mating, was particularly high in the four strains when compared to that of other breeds, indicating<br />
heterozygosity deficiency. Maximum Nei’s genetic distance was observed between Tuxedo and Golden<br />
strains; whereas the minimum Nei’s genetic distance was observed between Pharach and Tuxedo<br />
strains. Tuxedo strain revealed bottleneck event under three models of microsatellite evolution for sign,<br />
standardized differences and Wilcoxon sign rank tests. The power of the microsatellite marker as a<br />
useful tool for evaluating genetic variation within and between Japanese quail strains was also noted.<br />
Key word: Bottleneck, genetic distance, genetic variation, microsatellite, Japanese quail.<br />
INTRODUCTION<br />
The Japanese quail, originally domesticated around the<br />
11th century as a pet song bird (Howes, 1964; Crawford,<br />
1990), is valued for its eggs and meat. It is also a<br />
valuable laboratory species because of its small body<br />
size, rapid generation interval and high prolificacy (Mills<br />
et al., 1997). Japanese quail is phylogenetically closely<br />
related to the chicken (Stock and Bunch, 1982). Both<br />
species have a karyotype of 2n = 78 chromosomes and a<br />
similar genome length of 1.2 × 10 9 bp, consisting of<br />
morphologically distinct macrochromosomes (1–8 and<br />
the ZW sex chromosomes) and cytologically indistinguishable<br />
microchromosomes (Shibusawa et al., 2001).<br />
*Corresponding author. E-mail: h_emrani@asri.ir. Tel: +98-261-<br />
4430010-14 (465). Fax: +98-261-4413258.<br />
Abbreviations: PIC, Polymorphism information content; PCR,<br />
polymerase chain reaction; HWE, Hardy-Weinberg equilibrium.<br />
Thus, the Japanese quail has been recommended as a<br />
model species for poultry (Baumgartner, 1994; Mills et<br />
al., 1997; Kayang et al., 2004). Recently, 100 microsatellite<br />
markers were developed for Japanese quail<br />
(Kayang et al., 2000, 2002) and used to build the first<br />
microsatellite linkage map, which spans 576 cM and<br />
contains 58 loci assigned to 12 linkage groups (Kayang<br />
et al., 2004). Microsatellites are valuable genetic markers<br />
due to their dense distribution in the genome, great<br />
variation, co-dominant inheritance and easy genotyping.<br />
They were extensively used in parentage testing, linkage<br />
analyses, population genetics and bottleneck studies<br />
(Goldstein and Pollock, 1997).<br />
Genetic diversity in populations and the evolutionary<br />
forces that affect it are central to both evolutionary<br />
(Wright, 1931) and conservation biology (Frankham,<br />
1995b). When populations undergo temporary large<br />
reductions in size of so-called population bottlenecks (Nei<br />
et al., 1975), they lose genetic diversity through random<br />
drift. Identifying populations that have experienced a
4290 Afr. J. Biotechnol.<br />
severe reduction in size (a bottleneck) is important<br />
because bottlenecks can increase demographic stochasticity,<br />
rates of inbreeding, loss of genetic variation and<br />
fixation of mildly deleterious alleles, thereby reducing<br />
evolutionary potential and increasing the probability of<br />
population extinction (Frankel and Soule, 1981;<br />
Frankham, 1995a, b; Rall et al., 1988; Bryant et al., 1986;<br />
Goodnight, 1987; Luikart et al., 1998). In this study, the<br />
bottleneck and genetic diversity within and among four<br />
strains of Japanese quail was analyzed (Panda, Pharach,<br />
Golden and Tuxedo) by 12 microsatellite markers as<br />
recom-mended by Kayang et al. (2002).<br />
MATERIALS AND METHODS<br />
Sampling and DNA extraction<br />
Japanese quail strains, reared at Bonab Research Center,<br />
Northwest of Iran, were used as the experimental animals. These<br />
strains were reared for 17 generations and were phenotypically<br />
selected for high weight gain. Any new individual has not been<br />
introduced to the strains.<br />
Whole blood samples were randomly collected from 200<br />
individual belonging to four strains: 70 individuals from Pharach<br />
strain, 40 individuals from Panda strain, 50 individuals from Tuxedo<br />
strain and 40 individuals from Golden strain. About 200 µl of blood<br />
per individual was collected in 0.5 mM EDTA (pH 8). Genomic DNA<br />
was extracted by the salting out procedure (Miller et al., 1988).<br />
Microsatellite primers<br />
12 microsatellites with high polymorphism information content (PIC)<br />
value, indicating the informative of the markers, recommended by<br />
Kayang et al. (2002) were used in this study. The primers were<br />
encoded by GUJ0001, GUJ0021, GUJ0023, GUJ0034, GUJ0041,<br />
GUJ0049, GUJ0052, GUJ0055, GUJ0059, GUJ0070, GUJ0097<br />
and GUJ0099 characters. Primers were synthesized by TIBMO-<br />
LBIOL Company, Germany.<br />
PCR amplification<br />
Genomic DNA was amplified by PCR containing 50 ng of template<br />
DNA, 2.5 µl of 10 x buffer (10 Mm Tris, 50 mM Kcl, 0.1% gelatin,<br />
pH 8.4), 1.5 to 2.0 mM MgCl2 (as optimized for each marker), 200<br />
µM of each dNTPs , 1 µl of 5 pmol/µl each for forward and reverse<br />
primers and 1 u of Taq DNA polymerase; ddH2O was added to the<br />
volume of 25 µl. Reactions were carried out on a thermal cycler<br />
(Biometra) using an initial 2.5 min denaturation at 95°C, followed by<br />
30 cycles of denaturation at 95°C for 1 min, annealing at 50 to 62°C<br />
for 30 s, extension at 72°C for 30 s, and a final extension step at<br />
72°C for 5 min. The PCR products were electrophoresed on 8%<br />
denatured urea-polyacrylamide gel and bands visualized by rapid<br />
silver staining method (Sanguinetti et al., 1994). The gel was<br />
photographed using Gel-Doc XR (BioRad). Patterns of the different<br />
genotypes for each microsatellite locus were analyzed using Gel-<br />
Pro analyzer, version 3.1 for windows TM, which determines the<br />
allele's sizes in each animal.<br />
Statistical analysis<br />
Allelic frequencies were estimated using genotype counting. Hardy-<br />
Weinberg equilibrium (HWE) based on likelihood ratio were tested<br />
for different locus-population combinations by POPGENE software<br />
Version 1.31 (Yeh et al., 1999). Nei’s (1972) standard genetic<br />
distance and Reynolds genetic distance (Reynolds et al., 1983)<br />
matrices were calculated by tools for population genetic analysis<br />
(TFPGA) software version 1.3 (Miller, 1997) and dendrograms were<br />
constructed using unweighted pair-group method with arithmetic<br />
mean (UPGMA) by TFPGA software (Miller, 1997) with 1000<br />
bootstrap replications. Polymorphism criteria such as PIC<br />
(Botestein et al., 1980) and the number of observed and effective<br />
alleles (Hedrick, 1999) were also estimated by HET software<br />
version 1.8 (Ott, 2001) and POPGENE software packages, respectively<br />
with assumption of Hardy-Weinberg equilibrium. Average<br />
expected heterozygosity was calculated by POPGENE software<br />
(Yeh et al., 1999).<br />
To accomplish the goal of finding evidence of fluctuations in<br />
population sizes, bottleneck software package was used (Cornuet<br />
and Luikart, 1996) to detect deviations from mutation drift<br />
equilibrium. This method consisted three excess heterozygosity<br />
tests developed by Cornuet and Luikart (1996): (i) sign test, (ii)<br />
standardized differences test and (iii) a Wilcoxon sign-rank test. The<br />
probability distribution was established using 1000 simulations<br />
under three models: infinite allele model (IAM), stepwise mutation<br />
model (SMM) and two-phase model of mutation (TPM) by using<br />
Bottleneck v1.2.02 Software (http://www.ensam.inra.fr/URLB).<br />
RESULTS<br />
All loci were polymorphic in the four Japanese quail<br />
strains, except GUJ0001 and GUJ0041, which were<br />
monomorphic in the Panda and Tuxedo, respectively.<br />
The number of alleles per locus varied from 9 (GUJ0059)<br />
to 3 (GUJ0001, GUJ0021 and GUJ0041). New alleles<br />
were found in several loci that have not been previously<br />
reported in the same loci for other Japanese quail by<br />
Kayang et al. (2002). Significant deviation from HWE in<br />
all population-locus combinations were observed except<br />
for GUJ0041 in Pharach strain, GUJ0023, GUJ0099,<br />
GUJ0021, GUJ0034, GUJ0041 and GUJ0097 in Panda<br />
strain and GUJ0034, GUJ0055, GUJ0049 and GUJ0070<br />
in Golden strain (P < 0.05). Effective number of alleles,<br />
PIC, observed heterozygosity and expected heterozygosity<br />
are given in Table 1. All the expected heterozygosities<br />
were larger than the observed heterozygosities,<br />
except for GUJ0097 in Tuxedo.<br />
Nei’s (1972) standard genetic distance (Da), corrected<br />
for bias, due to sampling of individual, and Reynolds<br />
genetic distance (D Reynolds) matrices (Reynolds et al.,<br />
1983) are reported in Table 2.<br />
The expected heterozygosity (HExp) ranged from 0.841<br />
(GUJ0070) in the Pharach strain to 0.486 (GUJ0052) in<br />
the Golden strain. Polymorphism criteria such as PIC<br />
values and number of alleles indicate high polymorphism<br />
at some of the loci. The PIC is a good standard for<br />
evaluating genetic markers. The highest and the lowest<br />
PIC values belonged to GUJ0059 in Golden (0.815) and<br />
GUJ0041 in Panda strain (0.427), respectively. Effective<br />
number of alleles is a reciprocal of gene homozygosity<br />
(Hartl and Clark, 1989). The highest and the least<br />
effective numbers of alleles were in GUJ0059 locus<br />
(6.04) in Pharach strain and in GUJ0041 locus (1.91) in
Table 1. Statistical results of 12 loci of quail strains.<br />
Microsatellite<br />
primer<br />
GUJ0001<br />
GUJ0021<br />
GUJ0034<br />
GUJ0041<br />
GUJ0049<br />
GUJ0059<br />
GUJ0070<br />
GUJ0097<br />
Genetic parameter of<br />
microsatellite loci<br />
Sample size (n)<br />
Pharach 70 Panda 40<br />
Tuxedo<br />
50<br />
Emrani et al. 4291<br />
Golden 40<br />
NO 5 1 3 4<br />
Ne 3.37 1.00 2.95 3.07<br />
PIC 0.641 0.000 0.586 0.613<br />
HObs 0.230 0.000 0.000 0.000<br />
HExp 0.717 0.000 0.676 0.687<br />
FIS 0.672 0 1 1<br />
NO 6 3 5 5<br />
Ne 4.48 2.57 3.66 3.58<br />
PIC 0.751 0.535 0.680 0.677<br />
HObs 0.526 0.666 0.428 0.055<br />
HExp 0.798 0.666 0.727 0.730<br />
FIS 0.322 -0.091 0.410 0.922<br />
NO 5 5 5 7<br />
Ne 3.10 4.57 3.20 4.78<br />
PIC 0.627 0.745 0.636 0.761<br />
HObs 0.200 0.750 0.250 0.571<br />
HExp 0.690 0.883 0.709 0.805<br />
FIS 0.705 0.040 0.636 0.277<br />
NO 6 3 1 6<br />
Ne 2.65 1.91 1.00 2.75<br />
PIC 0.590 0.427 0.000 0.568<br />
HObs 0.321 0.250 0.000 0.250<br />
HExp 0.629 0.491 0.000 0.646<br />
FIS 0.484 0.475 0 0.601<br />
NO 6 6 5 7<br />
Ne 4.00 4.26 3.50 4.00<br />
PIC 0.715 0.735 0.668 0.715<br />
HObs 0.600 0.666 0.400 0.583<br />
HExp 0.769 0.787 0.733 0.766<br />
FIS 0.200 0.129 0.441 0.222<br />
NO 5 4 6 8<br />
Ne 3.27 3.57 4.96 6.03<br />
PIC 0.650 0.762 0.767 0.815<br />
HObs 0.333 0.000 0.250 0.071<br />
HExp 0.709 0.757 0.815 0.849<br />
FIS 0.520 1 0.687 0.914<br />
NO 7 5 6 7<br />
Ne 6.04 4.00 4.88 4.57<br />
PIC 0.813 0.707 0.765 0.750<br />
HObs 0.300 0.333 0.600 0.500<br />
HExp 0.841 0.782 0.815 0.797<br />
FIS 0.640 0.555 0.245 0.360<br />
NO 6 6 6 6<br />
Ne 4.84 3.66 4.76 3.52<br />
PIC 0.762 0.690 0.758 0.685<br />
HObs 0.500 0.625 0.846 0.647<br />
HExp 0.807 0.7500 0.805 0.727<br />
FIS 0.369 0.139 - 0.071 0.096
4292 Afr. J. Biotechnol.<br />
Table 1. Contd.<br />
GUJ0055<br />
GUJ0052<br />
GUJ0023<br />
GUJ0099<br />
NO 5 4 4 4<br />
Ne 4.458 3.446 3.188 3.173<br />
PIC 0.740 0.658 0.637 0.489<br />
HObs 0.45 0.555 0.307 0.411<br />
HExp 0.783 0.730 0.699 0.547<br />
FIS 0.399 - 0.217 0.552 0.237<br />
NO 5 4 5 4<br />
Ne 3.913 3.677 4.083 1.933<br />
PIC 0.701 0.685 0.712 0.563<br />
HObs 0.166 0.666 0.642 0.166<br />
HExp 0.750 0.755 0.768 0.486<br />
FIS 0.864 - 0.077 0.148 0.589<br />
NO 5 4 4 4<br />
Ne 3.161 2.976 3.881 2.919<br />
PIC 0.647 0.616 0.694 0.597<br />
HObs 0.414 0.375 0.500 0.235<br />
HExp 0.689 0.685 0.755 0.667<br />
FIS 0.357 0.435 0.326 0.687<br />
NO 5 4 3 4<br />
Ne 4.304 3.521 2.945 3.085<br />
PIC 0.729 0.662 0.586 0.625<br />
HObs 0.607 0.666 0.143 0.388<br />
HExp 0.774 0.736 0.672 0.685<br />
FIS 0.209 - 0.014 0.785 0.234<br />
NO, observed number of alleles; Ne, effective number of alleles; PIC, polymorphism information content; HObs , observed<br />
heterozygosity; HExp, expected heterozygosity; FIS , is the inbreeding coefficient.<br />
Table 2. Distance matrices estimated by Da (above diagonal) and DReynolds (below diagonal)<br />
distance using 1000 bootstrap replications.<br />
Strain Pharach Panda Tuxedo Golden<br />
Pharach *** 0.307 0.247 0.571<br />
Panda 0.050 *** 0.402 0.743<br />
Tuxedo 0.044 0.071 *** 0.794<br />
Golden 0.112 0.142 0.128 ***<br />
Panda strain, respectively (Table 1). FIS of the inbreeding<br />
coefficient, measure the relative heterozygote deficit and<br />
non-random mating in samples. Its value ranges between<br />
-1 (all individuals heterozygote), 0 (random association of<br />
alleles) and 1 (all individuals homozygote). If inbreeding<br />
is avoided, F = 0; negative F indices are usually from<br />
selection in favor of the heterozygotes, whereas positive<br />
values indicate that the considered population has an<br />
inbreeding system of mating (Liu et al., 2008). The mean<br />
values of FIS estimates obtained 0.45, 0.3, 0.47 and 0.46<br />
for Pharach, Panda, Tuxedo and Golden strains, respectively<br />
indicating the high level of inbreeding in the four<br />
strains. This could be as a result of a bottleneck effect.<br />
Bottlenecks influence the distribution of genetic variation<br />
within and among populations, thus the genetic effects of<br />
reductions in population size requires evaluation. To characterize<br />
it, sign, standardized differences and Wilcoxon<br />
sign rank tests were utilized. The values of average<br />
heterozygosity (He) and their probabilities (H > He) in the<br />
sign test, under three models of microsatellite evolution<br />
(IAM, SMM and TPM) were calculated and used to<br />
measure the expected number of loci with heterozygosity<br />
excess (Table 3). If the probability values for each model<br />
were less than 0.05, then null hypothesis was rejected,<br />
indicating that the bottleneck event occurred in this<br />
model. For example, the expected number of loci with<br />
heterozygosity excess in Pharach strain was 7.10 and<br />
7.15 for TPM and SMM, under null hypothesis in the sign<br />
test, respectively (Table 3). The probability values were,<br />
0.0749 and 0.0792, respectively, meaning that the null
Table 3. Test for null hypothesis testing under three microsatellite evaluation models, the IAM, SMM<br />
and TPM.<br />
Parameter IAM TPM SMM<br />
Sign test: number of loci with heterozygosity excess (probability)<br />
Pharach Expected = 7.01(0.0151) * 7.10 (0.0749) 7.15(0.0792)<br />
Panda Expected = 6.37(0.0024) * 6.46(0.0028) * 6.59 (0.1179)<br />
Tuxedo Expected = 6.32(0.0023) * 6.49(0.0030) * 6.54(0.0032) *<br />
Golden Expected = 7.03(0.0690) 7.11 (0.2095) 7.07 (0.3649)<br />
Standardized differences test (Ti values and probability)<br />
Pharach 3.272 (0.00053) * 2.077 (0.0189) * 0.131 (0.4480)<br />
Panda 4.204 (0.00001) * 3.435 (0.0003) * 2.497 (0.0062) *<br />
Tuxedo 4.998(0.00001) * 4.490(0.0000) * 3.729 (0.0001*)<br />
Golden 2.666 (0.0038) * 1.461 (0.0720) -0.441 (0.3295)<br />
Wilcox sign-rank test (probability of heterozygosity excess)<br />
Pharach 0.03418* 0.04248* 0.2334<br />
Panda 0.00049* 0.00049* 0.00342*<br />
Tuxedo 0.00049* 0.00049* 0.00049*<br />
Golden 0.00122* 0.05225 0.67725<br />
*Rejection of null hypothesis/bottleneck.<br />
hypothesis was accepted when using sign test. These<br />
results indicate that, due to mutation-drift equilibrium, the<br />
Pharach strain has not undergone a recent genetic bottleneck.<br />
However, the expected number of loci with<br />
heterozygosity excess was 7.01 for IAM with probability<br />
of 0.0151 and thus rejected the null hypothesis, indicating<br />
bottleneck under this model.<br />
The standardized difference test for Pharach strain<br />
provided the Ti (probability) statistics equal to 3.272<br />
(0.00053), 2.077 (0.0189) and 0.131 (0.4480) for the IAM,<br />
TPM and SMM models, respectively. The probability<br />
values were less than 0.05 for IAM and TPM, thus<br />
hypothesis of mutation-drift equilibrium was accepted<br />
under SMM only. Using the Wilcoxon rank test, the<br />
probability values were 0.03418 (IAM), 0.04248 (TPM)<br />
and 0.2334 (SMM) under the three models, indicating<br />
that the null hypothesis was accepted under SMM model.<br />
Among the used tests, Wilcoxon rank test and standard<br />
difference test showed bottleneck event in golden strain<br />
under IAM. However other P values tests were not<br />
significant, demonstrating that the null hypothesis of<br />
mutation–drift equilibrium was fulfilled in this strain. The<br />
results of all the three tests together showed that the<br />
bottleneck event did not occur in this strain. Golden strain<br />
is a wild strain that was collected from its natural niche<br />
and suffered few selective breeding programs. Results<br />
revealed bottleneck event in Tuxedo strain under three<br />
models for sign, standardized differences and Wilcoxon<br />
sign rank tests.<br />
DISCUSSION<br />
Emrani et al. 4293<br />
The Hardy-Weinberg equilibrium test showed several<br />
deviant loci in the four strains. There were many causes<br />
of disequilibrium such as small population, selection at or<br />
near the genomic locus, non-random mating, genetic drift<br />
and inbreeding. In addition, the possible occurrence of<br />
null alleles could have led to false observation of homozygotes<br />
which could have accounted for more deviations<br />
from HWE. The mean observed heterozygosity for these<br />
four strains were 0.387, 0.462, 0.364 and 0.325 for<br />
Pharach, Panda, Tuxedo and Golden, respectively. In all<br />
the four strains, the heterozygosity observed was lower<br />
than that reported for wild Japanese quail (0.66) (Chang<br />
et al., 2005). The average PIC of the 12 microsatellite loci<br />
in the four strains was close to the results of Kayang et<br />
al. (2002). Based on the classification of Botstein et al.<br />
(1980), PIC > 0.5 is highly informative, 0.25 < PIC < 0.5 is<br />
middle informative and PIC < 0.25 is slightly informative.<br />
In this study, all loci were highly informative (PIC > 0.5),<br />
except GUJOO41 (PIC = 0.427) in Panda strain. Reynolds<br />
distance were used to estimate pairwise genetic<br />
distance between the breed. This measure is recommended<br />
by Eding and laval (1999) for population with<br />
short divergence time. Maximum Nei’s genetic distance<br />
was observed between Tuxedo and Golden strains;<br />
whereas the minimum Nei’s genetic distance was<br />
observed between Pharach and Tuxedo strains. Both<br />
cluster resulted from Nei’s standard genetic distance and
4294 Afr. J. Biotechnol.<br />
a<br />
b<br />
Figure 1. UPGMA showing the genetic relationships among strains using<br />
Nei’s standard genetic distance (a) and Reynolds genetic distance (b).<br />
Reynolds genetic distance revealed two branches. One<br />
branch included Pharach, Tuxedo and Panda and<br />
another branch was consisted of Golden. Pharach and<br />
Tuxedo grouped closely (Figure 1). It was expected to<br />
find high genetic distance between Golden and other<br />
strains. Golden is the most prolific type among the<br />
Japanese quail strains. The mean values of FIS among<br />
the four strains were higher than that of the population<br />
studied by Kim et al. (2007), indicating a certain level of<br />
heterozygote deficiency. Significant heterozygote deficiencies<br />
have been reported in chicken breeds (Qu et al.,<br />
2006; Liu et al., 2008). The heterozygote deficiency (FIS ><br />
0) might be attributed to a number of factors, namely,<br />
sample relatedness, linkage with loci under selection<br />
(genetic hitchhiking), population heterogeneity, null<br />
alleles (non-amplifying alleles) and inbreeding<br />
(Shahsavarani and Rahimi-mianji, 2010). Null alleles<br />
were most unlikely segregated at all the loci. The most<br />
plausible explanation for the heterozygote deficiency was<br />
inbreeding in the four strains. These four closed strains<br />
especially Tuxedo strain were under selective breeding<br />
program for 17 generations. When a breeder conducts a<br />
selective breeding program, his primary objective is to<br />
alter, not conserve gene and genotypic frequencies in<br />
order to improve the population. Inbreeding and genetic<br />
drift are inevitable during a selective breeding program,<br />
because each act of selection creates a bottleneck event<br />
that accelerate the accumulation of inbreeding and<br />
magnify genetic drift. There are some techniques that can<br />
be used to moderate inbreeding and finally bottleneck so<br />
that it does not counteract selection. Managing a<br />
population to minimize the effect of bottleneck event can
e accomplished only by managing the number of<br />
effective population (Ne). Bottleneck reduces the Ne and<br />
make it difficult, if not impossible, to achieve genetic<br />
goals. Unfortunately, it is difficult to maintain a constant<br />
Ne generation after generation because Ne can decline<br />
due to diseases, selective breeding program, etc (Tave,<br />
1999). One way to maintain a constant Ne generation<br />
after generation can be pedigree mating. The weak<br />
performance of some economic traits (meat and number<br />
of eggs) in these strains, especially Tuxedo can be as a<br />
result of bottleneck event. Breeding strategies should<br />
therefore be designed to amplify the population size and<br />
simultaneously avoid inbreeding in these four strains. The<br />
information generated in this study will greatly aid in the<br />
establishment of effective breeding for these four strains<br />
and may further be utilized for studying differentiation and<br />
relationships among different quail strains.<br />
ACKNOWLEDGEMENTS<br />
This work was financially supported by the Animal Science<br />
Research Institute of Iran. The authors would like to<br />
acknowledge Dr. Ali Akbar Masoudi for his valuable input<br />
and assistance in the preparation, analysis and<br />
interpretation of this study.<br />
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African Journal of Biotechnology Vol. 10(20), pp. 3978-3985, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2334<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Characterization of a chestnut FLORICAULA/LEAFY<br />
homologous gene<br />
Tao Liu 1 *, Yun-qian Hu 2 and Xiao-xian Li 2<br />
1 Faculty of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan, 650201, China.<br />
2 Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan, 650204, China.<br />
Accepted 24 March, 2011<br />
The FLORICAULA/LEAFY (FLO/LFY) homologues’ genes are necessary for normal flower development<br />
and play a key role in diverse angiosperm species. In this paper, an orthologue of FLORICAULA/LEAFY,<br />
CmLFY (chestnut FLO/LFY), was isolated from the inflorescence of chestnut trees. Its expression was<br />
detected in various tissues. Furthermore, the flowering effectiveness of the gene was assessed with<br />
transgenic Arabidopsis. CmLFY protein showed a high degree of similarity to PEAFLO (78%), which is a<br />
homologue of FLO/LFY from pea. RT-PCR analysis showed that, CmLFY expressed at high levels in<br />
inflorescences, but not in young leaves, fruits or stems. The transgenic Arabidopsis with<br />
over-expressed CmLFY showed accelerated flowering, which supports that CmLFY encodes a functional<br />
orthologue of the FLORICAULA/LEAFY genes of angiosperms despite its sequence divergence. These<br />
results suggest that CmLFY is involved in inflorescence development in chestnut.<br />
Key words: Chestnut, homologue, FLORICAULA/LEAFY.<br />
INTRODUCTION<br />
Two developmental programmes can be distinguished<br />
during the life cycle of flowering plants. During the initial<br />
vegetative phase, the apical meristem produces leaves<br />
and lateral shoot. After floral induction, the plant enters<br />
into a reproductive phase and the apical meristem chan-<br />
ges its developmental pattern and initiates the production<br />
of flowers. In Antirrhinum and Arabidopsis this transition<br />
from vegetative to reproductive development pattern<br />
requires the establishment of floral meristem identity in<br />
the lateral meristems of the inflorescence, a process<br />
which has been demonstrated to be controlled by the<br />
homo- logous genes FLORICAULA (FLO) and LEAFY<br />
(LFY), respectively (Ma, 1998; Pidkowich et al., 1999).<br />
LFY is a transcriptional regulation gene thought to play a<br />
primary role in determining flower meristem identity.<br />
FLORICAULA (FLO), a LFY homologue in snapdragon,<br />
has been shown to have almost the same role. Loss func-<br />
* Corresponding author. E-mail: beixue2007@gmail.com. Tel:<br />
+86-871 5227721.<br />
Abbreviations: PCR, Polymerase chain reaction; RT-PCR,<br />
reverse transcriptase-PCR; RACE, rapid amplified cDNA ends.<br />
tion mutants of these genes result in the conversion of<br />
flowers into indeterminate secondary shoots (Coen et al.,<br />
1990; Weigel et al., 1992). The over-expression of LFY<br />
under a constitutive promoter in Arabidopsis caused early<br />
flowering and converted all lateral shoots into solitary<br />
flowers. In heterologous plants such as aspen, LFY has<br />
been shown to have effects on the acceleration of flower-<br />
ing and induction of ectopic flowers (Weigel and Nilsson,<br />
1995; Rottman et al., 2000), suggesting a conservation of<br />
LFY function across long phylogenetic distances within<br />
angiosperms. These reports strongly suggest that, the<br />
ability to control the expression of LFY or of its orthologs<br />
from other plants, could make it possible to artificially<br />
induce various plants to blossom and even skip or shorten<br />
the juvenile phase of woody plants.<br />
Flowering is an essential stage for fruit production and<br />
thus, an understanding of the genetic mechanisms under-<br />
lying the flowering event is important for efficient fruit<br />
production. During the last decade, molecular mecha-<br />
nisms of flowering have been studied extensively in<br />
herbaceous ‘‘model’’ plants such as Arabidopsis and<br />
Antirrhinum. In contrast, studies on the molecular<br />
mechanisms of flowering in fruit trees have just begun.<br />
Although, several flowering-related FLO/LFY orthologs<br />
have been cloned from kiwifruit (Actinidia deliciosa)
(Walton et al., 2001), grapevine (Vitis vinifera) (Carmona<br />
et al., 2002), and apple (Malus domestica) (Kotoda et al.,<br />
2002), their specific role in the induction of the<br />
characteristic features of tree reproductive development is<br />
still being elucidated.<br />
Trees are characterized by an extended adolescence.<br />
This is particularly important for commercial fruit/nut tree<br />
growers and breeders, because prolonged juvenile<br />
periods delay harvesting and the evaluation/breeding of<br />
new strains. Chestnut (Castanea mollissima) is one of the<br />
most commercially valuable fruit trees, with production<br />
once only annually. Chestnut has an extended juvenile-<br />
vegetative phase, during which vegetative growth is<br />
maintained. Attempts to exploit existing knowledge of the<br />
genetic control of flower development in Arabidopsis to<br />
engineer nut trees could have significant economic and<br />
scientific implications for the tree fruit industry. As yet, no<br />
studies have reported on the molecular mechanism<br />
underlying the development of chestnut flowers. Recently,<br />
MADS-box genes have been cloned and characterized in<br />
chestnut by us (Liu et al., 2006). Our results suggest that<br />
these chestnut MADS genes may be involved in floral<br />
organ and fruit development in chestnut. In this paper, we<br />
identified a chestnut FLORICAULA/LEAFY homologous<br />
gene involved in floral development. We described the<br />
isolation and characterization of CmLFY (DQ270548).<br />
The function of CmLFY was demonstrated by transgenic<br />
Arabidopsis, as expected for normal plants, transgenic<br />
plants showed earlier flowering phenotype, indicating that<br />
CmLFY may play a similar role of promoting flowering as<br />
the FLORICAULA/LEAFY genes in flower development.<br />
MATERIALS AND METHODS<br />
Plant materials<br />
The chestnut and Arabidopsis ecotype Columbia (Col) were used in<br />
this study. Chestnut examples were collected from Kunming,<br />
Yunnan, People’s Republic of China and were frozen in liquid<br />
nitrogen immediately and stored at -80°C.<br />
RNA extraction and cDNA cloning<br />
Total RNA was extracted from the inflorescences using a modified<br />
CTAB method (Zeng and Yang, 2002). The cDNA was prepared<br />
using Smart cDNA library construction kit (BD Biosciences Clontech,<br />
American). Full-length CmLFY cDNA was obtained by the 5′ and 3′<br />
rapid amplified cDNA ends (RACE) method (Chenchik et al., 1996).<br />
The first amplified chestnut cDNA contained about 440 bp between<br />
primers CmS and CmA, designed from Arabidopsis thaliana LFY<br />
(Weigel et al., 1992) and Antirrhinum majus FLO (Coen et al., 1990)<br />
cDNA sequences. The 3′ RACE was carried out between cassette<br />
CDSIII/3’ primer and CmS2 primer. The DNA fragment amplified<br />
was cloned into PMD18-T vector (Takara, Japan). The 5′ RACE was<br />
carried out between cassette CDSIII/5’ primer and CmA2 primer.<br />
Full-length cDNAs were amplified with the sense primer LFY-ORF5<br />
and antisense primer CDSIII/3’ primer. These clones were sequenced<br />
completely by the dideoxy method using ABI3730 automated<br />
sequencer (ABI Company, USA).<br />
Phylogenetic comparisons<br />
Liu et al. 3979<br />
Protein sequences of different LFY homologs were retrieved from<br />
GenBank and aligned with CLUSTAL. Phylogenetic relationships<br />
among these genes were inferred by NJ analysis (MEGA Ver.3.1).<br />
Plant transformation<br />
The full length CmLFY (including start codon ATG) was amplified<br />
between LFY-ORF5 and LFY-ORF3 (Figure 1). Then, the amplified<br />
cDNA was blunted and KpnI and SalI linkers were ligated both ends,<br />
cloned into a pCambia2301-101 binary vector in a sense-oriented<br />
manner under the CaMV 35S promoter. An Agrobacterium<br />
tumifaciens GV3101 strain (Van-Larebeke et al., 1974) was used for<br />
the transformation of the A. thaliana Columbia ecotype by the<br />
floral-dip method (Clough and Bent, 1998). For the selection of<br />
transformed plants, resultant seeds were planted on a 1/2MS culture<br />
medium containing kanamycin (50 µg mL --1 ) as selective antibiotics<br />
and then, transferred to 22°C under LD conditions (16 h light, 8 h<br />
dark). Transformants were identified as kanamycin-resistant when<br />
seedlings in the medium produced green leaves and well-<br />
established roots. Resistant transformants were transplanted to<br />
moistened potting soil composed of vermiculite and perlite (1:1<br />
(v/v --1 )) after two to four adult leaves had developed and their flower<br />
phenotypes were observed under long-day conditions.<br />
RNA analysis of transgenic plants by RT-PCR<br />
Detection of CmLFY transcripts was performed by RT-PCR. RNA<br />
was isolated from whole plants of Arabidopsis by a method using<br />
CTAB described previously described. CmLFY specific transcripts<br />
were identified using 1 µg of total RNA as a template and the<br />
following primers: a sense primer LFY-ORF5 and an antisense<br />
primer LFY-ORF3 for CmLFY (Table 1), giving rise to about 1300 bp<br />
long PCR product. PCR reactions were run for 35 cycles at 58°C for<br />
CmLFY. The PCR products were run on 1.5% (w/v --1 ) agarose gels<br />
stained with ethidium bromide.<br />
RESULTS<br />
Cloning and sequence analyses of CmLFY<br />
A comparison of Arabidopsis LFY (Weigel et al., 1992)<br />
and Antirrhinum FLO (Coen et al., 1990) showed the<br />
presence of several conserved regions. Two of these<br />
regions, CmS and CmA, were used to design degenerate<br />
oligo- nucleotide primers for RT-PCR. Using these<br />
primers with cDNA prepared from an inflorescence of<br />
chestnut, we obtained a PCR amplified fragment<br />
containing both primers and other conserved sequences.<br />
This fragment was named CmLFY (chestnut<br />
FLORICAULA/LEAFY homologue). To obtain a full-length<br />
CmLFY cDNA, several primers for 5’/3’ RACE were<br />
designed. Primer sites and the nucleotide sequences of<br />
primers used for cloning were shown in Figure 1 and<br />
Table 1, respectively.<br />
The coding region of CmLFY is 1,161 bp, encoding<br />
386 amino acids. The deduced amino acid sequence of<br />
CmLFY cDNA has 78% homology with PEAFLO, 79%<br />
with PtFL and 59.9% with PlaraLFY. NLF, PRFLL, PEAFLO,
3980 Afr. J. Biotechnol.<br />
Table 1. Primer sequences used in PCR cloning of CmLFY.<br />
Primer Oligonucleotide<br />
Primers for an internal fragment<br />
CmS sense primer<br />
CmA antisense primer<br />
Cassette primers<br />
CDSIII/5’ primer<br />
CDSIII/3’ primer<br />
3’ RACE primer<br />
CmS2 primer<br />
5’ RACE primer<br />
CmA2 primer<br />
CmLFY specific primers<br />
LFY-ORF5<br />
LFY-ORF3<br />
5’- GGGAGCACCCGTTCATTGTGACTG -3’<br />
5’-GA/CAGCTTG/TGTG/TGGGACATACCAGAC-3’<br />
5’-AAGCAGTGGTATCAACGCAGAGT-3’<br />
5’-ATTCTAGAGGCCGAGGCGGCCGACATG-3’<br />
5’-CGGCCTTGATTACCTCTTCCATCTC -3’<br />
5’- CACCGCGCTCCTTGGCAATGTTCTGT -3’<br />
5’-GGTACCAAGCTAGCTTCATTGATG -3’<br />
5’-CGGTCGACTAGAAATGCAAATTTTTCTC-3’<br />
Figure 1. Nucleotide and deduced amino sequences (single-letter code) of CmLFY cDNA. The asterisk (*) indicates a stop<br />
codon. Arrows above the sequences indicate the primers used for RACE-PCR.<br />
ELF1 and CmLFY lacked a proline-rich region in<br />
the variable region (roughly the first 40 amino acids). To<br />
more closely determine the evolutionary relationship<br />
between FLO/LFY-like proteins, a phylogenetic tree was<br />
con- structed (Figure 2b). This tree showed that, the<br />
topology of these genes seems to be concordant with the<br />
topology of the species phylogeny and suggests that<br />
CmLFY is an angiosperm ortholog of FLO/LFY-like genes.
a<br />
Liu et al. 3981
3982 Afr. J. Biotechnol.<br />
b<br />
Expression patterns of CmLFY<br />
Figure 2. (a) Sequence comparison of FLO/LFY-like proteins (accession numbers in<br />
parentheses): NLY and PRFLL from P. radiata (U76757 and U92008, respectively); CmLFY<br />
from chestnut (DQ270548); BOFH from B. oleracea (Z18362); FLO from A. majus (M55525);<br />
LFY from A. thaliana (M91208); NFL1 and NFL2 from N. tabacum (U16172 and U16174,<br />
respectively); ALF from P. hvbrida (AF030171); PtFL from P. balsamifera (U93196); RFL from<br />
O. sativa (AB005620); TOFL from L.esculentum (AF197934); PEAFLO from P. sativum<br />
(AF010190); ClLFY from C. glaziovii (AY633622); ELF1 from Eucalyptus globules (AF034806);<br />
JunefLFY from J. effusus (AF160481) and PlaraLFY from P. hispanica (AF106842). Black<br />
boxes indicate identical amino acids, shaded boxes indicate amino acids with similar<br />
properties, and dots indicate gaps introduced to optimize alignment. c1 and c2, conserved<br />
regions; v1 and v2, variable regions; (b) Protein sequence comparisons of CmLFY to other<br />
LFY/FLO homologs. The deduced amino acid sequence of CmLFY was compared with some<br />
published LFY homologs. Box characters represent chestnut protein.<br />
CmLFY mRNA expression was analyzed in various<br />
tissues by RT-PCR. Total RNA was isolated and PCR<br />
primers specific to CmLFY were used to detect the<br />
expression patterns in several tissues. The result showed<br />
that CmLFY mRNA was expressed in inflorescence<br />
tissues, but not in young leaves, fruits and stems (Figure<br />
3). The RT-PCR of CmLFY -specific primers resulted in<br />
amplification of a single band. Increasing the PCR cycle<br />
did not change these expression patterns. The amplified<br />
DNA fragments were the expected length.<br />
Ectopic expression in Arabidopsis<br />
To further verify the function of CmLFY, we constructed a<br />
binary vector pCambia2301-101-CmLFY containing full-<br />
length CmLFY coding area inserted in the sense-oriented<br />
direction under the control of the Cauliflower 35S<br />
promoter. The pCambia2301-101-CmLFY was introduced<br />
into wild- type Arabidopsis plants by<br />
Agrobacterium-mediated transformation. Fourteen<br />
independent transgenic plants that survived on kanamycin<br />
were identified (Table 2). Five (lines one-five) of them<br />
flowered earlier than the wild-type plants by five-seven
Figure 3. Expression pattern of CmLFY gene revealed by RT-PCR<br />
analysis. Lane 1: inflorescences; Lane 2: stem; Lane 3: tender leaves with<br />
no flowers; Lane 4: tender leaves with flowers; Lane 5: fruit. rRNA is used<br />
as RNA standard. Amplification of CmLFY was performed for 30 cycles.<br />
Table 2. Comparison of flowering time, number of rosette leaves in T1 transgenic and wild-type Arabidopsis (Col)<br />
plants in LD conditions.<br />
Transgenic line Days to flowering a Rosette leaves at time of flowering b Note<br />
1 18 4 Early flowering<br />
2 16 3 Early flowering<br />
3 18 4 Early flowering<br />
4 20 5 Early flowering<br />
5 20 4 Early flowering<br />
6 24 7<br />
7 24 8<br />
8 25 9<br />
9 22 8<br />
10 24 8<br />
11 25 8<br />
12 26 9<br />
13 26 10<br />
14 25 8<br />
wt c 25.5 d 9.5 d (n=10) e<br />
v-wt f 26 d 9.5 d (n=8) e<br />
a Days to flowering is defined as the time when flower primordial were first visible to the naked eye; b rosette leaves were<br />
counted on the day that flower primordial were first visible; c w wild-type Arabidopsis Columbia plants; d Mean number; e<br />
Number of plants;<br />
f vector-transformed plants.<br />
days and produced only four to five rosette leaves when<br />
they flowered (Figure 4 and Table 2). Most of the T1<br />
transgenic plants could self-pollinate and grow siliques<br />
normally and the resultant seeds also had the ability to<br />
germinate except for the lines 1, which is so small that it<br />
was difficult to obtain its seeds. The early flowering were<br />
inherited in the next generation and co- segregated with<br />
the kanamycin-resistant genes. The expression of the<br />
CmLFY mRNA in T1 plants was confirmed by RT-PCR, all<br />
putative transformed lines showed the expected products<br />
of CmLFY. No amplifi- cation was observed for the cDNA<br />
prepared from non-transformed Arabidopsis plants and<br />
vector-trans- formed Arabidopsis plants. The transgenic<br />
plants are stably transformed and the siblings which<br />
segregate without the transgene had no amplification of<br />
CmLFY and flowered at the same time as wild-type<br />
plants.<br />
DISCUSSION<br />
Liu et al. 3983<br />
Despite the importance of understanding the regulation of<br />
the flowering process in woody perennials for the<br />
management and improvement of woody species, very<br />
little is known about the underlying molecular<br />
mechanisms. Regulation of flowering in woody perennials<br />
shows remarkable differences in contrast to herbaceous<br />
species, that is, long juvenile phases, bud dormancy and<br />
the alternating vegetative and reproductive development<br />
according to the season.<br />
To investigate the molecular mechanism in chestnut<br />
flowering, we cloned CmLFY, a putative homologue<br />
of Arabidopsis LFY. A comparison (Figure 2a) of amino<br />
acid sequences with Arabidopsis LFY (Weigel et al.,<br />
1992), Antirrhinum FLO (Coen et al., 1990) and other<br />
FLO/LFY homologs (Brassica oleracea. BOFH.
3984 Afr. J. Biotechnol.<br />
Figure 4. Transgenic and wild-type Arabidopsis plants grown under LD<br />
photoperiods for 20 days. Arabidopsis plants were grown in one-half strength of<br />
medium for 9 days and transferred to potting soil. (a) wild-type Arabidopsis<br />
Columbia. (b) Transgenic Arabidopsis carrying a 35S::CmLFY gene.<br />
Anthony et al., 1993; Nicotiana tabacum. NFL. Kelly et al.,<br />
1995; Oraza sativa. RFL. Kyozuka et al., 1998; Pinus<br />
radiata. NLY. Mouradov et al., 1998 ; Petunia hybrida. ALF.<br />
Souer et al., 1998; Eucalyptus globulus, ELF1, Southerton<br />
et al., 1998 ; Lycopersicon esculentum. TOFL.<br />
Molinero-Rosales et al., 1999; Juncus effusus. JunefLFY.<br />
Frohlich and Parker., 2000; Platanus hispanica. PlaraLFY.<br />
Frohlich and Parker., 2000; Populus balsamifera. PTFL.<br />
Rottmann et al., 2000; Cedrela glaziovii. ClLFY. Marcelo<br />
and Adriana, 2006) showed the presence of conserved<br />
regions including two large conserved regions (c1 and c2)<br />
and two shorter regions of lower similarity (variable<br />
regions v1 and v2). These domains are typical markers for<br />
transcriptional acti- vators and may be functionally<br />
important for FLO/LFY-like proteins (Weigel et al., 1992;<br />
Coen et al., 1990). The phylogenetic tree showed that the<br />
topology of these genes seems to be concordant with the<br />
topology of the species phylogeny and suggests that<br />
CmLFY is an angiosperm ortholog of FLO/LFY-like genes<br />
(Figure 2b).<br />
The expression of CmLFY was only found in<br />
inflorescences, but not in tissues such as fruits, stems and<br />
tender leaves (Figure 3). It supports that the CmLFY is a<br />
functional homologous gene of chestnut. In the species<br />
such as apple, kiwifruit and grape, the expression levels<br />
of their FLO/LFY homologs increase in the proliferating<br />
inflorescence meristems generating inflorescence<br />
branches, with the highest levels detected in young floral<br />
meristems (Walton et al., 2001; Carmona et al., 2002).<br />
Therefore, the high levels of CmLFY expression in inflo-<br />
rescence suggests that CmLFY plays a role during chest-<br />
nut reproductive development as it has been suggested<br />
for most FLO/LFY-like genes studied.<br />
To further demonstrate the function of CmLFY, we<br />
have produced transgenic Arabidopsis plants expressing<br />
CmLFY. They flowered earlier and had a shorter<br />
inflorescence and reduced number of rosette leaves<br />
compared with the controls (Figure 3). Based on the result<br />
that several transgenic Arabidopsis with 35S::CmLFY<br />
flowered earlier than the controls, the expression of<br />
CmLFY should have some relationship with the early<br />
flowering phenotypes, although, the mechanism of<br />
flower-bud formation in chestnut might be different from<br />
that in Arabidopsis. Of course, to be more certain that the<br />
CmLFY gene functions similarly to LFY, LFY mutant-<br />
rescue experiments will be required. The CmLFY gene<br />
was not expressed strongly in transformants showing<br />
early flowering relative to those that were wild type in<br />
appearance, which suggests that the severity of pheno-<br />
type in transformants does not depend solely on the level<br />
of gene expression in this case, although the exact cause<br />
is still unknown. Some reports are available that describe<br />
transgenic Arabidopsis plants expressing LFY homologs<br />
from other plant species. For example, Arabidopsis<br />
transgenic lines expressing NEEDLY from Radiata pine<br />
and ELF1 from Eucalyptus showed early flowering<br />
(Mouradov et al., 1998; Southerton et al., 1998). Until now,<br />
however, our ability to manipulate fruit/nut tree strains<br />
through genetics has been limited by the extended<br />
maturation period of these plants. Both taking long time to<br />
flowering and the fact that, no characterized flowering<br />
mutants have been described in this genus make genetic<br />
studies in chestnut more difficult. Nevertheless, advances<br />
in the establishment of transformation protocols for
chestnut may allow us to use reverse genetic approaches<br />
and to define more clearly the role played by CmLFY in<br />
the reproductive development.<br />
The breeding of fruit trees of chestnut often requires<br />
more than 20 years, including periods of cross pollination,<br />
seedling selection and regional trials, to produce varieties<br />
that meet the demands of consumers. One of the limit<br />
factors of chestnut breeding is the long juvenile phase of<br />
at last several years. It had been found that transgenic<br />
approaches of LFY introducing could reduce the juvenile<br />
phase of Populus and the transgenic poplar flowered in 5<br />
months after regeneration (Weigel and Nilsson, 1995).<br />
Therefore, probably we can anticipate that these techni-<br />
ques are likely applicable to fruit trees of chestnut in future<br />
years and believed that, the transgenic approach would<br />
be a useful breeding strategy for reducing the time<br />
required for generation among woody plants. However,<br />
the efficiency of producing early flowering transgenic lines<br />
with Arabidopsis LFY gene seems to be low in woody<br />
plants in some case (Pena et al., 2001). This suggests<br />
that regulatory genes such as LFY do not always function<br />
beyond species as well as expected. Since environmental<br />
and genetic factors controlling flower development in<br />
chestnut have not been made clear so far, the CmLFY<br />
gene could at least, be one of the tools available for<br />
studying the mechanism of flower development in tree<br />
fruits such as the chestnut.<br />
ACKNOWLEDGMENTS<br />
This research was financially supported by the Faculty of<br />
Agronomy and Biotechnology (SJCX09012 to T Liu) and<br />
the National Natural Science Foundation of China (NSFC,<br />
30800062 to XX Li).<br />
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LEAFY controls floral meristem identity in arabidopsis. Cell, 69:<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4303-4312, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.2361<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
Productive performance of laying hens fed wheat-based<br />
diets included olive pulp with or without a commercial<br />
enzyme product<br />
M. Zarei 1 , M. Ehsani 2 and M. Torki 2 *<br />
1 Member of Young Researchers Club of Islamic Azad University, Aligudarz Branch, Iran.<br />
2 Department of Animal Science, Agriculture Faculty, Razi University, Imam Avenue, Kermanshah, Iran.<br />
Accepted 21 March, 2011<br />
Olive pulp (OP) is the remainder of olive cake (the raw material resulting from extraction of olive oil)<br />
after the removal of the seed fractions. It can be achieved by sieving the dry olive cake to separate most<br />
of the seeds. To assess effects of dietary inclusion of OP supplementing with a cocktail commercial<br />
enzyme on performance of laying hens and egg quality characteristics, one hundred and twenty 80week-old<br />
Lohmann LSL-Lite hens, with an average laying rate of 80.3 ± 3.8% (late production phase)<br />
and 1450 ± 14 g live body weight, were divided in 20 cages (n = 6). Hens in 5 cages (replicates) were<br />
randomly assigned to feed on one of the 4 experimental diets. Based on a 2×2 factorial arrangement, 4<br />
iso-caloric and iso-nitrogenous diets (ME =2720 kcal/kg and CP=150 g/kg) including OP (0.0 and 90.0<br />
g/kg) and a commercial cocktail enzyme with mostly xylanase activity (Nutrase®, 0.0 or 0.9 g/kg) were<br />
formulated. To determine blood biochemical parameters and differentiable count of white blood cells,<br />
one hen per replicate was bled via wing vein on day 35 of trial. To determine egg quality parameters, all<br />
eggs during three frequent days were collected on week 4 of trial. Collected data of feed intake (FI), egg<br />
production (EP), egg mass (EM) and calculated feed conversion ratio (FCR), blood biochemical<br />
parameters and egg quality traits during 6 week trial period was analyzed based on completely<br />
randomized design. Hens fed the OP-included diet shown decreased EP compared with hens fed the<br />
control diet during week 3 of experimental period (p ≤ 0.05). There was no significant difference<br />
between hens fed the OP-included diet and the control diet in terms of FI and EM. In addition, the same<br />
trend was observed in terms of enzyme effect on FI and EM. Dietary enzyme supplementation improved<br />
FCR compared with hens fed the control diet during week 6 of the experimental period (p ≤ 0.05).<br />
Including OP in the diets of hens decreased the blood level of triglycerides (p ≤ 0.05). Including OP in<br />
the diets of hens increased the yolk index (p ≤ 0.05). From the results of this investigation, it can be<br />
concluded that including OP in diets of laying hens up to 9% would not have deleterious effects on<br />
bird's productive performance. In addition, dietary supplementation with a cocktail enzyme with mainly<br />
xylanase activity improved FCR in hens.<br />
Key words: Olive pulp, enzyme, laying hens, performance, egg quality characteristics, blood parameters.<br />
INTRODUCTION<br />
The biggest single expense in any system of poultry pro-<br />
duction is feed, accounting for up to 70% of total<br />
*Corresponding author. E-mail: torki@razi.ac.ir. Tel:<br />
00989133277266.<br />
Abbreviations: AME, Available energy; NSP, non-starch<br />
polysaccharides; OP, olive pulp; FI, feed intake; EP, egg<br />
production; EM, egg mass; FCR, feed conversion ratio.<br />
production cost. In order to reduce feeding costs, attempts<br />
have been made to use agricultural and industrial byproducts<br />
as feed ingredients. Improved utilization of crop<br />
residues and by-products to be used in animal feeding<br />
deserves more attention. Examples of crop residues and<br />
agricultural by-products in Iran are; cereal bran, citrus<br />
pulp, tomato pulp, poultry litter and olive pulp (OP). Olive<br />
pulp is the remainder of olive cake (the raw material<br />
resulting from extraction of olive oil) after the removal of<br />
the seed fractions. OP is considered as a good source of
4304 Afr. J. Biotechnol.<br />
calcium, copper and cobalt but poor in phosphorus,<br />
magnesium and sodium and with fair levels of manganese<br />
and zinc (Harb, 1986). Due to low nutritive value<br />
(low in energy, digestible proteins and minerals and high<br />
in lignin), OP is seldom integrated into poultry feeding. In<br />
addition, a xyloglucan, one of the non-starch polysaccharides<br />
(NSP) which has anti-nutritive effects on<br />
monogastrics such as poultry and pigs, from OP has<br />
been reported by Gil-Serrano and Tejero-Mateo (1988).<br />
Coimbra et al. (1995) also showed the occurrence of the<br />
xylan-xyloglucan complexes in the OP cell walls. In<br />
addition, Rosa´rio and Domingues (2002) extracted<br />
glucuronoxylans with a xylose/glucose ratio of 7: 1 from<br />
OP.<br />
Although starch is the predominant carbohydrate in<br />
cereals, some of the constituent carbohydrates are watersoluble<br />
NSP, which are considered the major antinutritive<br />
factors in cereals and other varieties of feed ingredients<br />
(Bedford and Classen, 1992; Campbell and Bedford,<br />
1992). The common NSP include β-glucans, arabinoxylans<br />
and fructans (Classen and Bedford, 1991). The<br />
structures of NSP differ between grains and also between<br />
varieties of the same grain (Annison, 1990). The similarity<br />
between the types of NSP is usually the presence of β (1<br />
→ 4) backbones with or without the presence of β (1 →<br />
3) side linkages (Iji, 1999). The effects of NSP upon feed<br />
utilization and poultry productivity have been discussed in<br />
many reports. Salih et al. (1991) reported that, NSP<br />
reduced feed intake and decreased broiler chicken<br />
performance. The most noticeable effect of NSP in<br />
poultry diets is an increase in the viscosity of digesta and<br />
the excretion of sticky droppings. This is considered to be<br />
one of the greatest influences of NSP on broiler chicken<br />
productivity (Smits and Annison, 1996) and it is<br />
necessary to use exogenous enzymes to overcome these<br />
problems.<br />
Poultry naturally produces enzymes to aid the digestion<br />
of feed nutrients. However, they do not have enzyme to<br />
break down fiber completely and need exogenous enzymes<br />
in feed to aid digestion. Enzymes are used mainly to<br />
achieve consistency in performance and to reduce<br />
formulation costs by easing constraints on the inclusion<br />
level of some ingredients, such as wheat and barley.<br />
Hence, the supplementation of microbial enzymes in<br />
poultry diets is now common practice in many countries<br />
where the predominant cereals are wheat or barley.<br />
Improving poultry performance by dietary manipulation<br />
has been the goal of nutritionists. Enzyme has been<br />
reported to alleviate the negative effects of arabinoxylans,<br />
present in wheat as the dominant cell-wall constituent,<br />
by lowering gut viscosity and increasing protein,<br />
fat and starch digestibility (Bedford and Morgan, 1996).<br />
Moreover, an improvement in overall protein digestibility<br />
of dietary components other than wheat, such as soybean,<br />
has also been reported (Bedford and Morgan,<br />
1996). Pan et al. (1998) demonstrated that, commercial<br />
enzyme mixtures could promote metabolizable energy<br />
particularly utilized in wheat based rations. Many years of<br />
intensive research, both in official trials as well as in field<br />
trials, have shown that its bacterial origin gives it some<br />
very distinctive and valuable characte-ristics, distinguishing<br />
it clearly from xylanase. It reaches its highest<br />
efficiency in rations with a high inclusion of wheat and<br />
wheat by-products. Many years of practical experience<br />
have shown that, it also has an important effect on<br />
improving the digestibility of other grains, such as barley,<br />
corn, rice, rye and sorghum. Improved starch digestibility,<br />
for instance, has been reported to account for up to 35%<br />
of the improvement in available energy (AME) as a result<br />
of xylanase supplementation (Carre et al., 1992).<br />
Xylanase has been reported to alleviate the negative<br />
effects of arabinoxylans, present in wheat as the<br />
dominant cell-wall constituent, by lowering gut viscosity<br />
and increasing protein, fat and starch digestibility<br />
(Bedford and Morgan, 1996).<br />
The objectives of this study were to investigate the<br />
effects of dietary inclusion of OP and enzyme supplementation<br />
on performance of the laying hens fed with<br />
wheat-based diets.<br />
MATERALS AND METHODS<br />
All procedures used in this six-week experiment were approved by<br />
the Animal Ethics Committee of Razi University and complied with<br />
the “Guidelines for the Care and Use of Animals in Research". A<br />
total number of one hundred and twenty 80-week-old Lohmann<br />
LSL-Lite hens with an average egg production rate of 80.3 ± 3.8%<br />
(late laying phase) and 1450 ± 14 g live body weight, were obtained<br />
from a commercial supplier. After a week of adaptation, the hens<br />
were randomly allocated to one of four experimental diets. Hens<br />
were semi-randomly distributed between 20 cages (n=6) with<br />
almost same average body weight and egg production throughout<br />
the cages. The hen distribution was based on the previous production<br />
records of birds during one month before selecting them<br />
from their original flock and transferring to the experimental cages.<br />
Hens in 5 cages (replicates) were assigned to feed on one the 4<br />
experimental diets. Four iso-caloric and iso-nitrogenous diets (ME<br />
=2720 kcal/kg and CP=150 g/kg) including OP (0.0 and 90.0 g/kg)<br />
and a commercial cocktail enzyme with mostly xylanase activity<br />
(Nutrase ® , 0.0 or 0.9 g/kg) were formulated (Table 1).<br />
The approximate analysis of the OP used in this study is; dry<br />
matter (DM=93%), crude protein (CP= 6.06%), ether extract (EE%=<br />
7.6%), crude fiber (CF%= 48.2), ash (7.4%), calcium (Ca%=0.6)<br />
and total phosphorous (P%= 0.07). Fatty acid composition (%) of<br />
oils from OP used in this study is; total saturated fatty acids (∑SFA=<br />
15.01%), total monounsaturated fatty acids (∑MUFA= 71.38%) and<br />
total polyunsaturated fatty acids (∑PUFA= 9.10%). Nutrase® xyla is<br />
a bacterial endo-1,4-beta-xylanase, designed for use in pig and<br />
poultry rations with a high content of arabinoxylans. Nutrase® xyla<br />
is produced by Bacillus subtilis, which makes it today, the only EU<br />
registered bacterial xylanase preparation available on the market.<br />
The hens were housed in laying cages made from galvanized<br />
metal wire which provided approximately 430 cm 2 /hen. The cages<br />
were located in a windowless and environmentally controlled room<br />
with the room temperature kept at 21-23°C and the photoperiod set<br />
at 16 h of light (incandescent lighting, 10 lux) and 8 h dark. Each<br />
cage had a nipple waterier. Water was available ad libitum throughout<br />
the experiment. Feed consumption was measured on a weekly<br />
basis. To determine blood biochemical parameters and differentiable
Table 1. Ingredients and composition of the experimental diets.<br />
Olive pulp (g /100 g) 0.0 9.0<br />
Enzyme (g /100 g) 0.00 0.09 0.00 0.09<br />
Feed ingredients g / 100 g diet<br />
Wheat 62.78 62.78 58.06 58.07<br />
Fish meal 5.00 5.00 5.00 5.00<br />
Soybean meal 8.80 8.80 8.78 8.78<br />
Dried tomato pomace 3.50 3.50 3.50 3.50<br />
Oil 5.03 5.03 5.03 5.03<br />
Olive pulp 0.00 0.00 9.00 9.00<br />
Enzyme - Nutrease® 0.00 0.09 0.00 0.09<br />
Dicalcium phosphate 1.64 1.64 1.64 1.64<br />
Limestone 8.07 8.07 8.07 8.07<br />
Common salt 0.16 0.16 0.17 0.17<br />
Vit. and Min. Premix 1 0.25 0.25 0.25 0.25<br />
Sand 4.38 4.29 0.10 0.00<br />
DL-Methionine 0.15 0.15 0.15 0.15<br />
Calculated analyses<br />
ME (Kcal/kg) 2720 2720 2720 2720<br />
Crude protein (%) 15 15 15 15<br />
Ether extract (%) 6.65 6.65 6.65 6.65<br />
Crude fiber (%) 4.37 4.37 4.37 4.37<br />
Calcium (%) 3.67 3.67 3.67 3.67<br />
Available P (%) 0.33 0.33 0.33 0.33<br />
Lys (%) 0.68 0.68 0.68 0.68<br />
Met (%) 0.38 0.38 0.38 0.38<br />
Met and Cys (%) 0.61 0.61 0.61 0.61<br />
1 The vitamin and mineral premix provide the following quantities per kilogram of diet:<br />
Vitamin A, 10,000 IU (all-trans-retinal); cholecalciferol, 2,000 IU; vitamin E, 20 IU (αtocopheryl);<br />
vitamin K3, 3.0 mg; riboflavin, 18.0 mg; niacin, 50 mg; D-calcium<br />
pantothenic acid, 24 mg; choline chloride, 450 mg; vitamin B12, 0.02 mg; folic acid, 3.0<br />
mg; manganese, 110 mg; zinc, 100 mg; iron, 60 mg; copper, 10 mg; iodine, 100 mg;<br />
selenium, 0.2 mg; antioxidant, 250 mg.<br />
count of white blood cells, one hen per replicate was bled via wing<br />
vein on day 35 of trial. To determine egg quality parameters, all<br />
eggs during three frequent days were used. Collected data of feed<br />
intake (FI), egg production (EP), egg mass (EM) and calculated<br />
feed conversion ratio (FCR) during 6 week trial period was analyzed<br />
based on a 2×2 factorial arrangement of treatments and completely<br />
randomized design using GLM procedure of SAS. All statements of<br />
significance are based a probability of less than 0.05. The mean<br />
values were compared by Duncan’s multiple range test.<br />
RESULTS AND DISCUSSION<br />
Effects of dietary OP inclusion and enzyme supplementation<br />
on EP, FI, FCR, EW and EM are presented in<br />
Tables 2 to 6, respectively. There was no significant<br />
interaction between dietary OP inclusion and enzyme<br />
supplementation on the measured productive performance<br />
parameters throughout the experimental period (p<br />
> 0.05). Egg production (%) was not significantly affected<br />
by dietary OP inclusion (p > 0.05), except for week 3 of<br />
Zarei et al. 4305<br />
trial period. Hens fed with the OP-included diet shown<br />
decreased EP compared with hens fed with the control<br />
diet during week 3 of experimental period (p ≤ 0.05); but<br />
the overall EP during the whole trail period (weeks 1 to 6)<br />
was not significantly affected. There was no significant<br />
difference between hens fed with OP-included diet and<br />
the control diet in terms of FI (Table 3) and EM (Table 6).<br />
In addition, the same trend was observed in terms of<br />
enzyme effect on FI and EM. Feed conversion ratio was<br />
not significantly affected by dietary OP inclusion (p ><br />
0.05). Including OP in diet improved EW on weeks 4 and<br />
6 as well as the overall trial period (weeks 1 to 6). Studies<br />
concerning feeding with OP to monogastrics are limited<br />
(Tortuero et al., 1989). Several research studies were<br />
conducted to investigate the feasibility of utilizing OP in<br />
broiler rations. The proportion of OP in its rations is<br />
variable. There seems to be a limit between 50 and 100g/<br />
kg (Abo Omar, 2000; Rabayaa, 2000). Diets with different<br />
levels of fiber showed certain influence on gastro-
4306 Afr. J. Biotechnol.<br />
Table 2. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on egg production (%) of laying hens.<br />
Treatment<br />
Enzyme (g /100 g)<br />
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6<br />
0.00 67.85 75.71 79.28 84.04 86.42 83.80 79.52<br />
0.09 70.47 72.61 79.52 85.71 86.43 88.33 80.51<br />
Olive pulp (g /100 g)<br />
0.00 71.42 76.42 84.28 a 85.95 87.14 85.47 81.78<br />
9.00 66.90 71.90 74.52 b 83.81 85.71 86.66 78.25<br />
Olive pulp Enzyme<br />
0.00 0.00 69.52 75.71 83.81 84.76 86.66 82.85 80.55<br />
0.00 0.09 73.33 77.14 84.76 87.14 87.62 88.09 83.01<br />
9.00 0.00 66.19 75.71 74.76 83.33 86.19 84.76 78.49<br />
9.00 0.09 67.61 68.09 74.28 84.28 85.24 88.57 78.01<br />
SEM 5.83 5.36 3.84 3.32 3.16 2.26 3.09<br />
CV 19.91 17.06 11.41 9.24 8.64 6.21 9.14<br />
Source of variation Probability<br />
Enzyme 0.676 0.591 0.954 0.641 0.999 0.076 0.765<br />
Olive pulp 0.473 0.435 0.028 0.549 0.674 0.625 0.296<br />
Enzyme × Olive pulp 0.849 0.435 0.862 0.841 0.779 0.770 0.659<br />
a-b Means within a column (within main effects) with no common superscript differ significantly (p < 0.05); SEM= standard error of means.<br />
Table 3. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on feed intake (FI, g/ hen/ day) of<br />
laying hens.<br />
Treatment<br />
Feed intake (g/ hen/ day)<br />
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6<br />
Enzyme (g /100 g)<br />
0.00 105.66 116.55 117.72 118.22 116.89 118.13 115.53<br />
0.09 111.25 109.91 113.57 112.79 114.14 115.40 112.84<br />
Olive pulp (g /100 g)<br />
0.00 110.78 114.97 117.16 114.92 114.49 116.32 114.77<br />
9.00 106.13 111.49 114.13 116.09 116.54 117.21 113.60<br />
Olive pulp Enzyme<br />
0.00 0.00 109.95 116.88 119.14 116.45 114.81 117.19 115.74<br />
0.00 0.09 111.62 113.07 115.19 113.40 114.16 115.45 113.81<br />
9.00 0.00 101.38 116.23 116.30 120.00 118.97 119.07 115.33<br />
9.00 0.09 110.88 106.76 111.95 112.19 114.12 115.35 111.87<br />
SEM 4.18 4.00 2.96 2.52 3.30 1.61 2.29<br />
CV 9.11 8.34 6.05 5.16 4.71 3.26 4.75<br />
Source of variation Probability<br />
Enzyme 0.224 0.135 0.203 0.058 0.275 0.128 0.284<br />
Olive pulp 0.307 0.422 0.346 0.667 0.410 0.606 0.634<br />
Enzyme × Olive pulp 0.388 0.511 0.949 0.385 0.400 0.569 0.756<br />
SEM, Standard error of means.
Zarei et al. 4307<br />
Table 4. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on feed conversion ratio (FCR: g feed:<br />
g egg) of laying hens.<br />
Treatment<br />
Enzyme (g /100 g)<br />
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6<br />
0.00 2.58 2.49 2.39 2.24 2.16 2.26 a 2.35<br />
0.09 2.57 2.48 2.30 2.12 2.14 2.11 b 2.29<br />
Olive pulp (g /100 g)<br />
0.00 2.57 2.47 2.25 2.16 2.14 2.21 2.30<br />
9.00 2.59 2.51 2.44 2.20 2.17 2.15 2.34<br />
Olive pulp Enzyme<br />
0.00 0.00 2.64 2.53 2.31 2.20 2.15 2.31 2.36<br />
0.00 0.09 2.49 2.40 2.18 2.11 2.12 2.12 2.24<br />
9.00 0.00 2.50 2.45 2.46 2.27 2.18 2.21 2.34<br />
9.00 0.09 2.68 2.56 2.42 2.13 2.16 2.10 2.34<br />
SEM 0.16 0.13 0.10 0.08 0.07 0.06 0.07<br />
CV 14.68 12.74 10.23 9.15 8.73 6.57 7.46<br />
Source of variation Probability<br />
Enzyme 0.944 0.950 0.419 0.213 0.770 0.035 0.451<br />
Olive pulp 0.907 0.776 0.094 0.629 0.717 0.380 0.596<br />
Enzyme × Olive pulp 0.353 0.414 0.680 0.775 0.934 0.562 0.466<br />
SEM, Standard error of means.<br />
intestinal tract weight, length and content (Abo Omar et<br />
al., 1994; Abo Omar, 1995). The different levels of olive<br />
cake had no effect on carcass cuts of broilers carcass<br />
when fed with OP at levels up to 100 g/kg (Abo Omar and<br />
Gavoret, 1995). Rabayaa et al. (2001) who incorporated<br />
OP in four of the experimental groups at rates of 2.5, 5,<br />
7.5 and 10% in both starter and finisher feeds to replace<br />
similar rates of yellow corn reported that weight gain of<br />
chicks was the same in chicks consuming up to 7.5% of<br />
OP. However, weight gain of chicks fed the level of 10%<br />
OP had the lowest significant weight gain. In addition,<br />
similar trends were observed in chicks for F and feed<br />
conversion efficiency.<br />
Dietary enzyme supplementation improved FCR compared<br />
with hens fed with control diet during week 6 of the<br />
experimental period (p ≤ 0.05). Although enzyme addition<br />
decreased FCR during the whole trail period, this effect<br />
was significant only on week 6 (Table 4). Enzyme<br />
supplementation did not have significant effect on EW<br />
(Table 5). Xylans are the principal NSP of wheat and high<br />
levels of wheat in poultry diets can increase the viscosity<br />
of the gut contents, which impedes the circulation and<br />
absorption of nutrients, causing reduced feed intake, BW<br />
gain and feed efficiency (Annison and Choct, 1991).<br />
Chickens do not produce some enzymes, such as galactosidases<br />
(xylanase/β-glucanase); thus, corn-soybean-<br />
based diets without supplemented enzymes such as xylanases<br />
and pectinases might result in gas accumulation in<br />
the gut and diarrhea (Jaroni et al., 1999a; Wu et al.,<br />
2005). The endosperm cell wall of wheat is mainly composed<br />
of arabinoxylans. The addition of exogenous<br />
enzymes to wheat-based diets increases digestibility of<br />
nutrients in cockerels (Carré et al., 1990). It may also<br />
provide additional dietary energy as well as short chain<br />
fatty acids and oligosaccahrides (Iji, 1999). Such phenomena<br />
are associated with cellular proliferation and<br />
improved gut health (Iji, 1999). Xylanase is used<br />
extensively in wheat-based diets to counteract the effects<br />
of NSP in broiler (Bedford and Schulze, 1998). In addition,<br />
xylanase has been used in combination with other<br />
enzyme(s) (xylanase-based cocktail enzyme) in layer<br />
diets containing wheat or wheat and barley (Brenes et al.,<br />
1993; Pan et al., 1998; Scott et al., 1999, Francesch et<br />
al., 1995; Oloffs et al., 1998; Salobir, 1998; Jaroni et al.,<br />
1999a,b; Mathlouthi et al., 2002) and broiler diets<br />
(Bedford and Schulze, 1998) to counteract the effects of<br />
NSP; however, the results obtained with different experimental<br />
conditions and diets have been inconclusive. The<br />
degree of improvement obtained by adding enzymes to<br />
the diet depends on many factors including the level of<br />
antinutritive factor in the diet, the spectrum and concentration<br />
of enzymes used, the type of animal and the
4308 Afr. J. Biotechnol.<br />
Table 5. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on average egg weight (g).<br />
Treatment<br />
Enzyme (g /100 g)<br />
Week 1 Week 2 Week 3<br />
Egg weight (g)<br />
Week 4 Week 5 Week 6 Weeks 1 to 6<br />
0.00 62.29 62.80 63.07 62.77 62.81 42.53 59.38<br />
0.09 61.96 61.93 62.34 62.15 61.85 42.19 58.74<br />
Olive pulp (g /100 g)<br />
0.00 61.80 61.86 62.25 61.89 b 61.72 41.40 b 58.48 b<br />
9.00 62.46 62.87 63.16 63.03 a 62.94 43.32 a 59.63 a<br />
Olive pulp Enzyme<br />
0.00 0.00 61.95 62.05 62.10 62.00 61.90 41.37 58.56<br />
0.00 0.09 61.64 61.66 62.39 61.78 61.54 41.43 58.41<br />
9.00 0.00 62.63 63.54 64.04 63.55 63.73 43.69 60.19<br />
9.00 0.09 62.28 62.21 62.29 62.52 62.16 42.95 59.07<br />
SEM 0.61 0.68 0.71 0.51 0.72 0.32 0.52<br />
CV 2.35 2.58 2.67 1.93 2.75 1.83 2.07<br />
Source of variation Probability<br />
Enzyme 0.620 0.249 0.344 0.266 0.226 0.345 0.260<br />
Olive pulp 0.326 0.176 0.240 0.050 0.130 0.045 0.049<br />
Enzyme × Olive pulp 0.976 0.525 0.194 0.465 0.442 0.266 0.390<br />
SEM, Standard error of means.<br />
age of the animal (young animals tend to respond better<br />
to enzymes than older animals), type of gut micro flora<br />
present and the physiology of the bird. Older birds,<br />
because of the enhanced fermentation capacity of the<br />
micro flora in their intestines, have a greater capacity to<br />
deal with negative viscosity effects (Choct et al., 1995).<br />
Typically, enzymes added to layer feed appear to have<br />
little effect on egg mass but improve feed efficiency<br />
(Benabdeljelil and Arbaoui, 1994). Mathlouthi et al.<br />
(2002) who determined the effects of xylanase on true<br />
metabolizable energy values of wheat, barley and wheat<br />
bran as well as performance of laying hens fed with<br />
wheat-, barley- or wheat bran-based diets reported<br />
improved egg mass of layers fed with wheat- or wheat<br />
bran-based diets supplemented by xylanase. In addition,<br />
xylanase supplementation improved egg production, egg<br />
mass and feed conversion ratio of layers fed with low<br />
energy diet. It did improve the feed conversion ratio of<br />
layers fed with high-energy diet. Xylanase increased the<br />
TME values for wheat and barley and did not affect the<br />
TME value of wheat bran (Mathlouthi et al., 2002).<br />
Senkoylu et al. (2009) who investigated the effects of<br />
30% whole-wheat inclusion in a standard layer diet<br />
supplemented with xylanase, on laying performance,<br />
digestive organs and ileal mucosa development and<br />
reported that xylanase supplementation to whole wheat<br />
significantly improved egg production and feed<br />
conversion rate compared with the ground wheat and<br />
whole wheat fed groups. Pirgozliev et al. (2010) who examined<br />
the effect of dietary xylanase on the availability of<br />
nutrients for laying hens when fed on wheat-rye-soybased<br />
diets and reported that, the AME and nitrogen<br />
metabolisability coefficients of xylanase-supplemented<br />
diets were greater than the control diet. In addition, they<br />
reported supplementary xylanase significantly improved<br />
the coefficients of metabolisability of indispensable, dispensable<br />
and total amino acids. Their data suggested<br />
that, the use of a xylanase might improve the metabolisability<br />
of some nutrients, but that such effects might<br />
not always benefit production parameters. Feed intake<br />
and feed conversion ratio for egg production were not<br />
affected by xylanase (Pirgozliev et al., 2010). Diet<br />
supplementation with an enzyme cocktail providing 7 U/g<br />
of α-1, 6-galactosidase and 22 U/g of β-1, 4-mannanase<br />
significantly improved feed conversion of Lohmann<br />
Brown-Lite laying hens (Han et al., 2010).<br />
As it is shown in Table 7, among the blood biochemical<br />
parameters (cholesterol, high density lipoprotein, low<br />
density lipoprotein and triglycerides), only blood level of<br />
triglycerides was affected by dietary OP inclusion.<br />
Including OP in the diets of hens decreased the blood<br />
level of triglycerides (p ≤ 0.05); however, there was no<br />
significant difference between blood levels of cholesterol,<br />
high density lipoprotein and low density lipoprotein in
Zarei et al. 4309<br />
Table 6. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on egg mass (g/ hen/ day) of laying<br />
hens.<br />
Treatment<br />
Egg mass (g/ hen/ day)<br />
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6<br />
Enzyme (g /100 g)<br />
0.00 42.17 47.60 49.99 53.01 54.29 52.43 49.92<br />
0.09 43.59 44.92 49.64 53.33 53.42 54.72 49.94<br />
Olive pulp (g /100 g)<br />
0.00 43.98 47.27 52.51 53.47 53.80 52.70 50.62<br />
9.00 41.78 45.26 47.13 52.86 53.91 54.46 49.23<br />
Olive pulp Enzyme<br />
0.00 0.00 42.97 47.07 52.11 53.04 53.64 50.90 49.96<br />
0.00 0.09 45.00 47.47 52.90 53.90 53.97 54.50 51.29<br />
9.00 0.00 41.38 48.14 47.88 52.97 54.95 53.97 49.88<br />
9.00 0.09 42.19 42.38 46.38 52.76 52.88 54.95 48.59<br />
SEM 3.52 3.54 2.53 2.09 2.04 1.41 2.02<br />
CV 19.39 18.04 11.98 9.07 8.93 6.19 9.53<br />
Source of variation Probability<br />
Enzyme 0.708 0.483 0.895 0.883 0.691 0.142 0.992<br />
Olive pulp 0.562 0.597 0.061 0.781 0.961 0.252 0.522<br />
Enzyme × Olive pulp 0.872 0.420 0.674 0.806 0.584 0.391 0.545<br />
Table 7. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on serum<br />
biochemical parameters (Cholesterol, High density lipoprotein, Low density lipoprotein and<br />
Triglycerides) of laying hens.<br />
Blood biochemical parameters<br />
Treatment CHOL 1 Enzyme<br />
(mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl)<br />
0.00 144.63 59.875 61.25 1463.9<br />
0.09 137.88 66.125 58.37 1270.9<br />
Olive pulp<br />
0.00 156.75 62.62 64.00 1749.9 a<br />
9.00 125.75 63.37 55.62 984.9 b<br />
SEM 21.44 8.36 8.28 346.82<br />
CV 30.36 26.55 27.67 50.73<br />
Source of variation Probability<br />
Enzyme 0.758 0.469 0.734 0.588<br />
Olive pulp 0.173 0.930 0.331 0.047<br />
Enzyme<br />
pulp<br />
× Olive 0.810 0.815 0.964 0.776<br />
1-Cholesterol, triglycerides, high density lipoprotein, low density lipoprotein; a-b means within a column<br />
(within main effects) with no common superscript differ significantly (p < 0.05). SEM= Standard error of<br />
means.
4310 Afr. J. Biotechnol.<br />
Table 8. Effect of dietary inclusion of guar meal (0, 25 and 50 g/kg) and enzyme supplementation (0 and 0.4 g/kg) on<br />
differentiable counts of white blood cells (%, heterophil, lymphocyte, monocyte, eosinophil and basophil) of laying hens.<br />
Treatment<br />
Enzyme (g /100 g)<br />
Heterophil<br />
Differentiable counts of white blood cells (%)<br />
Lymphocyte Monocyte Eosinophil Basophil<br />
0.00 28.50 67.37 1.00 0.37 2.75<br />
0.09 25.12 70.87 1.37 0.00 2.62<br />
Olive pulp (g /100 g)<br />
0.00 23.12 72.50 1.25 0.25 2.87<br />
9.00 30.50 65.75 1.12 0.12 2.50<br />
SEM 3.72 3.83 0.48 0.28 1.05<br />
CV 27.72 11.08 81.53 298.14 78.38<br />
Source of variation Probability<br />
Enzyme 0.381 0.378 0.453 0.204 0.907<br />
Olive pulp 0.070 0.103 0.800 0.662 0.728<br />
Enzyme × Olive pulp 0.317 0.216 0.453 0.662 0.563<br />
Table 9. Effect of dietary inclusion of guar meal (0, 25 and 50 g/kg) and enzyme supplementation (0 and 0.4g/kg) on<br />
egg quality (egg index, yolk index, Haugh unit, eggshell weight and eggshell thickness) of laying hens.<br />
Egg quality characteristics<br />
Treatment<br />
Enzyme (g /100 g)<br />
Egg index Yolk index Haugh unit Egg shell weight Egg shell thickness<br />
0.00 74.60 41.83 70.06 6.87 40.00<br />
0.09 74.97 42.03 70.78 6.48 37.40<br />
Olive pulp (g /100 g)<br />
0.00 74.23 40.56 b 68.41 6.53 38.60<br />
9.00 75.34 43.29 a 72.44 6.82 38.80<br />
SEM 1.38 2.39 2.90 0.30 1.78<br />
CV 4.36 5.65 9.73 11.65 10.91<br />
Source of variation Probability<br />
Enzyme 0.798 0.853 0.817 0.281 0.187<br />
Olive pulp 0.455 0.020 0.207 0.423 0.917<br />
Enzyme × Olive pulp 0.946 0.092 0.312 0.612 0.131<br />
a-b Means within a column (within main effects) with no common superscript differ significantly (P < 0.05); SEM, standard<br />
error of means.<br />
hens fed with control and the OP-included diets. There<br />
was no significant effect of enzyme supplementation on<br />
the analyzed blood biochemical parameters (p > 0.05).<br />
Differentiable count of white blood cells was not<br />
significantly affected by dietary OP inclusion and enzyme<br />
supplementation (Table 8). There is no record in the<br />
literature presenting the effects of diet inclusion of OP on<br />
blood biochemical parameters of laying hens.<br />
As it is shown in Table 9, among the egg quality cha-<br />
racteristics (egg index, yolk index, haugh unit, eggshell<br />
weight and eggshell thickness); only yolk index was<br />
affected by dietary OP inclusion. Including OP in the diets<br />
of hens increased the yolk index (p ≤ 0.05); however,<br />
there was no significant difference between the other egg<br />
quality traits in hens fed with control and the OP-included<br />
diets. There was no significant effect of enzyme supplementation<br />
on the analyzed egg quality characteristics (p ><br />
0.05). Gunawardana et al. (2009) evaluated the effects of
Rovabio (a natural mixture of enzymes produced by the<br />
organism Penicillium funiculosum), dietary energy and<br />
protein on performance, egg composition, egg solids and<br />
egg quality of commercial Leghorns in phase 2 and reported<br />
dietary protein significantly increased feed<br />
consumption but decreased yolk color. As dietary energy<br />
increased, feed consumption decreased and yolk color<br />
increased. They also found a significant interaction<br />
among dietary protein, energy and Rovabio on egg<br />
production, BW, egg mass, feed conversion and yolk<br />
solids. Egg weight of hens fed with diets supplemented<br />
with enzyme was significantly greater than that of hens<br />
fed with diets without enzyme during weeks 3 and 4.<br />
However, enzyme did not significantly influence average<br />
egg weight (Gunawardana et al., 2009). In the research<br />
study by Pirgozliev et al. (2010) the yolk color of the birds<br />
receiving xylanase was darker than the yolk of the birds<br />
given the control diet. In addition, birds receiving<br />
xylanase had a significantly higher weight gain than those<br />
fed on the unsupplemented diet. Dirty and cracked eggs<br />
in their study were not affected by xylanase (Pirgozliev et<br />
al., 2010). The improvement in feed conversion for<br />
Lohmann Brown-Lite laying hens fed with enzyme<br />
cocktail (providing 7 U/g of α-1, 6-galactosidase and 22<br />
U/g of β-1, 4-mannanase) supplemented diet was accompanied<br />
by an increase in albumen weight and percentage<br />
and a decrease in the percentage of yolk and the ratio of<br />
yolk to albumin in the eggs (Han et al., 2010).<br />
Conclusions<br />
In conclusion, OP can be inserted in laying hens diets up<br />
to 9% with no adverse effect on bird's performance.<br />
Dietary inclusion of OP would have beneficial effect on<br />
laying hens' performance in terms of egg weight, yolk<br />
index and blood level of triglycerides. In this investigation,<br />
dietary supplementation with a cocktail enzyme with<br />
mainly xylanase activity improved FCR in hens.<br />
Acknowledgment<br />
Appreciation is expressed to Research Sector of Islamic<br />
Azad University, Aligudarz Branch, Iran, for financial<br />
support of this research.<br />
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availability. Poult. Sci. 77: 83-89. PMID: 9469756.<br />
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Palestine.<br />
Rabayaa E, Abo Omar JM, Othman RA (2001). Utilization of olive pulp<br />
in broiler rations. An-Najah Univ. J. Res. 15: 134-144.<br />
Salobir J (1998). Effect of xylanase alone and in combination with βglucanase<br />
on energy utilization, nutrient utilization and intestinal<br />
viscosity of broilers fed diets based on two wheat samples. Arch.<br />
Geflügelk. 62: 209-213.<br />
Salih ME, Classen HL, Campbell GL (1991). Response of chickens feed<br />
on hull-less barley to dietary β-glucanase at different ages. Anim.<br />
Feed Sci. Technol. 33: 139-149.<br />
Smits CHM, Annison G (1996). Non-starch polysaccharides in broiler<br />
nutrition: towards a physiological valid approach to their<br />
determination. Wolrd’s Poult. Sci. J. 52: 203-221.<br />
Pirgozliev V, Bedford MR, Acamovic T (2010). Effect of dietary xylanase<br />
on energy, amino acid and mineral metabolism, and egg production<br />
and quality in laying hens. Br. Poult. Sci. 51(5): 639-647.<br />
Rosa´rio M, Domingues M (2002). Structural characterisation of<br />
underivatised olive pulp xylo-oligosaccharides by mass spectrometry<br />
using matrix-assisted laser desorption/ionisation and electrospray<br />
ionization. Rapid. Commun. Mass Spectrom. 16: 2124-2132.<br />
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formulation, and restriction on the performance of laying hens. Can.<br />
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Senkoylu N, Samli HE, Akyurek H, Okur AA, Kanter M (2009). Effects of<br />
whole wheat with or without xylanase supplementation on<br />
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Tortuero F, Riopérez J, Luisa Rodríguez M (1989). Nutritional value for<br />
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African Journal of Biotechnology Vol. 10(20), pp. 4313-4317, 16 May, 2011<br />
Available online at http://www.academicjournals.org/AJB<br />
DOI: 10.5897/AJB10.1883<br />
ISSN 1684–5315 © 2011 <strong>Academic</strong> <strong>Journals</strong><br />
Full Length Research Paper<br />
An investigation on natural radioactivity from mining<br />
industry #<br />
E. Esmeray* and M. E. Aydin<br />
Selcuk University, Environmental Engineering Department, Konya, Turkey.<br />
Accepted 25 March, 2011<br />
The environmental problem of naturally occurring radioactive materials (NORM) is omnipresent on<br />
earth and their radioactivity may become concentrated as a result of human activities. Various<br />
industries produce concentrated radioactivity in their by-products. Mining originating industries such<br />
as the coal industries, petroleum extraction and processing and natural gas, mining enrichment waste,<br />
phosphate, etc have been well known and widely investigated. The Environmental Protection Agency<br />
(EPA) describes NORM wastes from the mining and processing of three categories of metals: Rare<br />
earth metals, special application metals and metals produced in bulk quantities by industrial extraction<br />
processes. Moreover, NORM has a lot of negative effects on the natural resources (water supplies,<br />
soils, air, etc.) and living organisms (human, animals, plants, microorganisms, etc.). In this study, we<br />
investigated NORM levels that originated from mining industry and the concentration of NORM in<br />
drinking water supplies. NORM parameter of gross alpha and gross beta were also in this study,<br />
seasonal changes in gross alpha and gross beta were investigated. The obtained results showed that,<br />
natural activity concentrations of α- and β-emitting radionuclides in all water samples did not exceed<br />
World Health Organisation (WHO) and Turkish Standards of Drinking Water (TS 266) recommended<br />
levels (Table 1). Concentrations ranging from 0.0062 Bq/l to 0.79 Bq/l and from 0.004 to 0.18 Bq/l were<br />
observed for the gross α and gross β activities, respectively. For all samples, the gross β activities were<br />
higher than the corresponding gross α activities.<br />
Key words: Natural radioactivity, mining industries, gross alpha, gross beta.<br />
INTRODUCTION<br />
Radiation of natural origin at the earth’s surface consists<br />
of two components namely cosmic rays and radiation<br />
from the radioactive nuclides in the earth’s crust. The<br />
latter component is the terrestrial radiation, which mainly<br />
originates from the so-called primordial radioactive<br />
nuclides that were made in the early stage of the formation<br />
of the solar system. Uranium, thorium and potassium<br />
are, however, the main elements contributing to natural<br />
terrestrial radioactivity (UNSCEAR, 2000).<br />
Naturally-occurring radioactive material (NORM) is the<br />
*Corresponding author. E-mail: eesmeray@selcuk.edu.tr. Tel:<br />
+90-332-2232080, +90-542-5629808.<br />
#This study has been presented as poster at the VIth<br />
International Conference of Modern Management of Mine<br />
Producing, Geology and Environmental Protection (SGEM),<br />
Albena-Varna, Bulgaria, 12-16 June 2006.<br />
term used to describe materials that contain radionuclides<br />
that exist in the natural environment. Long-lived<br />
radioactive elements of interest include uranium, thorium<br />
and potassium and any of their radioactive decay<br />
products, such as radium and radon. These elements<br />
have always been present in the earth’s crust and within<br />
the tissues of all living species. Studies of terrestrial<br />
natural radiation are of great importance for various<br />
reasons. They serve as useful tracers for atmospheric<br />
variation studies. It is usually realized that natural environmental<br />
radiation mainly depends on geological and<br />
geographical conditions. Also, an understanding of<br />
natural radioactivity was used to determine the age of the<br />
earth using the radioactive decay of 238 U to 206 Pb and<br />
235 U to 207 Pb. Each isotope has, however, a unique decay<br />
rate so that the ratio of the number of daughter atoms to<br />
parent isotopes in rocks and minerals depends on the<br />
time which has elapsed since the system became closed<br />
(Jamal, 2002). Many of the mining and processing
4314 Afr. J. Biotechnol.<br />
Figure 1. A map of the study area and location of the sampling points.<br />
(physical, chemical and termal): uranium, thorium, gold,<br />
silver, copper, nickel, iron, aluminium, molybdenum, tin,<br />
titanium, vanadium, etc. industries such as aforementioned<br />
cause natural radiation.<br />
Extraction of minerals<br />
This was undertaken by conventional underground or<br />
open pit mining techniques or acid leaching. Mineral processing<br />
can involve dry techniques including electrostatic<br />
or magnetic separation or wet techniques such as acid or<br />
alkaline leaching and chemical flotation or electrical or<br />
furnace smelting. All of these processes can affect the<br />
concentrations of radionuclides in both waste and product<br />
streams.<br />
Mineral sands production<br />
Mineral sands production (including ilmenite, leucoxene,<br />
rutile, zircon, monazite and xenotime) leads to several<br />
waste materials from primary processing of ore. Some of<br />
these waste materials are returned to the mined out pit<br />
for disposal.<br />
In copper production<br />
Wastes containing NORM arise in tailings from the<br />
flotation stage and furnace slags from the smelting stage.<br />
In tin and tantalum production, dry and wet separation<br />
stages produce tailings slurry that is further treated and<br />
disposed off in a tailings dam close to the mine site.<br />
Tantalum products also contain low levels of NORM. Iron<br />
and steel production wastes include blast furnace slags<br />
and dusts and fumes from the sinter plant and off gas<br />
cleaning in the blast furnace operation.<br />
In phosphate ore processing<br />
NORM is found in tailings from fertiliser production that<br />
are normally used as backfill at the mine site and also in<br />
fertilizer product at levels which do not cause any<br />
significant increase in the uranium and thorium levels in<br />
soil treated with fertilizer. In phosphoric acid production,<br />
the majority of the NORM is left in the phosphogypsum,<br />
which can be stockpiled on site or disposed of as landfill.<br />
Phosphogypsum may also be used in fertilizer, soil<br />
conditioner, building material (plasterboard, cement<br />
aggregate) and in road construction (ARPANSA, 2004).<br />
A lot of studies can be found on natural radioactivity in<br />
earth soil and water samples. In this study, natural radioactivities<br />
in water samples were investigated from mining<br />
origin.<br />
MATERIAL AND METHODS<br />
Site description<br />
The study area at the Konya city in Turkey is shown in Figure 1. It is
Table 1. Drinking water standards of gross alpha and gross beta (WHO, 2006; TS266, 2005).<br />
Radionuclide WHO (WHO,2006) Institute of Turkish Standards, (ITS 266, 2005)<br />
Gross alpha 0.01 Bq/l (2.7 pCi/l) 0.0037 Bq/l (0.1 pCi/l)<br />
Gross beta 0.1 Bq/l (27 pCi/l) 0.037 Bq/l (1 PCi/l)<br />
in the Turkey’s central Anatolia region. Konya has a lot of mine<br />
resources. These are barite, bentonite, mercury, porcelain clay,<br />
clay, limestone, chrome, meerschaum, marble, talcum, asbestos,<br />
lignite, etc. (MTA, 2010). Naturally, these mining resources and<br />
their processing activity lead to natural radiation on nearest water<br />
supplies and soil.<br />
Sampling<br />
In this study, gross alpha and gross beta activities were measured<br />
for the samples which have been taken from eight different drinking<br />
water resource (1 surface water, 6 well water, 1 tap water) in Konya<br />
city for two different seasons (winter and summer). Sampling points<br />
are shown in Figure 1. The values of measurements of the samples<br />
which were taken in the Centrum of Konya show that there is no<br />
natural radiation problem in water resources. Although, some<br />
measurements result to higher gross alpha parameter according to<br />
TS 266 (Turkish Standards of drinking water), but are still lower<br />
than WHO standards, so this causes no problem. Furthermore,<br />
gross beta activity values are fairly low according to TS 266 also,<br />
WHO standards, so beta radioactivity causes no danger (TS 266,<br />
2005). Drinking water standards of gross alpha and gross beta are<br />
shown in Table 1.<br />
Determination of natural radioactivity<br />
Primarily, the aim of this study was to determine the level of natural<br />
radioactivity in different drinking water supplies in Konya city. Eight<br />
sampling sites for drinking water supplies have been selected in<br />
Konya city. Figure 1 gives a detailed description of all sampling<br />
sites. Sample bottles were rinsend with pure water, then filled<br />
completely to minimize headspace. The sample bottles were<br />
cleaned using a modified procedure before collection. (Laxen and<br />
Harrison, 1981). Whole drinking water samples were collected by 1<br />
L capacity of polypropylene bottles and acidified pH 2 with pure<br />
HNO3 until analysis. Measurements of radioactivity level in all water<br />
samples were performed by the method of EPA using the gross<br />
alpha and gross beta counting system Epa method 900 (EPA,<br />
1980). An aliquot of a preserved drinking water sample is evaporated<br />
to a small volume and transferred quantitatively to a tared<br />
2-inch stainless steel counting planchet. The sample residue is<br />
dried to constant weight, re-weighed to determine dry residue<br />
weight, then, counted for alpha and/or beta radioactivity. Counting<br />
efficiencies for both alpha and beta particle activities were selected<br />
according to the amount of sample solids from counting efficiency<br />
vs sample solids standard curves (EPA, 1980). Gross α and βactivities<br />
were determined by means of a Tennelec LB 1000 low<br />
background proportional counter subsequently (Tennelec, 1989).<br />
The counter was calibrated with 241 Am and 90 Sr– 90 Y standards by<br />
using potentials of 1100 and 1600 V corresponding to the α and βplateaus,<br />
respectively. The total yields obtained for the α and βcountings<br />
were 16 and 34%, respectively.<br />
Calculations<br />
Equation 1: The following equation was used to calculate the alpha<br />
radioactivity:<br />
Esmeray and Aydin 4315<br />
Where, A is the net alpha count rate (gross alpha count rate minus<br />
the background count rate) at the alpha voltage plateau; C is the<br />
alpha efficiency factor read from the graph of efficiency versus mg<br />
of water solids per cm 2 of planchet area, (cpm/dpm); V is the<br />
volume of sample aliquot, (ml) and 2.22 is the conversion factor<br />
from dpm/pCi (EPA, 1980).<br />
Also, equations that were used to calculate beta radioactivity are<br />
given as:<br />
Equation 2: Is used if there are no significant alpha counts when<br />
the sample is counted at the alpha voltage plateau, the beta activity<br />
can be determined from the following equation:<br />
Where, B is the net beta count rate (gross count rate minus the<br />
background count rate at the beta voltage plateau); D is the beta<br />
efficiency factor read from the graph of efficiency versus mg of<br />
water solids per cm 2 of planchet area, (cpm/dpm); V is the volume<br />
of sample aliquot, (ml) and 2.22 is the conversion factor from<br />
dpm/pCi (EPA, 1980).<br />
Equation 3: Is used when counting beta radioactivity in the<br />
presence of alpha radioactivity by gas-flow proportional counting<br />
systems (at the beta plateau), alpha p<strong>articles</strong> are also counted.<br />
Since alpha p<strong>articles</strong> are more readily absorbed by increasing<br />
sample thickness than beta p<strong>articles</strong>, the alpha/beta count ratios<br />
vary with increasing sample thickness.<br />
Therefore, it is necessary to prepare a calibration curve by<br />
counting standards containing americium-241 with increasing<br />
thickness of solids on the alpha plateau and then, on the beta<br />
plateau, plotting the ratios of the two counts versus density<br />
thickness.<br />
The alpha amplification factor (E) from that curve is used to<br />
correct the amplified alpha count on the beta plateau. When<br />
significant alpha activity is indicated by the sample count at the<br />
alpha voltage plateau, the beta activity of the sample can be<br />
determined by counting the sample at the beta voltage plateau and<br />
calculating the activity from the following equation:<br />
Where, B (as defined in equation 2); D = (as defined equation 2); A<br />
= (as defined in equation 2); E is the alpha amplification factor read<br />
from the graph of the ratio of alpha counted at the beta<br />
voltage/alpha counted at the alpha voltage versus sample density<br />
(1)<br />
(2)<br />
(3)
4316 Afr. J. Biotechnol.<br />
Table 2. Winter season, activity concentrations of gross alpha and gross beta in water<br />
supplies samples in Konya city. (Esmeray and Aydın,2006)<br />
Sample location α Radioactivity (Bq/l) β Radioactivity (Bq/l)<br />
Apa Dam 0.072 ± 0.0023 0.20 ± 0.004<br />
Anit 0.079 ± 0.0026 0.08 ± 0.004<br />
Kongaz II 0.007 ± 0.0021 0.06 ± 0.004<br />
Kas III 0.0062 ± 0.0026 0.05 ± 0.004<br />
Forestry directorate 0.052 ± 0.0025 0.10 ± 0.004<br />
City center (KOSKI) 0.007 ± 0.0020 0.01 ± 0.003<br />
Marangozlar 0.014 ± 0.0022 0.09 ± 0.004<br />
Meram II 0.0077 ± 0.0011 0.18 ± 0.004<br />
Table 3. Spring Season, Activity concentrations of gross alpha and gross beta in<br />
water supplies samples in Konya City. (Esmeray and Aydın,2006)<br />
Sample location α Radioactivity (Bq/l) β Radioactivity (Bq/l)<br />
Apa Dam 0.018 ± 0.0019 0.11 ± 0.004<br />
Anit 0.035 ± 0.0023 0.07 ± 0.003<br />
Kongaz II 0.006 ± 0.0019 0.09 ± 0.004<br />
Kas III 0.0012 ± 0.0018 0.04 ± 0.003<br />
Forestry directorate 0.008 ± 0.0023 0.13 ± 0.004<br />
City center (KOSKI) 0.006 ± 0.0017 0.04 ± 0.003<br />
Marangozlar 0.065 ± 0.0028 0.18 ± 0.004<br />
Meram II 0.007 ± 0.0021 0.18 ± 0.004<br />
thickness; V is the volume of sample aliquot, (ml) and 2.22 is the<br />
conversion factor from dpm/pCi (EPA, 1980).<br />
Finally, all results were converted between units (pCi/l) to (Bq/l)<br />
and used for experimental results. (Esmeray, 2005)<br />
RESULTS AND DISCUSSION<br />
Activity concentrations of gross alpha and gross beta in<br />
drinking water in Konya are shown in Tables 2 and 3. As<br />
shown in Table 2, Apa Dam, Anit and Forestry Directorate’s<br />
gross alpha activity concentrations are over than<br />
the standards of ITS and nevertheless, all results of<br />
gross beta activity concentrations are permissible limits<br />
for both WHO and ITS regulations for drinking water.<br />
Only Marangozlar’s gross alpha activity concentration is<br />
more than ITS standards, as shown in Table 3, but all<br />
other gross alpha activity concentrations are permissible<br />
limits for both WHO and ITS regulations for drinking<br />
water standards. In addition, all gross beta and gross<br />
alpha activity concentrations are generally good and<br />
acceptable for WHO and ITS’s recommended levels of<br />
drinking water (Table 1).<br />
All experimental results show that, the gross alpha<br />
activity ranges between (0.0062 (Meram II) and 0.79<br />
(Anıt) Bq.l -1 -winter season) (0.0012 (Kas III) and 0.065<br />
(Koski) Bq.l -1 -spring season). Besides, gross beta activity<br />
lies between (0.001(Koski) and 0.2 (Apa Dam) Bq.l -1 -<br />
winter season) (0.004 (Koski, Kas III) and 0.18 (Meram II,<br />
Marangozlar) Bq.l -1 -spring season). Activity concentrations<br />
of gross alpha and gross-beta in water supplies<br />
in Konya are shown in Table 3 (winter and spring<br />
season).<br />
As shown in Tables 2 and 3, the lowest average were<br />
0.0062 ± 0.0026 Bq/l (gross alpha) and 0.01 ± 0.003 Bq/l<br />
(gross beta) in winter and the highest seasonal average<br />
were 0.079 ± 0.0026 Bq/l (gross alpha) and 0.20 ± 0.004<br />
Bq/l (gross beta) in winter. Also, the lowest average were<br />
0.0012 ± 0.0018 Bq/l (gross alpha) and 0.04 ± 0.003 Bq/l<br />
(gross beta) in spring and the highest seasonal average<br />
were 0.065 ± 0.0028 Bq/l (gross alpha) and 0.18 ± 0.004<br />
Bq/l (gross beta) in spring. Evaporation conditions, temperature<br />
changes, solubility of radioactive materials can<br />
be causes of seasonal changes in gross alpha and gross<br />
beta activities in Konya. Besides, the concentration of<br />
radioactivity increases in spring and summer due to high<br />
evaporation rates and the increase of solubility of salts<br />
due to the higher temperature of the water (Hosseini,<br />
2007). Additionally, high levels of natural radioactivity in<br />
drinking water are accompanied with potential health<br />
risks for the population by increasing the radiation dose.<br />
Hence, water must be purified before using it. There are<br />
several methods known to remove radioactivity from water
such as aeration to remove granular activated<br />
carbon (GAC), ion exchange methods (IX), reverse<br />
osmosis (RO) to remove “Gross alpha and gross beta,<br />
uranium etc.” and various adsorption methods to remove<br />
other natural radioactivity sources (Esmeray and Aydin,<br />
2008).<br />
Conclusions<br />
The levels of gross alpha and gross beta activity in Konya<br />
region located in central anatolia of Turkey have been<br />
studied. Experimental results observed show that, gross<br />
alpha and gross beta activity concentrations are lower<br />
than standards of WHO and ITS (WHO, 2006; ITS,<br />
2001). This kind of the study must be continued for other<br />
parts of Konya and Turkey. The results and data obtained<br />
in this work are a baseline which can be used to evaluate<br />
possible future changes.<br />
ACKNOWLEDGEMENT<br />
The authors thank the Selcuk University Research Fund<br />
(BAP) for its financial support of the work undertaken<br />
here (2004/70), that this paper has been derived from a<br />
part of MSc. thesis of Ertugrul ESMERAY.<br />
REFERENCES<br />
ARPANSA (2004). Naturally-Occurring Radioactive Material (NORM) in<br />
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EPA (Environmental Protection Agency) (1980). Prescribed Procedures<br />
for Measurement of Radioctivity in Drinking Water, (EPA/600/4-80-<br />
032),<br />
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Esmeray E (2005).Natural radioactivity in various water supplies of<br />
Konya, Turkey, MSc. Thesis, Selcuk university graduate school of<br />
natural and applied sciences, Konya.<br />
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Management of Mine Producing, Geology and Environmental<br />
Protection (SGEM 2006), Albena-Varna, Bulgaria, 12-16 June.<br />
Esmeray E, Aydın ME (2008). Comparison of natural radioactivitiy<br />
removal methods for drinking water supplies, J. Int. Environ. Appl.<br />
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Hosseini SA (2007). Radioactivity monitoring in drinking water of<br />
Zahedan, Iran, Iran. J. Radiat. Res. 5(2): 97-100.<br />
ITS (2001). Institution of Turkish Standards. Annual Progress Report.<br />
Jamal A-J (2002). Population doses from terrestrial gamma exposure in<br />
areas near to old phosphate mine, Russaifa, Jordan, Radiation<br />
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Laxen DPH, Harrison RM (1981). Cleaning methods for polyethene<br />
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MTA (2010). General directorate of mineral research and expolaration<br />
web site, mineral maps of provinces chapter,<br />
http://www.mta.gov.tr/v1.0/turkiye_maden/il_maden/pdf_2010/konya.pdf<br />
Tennelec, Inc, (1989). Manual Phoswich low Background<br />
Alpha/Beta/Gammacounting system. Oak Ridge, USA, 1.<br />
TS 266 (2005). Turkish Drinking Water Standards.<br />
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Atomic Radiations) (2000). Sources and effects of ionizing radiation.<br />
Report to the General Assembly, United Nations, New York.<br />
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World Health Organization.
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