articles - Academic Journals

African Journal of
Biotechnology
Volume10 Number 20 16 May, 2011
ISSN 1684-5315

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George Nkem Ude, Ph.D
Plant Breeder & Molecular Biologist
Department of Natural Sciences
Crawford Building, Rm 003A
Bowie State University
14000 Jericho Park Road
Bowie, MD 20715, USA
N. John Tonukari, Ph.D
Department of Biochemistry
Delta State University
PMB 1
Abraka, Nigeria
Prof. Dr. AE Aboulata
Plant Path. Res. Inst., ARC, POBox 12619, Giza,
Egypt
30 D, El-Karama St., Alf Maskan, P.O. Box 1567,
Ain Shams, Cairo,
Egypt
Dr. S.K Das
Department of Applied Chemistry
and Biotechnology, University of Fukui,
Japan
Prof. Okoh, A. I
Applied and Environmental Microbiology Research
Group (AEMREG),
Department of Biochemistry and Microbiology,
University of Fort Hare.
P/Bag X1314 Alice 5700,
South Africa
Dr. Ismail TURKOGLU
Department of Biology Education,
Education Faculty, Fırat University,
Elazığ,
Turkey
Prof T.K.Raja, PhD FRSC (UK)
Department of Biotechnology
PSG COLLEGE OF TECHNOLOGY (Autonomous)
(Affiliated to Anna University)
Coimbatore-641004, Tamilnadu,
INDIA.
Dr. George Edward Mamati
Horticulture Department,
Jomo Kenyatta University of Agriculture
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Bahnay, Al-bagour, Menoufia,
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Department of Food Science & Biotechnology,
Kyungpook National University
Daegu 702-701,
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DoD Biotechnology High Performance Computing
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Dr. Muhammad Akram
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Hamdard Al-Majeed College of Eastern Medicine,
Hamdard University,
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Dr. M.MURUGANANDAM
Departtment of Biotechnology
St. Michael College of Engineering & Technology,
Kalayarkoil,
India.
Dr. Gökhan Aydin
Suleyman Demirel University,
Atabey Vocational School,
Isparta-Türkiye,
Dr. Rajib Roychowdhury
Centre for Biotechnology (CBT),
Visva Bharati,
West-Bengal,
India.
Dr.YU JUNG KIM
Department of Chemistry and Biochemistry
California State University, San Bernardino
5500 University Parkway
San Bernardino, CA 92407

Editorial Board
Dr. Takuji Ohyama
Faculty of Agriculture, Niigata University
Dr. Mehdi Vasfi Marandi
University of Tehran
Dr. FÜgen DURLU-ÖZKAYA
Gazi Üniversity, Tourism Faculty, Dept. of Gastronomy
and Culinary Art
Dr. Reza Yari
Islamic Azad University, Boroujerd Branch
Dr. Zahra Tahmasebi Fard
Roudehen branche, Islamic Azad University
Dr. Tarnawski Sonia
University of Neuchâtel – Laboratory of Microbiology
Dr. Albert Magrí
Giro Technological Centre
Dr. Ping ZHENG
Zhejiang University, Hangzhou,
China.
Prof. Pilar Morata
University of Malaga
Dr. Greg Spear
Rush University Medical Center
Dr. Mousavi Khaneghah
College of Applied Science and
Technology-Applied Food Science, Tehran,
Iran.
Prof. Pavel KALAC
University of South Bohemia,
Czech Republic.
Dr. Kürsat KORKMAZ
Ordu University, Faculty of Agriculture,
Department of Soil Science and Plant nutrition
Dr. Tugay AYAŞAN
Çukurova Agricultural Research Institute, PK:01321,
ADANA-TURKEY.
Dr. Shuyang Yu
Asistant research scientist, Department of Microbiology,
University of Iowa
Address: 51 newton road, 3-730B BSB bldg.Tel:+319-335-
7982, Iowa City, IA, 52246,
USA.
Dr. Binxing Li
E-mail: Binxing.Li@hsc.utah.edu
Dr Hsiu-Chi Cheng
National Cheng Kung University and Hospital.
Dr. Kgomotso P. Sibeko
University of Pretoria,
South Africa.
Dr. Jian Wu
Harbin medical university ,
China.

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African Journal of Biotechnology
International Table of Contents: Journal Volume of Medicine 10 Number and Medical 20 16 May, Sciences 2011
ences
Research Articles
GENETICS AND MOLECULAR BIOLOGY
ARTICLES
Highly heterogeneous Ty3/Gypsy-like retrotransposon sequences in the
genome of cassava (Manihot esculenta Crantz) 3951
Michael A. Gbadegesin and John R. Beeching
Genetics of trichomes and its association with fibre and agronomic traits
in cotton 3964
Nausherwan Nobel Nawab, Qamar Shakil, Shahid Niaz, Muhammad
Munir Iqbal, Muhammad Ahsan Asif and Iftikhar Ahmad Khan
Quick and sensitive determination of gene expression of fatty acid synthase
in vitro by using real-time polymerase chain reaction amplification (PCR) 3973
Zuo-Hua Liu, Jin-Long Yang, Fei-Yun Yang, Ding-Biao Long, Ren-Yong Jia,
Kang-Cheng Pan, and Dai-Wen Chen
Characterization of a chestnut FLORICAULA/LEAFY homologous gene 3978
Tao Liu, Yun-qian Hu and Xiao-xian Li
Overexpression of a foxtail millet Acetyl-CoA carboxylase gene in maize
increases sethoxydim resistance and oil content 3986
Zhigang Dong, Huji Zhao,Junguang He, Junling Huai, Heng Lin, Jun Zheng,
Yunjun Liu and Guoying Wang

Table of Contents: Volume 10 Number 20 16 May, 2011
ences
ARTICLES
Variation in nodulation and growth of groundnut (Arachis hypogaea L.)
on oxisols from land use systems of the humid forest zone in southern
Cameroon 3996
Laurette Ngo Nkot, Dieudonné Nwaga, Albert Ngakou, Henri Fankem and
François-Xavier Etoa
Comparison of some anthropometric and biologic parameters in two
groups of Tunisian infants (0 to 2 years) 4005
M. Kamel, A. Barkia, M. Hamdaoui, H. Ketata, M. Kassis, M. Nasri and
A. Aouidet
Isolation and molecular characterization of RcSERK1: A Rosa caninagene
transcriptionally induced during initiation of protocorm-like bodies 4011
Xu Kedong, Liu Qinglin, Yang Huifang, Zeng Li, Dong Lili, Liu Fengluan,
Bi Ling, Ma Nan and Zhao Liangjun
Molecular cloning and expression of the luciferase coding genes of
Vibrio fischeri 4018
Golnaz Asaadi Tehrani, Sina Mirzaahmadi, Mojgan Bandehpour, Faramarz Laloei,
Akram Eidi, Toraj Valinasab and Bahram Kazemi
Study of polymorphism of leptin gene receptor in Mazandaran fowls 4024
H. A. Abbasi, S. Gharahveysi and R. Abdullahpour
Detection of genomic instability in hypospadias patients by random
amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) method 4029
Adnan A. Alsulaimani, Nabil S. Awad and Adel E. El-Tarras
cDNA, genomic sequence cloning and overexpression of cytochrome c
oxidase gene (COX6b1) from the Ailuropoda melanoleuca 4033
Jun Li, Yiling Hou, Wanru Hou, Bing Sun and Xiulan Su

ences
Table of Contents: Volume 10 Number 20 16 May, 2011
PLANT AND AGRICULTURAL TECHNOLOGY
ARTICLES
Effect of plant growth regulators (PGRs) on micropropagation of a
vulnerable and high value medicinal plant Hedychium spicatum 4040
Dinesh Giri and Sushma Tamta
Genotype-dependent responses of wheat (Triticum aestivum L.) seedlings
to drought, UV-B radiation and their combined stresses 4046
Liheng He, Xiaoyun Jia, Zhiqiang Gao and Runzhi Li
The effects of the regulated deficit irrigation on yield and some yield
components of common bean (Phaseolus vulgaris L.) under semi-arid
conditions 4057
Mehmet Simsek, Nuray Comlekcioglu and Irfan Ozturk
Molecular cloning of a novel GSK3/shaggy-like gene from Triticum
monococcum L. and its expression in response to salt, drought and other
abiotic stresses 4065
Yang Xian-Guang, Fan Jin-Yu and Deng Chuan-Liang
Some quality traits and neurotoxin β-N-oxalyl-L-α,β-diaminopropionic acid
(β-ODAP) contents of Lathyrus sp. cultivated in Turkey 4072
Ugur Basaran, Ozlem Onal Asci, Hanife Mut, Zeki Acar, Ilknur Ayan
Optimization of a plant regeneration protocol for broccoli 4081
Ke. Huang, Qiuyun Wu, Juncheng Lin and Jingui Zheng
Evaluation of bread wheat genotypes for salinity tolerance under saline
field conditions 4086
Munir Ahmad, Muhammad Munir, Armghan Shahzad, M. Shahid Masood
and Muhammad Iqbal

Table of Contents: Volume 10 Number 20 16 May, 2011
ences
ARTICLES
Antioxidant enzymes activities during secondary somatic embryogenesis in
Persian walnut (Juglans regia L.) 4093
M. Jariteh, H. Ebrahimzadeh, V. Niknam, K. Vahdati and R. Amiri
Sugar beet genotype effect on potential of bioethanol production using
Saccharomyces cerevisiae fermentation 4100
Parviz Mehdikhani, Hrachya Hovsepyan and Mahmood Rezazadeh Bari
Modeling and experiment to threshing unit of stripper combine 4106
Xu Lizhang and Li Yaoming
ENVIRONMENTAL BIOTECHNOLOGY
Yield responses of forage sorghums to salinity and irrigation frequency 4114
Siti Aishah, H. A. R. Saberi, R. A. Halim and A. R. Zaharah
Isolation and characterization of a novel sulfur-oxidizing
chemolithoautotroph Halothiobacillus from Pb polluted paddy soil 4121
Jiyan Shi, Huirong Lin, Xiaofeng Yuan and Yidong Zhao
Biomonitoring of some heavy metal contaminations from a steel plant by
above ground plants tissue 4127
M. Ataabadi, M. Hoodaji and P. Najafi
Isolation, purification and identification of bacteria from the shoes worn
by children 4133
Hui Li, Changqing Zhao, Jin Zhou, Huanhuan Shao and Wuyong Chen

Table of Contents: Volume 10 Number 20 16 May, 2011
ences
ARTICLES
Analyzing the two-dimensional plot of the interannual climate variability
for detection of the climate change in the Large Karoun River Basin, Iran 4138
Narges Zohrabi, Ali Reza Massah Bavani, Hossein Sedghi and Abdol RasolTelvari
INDUSTRIAL MICROBIOLOGY
Screening and optimization of extracellular lipases by Acinetobacter species
isolated from oil oil-contaminated soil in South Korea 4147
Periasamy Anbu, Myoung-Ju Noh, Da-Hye Kim, Jun-Seok Seo, Byung-Ki Hur
and Kyeong Ho Min
Antifungal activity of selected plant leaves crude extracts against a pepper
anthracnose fungus, Colletotrichum capsici (Sydow) butler and bisby
(Ascomycota: Phyllachorales) 4157
Lucy Johnny, Umi Kalsom Yusuf and R. Nulit
Isolation, identification and application in lignin degradation of an
ascomycete GHJ-4 4166
Huiju Gao, Yanwen Wang, Wenting Zhang, Weile Wang and Zhimei Mu
Improved taxol production in Nodulisporium sylviforme derived from
inactivated protoplast fusion 4175
Kai Zhao, Lixin Sun, Xi Ma, Xiuliang Li, Xin Wang, Wenxiang Ping, Dongpo Zhou
Production of ethanol from mango (Mangifera indica L.) peel by
Saccharomyces cerevisiae CFTRI101 4183
Lebaka Veeranjaneya Reddy, Obulam Vijaya Sarathi Reddy and
Young-Jung Wee

Table of Contents: Volume 10 Number 20 16 May, 2011
ences
ARTICLES
Secretory expression of Rhizopus oryzae α-amylase in Kluyveromyces lactis 4190
Song Li, Wei Shen, Xianzhong Chen, Guiyang Shi and Zhengxiang Wang
FOOD TECHNOLOGY
Volatile compounds of maari, a fermented product from baobab (Adansonia
digitata L.) seeds 4197
Charles Parkouda, Brehima Diawara, Samuel Lowor, Charles Diako, Firibu Kwesi
Saalia, Nana T Annan, Jan S. Jensen, Kwaku Tano-Debrah and Mogens Jakobsen
Antioxidant activities of Rosmarinus officinalis L. essential oil obtained by
hydro-distillation and solvent free microwave extraction 4207
Okoh O. O., Sadimenko A. P. and Afolayan A. J.
APPLIED BIOCHEMISTRY
Effects of bagging on sugar metabolism and the activity of sugar metabolism
related enzymes during fruit development of Qingzhong loquat 4212
Zhaojun Ni, Zhen Zhang, Zhihong Gao, Linping Gu and Leifang Huang
Anti-inflammatory evaluation of immature fruit and seed aqueous extracts
from several populations of Tunisian Citrullus colocynthisSchrad
Belsem Marzouk, Zohra Marzouk, Ehsen Haloui, Manel Turki,
Abderrahman Bouraoui, Mahjoub Aouni and Nadia Fenina
MEDICAL AND PHARMACEUTICAL BIOTECHNOLOGY
Effects of artemether on the plasma and urine concentrations of some
electrolytes in rats 4226
R.O. Akomolafe, I.O. Adeoshun, J.B. Fakunle, E.O. Iwalewa, A.O. Ayoka,
O.E. Ajayi, O.M. Odeleye and B.O. Akanji

ences
ARTICLES
Expression of biological active VHH camelid single domain antibody in
transgenic tobacco 4234
Behnaz Korouzhdehy, Mehdi Dadmehr ,Issa Piri, Fatemeh Rahbarizadeh and
Mahmood Solouki
Aloe arborescens aqueous gel extract alters the activities of key hepatic
enzymes and blood concentration of triglycerides, glucose and insulin in
alloxan-induced diabetic rats 4242
Motetelo Alfred Mogale, Sogolo Lucky Lebelo, Leshweni Jeremia Shai and
Jacobus Nicholaas Eloff
Increased vascular endothelial growth factor (VEGF) expression in rats with
spinal cord injury by transplantation of bone marrow stromal cells 4249
Deshui Yu, Libo Liu, Xiaodong Zhi, Yang Cao and Gang lv
In-ovo evaluation of the antiviral activity of methanolic root-bark extract
of the African Baobab (Adansonia digitata Lin) 4256
Sulaiman, Lanre K., Oladele, Omolade A., Shittu, Ismaila A., Emikpe,
Benjamin O., Oladokun, Agnes T. and Meseko, Clement A
ENTOMOLOGY
Table of Contents: Volume 10 Number 20 16 May, 2011
Effect of gamma radiation on different stages of Indian meal moth Plodia
interpunctella Hübner (Lepidoptera: Pyralidae) 4259
Hosseinzadeh Abbas, Shayesteh Nouraddin, Zolfagharieh Hamid Reza,
Bernousi Iraj, Babaei Mohammad, Zareshahi Hasan, Ahari Mostafavi Hossein,
Fatollahi Hadi
FISHERY SCIENCE
Body composition of freshwater Wallago attu in relation to body size,
condition factor and sex from southern Punjab, Pakistan 4265
Muhammad Yousaf, Abdus Salam and Muhammad Naeem

Table of Contents: Volume 10 Number 20 16 May, 2011
ences
BIOTECHNIQUES
ARTICLES
Rhizopus stolonifer exhibits dimorphism 4269
C. O. Omoifo
Effect of extracellular calcium chloride on sporangiospore-yeast
transformation of Rhizopus stolonifer 4276
C. O. Omoifo
ANIMAL SCIENCE
Genetic variation and bottleneck in Japanese quail (Coturnix japonica)
strains using twelve microsatellite markers 4289
Hossein Emrani, Cyrus Amirinia and Mohammad Ali Radjaee Arbabe
Evaluation of the effects of bovine herpesvirus-1 VP22 gene adjuvant in
the inhibin DNA vaccine for improving follicular development and litter size
in mice 4296
Han Li, Zhen YanHong, Sang Lei, Bai LiYa, Cai KaiLai, Guo AiZhen and Yang LiGuo
Productive performance of laying hens fed wheat-based diets included olive
pulp with or without a commercial enzyme product 4303
M. Zarei, M. Ehsani and M. Torki
ENVIRONMENTAL RADIOACTIVITY
An investigation on natural radioactivity from mining industry # 4313
E. Esmeray and M.E. Aydin

African Journal of Biotechnology Vol. 10(20), pp. 3951-3963, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1315
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Highly heterogeneous Ty3/Gypsy-like retrotransposon
sequences in the genome of cassava (Manihot
esculenta Crantz)
Michael A. Gbadegesin 1 * and John R. Beeching 2
1 Department of Biochemistry, University of Ibadan, Ibadan 200005, Nigeria.
2 Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, U. K.
Accepted 2 March, 2011
The use of PCR has enabled the survey of transposable elements in many plants; thereby making the
study of their diversity and applications possible in species where the full genome sequence data are
not yet available. In the present study, we used PCR primers anchored on the conserved domain of
reverse transcriptase and endonuclease to amplify the Ty3/Gypsy-like polyprotein fragment from the
genome of cassava (Manihot esculenta Crantz). The PCR product was cloned and sequenced.
Sequence analysis of individual clones clearly identified the conserved domain of the polyprotein
enzymes and showed the cassava Ty3/Gypsy-like retrotransposon, Megyp (for Manihot esculenta
gypsy-like), sequences to be highly heterogeneous. Some Megyps clustered with other plants’
Ty3/Gypsy-like retrotransposons, while some clustered with Gypsy of Drosophila melanogaster and
Ty3-2 of Saccharomyces cerevisiae in the comparative multiple sequence analysis. This suggests that
the later belong to the retrovirus lineage of this group of elements. Southern analysis showed that, the
Megyps and analogues were highly repeated within the genomes of cassava cultivars.
Key words: Cassava, transposable-elements, retrotransposons, retroviruses, Manihot esculenta, Ty3/Gypsy.
INTRODUCTION
There are two major super-families of transposable
elements (TEs) based on their transposition intermediate
and transposition mechanisms (Finnegan, 1992). DNA
TEs (Class II elements) move by excision and reintegration
via a DNA intermediate. They transpose by a ‘cut
and paste’ mechanism mediated by a transposase that
recognises their short terminal inverted repeated
sequences (TIRs). On the other hand, retrotransposons
or retro-elements (Class I elements) move and amplify
through RNA intermediates, which are reverse transcribed
before their integration into the nuclear genome.
They have been divided into two principal groups, the
long terminal repeat (LTR) retrotransposons and the non-
LTR retrotransposons.
Non-LTR retrotransposons lack LTRs and are trans-
cribed from an internal promoter. They are subdivided
*Corresponding author. E-mail: magbadegesin@yahoo.com.
Tel: +234 2 7504769. Fax: +44 8712 564876.
into long interspersed nuclear elements (LINEs) and
short interspersed nuclear elements (SINEs). The LTR
retrotransposons are further divided into two groups
Ty1/copia and Ty3/gypsy. These were so named after the
elements first described in Saccharomyces cerevisiae
(Ty1 and Ty3) and Drosophila melanogaster (Copia and
Gypsy). Transcription of LTR retrotransposons starts at
the 5’ LTR and ends at the 3’ LTR. The LTRs usually
contain the regulatory sequences for promoting and
terminating transcription of the element.
The use of PCR primers based on the highly conserved
amino acid sequence of enzymes domains has proved
highly successful in the survey of transposons in many
plants (Flavell et al., 1992; Hirochika and Hirochika,
1993; Suoniemi et al.,1998; Vershhinin et al., 2002;
Staginnus et al., 2001). It is making the study of transposable
elements diversity, abundance and applications
possible in species where full genome sequence data are
not yet available.
Although Ty1/copia-like elements have been reported
in many higher plants, fewer Ty3/gypsy-like retrotran-

3952 Afr. J. Biotechnol.
pol
LTR gag pr
RT RNaseH endo LTR
PBS ORF3 PPT
Figure 1. Structural organisation of Ty3/gypsy-like
retrotransposons. They are bounded at their termini by long
terminal repeats (LTRs). The primer binding site (PBS) and
polypurine tract (PPT) is represented as grey rectangles. In
between PBS and PPT (coloured boxes), are the two open
reading frames (ORFs) with coding potential for the structural
and enzymatic proteins needed for the retrotransposition
cycle: the group antigenic glycoprotein (gag) domain coding
for the protein that forms the nucleocapsid core; the protease
(pr) domain encoding the proteins necessary for the
maturation of the different proteins; the reverse transcriptase
(RT) domain encoding the enzyme responsible for the
creation of a DNA copy from the genomic RNA template; the
ribonuclease H (RNaseH) domain encoding the enzyme for
degradation of RNA hybridised to the first strand DNA; the
endonuclease (endo) domain, encoding proteins necessary
for the integration of the DNA copy into the host genome.
Most of these proteins are encoded as polyproteins (pol)
sometimes with overlapping ORFs and are processed into
individual components by pr. In addition, a third open reading
frame (ORF3) encoding an env-like activity is frequently found
in Ty3/gypsy retrotransposons. The block arrow heads indicate
the position of the forward and reverse primers for the
PCR.
sposon sequences or elements have been identified in
plant species (Su and Brown, 1997). Ty3/gypsy-like
retrotransposons share common features with Ty1/copialike
elements but the order of the domains between the
two long terminal repeats (LTRs) in Ty3/gypsy–like
elements resembles those of the retroviruses (LTR-gagpr-rt-RNaseH-endo-LTR)
(Figure 1). Some members of
Ty3/gypsy superfamily also sometimes contain an
additional open reading frame (ORF3) encoding an envlike
gene.
Cassava (Manihot esculenta Crantz) is the world’s sixth
most important crop in terms of production (Mann, 1997)
and the staple food of over 500 million people in the
tropical regions of the world. It however, has been grossly
understudied. In this study we isolated, cloned, sequenced
and analysed cassava polyprotein fragment unique
to Ty3/gypsy-like retrotransposons using degenerate
PCR primers. Cassava Ty3/gypsy-like retrotransposons
have been named Megyp for M. esculenta gypsy-like.
The diversity and organization of Megyp within the
cassava genome and their relationship to those of other
plants are also analyzed. The nucleotide sequences
described here have been submitted to the Genbank
database and given the accession numbers AY946154 -
AY946199.
MATERIALS AND METHODS
Plant material and DNA isolation
Using the method of Dellaporta et al. (1983), DNA was extracted
from young leaf samples of cassava cultivars grown in the tropical
glasshouse at the University of Bath. The growth conditions include
temperature at 22 to 28°C, relative humidity of 40 to 80% and a
minimum light period of 12 h per day under day light, supplemented
with 400 W Phillips high-pressure sodium lights when necessary.
PCR Amplification of polyprotein fragment of Megyp
sequences and cloning
The PCR method used was as described by Suoniemi et al. (1998)
with some modifications as described by Gbadegesin et al. (2008).
Amplified DNA bands were gel purified (Qiagen, ‘Qiaquik’), ligated
into pGEM ® -T Easy vector (Promega) and used to transform
competent Escherichia coli DH5α according to standard procedures
(Sambrook et al., 1989).
DNA gel blot analysis
Restriction digestions of genomic DNA (5 µg each) were carried out
using buffer and reaction conditions specified by the manufacturer
(Promega). Blotting and hybridisation were performed using
standard procedures (Sambrook et al., 1989).
Sequence and phylogenetic analyses
DNA molecules were sequenced on an ABI 337 automated dye
primer sequencer using universal primers for the cloning vector.
The first line of sequence identification was by using BLASTN and
TBLASTX searches against the GenBank non-redundant database
at the default parameters (Altschul et al., 1990). The sequence
fragments were assembled using the Vector NTI program.
Consensus sequence data were aligned using CLUSTAL W
(version 1.82) (Higgins et al., 1994). The PHYLIP program package
version 3.63 (Felsenstein, 2004), available from the author at
Department of Genetics, University of Washington, Seattle,
Washington, was used for phylogenetic analysis. Consensus
NEIGHBOR–joining trees (Saitou and Nei, 1987) were derived from
equally parsimonious trees using the extended majority rule in the
CONSENSE. Unless otherwise stated, distance matrices for
phylogenetic analyses based on nucleotide sequences data were
computed using DNADIST according to the Kimura 2-parameter
model (Kimura, 1980). Trees were drawn using TREEVIEW program
version 1.6.6 available from the author, Roderic D.M. Page of
the Taxonomy Unit, Department of Zoology, University of Glasgow.
RESULTS
PCR amplification of cassava Ty3/Gypsy-like
retrotransposon polyprotein fragment, cloning and
sequence analysis
PCR was carried out as described in the materials and
methods section. The amplified products were analysed
by electrophoresis on ethidium bromide stained 0.8%

Figure 2. PCR amplification of Ty3/gypsy-like polyprotein
fragment from cassava genomic DNA. PCR product was run
on a 0.8% agarose gel stained with ethidium bromide. The
size marker (lane M) is bioline DNA 100 bp ladder, while the
PCR product is shown in the right lane
agarose gel. Approximately 1.6 kb cassava DNA was
amplified (Figure 2). Amplified DNA was purified and
cloned as described earlier.
A clone was selected at random and sequenced from
both ends using T7 and SP6 primers. The sequence was
then submitted to BLASTN and TBLASTX searches
against the GenBank non-redundant database using the
default parameters. The searches confirmed that, a
Ty3/gypsy-like polyprotein fragment had been amplified
in the PCR experiments. The cassava element was 67%
identical (within the region of the alignment) to the
Ty3/gypsy-like retrotransposon polyprotein of Olea
europaea at the amino acid sequence level (Figure 3).
Thirty-six (36) clones (named Megyp1, Megyp2….
Megy36) in total were randomly selected.
Sequence and phylogenetic analysis
The selected clones were partially sequenced in both
directions using the T7 and SP6 primers. The sequences
were assembled using the Vector NTI contig assembly
program. The NTI contig assembly allowed visualization
and removal of vector sequences. The vector free sequence
data were submitted to BLASTN and TBLASTX
searches as before. Sequencing from the 5’ end gave 26
Megyp clones with good sequences of which 20 (77%)
showed clear homology to the polyprotein of Ty3/gypsylike
retrotransposons. However, sequencing from the 3’
end gave 28 clones with good sequences of which 26
(93%) showed clear homology to the polyprotein of
Ty3/gypsy-like retrotransposons (in most cases E-value
Gbadegesin and Beeching 3953
were in the region of e -63 ). These data show that, cassava
Ty3/gypsy-like retrotransposons are more diverged at the
5’ end of the amplified polyprotein fragment compared
with the 3’ end. Overall, the use of PCR primers
anchored on RT and endonuclease domains proved
useful and efficient for the isolation and characterisation
of this group of cassava retroelements.
The deduced translations of the Megyps left and right
nucleotide sequences were obtained using ORF finder
(www.ncbi.nlm.nih.gov/gorf/) (data not shown). Twelve
(70.6%) of the clones having good left and right sequences
and clear homology to Ty3/gypsy-like retrotransposon,
contain neither a frame shift nor a nonsense
mutation, while five (29.4%) have these mutations within
the sequences analysed. While it is possible to say that
the latter group could be defective enzymes, full
sequence data would be necessary to conclude that the
former code for functional enzymes.
Two of the clones with uninterrupted open reading
frames within the left and right sequences, Megyp5 and
Megyp28, were fully sequenced. They contain no stop or
frame shift mutations within the RT-RNaseH-endonuclease
sequences analysed. The nucleotide sequences
and deduced translation of these clones are shown in
Figure 4a, b. The two shared 88% sequence identity at
the nucleotide sequence level and 89% identity at the
level of amino acid sequence. The 5’ (RT) ends of the
Megyps are more diverged than the 3’
(ENDONUCLEASE) ends and the Megyp5 and Megyp28
nucleotide sequences did not align in the first 15
nucleotide base positions (data not shown). However, the
presence of the highly conserved block YAKFSKCEF of
the RT domain characteristic of Ty3/gypsy retrotransposons
(highlighted grey in Figures 4a, b) is a quick
check and provides strong evidence for it being part of
the polyprotein sequence in all of the cassava Ty3/gypsylike
retrotransposons.
To determine the relatedness of the cassava Ty3/
gypsy–like retrotransposons to each other the nucleotide
sequences (with the primer regions removed) for the 17
Megyps (left and right fragments for 15; full ~1.6 kb
fragment sequences for Megyps 5 and 28) were aligned
using CLUSTAL W) (Higgins et al., 1994). The aligned
nucleotide sequences were used to compute a distance
matrix using DNADIST of the PHYLIP package version
3.63 (Felsenstein, 2004), according to the Kimura 2parameter
model (Kimura, 1980). Trees were then produced
using the neighbor-joining method.
This method is based on all pairwise comparisons in
which positions for which there was no sequence data,
for example, the central regions for all sequences other
than Megyp5 and 28, were treated as missing data rather
than as gaps (Felsenstein, 2004).
Using an extended majority rule in the CONSENSE
program from the PHYLIP package, a consensusunrooted
tree was derived from 100 equally parsimonious
trees. The consensus tree was drawn using TREEVIEW

3954 Afr. J. Biotechnol.
Me: 55 CKIYQRVKLEHQKPAGMLNPLPIPEWKWENVVMDFVVGLPATSNRLNSIWVIVDRLTKSA 234
C + Q+VK+EHQKPAG LNPL IPEWKWEN+ MDFVVG P ++ N+IWV+VDRLTKSA
Oe: 1535 CMVCQQVKVEHQKPAGWLNPLDIPEWKWENITMDFVVGFPKSAIGNNAIWVVVDRLTKSA 1714
Me: 235 HFIPVRSGYSVDKLAQVYVEEIIRLHGAPVSIVSDRRLQFTSRSWRSLQNAMGTRLDLST 414
HF+PV+ +S+D+LAQ+Y+++++RL G PVSIVSDR L+FTS+ W+SLQ AMGT+L+ ST
Oe: 1715 HFLPVKMTFSLDQLAQLYIKDVVRLCGVPVSIVSDRDLRFTSKFWKSLQGAMGTKLNFST 1894
Me: 415 AFHPQTDGQSER 450
A+HPQTDGQSER
Oe: 1895 AYHPQTDGQSER 1930
Figure 3. Alignment of cassava (Me) amino acid sequence with Olea europaea (Oe) amino acid sequence of partial
polyprotein gypsy-like retrotransposon (gi, 7283091). The two sequences show 67% identity and 86% homology.
(version 1.6.6) as shown in (Figure 5). Three families of
Megyps, I, II and III emerged from the phylogenetic
analysis (Figure 5). There are seven, six and four clones,
respectively in these families.
The predicted amino acid sequences of the plant
Ty3/gypsy-like polyprotein listed in Table 1 and that of
Gypsy were aligned with those of Megyps (representative
cassava Ty3/gypsy-like retrotransposons) using the
CLUSTAL W programme (1.82) and colour shaded in
GENDOC as shown in Figure 6. The alignment reveals
blocks of residues previously identified as highly conserved
(Barber et al., 1990; Kulkosky et al., 1992; Springer
and Britten, 1993; Xiong and Eickbush, 1990). There is
highly conserved block YAKFSKCEF (box a) that
includes the invariant lysine (underlined) of reverse transcriptase
(Barber et al., 1990).
The conserved TDAS motif that defined the RNase H
region in most other Ty3/gypsy-like retrotransposons
(Springer and Britten, 1993) is present in most cassava
elements as CDAS (box b). In both cases, a key activesite
aspartate (Campbell and Ray, 1993) is conserved.
Also, conserved in the two fully sequenced cassava
Ty3/gypsy POL fragments, Megyp5 and 28, is the motif
N-3-DXL (box c) known to be essential in RNase H
catalysis (Campbell and Ray, 1993).
The N-terminal DNA-binding domain of integrase
(Kedar and Khan, 1990) is revealed as a conserved X-6-
H-29-C-2-C motif (box d), from which all the cassava
elements lack the first four upstream amino acids, a
feature shared with many other published sequences of
Ty3/gypsy POL (box d, Figure 6). The highly conserved
N-terminal GLLQPLPI motif (box e) of integrase is
present in all the Megyps as homologous GMLNPLPI.
Also present in the aligned Megyps is a D-60-D-35-E
motif of integrase domain, where E is part of the 3’ primer
sequence (not included in the alignment). The D,D-35-E
motif is completely conserved in retroviral and retrotransposon
integrases and is essential for enzymatic
activity (Baker and Luo, 1994; Kulkosky et al., 1992).
Overall, the amino acid sequences of the predicted
translation of cassava Ty3/gypsy-like POL compared well
with other Ty3/gypsy elements. This therefore confirmed
them again as authentic Ty3/gypsy-like polyprotein sequences.
Three families of Megyps and other plants Ty3/gypsylike
retrotransposons emerged from the subsequent
phylogenetic analysis (Figure 7). The cassava elements
on the tree are indicated with arrowheads. These
analyses revealed a high level of heterogeneity of
Megyps among the reported plant Ty3/gypsy group retrotransposon
using a PCR based assay. There are two
monophyletic families (I and II) consisting of cassava
Ty3/gypsy-like retrotransposons. The two clades were
supported by bootstrap values of 49 and 45%,
respectively, in the extended majority rule consensus tree
(Figure 7). The third clade (III) supported by 100%
bootstrap value consists of Gypsy of Drosophila
melanogaster and the Ty3/gypsy-like retrotransposons of
Arabidopsis thaliana rAt1, Ananas comosus, Oryza
sativa, Hordeum vulgare rHv1, Lilium henryi del and one
cassava element, Megyp18 and at 63% bootstrap, a
second cassava element (Megyp 22) is included in this
clade. The association within the sequences in this clade
is very robust as shown by the high values of the
bootstrap. Surprisingly, Megyp18 associated closely with
Gypsy and Ty3-2 in clade III (Figure 7) Gypsy, like other
retrovirus-like Ty3/gypsy retrotransposons, is known to
encode env-like activity. Further studies would be
required to classify the Megyps in this grouping as
members of these endogenous retroviruses.
Study on the genomic organization and diversity of
Ty3/Gypsy-like retrotransposons in cassava cultivars
A representative cassava Ty3/gypsy-like polyprotein
fragment, Megyp5, was used to probe Southern blots of
restriction digests of genomic DNA from a range of

A
Gbadegesin and Beeching 3955
1 ctggggttggtcttgcagactttgagggaacatggcttgtatgccaagttctctaaatgt
L G L V L Q T L R E H G L Y A K F S K C
61 gagttctggctgaggagcatttcgttcttggggcatgtagtgtcagagaatggtattgag
E F W L R S I S F L G H V V S E N G I E
121 gtagaccccaagaagacaaaaactgtggctaactggcctagacccacttcagtaacagag
V D P K K T K T V A N W P R P T S V T E
181 attagaagtttcttgggtttggcaggttactacaggaggttcgttcaggacttctcaaag
I R S F L G L A G Y Y R R F V Q D F S K
241 atagtagctcctctgaccagactgaccaggaagaatcagaagtttctgtggaccgacctg
I V A P L T R L T R K N Q K F L W T D L
301 tgcgaggagagtttcgaagagcttaagaagaggttgacttcagcaccagtgttagctctg
C E E S F E E L K K R L T S A P V L A L
361 ccatctagtgatgaggactttacagtcttttgtgatgcgtcccatatgggactgggttgt
P S S D E D F T V F C D A S H M G L G C
421 gtactgatgcagaatgagagggtgatcgcttatgcttctaggcagctgaagaagcatgag
V L M Q N E R V I A Y A S R Q L K K H E
481 ttgaattaccccacacatgaccttgagatggcagcagtaatctttgtactcaagatgtgg
L N Y P T H D L E M A A V I F V L K M W
541 aggcattacctctatggggtgaaatgtgagatctttacagatcataagagcctgcagtac
R H Y L Y G V K C E I F T D H K S L Q Y
601 atcttgagtcagagggatctgaatctgaggcagaggaggtgggtggagctgctgagtgac
I L S Q R D L N L R Q R R W V E L L S D
661 tatgattgcaagattcagtatcatccgggtaaggcgaatgtcgtggcagacgccctaagc
Y D C K I Q Y H P G K A N V V A D A L S
721 cggaagtcactaggcagtctatcccacatcgcggcagagaggagaccagtggtgaaggaa
R K S L G S L S H I A A E R R P V V K E
781 ttctacaagcttattgaggaaggtctacagttggagttgtctggtacaggtgccttagtg
F Y K L I E E G L Q L E L S G T G A L V
841 gcccagatgagagtagcacccatgtttctggagcaggtggctcagaaacagcatgaggac
A Q M R V A P M F L E Q V A Q K Q H E D
901 ccggagttagtgaaggttgccaggactgttcagtcaggcaaggatagcgagtacagattc
P E L V K V A R T V Q S G K D S E Y R F
961 gacagtaaagggatcctccgctatgggagcagactatgtgtaccagatgacattgggcta
D S K G I L R Y G S R L C V P D D I G L
1021 aaaggagacattatgagagaggctcataatgcaagatacagcattcaccctggagccact
K G D I M R E A H N A R Y S I H P G A T
1081 aagatgtatcaagatttgaagaaagtttattggtggccagcgatgaagaaagaagtggca
K M Y Q D L K K V Y W W P A M K K E V A
1141 cagttcgtgtcagcctgcgaagtgtgtcagagggtgaagctggaacatcagaagccggct
Q F V S A C E V C Q R V K L E H Q K P A
1201 ggaatgcttaacccgctacctatcccagaatggaaatgggagaatatagctatggacttc
G M L N P L P I P E W K W E N I A M D F
1261 gtagtggggttaccggcggcgtccaacagagtggactccatatgggtgattgtggacaga
V V G L P A A S N R V D S I W V I V D R
1321 ctcaccaaatctgctcacttcattcctgtcaggagtggctactctgtagacaagttggcg
L T K S A H F I P V R S G Y S V D K L A
1381 caggtgtatgtagatgagatcgtcaggctgcatggggttcctgtttcgatagtgtcagat
Q V Y V D E I V R L H G V P V S I V S D
1441 agagggccccagttcacctccagattttggcggagtctgcagaatgccatgggtactagg
R G P Q F T S R F W R S L Q N A M G T R
1501 ttggatttcagtactgccttc 1521
L D F S T A F
Figure 4. (A) Nucleotide sequence and deduced translation of Megyp5. The primer sequences are
omitted. The highly conserved amino acid sequence block YAKFSKCEF of RT domain characteristic of
Ty3/gypsy retrotransposons is highlighted grey. Recognition enzyme sequences are shown in bold
face for Eco RI (underlined), Hind III (oval) and Bgl II (box) used in Southern analysis of cassava; (B)
Nucleotide sequences and deduced translations of Megyp28. Primer sequences are omitted. The
highly conserved amino acid sequence block YAKFSKCEF of RT domain characteristic of Ty3/gypsy
retrotransposons is highlighted grey. Restriction enzymes sequences are shown as detailed in Figure
4a.

3956 Afr. J. Biotechnol.
B
1 ctgaggataatattacagaccttgagggaacatggcttgtatgccaagttctccaagtgt
L R I I L Q T L R E H G L Y A K F S K C
61 gagttctggttaaggagcatatcattcttggggcatatagtgtcagagaatggaatagag
E F W L R S I S F L G H I V S E N G I E
121 gtagaccccaagaagatagaagctgtgactaactggccaagacccacctcagtgacagag
V D P K K I E A V T N W P R P T S V T E
181 atcagaagcttcttgggtttggctggctactacaggaggttcgttcaggacttctctaag
I R S F L G L A G Y Y R R F V Q D F S K
241 attgcagctcctttaaccagattaaccagaaagaatcagagattcgagtggaccgatcag
I A A P L T R L T R K N Q R F E W T D Q
301 tgtgaagaaagtttcgaagagcttaagaagaggttgacttcagcaccagtgttagctctg
C E E S F E E L K K R L T S A P V L A L
361 ccaaacagcaatgaggatttcacagtgttctgtgatgcatccagagtaggcctgggttgt
P N S N E D F T V F C D A S R V G L G C
421 gtgttgatgcagaatggtaaggtgatcgcttatgcttctagacagccgaagaggcatgag
V L M Q N G K V I A Y A S R Q P K R H E
481 ttgaattaccccacacacgacctggaaatggcagcagttatctttgccctcaagatgtgg
L N Y P T H D L E M A A V I F A L K M W
541 aggcattacctctatggggtaaaatgtgagatcttcacagatcataagagcctgcagcac
R H Y L Y G V K C E I F T D H K S L Q H
601 atcttgaaccagagagagctgaacttgaggcagaggagatgggtagaactgttgagtgac
I L N Q R E L N L R Q R R W V E L L S D
661 tacgattgcaagatccagtaccatccgggtaaggctaatgtagtagctgatgccttaagc
Y D C K I Q Y H P G K A N V V A D A L S
721 cggaaatcacttggcagtctatcccacatcacggcagagaggagaccggtggtgaaggag
R K S L G S L S H I T A E R R P V V K E
781 ttttataagctcattgaggagggtctacagatggagttgtctggtacaggtgctttgatt
F Y K L I E E G L Q M E L S G T G A L I
841 gcacagatgaaagtaacccccgtgtttctggagcaagtggctcagaaacagcacgaggac
A Q M K V T P V F L E Q V A Q K Q H E D
901 ccagagttagtgaagattgccaggactgttcagtcaggcaaagatagtgagttcagattt
P E L V K I A R T V Q S G K D S E F R F
961 gatgataaggggatcctccgctatgggaacagactatgtgtaccagatgacatcgggcta
D D K G I L R Y G N R L C V P D D I G L
1021 aaaggagacattatgagagaggctcataatgcaaggtacagtgttcaccctggagccacc
K G D I M R E A H N A R Y S V H P G A T
1081 aagatgtaccaggatctgaagggagtgtattggtggccagctatgaagagggaagtggca
K M Y Q D L K G V Y W W P A M K R E V A
1141 cagttcgtgtcagcctgcgaaatatgtcagagggtgaagctggaacatcagaagccggct
Q F V S A C E I C Q R V K L E H Q K P A
1201 ggaatgcttaacccactgccgattccagagtggaaatgggagaacatagctatggatttt
G M L N P L P I P E W K W E N I A M D F
1261 gtagtggggttaccggcaacatccaacagactagactccatatgggtgattgtggacaga
V V G L P A T S N R L D S I W V I V D R
1321 ctcaccaaatctgctcacttcatccctgttaggagcaactactctgtggataagttagcg
L T K S A H F I P V R S N Y S V D K L A
1381 caggtttatgtggatgaagttgtcaggctgcatggggtcccagtttctatagtgtcagat
Q V Y V D E V V R L H G V P V S I V S D
1441 agagggccccagttcacctccaggttttggcggagtctgcagaatgctatgggtaccagg
R G P Q F T S R F W R S L Q N A M G T R
1501 ttggatttcagtactgccttc 1521
L D F S T A F
Figure 4. Contd.

Gbadegesin and Beeching 3957
Figure 5. Phylogenetic analysis of 17 cassava Ty3/gypsy-like retrotransposons (Megyps). The tree is based on 17
nucleotide sequences of pol gene fragments (Megyps): 15 are partial sequences from the two ends of the ~1.6 kb gene
fragments, while Megyps 5 and 28 were full 1.6 kb length. This is a consensus neighbor-joining unrooted tree
constructed with the PHYLIP package from the distance matrix following the Kimura 2-parameter model (Kimura, 1980).
Bootstrap values (100 replicates) are shown.
Table 1. Sources of polyprotein amino acids sequences used in comparative
phylogenetic analysis with the 16 cassava Megyps amino acid sequences.
Locus or sequence name Source species Gi number
A. comosus Ananas comosus 2995405
O. sativa Oryza sativa 37532428
Del Lilium henryi 19442
rHv1 Hordeum vulgare 3413486
rAt1 right Arabidopsis thaliana 3413430
rAt1 left Arabidopsis thaliana 3413431
Ty3-2 Saccharomyces cerevisiae 1084606
Gypsy Drosophila melanogaster 130583
The table shows the name of Ty3/gypsy retrotransposons and the GI (Geneinfo
identifier) number of corresponding polyprotein as well as the name of the source
organisms. The romani elements are rHv1 and rAt1.

3958 Afr. J. Biotechnol.
Figure 6. Alignment of the predicted amino acid sequences for 16 polyprotein fragments of cassava Ty3/gypsy-like retrotransposons
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
was represented by either translation of full ~1.6 kb or partial sequences from the two ends of the POL fragments. Colour blocking
indicates sequence conservation. Black = 100% identity, deep grey = > 80% identity, light grey = > 60% identity and non-shaded = <
60% identity. Letters in bold orange colour below the alignment and boxed regions labelled with small letter a-e indicate the key residues
conserved in all related enzymes as explained in the text.
cassava cultivars. Following high stringency washes (0.2
X SSC, 0.1% SDS, 65°C), strong signals were observed
in all the digests (Figure 8) and the autoradiograph
required a short exposure time. This showed that, the

Figure 6. Contd.
Megyp5 sequence and its homologues were highly
repeated within these genomes. The probe contained
one each of the Bgl II, Eco RI and Hind III recognition
sites (Figure 4a), which could explain the presence of
Gbadegesin and Beeching 3959
two bands in the DNA digestions by each of these
enzymes. However, multiples of two hybridising bands
were observed for each of the three enzymes (Figure 8),
indicating that multiple copies of Megyp5 and relatives

3960 Afr. J. Biotechnol.
Figure 6. Contd.
were integrated in the genome. Many of the bands are
very strong and distinct but there are few weak ones
suggesting that Megyp5 is cross hybridising with
sequences highly homologous to the probe, represented
by the strong major bands, as well as related diverged
fragments, seen as weak signals. The cultivars showed
no clear polymorphism of hybridisation fragments with
Megyp5 probe used (Figure 8).
DISCUSSION
The detection of Ty3/gypsy-like retrotransposons using
heterologous primers based on conserved domain of RT
in PCRs has not been efficient due to the relatively high
sequence heterogeneity among these elements (Su and
Brown, 1997). The main problem with the use of these
primers alone has been in the frequent amplification of
other sequences (other retroelements, transposons and
non-transposons), in addition to the desired Ty3/gypsy
sequences. For instance, of forty four sequenced clones
following genomic DNA PCR amplification using primers
based on the conserved RT domain in Brassica sp, only
twenty were similar to any of the known transposon types
and just fifteen were Ty3/gypsy-like of all known lineages
(Alix and Heslop-Harrison, 2004). In contrast, the use of
degenerate PCR primers anchored on both the integrase
and reverse transcriptase (Suoniemi et al., 1998) has
proved a more robust and reliable tool, as the design of
these primers exploited the differences in the domainorder
between the Ty1/copia and Ty3/gypsy groups of
retrotransposons. The usefulness of these primer sets
has been confirmed here by the isolation and characterisation
of the polyprotein fragment diagnostic of
Ty3/gypsy-like retrotransposon in cassava. This has
enabled a study of diversity of this group of retrotran-

Gbadegesin and Beeching 3961
Figure 7. Comparative phylogenetic analysis of 16 cassava Ty3/gypsy-like retrotransposons (Megyps) with other eight
from other organisms. The tree is based on predicted amino acid sequences of pol gene fragments. This is a consensus
neighbor-joining unrooted tree constructed with PHYLIP package. Distance matrix used the Jones-Taylor-Thornton model
(Jones et al., 1992). Three groups of Ty3/gypsy-like retrotransposons were revealed. The cassava elements are indicated
with arrowheads. Fourteen of them clustered into two monophyletic groups but Megyp18 and Megyp22 associated with
Gypsy and Ty3-2 group. The identities of other sequences used in comparative analyses with cassava’s are as in Table 2.
Bootstrap values (100 replicates) = > 45% is shown.

3962 Afr. J. Biotechnol.
23.1
9.4
6.56
4.36
2.3
2.0
0.56
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
Bgl II
EcoR I
Hind III
Figure 8. Southern blot analysis of Ty3/gypsy-like polyprotein of 12 cassava cultivars. 10 µg of genomic DNA from each
of the cassava cultivars; lanes 1 (MGA1); 2 (MNGA2); 3 (MDOM5); 4 (MNGA19); 5 (MCOL22); 6 (CMC40); 7 (MVEN77);
8 (CG402); 9 (SM627); 10 (SM985); 11 (SM1088); 12 (CM2177) were digested with Bgl II, Eco RI or Hind III. The
digested DNAs were separated on 0.8% agarose gels, transferred to nylon membrane and hybridised with the Megyp5
probe. Hind III-digested lambda DNA was used as a DNA size marker (M).
sposons in this important food crop. This approach has
also provided better information from all the conserved
domains for better resolution of the Ty3/gypsy group than
could be afforded by the use of individual enzymatic
domains (Springer and Britten, 1993; Wright and Voytas,
1998; Xiong and Eickbush, 1990).
Alignments of the nucleotide sequences of cassava
Ty3/gypsy clones (Megyps) and subsequently, the
inferred phylogenetic tree led to the identification of
diverse members of this group of elements in cassava. In
addition, the predicted translation of Megyps, aligned with
other plant Ty3/gypsy sequences, revealed the presence
of conserved residues established to be critical for
enzymatic activity of integrase, reverse transcriptase and
RNase H (Campbell and Ray, 1993; Kedar and Khan,
1990; Baker and Luo, 1994; Kulkosky et al., 1992) and
proving that they represent authentic Ty3/gypsy-like
retrotransposon sequences and suggesting that they
were probably derived from recently active elements.
Phylogenetic analysis of cassava and other plant
Ty3/gypsy polyproteins revealed a level of heterogeneity
in cassava elements that has not been reported in many
of plant Ty3/gypsy group retrotransposons. Most cassava
Ty3/gypsy-like retrotransposons are clustered into
monophyletic sub-groups (Figure 7). The groupings were
supported by bootstrap values of 49 and 45%. The low
bootstrap values are most probably due to the
heterogeneity of the cassava sequences. Megyp18
clustered closely with Ty3-2 retroelements (Figure 7)
suggesting that, Megyp18 represents the retrovirus
lineage of Ty3/gypsy retrotransposons in the genome of
cassava.
The findings in this study also support the suggestion
23.1
9.4
6.56
4.36
2.3
2.0
0.56
that, the use of primers based on conserved domains of
RT, which have proved inefficient for the isolation of
plants Ty3/gypsy-like retrotransposons, could be the
limiting factor in the study of the diversity and heterogeneity
among plants Ty3/gypsy-like retrotransposons. In
fact, the availability of the whole genome sequence has
revealed the presence of seven families of Ty3/gypsy-like
retrotransposons in A. thaliana (Wright and Voytas, 2002).
Availability of more Ty3/gypsy-like sequences from other
plants may give a better picture of the diversity of this
group of retrotransposons among plants. In their study,
Wright and Voytas (2002) further used primers specific
for the conserved domains of RT of endogenous retroviruses
lineage of Ty3/gypsy-like retrotransposons to
make a survey of this family of retrotransposons among
plants. The PCR assay revealed that, they are almost
universally present in genome of dicots and old-world
monocots (Wright and Voytas, 2002). Their ubiquitous
nature and potential for horizontal transfer by infection
implicates these retrotransposons as important vehicles
for plant genome evolution (Wright and Voytas, 2002).
Also of interest is the fact that, Ty3/gypsy-like
retrotransposons from a single or related plant species
were clustered in a subfamily indicating that, sequence
divergence during vertical transmission has a major
influence on the evolution of this group of retrotransposons
in plants. The presence of more than one family
of Ty3/gypsy-like retrotransposons in one plant species
indicates that, the retrotransposons of a family could
evolve independently within a species without affecting
the evolution of the members of other families. Southern
hybridisation supports the diversity identified by sequencing
and highlights that, multiple copies of Megyps are

integrated in the genome of all cassava cultivars tested.
However, these cultivars have the same pattern of
hybridisation with the three different restriction enzymes
(Bgl II, Eco RI or Hind III) digestion of the genomic DNA.
This suggests that, there are no recent retrotransposition
activities among these cassava elements. However, a
unique distribution of Ty3/gypsy-like sequences was
found for each of the four basic genomes of Hordeum
genus except for the subspecies H. vulgare and H.
spontaneum that were reported to show no polymorphism
of hybridisation fragments with all the Ty3/gypsy clones
used as probe (Vershinin et al., 2002). Also, study on the
genomic organization of the Ty3/gypsy-like retrotransposons
of different oil palm species and accessions by
southern hybridisation revealed minor differences (Kubis,
et al., 2003).
ACKNOWLEDGEMENTS
The first author would like to acknowledge funding from
the Commonwealth Scholarship Commission, UK. This
publication is part of an output from a research project
funded by the United Kingdom, Department for International
Development (DFID) for the benefit of developing
countries: R8156 Crop Post-Harvest Programme.
The views expressed are not necessarily those of DFID.
This work has been carried out in compliance with the
current laws governing genetic experimentation in the
U.K.
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African Journal of Biotechnology Vol. 10(20), pp. 3964-3972, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1752
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Genetics of trichomes and its association with fibre and
agronomic traits in cotton
Nausherwan Nobel Nawab 1* , Qamar Shakil 1 , Shahid Niaz 2 , Muhammad Munir Iqbal 1 ,
Muhammad Ahsan Asif 1 and Iftikhar Ahmad Khan 1
1 Department of Plant Breeding and Genetics, University of Agriculture, Faisalabad, Pakistan.
2 Vegetable Research Institute, Faisalabad, Pakistan.
Accepted 1 April, 2011
Hairiness/trichomes act as an important insect non-preference trait. The inheritance of this trait was
studied in three cross combinations involving a common pilose parent with three sparse hairy parents
which showed a theoretical monohybrid ratio of 1:2:1, showing incomplete dominance in F2. The
positive correlation of number of trichomes with lint percentage and micronaire suggested that the
selection for pilose hairiness would be helpful in enhancing the lint percentage and fibre fineness. The
number of trichomes had no effect on the expression of the traits like number of monopodial branches,
number of sympodial branches, number of bolls, boll weight and seed cotton yield which proved no
chance of undesirable linkage of these traits with number of trichomes.
Key words: Gossypium hirsutum L., trichomes, inheritance, fibre traits, agronomic traits.
INTRODUCTION
The sustainability of cotton production worldwide has
been affected due to the piercing, sucking insect pests
and bollworms which are a serious threat to the cotton
crop. Insect pests constitute a major factor in production
in all over the cotton growing areas of the world. In recent
times, insect control has been totally based on the use of
chemical insecticides. During the 1970’s and 1980’s, the
use of insecticides increased tremendously in almost all
cotton producing countries of the world including
Pakistan.
In Pakistan, during 2007- 2008 a total of 28 thousand
tonnes of insecticides was imported which measures the
foreign exchange of 74.5 million US dollars (GOP, 2007-
08). Little emphasis was placed on the plant genetic
resistance as a means of suppressing insect pests.
Painter (1951) noted that there were no efforts made to
develop genetic resistance to cotton insect pests. Genetic
resistance in the form of resistant varieties is an effective
means of minimizing yield losses caused by insect pests
and also leads to reduction in the use of insecticides
(Vaden Bosch, 1972; Van Dinther, 1972; Maxwell et al.,
1972; Bhatti et al., 1976). In the present scenario, by failing
*Corresponding author . E-mail: nnnawab24a@gmail.com. Tel:
0092-41-2652518 or 0092-321-766-2112.
to cope with all other possible means for insect control,
research has been accelerated on the host plant
resistance.
Hairiness/trichomes act as an important insect nonpreference
trait. The role of trichomes in plant defence
was evaluated by Levin (1973). It is clear that trichomes
play a role in plant defence, especially with regard to
phytophagous insects. Hairiness has been reported to
have a resistance against the sucking insect pests of cotton
(Lee, 1985). The degree of hair or trichome density on
the leaves of Gossypium species and cultivars is related
to varying degrees of resistance/susceptibility to sucking
pests, like whiteflies (Meagher et al., 1997), aphids and
jassids (Jenkins, 1989; Watson, 1989), or the boll weevil
(Thomson and Lee, 1980; Percy and Kohel, 1999). The
degree of jassid resistance had definite correlation with
the pilosity of the plant. The more tufted types were less
prone to jassid attack (Sikka et al., 1966).
The genetics of the insect resistance traits in cotton
have been reported to be governed by oligogenes
(Endrizzi et al., 1984). Knight (1952) identified two genes
for hairiness in cotton whereas, Niles (1980) reported that
the increase in the plant hairiness is governed by two
major genes and a complex of modifier genes. The major
gene was designated as H1 for sparse hairiness. A
second major gene, H2 controlled the finely dense
pubescence in an upland mutant designated as ‘pilose’.

In the F1 populations, both H1 and H2 showed incomplete
dominance (Simpson, 1947; Niles, 1980; Rahman and
Khan, 1998).
From industrial point of view, agronomic and fibre
quality traits hold a special position. The effect of pilosity
on agronomic traits was studied by Lee (1984) but no,
significant inferences were inferred for the trichome count
per cm, lint percentage and boll weight. The physical
parameters required for good spinning performance of
cotton fibre is a challenge for breeders. The pilose
condition is associated with decreased fibre length and
increased micronaire (Simpson, 1947; Lee, 1964, 1984).
But Kloth (1993) discovered a pilose like plant with
unexpectedly low micronaire among the homozygous
pilose plants.
Breeding through conventional tools has not lost its
significance even in the presence of modern tools of
science. Keeping in view, the extensive use and consumption
of cotton and its products, there is a need for
breeding varieties by incorporating the gene for hairiness
into promising cotton genotypes, which in turn will not
only minimize possible insect pest attack and insecticide
usage but also helps to improve yield and fibre quality
attributes. The information reported herein, would be
useful in determining the genetics for trichomes and its
effects on agronomic and fibre traits.
MATERIALS AND METHODS
Four cotton genotypes, Acala 63-74, FH 1000, CIM 446 and HRVO-
1 were selected and selfed for four generations by growing twice a
year, in a glasshouse and field during 2003 to 2004. The selected
parents with contrasting traits (Table 1) were planted in 300 × 300
mm earthen pots, containing a mixture of equivalent proportion of
sand, soil and farmyard manure, during November, 2004 in a
glasshouse. Temperature in the glasshouse was maintained at 30 ±
2°C during the day and 25 ± 2°C at night by using built in steam
heaters. The plants were exposed to natural sunlight supplemented
with artificial lighting, for a photoperiod of 16 h (ICAC, 2007). Three
crosses were attempted to obtain F0 seed during February to
March, 2005. The selfed seed of the parents was obtained by
covering their floral buds with butter paper bags. The crossing
scheme is given in Table 2. The F1 and their parents were sown
during the normal crop season of 2006 in two different sets planted
at two different locations. The F1 plants of each cross were divided
in three groups for developing BC1, BC2 and F2 for each
combination. The fresh F0, BC1 and BC2 seed was developed by
manual crossing, while the seed for F2 generations was produced
for each of the four combinations through manual selfing. The
experimental field was fertilized with N-P-K at the rate of 100-75-00
Kg/ha. The experiment in the field was laid out in a randomized
complete block design with three replications of each of the six
generations of the three crosses. The length of the plot was
maintained at 4.5 m, accommodating approximately 15 plants
spaced 300 mm apart.
The distance between the rows was 750 mm. A single plot per
replication was assigned to each of the parents and their respective
F1, while four plots per replication were assigned to each of the
backcrosses and eight plots per replication were assigned to raise
the F2 population of each cross. Ten guarded plants were selected
randomly in the parents and their F1, while fifty and thirty plants in
each replication were selected in F2 and backcross generations,
Nawab et al. 3965
respectively, to record the data during 2006. At maturity, the data
on various agronomic traits viz; plant height, number of monopodial
branches, number of sympodial branches, number of bolls, seed
cotton yield, boll weight and lint percentage were recorded. Fibre
quality characteristics like fibre length, fibre strength, fibre fineness,
fibre elongation percentage and fibre uniformity ratio of each plant
of a generation were measured using Spin lab high volume
instrument (HVI-900-A), M/S Zellweger Uster, Switzerland,
available in the Department of Fibre Technology, University of
Agriculture, Faisalabad, Pakistan. HVI measures fibre quality
characteristics according to the international trading standard. A
minimum of 10 g sample of lint from each of the guarded plants in
each generation was pre-conditioned to moisture applicability for at
least four to five hours prior to testing in the HVI. Fibre length was
measured, on the basis of the fibrograph. The samples were
prepared at fibro sampler in the form of fibre comb and the
fibrograph-910 brushed the sample fibres automatically by vacuum
action, and optical density of the sample was displayed on the
screen. The mean fibre length was derived according to the ASTM
(1977a) standard through the procedure laid down by Hunter
(1991). The average fibre length was calculated in mm for further
analysis. Fibre bundle strength was determined by Pressley
strength tester using the flat bundle method as specified by ASTM
standard (1977b). The fibre elongation was measured on HVI-900-
A. When a sample was moved into an optical sensor, where the
test for length and strength were performed, the percentage
increase in the length before fibre breakage was measured
(Anonymous, 1992a, b). For fibre uniformity ratio, the sample was
moved into the optical sensor of HVI-900-A and the reading of
optical density of the sample was displayed on the screen. The
uniformity ratio of fibre was measured according to ASTM standard
(1977a). Fibre fineness of micronaire value was measured on
fibrofine-920 device of HVI-900- A. When lid of the chamber
containing a sample is closed, the sample is compressed to a fix
and known volume. The sample was weighed on an electric
balance before placing in the test chamber. This balance
transmitted the mass through the control processor. This mass was
accepted if the weight was between 8.5 and 11.5 g of cotton. The
measured values of mass and pressure calculated were in fineness
value according to ASTM standard (1977c).
Trichome density studies
Trichomes represent the presence of small hairs on the cotton plant
(Figure 1). The trichome density on leaves was estimated following
the method proposed by Bourland et al. (2003). Three leaves at
random, each from upper, middle and lower portion of five plants in
parents and their F1’s and thirty and fifty plants were selected in
BC’s and F2’s in all the three replications were used to assess for
the quantitative measure of trichomes on the abaxial leaf surface.
Observations pertaining to the number of trichomes were recorded
with the help of an index card within an area of 1 mm 2 (Figure 2) lay
over the abaxial side of each leaf from three different positions and
averaged. The resultant mean values for number of trichomes from
three different portions of the plant were worked out as the final
reading for average number of trichomes per unit area. Trichomes
in the 1 mm 2 area were counted with the aid of high magnifying
power microscope (Olympus Z61).
On the basis of the trichome counts made for the leaves, a rating
scale was developed to categorize the trichome density on abaxial
surface of leaves by using a scale of 1 for sparsely (non) hairiness,
2 for moderate number of trichomes, 3 for (pilose) hairiness (Lee,
1968; Wright et al., 1999; Bourland et al., 2003; Stiller et al., 2004;
Lacape and Nguyen, 2005).
Chi-squared values and probabilities of goodness of fit of the
segregation ratios of F2 and backcross generations were tested
against theoretical ratio (Harris, 1912). Phenotypic and genotypic

3966 Afr. J. Biotechnol.
Table 1: Distinctive morphological features of the upland cotton accessions
S.No. Variety/ Accession Parentage Hairiness state Origin
1 Acala 63-74 - Glabrous USA.
2 CIM 446 CP 15/2 × S 12 Lightly hairy CCRI, Multan, Pakistan.
3 FH 1000 S 12 × CIM 448 Lightly hairy CRI, Faisalabad, Pakistan.
4 HRVO-1 B-557/2/Gambo Okra/Rajhans/3/Rajhans Pilose hairiness CRI, Faisalabad, Pakistan.
* CCRI = Central Cotton Research Institute, Multan. Pakistan. * CRI = Cotton Research Institute, Faisalabad. Pakistan.
Table 2: Scheme of crossing
S.No. Cross Trait Considered
1 HRVO-1 × FH 1000 Pilose hairiness × Lightly hairy
2 HRVO-1 × CIM 446 Pilose hairiness × Lightly hairy
3 HRVO-1 × Acala 63-74 Pilose hairiness × Glabrous
(a) Pilose hairiness (P1)
H2H2
Figure 1. Inheritance of leaf trichome trait
(c) Intermediate class of hairiness (F1)
H2h2
(b) Normal hairiness (P2)
h2h2

correlation coefficients between trichome counts and agronomic
and fibre traits were determined using the F2 data. Phenotypic
correlation coefficients were calculated following Dewey and Lu
(1959) using Minitab computer programme. The genetic
correlations (rg) between two characters X and Y were calculated
following Falconer (1981).
RESULTS
Genetic studies
The mean squares from the analysis of variance for
agronomic/fibre and number of trichomes for six generations
in three cotton crosses were tested at two different
locations and are presented in Table 3. Phenotypic
classes were developed as evident from Table 4, on the
basis of the trichome count ratings on the abaxial leaf
surface in the three crosses involving pilose hairy and
normal/sparse hairy plants. The inheritance pattern for
trichomes/hairiness in three different cross combinations
was studied on the basis of the F2 and test/back cross
population data.
In all the three cross combinations, leaf trichomes
segregated into three distinct classes (Figure 3). Nonsignificant
chi-squared values were observed for the
segregating ratios in F2 and backcross generations of the
three crosses. Observations of 1 sparse hairiness : 2
intermediate class of hairiness : 1 pilose hairiness on
leaves were observed in the F2 populations of the three
crosses. In the backcrosses with parent-I, ratios of 1 pilose :
1 intermediate, leaf hairiness were obtained. Similarly, in
the backcrosses with parent-II, ratios of 1 sparse : 1
Figure 2 Trichome counts on the abaxial side of cotton leaf with an
area of 1 mm2 of an index card.
intermediate, leaf hairiness were observed.
Phenotypic and genotypic correlations
Nawab et al. 3967
In all the three crosses (Table 5), there existed positive
and significant correlation of number of trichomes with lint
percentage. For fibre length and fibre strength, there was
highly significant and negative correlation with number of
trichomes. A significant and positive correlation of
number of trichomes with micronaire value meant a
negative correlation of number of trichomes with fibre
fineness in all of the three cross combinations. In the
case of fibre uniformity ratio and fibre elongation, a
negative association with number of trichomes was
observed in the crosses.
The study of number of trichomes were negatively but
significantly correlated with plant height in the cross
HRVO-1 × CIM 446. Non-significant correlations of
number of trichomes with number of monopodial
branches, number of sympodial branches, number of
bolls, seed cotton yield and boll weight were recorded.
DISCUSSION
Genetic studies
Highly significant differences among different generations
for all characters studied (Table 3) were observed in all
the three crosses. Significant differences among different
generations indicated the existence of genetic variability.

3968 Afr. J. Biotechnol.
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
tested at two different locations.
Source D.F
Replication 2 1.62
(1.62)
Generation 5 5.46
(5.44)
Location 1 0.40
(0.40)
G × L 5
1.37
(1.37)NS
HRVO-1 × FH 1000 Probability (F-value)
PH NMB NSB NBP BWt SCY L% FL FS FE U % Mic T 0.05 0.01
0.010
(0.47)
0.113
(5.33)
0.002
(0.12)
0.022
(1.06)NS
0.38
(1.05)
56.96
(156.7)
0.092
(0.25)
0.298
(0.82)
NS
1.20
(3.37)
66.64
(187.3)
0.059
(0.17)
0.144
(0.40)
NS
0.004
(0.93)
0.88
(190.7)
0.004
(0.82)
0.002
(0.47)
NS
1.02
(3.39)
2561.12
(8485.1)
1.28
(4.23)
0.15
(0.49)
NS
0.91
(3.03)
40.95
(135.6)
1.00
(3.33)
0.044
(0.15)
NS
0.003
(0.16)
5.56
(357.9)
0.010
(0.66)
0.009
(0.58)
NS
0.02
(0.06)
2.98
(9.84)
0.25
(0.82)
0.26
(0.87)
NS
0.004
(0.45)
0.082
(9.4)
0.001
(0.02)
0.001
(0.09)
NS
1.39
(1.21)
82.78
(72.25)
0.001
(0.00)
0.002
(0.001)
NS
0.009
(3.0)
0.352
(140.7)
0.000
(0.04)
0.001
(0.25)
NS
96.68
(1.70)
28576.09
(504.4)
40.19
(0.71)
48.58
(0.85)
NS
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
HRVO-1 × CIM 446
Replication 2 1.12
(0.55)
Generation 5 62.98
(31.10)
Location 1 7.70
(3.80)
G × L 5 1.42
(0.70)
NS
0.00
(0.03)
0.023
(24.04)
0.004
(4.20)
0.001
(0.56)
NS
0.18
(0.07)
17.82
(7.26)
5.53
(2.25)
6.45
(6.10)
NS
0.235
(0.64)
25.95
(71.3)
0.340
(0.93)
0.280
(0.57)
NS
0.00
(0.22)
0.650
(335.66)
0.009
(4.25)
0.003
(1.75)
NS
3.29
(3.33)
1210.5
(1215.8)
3.32
(3.36)
1.26
(1.27)
NS
0.16
(1.30)
9.74
(78.97)
0.82
(6.66)
0.175
(1.42)
NS
0.24
(4.70)
12.54
(3.2)
0.032
(0.42)
0.071
(0.95)
NS
0.43
(3.08)
6.30
(45.70)
0.25
(1.80)
0.23
(0.17)
NS
0.035
(1.12)
0.60
(19.32)
0.051
(1.61)
0.036
(1.14)
NS
0.188
(3.36)
4.64
(83.65)
0.095
(1.71)
0.025
(0.45)
NS
0.00
(0.08)
0.69
(362.8)
0.00
(0.18)
0.001
(0.38)
NS
4.62
(0.98)
39653.05
(8433.6)
20.00
(4.25)
0.78
(0.17)
NS
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
HRVO-1 × Acala 63-74
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
(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)
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
(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)
Location 1 92.18
(16.79)
G × L 5 3.36
(0.61)
NS
0.06
(0.90)
0.001
(0.02)
NS
0.67
(4.24)
0.29
(1.85)
NS
4.16
(4.24)
1.19
(1.22)
NS
0.002
(0.63)
0.006
(1.57)
NS
0.16
(0.04)
2.00
(0.47)
NS
0.63
(1.43)
0.204
(6.80)
0.10
(2.40)
0.001
(0.02)
0.14
(2.63)
0.041
(20.30)
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
0.162
(0.37)
NS
0.022
(0.74)
NS
0.02
(0.51)
NS
0.029
(0.98)
NS
0.04
(0.78)
NS
0.00
(0.21)
NS
30.14
(0.62)
3.95
(0.08)
NS
3.44 5.72
2.66 3.99
4.30 7.95
2.66 3.99
3.44 5.72
2.66 3.99
4.30 7.95
2.66 3.99
3.44 5.72
2.66 3.99
4.30 7.95
2.66 3.99
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 =
fibre length, FS = fibre strength, FE = fibre elongation, U% = fibre uniformity ratio, FF = fibre fineness.

Number of plants
Number of plants
Number of plants
80
70
60
50
40
30
20
10
80
70
60
50
40
30
20
10
0
0
80
70
60
50
40
30
20
10
0
P 2
a) HRVO x FH 1000
P 2
B 2
F 1
F 2
40 60 80 100 120 140 160 180 200 220 240
b) HRVO x CIM 446
B 2
F 1
F 2
40 60 80 100 120 140 160 180 200 220 240
c) HRVO x Acala 63-74
P 2
B 2
40 60 80 100 120 140 160 180 200 220 240
Figure 3. Frequency distributions for number of leaf trichomes in F2 generations of three crosses..
B 1
B 1
F 2
F 1
B 1
Nawab et al. 3969
P 1
P 1
P 1

3970 Afr. J. Biotechnol.
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
trichomes trait
Cross Gen. Expected
Ratios
HRVO-1 × FH 1000
HRVO-1 × CIM 446
HRVO-1 × Acala 63-74
Sparse
Hairy
Observed value Expected value
Intermediate
Hairy
Pilose
Velvet
Sparse
Hairy
Intermediate
Hairy
Pilose
Velvet
χ 2 Prob.
F2 1: 2:1 34 79 37 37.5 75 37.5 0.55 0.90-0.75
BC1 1:1 - 53 37 - 45 45 2.84 0.10-0.05
BC2 1:1 36 54 - 45 45 - 3.60 0.10-0.05
F2 1: 2:1 41 69 40 37.5 75 37.5 0.97 0.75-0.50
BC1 1:1 - 37 53 - 45 45 2.84 0.10-0.05
BC2 1:1 48 42 - 45 45 - 0.40 0.75-0.50
F2 1: 2:1 35 74 41 37.5 75 37.5 0.50 0.90-0.75
BC1 1:1 - 47 43 - 45 45 0.18 0.75-0.50
BC2 1:1 52 38 - 45 45 - 2.18 0.25-0.10
Table 5: Genotypic (upper value) and phenotypic (lower value) correlations along with the probability values given in parenthesis for
number of trichomes and agronomic/fibre traits in three cotton crosses.
Trait 1 2 3
Number of Trichomes
L %
0.85**
(0.00)
0.81**
(0.00)
FL
-0.96**
(0.00)
-0.71**
(0.00)
FS
-0.98**
(0.00)
-0.86
(0.00)
FE
-0.83**
(0.00)
-0.71**
(0.00)
U% -0.83**
(0.00)
-0.73**
(0.00)
FF
0.69*
(0.040)
0.56*
(0.048)
0.92**
(0.00)
0.91**
(0.00)
-0.95**
(0.00)
-0.95**
(0.00)
-0.98**
(0.00)
-0.96**
(0.00)
-0.95**
(0.00)
-0.74**
(0.00)
-0.97**
(0.00)
-0.92**
(0.001)
0.62*
(0.042)
0.60*
(0.040)
0.90**
(0.00)
0.88**
(0.00)
-0.75**
(0.00)
-0.72**
(0.00)
-0.90**
(0.00)
-0.80**
(0.00)
-0.88**
(0.00)
-0.72**
(0.00)
-0.86**
(0.00)
-0.82**
(0.00)
0.63*
(0.045)
0.60*
(0.040)
Trait 1 2 3
Number of Trichomes
PH
-0.35
(0.064)
-0.34
(0.063)
0.16
NMB
(0.133)
0.15
(0.149)
NSB -0.18
(0.105)
-0.18
(0.105)
NBP -0.29
(0.07)
-0.28
(0.068)
SCY 0.27
(0.22)
0.26
(0.18)
BWt
0.17
(0.12)
-0.18
(0.11)
-0.88**
(0.00)
-0.84**
(0.00)
-0.07
(0.32)
-0.06
(0.34)
0.38
(0.07)
0.30
(0.10)
-0.34
(0.09)
-0.23
(0.058)
0.22
(0.06)
0.18
(0.10)
0.15
(0.14)
0.12
(0.20)
L% = Lint percentage, FL = Fibre length, FS = Fibre strength, FE = Fibre elongation, U% = Fibre uniformity ratio,
FF = Fibre fineness, 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.
1 = HRVO-1 × FH-1000, 2 = HRVO-1 × CIM-446, 3 = HRVO-1 × Acala 63-74.
*P < 0.05 = Significant, **P < 0.01 = Non-significant.
However, the non-significant values of the generation ×
location interactions (G × L) in the three cross combina-
-0.12
(0.20)
-0.11
(0.22)
-0.29
(0.08)
-0.28
(0.09)
-0.32
(0.08)
-0.29
(0.061)
-0.26
(0.10)
-0.21
(0.07)
-0.35
(0.25)
-0.32
(0.19)
-0.22
(0.072)
-0.24
(0.081)
tions further confirmed the authenticity of the results as
these results were independent of the environmental

influence. The intermediate (H2h2) progeny in F1 was due
to incomplete dominance. The two homozygous extremes
for trichomes/hairiness : pilose hairy (H2H2) and
sparse/normal hairy (h2h2) were easily distinguishable
(Figure 3). The F2 data regarding number of trichomes
were categorized into three main classes (Table 4):
pilose hairy (H2H2), sparse/normal hairy (h2h2) and intermediate
hairy (H2h2) (Simpson, 1947). The major gene is
designated as H1 for sparse hairing. A second major
gene, H2 controls the finely dense pubescence in an
upland mutant designated as ‘pilose’. In the F1 populations,
both H1 and H2 showed incomplete dominance
(Niles, 1980; Rahman and Khan, 1998). The segregation
in the backcrosses with parent-I and parent-II also fit to
the theoretical ratio of 1:1 which further confirmed the
incomplete dominance pattern of inheritance. The nonsignificant
chi-square values in F2 for trichomes in all the
crosses fit well against the theoretical monohybrid ratio of
1:2:1.This ratio is in proximity to the understanding gotten
from Figure 3a, suggesting that inheritance of this trait
can be manipulated easily in a breeding programme.
However, a very small proportion of plants fell in another
interme-diately resembling hairiness category. This
phenotypic expression of the intermediate hairy state in
hetero-zygous condition was probably affected by the
genetic background of the parents indicating, modifying
gene effects (Falconer and Mackay, 1996; Rieseberg et
al., 1996; Xu et al., 1997; Rahman and Khan, 1998;
Schwarz-Sommer et al., 2003).
Phenotypic and genotypic correlations
Correlation in plant breeding is a useful tool of indirect
selection of the secondary trait with the improvement in
the primary trait. Correlation coefficient is a statistical
measure which is used to find out the degree and
direction of the relationship between two or more variables.
A positive value gives the indication of the same direction
of the two variables in question and vice-versa. Fibre
quality traits hold a special position from the cotton
spinning industry. It was therefore, necessary to study the
effect of this insect non-preference trait (trichomes) on
the fibre quality determining traits. Genotypic correlations
indicated correlation between the two characters (Table
5). Similar type of magnitude was observed by Dhanda et
al. (1984) and Tyagi (1987) for genetic correlations. The
higher values of phenotypic correlation coefficients than
the genotypic correlation coefficients where ever present,
indicating that the correlation between the two characters
was not only due to genes, but environment also played
its role in the expression of the character. The fibre
fineness is recorded in micronaire value. This means that
the higher the magnitude of micronaire value, the lesser
will be the fineness of the fibre and vice-versa. Significant
correlation between the two characters gave dependency
of the two characters, while non-significant correlation
indicated the independent nature of the two characters
Nawab et al. 3971
under study as suggested by Singh and Narayanan
(2000).
The positive correlation of number of trichomes with lint
percentage in all of three crosses (Table 4) suggested
that the selection for pilose hairiness would be helpful in
enhancing the lint percentage. This result is in line with
that of Lee (1984) and Kloth (1993). The negative
correlation of fibre length, fibre strength, fibre uniformity
ratio, fibre elongation and micronaire value (positive
correlation of number of trichomes with fibre fineness)
with number of trichomes in the crosses corroborated to
the findings of Lee (1964, 1984). The negative correlation
of hairiness/trichomes with these mentioned traits determining
fibre quality gave an understanding that the
presence of the genes controlling these traits might be
present on the same chromosome. Reports are also
available regarding the pilosity with shorter fibre (Kohel et
al., 1967, Lee, 1964, 1984). Association of hairiness with
short fibre length was supposed to be gene pleiotropy as
it was proposed by Simpson (1947) but the later studies
by Kloth (1993) proved this association to be a linkage
between the genes affecting hairiness and fibre quality
determining traits.
The negative correlation of trichomes with plant height
in HRVO-1 × CIM 446 suggest that increase in hairiness
state would decrease the plant height but otherwise,
there would be no effect on other morphological traits.
Yield determining traits like number of monopodial
branches, number of sympodial branches, number of
bolls, boll weight and seed cotton yield are components
of an efficient breeding programme. Non-significant
correlations of these traits with number of trichomes/
hairiness revealed that hairiness or number of trichomes
had no effect on the expression of these agronomic/yield
related traits. In contrast to the effect of number of
trichomes on fibre related traits, a well documented proof
is available but the effect of the same on yield
determining traits was proved in the present manuscript
as a step forward in the research on trichomes. Incorporation
of the gene for hairiness into commercial cotton
cultivars will not only limit the population of the sucking
insects but also result in the improvement of the fibre
attributes without disturbing the major traits relating to
yield (number of monopodial branches, number of
sympodial branches, number of bolls, boll weight and
seed cotton yield).
Keeping in view, the bio-safety requirements of the
emerging era and the available knowledge of the
naturally conferring resistance against insects, the
present studies were performed to investigate the genetics
of hairiness/trichomes in relation with the agronomic and
fibre related traits in cotton. The main conclusions from
this work are the non-significant chi-square values in F2
for trichomes in all the crosses which fit well against the
theoretical monohybrid ratio of 1:2:1. The positive
correlation of number of trichomes with lint percentage
and micronaire (negative correlation of number of
trichomes with fibre fineness) in all the three crosses

3972 Afr. J. Biotechnol.
suggested that the selection for pilose hairiness would be
helpful in enhancing the lint percentage and fibre
fineness.
The present findings elaborated the novelty of the
research with a confidence that incorporation of the gene
for pilosity had no effect on the expression of these
agronomic/yield related traits.
Acknowledgement
The authors are thankful to Dr. Muhammad Shafique
Tahir, Assistant Botanist and Dr. Noor-Ul-Islam, Director,
Cotton Research Institute, Ayub Agricultural Research
Institute, Faisalabad, Pakistan for providing the
germplasm as well as support in basic field
operations.
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African Journal of Biotechnology Vol. 10(20), pp. 3973-3977, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2304
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Quick and sensitive determination of gene expression
of fatty acid synthase in vitro by using real-time
polymerase chain reaction amplification (PCR)
Zuo-Hua Liu 1,2 , Jin-Long Yang 1,2 , Fei-Yun Yang 1 , Ding-Biao Long 1 , Ren-Yong Jia 2 , Kang-
Cheng Pan 2 * and Dai-Wen Chen 3
1 Chongqing Academy of Animal Science, Chongqing, 402460, Chongqing China.
2 College of Veterinary Medicine of Sichuan Agricultural University, Yaan 625014, Sichuan Province, China.
3 Institute of Animal Nutrition, Sichuan Agricultural University, Yaan 625014, Sichuan Province, China.
Accepted 17 March, 2011
Obesity results from an imbalance between energy intake and energy expenditure, which leads to a
pathological accumulation of adipose tissue, but the underlying mechanism at gene level, is far from
being elucidated. The objective of this study was to investigate the correlation between mRNA express
from fatty acid synthase (FAS) with a different glucose level in primary adipocytes by real-time
polymerase chain reaction amplification (PCR), which can aid in the understanding of the mechanism of
obesity in vitro. By using the following formula, this study was able to quantify the mRNA expression of
FAS of unknown samples: Y = -3.156X + 41.21 (Y = threshold cycle, X = log starting quantity). The high
concentrations of glucose group significantly improved the mRNA expression of FAS (P < 0.01) rather
than 0.25 and 0% concentrations of glucose. These results provide significant data that confirm an
association between different glucose level and FAS expression in preadipocytes. The glucose
concentration of the high group substantially augmented the mRNA expression of FAS.
Key words: Expression, fatty acid synthase, lipid deposition, real-time polymerase chain reaction amplification
(PCR).
INTRODUCTION
Excessive weight gain can be attributed to the interactions
among environmental factors (dietary intake and
physical activity), genetic predisposition and the
individual behaviors (Campion et al., 2009; Lomba et al.,
2010). Thus, genetic predisposition is an important determinant
of the increasing prevalence of metabolic
syndrome and associated complications such as obesity
(Stone et al., 2002; Speakman, 2007; Dorn et al., 2010).
Obesity is strongly associated with the metabolic
syndrome and related to many diseases such as
hypertension, heart disease and diabetes. Obesity results
from an imbalance between energy intake and energy
expenditure, leading to a pathological accumulation of
adipose tissue (Kopelman, 2000). It has a heterogeneous
*Corresponding author. E-mail: 391556428@qq.com. Tel: +86-
23-46792362. Fax: +86-23-46792362.
phenotype with the involvement of multiple genes and
their interactions with non-genetic factors (Vladimir et al.,
1997). The accumulation of fat is caused by hypertrophy
and hyperplasia of adipocytes. Therefore, an understanding
of the molecular basis of hyperplasia as well as
hypertrophy would contribute to the establishment of
medical treatments for preventing health risks that can
cause serious illnesses and death (Hishida et al., 2007).
One explanation for the increase in the number of
adipocytes is that, the uptake of exogenously derived
fatty acids (FA) and de novo of biosynthesis of FA are
increased and the hydrolysis of acylglycerols is
decreased. Among the different mechanisms that could
lead to fat deposition and obesity, the epigenetic
regulation of gene expression has emerged in recent
years as a potentially important contributor (Campion et
al., 2009; Lomba et al., 2010). Recent studies have revealed
that, several proteins such as fatty acid synthase
(FAS), a key enzyme regulating lipid metabolism, play

3974 Afr. J. Biotechnol.
Table 1. Details of the primer pairs and cDNA concentration used for the FQ-PCR.
Name 5′ sequence 3′ Amplicon size (bp) cDNA (µM)
Forward(FAS -F) AGCCTAACTCCTCGCTGCAAT 196 1
Reverse(FAS -R) TCCTTGGAACCGTCTGTGTTC 1
TaqMan probe(FAS -P) (FAM) TCCTGCGGCATCCACGAGACCACC (Eclipse) 1.2
The cDNA concentration refers to the final concentration of cDNA equal to reverse-transcribed RNA in each PCR.
important roles in adipocyte metabolism (Lomba et al.,
2010; Marie et al., 2010). The events occurring during the
stages of adipocyte meta-bolism are relatively well
characterized, but the molecular mechanisms underlying
the stages of adipogenesis remain unknown.
This study reported a fluorescent quantitative real-time
polymerase chain reaction amplification (FQ-PCR) assay
for quantifying FAS gene expression at different glucose
levels in primary adipocytes. The results of this study
provide interesting data that may help understand the
molecular mechanisms underlying the stages of adipogenesis.
MATERIALS AND METHODS
Isolation of adipocyte precursors from pig adipose tissue
Epididymal fat pads of healthy pigs (age, 18 to 20 days; Landrace)
were removed under sterile conditions and transferred to ice-cold
Dulbecco's modified eagle medium containing penicillin, streptomycin
and fungizone (Sangon Biological Engineering Technology
and Services Co., Ltd, Shanghai, China). Preadipocytes were
isolated as described by Janke et al. (2002).
Cells were cultured with different glucose contents
Preadipocytes were incubated at 37°C in a humidified atmosphere
with 5% CO2 as described by Janke et al. (2002). The cells were
stimulated to proliferate over 24 h and then, glucose was added at
various concentrations to the culture medium (glucose-free
adipocyte medium, supplemented with 1 µM insulin; 0.25% glucose
adipocyte medium, supplemented with 1 µM insulin and 0.5%
glucose adipocyte medium, supplemented with 1 µM insulin).
Gene expression analyzed using FQ-PCR
RNA isolation and FQ-PCR
Preadipocytes (1 ml) were trypsinized with 1 ml digestive, washed
once with PBS, and pelleted (380 g, 10 min) after glucose was
added at various concentrations to the culture medium at 2, 4, 8, 16
and 32 h. Total RNA was extracted with TRIzol (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions. An ABI
PRISM 7000 sequence detection system (Applied Biosystems,
Foster City, CA) was used for FQ-PCR. The reaction mixture was
prepared using the universal PCR master mix (Applied Biosystems)
according to the manufacturer’s instructions. The mixture was
incubated at 50°C for 5 min and at 94°C for 40 s and then, the PCR
was performed by 34 cycles at 94°C for 40 s and at 59°C for 40 s,
72°C for 40 s and an additional extension period at 72°C for 2 min.
Melting curve from 65 to 95°C read every 0.5°C hold 5 s. Relative
standard curves were generated in each experiment to calculate
the input amounts of the unknown samples. Sensitivity and
specificity analysis of the assay was as described by Yang et al.
(2009).
Specific RT-PCR primer and probe design
Expression analysis was performed for the genes encoding FAS
published sequences at GenBank. The oligonucleotide primer set
for the gene was designed using Primer Premier 5.0 software. The
sequences and concentrations of primers and cDNA are shown in
Table 1. The specificity of all the primers was tested by searching
the homologous DNA sequences against the GenBank BLAST
database.
Preparation of standard templates
The conventional RT-PCR was performed for standard RNA using
the primers (FAS-F, FAS-R) mentioned earlier. The standard template
was prepared as described by Yang et al. (2008).
Establishment of the FQ-PCR standard curve
Standard plasmids (1 × 10 9 copies/µl) were used to establish the
standard curve .The Primers (FAS-F, FAS-R and FAS-P) were used
for this amplification and their concentrations ranged from 1 × 10 8 to
1 × 10 2 gene copies/µl. A regression curve was constructed by
plotting the threshold cycle (Ct) values versus the logarithm of the
RNA copy number. Analysis of the measures was undertaken after
each run to verify identical amplification efficiencies and conditions
between runs.
Finally, based on the data for generating the standard curve with
the software of the iCycler IQ Detection System (Bio-Rad, USA), Ct
values for each sample were determined from the point at which
fluorescence breached the threshold fluore-scence line (Yang et al.,
2008; Yang et al., 2009).
Statistical analysis
Data were expressed as means ± SDs. Statistical significance of
the differences was determined using Student’s t-test. P values less
than 0.01 were considered statistically significant (Kang et al.,
2010).
RESULTS
Standard curve of the established FQ-PCR
The standard curve of FQ-PCR was constructed using

Liu et al. 3975
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
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
threshold (Ct) values are presented on the y-axis. Each dot represents the result of triplicate amplification of each dilution. The correlation
coefficient and the slope value of the regression curve were calculated and are indicated.
serially diluted plasmids with tenfold dilution, ranging from
10 8 to 10 2 copies as templates. The correlation coefficients
of the standard curve was 0.9998 and the PCR
efficiencies was greater than 0.95 (Figure 1). The high
PCR reaction efficiencies indicated that, the FQ-PCR
assay was suitable for genomic DNA quantification. The
good linearity between DNA quantities and fluorescence
values (Ct) indicated that, the assay was well suitable for
quantitative measurements. By using the following
formula, this study could quantify the mRNA expression
of FAS of unknown samples: Y = -3.156X + 41.21 (Y =
threshold cycle, X = log starting quantity).
Sensitivity, specificity analysis of the established FQ-
PCR
Ten-fold dilutions of the standard templates were tested
by the established FQ-PCR assay to evaluate the sensitivity
of the system and the detection limit was found to
be 1.0 × 10 1 copies/reaction (data no shown).
The test was performed using DNA from standard RNA
and several other bacteria as templates to examine its
specificity; the result of this analysis showed that, none of
the bacteria yielded any amplification signal, suggesting
that the established FQ-PCR assay was highly specific
(data no shown).
Dynamic changes in FAS gene expression in
adipocyte
The dynamic changes in FAS gene expression in adipocytes
after glucose was added at various concentrations
to the culture medium were intermittently determined over
a 32 h period by FQ-PCR. The study results revealed
that, the copy numbers of FAS of samples peaked at 2 h
when the glucose concentration was 0, 0.25 and 0.5%,
they were 4.05 ± 0.86, 4.84 ± 0.62, 5.75 ± 0.88 (log
copies/µl), respectively. The glucose concentration of the
high group substantially augmented the mRNA
expression of FAS (P < 0.01) (Figure 2).
DISCUSSION
FQ-PCR has become a potentially powerful alternative
for quantifying mRNA expression because it is simpler,
more rapid, more reproducible and more sensitive than
other methods (Shimazu et al., 2009; Brym et al., 2007;
Calikoglu et al., 2006). In this study, the applicability of
FQ-PCR for the quantification of FAS mRNA expression

3976 Afr. J. Biotechnol.
Figure 2. Dynamic changes in FAS gene expression in adipocytes with different glucose concentration in different
time.
was clearly established by showing that, it has remarkable
sensitivity and high-throughput potential, which are
beyond the scope of other assay methods.
FAS catalyze the last step in the fatty acid biosynthesis
and thus, it is believed to be a major determinant to
generate fatty acids by de novo lipogenesis (Dorn et al.,
2010). It is clear that some genes would show an
increase or decrease in response to glucose. The aim of
this study was to analyze the expression of FAS, which is
a central enzyme of lipogenesis with a different glucose
level. This study revealed a significant correlation of FAS
expression with the high degree of glucose in primary pig
preadipocytes in vitro. Surprisingly, the copy numbers of
FAS peaked at 2 to 8 h at various concentrations of
glucose. At 0.5% concentration, glucose significantly augmented
the mRNA expression of FAS (P < 0.01) when
compared with 0.25 and 0% concentrations. These
results finding is just an initiation of FAS in obesity to be
elucidated. Further studies are required to unravel the
relationship between nutrient and hormone and other
gene such as hormone-sensitive lipase.
Conclusion
In conclusion, the study results confirm the association
between different glucose levels and FAS expression in
preadipocytes. At 0.5% concentrations, glucose significantly
augmented the mRNA expression of FAS (P <
0.01) rather than at concentrations of 0.25 and 0%. However,
further research is needed to validate this conclusion.
ACKNOWLEDGEMENTS
This study was supported by The Chongqing Natural
Science Funds (No. 2008BA1013); The Chongqing
Natural Science Funds (No. 2009BA1078).
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African Journal of Biotechnology Vol. 10(20), pp. 3978-3985, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2334
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Characterization of a chestnut FLORICAULA/LEAFY
homologous gene
Tao Liu 1 *, Yun-qian Hu 2 and Xiao-xian Li 2
1 Faculty of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan, 650201, China.
2 Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan, 650204, China.
Accepted 24 March, 2011
The FLORICAULA/LEAFY (FLO/LFY) homologues’ genes are necessary for normal flower development
and play a key role in diverse angiosperm species. In this paper, an orthologue of FLORICAULA/LEAFY,
CmLFY (chestnut FLO/LFY), was isolated from the inflorescence of chestnut trees. Its expression was
detected in various tissues. Furthermore, the flowering effectiveness of the gene was assessed with
transgenic Arabidopsis. CmLFY protein showed a high degree of similarity to PEAFLO (78%), which is a
homologue of FLO/LFY from pea. RT-PCR analysis showed that, CmLFY expressed at high levels in
inflorescences, but not in young leaves, fruits or stems. The transgenic Arabidopsis with
over-expressed CmLFY showed accelerated flowering, which supports that CmLFY encodes a functional
orthologue of the FLORICAULA/LEAFY genes of angiosperms despite its sequence divergence. These
results suggest that CmLFY is involved in inflorescence development in chestnut.
Key words: Chestnut, homologue, FLORICAULA/LEAFY.
INTRODUCTION
Two developmental programmes can be distinguished
during the life cycle of flowering plants. During the initial
vegetative phase, the apical meristem produces leaves
and lateral shoot. After floral induction, the plant enters
into a reproductive phase and the apical meristem chan-
ges its developmental pattern and initiates the production
of flowers. In Antirrhinum and Arabidopsis this transition
from vegetative to reproductive development pattern
requires the establishment of floral meristem identity in
the lateral meristems of the inflorescence, a process
which has been demonstrated to be controlled by the
homo- logous genes FLORICAULA (FLO) and LEAFY
(LFY), respectively (Ma, 1998; Pidkowich et al., 1999).
LFY is a transcriptional regulation gene thought to play a
primary role in determining flower meristem identity.
FLORICAULA (FLO), a LFY homologue in snapdragon,
has been shown to have almost the same role. Loss func-
* Corresponding author. E-mail: beixue2007@gmail.com. Tel:
+86-871 5227721.
Abbreviations: PCR, Polymerase chain reaction; RT-PCR,
reverse transcriptase-PCR; RACE, rapid amplified cDNA ends.
tion mutants of these genes result in the conversion of
flowers into indeterminate secondary shoots (Coen et al.,
1990; Weigel et al., 1992). The over-expression of LFY
under a constitutive promoter in Arabidopsis caused early
flowering and converted all lateral shoots into solitary
flowers. In heterologous plants such as aspen, LFY has
been shown to have effects on the acceleration of flower-
ing and induction of ectopic flowers (Weigel and Nilsson,
1995; Rottman et al., 2000), suggesting a conservation of
LFY function across long phylogenetic distances within
angiosperms. These reports strongly suggest that, the
ability to control the expression of LFY or of its orthologs
from other plants, could make it possible to artificially
induce various plants to blossom and even skip or shorten
the juvenile phase of woody plants.
Flowering is an essential stage for fruit production and
thus, an understanding of the genetic mechanisms under-
lying the flowering event is important for efficient fruit
production. During the last decade, molecular mecha-
nisms of flowering have been studied extensively in
herbaceous ‘‘model’’ plants such as Arabidopsis and
Antirrhinum. In contrast, studies on the molecular
mechanisms of flowering in fruit trees have just begun.
Although, several flowering-related FLO/LFY orthologs
have been cloned from kiwifruit (Actinidia deliciosa)

(Walton et al., 2001), grapevine (Vitis vinifera) (Carmona
et al., 2002), and apple (Malus domestica) (Kotoda et al.,
2002), their specific role in the induction of the
characteristic features of tree reproductive development is
still being elucidated.
Trees are characterized by an extended adolescence.
This is particularly important for commercial fruit/nut tree
growers and breeders, because prolonged juvenile
periods delay harvesting and the evaluation/breeding of
new strains. Chestnut (Castanea mollissima) is one of the
most commercially valuable fruit trees, with production
once only annually. Chestnut has an extended juvenile-
vegetative phase, during which vegetative growth is
maintained. Attempts to exploit existing knowledge of the
genetic control of flower development in Arabidopsis to
engineer nut trees could have significant economic and
scientific implications for the tree fruit industry. As yet, no
studies have reported on the molecular mechanism
underlying the development of chestnut flowers. Recently,
MADS-box genes have been cloned and characterized in
chestnut by us (Liu et al., 2006). Our results suggest that
these chestnut MADS genes may be involved in floral
organ and fruit development in chestnut. In this paper, we
identified a chestnut FLORICAULA/LEAFY homologous
gene involved in floral development. We described the
isolation and characterization of CmLFY (DQ270548).
The function of CmLFY was demonstrated by transgenic
Arabidopsis, as expected for normal plants, transgenic
plants showed earlier flowering phenotype, indicating that
CmLFY may play a similar role of promoting flowering as
the FLORICAULA/LEAFY genes in flower development.
MATERIALS AND METHODS
Plant materials
The chestnut and Arabidopsis ecotype Columbia (Col) were used in
this study. Chestnut examples were collected from Kunming,
Yunnan, People’s Republic of China and were frozen in liquid
nitrogen immediately and stored at -80°C.
RNA extraction and cDNA cloning
Total RNA was extracted from the inflorescences using a modified
CTAB method (Zeng and Yang, 2002). The cDNA was prepared
using Smart cDNA library construction kit (BD Biosciences Clontech,
American). Full-length CmLFY cDNA was obtained by the 5 and 3
rapid amplified cDNA ends (RACE) method (Chenchik et al., 1996).
The first amplified chestnut cDNA contained about 440 bp between
primers CmS and CmA, designed from Arabidopsis thaliana LFY
(Weigel et al., 1992) and Antirrhinum majus FLO (Coen et al., 1990)
cDNA sequences. The 3 RACE was carried out between cassette
CDSIII/3’ primer and CmS2 primer. The DNA fragment amplified
was cloned into PMD18-T vector (Takara, Japan). The 5 RACE was
carried out between cassette CDSIII/5’ primer and CmA2 primer.
Full-length cDNAs were amplified with the sense primer LFY-ORF5
and antisense primer CDSIII/3’ primer. These clones were sequenced
completely by the dideoxy method using ABI3730 automated
sequencer (ABI Company, USA).
Phylogenetic comparisons
Liu et al. 3979
Protein sequences of different LFY homologs were retrieved from
GenBank and aligned with CLUSTAL. Phylogenetic relationships
among these genes were inferred by NJ analysis (MEGA Ver.3.1).
Plant transformation
The full length CmLFY (including start codon ATG) was amplified
between LFY-ORF5 and LFY-ORF3 (Figure 1). Then, the amplified
cDNA was blunted and KpnI and SalI linkers were ligated both ends,
cloned into a pCambia2301-101 binary vector in a sense-oriented
manner under the CaMV 35S promoter. An Agrobacterium
tumifaciens GV3101 strain (Van-Larebeke et al., 1974) was used for
the transformation of the A. thaliana Columbia ecotype by the
floral-dip method (Clough and Bent, 1998). For the selection of
transformed plants, resultant seeds were planted on a 1/2MS culture
medium containing kanamycin (50 g mL --1 ) as selective antibiotics
and then, transferred to 22°C under LD conditions (16 h light, 8 h
dark). Transformants were identified as kanamycin-resistant when
seedlings in the medium produced green leaves and well-
established roots. Resistant transformants were transplanted to
moistened potting soil composed of vermiculite and perlite (1:1
(v/v --1 )) after two to four adult leaves had developed and their flower
phenotypes were observed under long-day conditions.
RNA analysis of transgenic plants by RT-PCR
Detection of CmLFY transcripts was performed by RT-PCR. RNA
was isolated from whole plants of Arabidopsis by a method using
CTAB described previously described. CmLFY specific transcripts
were identified using 1 g of total RNA as a template and the
following primers: a sense primer LFY-ORF5 and an antisense
primer LFY-ORF3 for CmLFY (Table 1), giving rise to about 1300 bp
long PCR product. PCR reactions were run for 35 cycles at 58°C for
CmLFY. The PCR products were run on 1.5% (w/v --1 ) agarose gels
stained with ethidium bromide.
RESULTS
Cloning and sequence analyses of CmLFY
A comparison of Arabidopsis LFY (Weigel et al., 1992)
and Antirrhinum FLO (Coen et al., 1990) showed the
presence of several conserved regions. Two of these
regions, CmS and CmA, were used to design degenerate
oligo- nucleotide primers for RT-PCR. Using these
primers with cDNA prepared from an inflorescence of
chestnut, we obtained a PCR amplified fragment
containing both primers and other conserved sequences.
This fragment was named CmLFY (chestnut
FLORICAULA/LEAFY homologue). To obtain a full-length
CmLFY cDNA, several primers for 5’/3’ RACE were
designed. Primer sites and the nucleotide sequences of
primers used for cloning were shown in Figure 1 and
Table 1, respectively.
The coding region of CmLFY is 1,161 bp, encoding
386 amino acids. The deduced amino acid sequence of
CmLFY cDNA has 78% homology with PEAFLO, 79%
with PtFL and 59.9% with PlaraLFY. NLF, PRFLL, PEAFLO,

3980 Afr. J. Biotechnol.
Table 1. Primer sequences used in PCR cloning of CmLFY.
Primer Oligonucleotide
Primers for an internal fragment
CmS sense primer
CmA antisense primer
Cassette primers
CDSIII/5’ primer
CDSIII/3’ primer
3’ RACE primer
CmS2 primer
5’ RACE primer
CmA2 primer
CmLFY specific primers
LFY-ORF5
LFY-ORF3
5’- GGGAGCACCCGTTCATTGTGACTG -3’
5’-GA/CAGCTTG/TGTG/TGGGACATACCAGAC-3’
5’-AAGCAGTGGTATCAACGCAGAGT-3’
5’-ATTCTAGAGGCCGAGGCGGCCGACATG-3’
5’-CGGCCTTGATTACCTCTTCCATCTC -3’
5’- CACCGCGCTCCTTGGCAATGTTCTGT -3’
5’-GGTACCAAGCTAGCTTCATTGATG -3’
5’-CGGTCGACTAGAAATGCAAATTTTTCTC-3’
Figure 1. Nucleotide and deduced amino sequences (single-letter code) of CmLFY cDNA. The asterisk (*) indicates a stop
codon. Arrows above the sequences indicate the primers used for RACE-PCR.
ELF1 and CmLFY lacked a proline-rich region in
the variable region (roughly the first 40 amino acids). To
more closely determine the evolutionary relationship
between FLO/LFY-like proteins, a phylogenetic tree was
con- structed (Figure 2b). This tree showed that, the
topology of these genes seems to be concordant with the
topology of the species phylogeny and suggests that
CmLFY is an angiosperm ortholog of FLO/LFY-like genes.

a
Liu et al. 3981

3982 Afr. J. Biotechnol.
b
Expression patterns of CmLFY
Figure 2. (a) Sequence comparison of FLO/LFY-like proteins (accession numbers in
parentheses): NLY and PRFLL from P. radiata (U76757 and U92008, respectively); CmLFY
from chestnut (DQ270548); BOFH from B. oleracea (Z18362); FLO from A. majus (M55525);
LFY from A. thaliana (M91208); NFL1 and NFL2 from N. tabacum (U16172 and U16174,
respectively); ALF from P. hvbrida (AF030171); PtFL from P. balsamifera (U93196); RFL from
O. sativa (AB005620); TOFL from L.esculentum (AF197934); PEAFLO from P. sativum
(AF010190); ClLFY from C. glaziovii (AY633622); ELF1 from Eucalyptus globules (AF034806);
JunefLFY from J. effusus (AF160481) and PlaraLFY from P. hispanica (AF106842). Black
boxes indicate identical amino acids, shaded boxes indicate amino acids with similar
properties, and dots indicate gaps introduced to optimize alignment. c1 and c2, conserved
regions; v1 and v2, variable regions; (b) Protein sequence comparisons of CmLFY to other
LFY/FLO homologs. The deduced amino acid sequence of CmLFY was compared with some
published LFY homologs. Box characters represent chestnut protein.
CmLFY mRNA expression was analyzed in various
tissues by RT-PCR. Total RNA was isolated and PCR
primers specific to CmLFY were used to detect the
expression patterns in several tissues. The result showed
that CmLFY mRNA was expressed in inflorescence
tissues, but not in young leaves, fruits and stems (Figure
3). The RT-PCR of CmLFY -specific primers resulted in
amplification of a single band. Increasing the PCR cycle
did not change these expression patterns. The amplified
DNA fragments were the expected length.
Ectopic expression in Arabidopsis
To further verify the function of CmLFY, we constructed a
binary vector pCambia2301-101-CmLFY containing full-
length CmLFY coding area inserted in the sense-oriented
direction under the control of the Cauliflower 35S
promoter. The pCambia2301-101-CmLFY was introduced
into wild- type Arabidopsis plants by
Agrobacterium-mediated transformation. Fourteen
independent transgenic plants that survived on kanamycin
were identified (Table 2). Five (lines one-five) of them
flowered earlier than the wild-type plants by five-seven

Figure 3. Expression pattern of CmLFY gene revealed by RT-PCR
analysis. Lane 1: inflorescences; Lane 2: stem; Lane 3: tender leaves with
no flowers; Lane 4: tender leaves with flowers; Lane 5: fruit. rRNA is used
as RNA standard. Amplification of CmLFY was performed for 30 cycles.
Table 2. Comparison of flowering time, number of rosette leaves in T1 transgenic and wild-type Arabidopsis (Col)
plants in LD conditions.
Transgenic line Days to flowering a Rosette leaves at time of flowering b Note
1 18 4 Early flowering
2 16 3 Early flowering
3 18 4 Early flowering
4 20 5 Early flowering
5 20 4 Early flowering
6 24 7
7 24 8
8 25 9
9 22 8
10 24 8
11 25 8
12 26 9
13 26 10
14 25 8
wt c 25.5 d 9.5 d (n=10) e
v-wt f 26 d 9.5 d (n=8) e
a Days to flowering is defined as the time when flower primordial were first visible to the naked eye; b rosette leaves were
counted on the day that flower primordial were first visible; c w wild-type Arabidopsis Columbia plants; d Mean number; e
Number of plants;
f vector-transformed plants.
days and produced only four to five rosette leaves when
they flowered (Figure 4 and Table 2). Most of the T1
transgenic plants could self-pollinate and grow siliques
normally and the resultant seeds also had the ability to
germinate except for the lines 1, which is so small that it
was difficult to obtain its seeds. The early flowering were
inherited in the next generation and co- segregated with
the kanamycin-resistant genes. The expression of the
CmLFY mRNA in T1 plants was confirmed by RT-PCR, all
putative transformed lines showed the expected products
of CmLFY. No amplifi- cation was observed for the cDNA
prepared from non-transformed Arabidopsis plants and
vector-trans- formed Arabidopsis plants. The transgenic
plants are stably transformed and the siblings which
segregate without the transgene had no amplification of
CmLFY and flowered at the same time as wild-type
plants.
DISCUSSION
Liu et al. 3983
Despite the importance of understanding the regulation of
the flowering process in woody perennials for the
management and improvement of woody species, very
little is known about the underlying molecular
mechanisms. Regulation of flowering in woody perennials
shows remarkable differences in contrast to herbaceous
species, that is, long juvenile phases, bud dormancy and
the alternating vegetative and reproductive development
according to the season.
To investigate the molecular mechanism in chestnut
flowering, we cloned CmLFY, a putative homologue
of Arabidopsis LFY. A comparison (Figure 2a) of amino
acid sequences with Arabidopsis LFY (Weigel et al.,
1992), Antirrhinum FLO (Coen et al., 1990) and other
FLO/LFY homologs (Brassica oleracea. BOFH.

3984 Afr. J. Biotechnol.
Figure 4. Transgenic and wild-type Arabidopsis plants grown under LD
photoperiods for 20 days. Arabidopsis plants were grown in one-half strength of
medium for 9 days and transferred to potting soil. (a) wild-type Arabidopsis
Columbia. (b) Transgenic Arabidopsis carrying a 35S::CmLFY gene.
Anthony et al., 1993; Nicotiana tabacum. NFL. Kelly et al.,
1995; Oraza sativa. RFL. Kyozuka et al., 1998; Pinus
radiata. NLY. Mouradov et al., 1998 ; Petunia hybrida. ALF.
Souer et al., 1998; Eucalyptus globulus, ELF1, Southerton
et al., 1998 ; Lycopersicon esculentum. TOFL.
Molinero-Rosales et al., 1999; Juncus effusus. JunefLFY.
Frohlich and Parker., 2000; Platanus hispanica. PlaraLFY.
Frohlich and Parker., 2000; Populus balsamifera. PTFL.
Rottmann et al., 2000; Cedrela glaziovii. ClLFY. Marcelo
and Adriana, 2006) showed the presence of conserved
regions including two large conserved regions (c1 and c2)
and two shorter regions of lower similarity (variable
regions v1 and v2). These domains are typical markers for
transcriptional acti- vators and may be functionally
important for FLO/LFY-like proteins (Weigel et al., 1992;
Coen et al., 1990). The phylogenetic tree showed that the
topology of these genes seems to be concordant with the
topology of the species phylogeny and suggests that
CmLFY is an angiosperm ortholog of FLO/LFY-like genes
(Figure 2b).
The expression of CmLFY was only found in
inflorescences, but not in tissues such as fruits, stems and
tender leaves (Figure 3). It supports that the CmLFY is a
functional homologous gene of chestnut. In the species
such as apple, kiwifruit and grape, the expression levels
of their FLO/LFY homologs increase in the proliferating
inflorescence meristems generating inflorescence
branches, with the highest levels detected in young floral
meristems (Walton et al., 2001; Carmona et al., 2002).
Therefore, the high levels of CmLFY expression in inflo-
rescence suggests that CmLFY plays a role during chest-
nut reproductive development as it has been suggested
for most FLO/LFY-like genes studied.
To further demonstrate the function of CmLFY, we
have produced transgenic Arabidopsis plants expressing
CmLFY. They flowered earlier and had a shorter
inflorescence and reduced number of rosette leaves
compared with the controls (Figure 3). Based on the result
that several transgenic Arabidopsis with 35S::CmLFY
flowered earlier than the controls, the expression of
CmLFY should have some relationship with the early
flowering phenotypes, although, the mechanism of
flower-bud formation in chestnut might be different from
that in Arabidopsis. Of course, to be more certain that the
CmLFY gene functions similarly to LFY, LFY mutant-
rescue experiments will be required. The CmLFY gene
was not expressed strongly in transformants showing
early flowering relative to those that were wild type in
appearance, which suggests that the severity of pheno-
type in transformants does not depend solely on the level
of gene expression in this case, although the exact cause
is still unknown. Some reports are available that describe
transgenic Arabidopsis plants expressing LFY homologs
from other plant species. For example, Arabidopsis
transgenic lines expressing NEEDLY from Radiata pine
and ELF1 from Eucalyptus showed early flowering
(Mouradov et al., 1998; Southerton et al., 1998). Until now,
however, our ability to manipulate fruit/nut tree strains
through genetics has been limited by the extended
maturation period of these plants. Both taking long time to
flowering and the fact that, no characterized flowering
mutants have been described in this genus make genetic
studies in chestnut more difficult. Nevertheless, advances
in the establishment of transformation protocols for

chestnut may allow us to use reverse genetic approaches
and to define more clearly the role played by CmLFY in
the reproductive development.
The breeding of fruit trees of chestnut often requires
more than 20 years, including periods of cross pollination,
seedling selection and regional trials, to produce varieties
that meet the demands of consumers. One of the limit
factors of chestnut breeding is the long juvenile phase of
at last several years. It had been found that transgenic
approaches of LFY introducing could reduce the juvenile
phase of Populus and the transgenic poplar flowered in 5
months after regeneration (Weigel and Nilsson, 1995).
Therefore, probably we can anticipate that these techni-
ques are likely applicable to fruit trees of chestnut in future
years and believed that, the transgenic approach would
be a useful breeding strategy for reducing the time
required for generation among woody plants. However,
the efficiency of producing early flowering transgenic lines
with Arabidopsis LFY gene seems to be low in woody
plants in some case (Pena et al., 2001). This suggests
that regulatory genes such as LFY do not always function
beyond species as well as expected. Since environmental
and genetic factors controlling flower development in
chestnut have not been made clear so far, the CmLFY
gene could at least, be one of the tools available for
studying the mechanism of flower development in tree
fruits such as the chestnut.
ACKNOWLEDGMENTS
This research was financially supported by the Faculty of
Agronomy and Biotechnology (SJCX09012 to T Liu) and
the National Natural Science Foundation of China (NSFC,
30800062 to XX Li).
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African Journal of Biotechnology Vol. 10(20), pp. 3986-3995, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.053
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Overexpression of a foxtail millet Acetyl-CoA
carboxylase gene in maize increases sethoxydim
resistance and oil content
Zhigang Dong 1,2# Huji Zhao 1# Junguang He 1 , Junling Huai 1 , Heng Lin 1 , Jun Zheng 3 , Yunjun
Liu 3 and Guoying Wang 1,3*
1 State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, China.
2 Pomology Research Institute, Shanxi Academy of Agricultural Sciences, Taigu 030815, China.
3 Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Accepted 11 April, 2011
Acetyl-coenzyme A carboxylase (ACCase) plays a crucial role in fatty acid metabolism in plants,
catalyzing the carboxylation of acetyl-CoA to produce malonyl-CoA. In grasses, the plastids ACCases
are critical determinants of plant sensitivity to herbicide classes cyclohexanediones (CHDs) and
aryloxyphenoxy propionates (APPs). We transformed the full-length cDNA of a plastid ACCase gene
from foxtail millet (Setaria italica) line Chum BC6-1, which is highly resistant to sethoxydim, into maize
(Zea mays L.) drived by a ubiquitin promoter. The substantial results indicated that the sethoxydim
resistance of transgenic plants is increased, with a damage index of 33%, much lower than that of 97%
in the untransformed plants. Additionally, the oil content of transgenic maize seeds was 24 to 65%
higher than that of untransformed seeds. These results indicate that the ACCase transgenic plants can
be used to develop new maize hybrids that are tolerant to herbicide CHDs with high oil content.
Key words: Zea mays, Acetyl-coenzyme A carboxylase (ACCase), oil content, transgenic plants,
Agrobacterium tumefaciens
INTRODUCTION
Acetyl-coenzyme A carboxylase (ACCase) was first
discovered nearly 50 years ago and has been studied
extensively over the years (Wakil et al., 1983). Two forms
of ACCase exist in plants. One is located in the plastids,
the primary sites of plant fatty acid synthesis. This form is
a “prokaryotic-type” multi-subunit enzyme and the other
form, usually located in the cytoplasm, is a “eukaryotictype”
multifunctional enzyme. In the grass family (including
wheat, rice and maize), however, the “eukaryotic-type” is
located in both the cytosol and plastids “(Alban et al.,
1994; Konishi and Sasaki, 1994; Gornicki et al., 1997;
*Corresponding author. E-mail: gywang@caas.net.cn.
Abbreviations: ACCase, Acetyl-CoA carboxylase; CHDs,
cyclohexanediones; APPs, aryloxyphenoxy propionates; HR,
high resistance; HS, high sensitivity.
#These authors contributed equally.
Zhao et al., 2004; Tong L, 2005).
Plant ACCases have attracted particular attention
because the grass plastid ACCase is the active site of
three chemically distinct classes of herbicides,
aryloxyphenoxypropionates (APPs, also known as
FOPs), cyclohexanediones (CHDs, also known as DIMs)
and phenylpyrazolin. These classes of compounds
reduce ACCase activity in meristematic tissue and kill
sensitive plants by inhibiting their fatty acid biosynthesis.
It has been reported that, grass ACCase in plastids is
sensitive to these herbicides, but dicot and nongraminaceous
monocot ACCases exhibit less sensitivity
(Herbert et al., 1997; Christoffers et al., 2002). When
these herbicides are used to kill weed grasses, they also
kill the crop grasses, thus, greatly limiting their
application. An alternative approach would be to produce
grass crops highly tolerant to these herbicides.
There are some reports indicating that the sensitive
form of the enzyme has an Ile residue, while the resistant
form has a Leu residue at the putative herbicide-binding

site (Zagnitko et al., 2001). Additionally, a single Ile to
Leu replacement at an equivalent position changes the
wheat plastid ACCase from sensitive to resistant
(Zagnitko et al., 2001). Furthermore, the cDNA (foxACC-
R gene) of the foxtail millet (Setaria italica) line Chum
BC6-1, which has been reported to show high resistance
to sethoxydim, has the same Leu reside in this position
compared with an Ile residue in sensitive lines (Wang T
et al., 1997; Zhao et al., 2004).
In this study, we transformed the foxACC-R gene into
the maize inbred line Zong 3 using Agrobacterium
tumefaciens mediated transformation (Zhang et al.,
2001). Southern blot assay, RT-PCR detection and the
test of sethoxydim tolerance on transgenic plants indicated
that the foxACC-R gene was stably integrated into
the maize genome; also, all these plants with the higher
oil contents. Our objective was to develop maize lines
that were highly resistant to sethoxydim; therefore, these
transgenic lines can be incorporated into maize breeding
programs in the future.
MATERIALS AND METHODS
Plasmid construction
The binary vector pCAMBIA3301 (Center for the Application of
Molecular Biology to International Agriculture, Canberra, ACT,
Australia) was used as a backbone for the construction of a plant
expression vector. The bar gene, 35S promoter and GUS reporter
gene were replaced by the foxACC-R (AY219175) driven by the
ubiquitin promoter from maize to construct a marker-free p3301-
ACCase vector (Figure 1).
Maize transformation
The approach used to generate transgenic maize plants was
adapted from that described by Yang et al., 2001 and Zhang et al.,
2001 The size of immature embryos are 1.0 to 2.0 mm, embryos
were isolated from the maize inbred line Zong 3 and put into liquid
infection medium (D basal medium (Duncan et al.,1985), 68.5 gl –1
Suc, 36.0 gl –1 Glc, 100 µM AS (Sigma), pH 5.2) in 2-ml Eppendorf
tubes (20 to 100 embryos per tube). Then, they were washed twice
with this medium. The final wash was removed and 1.5 ml of A.
tumefaciens suspension was added. The tubes were gently
inverted 20 times and then, rested upright for five minutes with the
embryos submerged. After infection, the embryos were transferred
to the surface of co-cultivation medium (D basal medium, 20 gl –1
Suc, 10 gl –1 Glc, 0.85 mgl –1 silver nitrate, 100 µM AS, 8 gl –1 agar, pH
5.8). The embryos were incubated in the dark for three days at
25°C, after which they were transferred to resting medium (D basal
medium, 20 gl –1 Suc, 10 gl –1 Glc, 0.85 mgl –1 silver nitrate, 250 mgl –1
cefotaxime, 8 gl –1 agar, pH 5.8) for seven days. Then, the embryos
were moved to selection medium which was identical to resting
medium with the addition of 5 µM sethoxydim and maintained for
two weeks in the dark at 25°C. The concentration of sethoxydim
was then increased to 10 µM for another three successive
selections. Sethoxydim-resistant calli were placed on MS medium
for regeneration. The shoots of 2 to 3 cm in height were moved to
1/2 MS rooting medium (Murashige and Skoog, 1962). Calli for
regeneration and regenerated plantlets were grown in a growth
chamber at 28°C under fluorescent white light in 16:8 h light: dark
cycle.
PCR analysis of transgenic maize plants
Dong et al. 3987
Genomic DNA was isolated from leaves of transgenic maize and
untransformed plants by the SDS method for PCR analysis (Sabelli
and Shewry, 1995). Primers for the ACCase gene (ACCase-S 5’-
CAATGTGTATGCTACATGTTTTTGT-3’ and ACCase-A 5’-
ACCCCTGCAAAACCAGA-3’) were used for PCR analysis, it is
specific for foxtail millet and there was no corresponding band in
the maize genome (Zhao et al., 2004). The PCR products
were subjected to 0.8% agarose gel electrophoresis and stained by
EtBr to visualize the expected band.
Southern blotting
Southern blot analysis was carried out by standard procedures as
described by Sambrook and Russell (2001). DNA samples isolated
from maize leaves of transgenic and untransformed plants were
purified, concentrated and quantified. About 50 µg of genomic DNA
was digested with 40 U of EcoRI (Promega) at 37°C overnight. The
digested DNA was separated by electrophoresis on a 0.8% agarose
gel and blotted on Hybond- N+ nylon membrane (Amersham
Pharmacia) in 0.4 mol/l NaOH. The probe (ACCase-S 5’-
CAATGTGTATGCTACATGTTTTTGT-3’ and ACCase-A 5’-
ACCCCTGCAAAACCAGA-3’) was random -primed labeled with ( -
32P) dCTP. Southern hybridization was carried out overnight at
42°C. The hybridized filters were washed in 2 × SSC with 0.5%
SDS at 42°C for 30 min, twice in 1×SSC with 0.1% SDS at 65°C for
30 min and twice in 2×SSC at 65°C for 10 min then exposed to xray
film (Fuji) at -70°C.
Semi-quantitative RT-PCR analysis of transgenic maize plants
Total RNA of the transgenic and untransformed plants was
extracted according to the Trizol reagent (Invitrogen) protocol. All
preparations of total RNA were evaluated by denatured agaroseglyoxal
gel electrophoresis. First-strand cDNAs were synthesized
using ImProm-II TM reverse transcriptase (Promega) and the
ACCase transcript was amplified using foxtail millet ACCase gene
primers (ACCase-S 5’- CAATGTGTATGCTACATGTTTTTGT-3’
and ACCase-A 5’- ACCCCTGCAAAACCAGA-3’). At the same time,
maize tubulin cDNA (Tubulin-S 5’-
GCTATCCTGTGATCTGCCCTGA-3’ and Tubulin-A 5’-
CGCCAAACTTAATAACCCAGTA-3’) was also amplified as a
control.
Determination of oil content in transgenic maize seeds
For the oil content measurements, the transgenic and
untransformed plants were grown in the same environment and
harvested at the same time. Using a near infrared reflectance
spectroscopy (NIRS) instrument (BRUKER, VECTOR22/N), 20
intact seeds in a revolving sample with a diameter of 50 mm were
scanned 64 times at a wavelength region of 80 to 240 nm to
calculate the average spectrum. In order to eliminate the influence
of seed size and seed uniformity on the spectrum, each sample
was replicated three times. Means for transgenic and
untransformed plants were compared by using the two-sample ttest
at the 5% confidence level as detected by LSD tests. The T0
plants of PCR positive were self-pollinated to produce T1 seeds,
and T1, T2 plants that were highly resistant to sethoxydim were selfpollinated
to produce T3 seeds.
Sethoxydim spraying test
On a sunny day (>25°C), six-leaf stage T1 plants were painted with

3988 Afr. J. Biotechnol.
Figure 1. Structure of the expression vector p3301-ACCase. ACCase. The ubiqutin promoter was fused
with the foxACC-R R gene. Horizontal lines represent short pieces of polylinker DNA that connects
the gene elements. The plasmid vector pCAMBIA3301 was as the backbone, and for the future
application, ation, the bar gene, the 35S promoter and GUS reporter gene were deleted. Relevant
restriction endonuclease sites are indicated.
a 625 mg/l solution of sethoxydim (12.5%; Nisso, Japan). Four Four- to
six-leaf stage T2 plants were sprayed in the field with a
concentration of 1250 mg/l sethoxydim. The reaction of the plants
to sethoxydim was recorded one week later. The degree of
herbicide damage was evaluated according to the following criteria:
level 0, no obvious chemical ical damage symptoms; level I, leaves
show temporary chemical damage spots or slight growth
suppression; level II, leaves show heavy chemical damage
spots,loss loss of green coloration, shrinkage, abnormal growth or
obvious growth suppression, but normal growth may be restored;
level III, the growing point dies or continued growth is seriously
suppressed; level IV, partial or the complete plant death.
The damage index was s calculated according the following
formula: damage degree index = ( (damage ge level * number)/ total
number * highest level) * 100% (Jiang, 1987). ). The resistance of
maize to the herbicide was divided into five grades according to the
damage degree index: HR (highly resistant) (index 20); R
(resistant) (20

Comparison of oil content of transgenic and wildtype
plants
The oil content of seeds was not determined for T0 plants
of PCR positive lines due to the potential epigenetic
variation generated during plant tissue culture. Starting
from T1 plants, oil content was measured and compared
with control plants. From the results of the comparison of
T1, T2 and T3 generation plants (Table 2; Figure 7), all
positive plants of each line had significantly higher oil
content than controls. Lines 40 and 46 had 54 to 65%
higher oil content than the controls in the T3 generation.
Sethoxydim tolerance assay of transgenic plants
To determine the response of the transgenic plants to
Figure 2. Alignment of the amino acid sequence of
the CT domain of ACCase genes. The isoleucine
/leucine is indicted in bold. ACCase sequences were
obtained from GeneBank, left is the accession no.
The species is listed in Table 1.
Dong et al. 3989
sethoxydim, the fifth leaf of seedlings at the six-leaf stage
was painted with sethoxydim at a concentration of 625
mg/l. Seven days after treatment, the transgenic
seedlings showed normal growth (Figure 8a), while the
untransformed plants wilted, turned brown and died in
two weeks (Figure 8b).
T2 plants were sprayed with 1250 mg/l sethoxydim (the
commercial concentration) in the field. Seven days later,
the transgenic plants all showed normal growth, while the
untransformed plants wilted (Figure 8c, d and e). The
degree of herbicide damage was evaluated for every
plant of the T2 generation (Table 3). The damage index of
lines 40, 46 and 59 was lower than 20% and that of
lines6, 17 and 76 were between 20 and 33% and the
control had a damage index of 97%.
These results showed that the foxACC-R transgenic
plants were more resistant than the control.

3990 Afr. J. Biotechnol.
DISCUSSION
Table 1. The species chosen for the analysis and their accession number.
Accession number Species name Special residue
AF025469 Caenorhabditis elegans L
U39321 Triticum aestivum (bread wheat)-Acc2 L
AF029895 Triticum aestivum (bread wheat)-Acc1 I
U19183 Zea mays I
AY219175 Setaria italica (foxtail millet)-R L
AY219174 Setaria italica (foxtail millet)-S I
AF330145 Toxoplasma gondii L
AF072737 Avena sativa (oat) I
AF359513 Lolium rigidum-S I
AF359514 Lolium rigidum-R L
AJ132890 Bos taurus (cattle) L
NM-198839 Homo sapiens (human)-ACC1 L
NM001093 Homo sapiens (human)-ACC2 L
J03541 Gallus gallus (chicken) L
J03808 Rattus norvegicus (Norway rat) L
L20784 Cyclotella cryptica L
L25042 Medicago sativa L
L42814 Glycine max (soybean) L
M92156 Saccharomyces cerevisiae L
P78820 Schizosaccharomyces pombe L
X77576 Brassica napus (rape) L
X99102 Hordeum vulgare subsp. vulgare L
Y15996 Emericella nidulans L
Z46886 Ustilago maydis L
CM000142 Oryza sativa Japonica I
EU660897 Aegilops tauschii I
EU660891 Aegilops tauschii L
CM000147 Oryza sativa Japonica L
EQ973783 Ricinus communis (castor bean) L
AF062308 Arabidopsis thaliana-ACC1 L
AF062308 Arabidopsis thaliana-ACC2 L
CM000338 Populus trichocarpa L
CM000341 Populus trichocarpa L
In plants, ACCase is a proven target for herbicide action.
Genetic analyses have identified several single mutation
sites in the CT domain that determine the herbicide
resistance (Nikolau et al., 2003). A single-site Ile-to-Leu
mutation at the position equivalent to residue 1705 in
yeast ACC which represents an extremely subtle change
in the side chain of the amino acid, is sufficient to confer
resistance to both APPs and CHDs in the ACC enzyme
of many grasses, while those that are resistant to these
herbicides have a Leu residue at this position (Zagnitko
et al., 2001; Delye et al., 2002). The second single-site
mutation that confers resistance to herbicides is an Ile-to-
Asn change (equivalent to residue 1967 in yeast ACC),
again in the CT domain of ACC (Delye et al. 2003, 2004).
Mutations at three additional sites in the CT domain have
been identified from resistant weeds (Delye et al., 2005).
An Asp/Gly change (equivalent to residue 2004 in yeast
ACC) confers resistance to both APPs and CHDs,
whereas a Trp/Cys and a Gly/Ala change (1953 and 2022
in yeast ACC, respectively) confer resistance to APPs
only. From our results and analysis, we concluded that
the change from Leu to Ile at residue 1705 is critical for
the interaction of ACCase with CHDs. Interestingly, our
phylogenetic analysis (Figure 9) of ACCase genes of

Figure 3. Production of transgenic maize plants. (A) Sethoxydimresistant
calli with the white growing point; (B) plantlet regeneration
from the sethoxydim-resistant calli; (C) plantlets in 1/2 MS rooting
medium; (D) regenerated plantlets in a growth chamber.
Dong et al. 3991
Figure 4. PCR detection of ACCase band (195 bp) in the posterity. M: DNA marker; lane 1, 6, 9, 17, 25, 33,40, 46,
59 and 76 represents the transgenic lines; lane CK- represent the untransformed plant Zong3; lane CK+ represent
the plasmid p3301-ACCase.
Table 2. The oil contents of T1, T2 and T3 generation plants.
Line
Oil content of T1
generation (%)
Average oil content of T2 generation
(%)
Average oil content of T3
generation (%)
6 3.56±0.05 de 3.61±0.08 c 4.29±0.21 c
17 3.86±0.13 cd 4.30±0.25 b 4.47±0.35 bc
40 5.03±0.43 b 5.49±0.36 a 5.34±0.64 a
46 5.62±0.30 a 5.98±0.44 a 5.71±0.20 a
59 4.10±0.29 c 5.52±0.32 a 5.07±0.31 ab
76 5.09±0.18 b 4.54±0.39 b 4.58±0.43 bc
Z3 3.39±0.19 e 3.51±0.37 c 3.47±0.21 d
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,
40, 46, 59 and 76 represented independent transgenic lines, Z3 is untransformed inbred line. Means with standard errors were
calculated from three replicates. Different letters represent significant difference at 5% confidence level as detected by LSD tests

3992 Afr. J. Biotechnol.
Figure 5. Southern blot analysis of TT2
transgenic maize plants.
Aliquots of 50 µg of EcoRI -digested total maize leaf DNA was
fractionated on 0.8% agarose gel, blotted to nylon membrane and
probed with
plasmid p3301
plant.
32 P-labeled 195bp ACCase gene fragment. CK+ is the
plasmid p3301-ACCase (16.37 kb) and CK- is the untransformed
Figure 6. Detection of levels of the foxtail millet ACCase
transcripts by semi semi-quantitative RT-PCR. Upper bands are the
PCR fragments specific for Accase Accase. Lower bands are the maize
actin PCR fragments used as the control. 6, 17, 40, 46, 59 and 76
are the transgenic lines, Z3 is the untransformed plant.
Figure 7. The ratio of oil content of the TT1,
T2 and T3 generation plants. The data ata bars
indicate the ratio of oil content of transgenic lines to untransformed control.

Table 3. The damage index of T3 generation transgenic plants by herbicide.
Parameter
Line
The damage index (%) 30.00 22.22 16.67 14.28 13.33 32.14 96.67
The damage index is the mean value of fourteen to fifteen plants plants.
Figure 8. The sethoxydim resistance evaluation in the field. The fifth leaf of seedlings with 4 to 6 true leaves was pained by
625 mg/l of sethoxydim. Seven days after treatment, the WT plants turned wilt and brown (B), while the transgenic seedlings
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
untransformed control. (E) Left line is the transgenic plants and right line is untransformed plants.
these eukaryotic and prokaryotic types indicates that, all
types that hat have an Ile residue at this position are grouped
into one subfamily, except for AF359514 and AY219175,
which show a leu/Ile substitution at this position. These
results suggest that the CT domain of ACCases in this
subfamily has a special structure that t can bind the
herbicide and that this particular mutation may interfere
with herbicide binding, leading to herbicide resistance.
However, the exact mechanism is not clear. clear.ACCase has
been identified as an important regulatory enzyme for
plant fatty acid synthesis by three in vivo approaches.
Analysis of substrate and product pool sizes has
implicated ACCase in the light/dark regulation of fatty
acid synthesis in spinach leaves (Post-Beittenmiller
et
al.,1992) and chloroplasts (Post-Beittenmiller Beittenmiller et al.,
1991). The total oil content of seeds is increased
approximately 5% by the expression of Arabidopsis
ACCase in rapeseed plastids (Keith et al., 1997). In this
study, over-expression of the plastid foxACC foxACC-R gene
from a foxtail millet increased oil content from 24 to 65%
in the T3 generation over that of the control maize inbred
Transgenic line
6 17 40 46 59 76
Dong et al. 3993
Control
line Zong3 (Table 2). This result is consistent with that
found in tobacco suspension cells, where ACCase is the
apparent site of feedback inhibition of fatty acid synthesis
when supplemented with exogenous fatty acids (Shintani
and Ohlrogge, 1995).
Sethoxydim is a post-emergence emergence herbicide used to
eliminate weeds in dicotyledonous crops. It is absorbed
by the leaves and transported throughout the phloem
towards the apical meristem. stem. Systemic herbicides require
from several days to a few weeks to move throughout the
vascular system of a treated plant. In monocots, necrotic
zones develop in newly formed leaves, resulting in
growth inhibition and cell death in one or two weeks. In
dicots, effects of post-emergence emergence herbicides have been
reported under field conditions (Griffin and Habetz, 1989;
Hart and Roskamp, 1998), but plants usually recover
from injury, depending on growth conditions and season
(Kapusta et al., 1986).
Our results s indicate that the full-length full cDNA of
foxACC-R, when introduced duced into maize, leads to high
resistance to the herbicide sethoxydim; the damage indices
Z3

3994 Afr. J. Biotechnol.
Figure 9. Phylogenetic analysis of the ACCase genes. The amino acid sequences of the
ACCase gene were aligned by ClustalX1.83 and the phylogenetic tree was constructed
using the NJ method in MEGA 3.1. All the types which have Ile residue were grouped into
one subfamily, except for the AF359514 and AY219175 which is leucine /isoleucine
substitution in this position.
of all transgenic lines were lower than 33%, while that of
the control was 97%.
In summary, over-expression of the full-length cDNA of
the foxACC-R gene in maize leads to increased
resistance to sethoxydim. These lines might be used to
develop new maize hybrids with herbicide tolerance in
the near future.
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African Journal of Biotechnology Vol. 10(20), pp. 3996-4004, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB11.106
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Variation in nodulation and growth of groundnut
(Arachis hypogaea L.) on oxisols from land use
systems of the humid forest zone in southern
Cameroon
Laurette Ngo Nkot 1 *, Dieudonné Nwaga 2 , Albert Ngakou 3 , Henri Fankem 1 and François-Xavier
Etoa 4
1 Department of Plant Biology, Faculty of Science, University of Douala. P.O. Box 24157, Douala, Cameroon.
2 Laboratory of Soil Microbiology, Biotechnology Centre, University of Yaoundé I. P.O. Box 812 Yaoundé, Cameroon.
3 Department of Biological Sciences, Faculty of Science, University of Ngaoundéré. P.O. Box 454, Ngaoundéré,
Cameroon.
4 Department of Biochemistry, Faculty of Science, University of Yaoundé I. P.O. Box: 812 Yaoundé, Cameroon.
Accepted 11 April, 2011
Improving biological nitrogen fixation through legume nodulating bacteria (LNB) inoculation requires
knowledge on the abundance and effectiveness of indigenous population in the ferralsols. Nodulation
of groundnut was examined under pots experiment in four location sites of the Humid-forest zone:
Bertoua in the East; Ebolowa in the South; Bokito and Yaoundé in the Centre Regions of Cameroon and
within each of the locations, in four land use systems (LUSs) of different levels of disturbance: mixed
farming (1 to 3 years old); fallows (3 to 5 years old); cocoa plantation (> 20 years old); forest (> 30 years
old). Results indicate that, soils under investigation are acidic with pH ranging from 3.68 in the Ebolowa
forest to 6.92 in mixed farming at Bokito. Groundnut formed nodules in all the four LUSs. Soils from
plantations and forests were poorly nodulated, whereas those from mixed farming and fallows were
highly nodulated, with a positive and significant correlation (r 0.406; p < 0.0001) observed between
nodulation and plant biomass in all the studied sites. These results suggest a high variation in
groundnut nodulating bacteria density in soils from diverse LUSs of the humid forest zone of
Cameroon; although, there was a site effect.
Key words: Acid soil, Arachis hypogaea, biomass, land use systems, multilocal, nodulation.
INTRODUCTION
Many soils in the tropics are fragile, with high acidity and
aluminium toxicity, high phosphorus fixation and low soil
biodiversity (Cardoso and Kuyper, 2006). Nitrogen and
phosphorus are the most limiting nutrients for crop
production in these ferral soils (Dogbe et al., 2002). In
Cameroon, acid soils cover more than 80% of arable
lands (The, 2000). Synthetic fertilizers for improving soil
*Corresponding author. E-mail: lnkot@yahoo.fr. Tel: (00 237) 99
92 77 16 or (00 237) 33 30 31 07. Fax: (00 237) 33 42 76 12.
fertility are rarely available to most farmers. In addition,
these fertilizers may induce soil acidification and become
less efficient after many cropping years (Bado, 2002),
leading to a high dependence of soil to N fertilizer for
optimum yield (Fening and Danso, 2002). Since fallow
practice to restore soil fertility is no longer possible
because of land scarcity, there is a need of more efficient
practices.
Four land use systems (LUSs) are common in humid
forest zone in Cameroon: forest; cocoa or coffee plantation;
fallow and mixed farming. Forest conversion to
farms or plantations is increasing in response to popula-

tion growth (Van Noordwijk et al., 2002). Cocoa
(Theobroma cacao) plantation, is one of the dominant
LUS, has served in the past as an economically-attractive
land use but has been rejected by many farmers because
of low and unpredictable prices on the world markets.
Hence, farmers were forced to abandon cocoa or coffee
plantations toward other income-generating opportunities
such as food crops. Forest and some old plantations are
used for annual crops such as groundnut in a mixed
farming system. Under conditions of low external input,
the use of biological nitrogen fixation appears to be a
cheaper and environmentally friendly source of N for
plants (Peoples et al., 1995). According to Bogino et al.
(2006) a high degree of nodulation and nitrogen fixation
in groundnut is provided by indigenous LNB in some soils
from Argentina. Groundnut (Arachis hypogaea L.) is an
important legume crop that provides food for human
subsistence and other products. In Cameroon, groundnut
is the major legume crop in slash and burn, traditional
agricultural food production system. It is grown on
250.000 ha with more than half of the production area in
the northern part of the country (Ntoukam et al., 1996).
The yield is low, with an average 0.85 t.ha -1 compared
with 1.05 t.ha -1 in Senegal and 1.03 t.ha -1 in Nigeria
(Anonymous, 2000). In sub-Saharan Africa, cropping
systems, slash and burn agriculture and acid soil infertility
could be major limiting factors for this crop yield. In
addition, the density of indigenous LNB in diverse land
use systems may affect nodulation and legume performances.
According to Nwaga et al. (2010), land use
changes and cropping practices such as burning may
affect soil functioning in the humid forest, along with
diversity and occurrence of beneficial micro-organisms.
When comparing indigenous versus selected rhizobia
from USA, yield diversity of 0.33 to 1.48 t/ha was noticed
according to groundnut genotypes and 0.27 to 0.76 t/ha
according to rhizobia x genotype interactions in northern
Cameroon low rainfall regions (670-1068 mm)
(Mekontchou et al., 2007). It is therefore, important to
select acid LNB strains with high symbiotic effectiveness
to improve legume production in tropical acid soils. One
of the major problems to improve A. hypogaea in this
region is ‘flat pod phenomenon’ or ‘empty seeds
phenomenon’ of groundnut. Few data are available on
the nodulation and plant growth of legumes such as A.
hypogaea in the Congo Basin zone in Central Africa.
Therefore, the objective of this study was to assess under
greenhouse conditions, nodulation and plant growth of A.
hypogaea on acidic soils from diverse land use systems
in the humid forest zone of southern Cameroon.
MATERIALS AND METHODS
Study sites, experimental design and treatments
Experiments were carried out in soil samples (2 kg plastic bags)
collected from four location sites of the humid forest zone: Bertoua
in the East; Ebolowa in the South; Bokito and Yaoundé in the
Ngo Nkot et al. 3997
Centre regions of Cameroon (Figure 1). Within each site, soils were
collected in four replicates in the four different land use systems
(LUSs): mixed farming, fallow (Chromolaena odorata), cocoa or
coffee plantation and forest. The experimental design was thus, a
split plot (4 x 4) x 4 with the 4 different land uses representing the
main factors and the 4 study sites the sub-factors.
Soil sampling and analysis
A total of 64 soil samples were collected in four location sites
according to Swift et al. (2001). A composite soil sample was a
mixture of ten sub-samples all collected from a LUS of the same
location site. Aseptic precautions were taken during sampling and
handling of each soil to avoid contamination. Soil sample were
taken from sites that had no previous history of LNB inoculation.
The 64 composite samples were taken from the top 0 to 20 cm soil
depth, from which the un-decomposed plant material were removed
by hand. The chemical analysis of the soil was conducted using
standard methods (Anderson and Ingram, 1993).
Assessment of nodulation and growth of A. hypogaea
Groundnut seeds of the A26 variety were obtained from the Institute
of Agricultural Research and Development (IRAD). Nodulation was
assessed by growing germinated seed in perforated plastic bags
containing more than 2 kg of soil samples. Seeds were surface
sterilized in 3.3% calcium hypochlorite solution for 3 min and
washed thoroughly with sterile water, re-suspended in 70% alcohol
for 3 min and washed thoroughly with sterile water. The seeds were
incubated to germinate in sterile Petri dishes containing 0.9% (w/v)
water agar for 2 to 3 days at 28°C. After germination, six seedlings
were sown in each plastic bag. Two weeks after germination, the
number of plants was thinned to four per plastic bag, thus, sixteen
per treatment.
Plants were watered and harvested 6 weeks after sowing. The
root systems of individual plants were washed separately, their
nodules picked and counted from sixteen plants per treatment.
Plant shoot and roots systems were let to dry at 70°C for more than
72 h in an air dry oven and then weighed until constant mass (Athar
and Johnson, 1996).
Statistical analysis
Data were subjected to analysis of variance (ANOVA). Means were
separated between treatments with the least significant difference
(LSD), using the Statgraphic plus, version 5.0 (SIGMA PLUS)
computer package. Comparisons were made among treatments of
the same location site. A p-value < 0.05 was generally used to
evaluate significance, although, higher levels were considered for
p-value < 0.0001. The statistical package for social sciences
(SPSS) was used to assess the correlation between the nodulation
and growth parameters.
RESULTS
Differences in the chemical components of studied
soils
The chemical properties of soils under study are shown in
Table 1. The soil aluminium content was consistently
lower in the fallow (0.34 meq/100 g) than in other LUSs at
Bertoua (p = 0.025), whereas at Bokito, it was

3998 Afr. J. Biotechnol.
Figure 1. Localisation of Cameroon in Africa and distribution of the investigated sites in
humid forest zones.
significantly lower (p = 0.013) in the forest (0.28 meq/100
g) than the plantation (0.49 meq/100 g). At Ebolowa, the
soil aluminium content was significantly lower in the
fallow (0.32 meq/100 g; p = 0.002). In contrast, no significant
difference was found between the soil aluminium
contents of different LUSs in Yaoundé. The lower
aluminium values was 0.20 meq/100 g and the higher
values was 0.52 meq/100 g.
Soil pH of the four sites of the humid forest zone
ranged from 3.96 in the forest at Ebolowa to 6.00 in
mixed farming at Bertoua and did not show significant
differences between the LUSs in any of the studied sites.
Ebolowa soils were more acid than those of Bertoua,
Bokito and Yaoundé. Soil organic carbon ranged from
0.88 to 2.18%, while soil organic matter varied from 1.51
to 3.03%, with a mean value of 2.59%. At Bokito, the soil
organic carbon was similar in forest, mixed farming and
fallow, but was significantly higher (p = 0.017) than in
plantation. Similarly, the soil organic matter did not significantly
vary between the Ebolowa mixed farming and
forest, but was significantly higher (p = 0.05) than those
of the fallow and plantation. The total mean soil N value
was low and ranged between 0.11 and 0.15% with little or
no site variations. Plantation had the most acid LUS soil
with an average of 4.85.
Nodulation of groundnut in different LUSs
All the soils used for nodulation experiment had
indigenous rhizobia that were able to nodulate groundnut.
The mean nodule number varies from 13 in the
plantations to 94 nodules in the fallow, depending on site
and LUSs. A highly significant correlation was found
between nodule number and groundnut growth.
The number of nodules per plant was significantly
higher (p < 0.0001) in the fallow than in other LUSs in all
the experimental sites, with the maximum number of
nodules observed at Bokito (94 nodules). This nodulation
status (>75 nodules/root system) is characterized as
good according to Amijee and Giller (1998). There was
no significant difference between the number of nodules
formed in the plantation and forest at Bertoua, mixed
farming, plantation and forest at Bokito and Yaoundé,
mixed farming and plantation at Ebolowa. The number of
nodules formed by A. hypogaea in all the LUSs at
Ebolowa was lower than that of any of the sites, with the
lowest accounting for plantation (13 nodules). This nodu-

Ngo Nkot et al. 3999
Table 1. Variation of the soil chemical parameters under different land uses of the experimental sites in the humid forest zone of
southern Cameroon.
Site Chemical parameter
Bertoua
Bokito
Ebolowa
Yaoundé
Farm Fallow
Land use
Plantation Forest
p-value LSD (5%)
Al 3+ (meq/100 g) 0.47 b 0.34 a 0.40 ab 0.39 ab 0.025 0.127*
C/N (ratio) 8.60 a 12.04 a 9.40 a 8.65 a 0.561 ns
pH (H2O) 6.00 a 5.97 a 5.71 a 5.54 a 0.285 ns
N (%) 0.12 a 0.11 a 0.11 a 0.11 a 0.846 ns
OM (%) 1.77 a 2.05 a 1.78 a 1.60 a 0.549 ns
OC (%) 1.03 a 1.20 a 1.03 a 0.93 a 0.535 ns
Al 3+ (meq/100 g) 0.45 ab 0.45 ab 0.49 b 0.28 a 0.013 0.20**
C/N (ratio) 8.14 a 8.70 a 7.99 a 9.54 a 0.736 ns
pH (H2O) 5.68 a 5.71 a 5.15 a 5.68 a 0.830 ns
N (%) 0.125 a 0.112 a 0.115 a 0.130 a 0.450 ns
OM (%) 1.72 ab 1.67 ab 1.51 a 2.12 b 0.097 0.61*
OC ( %) 1.00 ab 0.97 ab 0.88 a 1.22 b 0.017 0.34**
Al 3+ (meq/100 g) 0.52 c 0.32 a 0.37 ab 0.44 bc 0.002 0.122**
C/N(ratio) 11.62 a 18.89 b 15.37 ab 17.92 b 0.041 6.29*
pH(H2O) 4.09 a 4.02 a 4.14 a 3.96 a 0.783 ns
N (%) 0.14 c 0.11 a 0.13 bc 0.12 ab 0.005 0.01**
OM (%) 2.93 bc 2.15 a 2.23 ab 3.03 c 0.05 0.77*
OC (%) 1.68 a 2.15 a 2.02 a 2.18 a 0.222 ns
Al 3+ (meq/100 g) 0.21 a 0.20 a 0.25 a 0.20 a 0.488 ns
C/N (ratio) 12.14 a 8.94 b 8.93 b 8.52 b 0.081 3.20*
pH (H2O) 4.75 a 5.15 a 4.39 a 5.26 a 0.325 ns
N (%) 0.125 a 0.127 a 0.145 a 0.135 a 0.369 ns
OM (%) 2.55 a 1.95 a 2.24 a 2.04 a 0.468 ns
OC (%) 1.48 a 1.13 a 1.30 1.17 a 0.463 ns
Values in a row followed by the same letter within a studied site are not significantly different at 5% level (ns: non-significant; *,
significant; **, highly significant).
lation status (25 nodules/root system) was characterized
as poor according to Amijee and Giller (1998). Poor
nodulation occurs only in plantation and forest, while
moderate nodulation (25 to 75 nodules/root system were
obtained in fallow and mixed farming. The number of
nodules per plant was higher at Bokito and the lowest
one at Ebolowa.
The dry weight of nodules harvested from plants at
Bokito was significantly higher than that of any other
studied site (Figure 2). Although, there were some
differences in the nodules dry weight between LUSs,
which was not statistically significant between the sites.
Growth of A. hypogaea in different LUSs
The highest shoot dry weights of A. hypogaea were
observed in Bokito soils, followed by Bertoua soils;
whereas the shoot dry weights of plants in plantations
were lower than those of other LUSs (Table 2). Fallow
significantly improved the fresh and dry weight of root
and shoot systems of groundnut in Bertoua (p < 0.0006),
Bokito (p < 0.0009) and Ebolowa (p < 0.007) compared
with mixed farming, plantation and forest. In Yaoundé in
contrast, whereas the fresh weight of shoot system was
significantly greater in the forest (p < 0.0001) than in
other land uses, the fresh weight of the root system was
similar in mixed farming and fallow, but was at the same
time significantly enhanced (p < 0.0001) than in
plantation and forest.
There was no significant difference (p = 0.321) between
the land uses as far as the dry weight of root system is
concerned. The greatest plant fresh weight system was
registered in a forest of Yaoundé (11.16 g), while the
smallest was observed in a plantation of Ebolowa (4.03
g). The dry weight of the root system was generally not
high, ranging from 0.11 g in plantations (Bokito) to 0.32 g
in mixed farming (Yaoundé).
At Bokito, highly significant correlations were found

4000 Afr. J. Biotechnol.
Table 2. Effect of LUSs on the biomass of A. hypogaea in four sites in humid forest zone of southern
Cameroon.
Study site Parameter
Bertoua
Bokito
Ebolowa
Yaoundé
Land use
Farm Fallow Plantation Forest
p-value LSD 5%
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
from humid forest soils in southern Cameroon.
Experimental site
Bertoua
Bokito
Ebolowa
Yaoundé
Shoot and root biomass
Fwrs Fwss Dwrs Dwss
r = 0.579*** r = 0.774*** r = 0.055 ns r = 0.635***
p < 0.0001 p < 0.0001 p = 0.665 p < 0.0001
r = 0.468*** r = 0.692 *** r = 0.461*** r = 0.474***
p < 0.0001 p < 0.0001 p < 0.0001 p < 0.0001
r = 0.081 ns r = 0.503*** r = - 0.010 ns r = 0.406***
p = 0.522 p < 0.0001 p = 0.935 p< 0.001
r = -0.133 ns r = 0.698*** r = -0.069 ns r = 0.558***
p = 0.296 p < 0.0001 p = 0.589 p < 0.0001
Fw, Fresh weight; Dw, dry weight; ns, non significant; rs, root system; ss, shoot system; ***, very highly
significant.
between nodule number and respectively the fresh
weight root system (r = 0.468; p < 0.0001) and the dry
weight root system (r = 0.461; p < 0.0001). Other significant
and positive correlations were observed between
the number of nodules and the dry weight of shoot
system (r = 0.474; p < 0.0001) (Table 3). In contrast,
there was no significant correlation between the number
of nodules and the dry weight of root system at Bertoua,
between the number of nodules and the dry weight of
root system at Ebolowa and between the number of
nodules and the dry weight of root system at Yaoundé.
The positive correlation observed between nodulation
and dry weight shoot growth of groundnut suggests that
the two parameters evolve together.
DISCUSSION
The soils of all the four LUSs were acidic with high
aluminium content in most sites except Yaoundé. Since
these mixed farming and fallow soils generally have
never received inorganic fertilizer, the low pH value is
assigned to growing lands. Except in Yaoundé, LUSs
significantly influence the soil aluminium. The lower
aluminium values, 0.20 meq/100 g is from the fallow and
the higher values, 0.52 meq/100 g is from the mixed
farming. In Bertoua and Ebolowa, the soil aluminium
content was significantly lower in the fallow when
compared with other LUSs. The low aluminium content in
fallow could be due to the residual effect of the wood ash
from the slash and burn practice. Agoumé and Birang
Ngo Nkot et al. 4001
(2009) also reported that, aluminium saturation was
significantly affected by the LUSs with the lowest
aluminium saturation obtained in soils of C. odorata
fallows. They noted that, soils of fallows presented a
higher fertility level when compared with those of the
secondary forests and cocoa plantations.
The different LUSs did not significantly influence soil
acidity in all the experimental sites. Soils under
investigation have pH values ranging from 3.96 to 6.00.
Ebolowa soil was the most acidic of all (pH 4.07), in
agreement with other results from which the soils of the
southern region of Cameroon were acid with pH ranging
from 3.69 to 4.12 (Fankem et al., 2006).
All the groundnut plants examined formed effective root
nodules, suggesting that all these soils contain
indigenous LNB able to nodulate A. hypogaea. The cross
section of some nodules showed the red coloration,
suggesting the presence of leghemoglobin, related to
nitrogen efficiency (Abdel-Wahab et al., 2002). In all the
experimental soils, no rhizobia inoculant strains were
used. Nodules were thus, formed by indigenous LNB.
These results are in accordance with other studies that
showed that, nodulation failure is rare for many tropical
legumes such as A. hypogaea, a promiscuous species
belonging to cowpea miscellany group (Alwi et al., 1989).
Moderate to higher number of nodules, from 30 to 94
per plant was formed in the mixed farming and fallows in
soils from humid forest zone in southern Cameroon. But
can we consider that, natural nodulation in these LUSs is
adequate? Assessments of more mixed farming
nodulation and inoculation experiments are needed to

4002 Afr. J. Biotechnol.
confirm this. Some results reported that, nodule number
from indigenous rhizobia was higher in farm previously
planted with groundnut than in other farms where
groundnut was not previously planted (Bogino et al.,
2006). The density of LNB in these soils can be
compared with nodulation response of groundnut in the
different LUSs to explain the results. The comparison
supports the observation that, when LNB density in these
LUSs is high, the nodulation is also high. LNB density is
generally lower in cocoa or coffee plantations and forest
soils than in fallows or mixed farming of southern
Cameroon (Ngo Nkot, 2009). This could be the
consequence of farming practices management involving
pesticides in plantations and a legume for LNB in mixed
farming and fallows. Thakuria et al. (2009) also observed
a gradual increase in nitrogenase activities in roots of rice
and pea under Azospirillum/Rhizobium alone integrated
nutrient management (INM) or Azospirillum/Rhizobium
plus phosphate-solubilizing bacteria dual INM plots in
acidic soils, which could be a result of either population
build-up of introduced bio-inoculants in the rhizosphere or
better soil environment for the N fixers. The highest level
of rhizobia genetic diversity was recorded in soils under
cocoa plantation when compared with soils under mixed
farming with groundnut (Ngo Nkot et al., 2008).
Soils from cocoa or coffee plantations and forests in
southern Cameroon had the lowest number of
groundnuts nodules, respectively 32 and 42 per plant.
This suggests that, LNB were less abundant in these
LUSs. The lack of response to inoculation has been
previously demonstrated to be attributed to other factors
such as inadequate nodulation, unfavourable conditions
for survival of introduced LNB strains and inability of
inoculant strains to compete with indigenous ones for
nodule sites (Bogino et al., 2006). The mean number of
nodules per LUS was very low in cocoa/coffee plantations,
probably because these soils had been under cocoa or
coffee plantations for the past 20 years at least, excluding
legumes but regularly using fungicides and insecticides
for disease and pest control. Ciani and Diriye (1995)
noted the absence of LNB in soils under banana and
papaya plantations compared with soil were leguminous
plants such as groundnut and cowpea were cultivated.
Soils under plantations were the most acid of all the
LUSs (pH 5.83 in average). By decreasing the LNB
population density, acidity may delay the initiation of
infection process and hence, the appearance of
subsequent nodules. The decline of LNB in plantation
may be the consequence of the lower soil pH. These
findings are similar to other results reported by Cheng
(2003), who showed that, Medicago murex grown in acid
soil (pH 4.3) produced fewer nodules (2.3/plant) than
plants grown at pH 7.0 (8.2/plant) 41 days after sowing.
According to Brady and Weil (2002), the low pH in acid
soils leads to an increased availability of aluminium and
manganese which become toxic, as well as a low
concentration of phosphate, calcium and molybdenum.
The lack of adequate number of Bradyrhizobium
japonicum in Nigerian soils was shown to limit nodulation
and nitrogen fixation of soybean, thus, decreasing the
yield (Broomfiel and Ayanaba, 1980). The frequently low
number of nodules observed in forest soils has been
associated with a low requirement for N, which is
considered not to be a limiting factor under these
conditions (Zilli et al., 2004). Tematio et al. (2001)
showed that, soils in the forest are not very fertile. The
fertility is limited by soil acidity, the low amount of cations
exchanged capacity, the aluminium toxicity and the low
available phosphorus. In such conditions, LNB growth
was reduced (Hussein, 2000; Zahran, 1999). Selection of
acid tolerance rhizobial strains of Phaseolus vulgaris L.
done by Gutierrez and Barraquio (2010) yielded that only
36 acid-tolerant strains over 189 (19%) were isolated in
Philippines. Some local strains of rhizobia have been
previously shown to be more acid tolerant than a Chinese
one (Nwaga and Ngo Nkot, 1998). Further studies are
needed to characterize, assess LNB density and select
A. hypogaea strains adapted for acidic oxisols. According
to Lindström et al. (2010) a challenge for agriculture is to
match rhizobia and legume crops for optimal performance
either by having plant genotypes adapted to local
rhizobial populations or by inoculating effective strains
adapted to prevailing environmental conditions and good
competitive ability against local, less effective strains.
Nodules were present in large number at Bokito grey
soils compared with Ebolowa reddish soil sites. This poor
nodulation at Ebolowa clay soils might indicate its
contribution for poor plant growth and low yield of A.
hypogaea. Alemayehu (2010) also find poor nodulation of
Vicia faba in Ethiopian soils and conclude that, poor BNF
may partially be responsible for reduction of faba bean
nodulation.
In Bokito sandy loam soils, the cropping systems are
more diversified including A. hypogaea in the farms
together with other legumes such as Vigna unguiculata,
while in fallows, Crotalaria juncea was present. The
improved soil fertility of Bokito site, due to the presence
of more local LNB can account for the large density of
nodules observed in this site. Remarkable increases in
groundnut yield have been achieved through inoculation
with Bradyrhizobium sp. mainly in areas cultivated with
groundnut for the first time (Lanier et al., 2005).
Inoculation of groundnut by LNB provided contrasting
responses on two sites in central region of Cameroon;
since yield increases of 169% on a relatively fertile clayey
loam soil from Yaoundé and of only 5% on a mixed
farming low-fertility sandy loam soil from Bokito (Mandou
et al., 2002; Nwaga et al., 2010). A synergistic effect of
inoculation and molybdenum seed treatment was also
noticed, since this dual treatment resulted in a 288% yield
increase in the Yaoundé site and a 21% increase in the
Bokito site, when compared with the untreated control.
The contrasting response to inoculation in the two study
sites may be related to the density of indigenous LNB
populations, since the Bokito soil contained 17,000
cells/g and the Yaoundé soil only 170. Increase yield of

82% has been observed for A. hypogaea after rhizobia
inoculation (Betsama, 1999). An inoculation response is
extremely unlikely when native LNB soil bacteria density
is more than 1,000 cells g -1 (Thies et al., 1991; Mafongoya
et al., 2004). Some preliminary results indicate that, soil
infertility and low density of LNB could be one of the
causes of low pod filling of groundnut.
The ability of A. hypogaea to nodulate in all the soils
examined is an indication that the compatible LNB were
present in these soils, but in diverse density. The growth
of groundnut expressed in terms of shoot and root weight
basis was better at Bokito and Bertoua than Yaoundé
and Ebolowa soils, confirming the relation with nodulation
results. Once again, growth was more improved in fallow
than mixed farming, plantation and forest in all the
experimental sites. The positive correlation observed
between nodulation and dry weight, shoot growth of
groundnut reflect that the LNB symbiosis enhance the
plant response by providing increase nitrogen for the
growth. Similar results were reported in sub Saharan
Africa mixed farming inoculated with strains of LNB for
cowpea (Ngakou et al., 2007), soybean (Megueni et al.,
2006) and many other tropical legumes (Nwaga et al.,
2010). Alemayehu (2010) also reported a positive corre-
lation between nodulation and shoot height of V. faba.
According to Lindström et al. (2010) inoculation is
recommended if the field has no history of legume
cultivation, especially if the plant is exotic to a new
environment or if the soil is acidic, saline or otherwise
hostile to rhizobia. In this study, inoculation is more likely
to provide significant increase in nodulation, nitrogen
fixation and seed yield in plantations and forest where
soils are acidic.
Conclusion
Various soils of the humid forest zone of Cameroon
contain LNB able to nodulate groundnut, but, are this
enough for optimal nitrogen fixation and seeds production
of A. hypogaea? Nodulation and growth of A. hypogaea
closely depend on the land use system type and much
more on the sites. An important variation was noticed on
groundnut nodulation and growth according to site and
land use. Further studies are needed to characterize
major factors involved such as rhizobia density and select
acidity tolerant strains adapted to environmental
conditions of the humid forest zone of Cameroon.
Investigations on these isolates could provide additional
information on their symbiotic effectiveness with the aim
of identifying very effective indigenous nitrogen fixing
strains for local production of a specific groundnut
inoculant.
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African Journal of Biotechnology Vol. 10(20), pp. 4005-4010, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.861
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Comparison of some anthropometric and biologic
parameters in two groups of Tunisian infants (0 to 2
years)
M. Kamel 1 *, A. Barkia 1,2 , M. Hamdaoui 3 , H. Ketata¹, M. Kassis 4 , M. Nasri 2 and A. Aouidet 3
1 Ecole Supérieure des Sciences et Techniques de la Santé, de Sfax, boite postale 1099 Sfax 3038, Tunisie.
2 Laboratoire de Génie Enzymatique et de Microbiologie, Ecole des Ingénieurs, Sfax, Tunisie.
3 Ecole Supérieure des Sciences et Techniques de la Santé, Tunis, Tunisie
4 Faculté de Médecine, Sfax, Tunisie.
Accepted 17 December, 2010
Malnutrition still remains a preoccupying health problem that affects the most vulnerable age group
(less than five years). The aim of this research is to establish the nutritional profile of the Tunisian
infants less than 2 years, to specify the principal deficiencies and the possible origins of these
deficiencies. In our transverse exploratory study carried out in a period of 12 months, two groups of
infants less than 2 year old; a control group (n = 18) and a malnourished group (n = 18) were used. Our
data consolidate the impact of pregnant women’s nutritional state, breastfeeding on the infant and the
infant growth. In comparison with the control group, the malnourished group showed a significant
reduction in weight/age, weight/height (p < 0.001), a significant reduction of the average values of
cholesterol associated with high density lipoproteins (Chol-HDL) and the ratio of cholesterol associated
with high density lipoproteins to cholesterol associated with low density lipoproteins (Chol-HDL / Chol-
LDL). This shrinkage showed a positive correlation with the weight/height ratio which is an indicator of
malnutrition gravity. The results of this study confirmed the existence of a remarkable change of the
biological profile among malnourished infants. In addition, the study shows the necessity to target and
develop means to fight against factors favoring malnutrition.
Key words: Malnutrition, infants, breastfeeding, protein, lipoprotein.
INTRODUCTION
In Tunisia, protein energy malnutrition (PEM) in preschool
children is quite common, especially in rural areas (El Ati
et al, 2002). According to the available epidemiological
data, it was noticed that the prevalence of PEM varied
between 11.2 and 23.4% in rural areas and 10.4 and
6.2% in urban areas (El Ati et al., 2002). PEM affects
various significant functions such as the physical growth,
mental development, training capacity, the organism deence
capacity, in particular the immunizing response and
*Corresponding author. E-mail : kamelmed2006@yahoo.fr.
Abbreviations: PEM, Protein energy malnutrition; EDTA,
ethylenediamine tetra-acetic acid; apo, apolipoproteins; TG,
triglycerides.
antioxidant processes (Ahmed et al., 2009; Chen et al.,
2009; Moynihan et al., 2009). The danger of malnutrition
is especially serious among the young population, particularly
children younger than 5 years.
A well regulated diet must satisfy not only the protein
energetic needs, but also those various micro nutriments
(vitamins, zinc, etc.), which interact with the pathogenneses
of various diseases that are established. For
example, it was reported that a deficit in zinc, selenium,
iron, vitamins A, E and acid folic is able to alter the
immunizing responses (Makonnen et al., 2003; Rubhana
et al., 2004). Similarly, it was shown that there existed a
significant relation between diarrhoea, measles, anaemia
and deficit in zinc, vitamin A and iron, respectively
(Rahman et al., 2002; Muller et al., 2003).
Malnutrition is also associated with biological disturbance.
It is particularly accompanied by a reduction in

4006 Afr. J. Biotechnol.
various proteins serum (albumin, prealbumin, retinol binding
and apolipoproteins) and a reduction in the total
cholesterol (chol), Chol-high density lipoprotein (HDL)
and the ratio of Chol-HDL to Chol-low density lipoprotein
(LDL).
The aim of this study is to look for ways of establishing
the nutritional profile of the Tunisian infants of less than
two years, examine the effect of malnutrition on this
profile and specify the principal deficiencies affecting the
young infants.
MATERIALS AND METHODS
Subjects
In our transverse exploratory study carried out during a period of 12
months (from January to September, 2007), thirty six infants under
2 years were recruited and divided into two groups: The first group
is a control (n = 18), aged 11.3 ± 9.4 months, they were recruited
among those frequenting the services of University Hospital Centre
(UHC) of Sfax (large city in the south of Tunisia) through the regular
postnatal control. They had a Z score for both their weight and
height (between – 1 DS and + 1 DS).
The second group (n = 18), malnourished infants aged 7.8 ± 6.6
months, was recruited among those hospitalized in the service of
paediatrics of this hospital. All the malnourished cases had a Z
score < -2 D S (Pelletier et al., 1995). The protocol of research was
made and approved by the ethical committee of the centre (UHC)
and accepted by parents who gave written consent to the inclusion
of their children in the study.
Socio-economic, anthropometric and dietary assessments
In the two groups, epidemiological characteristics (age and daily
food consumption), clinics and anthropometrics parameters (height
and weight) were noted. A socioeconomic survey, using a type
closed standard questions was investigated. Moreover, the daily
food intake of mothers was recorded according to the 24 h dietary
recall method. The team also measured the index of body weight
mass and the brachial perimeter.
Blood collection
Blood samples were taken from malnourished infants before any
treatment. In both groups, fasting blood was drawn in vacationer
tubes containing ethylenediamine tetra-acetic acid (EDTA) and
centrifuged at 3000 rpm for 10 min, then the plasma was removed
and conserved at 80°C until the analysis of different biological
parameters were done.
Plasma biological parameters
Total proteins were quantified using the Biuret method (Biuret and
Layne, 1957). The albumin, prealbumin, apolipoproteins AI (apo-AI)
and B (apo-B) were determined by immunoturbidimetry on automates
COBAS INTEGRA 400 (Roche Diagnostic Gmb H,
Mannheim, Germany). The serum zinc and iron were determined
after dilution of the sample in ultra-pure water by atomic absorption
spectrophotometry using titrisol standard solution (Merck, Darmstadt,
Germany) and PERKIN EL MER MODEL 305 B.
Vitamins
The serum vitamins A and E were realized using reversed-phase
height-pressure liquid chromatography (RP-HPLC) according to the
method of Jacob and Elmadfa (1995). Briefly, plasma proteins were
precipitated with ethanol and lipids were extracted with n-hexane.
After evaporation, the dry residue was redissolved with 150 µl of
methanol-dichlormethane (85:15 v/v), mixed and then an amount of
100 µl of this solution was injected into a guard column (Merck
LiChrospher 100 RP18 (10 µm), 250 x 4 mm). Samples were run at
a flow rate of 1.0 ml/min on a Dionex HPLC system (Summit TM
HPLC, USA). Absorption was monitored at 325 nm for retinol, at
295 nm for tocopherols, at 450 nm for carotenoids and at 270 nm
for coenzyme Q10. Concentrations were calculated from areas
under the curve using an external calibration curve. The vitamins
were determined by spectrophotometer detector in 340 nm for the
retinol and the retinal- acetate and in 292 nm for the alphatocopherol.
Lipoproteins
The fraction of HDL lipoproteins was obtained after precipitation
with phosphotungstic acid containing the apo B (LDL), by using the
case marketed by Bio Merieux (France). The total cholesterol,
triglycerides (TG) and HDL were determined by enzymatic methods
by using the automat RA 1000 analyser (Bayer Diagnostic, München,
Germany). The cholesterol LDL were calculated by using the
formula of Friedward:
Chol LDL = CT- (Chol LDL + Chol VLDL)
The very low density lipoproteins were calculated by dividing the
rates of TG by 5. All our subjects had concentrations of TG lower
than 3 g/l, which is a necessary condition for the application of Friedewald
et al., (1972) formula.
Statistical assessment of the data
The average and standard deviation of the different parameters
studied were calculated. Students’ t-test was used to assess the
differences between the average values of these studied parameters
in the groups. Statistical significance was considered for p
values

Table 1. Comparison to the average values of the anthropometric parameters in the two groups of
infants.
Parameter Control (C) Malnourished (M) Significances (P)
Age (months) 11.32 ± 9.37 7.83 ± 6.62 NS
Weight at birth ( kg) 3.14 ± 0.28 2.74 ± 0.45 P < 0.05
Weight (kg) 8.06 ± 2.86 5.13 ± 2.23 P < 0.02
Weight of reference (kg) 7.62 ± 2.53 7.62 ± 2.54 NS
Height at birth (cm) 50.39 ± 0.92 49.89 ± 0.96 NS
Height (cm) 66.43 ±10.10 61.08 ± 9.56 NS
Height of reference (cm) 67.39 ± 9.98 67.79 ± 9.54 NS
Weight/height 0.12 ± 0.03 0.08 ± 0.02 P < 0.001
Height/age 18.17 ±17.52 16.56 ±15.67 NS
ns: No significant difference
Table 2. Comparison of the mean values of the biological parameters in control and malnourished
groups.
Parameter Control (C) Malnourished (M) Significances(P)
Apolipoprotein AI (g/l) 1.00 ± 0.19 0.77± 0.13 P < 0.001
Apolipoprotein B (g/l) 0.59 ± 0.27 0.54 ± 0.09 NS
Proteins (g/l) 60.71±10.09 49.25 ± 9.85 P < 0.001
Albumin (g/l) 30.80 ± 5.86 24.62 ± 5.22 P < 0.001
Prealbumin (g/l) 0.14 ± 0.07 0.11 ± 0.05 NS
Vitamin A (µg/ml) 0.34 ± 0.16 0.21 ± 0.09 P < 0.01
Vitamin E (µg/ml) 10.07 ± 3.79 4.72 ± 4.14 P < 0.001
Iron (mg/ l) 40.91 ± 3.40 23.55 ± 3.65 P

4008 Afr. J. Biotechnol.
Table 3. Correlate coefficients of weight/height ratio with other
parameters.
Parameter Weight/height
Age 0.69 (***)
Weight at birth 0.37 (*)
Apolipoprotein AI ns
Apolipoprotein B ns
Proteins 0.68 (***)
Albumin 0.49 (***)
Prealbumin 0.70 (***)
Vitamin A 0.66 (***)
Vitamin E 0.77 (***)
Zinc ns
Iron 0.67 (***)
Total cholesterol 0.37 (*)
Triglycerides 0.48 (***)
Chol- HDL ns
Chol-LDL 0.37 (*)
Chol-HDL /Chol-LDL ratio -0.04
Chol-HDL; Cholesterol associated with high density lipoproteins;
Chol-LDL: cholesterol associated with low density lipoproteins;
Chol-HDL /Chol-LDL: ratio of Chol-HDL to Chol-LDL.
*p < 0.05, ** p < 0.01, ***p < 0.001, ns: no significant difference.
decrease in vitamin A, zinc (p < 0.01), Chol-HDL / Chol-
LDL ratio (p < 0.02), Apo-AI, total proteins, albumin,
vitamin E, HDL and iron (p < 0.001).
In our study, we examined the relationship between the
ratio weight/height (at the moment of recruitment) with
the various examined parameters. Our data (Table 3)
show that, some parameters correlated significantly with
the ratio weight/height at the moment of recruitment,
except the Apo AI, Apo-B, zinc and Chol-HDL.
Our results show that the malnutrition of the infants
induced a delay of the growth and perturbation of the
biological parameters. Nutritional status was assessed
using weight-for-height as an indicator to the present
state of nutrition and height-for-age as an indicator of
past nutrition. The growth of our groups was evaluated
from the study of the ratios weight/height and height/age.
The decrease in the first ratio is due to a fast thinning,
whereas the decrease in the second ratio can be
explained by a delay in the height growth. Only the first
ratio has been found to be significantly decreased in the
malnourished group, comparatively with the control
group. The non variation of the second ratio can be explained
by the age of our subjects which was of few
months (7.83 ± 6.62 months).
The borderline grade weight/height ratio among malnourished
infants is probably attributable to the less
privileged socioeconomic conditions of the community
they belonged to. In addition, the abnormal state of our
patients would be, in part, due to a prenatal origin as it is
suggested by their weight at birth, less than that found in
the control group. This hypothesis is confirmed by the
existence of a positive correlation between their weight at
birth and the weight/height relationship at the time of their
admission in the hospital.
The interaction between the nutritional state of the
mother and that of the foetus has been established. It has
been notably reported that a mother’s iron deficiency is
explained, within the newborn baby, by an increasing
anaemia (Sibeko et al., 2004). Besides, it has been
proved that the satisfaction of the needs of the foetus for
calcium requires an adequate amount of calcium and
vitamin D by pregnant women. In the default case, the
infant would have been born with a deficit of calcium and
a weak reserve of vitamin D. This relationship between
mother-infant also appeared clearly after the quantifycations
of vitamins A and E in the mother’s blood as well
as in that of the umbilical cord (Schulpis et al., 2004).
The alimentary survey realized in our subjects revealed
that, many postnatal factors were thought to aggravate
the delay of the growth and biological disturbances. The
absence of breast-feeding in our patients (94%) and the
contribution of an insufficient need of artificial milk clearly
showed a nutritional deficit. Besides, the great loss in
weight and height should also be taken into consideration
since our patients suffered from gastro-intestinal
problems. These problems could result from inadequate
complementary foods taken by babies in/before due time
(before 4 to 6 months), due to the insufficient amounts of
milk they have had. Other factors could interfere in the
growth, especially the problems of infection as a result of
the bad hygienic conditions. Such state was also likely to
occur in underfed babies who are protected by various
defensive factors existing in the mother’s milk (IgA,
lysozyme, macrophages and granulocytes) (Hanson and
Soderstrom, 1981).
Therefore, it was of much importance to study parameters
relating to oxidative stress (antioxidant vitamins
and lipid peroxidation products). In fact, in malnourished
infants, levels of plasma antioxidant vitamins A and E did
significantly differ from levels observed in a similar control
group. In addition, plasma levels of antioxidants were
significantly lower in these malnourished infants. These
observations are in agreement with other studies (Golden
and Ramadath, 1987; Houssaini et al., 1977; Mc Laren et
al., 1969). From the course of this study, it is evident that
there is a significant correlation between enzymatic antioxidants
and the antioxidant vitamin levels that reflect the
pathological features of nutritional program rehabilitation.
Therefore, there should be special emphasis on supplying
antioxidant agents during any nutritional rehabilitation
programme such as vitamins (A, E and C) and trace elements
(calcium, magnesium, potassium and iron). Consideration
should also be given to supplying therapeutic
enzymatic antioxidants in order to reduce free radical

generation and enhance antioxidant status. These measures
may help to reduce the mortality and morbidity
associated with malnutrition.
Other studies attempted to investigate the combination
of several antioxidants as supplements (zinc, copper,
selenium, vitamin A and E, etc.). It appears that, anti-oxidants
work together to help protect the body from
potentially harmful effects of free radicals (Gauche and
Hausswirth, 2006). On the other hand, the malnourished
group compared to the control group, showed a significant
reduction of other various plasmatic parameters as
vitamins A and E, proteins, apo-AI and trace element
(Table 3). In accordance with our findings, a reduction of
serum concentrations of albumins and vitamin E has
been observed in Moroccan infants suffering from severe
under nutrition (Squali et al., 2001). However, the
concentration of TG which increases in these babies was
found to be stable in our patients. A study on young
Tunisian population suffering from stagnation reported a
reduction of the apo-AI but not in the cholesterol, triglycerides,
chol-LDL or chol-HDL concentrations (Sibeko et
al., 2004).
The alteration of the biological profile observed in the
malnourished group can result from the insufficient food,
the over loss and an unsatisfactory absorption. This can
be the result, notably of a quantitative imbalance of feeding.
A deficit in vitamin D (a favourable factor for calcium
absorption) or an excess in phosphorus (accelerating
calcium) can, for example, lead to a hypocalcaemia
despite the resulted sufficient calcium. The resulted irons
under a form of weak absorptions (such as vitamin C)
can produce hyposideremy, even if the resulted iron is
sufficient.
Other markers of nutritional status, the haemoglobin
and albumin levels were also reduced in malnourished
group. At this stage, it is interesting to notice that, the
mothers of these infants consume a lot of tea, which can
aggravate anaemia, as reported in other surveys
(Cournot and Hercberg, 1993; Hamdaoui et al., 1995).
The change in the iron rate, observed in the malnourished
group would then have started since the foetal
stage as a result of an interaction of the mother’s state
with that of foetus. This hypothesis could be seen as
decrease in the rates of haemoglobins found in the
mothers of the malnourished young.
Our results show the existence of the relationship between
the growth delay and the biological disturbance.
Indeed, in the malnourished group, the ratio of weight/
height correlated positively with some biological parameters
as protein, albumin, prealbumin, vitamin A, vitamin
E, iron, total Chol, TG and LDL Chol. Such effects were
reported by other studies concerning young malnourished
subjects. These people particularly showed a low rate of
apo-AI (13 and 23) and a stable level of these parameters
(Sibeko et al., 2004; Slimane et al., 1992; Truswell,
1975). However, their apo-B remained unchanged
(Sibeko et al., 2004).
Conclusion
Kamel et al. 4009
The results of this study confirmed the impact of pregnant
women nutritional state and breastfeeding on the infant
and infant growth. Our data showed a significant alteration
in the level of several anthropometric and biological
parameters in the young malnourished when compared
to the control. Such effects show the necessity of establishing
a program to fight against the factors favouring
malnutrition in our country. These results also show that,
it is necessary to take the supplementation of antioxidants
agents such as vitamins (A, E and C) and trace
elements (calcium, magnesium, potassium and iron)
during any nutritional rehabilitation program into consideration.
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African Journal of Biotechnology Vol. 10(20), pp. 4011-4017, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1520
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Isolation and molecular characterization of RcSERK1: A
Rosa canina gene transcriptionally induced during
initiation of protocorm-like bodies
Xu Kedong 1 , Liu Qinglin 2 , Yang Huifang 1 , Zeng Li 3 , Dong Lili 1 , Liu Fengluan 1 , Bi Ling 1 , Ma
Nan 1 and Zhao Liangjun 1 *
1 Department of Ornamental Horticulture and Landscape Architecture, China Agricultural University, 2 Yuanmingyuan
West Road, Haidian District, Beijing 100193, People’s Republic of China.
2 Department of Landscape Architecture, Sichuan Agricultural University, Ya'an, Sichuan 625014, People’s Republic of
China.
3 Agricultural and Biological College of Shanghai Jiaotong University, Shanghai 200240, People’s Republic of China.
Accepted 20 January, 2011
A somatic embryogensis receptor-like kinase (SERK) gene was isolated from protocorm-like bodies
(PLBs) of Rosa canina by a rapid amplification of cDNA ends (RACE) approach and was designated as
RcSERK1. The RcSERK1 encodes a protein of 626 amino acid residues with a calculated molecular
mass of 68.79 kDa and theoretical isoelectric point of 5.65. The amino acid sequence of RcSERK1
shares all the characteristic features of a SERK protein, including the signal peptide (SP), the leucine
zipper (LZ), the five leucine-rich repeats (LRRs), the pro-rich domain containing the so-called Ser-Pro-
Pro (SPP) motif, the transmembrane domain (TM), the kinase domain and the C-terminal domain. The
transcripts of RcSERK1 were more enriched in PLBs than in rhizoids and callus, but not detected in
leaflets (incubated under dark and before producing callus) and the regenerated shoots. Subcellular
localization indicated that the fluorescence of RcSERK1-GFP was recorded in the plasma membrane.
We argue that RcSERK1 is a Leu-rich repeat receptor-like kinase (LRR-RLK) and plasma membrane
localization protein.
Keywords: somatic embryogensis receptor-like kinase (SERK)1, protocorm-like bodies (PLBs), Rosa canina,
RACE, RcSERK1.
INTRODUCTION
Somatic embryogensis receptor-like kinase (SERK)
genes encode leucine-rich repeat receptor-like kinases
*Corresponding author. E-mail: zhaolj5073@sina.com. Tel: +86-
10-62733315. Fax: +86-10-62733603.
Abbreviations: EX, Extracellular domain; LRRs, leucine-rich
repeats; LRR-RLK, leucine-rich repeat receptor-like kinase; LZ,
leucine zipper; PLBs, protocorm-like bodies; UTR, untranslated
region; SE, somatic embryogenesis; SERK, somatic
embryogenesis receptor-like kinase; SP, signal peptide; SPP,
serine-proline-proline; TDZ, thidiazuron; TM, transmembrane
domain; RACE, rapid amplification of cDNA ends; CaMV,
cauliflower mosaic virus; PCR, polymerase chain reaction; RT-
PCR, reverse transcriptase-PCR.
(LRR-RLKs) (Schmidt et al., 1997; Hecht et al., 2001),
and SERKs share the canonical structure of LRR-RLKs
but have a limited number of leucine-rich repeat (LRR)
motifs (Colcombet et al., 2005). The first SERK gene
identified was reported in carrot (Daucus carota) suspension
cultures where it was specifically expressed in
cells which developed into somatic embryos (Schmidt et
al., 1997; Nolan et al., 2009). SERK genes have been
isolated from several plant species including Arabidopsis
thaliana (Hecht et al., 2001), Zea mays (Baudino et al.,
2001), Medicago truncatula (Nolan et al., 2003),
Helianthus annuus (Thomas et al., 2004), Ocotea
catharinensis (Santa-Catarina et al., 2004), Dactylis
glomerata (Somleva et al., 2000), Citrus unshiu (Shimada
et al., 2005), Oryza sativa (Hu et al., 2005) and
Theobroma cacao (Santos et al., 2005). Ectopic expres-

sion of the full-length AtSERK1 cDNA under the control of
the cauliflower mosaic virus (CaMV) 35S promoter did
not result in any altered plant phenotype. However, seedlings
that overexpressed the AtSERK1 mRNA exhibited a
3- to 4-fold increase in efficiency for initiation of somatic
embryogenesis (Hecht et al., 2001). AtSERK1 and
AtSERK2 receptor kinases function together as an
important control point for sporophytic development controlling
male gametophyte production (Colcombet et al.,
2005). Fradin et al. (2009) indicated that tomato
SERK3/BAK1 physically associates with the RLP Ve1 to
initiate verticillium immunity.
Somatic embryogensis receptor-like kinase (SERKs)
form a sub-group among LRR-RLKs, which comprise the
largest sub-family of RLKs in plants, and are involved in
key plant developmental processes. SERK genes were
isolated in several plant species suggesting the ubiquitous
presence of a small SERK gene family in all plants,
and moreover, their functional conservation with a
specific role in embryogenesis, and possibly other developmental
processes (Nolan et al., 2009).
We have successfully established a high efficiency
somatic embryogenesis (SE) system of Rosa canina,
which is an important ornamental plant widely grown in
the world, whose protocorm-like bodies (PLBs) were
induced from rhizoids by thidiazuron (TDZ) using leaflets
of tissue cultured seedlings. PLBs were also considered
to be somatic embryos, with an intermediary callus formation,
which sometimes is a prerequisite for PLBs
formation (Tian et al., 2008). We have studied the
development process, morphological characteristics and
microscopic structure of PLBs when compared with that
of somatic embryos from other plants, and we found that
PLBs were embryo aggregates, which were coated by
cellular tissues (Tian et al., 2008).
In this study, we isolated and characterized a LRR-RLK
gene, designated as RcSERK1. And our analyses suggested
that RcSERK1 was orthologous to other plant
SERKs, and the possible role of RcSERK1 would be
crucial for the explanation of somatic embryogenesis
(SE) of R. canina.
MATERIALS AND METHODS
Plant materials
Leaflets, callus, rhizoids, PLBs and the regenerated shoots of R.
canina were collected separately. All samples of plants were frozen
immediately in liquid nitrogen and stored at -80°C for RNA extraction.
Isolation of the RcSERK1 gene
Polymerase chain reaction (PCR) was performed to obtain a partial
sequence of RcSERK1 by using the first strand cDNA of R. canina
as a template. With multiple sequence alignment of related SERK
base sequences, two degenerate primers corresponding to the
amino acid sequences, FKS1 sequence (5'-GTGAAY(C/T)
Kedong et al. 4012
CCTTGCACATGGTTY(C/T)CATGT-3') and RKS1 sequence (5'-
ATGGAR(A/G)TACAAGGAR(A/G)ACCCAR(A/G)GTH(A/T/C)ACA-
3') were used for the PCR of partial sequence. The AUAP primer
(5’-GGCCACGCGTCGACTAGTAC-3’) and another primer
corresponding to the FKS2 sequence (5'-TAATGGCAGCGTT
GCCTCAGTT-3') were used for 3’ RACE. Primers for 5' RACE
were: AAP sequence, 5'-GGCCACGCGTCGACTAGTA CGGGII
GGGIIGGGIIG-3' and RK5-1 sequence, 5'-TGGTGCTGCTTTA
CTATTTGCTGCC-3', for the first polymerase chain reaction (PCR).
And the second PCR primers were: AUAP and RK5-1.
The RACE reactions were performed according to the
manufacturer’s protocol (Invitrogen RACE cDNA amplification kit,
USA). We obtained a single full-length cDNA sequence by combining
the 5'-RACE fragment, a partial fragment and C-terminal
fragment. Finally, a pair of primers (FK1: 5'-TGGGGTGGTGGT
GAGAACAGGCTTTGG-3' and RK1: 5'-ACCGCCGCAAATGA
TACAAC TTGC-3') were then designed from the putative 5' and 3'
untranslated region (UTR) of the full-length cDNA sequence. An
1881bp putative RcSERK1 fragment was generated. The nucleotide
sequences of RcSERK1 reported in this paper have been
submitted to the GenBank under accession numbers: HM802242.
The resultant DNA fragments and RACE products were gel purified
and cloned into the pMD18-T vector (Takara) and sequenced
(Invitrogen, Beijing).
RNA isolation, DNase treatment and semi-quantitative reverse
transcription polymerase chain reaction (RT-PCR) assay
Total RNA from various R. canina tissues was extracted using
RN09-EASY spin Kit (Biomed, Beijing, China) according to the
manufacturer’s instructions. Total RNA preparations were subjected
to an on-column DNase digestion, while simultaneously performing
RNA clean-up, using a Qiagen RNase-Free DNase-Set and Qiagen
RNeasy RNA Clean-up Midi Kit (Qiagen, Germany). The first strand
cDNA was synthesized with 1 µg total RNA and 1 µl superscriptII
enzyme (Invitrogen, USA) according to the manufacturer’s protocol.
As a control, the 18s rRNA gene (Genbank accession number:
FM164424.1) was amplified from various R. canina tissues. The
primers used for detecting RcSERK1 gene expression were: forward
primer 5'-CGTCGCTCATCCCTTATGGATCAT-3' and reverse
primer 5'-AGAATTCGGATGAGGAGCTAATTC-3'. The PCR was
performed as follow: pre-denaturation at 94°C for 5 min, followed by
35 cycles of 45 s at 94°C, 45 s at 55°C, 2 min at 72°C for
RcSERK1, 28 cycles for 18s rRNA and a final extension of 10 min
at 72°C. The amplified products were resolved on a 1.2% agarose
gel and then detected by agarose gel electrophoresis. All RT-PCR
experiments were repeated at least three times.
Sequence alignment and phylogenetic tree analysis
The sequence alignment of RcSERK1 and other SERK amino acid
sequences were compared by DNAMAN (ver 5.2.2) and the
phylogenetic tree was constructed by neighbor-joining method with
MEGA program (ver 4.0).
Subcellular localization
The RcSERK1 open reading frame (ORF) were cloned into the Hind
III and SmaI sites of the pSAT6-GFP-N1 vector. This vector
contains a modified red-shifted (green fluorescent protein, GFP) at
NcoI-XbaI sites. The RcSERK1-GFP construct was transformed
into onion epidermal cells by particle bombardment as described
earlier (Wang and Fang, 2002). The transient expression of the
RcSERK1-GFP fusion protein was observed using confocal
microscopy.

4013 Afr. J. Biotechnol.
RESULTS
Figure 1. Nucleotide and deduced amino acid sequences of RcSERK1 (GenBank No. HM802242). The canonical
structure of the RcSERK1 proteins is the presence of the extracellular SPP motif in combination with precisely five
LRRs, which is underlined.
Isolation of the RcSERK1 gene from PLBs of R.
canina
Using the homologous regions of the SERK1 genes, a
partial cDNA was isolated from PLBs of R. canina by
using degenerate PCR. The full-length cDNAs were ob-
tained by employing rapid amplification of the 3'-cDNA
end (3'-RACE) and the 5'-cDNA end (5'-RACE), and were
designated as RcSERK1 (Genbank accession No.
HM802242). Sequence analysis showed that the
RcSERK1 cDNA was 2438 bp in length, including a complete
ORF of 1881 bp flanking with a 5'-UTR of 301 bp
and a 3'-UTR of 256 bp (Figure 1). The predicted protein
of RcSERK1 comprises 626 amino acids with a calculated

Kedong et al. 4014
Figure 2. The alignment of the identified RcSERK1 with other plant SERK proteins including the SP, LZ, five LRRs, SPP motif,
TM, kinase domain (DI to DXI) and the C-terminal domain. Positions containing identical residues are shaded in navy blue,
while conservative residues are shaded in pink.
molecular mass of 68.79 kDa and its theoretical isoelectric
point was 5.65.
As shown in the Figure 2 alignment, RcSERK1 belongs
to the LRR-type cell surface RLKs, which possess a
number of characteristic domains. These include an
extracellular domain (EX) containing a variable number of
LRR units immediately followed by a single transmembrane
domain (TM) and an intracellular kinase
domain responsible for phosphorylating downstream
proteins (Hecht et al., 2001). The amino acid sequence of
RcSERK1 shows a high percentage of identity with
AtSERK1 (88.52%) and DcSERK (77.32%). The
predicted protein structure of the RcSERK1 protein
shares all the characteristic features of that protein, and
starts at the N-terminus with a signal peptide (SP) followed
by a LZ, the five LRRs, a pro-rich domain called the
serine-proline-proline (SPP), a single TM, the 11
conserved subdomains of a Ser-Thr kinase and a Cterminal
leu-rich domain (Hanks et al., 1988). The hallmark
of the SERK proteins is the presence of the extracellular
Ser-Pro-Pro motif in combination with precisely
five LRRs (Albrechta et al., 2005). Upon comparison of

4015 Afr. J. Biotechnol.
Figure 3. Phylogenetic tree analysis of RcSERK1 and other plant SERK proteins. The tree
was constructed by neighbor-joining method with MEGA program (ver 4.0). Branch numbers
represent percentage of bootstrap values in 1000 sampling replicates and scale indicates
branch lengths. The accession numbers are as follows: CsSERK (Citrus sinensis)
(FJ851422), CuSERK (Citrus unshiu) (AB115767), RcSERK1 (R. canina) (HM802242),
StSERK (Solanum tuberosum) (EF175215), AtSERK1 (Arabidopsis thaliana) (NM_105841),
AtSERK2 (A. thaliana) (AF384969), AtSERK3 (A. thaliana) (AF384970), DcSERK (Daucus
carota) (DCU93048), CnSERK (Cocos nucifera) (AY791293), ZmSERK1 (Zea mays)
(NM_001111662), ZmSERK2 (Z. mays) (NM_001111663), OsSERK1 (Oryza sativa)
(AY652735) and PtSERK (Populus tomentosa) (DQ680855).
the amino acid sequences with other species, it was
found that RcSERK1 had very high identity including the
LZ, LRRs, SPP motif, TM, kinase domain and the Cterminal
domain, except for the SP (Figure 2).
We chose herbaceous and woody dicots and monocots,
with different families and genera, to investigate the
evolutionary relationship among plant SERK proteins. A
phylogenetic tree (Figure 3) was constructed using the
neighbor-joining method with the full-length amino acid
residues. The results showed that RcSERK1 was tightly
clustered with CsSERK and CuSERK, and StSERK were
grouped into a cluster, and henceforth, designated as
RcSERK1, whereas AtSERK1 and AtSERK2 formed
another cluster.
Expression analysis of RcSERK1
The expression profiles of RcSERK1 gene in various R.
canina tissues were investigated using a semi-quantitative
RT-PCR assay. Various tissues were respectively
collected, as described in materials and methods.
RcSERK1 mRNA was detected in PLBs, callus and
rhizoids, but not in leaflets and the regenerated shoots,
and mainly in the PLBs but weak in the callus and rhizoids
(Figure 4).
Localization of RcSERK1 in the plasma membrane
To examine subcellular localization of RcSERK1 protein,
the RcSERK1-GFP fusion protein was introduced into
onion epidermal cells by particle bombardment. As
shown in Figure 5, the RcSERK1-GFP fusion protein was
recorded in the plasma membrane, whereas the control
GFP alone was distributed throughout the cytoplasm.
These results show that the RcSERK1 protein is a plasma
membrane localized protein.
DISCUSSION
In this study, a LRR-RLK gene RcSERK1 has been
isolated from PLBs of R. canina, and its expression has
been investigated in leaflets, callus, rhizoids, PLBs and
the regenerated shoots. To our knowledge, this is the first

Figure 4. Expression patterns of RcSERK1 in different tissues.
Expression patterns of RcSERK1 in leaflets (LEA), callus (CAL), rhizoids
(RHI), PLBs (PLB) and the regenerated shoots (REG). Ethidium bromide
staining of PCR products using RcSERK1-specific primers with (top) and
without (middle) prior reverse transcription and the RT-PCR products with
18S rRNA-specific primers (bottom).
Kedong et al. 4016
Figure 5. RcSERK1 localizes to the plasma membrane. Onion epidermal cells were transformed with 35S::GFP and
35S::RcSERK1-GFP. Transformed cells in the bright light (A and E), in the dark for cell nucleuses stained with DAPI (B and F),
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
incubation for 20 h.
Report of cloning of a SERK gene in R. canina and the
study of its expression.
Sequence analysis of RcSERK1 revealed high levels of
similarity to other plant species of SERKs, and it contains
the signal peptide (SP), the leucine zipper (LZ), the five
leucine-rich repeats (LRRs), the pro-rich domain containing
two tandemly repeated SPP sequences, the TM,
the kinase domain and the C-terminal domain. Its sub-
cellular location was verified as a membrane protein,
consistent with previous reports (Shah et al., 2001), and
implied the role of RcSERK1 as a functional gene. The
sequence alignment and phylogenetic tree analysis both
were consistent with RcSERK1 being a functional SERK
orthologue, for the hallmark of the SERK proteins is the
presence of the extracellular SPP motif in combination
with precisely five LRRs.

4017 Afr. J. Biotechnol.
Furthermore, RcSERK1 clustered most closely with
SERK gene family members such as CsSERK1, CuSER-
K1, AtSERK1 and StSERK, which is implicated in
evoking somatic embryogenesis. Monitoring of SERK1
expression during progression of R. canina SE revealed
RcSERK1 expression not only in PLBs, but also in callus
and rhizoids, however, not detected in leaflets and the
regenerated shoots. It showed that RcSERK1 was
expressed at the beginning of the callus of leaves in vitro,
but under artificial stress-induced, such as dark and 2,4dichloro-phenoxyacetic
acid (2,4-D), and then
disappeared at the stage of protocorm-like bodies
(PLBs). So expression analysis suggests that the isolated
RcSERK1 could be a functional SERK orthologue and an
important embryogenic correlation factor. Transformation
of the RcSERK1 into plants and further analysis should
reveal its possible functions in SE of R. canina.
ACKNOWLEDGEMENTS
This research was supported by the Surface Project from
National Natural Science Foundation of China (Grant No.
30871733)
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African Journal of Biotechnology Vol. 10(20), pp. 4018-4023, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2363
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Molecular cloning and expression of the luciferase
coding genes of Vibrio fischeri
Golnaz Asaadi Tehrani 1 , Sina Mirzaahmadi 1 , Mojgan Bandehpour 2 , Faramarz Laloei 3 , Akram
Eidi 1 , Toraj Valinasab 3 and Bahram Kazemi 2,4 *
1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.
2 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran.
3 Iranian Fisheries Research Organization, Ecology research center of the Caspian Sea, Sari, Iran.
4 Department of Biotechnology Shahid Beheshti University of Medical Sciences, Tehran, Iran.
Accepted 7 April, 2011
Vibrio fischeri is a symbiotic marine bacterium and a nonpathogenic member of the Vibrionaceae which
produces luminescence by expressing the lux operon. The lux operon encoding luciferase (luxAB) and
proteins required to synthesize the aldehyde substrate (luxCDE), is controlled with luxR and luxI. In this
study, we amplified the chromosomal fragment contains luxAB of V. fischeri, amplified fragment cloned
into the pTZ57R vector and sequencing to confirmed the fragment. The sub cloning of luxAB gene was
carried out in the pETDuet-1 expression vector and expression procedures were performed in
Escherichia coli strain Nova blue. As a result, a 2046 bp fragment which contains the whole fragment of
luciferase coding genes and intergenic sequences were cloned in pETDuet-1 expression vector.
pETDuet luxAB recombinant plasmid was confirmed by restriction analysis; subsequently 76 kD
expressed protein was detected using SDS–PAGE and western blot using specific polyclonal antibody.
In this study, cloning of the luciferase coding genes was performed successfully, in which the
synthesized construct can be applied as a reporter cassette in prokaryotic systems and as a marker or
tag in the manipulation, and the control of gene expression in the fields of research, production, control
of microorganism and other biotechnological applications.
Key words: Vibrio fischeri, lux operon, luciferase, luminescence.
INTRODUCTION
Vibrio fischeri is a marine bioluminescence bacterium and
a nonpathogenic member of the Vibrionaceae, a large
family of marine γ –proteobacteria, (Thompson et al.,
2004) it lives both as a free living organism and also as a
symbiotic in the light-emitting organs of the Hawaiian
bobtail squid, Euprymna scolopes, where it produces
luminescence by expressing the lux operon (Nyholm and
Mcfall-Ngai, 2004; Ruby, 1999; Visick and Mcfall-Ngai,
2000). In seawater and free-living form, V. fischeri exists
at low cell densities and appear to be non-luminescent,
while in light organ symbiosis with fish and squid, where
the density of V. fischeri cells is high, it is usually lumine-
*Corresponding author. E-mail: Bahram_14@yahoo.com. Tel:
+9821 22439957. Fax: +98 21 22439956.
scent (Sitnikov et al., 1995). In the best studied luminous
bacterium, V. fischeri, there are at least eight lux genes
encoding the proteins essential for luminescence. The lux
regulon is organized in two operons with a divergent
transcription pattern. The seven genes (luxICDABEG),
followed by a transcriptional terminator comprise the
rightward operon, which encode the enzymes required for
the synthesis of the autoinducer (luxI) and the alpha
(luxA) and beta (luxB) subunits of the enzyme luciferase,
the luxC, luxD and luxE genes encode the enzymes
participating in the formation of the long-chain aldehyde,
luxG is not essential for luminescence but is believed to
increase the capacity of the cell to synthesize flavin
mononucleotide (FMN). The leftward operon consists of a
single gene, LuxR, encoding the transcriptional regulatory
protein (Figure 1) (Visick et al., 2000). Bacterial luciferase
is a heterodimeric enzyme of 77 kDa comprising α and β

Tehrani et al. 4019
Figure 1. Luciferase gene organization in v. fischeri: Structural genes contain, luxA and luxB, luxC, luxD, luxE and
regulatory genes contain, luxI and luxR.
Figure 2. The reaction of bacterial luciferase.
subunits with approximate molecular size of 40 and 37
kDa, respectively. Amino acid sequence alignment
between the two subunits reveals that, they share 32%
sequence identity. The α subunit contains 29 additional
amino acid residues inserted between residues 258 and
259 of the β subunit, indicating that the two genes have
arisen by gene duplication through the course of
evolution. There is a single active center in the luciferase
heterodimer that resides on α subunit and binds one
reduced flavin molecule. It seems that, β subunit is
essential for a high quantum yield reaction (Baldwin et
al., 1995; Fisher et al., 1996).
In the luminescence reaction, luciferase converts
aliphatic-aldehyde substrate, oxygen and reduced FMN
(FMNH2) into the corresponding aliphatic acid, water and
FMN, with the concomitant production of light (Figure 2).
In the absence of the aldehyde substrate, luciferase
catalyzes a reaction that yields no light and produces
oxygen radicals rather than water (Nelson et al., 2007).
Luminescence in V. fischeri is controlled by a
population density- responsive regulatory mechanism
called quorum sensing (Dunlap, 1999). At low cell
population density, LuxI synthesizes only basal levels of
the signal, 3-oxohexanoyl L-homoserine lactone (N3-oxo-
C6-HSL); however, with increasing cell density the signal
molecule accumulates to reach a threshold concentration
that can bind to LuxR. The LuxR-AHL complex directly
activates transcription of the luxICDABEG genes and
resulting light production. V. fischeri cells also synthesize
a second autoinducer molecule, octanoyl L-homoserine
lactone (C8- HSL), that under some conditions it may
stimulate transcription of the lux genes (Fuqua et al.,
1994; Milton, 2006; Ulitzur, 1998; Waters and Bassler,
2005). The V. fischeri transcriptional regulator, LitR also
participates in luminescence regulation by inducing the
transcription of luxR, particularly at low cell densities
(Lupp et al., 2003).
The aim of the current study was to clone the alpha
(luxA) and beta (luxB) subunits of the enzyme luciferase
gene in a suitable prokaryotic expression vector in order
to express and produce the desired protein in Escherichia
coli and it can be used as a reporter gene construct for
further biological and biotechnological applications.
Moreover, purification and testing the biological activities
along with comparison with other reporter gene systems
or other constructs which carry luciferase genes will be
the next goals of this research work.
MATERIALS AND METHODS
Bacterial strains and plasmids
V. fischeri strain, ATCC 7744 was kindly provided by Iranian
Research Organization for Science and Technology (IROST) which
was grown at 20 to 25°C. Nutrient media used for the growth of V.
fischeri was DSMZ medium 246 that contained artificial seawater
(750.0 ml), peptone (10.0 g), beef extract (10.0 g) and water (250.0
ml). The ingredients of artificial sea water were NaCl (28.13 g), KCl
(0.77 g), CaCl2 × 2H20(1.60 g), MgCl2 × 6H20(4.80 g), NaHCO3
(0.11 g), MgSO4 × 7H20(3.50 g) in 1000.0 ml distilled water. Cloning
vector pTZ57R (2.8 kb) and expression vector pETDuet-1(5.4 kb)
were obtained from Fermentase and Novagene, respectively.
Genomic DNA extraction, PCR and sequencing
Genomic DNA extraction was carried out by Phenol-chloroform
procedure and precipitated in ethanol (Sambrook and Russell,
2001). Sense and antisense oligonucleotide primers were designed
(Table 1) based on the nucleotide sequence data of luxA and luxB
obtained from Gene Bank (NC_006841.1 for luxA and
NC_006841.2 for luxB). PCR reaction mixture (30 µl) contained, 1
µg DNA 0.1 mM dNTPs, 1.5 mM MgCl2, 1X PCR buffer, 20 pmol of
each primers and 1.25 units Taq DNA polymerase (CinnaGen,
Iran).
PCR amplification was performed under the following condition:
denaturation step; 5 min at 94°C followed by 30 cycles of 40 s at
94°C, 60 s at 51°C in the annealing step and 60 s at 72°C for the
extension step. The resulting PCR product was extended for a
further 5 min at 72°C. The PCR product was analyzed on a 1.5%
agarose gel and purified by using DNA extraction kit (Fermentase
Lithuania) according to the manufacturer’s instructions and

4020 Afr. J. Biotechnol.
Table 1. Primers for amplifying lux genes by PCR.
subjected to sequencing using dideoxynucleotide chain termination
method.
Construction of recombinant plasmids pETDuet-luxAB
To construct luminescent E. coli, purified PCR product of luxAB
gene was ligated to a 3´ T tailed EcoRV digested pTZ57R (Gaastra
and Hansen, 1984) and transformed in E. coli TOP10 competent
cells as described previously (Hanahan, 1983). Recombinant
clones were confirmed by universal PCR and restriction enzyme
digestion analysis .The luxAB gene was released by BamH1 and
Kpn1 digestion and subcloned in to the pETDuet-1 expression
vector.
Protein expression
PETDuet-luxAB was transformed in E. coli Nova blue strain and
selected on LB agar containing 50 µg/lm of ampicilin. A bacterial
colony was inoculated into a medium (1.2% Bacto tryptone, 2.4%
yeast extract, 0.04% glycerol, 1% M9 salts (6.4% Na2HPO4-7H20,
1.5% KH2PO4, 0.025% NaCl, 0.05% NH4Cl)) and incubated
overnight at 37°C in a shaker incubator at 200 rpm. The overnight
cultured bacteria was inoculated into a 50 ml flask and incubated at
37°C on orbital shaker incubator at 200 rpm. Cultures in logarithmic
phase (at OD600 of 0.6) were induced with IPTG (0.5 mM) and
samples were collected before and after induction (0, 3 and 5 h),
cells were lysed in 2x sample buffer (100 mM Tris-HCl pH 8, 20%
glycerol, 4% SDS, 2% beta mercaptoethanol, 0.2% bromo phenol
blue) and separated by 12% SDS-PAGE. The gel was stained by
Coomassie brilliant blue R250 and gene expression analyzed in
comparison with uninduced control samples in parallel (Smith,
1984).
Western blot analysis using antibody
For western blot analysis, cell lysate was separated on 12% (v/v)
SDS-PAGE and electrophoretically transferred onto nitrocellulose
membrane, after UV cross linking for protein fixation; the membrane
was blocked with 3% BSA at room temperature. The bacterial
luciferase antibody (HRP) (Abcam, UK) was used as specific
antibody in 1:1000 of the protein band was performed by DAB
(Diamino benzoic acid) and H202 (Shewry and Fido, 1998).
RESULTS
A DNA fragment encoding the luxAB part of lux operon
was amplified by PCR using sense and antisense
primers, specific restriction sites for BamH1 and Kpn1
were introduced into 5´ end of forward and reverse
primer, respectively. The PCR product was analyzed
in1.5% agarose gel after electrophoresis (Figure 3).
LuxAB Primer
Forward (FluxA) GGATCCATGAAGTTTGGAAATATTTG
Reverse (RluxB) GGTACCTTAAGGCAGATTCTTTTC
The primers are presented from 5ʹ to 3ʹ ends; the introduced restriction
sites are underlined.
The PCR product of luxAB was purified from agarose gel
using Fermentase kit and ligated into cleaved pTZ57R
cloning vector. Following the confirmation of the cloned
luciferase coding genes by sequencing, using BamH1,
Kpn1 restriction enzymes pTZ57R/luxAB plasmid was
digested, a 2046 bp DNA fragment purified, ligated into
the BamH1, Kpn1 sites of an expression vector
(pETDuet-1) and transformed into E. coli TOP10;
recombinant plasmid was confirmed through restriction
digestion using BamH1 and Kpn1 enzymes and named
pETDuet -1/luxAB, (Figure 4); subsequently, transformation
procedure continued into E. coli expression
strains including BL21, Nova blue and JM109.
After induction with IPTG (1 mM), produced proteins
were run onto 12% SDS- PAGE gel, the three protein
bands 37, 40 and 76 kD were detected, which indicates
protein expression of luxB, luxA and luxAB genes,
respectively as shown in Figure 5. The best protein
expression results was obtained after transformation of
the recombinant plasmid onto Nova blue cells in comparision
with two other E. coli strains JM109 and BL21. The
gene segment was under the control of T7 promoter in
the expression vector and the expressed proteins were
accumulated in the cytoplasm.
The protein identity was verified by western blot
analysis. The assay revealed specifically recognition of
luciferase coding genes (luxAB) by bacterial luciferase
antibody (a rabbit polyclonal antibody to bacterial luciferase,
IgG HRP conjugated), whereas no reactivity was observed
in control sample (Figure 6).
DISCUSSION
Gene screening with an easily assayable product,
reporter genes, amplify the signal from the cell surface to
produce a rapid, highly sensitive, reproducible and easily
detectable response. The variety of reporter genes
available including β-galactosidase (lacZ), chloramphenicol
acetyltransferase (CAT), insect luciferase (luc),
bacterial luciferase (lux), alkaline phosphates (phoA), βlactamase
(bla), β-glucuronidase uidA (gusA, gurA),
green fluorescent protein (GFP) and their applications are
very broad in both in vitro and in vivo assays (Jiang et al.,
2008; Köhler et al., 2000; Naylor, 1999). Among all
studied reporter genes, bacterial luciferase are the most
abundant, widely distributed, extensively studied and the
best understood of all types of bioluminescent genes. It is

Figure 3. PCR amplification of the luxAB coding
region using FluxA and RluxB primers. Lane 1and
2 are the same PCR products; lane 3 is a DNA
ladder marker.
Figure 4. lane 1 and 3; the products of digestion with BamH1 and Kpn1; lane 2 and 4 uncut recombinant
plasmid; lane 5 molecular size marker 10000, the released fragment migrated below the 2500 bp fragment
of the DNA ladder.
an α/β heterodimer, a flavine monooxygenize which is
homologous in all bacterial luciferase, catalyzes the
oxidation of a long-chain aldehyde and releases energy
in the form of visible light. Yet luciferase coding genes
have been isolated and cloned from different bacterial
strains like Vibrio harveyi, V. fischeri, Photobacterium
phosphoreum, Photobacterium leiognathi and
Xenorhabdus luminescens in separate or fusion forms
(Meighen, 1994; Wilson and Hasting, 1998). The lux
Tehrani et al. 4021
genes have been transferred into E. coli and a multitude
of different prokaryotic species by transformation,
transduction, conjugation or even bacterial genome
integration, using a variety of different plasmid vectors
(Meighen, 1991).
Previous studies indicate that, the genes encoding
luciferase subunits can generate heterodimers in various
forms (AB1, AB2, BA1, BA2 and A+ B), but the enzyme
activity is decreased in comparison with wild type binary

4022 Afr. J. Biotechnol.
A+B construct, especially in the case of BA1 and BA2
forms which their activity reported to be about 2% of the
wild type (Olsson et al., 1989). In contrast, the current
synthesized construct pETDuet. LuxAB not only simplify
the use of lux system as a reporter enzyme in prokaryotic
cells, but also can provide a special condition in which
both lux genes express simultaneously, from a same
promoter (T7). Furthermore, it has been shown that
obtaining the high expression of the lux genes requires a
strong promoter and ribosome binding site (RBS) on the
expression plasmid that can increase the amount of
product, in which both are available in this effort using
pETDuet-1 vector.
In the previous researches, expression of luciferase
(luxAB) component of the lux system using multiple
plasmids (PB, pGMC12, pFIT001, Plx, pRS1105, pCVG)
about V. harveyi and in the fusion form (pCK218) about
V. fischeri have been reported (Greer and Szalay, 2002),
but in this study, we constructed a polycistronic lux
system by cloning the luxA, luxB and intergenic sequence
Figure 5. SDS PAGE analysis, lane 1 control cell lysate without
recombinant palsmid; Lane 3 and 4 cell lysate of E. coli Nova blue
containing pETDuet-1/luxAB in different sampling times (3 and 5 h,
respectively) after induction with IPTG; lane 2 before induction with
IPTG.
Figure 6. Western blot analysis of luxAB protein. Lane
1 induced culture containing Nova blue bacteria
harboring pETDuet-1 recombinant plamsids after 3 h
induction; lane 2 after 5 h induction; lane 3 extracted
protein from culture containing Nova blue strain, as
control.
as a 2046 bp fragment into a TA cloning vector
(pTZ57R), followed by subcloning the fragment into the
expression vector pETDuet-1 and successfully expressed
in Nova blue cells that accordingly, not have already
been expressed and reported. In addition to luxAB
protein (76 kD), free luxA and luxB subunits (40 and 37
kD) were also detected in different extracts. The amount
of free subunits, moreover the 76 kD protein, increased
with IPTG induction which confirm that these subunits
were also translated from the lux transcripts.
Finally, pETDuet-luxAB construct can now be applied
as a reporter cassette in prokaryotic systems, resulting in
luminous phenotype on addition of a fatty aldehyde
(decanal) to the cells. The expression of the lux genes in
different bacterial species provided a simple and
sensitive system for monitoring the growth and distribution
of the bacteria in the environment. By using the lux
genes as reporters of gene expression, the strength and
regulation of transcription from various promoters can be
readily monitored and also light emission can easily be

detected and measured. Furthermore, this reporter
cassette can be used as a marker or tag in the manipulation
and the control of gene expression in the fields of
research, production, control of microorganism and other
biotechnological applications.
ACKNOWLEDGEMENTS
This study was financially supported by Iranian Fisheries
Research Organisation, Ecology research center of the
Caspian Sea and was done in Cellular and Molecular
Biology Research Center- Shahid Beheshti University of
Medical Sciences. The authors are thankful for the
support and cooperation of the directors.
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African Journal of Biotechnology Vol. 10(20), pp. 4024-4028, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2466
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Study of polymorphism of leptin gene receptor in
Mazandaran fowls
H. A. Abbasi*, S. Gharahveysi and R. Abdullahpour
Department of Animal Sciences, Qaemshahr Branch, Islamic Azad University, Qaemshahr, Iran
Accepted 25 March, 2011
In chickens, leptin is expressed mainly in the liver and adipose tissue. In Iran, Mazandaran native fowls
are under recording and breeding programs, but according to the action modes and importance of the
leptin receptor, its polymorphisms can be related to economical traits such as body weight. In this
study, in order to identify allelic polymorphism in leptin gene receptor, a restriction fragment length
polymorphism (RFLP) method was used. Blood samples were collected randomly from 100 individuals.
The DNA extraction was based on a salting-out method, while an amplified polymerase chain reaction
technique was used. The quantity and quality of extracted DNA were examined using
spectrophotometric and agarose gel electrophoresis. A strategy, employing polymerase chain reaction,
was used to amplify a 374 bp fragment of 9 to 11 exon leptin gene receptor. Digestion of amplicons with
HaeIII revealed leptin gene receptor. The obtained results from restriction digestion showed none of the
polymorphism in leptin receptor gene, so all samples were monomorph due to the fact that there was
no mutation that was related to polymorphism.
Key words: Leptin gene receptor, PCR- RFLP, polymorphism, fowl, HaeIII.
INTRODUCTION
In farm animals, the control and prediction of fatness is of
a high economic interest. The exaggerated adipose
tissue development negatively affects the whole body
metabolism, production efficiency, reproduction and meat
quality. The word leptin comes from the Greek leptos,
meaning thin, referring to the anti-obesity effect which
was believed to be the primary physiological function of
the hormone. Leptin is the product of the ob gene,
discovered by Zhang et al. (1994) using the positional
cloning technique.
Leptin was first identified as the gene product found
deficient in the obese ob/ob mouse. The gene is located
on chromosome 6 in the mouse and chromosome 7 in
humans, and encodes a protein that shows a high degree
of homology between species. Mutations in this ob gene
* Corresponding author. E-mail: h.abbasi1389@gmail.com.
Abbreviations: PCR, Polymerase chain reaction; RFLP,
restriction fragment length polymorphism; LEP, leptin; LEPR,
leptin receptor; NPY, neuropeptide Y.
revealed the pivotal role of leptin in energy balance
(Zhang et al., 1994). The full coding sequence contains
167 amino acids (a.a.) and is composed of a 21-amino
acid signal peptide and a 146-amino acid circulating,
bioactive hormone. However, leptin is a 16-kDa hormone
that has been shown to play an important role in the
regulation of food intake, energy expenditure and hypothalamus
endocrine function in response to nutritional
changes (Friedman and Halaas, 1998; Elmquist et al.,
1999). Recent studies have demonstrated that leptin is
produced by other tissues, such as brain, pituitary gland,
skeletal muscles and stomach. In mammals, leptin is
expressed primarily in adipose tissue (Zhang et al., 1994)
and at a lower level in the placenta and stomach
(Masuzaki et al., 1997; Bado et al., 1998). Thus, it
represents an excellent candidate gene for polymorphism
investigation and association with economic traits in
livestock species. Organization of this gene is conserved
among mouse, human and bovine, presenting three
exons and two introns (Taniguchi et al., 2002). In bovine,
polymorphisms on LEP gene has been associated with
body fat, feed intake and milk yield (Buchanan et al.,
2002; Liefers et al., 2002; Nkrumah et al., 2005). Unlike

mammals, little is known about the avian LEP gene
function. In chicken, only its coding sequence was identified
and sequenced by Taouis et al. (1998). The leptin
signal is mediated through a species receptor localized in
the target tissues, and the leptin receptor (LEPR) belongs
to the class I cytokine receptor superfamily that shares
common structural features and signal transduction
pathways (Tartaglia et al., 1995). Leptin mediated its
central effect through specific receptors located in the
hypothalamus. Leptin receptors have been located on
neurons producing neuropeptide Y (NPY), and when
activated by leptin binding, it is hypothesized to function
in part by down regulating the production of hypohypothalamic
NPY (orexigenic effector) to inhibit ingestive
behavior (Schwartz et al., 1997).
Mammalian adipocytes produce and secrete more
leptin in bloodstream as fat storage increases (Maffei et
al., 1995), signalling the brain via leptin receptor
(Hakansson et al., 1996; Banks et al., 1996) and modulating
the hypothalamic neuropeptide system to suppress
appetite and increase energy expenditure (Rohner et al.,
1996; Kristensen et al., 1998). More interestingly, it has
been shown that chicken leptin gene expression is sensitive
to hormonal treatment in liver, but not in adipose
tissue (Ashwell et al., 1999). These observations are
thought to be due to the role of the avian liver as the
primary source of lipogenesis (Goodridge and Ball, 1967;
Leveille et al., 1968). The peripheral action of leptin in the
chicken is poorly documented except in the pancreas,
where it has recently been demonstrated that leptin has a
profound inhibitory influence upon insulin secretion in the
perfused chicken pancreas (Benomar et al., 2003).
Several studies have showed that exogenous administration
of leptin decreased feed intake in chicks, which
was similar to the one described in mammals, but the
anorexigenic effect within chicken hypothalamus was
mediated via selective neuropeptides, such as NPY and
orexin (Dridi et al., 2005).
In Iran, Mazandaran native fowls are under recording
and breeding programs, but according to the action
modes and importance of leptin gene receptor, its polymorphisms
can be related to economical traits such as
body weight. The aim of the present study is to investigate
the polymorphisms of leptin receptor gene in
Mazandaran native fowls using PCR-RFLP methodology.
Association between different allelic and genotypic forms
of gene and economical important traits can be found if
the polymorphisms are seen. Finding an association
between these caused an improved accuracy and genetic
gain in fowls.
MATERIALS AND METHODS
Experimental population
The study, which was conducted at the Native fowls breeding
station of Mazandaran located in the North of Iran, have been
established in 1988 with the objective of conserving the endan-
gered population of native fowls in rural areas. The location is
Abbasi et al. 4025
typically hot and semi-arid with yearly minimum and maximum
temperature ranges between 4 and 34°C, respectively. The station
has two main activities, namely extension and genetic improvement.
Genetic improvement is done by selecting the best 100 cocks
and 800 hens as parents of the next generations. Parents of each
generation are selected among 7000 pedigreed and performance
recorded birds produced by each generation. The extension part is
continuously producing and distributing 8 weeks old chicks among
rural communities with the aim of increasing the population of
native fowls in Northern provinces of Iran. Rearing chicks in a 30 to
60 days period and distributing them in rural areas to enhance meat
and egg production are quantitative goals of Mazandaran native
fowl breeding station.
Sample collection and DNA isolation
A total of 100 blood samples (5 ml) were collected in EDTA (1 mg
ml -1 ) treated tubes as an anticoagulant from randomly chosen
individuals. The chicks were sampled randomly from four rearing
salons, and each contains chicks with same hatching. Samples
were transferred to the laboratory with ice flask and stored at -20°C
for further analysis. DNA was isolated by standard salt procedure
described by Miller et al. (1988). The quality and quantity of the
extracted DNA was checked by spectrophotometer and agarose gel
electrophoreses.
DNA samples were adjusted to a concentration of 25 µl reaction
mixture, containing 200 ng/µl, and exactly 1/5 µl of the DNA samples
were used as template for polymerase chain reaction.
Primers design
One set of primers was designed using Primer3 primer design
software [(Rozen and Skaletzky, 2000) - www.frodo.wi.mit.edu/cgibin/primer3/primer3_www.cgi].
These primers were designed based
on the chicken sequence leptin gene receptor (GenBank–
NC006095.2), Gallus gallus chromosome 8 and reference assembly
(based on Gallus_gallus-2.1). The primers were analyzed on
NetPrimer (http://www.premierbiosoft.com/netprimer/netprlaunch/
netprlaunch.html), in order to avoid secondary structures, such as
hairpins and loops and primer dimmer. Specific primer pairs were
prepared from the Sina gene (Iran) company in a lyophilized form
and were solved in double steriled water and stored at -20°C. Blood
purified DNA showed better quantity and quality.
Leptin gene receptor amplification
The design primer, primer premier 3(2000) software, was used for
the DNA sequence in exons 9 to 11 leptin gene receptor, while the
Net Primer was used to compare the produced sequence and the
saved sequence. The sequences of the forward and reverse
primers for the amplification of the leptin gene receptor were: F 5´-
GTGTGATAGCTTTGAATGTTGGTG -3´ and R 5´- CTCTTCTG
TTGCCAGCTGTGAT -3´. The polymerase chain reaction for the
leptin gene receptor was performed in a 25 µl reaction mixture,
containing 3 mM MgCl 2 , 200 µM of each dNTPs, 0.4 µM of each
primers, 1X PCR buffer, 1U Taq polymerase (Cinagen, Iran) and
100 ng of genomic DNA template. The reaction mixture was placed
in a DNA thermal cycler (Perkin Elmer 9700). Thermal cycling
conditions included an initial denaturation step at 93°C for 3 min
followed by 33 cycles of 93°C for 45 sec, 57°C for 45 s, 72°C for 45
s and a final extension at 72°C for 4 min.
Restriction digest
For the PCR- RFLP assays, 15 U of the PCR products were digested

4026 Afr. J. Biotechnol.
Figure 1. Various samples obtained from PCR.
Figure 2. Schematic of the 374 bp sequence of leptin gene receptor.
with 5 units of HaeIII (Gibco BRL, life Technologies, USA) at 37°C
for at least 24 h, respectively. Digested products were separated by
electrophoresis on 1% agarose gel in 1×TBE (Tris-Boric acid-
EDTA) buffer at 85 V for 1 h. The 100 bp DNA ladder (Fermentas
SM0331) was used in each gel as a molecular size standard. The
gels were stained with ethidium bromide and the fragments were
visualized using UV transilluminator.
RESULTS AND DISCUSSION
One of the challenges of the poultry industry is to improve
chicken's carcass quality and reduce fat content, without
prejudicial effects on the genetic gains already obtained.
In poultry, the animal’s rapid growth rate has led to excessive
body fat associated with impairment of the total
body metabolism, disorders in the reproductive functions
and muscular development resulting in low performance
with high mortality.
Using an appropriate thermal program in PCR, the 374
400 bp
300 bp
bp segment was amplified without unspecific bands
(Figure 1). The specific primer pairs were designed from
sequence of leptin receptor gene in genbank NCBI site.
Using these primers, a 374 bp segment from exons 9 to
11, which started from nucleotide 55 to 429, was amplified
(Figure 2). The HaeIII restriction enzyme has a
restriction site of ((GGCC)) in the amplified segment and
this site was cut after the second ((G)) base. If the
enzyme cuts the segment, then two bands with lengths of
241 and 133 are seen after electrophoresis on the gel
(Figure 3). However, if the mutation did not occur in the
amplified segment, the HaeIII can not cut the segment
and as such, only one allele (allele B) would be seen, but
if the mutation occurs, two alleles (A and B) are seen
after restriction of digestion. It is shown in the samples
with two bands that the DNA was cut at both strands after
digestion. The digestion products showed that
Mazandaran native fowls were monomorph at the leptin
receptor gene, but the entire samples showed one band

Abbasi et al. 4027
300 bp
100 bp
Figure 3. Results of PCR-RFLP analysis for leptin gene receptor by restriction enzyme HaeIII on 1% agarose
gel and on ladder 100 bp (fermentas).
on agarose gel; therefore, all had BB genotype in this
locus. To confirm the accuracy of digestion, this process
was performed twice, and the cloning study revealed that
the leptin receptor gene has six isoforms such as the long
isoform. Other cloning studies showed three exons and
two introns in the leptin gene. Investigation of the mRNA
of the leptin receptor gene showed eighteen exons in it
(Almeida et al., 2003).
Nevertheless, the cloning study revealed an expression
of leptin gene in the chicken’s liver (Taose et al., 1998).
The polymorphisms of leptin receptor gene were
surveyed in Khoozestan native fowl population using
RFLP-PCR at 2009. It showed that change in the
restriction site of HaeIII generated different restricted segments.
Consequently, three genotypes and two alleles
were seen in Khoozestan fowls. The allelic frequencies of
leptin gene receptor in Khoozestan fowls were 31.19 and
61.81 for alleles A and B, and the genotypic frequencies
were 18.81, 24.75 and 54.44 for genotypes AA, AB and
BB, respectively. Results showed deviation from Hardy-
Weinberg equilibrium in the population of Khoozestan
fowls. Furthermore, the association study revealed that
allele A had positive effect on economical traits than
allele B. The observed and expected heterozygosities
were 0.243 and 0.568, respectively. However, the obtained
results from Mazandaran native fowls were
contrary to those of Khoozestan fowls. The frequency of
the wild type allele is higher than the mutant allele in
Mazandaran native fowls. This can be due to the
physiological role of allele B in Iranian fowls. Inbreeding
and family selection can be one of the major factors that
enhance the BB genotype in Mazandaran native fowls.
Reared chickens in the breeding station of Mazandaran
native fowls are prepared from the state native fowls'
breeding center, and in addition, it is a closed population
and therefore is open for disequilibrium factors.
Inbreeding coefficient is high in the closed population
which, in turn, causes a decrease of diversity in the population.
Increasing effective population size, controlling
mating and preparing independent populations with large
number of primitive individuals are necessary for preventing
decrease of diversity in Mazandaran native fowls.
Nonetheless, designed primers in this study were first
used in Iranian chickens.
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African Journal of Biotechnology Vol. 10(20), pp. 4029-4032, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2563
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Detection of genomic instability in hypospadias
patients by random amplified polymorphic DNApolymerase
chain reaction (RAPD-PCR) method
Adnan A. Alsulaimani 1 , Nabil S. Awad 2 * and Adel E. El-Tarras 2
1 College of Medicine, Taif University, Taif, Kingdom of Saudi Arabia.
2 College of Medicine, Biotechnology and Genetic Engineering Unit, Taif University, Taif, Kingdom of Saudi Arabia.
Accepted 18 March, 2011
Hypospadias is a urogenital malformation, and it is a common inborn disorder in male individuals. The
etiology of hypospadias is still unsolved. The present study is aimed to identify the genetic instability in
hypospadias patients. Random amplified polymorphic DNA (RAPD), a polymerase chain reaction (PCR)
based technique, was adopted using ten random primers in twelve cases and twelve controls. The
primer detectability on genomic instability in 12 samples ranged from 25% with primer OPA-01 to 66%
with OPA-08. Case 2 showed the highest genomic instability (80%). The lowest genomic instabamility
was (10%) case 6. The results determined numbers of genomic instabilities among hypospadias
patients. In addition, the RAPD-PCR technique is a powerful tool for detection of genomic instability in
hypospadias patients. Further larger studies are needed, which include low and high grade of patients
to: 1) Obtain RAPD markers useful for hypospadias early diagnosis; 2) investigate different genes
directly involved in the etiology of hypospadias; 3) analyze chromosomal instability among hypospadia
patients.
Key words: Hypospadias, random amplified polymorphic DNA (RAPD), genomic instability.
INTRODUCTION
Hypospadias is one of the most common congenital
disorders (Sutherland et al., 1996) characterized by
opening on the ventral side of the penis (Manson and
Carr 2003). Hypospadias occurs in 1 out of 300 live male
births (Kalfa et al., 2008). Different studies on the etiology
of hypospadias were conducted and most of them have
focused on a number of candidate genes that control
androgen action and metabolism including the SRD5A2,
HSD17B3 and the AR genes (Manson and Carr., 2003)
and CXorf6 gene mutations (Fukami et al., 2006; Kalfa et
al., 2008).
Measurement of genomic instability has been
performed by techniques like flow cytometry, fluorescent
*Corresponding author. E-mail: nabilfaris151@yahoo.com.
Abbreviations: CGH, Comparative genomic hybridization;
RAPD, random amplified polymorphic DNA; PCR, polymerase
chain reaction; EDTA, ethylenediaminetetraacetic acid.
in situ hybridization, comparative genomic hybridization
(CGH) and allelotyping, which, although informative, are
cumbersome to perform and hence, impractical in the
assessment of clinical cases (Basik et al., 1997).
Random amplified polymorphic DNA (RAPD) is a
polymerase chain reaction (PCR) based fingerprinting
technique that amplifies random DNA fragments with
single short primers of arbitrary nucleotide sequence
under low annealing stringency (Ong et al., 1998;
Williams et al., 1990). The applications of RAPD
technique have been found among several kinds of
organism including bacteria (Sahoo et al., 2010), fungi
(Motlagh and Anvari, 2010), plants (El-Tarras et al., 2004,
animals (Güneren et al., 2010), insects (Awad et al.,
2010) and humans (Saleh et al., 2010). The usefulness of
the RAPD technique for the detection of genomic
instability in various types of human tumors has been
widely documented (Singh and Roy, 2001; Papadopoulos
et al., 2002; Zhang et al., 2004; Xian et al., 2005; Saleh
et al., 2010). No evidence for an association between
genomic instability and hypospadias has been

4030 Afr. J. Biotechnol.
investigated. So the specific aims of this study are to
investigate the association between genomic instability
and hypospadias, as well as to evaluate the usefulness of
the RAPD technique for the detection of genomic
instability in hypospadias patients.
MATERIALS AND METHODS
Blood samples collection and DNA extraction
Whole blood samples of 12 hypospadias patients and 12 healthy
individuals were drawn from a peripheral vein into
ethylenediaminetetraacetic acid (EDTA) tubes and stored at -20°C
until DNA extraction step. Genomic DNA was extracted from blood
samples according to instructions of Blood DNA Preparation Kit
(Jena Bioscince; Germany).
DNA amplification
Total reaction volume of 25 ul of 2x superhot PCR Master Mix
(Bioron; Germany) was used and contains 10 Pmol of each 10
different arbitrary 10-mer primers and 25 to 50 ng of genomic DNA.
The names and sequences of these oligoprimers are listed in Table
1. The RAPD-PCR amplification reactions were performed in
Eppendorf ® thermal cycler using the following PCR program: 1
cycle at 94°C, 4 min; 35 additional cycles consisting of 94°C 5 s,
37°C 20 s and 72°C 20 s. After the amplification, the PCR reaction
products were electrophoresed with 100 bp ladder marker
(Fermentas, Germany) on 10 x 14 cm 1.5%-agarose gel (Bioshop;
Canada) for 30 min using Tris-borate- EDTA Buffer. The gel was
stained with 0.5 μg/ml of ethidium bromide (Bioshop; Canada).
Data analysis
All gels were visualized and documented using a GeneSnap 4.00-
Gene Genius Bio Imaging System (Syngene; Frederick, Maryland,
USA). Each RAPD-PCR amplified DNA fragment was assumed to
represent a single locus. The digital image files were analyzed
using Gene Tools software from Syngene. The densitometric
scanning of each based on its three characteristic dimensions was
carried out. Each band was recognized by its length, width and
intensity. Accordingly, the relative amount of each band was
measured and scored. The absence or addition of an amplified
product in at least one hypospadias sample and its corresponding
control sample was used as a criterion of genomic instability.
RESULTS AND DISCUSSION
During the present study, 12 hypospadias patients and
corresponding normal individuals were examined using
RAPD-PCR method to detect genomic instability which
appears as banding pattern changes between patients
and normal amplified DNA with ten different RAPD
primers. Results of genomic instability detected by
RAPD-PCR analysis are shown in Table 1 and Figure 1.
Among all studied cases, genomic instability was demonstrated
with at least one primer. Among all studied cases
with all primers, the detectability of genomic instability
ranged from 25% with primer OPA-01 to 66.6% with
OPA-08 primer. With all used primers, case 2 showed the
highest genomic instability (80%), whereas, t he lowest
genomic instability was (10) with case 6.
There are three types of hypospadias glandular or
coronal (mild), penile (moderate), scrotal and perineal
(severe) which are also called first, second and third
grade, respectively (Ghirri et al., 2009). The powerful
technique that detects genomic alteration correlated with
human tumor is microsatellites analysis (Odenthal et al.,
2009; Janavicius et al., 2010). However, this methodology
is time consuming and can only detect base-pair
expansion or contraction in specific microsatellite loci
(Ong et al., 1998). On the contrary, for genomic instability
analysis, it is important to investigate genetic alterations
in the entire genome besides microsatellite loci. In
contrast, the RAPD method can simply and rapidly detect
genetic alterations in the entire genome without
knowledge of specific DNA sequence information
(Papadopoulos et al., 2002; Ibrahim et al., 2010). In the
RAPD method, genetic alterations appeared as either
loss or gain of a band, shift of a band, or decrease or
increase of intensities of a band of cancer tissue DNA
relative to the corresponding normal tissue DNA (Maeda
et al., 1999). Obtained results indicated that RAPD-PCR
is an effective tool for identifying genetic alteration and
genomic instability which is in agreement with several
various studies (Ibrahim et al., 2010; Wang, 2001). Figure
1 shows the banding profiles of hypospadias and
corresponding normal DNAs and demonstrate the
detected genetic alteration by RAPD technique among
hypospadias patients in comparison with normal control
group. Banding shifts, missing bands and/or banding
intensity changes, which indicate genomic instability,
were demonstrated in this figure.
These results might be due to mutations that occurred
at the primer - template interaction sites (Maeda et al.,
1999). The summarized results which are illustrated in
Table 1 indicated that, there are differences in genetic
instability among the studied cases which ranged from
10% with case number 6 to 80% for case number 2.
These differences might be due to differences in studied
hypospadias types (mild, moderate or sever). Data in
Table 1 and Figure 1 reflects the ability of each primer to
detect genomic instability which range from 25% with
primer OPA-01 to 66.6% with OPA-08 primer. This
finding might be due to the fact that some loci in the DNA
or chromosome were apt to changes of nucleotide
sequences like the sequences which amplified by primer
OPA-08, while the sequences amplified by primer OPA-
01 would tend to remain stable among hypospadias
patients (Jianxun et al., 2002). Although, the number of
hypospadias samples used in this study is relatively
small, these results seem to indicate that there is a close
relationship between genomic instability detected by RAPD
analysis and this is in agreement with (Ong et al., 1998).
Conclusion
From the obtained results, it could be concluded that,

Alsulaimani et al. 4031
Table 1. Primer code, their sequences and % of primer detectability of genetic instability among hypospadias cases in comparison
with controls.
Primer
code
Primer sequence Samples Primer
detectability (%)
1 2 3 4 5 6 7 8 9 10 11 12
OPA-01 CAGGCCCTTC - + - - - - - - + - - + 25
OPA-02 TGCCGAGCTG - + - - + - - + + + - - 41.6
OPA-03 AGTCAGCCAC + + - - - - - + - + - - 33.3
OPA-04 AATCGGGCTG + - - + + + - - + + - - 50
OPA-05 AGGGGTCTTG + - - + - - - - + - + - 33.3
OPA-06 GGTCCCTGAC - + + + - - - + + - - + 50
OPA-07 GAAACGGGTG - + - + + - + - - + + - 50
OPA-08 GTGACGTAGG + + + + - - + + - + - + 66.6
OPA-09 GGGTAACGCC - + - + - - - + - - - + 33.3
OPA-10 GTGATCGCAG - + - + - - - + - - - + 33.3
Total
40 80 20 70 30 10 20 60 50 50 20 40
+: Genomic instability detected; -: no genomic instability detected.
Figure 1. Genetic alteration revealed from RAPD-PCR analysis of hypospadias (h) and corresponding
control (n). A, with primer OPA-02; B, with primer OPA-03 and C, with OPA-05.
among hypospadias patients, a number of genetic insta-
bilities represented as loss or gain, shift and/or decrease
or increase of intensities of a DNA bands were identified.
RAPD-PCR method is suitable for detecting genomic
changes among hypospadias patients. Further, deep
molecular genetic case control studies with high and low
hypospadias grade are needed to: 1) Determine and
identify specific DNA markers that could be used in early
detection and molecular diagnosis programs of
hypospadias; 2) investigate different genes directly
involved in the etiology of hypospadias and 3) analyze
chromosomal instability among hypospadias patients.
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African Journal of Biotechnology Vol. 10(20), pp. 4033-4039, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2564
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
cDNA, genomic sequence cloning and overexpression
of cytochrome c oxidase gene (COX6b1) from the
Ailuropoda melanoleuca
Jun Li 1,2 , Yiling Hou 1,2 , Wanru Hou 1,2 *, Bing Sun 1,2 and Xiulan Su 1,2
1 College of Life Science, China West Normal University, 637009, Nanchong, Sichuan, People’s Republic of China.
2 Key Laboratory of Southwest China Wildlife Resources Conservation (Ministry of Education), People’s Republic of
China.
Accepted 7 April, 2011
Cytochrome c oxidase (COX) is a component of the mitochondria respiratory chain. COX6b1 is one of
the COX small subunits encoded by nuclear genes. In currently study, the cDNA and the genomic
sequence of COX6b1 were successfully cloned from the Ailuropoda melanoleuca with the RT-PCR
technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned is 341 bp in size,
containing an open reading frame (ORF) of 261 bp encoding 86 amino acids. The length of the genomic
sequence is 4657 bp, which was found to possess 3 exons and 2 introns. Alignment analysis indicated
that the nucleotide sequence and the deduced amino acid sequence are highly conserved with other six
species studied. Topology prediction showed that there are one N-glycosylation site, one protein kinase
C phosphorylation site, two casein kinase II phosphorylation sites, two N-myristoylation sites and one
microbodies C-terminal targeting signal site in the COX6b1 protein of the A. melanoleuca. The COX6b1
gene was also overexpressed in Escherichia coli and the result indicated that COX6b1 fusion with the
HIS-tagged gave rise to the accumulation of an expected 13.46 kDa polypeptide. The expression
product obtained could be used to purify the protein and study its function further.
Key words: Cytochrome c oxidase subunit Vib polypeptide 1 (COX6b1), clone, overexpression, Ailuropoda
melanoleuca (Giant Panda), sequences analysis.
INTRODUCTION
Cytochrome c oxidase (COX; EC 1.9.3.1), the terminal
enzyme complex of the mitochondria respiratory chain, is
a multisubunit enzyme located in the mitochondrial inner
membrane. It oxidizes cytochrome c and transfers
electrons to molecular oxygen. Meanwhile, COX is an
important energy-generating enzyme critical for the
proper functioning of most cells, especially those of highly
oxidative organs, such as the brain (Capaldia, 1990). In
mammals, it is comprised of 13 protein subunits of which
three large subunits (subunit I to III) are encoded by the
mitochondrial genome (mtDNA), constituting the catalytic
core of the enzyme, and the remaining 10 subunits (IV,
*Corresponding author. E-mail: hwr168@yahoo.com.cn.
Tel/Fax: +86-0817-2568653.
Va, b, VIa, b, c, VIIa, b, c and VIII) are nuclear genes
(nDNA) products, which are synthesized on cytoplasmic
ribosomes and are imported into the mitochondria by
leading sequences (Cao et al., 1988; Barrientos et al.,
2002).
Study had confirmed that all 13 subunits are essential
for a functional holoenzyme (Liang et al., 2006). The
mtDNA-encoded subunits provide the catalytic core of
cytochrome-c oxidase, and the nuclear-encoded subunits
may be involved in the regulation and assembly of the
complex (Yanamura et al., 1988). As for COX6b1 gene,
Massa et al. (2008) found that it is associated with severe
infantile encephalomyopathy. Cui et al. (2010) also
pointed out that the loss of COX6b1 genes leads to
mitochondria dysfunction that could cause apoptosis of
the blastocyst-stage embryos.
Ailuropoda melanoleuca, a rare and endangered
species currently found only in China, has a very high

4034 Afr. J. Biotechnol.
ecological, scientific, economic, cultural and aesthetic
value. At present, the COX genes have been extensively
characterized in mammals (Bachman, 1995; Sacconi et
al., 2005; Wu et al., 2007; Du et al., 2007), whereas there
is a handful of report about the COX6b1 genes of A.
melanoleuca.
In this study, RT-PCR technique was used for
amplifying the cDNA of COX6b1 gene from the total RNA.
Touchdown-PCR technique was employed to amplify the
genomic sequence of the COX6b1 from extraction DNA
with the skeleton muscle of the A. melanoleuca. The
characteristics of this gene was identified by homologous
analysis according to the related sequences in GenBank,
and the overexpression of the gene was conducted in
Escherichia coli using pET28a plasmids. The research
can provide a scientific data to enrich the species
database.
MATERIALS AND METHODS
Skeletal muscle was collected from a dead A. melanoleuca at the
Wolong Conservation Center of the A. melanoleuca, Sichuan,
China. The collected skeletal muscle was frozen in liquid nitrogen
for DNA and RNA isolation.
DNA and RNA extraction
Approximately 500 mg muscle tissue of A. melanoleuca was ground
to fine powder in liquid nitrogen, and the powder was then
immediately transferred into a 2 ml centrifuge tube and carefully
added to 1 ml cold lysis buffer (100 mM Tris-HCl, pH 8.0, 100 mM
EDTA and 0.5% SDS). The mixture was incubated at 65°C at least
1 h with gently shaking occasionally. After incubation, the mixture
was centrifuged with 10000 rpm for 10 min at 4°C. Subsequently,
the supernatant was carefully transferred to a new 2 ml centrifuge
tube and an equal volume of chloroform : isoamylalcohol (24:1) was
added and mixed gently by inversion for 1 min, followed by keeping
for 10 min at room temperature and centrifugation at 10000 rpm for
10 min at 4°C. The aqueous phase was carefully transferred to a
fresh tube, and two third volumes of ice-cold isopropanol were
added. Finally, the DNA obtained was then dissolved in TE buffer
and kept at -20°C.
Total RNAs were isolated from the muscle tissue of A.
melanoleuca using the Total Tissue/Cell RNA Extraction Kits (Waton
Inc., Shanghai, China) according to the manufacturer's instructions.
RNA sample was dissolved in RNase-free water, and kept at -70°C.
DNA and RNA sample quality was determined by electrophoresis
on 1.0% agarose gel and quantification was performed spectrophotometrically.
Primers design, RT-PCR and cloning of cDNA sequence
The PCR primers were designed by Primer Premier 5.0, according
to the mRNA sequence of COX6b1 from Homo sapiens
(NM_001863), Bos taurus (NM_176675), Pongo abelii
(NM_001131741), Sus scrofa (NM-001097497), Mus musculus
(NM_025628) and Rattus norvegicus (NM_001145273). The
specific primers of cDNA sequence are as follows: COX6b1-F:
CTTTGCTGAGGGTCACATTG; COX6b1-R: GAGAGGTG [G/A]
GTGGAGCCAG.
Total RNAs were utilized to synthesis the first-stranded cDNAs
using a reverse transcription kit with Oligo dT as the primers
according to the manufacturer’s instructions (Promega). The 20 µl
of first-strand cDNA synthesis reaction system included 1 µg of total
RNAs, 5mM of MgCl2, 1mM of dNTPs, 0.5µg of Oligo dT15, 10U/µL
of RNase inhibitor, and 15 U of AMV reverse transcriptase, and was
incubated at 42°C for 60 min.
The first-strand cDNA synthesized was used as a template for
cds sequence amplification. The total reaction volume for DNA
amplification was 25 l. The reaction mixtures contained 1.5 mM of
MgCl2, 200 M of each of dATP, dGTP, dCTP and dTTP (Omega),
0.3 M of each primer, 5.0 units of Taq plus DNA polymerase
(Sangon Co, Shanghai, China). DNA amplification was performed
using a MJ Research thermocycler, Model PTC-200 (Watertown,
MA) with a program of 4 min at 94.0°C, followed by 30 cycles at
94.0°C for 30 s, 45°C for 30 s, and 72.0°C for 1 min, then ended
with the final extension for 10 min at 72.0°C. After amplification,
PCR products were separated by electrophoresis in 1.5% agarose
gel with 1× TAE (Tris-acetate-EDTA) buffer, stained with ethidium
bromide and visualized under UV light. The expected fragments of
PCR products were harvested and purified from gel using a DNA
estraction kit (Omega, China), and then ligated into a pET28a
vector at 16°C for 12 h. The recombinant molecules were
transformed into E. coli complete cells (JM109), and then spread on
the LB-plate containing 50 g/ml ampicillin, 200 mg/ml IPTG
(isopropyl-beta- D-thiogalactopyranoside) and 20 mg/ml X-gal.
Plasmid DNA was isolated and digested by PstI and ScaII to verify
the insert size. Plasmid DNA was sequenced by Huada
Zhongsheng Scientific Corporation (Beijing, China).
Cloning the genomic sequence of COX6b1
The PCR primers were designed based on the cDNA sequence of
the COX6b1 from the A. melanoleuca obtained earlier and complete
genomic sequences of the COX6b1 from four mammals studied
including H. sapiens, M. musculus, R. norvegicus and B. taurus.
The specific primers of genomic sequence are as follows: COX6b1-
F: AGAGTCAGCACCATGGCAG; COX6b1-R:
GAGAGGTGGGTGGAGCCAG.
The genomic sequence of the COX6b1 gene was amplified using
Touchdown-PCR with the following conditions: 94°C for 30 s, 62°C
for 45 s, 72°C for 2 min in the first cycle and the annealing
temperature deceased 1°C per cycle; after 10 cycles conditions
changed to 94°C for 30 s, 52°C for 45 s, 72°C for 2 min for another
25 cycles. The fragment amplified was also purified, ligated into the
clone vector and tansformed into the E. coli competent cells. Finally,
the recombinant fragment was sequenced by Sangon (Shanghai,
China).
Construction of the expression vector and overexpression of
recombinant COX6b1
PCR fragment corresponding to the COX6b1 polypeptide was
amplified from the COX6b1 cDNA clone with the forward primer, 5’-
CAGAATTCATGGCAGAAGACA (EcoR I)-3’ and reverse primer, 5’-
CGAAGCTTCAGATCTTCCCA (Hind III)-3’, respectively. The PCR
was performed at 94°C for 3 min; 35 cycles of 30 s at 94°C, 45 s at
55°C and 1 min at 72°C; 8 min at 72°C. The amplified PCR product
was cut and ligated into corresponding site of pET28a vector
(Stratagen). The resulting construct was transformed into E. coli
BL21 (DE3) strain (Novagen) and used for the induction by adding
IPTG at an OD600 of 0.6 and culturing further for 4 h at 37°C, using
the empty vector transformed BL21 (DE3) as a control. The
recombinant protein samples were induced after 0, 0.5, 1.5, 2, 2.5,
3, 3.5 and 4 h and then separated by SDS-PAGE and stained with
Commassie blue R250.

Data analysis
The sequence data were analyzed by GenScan
(http://genes.mit. edu/GENSCAN.html). Homology research of the
A. melanoleuca COX6b1 gene as compared with the gene
sequences of other species was performed using Blast 2.1
(http://www.ncbi.nlm.nih.gov/blast/). Open reading frame (ORF) of
the DNA sequence was searched by ORF finder
(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Protein structure of the
COX6b1 sequence cloned was deduced using predict protein
software (http:// cubic. Bioc. columbia.edu/predictprotein/). Multiple
sequence alignment was performed by DNAMAN 6.0. The
prediction of protein functional sites and biochemical characteristics
depend on the software ExPASy Proteomics Server. Protein tertiary
structure was simulated by SWISS MODEL
(http://swissmodel.expasy.org/). The construction of evolutionary
tree relies on MEGA4.0RESULTS
RESULTS
Analysis of the cDNA of COX6b1 from the A.
melanoleuca
The electrophoresis showed that the amplified cDNA
fragment was about 500 bp in size (Figure not shown).
The sequencing result indicated that the length of the
cDNA cloned is 436 bp, corresponding to the
eletrophoretic result. Blast research analysis revealed
that the COX6b1 cDNA sequence cloned share high
similarities with other mammals reported, including H.
sapiens, B. taurus, S. scrofa, P. abelii, M. musculus and
R. norvegicus, especially the similarity is up to 95.79%
with B. Taurus (Taber 1). On the basis of the high identity,
we concluded that we had cloned the cDNA encoding the
COX6b1 protein of A. melanoleuca. The COX6b1 cDNA
sequence has been submitted to Genbank (accession
number: HQ326486), which contained a 261-bp-length
ORF encoding 86 amino acids (Figure 1).
Analysis of the genomic sequence of COX6b1
The electrophoresis showed that about 5000-bp-length
DNA fragment was amplified with primers COX6b1 -F
and COX6b1 –R (Figure not shown). The sequencing
indicated that the length of the DNA fragment cloned is
4657 bp. The genomic sequence of the COX6b1 has
been submitted to Genbank (accession number:
HQ326485). The comparison of nucleotides sequence
between the genomic and cDNA sequences indicated
that genomic sequence cloned contains three exons and
two introns with software Lasergene. The distribution of
three exons is as follows: 13 to 118 bp; 1487 to 1587 bp;
4584 to 4637 bp (Table 2).
Comparison of the genomic sequences with some
vertebrates (including H. sapiens, B. taurus, M. mulatta,
P. abelii, M. musculus and X. tropicalis) was performed by
DNAMAN 6.0. The result demonstrated that their
sequences have high similarity. Nevertheless, the number
Li et al. 4035
and length of exons are different among the species
when they are compared (Table 2).
Prediction and analysis of protein functional sites in
COX6b1 protein of the A. melanoleuca
The results indicated that the COX6b1 protein sequence
of A. melanoleuca share over 85% similarities with six
species when they are compared, especially high
similarities with B. Taurus (96.51%) and S. scrofa
(97.67%). Primary structure analysis revealed that the
molecular weight of the putative COX6b1 protein is 10.16
kDa with a theoretical pI of 8.83. Topology prediction
showed that there are one N-glycosylation site, one
protein kinase C phosphorylation site, two casein kinase
II phosphorylation sites, two N-myristoylation sites and
one microbodies C-terminal targeting signalsite in
COX6b1 protein of A. melanoleuca (Figure 2). There was
complete consistent in the comparison of functional sites,
except that B. taurus has one more protein kinase C
phosphorylation site than other species related.
Overexpression of the COX6b1 gene in E. coli
The COX6b1 gene was overexpressed in E. coli, using
pET28a plasmids carrying strong promoter and
terminator sequences derived from phage T7. Therefore,
the COX6b1 gene was amplified individually by PCR and
cloned in a pET28a plasmid, resulting in a gene fusion
coding for a protein bearing a HIS-tag extension at the N
terminus. The product of expression was tested by SDS-
PAGE analysis of protein extracted from recombinant E.
coli BL21 strains (Figure 4). The results indicated that the
protein COX6b1 fusion with the N-terminally HIs-tagged
gave rise to the accumulation of an expected 13.46 kDa
polypeptide that formed inclusion bodies. Apparently, the
recombinant protein was expressed after an hour of
induction, and it reached the highest level at 3 h after
induction. These results suggested that the protein is
active and it is just the protein encoded by the COX6b1
from the A. melanoleuca. The expression product
obtained could be used to purify the protein and study its
function further.
DISCUSSION
COX is a key enzyme in the process of oxidative
phosphorylation of mitochondria respiratory chain, and its
damage can directly affect mitochondria function. Studies
has also shown that mutation in a nulear-encoded COX
subunit, COX6b1, will cause drial encephalomyopathy
due to complex IV deficiency (Massa et al., 2008). In this
study, we cloned successfully, COX6b1 gene from the A.
melanoleuca, which share high identity on the nucleotide
sequence and the deduced amino acid sequence with H.

4036 Afr. J. Biotechnol.
Figure 1. Nucleotide sequence and putative amino acid sequence of COX6b1 cDNA from the
A. melanoleuca. The asterisk (*) represents termination codon.
Figure 2. Comparison of functional sites based on the COX6b1 amino acid sequences among
the different species. : N-glycosylation site; : protein kinase C phosphorylation site; :
casein kinase II phosphorylation site; N-myristoylation site; : microbodies C-terminal
targeting signal; polymorphic site.

Li et al. 4037
Table 1. The comparison of gene sequence and encoding sequence with Cox6b1 among the A. melanoleuca, human and other
four species (%).
Parameter H. sapiens B. taurus P. abelii S. scrofa M. musculus R. norvegicu
cds similarity (100%) 90.42 95.79 89.27 95.02 91.57 91.19
aa similarity (100%) 86.05 96.51 84.88 97.67 95.35 95.35
Molecular
(kD)
weight
10.19 10.16 10.19 10.19 10.07 10.07
Isoelectric point (PI) 6.85 8.51 6.85 8.51 8.81 8.81
Table 2. The length comparision of the Cox6b1 genomic sequence between A. melanoleuca and other six.
Species Size (bp) Number of exons Join site in the CDS Accession number
A. melanoleuca 4657 3 13-118; 1487-1587; 4584-4637 HQ326485
H. sapiens 10562 3 3022..3127, 6349..6449, 10372..10425 NG_012193
B. taurus 8879 2 1644..1749, 8690..8744 NC_007316
M. mulatta 11268 3 2866..2974, 7119..7219, 11081..11134 NC_007876
P. abelii 9889 2 5274..5375,9697..9750 NC_012610
M. musculus 9178 3 1547..1652, 2637..2737, 8990..9043 NC_000073
X. tropicalis 10357 2 8856..>8957, 10160..10213 NW_003164399
Figure 3. ME evolution tree constructed based on cds sequence of variety species.
sapiens, B. taurus, P. abelii, S. scrofa, R. norvegicus and
M. musculus as well as on the deduced amino acid
sequence. The pI and molecular weight with COX6b1 of
A. melanoleuca are close to the compared six species
(Table 1). These results suggested that the gene we
cloned is COX6b1 gene of A. melanoleuca.
Schmidt et al. (2002) examined the amino acid
replacements for 16 genes that encode the proteins of
the COX holoenzyme in eight vertebrate species, and
found that COX6b1 have undergone acceleration in
amino acid replacement rates. They thought the mature
portions of these genes are likely to have undergone a
functionally significant change to adapt in nature. In this
study, a variety of polymorphic sites were found on the
nucleotide sequence, but they belong mostly to
synonymous mutation. Furthermore, a lot of amino acid
variation was also found by the comparison of amino acid
sequence (Figure 2). The result shows that the change of
A. melanoleuca and B. Taurus, S. scrofa in amino acid
appeared largely consistency. We also analyzed the
function sites of COX6b1 and found that the function sites
are greatly consistent in seven mammal species when
they are compared, except that the B. Taurus has one
more casein kinase II phosphorylation site (Figure 2).
These results are in agreement with the results obtained
by the ME (minimum evolution) evolutionary tree of
COX6b1 (Figure 3). In addition, Lu et al. (2010) found
that the untranslated region (UTR) polymorphisms of
COX6b1 subunit gene is pervasive and considered COX
subunit gene heterogeneity may mediate COX functional
variation. In this paper, the genomic sequence of
COX6b1 was compared by DNAMAN 6.0, and UTR

4038 Afr. J. Biotechnol.
Figure 4. Protein extracted from recombinant E. coli BL21 strains were analyzed by SDS-PAGE gel
stained with Commassie blue R250. Numbers on the right shows the molecular weight and the arrow
indicates the recombinant protein bands induced by IPTG with 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 and 4 h
(lanes 2 to 9), respectively. Lane 1 represents the products of the E. coli strains with the empty vectors.
polymorphisms of COX6b1 subunit gene were also
found.
As is well known, the biogenesis of cytochrome c
oxidase is a complex process controlled by two distinct
genomes. Meanwhile, NRF (nuclear respiratory factor)
also act as an important mediator of coordinated
regulation of all ten nuclear-encoded COX subunit genes
(Scarpulla, 2002; Shilpa et al., 2009). COX6b1 is one of
COX small subunits encoded by nuclear genes. Similarly,
NRF will also have an effect on COX6b1. Furthermore,
Taanman’s experiment in 1990 have demonstrated that
the steady-state levels of the COX6b transcript were
different in the tissues examined in human liver, skeletal
muscle, MOLT-4 cells and fibroblasts. In this paper, we
achieved only efficient express in a prokaryotic organism.
The gene’s transcription level needs further research.
The cDNA and the genomic sequence of COX6b1 gene
were cloned successfully from the skeleton muscle of the
A. melanoleuca, respectively, which were sequenced and
analyzed preliminarily. The cDNA of the COX6b1 gene
was also overexpressed in E. coli. The data will enrich
and supplement the information about COX6b1. In
addition, it will contribute to the protection for gene
resources and the discussion of the genetic
polymorphism.
ACKNOWLEDGEMENTS
This work was supported by the Key Chinese National
Natural Science Foundation (30470261), Application
Technology Project in Sichuan Province (2006J13-057),
Key Scientific Research Foundation of Educational
Committee of Sichuan Province (07ZA120), Key
Discipline Construction Project in Sichuan Province
(SZD0420), Sichuan Key Discipline Zoology Construction
Funds Subsidization Project (404001), Application
Infrastructure Projects in Sichuan Province (2009JY0061)
and Education Department Youth Fund Project of
Sichuan Province (092B088).
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African Journal of Biotechnology Vol. 10(20), pp. 4040-4045, 16 May, 2011
Available online at http://www.academicjournals.org
DOI: 10.5897/AJB10.857
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Effect of plant growth regulators (PGRs) on
micropropagation of a vulnerable and high value
medicinal plant Hedychium spicatum
Dinesh Giri 1 * and Sushma Tamta 2
1 Plant Tissue Culture Laboratory, Department of Botany, D.S.B. Campus, Kumaun University, Nainital, Uttarakhand,
263002, India.
2 Department of Biotechnology, Bhimtal Campus, Kumaun University, Nainital, Uttarakhand, India.
Accepted 15 March, 2011
A complete micropropagation protocol was developed by applying different plant growth regulators
(PGRs) of a vulnerable and high value aromatic medicinal plant, Hedychium spicatum. Three cytokinins,
6-benzyladenine (BA), kinetin (KN) and thidiazuron (TDZ) were used and among these, the lower
concentration of TDZ (1.0 µM) was found to be the most effective treatment in relation to induction of
high frequency shoot multiplication (83.33%), number of shoots per explant (3.86 shoots) and average
number of shoots per flask (19.33 shoots). Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and α-
naphthalene acetic acid (NAA) were the used auxins in this study for in-vitro rooting. Among these used
auxins, the lower concentration of IBA (2.5 µM) was the prominent plant growth regulator regarding in
vitro rooting. Well rooted and healthy plantlets were obtained after 2 months of hardening and
transferred to the field (1990 m) with 90.0% survival. On the basis of available literature, this is the first
and significant study regarding the comparative effect of different PGRs on in-vitro propagation study
of H. spicatum. This significant study could be useful for large scale propagation and ex-situ
conservation of this vulnerable Himalayan species.
Key words: Cytokinins, thidiazuron, shoot multiplication, in-vitro propagation, ex-situ conservation.
INTRODUCTION
Hedychium is one of the plant genus which is commonly
used in the preparation of indigenous medicine. There
are 50 species of Hedychium in tropical Asia (Kirtikar and
Basu, 1984), 37 species in India and 8 species in
Western Himalaya. Hedychium spicatum (Zingiberaceae),
commonly known as kapoor kachri, van- haldi or ginger
lilly, is distributed in subtropical Himalayas in the states of
Assam, Arunanchal Pradesh and Uttarakhand at an
altitude of 1000 to 3000 m. The root stock (rhizome) is
useful in asthma, pain, foul breath, hiccough, vomiting
and laxative, stomachache, carminative, stimulant tonic to
the brain, in liver complaints, diarrhea and pains (Kirtikar
and Basu, 1984).
Rhizome extracts showed antimicrobial activity against
*Corresponding author. E-mail: giri_gld@rediffmail.com.
gram-positive and negative bacteria (Bisht et al., 2006).
Recently, the crude extract of the rhizome has been used
in the preparation of an anticancerous drug, PADMA 28
(Nayab et al., 2004). Samant et al. (2007) reported that
H. spicatum is commercially exploited from its natural
area. According to the threat status of IUCN criteria, H.
spicatum has become vulnerable (Samant and Pant,
2006). It is also listed in near threatened category of the
essential oil bearing plants (Samant and Palni, 2000).
Hence, prioritization of this species needs to be done for
propagation and conservation. Therefore, the present
study was conducted to see the effect of different PGRs
on micropropagation of this species.
MATERIALS AND METHODS
Seeds of H. spicatum were collected in the month of October to
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
medium.
Treatment (µM)
Number of
shoot/ flask
Number of
shoot/ explant
Percentage shoot
multiplication
Giri and Tamta 4041
Average shoot
length (cm)
Control (0.0) 05.00±0.00 - - 1.80±0.65
BA (0.1) 05.66±0.88 1.13±0.17 53.33±6.66 3.92±0.30
(1.0) 07.00±0.57 1.40±0.11 53.33±6.66 3.653±0.52
(2.5) 08.66±1.45 1.73±0.29 43.33±23.33 3.214±0.41
(5.0) 07.00±1.54 1.40±0.23 40.00±11.54 2.568±0.07
(10.0) 05.66±0.33 1.13±0.06 40.00±11.54 2.632±0.36
(20.0) 05.66±0.66 1.13±0.13 40.00±11.54 2.236±0.34
KN (0.1) 07.66±1.76 1.46±0.40 76.66±8.81 4.94±0.36
(1.0) 07.33±0.88 1.46±0.17 53.33±6.66 4.00±0.35
(2.5) 07.00±1.52 1.40±0.30 73.33±13.33 4.54±0.95
(5.0) 07.66±1.45 1.46±0.35 56.66±20.27 3.90±0.17
(10.0) 07.33±0.33 1.13±0.26 40.00±11.54 3.52±0.23
(20.0) 05.33±0.33 1.15±0.07 26.66±6.66 3.59±0.83
TDZ (0.1) 10.66±1.20 2.13±0.24 83.33±12.01 1.52±0.08
(1.0) 19.33±4.91 3.86±0.98 83.33±12.08 1.10±0.15
(2.5) 16.66±0.88 2.66±0.54 63.33±14.52 2.01±0.34
(5.0) 14.00±6.50 2.80±1.13 50.00±20.81 1.41±0.08
(10.0) 08.33±1.76 1.16±0.35 56.66±20.27 1.53±0.03
(20.0) 10.33±2.90 2.06±0.58 40.00±11.54 1.99±0.16
Values are mean ± standard error, each treatment consisted of 15 explants (5 in each flask, 3 flasks per
treatment). Data were recorded after 4 weeks of inoculation. - = no shoot multiplication.
brought to the laboratory. Seeds were separated from spikes, air
dried for 3 days at room temperature and stored at 4°C in the dark.
After 65 days, seeds were taken out and washed under running tap
water along with a mild detergent and treated with a systemic
fungicide (Bavistin, 1% w/v, 15 min). Following washing with double
distilled water, seeds were transferred to laminar air flow cabinet.
Surface sterilization was carried out by using mercuric chloride
(HgCl2, 0.1% w/v, 3 min) followed by washing with sterile distilled
water (5 times). Seeds were inoculated (10 seeds per flask) on MS
(Murashige and Skoog, 1962) basal medium containing agar (0.8%
w/v) and transferred to the culture room. Three weeks after
inoculation, shoot tips (size approximately 1.5 cm) were excised
from in vitro germinated seedlings and inoculated on MS medium
supplemented with different concentrations (0.1 to 20 µM) of 6benzyladenine
(BA), kinetin (KN) and thidiazuron (TDZ) (Table 1)
for shoot multiplication. In vitro generated microshoots were used to
induce rooting. These microshoots (2.5 to 5.0 cm in length) were
inoculated on MS medium supplemented with different concentrations
of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and
α- naphthalene acetic acid (Table 2).
All cultures were raised in 250 ml erlenmeyer flasks (100 ml
medium in each flask). Sucrose (3.0%, w/v) was added as a carbon
source and medium was gelled with agar (0.8%, w/v). The pH of the
medium was adjusted to 5.8 before autoclaving (1.05 kg cm -2 ,
121°C and 20 min). Cultures were maintained in a culture room at
25 ± 1°C in 16/8 h light/dark photoperiodic cycle, with irradiance by
cool fluorescent tubes (Philips 40 W).
After rooting, the microshoots with well developed roots were
taken out from the culture flasks and roots were then washed
thoroughly with tap water to remove the adhering medium.
Following washing, these plantlets were transferred to thermocole
cups (8 cm height and 4 cm diameter) containing soil and farmyard
manure (3:1, v/v). Transparent polythene bags were inverted over
these thermocole cups (8 cm height and 4 cm diameter) to maintain
the humidity and watered on alternate days. Initially, thermocole
cups were kept inside a culture room for first four weeks and
gradually removed to polyhouse (25 ± 1°C) for further growth. After
2 months, these hardened plantlets were transferred to field (near
department nursery) to study the survival rate of these in vitro
raised plantlets.
Statistical analysis
Data were subjected to statistical analysis and standard error (SE)
and ANOVA were calculated following Snedecor and Cochran
(1967).
RESULTS AND DISCUSSION
Seeds started germinating within 15 days of inoculation
on MS basal medium. After one week of germination (3
weeks after inoculation), the explants (shoot tips, size
approximately, 1.5 cm) from in-vitro germinated seedlings
were excised and inoculated on MS medium supplemented
with different concentrations of BA, KN and TDZ
which resulted in multiple shoot formation. However,
number of shoots per explant, as well as number of
shoots per flask varied with the treatments (Table 1).
Each shoot tip when cultured on MS basal medium
(without PGRs), developed into a complete plantlet with
profuse root system. MS medium when supplemented

4042 Afr. J. Biotechnol.
Figure 1. Micropropagation of H. spicatum. A and B, Lower concentration of TDZ (1.0 µM) showed high frequency of shoot
multiplication after 4 weeks of inoculation in MS medium; C, effect of cytokinins on shoot length after 8 weeks of inoculation in
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
lower concentration of IBA (2.5 µM) after 8 weeks of root initiation; F, hardening of plantlets in a culture room; G, well rooted
plants after two months of hardening (photograph taken just before field transfer); H, field transfer of plantlets (at department
nursery); I, well grown plant in field after 3 months of field transfer.
with different concentrations of BA had best response in
2.5 µM in terms of the number of shoots per flask (8.66)
and number of shoots per explant (1.73). However,
percent shoot multiplication was maximum (53.33%) with
0.1 and 1.0 µM BA, and the average shoot length was
highest (3.92 cm) in lower concentration (0.1 µM) of BA.
When KN was used for shoot multiplication, all the used
concentrations (0.1 to 20 µM) had more or less same
effect for three parameters (number of shoots per flask,
number of shoots per explant and average shoot length)
(Table 1), but percent shoot multiplication was maximum
(76.66%) in lower concentration of KN (0.1 µM). Among
various concentrations of TDZ tested, 1.0 µM TDZ
produced maximum number of shoots (19.33 shoots per
flask, 3.86 shoots per explant) within four weeks (Table 1
and Figure 1A, B). Although other concentrations of TDZ
also gave good shoot multiplication, the percent shoot
multiplication was highest (83.33%) in lower concen-
tration of TDZ (0.1 and 1.0 µM). The shoots formed in
TDZ supplemented medium were short in length but the
shoot length was quite good in KN supplemented MS
medium (Figure 1C). Data were analyzed by using
ANOVA which showed that among the used cytokinins,
average number of shoots per flask, number of shoots
per explant and average length of shoots were highly
significant (p < 0.01 at 1%). However, only percent shoot
multiplication was insignificant (p < 0.01 at 1%) within
used concentrations of cytokinins (Table 2).
Effect of different auxins on in vitro induction of rooting
of microshoots of H. spicatum is summarized in Table 3.
All the used auxins (concentration ranges from 0.1 to 10
µM) were able to induce rooting in microshoots, including
control (auxin free MS medium) also. However, rooting
percentage in control was only 29.62% and it took 21
days to root in comparison with other treatments where
the differentiation of roots on the elongated shoots

Table 2. ANOVA summary.
Source Dependent variable df Mean square F-value
Cytokinins
Concentration
Average number of shoots per flask 2 215.73 16.25*
Average number of shoots per explant 2 7.39 12.40*
Percentage shoot multiplication 2 1220.63 2.51ns
Average shoot length (cm) 2 22.46 37.39*
Average number of shoots per flask 6 42.75 3.22 *
Average number of shoots per explant 6 4.77 8.02*
Percentage shoot multiplication 6 5039.68 10.37*
Average shoot length (cm) 6 2.775 4.62*
*Significant at 1% (p < 0.01) and ns, not significant; no, number; df, degree of freedom
Table 3. Effect of different auxins on in vitro rooting of micro-shoots of H. spicatum on MS medium.
Treatment
(µM)
Percentage
rooting
Time taken for root
emergence (days)
Average number of
root per shoot
Giri and Tamta 4043
Average length
of root (cm)
Control (0.0) 29.62±3.70 21.00±1.15 1.15±0.03 1.86±0.03
IAA (0.1) 40.73±3.70 14.00±0.57 2.74±0.41 2.20±0.20
(1.0) 51.84±3.70 15.33±0.66 3.15±0.26 3.30±0.52
(2.5) 66.66±0.00 10.66±0.88 3.40±0.87 3.15±0.30
(5.0) 55.55±6.41 16.33±1.45 2.54±0.05 2.56±0.46
(10.0) 33.33±0.00 15.00±0.57 2.17±0.12 1.78±0.33
NAA (0.1) 44.44±6.41 11.66±1.45 3.12±0.43 2.94±0.10
(1.0) 62.95±3.70 10.35±0.47 3.07±0.29 3.74±0.19
(2.5) 81.47±3.70 10.5±0.57 3.83±0.16 3.92±0.18
(5.0) 85.17±3.70 10.44±0.59 3.78±0.18 3.42±0.46
(10.0) 37.03±3.70 11.77±0.39 2.59±0.02 2.54±0.22
IBA (0.1) 59.25±3.70 11.00±0.57 3.86±0.16 3.30±0.20
(1.0) 70.36±3.70 10.33±0.88 4.07±0.27 4.31±0.38
(2.5) 96.29±3.70 10.24±0.58 5.25±0.08 4.87±0.39
(5.0) 92.58±3.70 10.00±0.57 5.06±0.47 5.26±0.44
(10.0) 55.55±6.41 12.00±0.57 3.27±0.46 3.01±0.25
Values are mean ± standard error (SE). There are nine explants (three microshoots per flask) in each treatment.
occurred over a period of 1 to 2 weeks (Table 3). Trend
for percent rooting was similar in all the three used
auxins, which means percent rooting increased with
increasing concentration of auxins up to certain extent
and then it decreased with increasing concentration of
auxin. In case of NAA, rooting percentage increased with
increasing concentration up to 5.0 µM, and 5.0 µM NAA
was able to induce 85.17% rooting, then rooting percentage
decreased to 37.03% as the concentration increased
to 10.0 µM. Similar trend was observed in microshoots
which were inoculated in IAA and IBA supplemented MS
medium. In these two auxin treatments, percentage
rooting increased only up to 2.5 µM concentration and
then it decreased with increasing concentration (Table 3).
Among different concentration of IAA, 2.5 µM concentration
enhanced maximum rooting percentage (66.66%)
within 10 days of inoculation. The average number of
roots per shoot was highest (3.40 roots/shoot) in 2.5 µM
IAA, while 1.0 µM showed maximum average length of
roots (3.3 cm). Percent rooting was improved up to
85.0% by using 5.0 µM of NAA within 10 days of inoculation,
however, the lower concentration of NAA (2.5 µM )
showed best response in relation to the average number
of roots per shoot (3.83 roots) and average length of
roots (3.92 cm). Among various concentrations of IBA,
the lower concentration (2.5 µM) showed maximum

4044 Afr. J. Biotechnol.
Table 4. ANOVA summary.
Source
Percentage rooting
Time taken for root
emergence (in day)
Average number of
root/shoot
Average length of
root (cm)
DF MS F-ratio DF MS F-ratio DF MS F-ratio DF MS F-ratio
Concentration 5 3791.0 75.37* 5 128.0 57.80* 5 10.3 52.9* 5 10.39 36.67*
Auxins 2 1982.0 39.40* 2 49.3 22.26* 2 7.40 38.0* 2 7.54 26.61*
Concentration x auxins 10 160.4 3.19* 10 6.0 2.74** 10 0.60 3.1* 10 0.64 2.28**
Error 36 50.29 36 2.2 36 0.19 36 0.28
*Significant at 0.01 level (at 1%); ** significant at 0.05 level (5%).
percent rooting (96.29%) and average number of roots
per shoot (5.25 roots), while the average length of roots
was 5.26 cm in 5.0 µM IBA supplemented medium. Out
of these three used auxins, IBA showed best response in
relation to percent rooting; average number of roots per
shoot and average length of roots (Table 3 and Figure
1D, E). Mean significance difference (at the 5% level)
computed by applying ANOVA showed a significant
difference among various parameters (percent rooting,
time taken for root emergence (in days), average number
of roots per shoot and average length of roots) (Table 4).
One month after rooting, these plantlets were hardened
under growth chamber for four weeks (Figure 1F). Well
rooted and healthy plantlets were obtained after 2 months
of hardening (Figure 1G) and transferred to the field condition
(1990 m) with ninety percent survival rate (Figure
1H and I).
Therefore, on the basis of results obtained in this
experiment, TDZ was found to be the most effective
cytokinin for shoot multiplication, however, shoot lengths
was better in KN supplemented medium. On the basis of
literature, it is known that TDZ act as potent regulator for
in vitro propagation system and as an effective mean of
induction of adventitious shoots in a number of plant
species (Huettman and Preece, 1993; Lu, 1993). It has
been known to promote cytokinins-like activity similar to
that of N 6 substituted adenine derivatives and has been
used in tissue culture studies since 1982 (Mok et al.,
1982; Siddique and Anis, 2007). TDZ stimulate cytokinins
activity and it is a better substitute of other cytokinins like
BA and KN that are generally used in tissue culture work
(Kannan et al., 2007; Vinayak et al., 2009). Sujatha and
Reddy (1998), also reported that a higher shoot
multiplication rate was obtained on TDZ supplemented
media. However, it concomitant with a mark reduction in
shoot length. Mok et al. (1987) showed that lower concentration
of TDZ (0.3 µM) was very effective in
multiplication of Broccoli (Brassica oleracea) and higher
concentration (10.0 µM) induced abnormal growth and
vitrification. TDZ stimulated shoot regeneration in a
concentration-dependent manner, and higher concentration
significantly reduced the height of newly formed
shoot (Tomsone et al., 2004). Present study indicates
that by using lower concentration of TDZ (1.0 µM), the
shoot multiplication frequency was the best. The high
efficiency of TDZ may be attributed to its ability to induce
cytokinin accumulation (Victor et al., 1999) or enhance
the accumulation and translocation of auxin within the
tissue (Murthy et al., 1998). The growth promoting activity
of thidiazuron was accompanied by high acid-phosphate
level (Mok et al., 1987) and is stable and biologically
active at low concentration than other cytokinins (Genkov
and Ivanova, 1995).
This significant study concluded that the lower concentration
of IBA was the prominent PGRs regarding in vitro
rooting. In the present study, the superiority of IBA over
other auxins regarding in vitro rooting is supported by the
result of other studies like Sreekumar et al. (2000) in
Hemidesmus indicus, Mala and Bylinsky (2004) in
Daphne cneorum, Kannan et al. (2006) in Caesalpinia
bonduc and Gantait et al. (2009) in Dendrobium
chrysotoxum. The present investigation suggests that the
IAA showed least response in root formation. In comparison
with other auxins, similar observation was reported
by Kannan et al. (2007). However, it was clear that
increasing the concentration of these auxins did not
responded well in the present study. The variation in
effectiveness of different auxin sources may be attributed
to their differential affinity to auxin receptors involved in
the rooting process, which may be cultivar specific
(Tereso et al., 2008) and depend on the plant type
(Nandgopal and Kumari, 2007). A disadvantage of micropropagation
is the high mortality rate during the transfer
of in vitro plantlets to ex vitro (Lovato et al., 1999). But in
the present study, a very good survival rate (90.0%) was
achieved in comparison to earlier studies like Badoni et
al. (2010), where they got only 40 to 50% survival rate,
and Koul et al. (2005) that reported 80 to 85% survival
rate. There were no previous literatures available on the
comparative study that applied these three different
auxins on in vitro rooting of this species. This significant
study shows that the lower concentration of IBA was the
prominent plant growth regulator regarding in vitro
rooting.
On the basis of available literature regarding micropropagation
study of H. spicatum (Koul et al., 2005;
Badoni et al., 2010), there was no such type of comparative
study using different plant growth regulators and

this would be the first report on this species. This
significant study concluded that, by using lower concentrations
of TDZ (1.0 µM) and IBA (2.5 µM), it could be
able to achieve a number of in vitro raised plantlets within
a limited period of time. Beside propagation, this study
would also become an ex situ conservation strategy of
this vulnerable Himalayan species.
ACKNOWLEDEGMENT
Authors are thankful to the head of department for
providing necessary facility in the department.
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African Journal of Biotechnology Vol. 10(20), pp. 4046-4056, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1968
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Genotype-dependent responses of wheat (Triticum
aestivum L.) seedlings to drought, UV-B radiation and
their combined stresses
Liheng He 1 , Xiaoyun Jia 1 , Zhiqiang Gao 1 * and Runzhi Li 2
1 Agronomy College, Shanxi Agricultural University, Taigu 030801, China.
2 Department of Plant and Soil Science, University of Kentucky, Lexington, KY 40546, USA.
Accepted 8 April, 2011
Experiments were conducted under controlled conditions to investigate the growth and physiologicalbiochemical
responses of wheat (Triticum aestivum L.) seedlings to UV-B, drought, and their combined
stresses. Both UV-B and drought treatments retarded seedling growth with UV-B having worse impact
on wheat plants. Chlorophyll content and relative water content (RWC) in leaves, as stress makers,
were significantly affected by UV-B and drought, respectively. The increased rate and amount of H2O2
were stress-different and genotype-dependent. Likely, the temporary expression patterns of antioxidant
enzymes (superoxide dismutase, SOD, and catalase, CAT) and compounds (proline, and ascobate acid,
AsA) exhibited differences under the tested stressful conditions in the two genotypes, indicating that
they play significant roles in plant responses to these stresses. Pre-application of either stress
reduced the damage caused by subsequent application of the other stress, and this induced defense
was greater by UV-B than by drought. Compared to the stress applied separately, the combined
application of drought and UV-B at the same time resulted in more adverse effects on the wheat
seedlings of the susceptible variety, and more positive effects on the tolerant wheat genotype. These
results provide novel insights into understanding the cellular and molecular mechanisms responsible
for plant tolerance to various stresses and their interactions.
Key words: Wheat (Triticum aestivum L.), UV-B radiation, drought, combined stress, antioxidant system,
genotype.
INTRODUCTION
The ongoing depletion of stratosphere ozone layer is
resulting in an increase of solar ultraviolet-B (UV-B)
radiation (280 to 320 nm) reaching the earth’s surface, as
measured in many geographic regions (McKenzie et al.,
2003). The enhanced levels of UV-B radiation has been
shown to have deleterious effects on biological organisms.
Plants exhibit different responses to UV-B irradiation
with respect to growth, production of dry matter and
biochemical changes (Kramer et al., 1991). Some plant
*Corresponding author. E-mail: gsshlh@hotmail.com or
gaozhiqiang1964@126.com. Tel: +86 354 6289789.
Abbreviations: SOD, Superoxide dismutase activity; CAT,
catalase activity.
species are unaffected by UV-B irradiation and several
are apparently stimulated in their growth, but most
species are sensitive and damaged (Teramura, 1983).
UV-B irradiation can also influence the physiological
responses of plants to the other environmental factors.
Elevated UV-B irradiation limited the ability of wheat, rice
and soybean to take advantage of the elevated CO2 in
photosynthesis (Teramura et al., 1990), but however,
enhanced heat tolerance of cucumber seedlings
(Teklemariam and Blake, 2003).
On the other hand, the effect of enhanced UV-B
radiation on plants can be modified by other co-occurring
stresses or by simply changing environmental factors like
atmospheric CO2 (Bjorn et al., 1997). Temperature
affected the extent of growth inhibition of cucumber
cotyledons irradiated with UV-B (Takeuchi et al., 1993).
Sensitivity of crop plants to UV-B is also influenced by

water regime, ambient levels of visible radiation and
nutrient status (Manetas et al., 1997; Balakumar et al.,
1993; Levizou and Manetas, 2001).
Water stress or soil drought is an important restricting
factor, which limits the productivity of many crops and
affects both quality and quantity of the yield. Drought
stress brings about a reduction in growth rate, stem
elongation, leaf expansion and stomatal movements
(Hsiao, 1973). It also affect various physiological and biochemical
processes governing plant growth and
productivity (Daie, 1988). Under field conditions plants
usually experience several stresses simultaneously. The
stresses may cause a variety of plant responses which
can be additive, synergistic or antagonistic.
In many cases, UV-B irradiation appears companying
with drought stress during crop plant growth seasons.
Elucidation of the interaction between drought and UV-B
stresses and their effects on plant growth and development
would help in understanding the mechanism
responsible for plant adaptation to changing environmental
conditions and developing agronomic systems for
crop productions. Although the responses of plants to
UV-B or drought have been intensively investigated,
evidence of study in interaction between UV-B exposure
and drought stress in plants had just emerged in the
latest years. The mechanisms of sensitivity or tolerance
of crop plants, either in growth and yield, to combined
stresses remain unknown. Particularly, genotypic effects
on plant responses to the two-stress combination are still
unclear.
Wheat (Triticum aestivum L.), as one of the crucially
important crops in the global food supply, is mainly
distributed in the mid-latitude regions in the northern
hemisphere (Lantican et al., 2005) where UV-B radiation
and drought naturally occur simultaneously or subsequently
during wheat growth seasons. Therefore, the
purpose of this study was to investigate and compare
their effect and interaction on some biochemical stress
markers and stress defense enzyme systems in seedlings
of winter wheat. We also evaluated the genotypic
differences in physiological response of wheat to UV-B
radiation and drought combined stress.
MATERIALS AND METHODS
Drought and UV-B treatments
Wheat (Triticum aestivum L.) varieties, Jinmai 47(drought-resistant)
and Shunmai 1718 (drought-susceptible) were used in this
experiment. After surface sterilization, healthy seeds were sown in
plastic pots (15 x 15 cm) containing vermiculite, and irrigated with ½
strength Hoagland’s solution. Wheat seedlings were grown in a
growth chamber (14/10 h photoperiod; 25/20°C day/night). 7 dayold
wheat seedlings were subjected to drought stress (+D) by 10%
polyethylene glycol (PEG6000), which provoked moderate water
stress (-0·5 MPa), to UV-B radiation (+UV), or to a combination (+D
+ UV), respectively. All stresses were applied throughout 20 days
for both genotypes.
As a source of UV-B radiation, a mercury lamp with a charac-
He et al. 4047
teristic emission in the range of 280 to 320 nm (HPQ 125 W,
Phillips, Eindhoven, The Netherlands) was used. The lamp
irradiation gave a photon flux density of 64·4 μmol m -2 s -1 and the
UV-B irradiation was about 80% of the total light emission. The
seedlings were transferred daily under the HPQ lamp for 2 h in the
middle of the day. The distance between the lamp and plants was
25 cm. The plants without UV-B irradiation treatment were kept
under a polyester film (0.13 mm Mylar Type D) (Du Pont Co,
Newton, CT, USA), which absorbs radiation below 320 nm. For the
treatments with UV-B irradiation, a cellulose acetate filter (0.13 mm)
was used to cut off the radiation below 280 nm, and plants received
49 kJm -2 d -1 biologically effective UV-B radiation.
Measurements of wheat seedling growth parameter
All measurements for seedling growth parameter were conducted at
the end of each stress treatment. Fresh weight of the above-ground
parts was measured, and after that plants were dried at 105°C to
constant weight for the determination of dry weight. Relative leaf
water content (RWC) was calculated according to the equation of
Fletcher et al., (1988). Leaf area was measured according to
Tsonev and Sergiev (1993) using a planar scanner and image plot
software.
Determination of proline, H2O2, chlorophyll and ascorbate
The fresh plant materials collected at various time points were
immediately used for the extraction and assay according to the
appropriate methods listed here.
Free proline was extracted, derivatized with acid ninhydrin, and
absorbance was read according to Bates et al. (1973) method using
L-proline as a standard. Content of proline was expressed as
µmol/g fresh weight. Total chlorophylls were extracted with 80%
acetone and were estimated according to Arnon (1949). Content of
the chlorophylls was expressed as mg/g fresh weight. Hydrogen
peroxide (H2O2) was measured according to Alexieva et al. (2001).
H2O2 was measured spectrophotometrically after reaction with KI.
The reaction mixture consisted 0.5 ml 0.1% trichloroacetic acid
(TCA) leaf extract supernatant, 0.5 ml of 100 mM K-phosphate
buffer and 2 ml reagent (1 M KI w/v in fresh double-distilled water
H2O). The blank probe consisted of 0.1% TCA in the absence of
leaf extract. The reaction was developed for 1 h in darkness, and
absorbance was measured at 390 nm. The amount (µmol/g fresh
weight) of hydrogen peroxide was determined using a given H2O2
standard curve.
Ascorbate content was determined according to the method
described by Foyer et al. (1983). Plant materials (0.5 g) were
ground in liquid nitrogen and then 2 mL of 2.5 M perchloric acid was
added. The crude extract was centrifuged at 4°C for 15 min at
15,000 g, and the supernatant was neutralized with saturated
K2CO3 using methyl orange as an indicator. Insoluble KClO4 was
removed by centrifugation and aliquots of the supernatant were
used for measuring ascorbate and dehydroascorbic acid (DHA)
contents. The reduced ascorbate (ascorbic acid, AsA) was
determined spectrophotometrically at 265 nm in 0.1 M NaH2PO4
buffer (pH 5.6), with 0.1 units of ascorbate oxidase. The total
ascorbate was determined after incubation in the presence of
30 mM DTT. The standard curve was prepared with AsA. DHA level
was obtained as the difference between AsA and total ascorbate.
The content was calculated as µmol/g fresh weight.
Enzyme extraction and assay
For enzymatic activities, assays were carried out using the crude
extract of the leaves as the enzyme source. Leaves were

4048 Afr. J. Biotechnol.
Table 1. Effect of drought and UV-B applied alone or in combination on growth parameters of wheat seedlings.
Parameter Control UV-B (+U) Drought (+D)
UV-Bdrought
(preU+D)
Drought-
UV-B
(preD+U)
Drought-
UV-B
(U+D)
Resistant genotype
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*
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*
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*
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
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*
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*
Susceptible genotype
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**
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**
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**
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**
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*
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
Values in brackets are percentage of control. The data are mean±SE (n=20). * and **, indicate significant difference between control and UV-B
radiation, Drought or their combined stress at P

Hundred percent of control(%)
300
250
200
150
100
50
0 2 4 6 8 10 2 4 6 8 10
Time of treatment (d)
Figure 1. The level of H2O2 in wheat seedlings under the successive stress (pred+u, preu+d) of UV-B
and drought. 100 percent (%) corresponds to the H2O2 level in the control plants without any stress.
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
the second stress (+U or +D), respectively. R1, drought-resistant genotype treated by preD+U
stresses; S1, drought-susceptible genotype treated by preD+U stresses; R2, drought-resistant
genotype treated by preU+D stresses; S2, drought-susceptible genotype treated by preU+D stresses.
Resistant genotype (R) and susceptible genotype (S) were indicated. Vertical bars are standard
errors (S.E.) of means.
reduction of chlorophyll content were considerable in
comparison with the control in Jinmai 47 (drought-tolerant
genotype). These data indicated that the stress
pretreatment induced plant defense response which
subsequently alleviated the negative effects of the other
stress. Moreover, plant responses to drought, UV-B and
the combined stress were genotype-dependent.
Time course of H2O2 content in wheat seedlings
under the stresses
H2O2 concentrations were maintained around 1.1 to 1.3
µmol g −1 FW in the control leaves at the beginning and
end of the experiment. However, the content of H2O2
increased significantly under stressful conditions following
seedling growth (Figure 1 and 2) compared to the
control plants without any stress. Moreover, the peak
value and time course of H2O2 level were different for
different stresses and genotypes.
In the pretreatments, UV-B induced H2O2 up to its peak
level at days 2 and 4 for Jinmai 47 and Shunmai 1718,
respectively; two days earlier than drought stress. The
maximum amount of H2O2 under UV-B was 31 and 47%
higher than that under drought stress for the two
genotypes, respectively. After the peak time, H2O2
R1
S1
R2
S2
He et al. 4049
content declined till the end (day 10) of the pretreatments,
at which H2O2 level was higher in the UV-B-stressed
plants than the drought-stressed plants for both varieties.
In the following second treatments, H2O2 was induced to
elevate again, and then reduced. The enhancement in
the drought-pretreated seedlings by UV-B was greater
than the UV-B-pretreated seedlings by drought. At the
end of the second stress, all the plants showed a lower
H2O2 level (P>0.01) than at the end of the pretreatments.
Furthermore, H2O2 level in the tolerant genotype was
significantly lower (P>0.01) than in the susceptible one.
Particularly, the pre-UV-B and following drought
treatments led to lower H2O2 compared to the pre-drought
and consequent UV-B stresses for both varieties.
Under the combined stress of UV-B and drought
applied simultaneously (Figure 2), H2O2 level quickly increased
to its maximum and then reduced through to the
end of the treatments. Comparison between the two
genotypes showed that the peak level and time were
37% lower and were 2 days earlier in the tolerant
genotype than in the susceptible one. At the end of the
combined stress, H2O2 level was 62% higher in the
susceptible variety than in the tolerant variety. Overall,
UV-B radiation caused more effects on H2O2 than the
drought treatment. Pretreatment of either stress reduced
the impact of subsequent application of the other stress.

4050 Afr. J. Biotechnol.
Hundred percent of control(%)
300
250
200
150
100
50
0
R
S
0 2 4 6 8 10
Time of treatment (d)
Figure 2. The level of H2O2 in wheat seedlings under the combined stress (U+D) of UV-B and drought.
100% corresponds to the H2O2 level in the control plants without any stress. Samples were collected at
0, 2, 4, 6, 8 and 10 days after the combined stress. R,Resistant genotype; S, Susceptible genotype.
Vertical bars are standard errors (S.E.) of means.
For susceptible genotype, the combined stress produced
more H2O2 than the drought or UV-B treatment alone,
while the H2O2 level induced by the combined stress was
higher than by the drought and lower than by UV-B stress
in the tolerant variety.
Temporary expression of antioxidative enzymatic
activity induced by the stresses
The activity of SOD and CAT experienced a slight
change in the control plants during the 20 days of growth,
and this variation was not significant. However, the three
enzymes displayed distinct patterns of activity expression
under the stressful conditions.
Following the pretreatment of drought (preD) and
subsequent UV-B stress (+U), SOD activity (Figure 3A)
increased to its first peak on day 2 and day 4 post preD in
Jinmai 47 and Shunmai 1718, respectively. The second
higher peak occurred on day 2 post +U for both genotypes.
After the second peak, SOD level slowly increased
till the end of the treatment. However, only one peak of
SOD activity was induced on day 2 and day 4 post
pretreatment of UV-B (preU) in Jinmai 47 and Shunmai
1718, respectively. Unlike in the preD+U treatments, the
subsequent drought stress (+D) did not significantly
enhance the additional SOD activity to the end of the
preU+D treatments. As shown in Figure 1A, SOD levels
at the peak and the end of the preU+D treatments were
both higher in the susceptible variety ( Shunmai, 1718)
than in the tolerant one (Jinmai 47).
CAT activity (Figure 3B) was also changed greatly
following the treatments. The first peak of CAT activity
occurred at day 2 or day 4 after the pretreatment of either
UV-B or drought. The subsequent stress of the other induced
more enhancement of CAT activity and resulted in
the second peak at different time for different stresses
and genotypes. In contrast to SOD, the increased CAT
level was much higher in the tolerant variety than in the
susceptible one, particularly at the peak time and the end
of the treatments. Moreover, CAT activity post the peak
was reduced rapidly in the susceptible variety while the
activity kept stable or just slightly declined in the tolerant
genotype. Again, UV-B radiation exhibited more effects
on CAT activity than drought stress.
The activity expression of SOD and CAT under the
combine stresses (Figure 4) was mostly like the pattern
under UV-B radiation alone. For tolerant genotype, there
were a higher level of CAT and a lower level of SOD in
the double-stress treatments.
Conversely, higher SOD and lower CAT were detected
in the double stresses for the susceptible variety.

SOD activity
Units/ mg protein
Catalase activity
(nmol H2O2 /mg protein min)
45
40
35
30
25
20
15
10
70
65
60
55
50
45
40
35
30
25
A
0 2 4 6 8 10 2 4 6 8 10
B
Time of treatment(d)
0 2 4 6 8 10 2 4 6 8 10
Time of treatment (d)
Figure 3. Changes in activities of antioxidant enzymes under the successive stresses (preD+U,
preU+D) of UV-B and drought. Samples were collected at 0, 2, 4, 6, 8 and 10 days after the first
stress (preD or preU) and then the second stress (+U or +D), respectively. R1, drought-resistant
genotype treated by preD+U stresses; S1, drought-susceptible genotype treated by preD+U
stresses; R2, drought-resistant genotype treated by preU+D stresses. S2: drought-susceptible
genotype treated by preU+D stresses. Resistant genotype (R) and susceptible genotype (S)
were indicated. Vertical bars are standard errors (S.E.) of means.
Changes in contents of antioxidative metabolites
under the stresses
Free proline in the drought-pretreated plants were observed
to increase by 1.16 and 0.5 folds greater than that
R1
S1
R2
S2
R1
S1
R2
S2
He et al. 4051
in the control at the end of the pretreatment for tolerant
and susceptible genotype, respectively (Figure 5). However,
pretreatment of UV-B just enhanced proline by a
smaller amount, and there was no significant difference
between the tolerant and susceptible genotypes. Sub-

4052 Afr. J. Biotechnol.
SOD activity
(Units/ mg protein)
Catalase activity
(nmol H2O2 /mg protein min)
45
40
35
30
25
20
15
10
5
0
80
70
60
50
40
30
20
10
0
A
0 2 4 6 8 10
B
Time of treatment (d)
0 2 4 6 8 10
Time of treatment (d)
Figure 4. Changes in activities of antioxidant enzymes under the combined stress of UV-B
and drought. Samples were collected at 0, 2, 4, 6, 8 and 10 days after the combined stress.
R3, drought-resistant genotype treated by the combined stresses; S3, drought-susceptible
genotype treated by the combine stresses. Vertical bars are standard errors (S.E.) of means.
sequent UV-B stress only induced proline level which
additionally accumulated a little in the drought-pretreated
plants, while subsequent drought stress resulted in a
larger enhancement of proline content in the UV-Bpretreated
plants. The combination of both stresses led to
a slightly lower proline content in comparison with the
drought application alone, and this depressed effect on
proline content was greater in the susceptible genotype
than in the tolerant one (Figure 5). The data showed that
drought stress had a much stronger impact than UV-B
R3
S3
R3
S3
stress in relation to proline accumulation regardless
applied together, separately, and successively.
All stresses increased ascorbic acid (AsA) concentration,
and did not cause much change in dehydroascorbic
acid (DHA) content compared to the control
(data not shown). As a result, a higher ASA/DHA ratio
was observed following the treatments (Figure 6). No
significant difference was detected between the two
genotypes. The percent increment of ASA/DHA ratio was
lower in the pretreatment of either UV-B (36%) or drought

AsA/DHA
Proline (µmol/g FW)
10
9
8
7
6
5
4
3
2
1
0
R S
control D preD+U U preU+D U+D
Treatment
Figure 5. Free proline content in winter wheat seedlings under UV-B, drought, and the
combined stresses. Samples were collected at the end of each treatment. D, Drought treatment
alone; U, UV-B treatment alone; U+D, UV-B and drought treatments applied simultaneously;
preD+U, drought treatment applied firstly and then UV-B treatment was applied; preU+D, UV-B
treatment was applied firstly and then drought treatment was applied. Values are means of six
replicates and standard errors (S.E.).
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
R S
control D preD+U U preU+D U+D
Treatment
Figure 6. The reduced-to-oxidized ascorbate ratio (AsA/DHA) in winter wheat seedlings under UV-B,
drought, and the combined stresses. Samples were collected at the end of each treatment. D, Drought
treatment alone; U, UV-B treatment alone; U+D, UV-B and drought treatments were applied simultaneously;
preD+U, Drought treatment was applied firstly and then UV-B treatment was applied; preU+D, UV-B
treatment was applied firstly and then drought treatment was applied. Values are means of six replicates
and standard errors (S.E.).
He et al. 4053

4054 Afr. J. Biotechnol.
(38%) than in the subsequent application of drought (81%)
or UV-B (77%) stress. This enhancement was more pronounced
under the combined stresses (Figure 6) so that
ASA/DHA ratio at the end of the treatment was higher in
the plants (3.82) by the combined stresses than those
when UV-B or drought stress was applied alone (2.33
and 2.36) and successively (3.16 and 3.14), indicating
that the combined stress exceeded the effect of a single
stress factor on ASA concentration.
DISCUSSION
With increasing depletion of stratospheric ozone and acelerating
global warming, UV-B irradiation and drought
stresses are becoming much more detrimental to
ecological systems and crop production in the world. The
mechanisms of crop plants to the combined stress of
these two factors remain unknown although the
interaction between UV-B and drought stress in plants
has emerged these years. Therefore, the present study
was conducted to investigate physiological responses of
different genotypes of winter wheat to the combined
stresses of UV-B and drought applied simultaneously and
successively.
Our data (Table 1) of wheat growth parameters measured
under the stresses tested displayed that pretreatment of
either UV-B or drought reduced the damage caused by
subsequent application of the other stress, conforming
the viewpoint that exposure of plants to a moderate
stress can induce a tolerance to a more severe stress
and such treatment also can improve tolerance to other
stresses (Wang et al., 2003). Moreover, the present study
showed that the tolerant variety exhibited better performance
than the susceptible one in this aspect, and the
UV-B-induced wheat plant protection against drought was
stronger than the drought-induced tolerance to UV-B
radiation. This result indicated that the expression of the
so-called cross-acclimation to stresses was related to the
plant genotypes and stress factors.
In comparison to the inhibited effects on plant growth of
UV-B or drought stress alone, the effect of the combination
of the two stress factors could be additive (more
damage) (Tian and Lei, 2007) or antagonistic (reduced
damage) (Sullivan and Teramura, 1990; Alexieva et al.,
2001). Our data further indicated that this combined
effect was genotype-dependent. For the tolerant variety,
the combined stress of UV-B and drought resulted in the
moderated injury to wheat seedlings, which was less than
the injury caused by the stresses applied individually.
However, the combined stress caused more severe
damage to wheat seedlings than stress factors applied
separately for the susceptible variety. Thus, the combined
application of drought and UV-B had more strong
adverse effects on wheat seedlings of the susceptible
genotype, but more positive effects on the tolerant wheat
genotype.
UV-B and drought stresses seemed to act in two different
ways, as it could be expected, drought signifi-cantly
influenced the RWC, and UV-B largely affected
chlorophyll of the treated leaves (Table 1). This finding
further support that chlorophyll content and RWC were
proposed as a typical maker for plant response to UV-B
and drought stresses, respectively (Teramura et al., 1991;
Correia et al., 1999; Santos et al. 2004). When UV-B
irradiation and drought stresses were applied simultaneously
or successively, the specific effects of UV-B on
chlorophyll and drought on RWC were slightly reduced
compared to the single stress, again indicating that
interaction between UV-B and drought alleviated, to
some extent, the negative effect of UV-B or drought alone.
Generation of reactive oxygen species (ROS) including
H2O2 is recognized as one of the early effects of various
biotic and abiotic stresses on plants (Bowler and Fluhr,
2000), and high accumulation of ROS can result in
oxidative damage in the absence of effective protective
mechanism (Alexieva et al., 2001). In our case, an
increase in H2O2 was observed in all the treated plants
compared to the control. However, the increase was
more evident in the UV-B treated plants than in the
drought-stressed plants (Figure 1), and thus UV-B
caused a more severe damage than the drought stress
on wheat seedlings measured as there was more obvious
reduction in growth (Table 1). Comparatively, the interaction
of UV-B and drought led to less damage on wheat
seedlings, which was evidenced by a lower level of the
peak value of H2O2 in the plants measured at the end of
the combined stresses applied simultaneously or successively
(Figure 2).
On the other hand, H2O2 received much attention as a
signal molecule in response to different stresses (Prasad
et al., 1994; Gong et al. 2001; Aroca et al., 2003). H2O2
mediated the regulation of transcription in response to
UV-B exposure as an important early upstream signal
(Brosche and Strid, 2003). Moreover, H2O2 was also
implicated in the gene expression related to cold
acclimation to resist freezing stress (Foyer et al. 1997).
Activation of endogenous protective mechanisms can in
turn tolerate or delete excess ROS burst. We found that
the enhanced H2O2 level under the stresses was followed
by the up-regulation of the enzyme activities (Figure 3).
This suggests that H2O2 may act more as a signal
molecule than directly inducing oxidative damage. In
other words, the increased H2O2 concentration by UV-B
or drought may trigger the cross-acclimation to tolerant
the subsequent stresses in winter wheat through stimulation
of the antioxidant defense systems.
SOD, the first antioxidant enzyme for scavenging ROS,
catalyzes the dismutation of superoxide into oxygen and
H2O2, while CAT reacts with H2O2 to produce water and
oxygen. In this study, it was observed that activities of the
two key antioxidant enzymes increased following all the
treatments compared to the control plants, but their
expression patterns were different for different genotypes

and stress conditions. For example, under the successive
stress conditions of first UV-B radiation and then drought,
SOD expression was “rapid up to the peak and then kept
stable” in both genotypes, whereas the peak level was
higher and occurred two days later in the susceptible
genotype than in the tolerant one (Figure 3A). Consequently,
SOD level was greater in the susceptible
genotype than in the tolerant one at the end of the
treatment. CAT expression pattern, however, was “rapid
up to peak, sustaining roughly stable, and then increasing
again” for the tolerant genotype or “decreasing” for the
susceptible one (Figure 3B). As a result, the CAT level
was higher in tolerant genotype than in the susceptible
one at the end of the treatments. Similarly, SOD and CAT
activities also increased following the successive
stresses of first drought and then UV-B except that the
enhancement rate was lower in the first stress than in the
second stress. Therefore, different levels of H2O2 under
all stresses, particularly higher H2O2 content in the susceptible
genotype than in the other may be due to the
differential expressions of activities of those enzymes
following the stress. Our inference is in agreement with
the report that cellular H2O2 concentration is the result of
the balance between its production and utilization (Bowler
et al., 1992).
Under the combined stresses of UV-B and drought
applied simultaneously, the two enzyme activities (Figure
4) were expressed like in the case under UV-B stress
alone, but SOD activity was lower and CAT was higher
than that under drought and UV-B stress alone for the
tolerant variety. Again, our data of the two enzyme
activities indicated that the interaction of UV-B and
drought stresses alleviated the negative effect of the
stress factors on the tolerant genotype, but the combined
stress led to more damage on the susceptible one.
Notably, many contradictory results about antioxidant
enzyme response to different stresses have emerged due
to the fact that the levels of enzyme responses depend
on the plant species, the developmental stage, the
organs, as well as on the duration and severity of the
stress (Wilson et al., 1993; Caldwell et al., 1983;
Chappell et al., 1994). In addition, the presence of
different enzyme isoforms expressed to a different extent
in different seedling growth conditions (stressed and unstressed),
and with different substrate affinity could be
not excluded.
In addition to the activation of antioxidant enzymes, the
enhancement of antioxidant metabolites such as proline
and ascorbate acid (AsA) in cells is also another defense
mechanism against the ROS burst caused by various
stresses (Monk et al., 1989). In many plants, free proline
accumulates in response to biotic and abiotic stresses,
including water stress (Day et al. 1993), extreme temperatures
(Strid et al., 1992), heavy metal toxicity (Long
et al., 1983), and UV-B irradiation (Schreiber et al., 1986).
Our data showed that drought stress had a much
stronger impact than UV-B stress in relation to proline
He et al. 4055
accumulation regardless applied together, separately,
and successively (Figure 5), confirming that proline is a
typical marker for osmotic stress, which is well-described
in water and salt stresses (Heuer, 1994; Fedina et al.,
2006). However, proline may exert a protective action on
UV-B stress because drought-treated wheat seedlings
accumulated higher level of proline, and appeared less
damaged by UV-B radiation compared to the control
plants (Table1). Consistence with our results, a positive
effect of proline accumulation on the reduction of the UV-
B induced damage is proposed in other reports
(Kurkdjian and Guern, 1989; Alexieva et al., 2001).
Ascorbate is a major primary antioxidant, reacting
directly with hydroxyl radicals, superoxide and singlet
oxygen, and is also a powerful secondary antioxidant, reducing
the oxidized form of a-tocopherol (Agrawal and
Rathore, 2007). In our study, foliar ascorbic acid
concentration was found to increase in all the stressful
conditions. Moreover, the rate of increase was lower in
the pretreatments of ether UV-B or drought than in the
subsequent stresses (Figure 6), suggesting that
enhancement of ASA content was not a rapid response
to the single stress of UV-B or drought. However, under
the combined stress of the two factors applied together,
ASA increase was faster and much higher (Figure 6),
indicating that the interaction between UV-B and drought
stresses generated a protective effect on plants. Unlike
the other parameters measured here, the time course
ASA level induced by the stress showed no significant
difference between the two genotypes.
In conclusion, pretreatment of either UV-B or drought
can increase wheat seedling tolerance to the subsequent
stress, and moreover, UV-B induced tolerance against
drought stress was stronger than the drought-induced
defense to UV-B stress evaluated as changes in plant
growth parameters and in amounts of stress markers.
The combined application of drought and UV-B together
brought out strong adverse effects on wheat seedlings of
susceptible variety, but more positive effects on the
tolerant wheat genotype. By scavenging excessive ROS,
antioxidant enzymes and compounds functioned importantly
in the plant responses to UV-B radiation, drought,
and their combined stresses. The expression patterns of
the enzymes and temporary changes of the antioxidants
tested following the treatments displayed differences to
some extent for different stress conditions and genotypes,
leading us to infer that the expression of plant defense
strategy to stress was differently regulated by a
complicate system consisting of plant genetic background,
physiological status, stress factors and their interactions
although the presence of a basic common response in
both genotypes under the stress conditions was observed.
Acknowledgements
This research was supported by the Earmarked Fund for

4056 Afr. J. Biotechnol.
Modern Agro-industry Technology Research System of
China, supported by Shanxi Province Foundation for
Returness, China.
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African Journal of Biotechnology Vol. 10(20), pp. 4057-4064, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2012
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
The effects of the regulated deficit irrigation on yield
and some yield components of common bean
(Phaseolus vulgaris L.) under semi-arid conditions
Mehmet Simsek 1 *, Nuray Comlekcioglu 2 and Irfan Ozturk 3
1 Department of Agricultural Structures and Irrigation, Faculty of Agriculture,Harran University, Sanliurfa, Turkey.
2 Department of Horticulture, Faculty of Agriculture, Harran University, Şanliurfa Sanliurfa, Turkey.
3 Department of Biometry Science, Faculty of Agriculture, Harran University, Sanliurfa, Turkey.
Accepted 28 March, 2011
During three consecutive years (2002 to 2004), common bean was grown to investigate the effects of
the regulated deficit irrigation (RDI) on the yield and yield parameters under semi-arid conditions. Field
experiments were conducted on a clay soil. The growing season of common bean was divided into two
phases: (1) Vegetative stage (V); from seed germination to the beginning of flowering and (2)
reproductive stage (R); from the flowering to the last fruit harvesting. The statistical design was a splitplot
with three replications, where the growth stage was the main factor of variation and the irrigation
was the secondary factor. The irrigation treatments consisted of all possible combinations of full
irrigation (T1 and T5 (V100 and R100: 100% of irrigation water (IW)/cumulative pan evaporation (CPE)) or
limited irrigation (T2: V75-R100, T3: V50-R100, T4: V25-R100, T6: V100-R75, T7: V100-R50 and T8: V100-R25) in two
phases. Fresh bean yield and pod weight (PWt), pod length (PL), pod width (PWh) and number of seed
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,
12.0 and 21.0% lower than the yields obtained from the control (18.36 and 18.40 t ha -1 ) treatments,
respectively. Results demonstrated that, vegetative stage was the more sensitive than the reproductive
stage to the deficit irrigation. The highest irrigation water use efficiency (IWUE) and water use efficiency
(WUE) were found in T6 and T7 treatments as 2.58 and 2.66 kg m -3 , respectively.
Key words: Phaseolus vulgaris L., class A pan, drip irrigation, deficit irrigation.
INTRODUCTION
Common bean is being consumed as a source of
proteins, calories, fibers and minerals in the developing
countries (Ramos et al., 1999; Singh et al., 1999). Also,
since it has an ability to bind atmospheric nitrogen, it
plays a significant role for the crop rotation and sustainable
cropping systems (Donald and Paulsen, 1997).
Common bean is planted on an area of 0.98 million ha
and has a production of 6.82 million t as fresh in the
world. Turkey is ranked second to China with a production
amount of 0.56 million t and a planted area of 0.07
*Corresponding author. E-mail: mehmetsimsek@harran.edu.tr,
simsek0154@gmail.com. Tel: +90 414 3183756. Fax: +90 414
3183682.
Million ha (FAO, 2008). Besides benefits of bean to the
human diet and environment, its production requires a
significant amount of water due to its relatively shallow
root system. Thus, the amount of irrigation applied at the
developmental (vegetative and reproductive) stages,
affect the bean production in that plant growth and yield
are often reduced by periods of water stress (Ramos et
al., 1999).
Efficient use of water in any irrigation systems is vital
especially in arid and semi-arid regions. Drought occurs
in many parts of the world in every year; often with
devastating effects on crop production (Ludlow and
Muchow, 1990; Tonkaz, 2006). As the water supply is
limited worldwide, there is a need of water saving in
irrigation and thus, deficit irrigation should be applied.
Drip irrigation, reduces deep percolation and evaporation

4058 Afr. J. Biotechnol.
and controls water status of the soil more precisely within
the crop root zone (Singandhupe et al., 2003). One
choice for improving irrigation water management is to
alter from furrow or sprinkler irrigation to drip irrigation.
Several alternative irrigation methods have been used
such as surface, drip and sprinkler irrigation systems for
the production of common bean. However, recently using
the drip irrigation has attracted a significant amount of
attention of researchers and farmers instead of using
surface and sprinkler irrigations either to increase the
efficient use of water or the yield (Hanson and May,
2004). Moreover, drip irrigation reduces deep percolation
and evaporation and controls water status of the soil
more precisely within the crop root zone (Singandhupe et
al., 2003). Numerous studies were carried out in the past
for the development and evaluation of irrigation scheduling
techniques under a wide range of irrigation
systems and management techniques, soil, climate and
crop conditions (Kang et al., 2000; Boutraa and Sanders,
2001; Wakrim et al., 2005; Zhang et al., 2006.)
The irrigation scheduling techniques is very critical to
use the drip irrigation most efficiently because while
excessive amount of irrigation causes a decrease in the
yield, inadequate irrigation results in a water stress and a
reduction in production (Sezen et al., 2005; Onder et al.,
2006). Wakrim et al. (2005) reported that, even though
irrigation increases the overall plant growth rate, excess
water might bring about the poor grain yields in common
bean. Also, this statement was in agreement with several
studies which showed that, implementations of limited
and excessive irrigation for common bean result in lower
yield as they create stress during plant development
(Nielsen and Nelson, 1998; Dapaah et al., 1999; Sezen
et al., 2005). The deficit irrigation techniques, including
regulated deficit irrigation (RDI), have been developed to
control excessive vegetative growth or water saving. RDI
is widely used in the horticultural industry since it results
in the more efficient use of irrigation water and often
improves the crop quality (Turner, 2001).
The meteorological-based irrigation scheduling approach,
such as implementations of pan evaporation replenishment,
cumulative pan evaporation (CPE) and ratio between
irrigation water and CPE was employed by many
researchers (Saudan et al., 2000; Ferreia and Carr 2002;
Simsek et al., 2005). This was due to its simplicity, data
availability and high degree of adaptability to the farmer
level (Imtiyaz et al., 2000 and Simsek et al., 2005).
The objective of this study was to determine the effects
of RDI on the yield and yield components of the fresh
common bean in the semi-arid conditions.
MATERIALS AND METHODS
Experimental site and climatic data
This study was conducted on a clay soil at the Agricultural
Experimental Research Field of the Harran University during the
growth periods of 2002, 2003 and 2004. The site is situated on the
37°08' N and 38°46' E and 464 m above sea level. The soil water
contents (w/w; %) of the soil at field capacity were 33.1, 33.2 and
33.7% and at permanent wilting point 21.6, 21.9 and 22.8%, in 0 to
30, 30 to 60 and 60 to 90 cm soil depths, respectively. The
corresponding bulk densities were 1.41, 1.45 and 1.44 Mg m 3 . The
study area was located in a semi-arid climate. The soil water
content was measured gravimetrically in each 0.3 m layer down to
0.9 m depth in two replicates per treatment. These measurements
repeated with intervals of 12 days were done six times for each
year. The lowest level of relative humidity and the highest
temperature and solar radiation occur in summer months. The
recorded maximum and the minimum temperatures were 32.7 and
11.7°C in August and November. The highest and lowest values of
relative humidity were measured as 72.2 and 32.2% for the months
of November and September, respectively (Table 1). The highest
and the lowest solar radiations were measured as 24.0 and 8.8 MJ
m -2 day -1 in August and November.
Crop evapotranspirations under varying irrigation regimes was
calculated using the water balance model (Garrity et al., 1982).
ETc=IW+P–D–R±∆S
Where, ETc is the seasonal crop evapotranspiration (mm); IW is the
total irrigation water applied (mm); P is the precipitation (mm); D is
the drainage (mm); R is the run-off (mm) and ∆S is the variation in
water content (mm) of the soil profile. All terms are expressed in
mm of water in the bean root zone. The effective root depth was
taken as 60 cm. Run-off was taken as to be nil since no run-off was
observed in drip irrigation system.
Total amount of irrigation water applied was calculated by using the
following equation (Doorenbos and Pruitt, 1975):
IW=AxEpan
Where, IW is the amount of irrigation water applied (L); A is the plot
area (m 2 ) and Epan is the amount of cumulative evaporation during
an irrigation interval (mm).
Crop management and irrigation treatments
The experiments were conducted in the field for consecutive 3
years. Common bean cv. Gina was examined in this study. The
seeds were sown in August 05, 11 and 08 in 2002, 2003 and 2004,
respectively. Seeds were spaced with 25 cm in a row and 70 cm
between rows, as used in traditional by the growers of this region.
Each plot was 6 m in length and five rows (21 m 2 ). The plants were
fertilized with 120, 100 and 120 kg ha -1 of N, P2O5, and K2O5,
respectively.
The total crop duration was divided into two phenological stages
in order to distinguish different plant growing stages and irrigation
treatments: Vegetative stage (V); from seed germination to
beginning of flowering and reproductive stage (R); from the first
flowering to the end of harvesting (Tables 2 and 3). The case study
involved four drip irrigation treatments applied, in two growth
stages. Full irrigations (T1 and T5: V100-R100 (IW/CPE ratio 100%))
were applied to the control treatments in the V and R stages, while
25, 50 and 75% limitations, were applied to the other plots and
compared to the control. RDI was studied with treatments T2: V75-
R100, T3: V50-R100, T4: V25-R100, T6: V100-R75, T7: V100-R50, and T8:
V100-R25 (Table 2). The CPE values were obtained by the use of
class A pan evaporometer located in the experimental site. Three
day CPE values were used for irrigation.
The water from water tank was delivered to the field by a submain
32 mm diameter polyethylene (PE), 16 mm diameter laterals
with in-line emitters located 0.4 m apart, surface drip laterals

Table 1. Some climatic data during the experiment.
2002
2003
2004
Long-run data
(1929 to 2004)
Year/month Ta(°C) RH(%) E0(mm d -1 ) Rs(MJ m -2 day -1 ) u2(m s -1 )
August 30.5 43.7 12.2 24.0 3.0
September 26.9 47.8 9.5 19,8 2.4
October 21.8 48.6 4.7 14,2 2.5
November 14.4 62.4 3.2 10.6 1.9
August 32.7 32.2 10.9 22.8 2.6
September 26.4 42.4 9.3 19.7 2.8
October 21.5 51.5 4.0 13.9 1.7
November 12.7 62 2.8 9.8 1.5
August 30.8 40.7 11.8 23.5 2.5
September 27.3 34.8 8.4 19.4 1.9
October 21.7 48.7 4.3 13.1 1.6
November 11.7 72.2 3.0 8.8 1.4
August 31.1 32.1 11.9 21.4 2.7
September 26.6 34.9 9.3 18.2 2.4
October 20.1 44.5 4.5 13.1 1.8
November 12.8 59.0 3.0 8.5 1.6
Simsek et al. 4059
Average of monthly values of air temperature (Ta), relative humidity (RH), evaporation from class “A” pan (ET0), solar radiation
(Rs), and wind speed at 2 m height (u2).
Table 2. Treatments details of irrigation regime in the different
growth stages.
Treatment
Irrigation regime
(CPE ‡ : 100, 75, 50 and 25%)
V R
T1 V100 R100
T2 V75 R100
T3 V50 R100
T4 V25 R100
T5 V100 R100
T6 V100 R75
T7 V100 R50
T8 V100 R25
‡ CPE: Cumulative pan evaporation.
spaced at 0.7 m intervals, one drip lateral each row and operating
at a constant pressure of 152 kPa with 3.0 L h -1 . Irrigation water
was checked by flow water meter in valves.
The harvest was conducted weekly and yield values were
measured for each plot and were calculated for hectare (t ha -1 ). Pod
weight (PWt), pod length (PL) and pod width (PWh) values of the
randomly chosen 50 pods and the values of the number of seed per
pod (NSPP) were determined for each fruit.
Water use efficiency (WUE) and irrigation water use efficiency
(IWUE)
WUE is the ratio between bean yield (t ha -1 ) and seasonal crop
Table 3. Durations of the two growth stages (in days) of
common bean in three years.
Growth stage 2002 2003 2004
V 41 40 42
R 58 58 52
Total duration 99 98 94
evapotranspiration (mm), as can be seen in the equation as follow:
Ya
WUE =
ET
c
If the yield Ya is expressed in kg and the water use ETc is
expressed in m 3 m -2 , then WUE has units of kg m -3 on a unit water
volume basis or g kg -1 when expressed on a unit water mass basis
(Stanhill, 1986; Howell et al., 1990). IWUE is obtained by using the
ratio of the yield per unit IW (mm) (equation).
Ya
IWUE =
IW
Experimental design and statistical analysis
The field experiments were set up with split plot design with three
replicates, where the growth stage was the main factor of variation
and the irrigation was the secondary factor. The data were analyzed

4060 Afr. J. Biotechnol.
using SAS statistical program (SAS Inst 1991). Analysis of variance
(ANOVA) test was conducted and significant differences among
treatments were determined using the TUKEY method. Square root
transformation was performed for number of seed per pod to
analysis of variance.
RESULTS
Effect of RDI on applied water and evapotranspiration
Irrigation treatments were initiated when the soil water
content at the effective root depth decreased to 50% of
the available soil water on August 26 (21 days after
sowing; 21 DAS) in the first, on August 31 (20 DAS) in
the second and on August 30 (22 DAS) in the third year.
The sowing day was considered as 0 day (0 DAS). The
final irrigation treatments were applied November 6,
2002, November 11, 2003 and November 6, 2004. A total
of nineteen irrigations were applied in the first two years,
while eighteen irrigations were applied in the third year.
When the mean of the three years were calculated, the
duration of the vegetative stage was 41 days while the
duration of the reproductive stage was 56 days (Table 3).
Total eight harvests were made in all the experimental
years.
Table 4 shows the values of fresh bean yield, IW and
ETC. The average values of IW and ETc were 721, 672,
623, 575, 721, 682, 642 and 603 mm and 742, 696, 651,
602, 747, 669, 607 and 558 mm for T1, T2, T3, T4, T5, T6,
T7 and T8, respectively. The lowest value was determined
for T8 being applied with 75% water deficit in the reproductive
stage. This was followed by the treatments on
which 50 and 25% water deficit was applied. These
values were higher than the values of IW and ETc
presented in the studies of Onder et al. (2006) and Sezen
et al. (2005). This might be resulted from the decrease in
the relative humidity, the temperature increase and
evaporation ratio increase due to the semi-arid conditions
in this study.
Irrigation water use efficiency and water use
efficiency
IWUE and WUE values were given in Table 4. The
highest IWUE was obtained as 2.58 kg m -3 in T6 whereas
the highest WUE was calculated as 2.66 kg m -3 in T7,
when the mean of the three years was examined. A direct
relation was observed between the depth of the applied
water and evapotranspiration. IWUE and WUE values
were decreased for RDI treatments in the V stage when
the applied and consumed water were decreased. However,
no significant differences in IWUE and WUE values
were observed for the RDI treatments belonging to the R
stage. The lowest mean IWUE and WUE were measured
as 1.88 and 1.80 kg m -3 , respectively on the T4. Wakrim
et al. (2005) determined the WUE values for common
bean between 1.91 and 2.92 kg m -3 which was well
correlated with our data. In addition, values of IWUE and
WUE found by the study of Onder et al. (2006) were in
agreement with our data, in that, values varied from 1.56
to 2.61 kg m -3 for IWUE and from 1.42 to 2.02 kg m -3 for
WUE. The values of IWUE and WUE for the V stage
were determined lower than the ones obtained for the R
stage. The reason was related with the decrease in the
yield in T2, T3 and T4 treatments.
Sezen et al. (2005) reported that, the IWUE and WUE
for Gina variety cultivated by drip irrigation were between
3.80 and 7.29 and 4.14 and 6.16 kg m -3 and the yield was
between 12.20 and 20.60 t ha -1 , respectively. The higher
values of IWUE and WUE had been attributed to the
higher relative humidity, caused by decrease in the water
consumption.
Yield-RDI relationship
As shown in Table 5, the highest mean yield was
observed in control treatment as more than 18 t ha -1 . The
yields of the T2, T3, T4, T6, T7 and T8 treatments were 27,
35, 41, 4, 12 and 21% lower than the yield obtained from
the control treatments, respectively. The lower yield in T2,
T3, and T4, when compared with control treatments, can
be attributed to the use of deficit of water as well as the
high temperature during V stage. The V stage was
defined as the period between the beginning of August
and the middle of September. The mean temperatures
were very high during this period and the relative
humidity was very low, whereas the evaporation ratio was
too high (Table 1). Nielsen and Nelson (1998) reported
that, the water stress lead to the growth of the shortest
plants with the least leaf area during the vegetative
growth stage of black bean. In this study, thin and short
plants and plants with lower number of branches were
observed visually in T2, T3 and T4. Water stress during
the R stage in T6, T7, and T8 can also lead to a significant
decrease in the yield by increasing the number of aborted
flowers and pods per plant. Wakrim et al. (2005) showed
that, the RDI treatments resulted in the mild stress which
affected significantly both plant vegetative and reproductive
growth. Similar decreases in the yield have
already been reported by Singh (1995) in common bean,
Karam et al. (2005) in soybean, Cakir (2004) and Payero
et al.(2006) in corn and Karam et al. (2007) in sunflower.
Application of adequate water during flowering and pod
development is the most significant factor in bean
irrigation. Similar responds were observed in this study
for the common bean that was cultivated in the semi-arid
zone. Similar trend in the yield was seen for the yield
components in all irrigation treatments. Water stress
combined with high temperature during flowering of the
bean brought about a decrease in all yield components.

Table 4. IWUE and WUE for RDI treatments in 2002, 2003 and 2004.
Treatment Fresh bean yield(t ha -1 )
2002
a IW(mm)
Simsek et al. 4061
b ETc(mm) IWUE(kg m -3 ) WUE(kg m -3 )
T1 18.37 786 797 2,34 2,30
T2 13.37 734 751 1,82 1,78
T3 12.17 683 704 1,78 1,73
T4 10.64 631 625 1,69 1,70
T5 18.40 786 811 2,34 2,27
T6 17.64 743 739 2,37 2,39
T7 16.04 700 664 2,29 2,42
T8 14.30 656 608 2,18 2,34
2003
T1 17.77 690 708 2.58 2.51
T2 12.84 643 677 2.00 1.90
T3 11.24 595 622 1.89 1.81
T4 10.57 548 589 1.93 1.79
T5 17.94 690 703 2.60 2.55
T6 17.27 652 623 2.65 2.77
T7 15.80 613 558 2.58 2.83
T8 13.90 575 530 2.42 2.62
2004
T1 18.94 687 721 2.76 2.63
T2 13.97 640 660 2.18 2.12
T3 12.27 593 628 2.07 1.95
T4 11.24 545 591 2.06 1.90
T5 18.80 687 728 2.74 2.58
T6 17.84 651 644 2.74 2.77
T7 16.64 615 600 2.71 2.77
T8 15.40 578 536 2.66 2.87
Average
T1 18.36 721 742 2.55 2.47
T2 13.39 672 696 1.99 1.92
T3 11.89 623 651 1.91 1.83
T4 10.82 575 602 1.88 1.80
T5 18.38 721 747 2.55 2.46
T6 17.58 682 669 2.58 2.63
T7 16.16 642 607 2.52 2.66
T8 14.53 603 558 2.41 2.60
a IW, Irrigation water; b ETc, evapotranspiration.
Yield components
The effects of year, growth stages and irrigation regimes
on considered yield components were significant (P <
0.001), whereas no significant differences were found
between the year for the NSPP and between irrigation
regime for PL (Table 6).
These differences could be attributed to the climate,
since it was considerably cold three weeks before the last
harvest time in the second year. The temperatures
dropped by 10°C, compared with other years. Sudden
climate change caused a drop in the yield and yield
components in 2003. The values obtained in the first and
the third years were similar, whereas the yield for the
second year was lower than the values found in other
years. The interactions between the growth stage and

4062 Afr. J. Biotechnol.
Table 5. The effects of regulated deficit irrigation regimes on common bean yield and its components.
Treatment Year T1 T2 T3 T4 T5 T6 T7 T8
2002 18.37±0.66
Fresh bean yield (t ha-1)
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
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
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
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
Pod weight (g)
Pod length (mm)
Pod width (mm)
Number of seed per pod†
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
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
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
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
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
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
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
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
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
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
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
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
2002
2003
2004
2.39±0.013 a
(5.7)
2.32±0.063 a
(5.4)
2.42±0.027 a
(5.8)
Average 2.37±0.024a
(5.6)
2.10±0.023 de
(4.4)
2.08±0.035 cd
(4.3)
2.12±0.023 bc
(4.5)
2.10±0.015 c
(4.4)
2.05±0.023 e
(4.1)
1.99±0.044 cd
(4.0)
2.03±0.021 cd
(4.1)
2.02±0.018 d
(4.1)
2.02±0.030 e
(4.05)
1.95±0.037 d
(3.8)
1.98±0.031 d
(3.9)
1.98±0.019 d
(3.9)
2.39±0.023 a
(5.7)
2.34±0.045 a
(5.5)
2.42±0.041 a
(5.9)
2.38±0.022 a
(5.7)
Number of seed per pod was subjected to square root transformation. Non transformed data are represented by numbers given in brackets.
2.31±0.044 ab
(5.3)
2.27±0.024 ab
(5.2)
2.38±0.007 a
(5.7)
2.32±0.022 ab
(5.4)
2.27±0.030 bc
(5.2)
2.25±0.026 ab
(5.1)
2.33±0.021 a
(5.4)
2.28±0.018 b
(5.2)
2.18±0.043 cd
(4.8)
2.12±0.023 bc
(4.5)
2.18±0.030 b
(4.8)
2.16±0.019 c
(4.7)

Simsek et al. 4063
Table 6. Analysis of variance for bean yield, pod weight, pod length, pod width and number of seed per pod (mean of
2002, 2003 and 2004).
Source of variation
Degree of
freedom
Mean square
FY PWt PL PWh NSPP
Year (Y) 2 5.665*** 7.693*** 630.712*** 22.141*** 0.029 ns
Y X R 6 0.345* 0.105 ns 8.464 ns 0.348 ns 0.006 ns
Growth stage (A) 1 167.445*** 28.880*** 1673.311*** 11.045*** 0.525***
Y X A 2 0.043 ns 0.024 ns 10.125 ns 0.755 ns 0.002 ns
Error 6 0.077 0.106 14.258 0.252 0.006
Irrigation regime (B) 3 107.211*** 16.941*** 2687.815 ns 29.585*** 0.305***
Y X B 6 0.085 ns 0.206 ns 18.782 ns 1.484* 0.001 ns
A X B 3 18.595*** 3.343*** 216.972*** 1.492* 0.054***
Y X A X B 6 0.192 ns 0.121 ns 10.753 ns 0.366 ns 0.001 ns
Error 36 0.248 0.187 12.875 0.465 0.002
Total 71 8.02 1.622 175.301 2.557 0.026
*Significant at P < 0.05; ***significant at P < 0.001; R: replication; RDI, regulated deficit irrigation; FY, fresh yield; PWt, pod
weight; PL, pod length; PWh, pod width; NSPP, number of seed per pod.
RDI were very significant at P < 0.001, for FY, PWt, PL
and NSPP, at P < 0.05 for the PWh (Table 6). Table 5
represents the yield components data of treatments.
Positive correlation was obtained between the yield and
the values of PWt, PL, PWh and NSPP. Similarly, Lyon et
al. (1995) reported that a positive correlation exists
between the seed number, seed weight and the yield.
The reason of the higher yield for the control treatments
was due to mainly increase the vegetative growth parameters.
Similar findings have been reported previously by
Demirtas et al. (2010) in soybean and Onder et al. (2006)
in green bean.
As expected, PWt increase had a significant effect on
the yield. In addition, PWt was significantly influenced by
irrigation level (Onder et al., 2006). The lowest value of
the PWt was found in the T4 on which 75% water stress
was applied in the V stage as 6.72 g, when the pod
weight values were examined. The yields were generally
found to be decreasing, for the treatments on which RDI
was applied on the V stage or R stage, as the PWt was
decreased (Table 5). The PWt values of the control were
observed to be higher for all the three years since water
stress was not applied. RDI regime applied in the V stage
caused a significant decrease in PWt, whereas no
important deviation was observed in the R stage (Table
5).
When the PL was examined for the RDI regime, the
values were decreased with the decreasing of irrigation
water. The longest PL (113.63 and 113.23 mm) was observed
in the T1 and T5, while the lowest PL value was
measured as 79.47 mm for T4.
RDI significantly affected the NSPP and displayed
similar effects on both growing stages. When the mean
values of the years were only considered, it was determined
that the yield increased with increasing the NSPP.
The highest and lowest NSPP were determined as 5.70
for T5 and 3.94 for T4 (Table 5).
Conclusions
The results of this study showed that, more positive
results could be obtained for the RDI regimes in the R
stage than the RDI regimes used in the V stage. It is
found that bean yield is very sensitive to the water stress.
Unless RDI is compulsory, it should not be applied in the
V stage because the sensitivity of the plant is considerably
high at this stage. However, excessive tempe-

4064 Afr. J. Biotechnol.
rature was a dominant factor in the decreasing of the
flowering and fruit set. Therefore, the seeding of the
common bean must be done in the first or second weeks
of the August. Climatic conditions particularly, cumulative
pan evaporation play the most significant role among
others. Therefore, irrigation techniques and programs
considering climatic parameters have become very significant.
The RDI regime with 25% deficit at reproductive
stage could be recommended to growers without a
significant decrease in the yield in semi arid zone.
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African Journal of Biotechnology Vol. 10(20), pp. 4065-4071, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2399
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Molecular cloning of a novel GSK3/shaggy-like gene
from Triticum monococcum L. and its expression in
response to salt, drought and other abiotic stresses
Yang Xian-Guang, Fan Jin-Yu and Deng Chuan-Liang*
School of Life Science, Henan Normal University, Xinxiang 453007, China.
Accepted 25 March, 2011
The glycogen synthase kinase 3 (GSK3)/SHAGGY-like kinases are nonreceptor serine/threonine protein
kinases that are involved in a variety of biological processes. Here, a novel GSK-3-like kinase encoding
cDNA was isolated from Triticum monococcum L. seedlings by reverse transcriptase polymerase chain
reaction (RT-PCR). Sequence analysis showed that the full length of cDNA consist of 1,543 bp with an
open reading frame of 1,068 bp, which encodes 355 amino acid residues. The deduced amino acid
sequence showed a high homology with shaggy-like kinases from Triticum aestivum, Zea mays,
Trifolium repens, Nicotine tabacum, Medicago sativa and Arabidopsis thaliana; therefore, the gene was
named TmGSK1 (Triticum monococcum Glycogen Synthase Kinase 1,GenBank Accession No.
DQ443471). Southern blot analysis indicated that there was only one copy of TmGSK1 in the einkorn
wheat genome. Quantitative real-time RT-PCR studies showed that the expression of TmGSK1 in the
einkorn wheat was induced by salt stress, mechanical wounding, ABA hormone, cold and drought.
These results suggest that cells accumulate more TmGSK1 mRNA response to those abiotic stresses.
TmGSK1 was shown to be a positive regulator commonly involved in the tolerance to salt, mechanical
injury, ABA hormone, cold and drought in einkorn wheat.
Key words: TmGSK1, abiotic stress, shaggy-like kinase, signal transduction, Triticum monococcum L.
INTRODUCTION
Abiotic stresses such as salinity, drought, extreme tem-
peratures, chemical toxicity and oxidative stress are
becoming particularly widespread in many regions. It is
estimated that salinity affects at least 20% of the world’s
arable land and more than 40% of irrigated land to various
degrees (Wang et al., 2003). These stresses reduce
average yields for most major crop plants by more than
50%. Further more, drought, salinity, extreme tempe-
ratures and oxidative stress are often interconnected, and
may affect the plants in a similar way (Katerji et al., 2004;
Almansouri et al., 2001). These diverse environmental
stresses often activate similar cell signaling pathways and
cellular responses such as the production of stress
*Corresponding author. E-mail: biol@htu.cn
Abbreviations: GSK-3, Glycogen synthase kinase-3; RT-PCR,
reverse transcriptase polymerase chain reaction; PCR,
polymerase chain reaction.
proteins, up-regulation of anti-oxidants and accumulation
of compatible solutes, leading to plant defense and/or
adjustment to adverse conditions (Bajguz and Hayat,
2009; Zhu, 2002; Knight and Knight, 2001). Various
abiotic stresses lead to the overproduction of reactive
oxygen species (ROS) in plants which are highly reactive
and toxic, and cause damage to proteins, lipids,
carbohydrates and DNA which ultimately results in
oxidative stress (Gill and Tuteja, 2010). Oxidative and
osmotic stress activates several protein kinases including
mitogen-activated protein kinases, which may mediate
osmotic homeostasis and/or detoxification responses. A
number of phospholipid systems are activated by osmotic
stress, generating a diverse array of messenger
molecules, some of which may function upstream of the
osmotic stress-activated protein kinases (Zhu, 2001) and
glycogen synthase kinase3/ shaggy like kinase.
Glycogen synthase kinase-3 (GSK-3) is a ubiquitously
expressed protein kinase. It was originally identified as a
regulator of glycogen synthesis in mammals. Animal
GSK-3 participates in glycogen metabolism and in both the

4066 Afr. J. Biotechnol.
Wnt/β-catenin and PI3K/AKT signaling pathways involved
in the regulation of several physiological processes,
including glycogen metabolism, protein synthesis, trans-
cription factor activity and developmental control.
Whereas, plant GSK-3-like kinases (GSKs) have only
recently entered the scene. In contrast to mammals,
which contain two genes, a multigene family encodes
plant GSKs. GSK-3-like genes have been isolated from
plants such as alfalfa (Pay et al., 1993), arabidopsis
(Rozhon et al., 2010; Tavares et al., 2002; Piao et al.,
1999; Dornelas et al., 1999; Jonak et al., 1995; Bianchi et
al., 1994), Petunia hybrida (Decroocq-Ferrant et al., 1995),
Nicotiana tabacum (Einzenberger et al., 1995), Triticum
aestivum (Chen et al., 2003), Oryza sativa (Koh et al.,
2007) and Zea mays (Zou et al., 2008). Present evidence
indicates that plant GSKs are involved in different
processes such as flower development (Dornelas et al.,
2000), wound responses (Jonak et al., 2000), salt stress
(Piao et al., 2001) and brassinosteroid signaling (Peng et
al., 2010; Li and Nam, 2002). Einkorn wheat is a rare
species that reduces the magnitude of population and the
area of distri- bution under anthropogenic activity. It is
closely related to cultivated wheat and has some
properties, for example, resistance to diseases, and thus
were used in selection to improve wheat varieties. Here,
we isolated a member of GSK family from einkorn wheat
and analyzed its expression patterns in response to
abiotic stresses by real time quantitative polymerase
chain reaction (PCR) approach.
MATERIALS AND METHODS
Plant material and stress treatments
Seed of the einkorn wheat (Triticum monococcum L.) were
surface-sterilized for 5 min in 1% (w/v) sodium hypochlorite and was
then washed in distilled water. The sterilized seeds were soaked in
distilled water at 30°C overnight, for germination. The germinated
seeds were transplanted into a pot containing vermiculite. The
seedings were irrigated with distilled water. Two-week-old plant was
treated with distilled water (as a control for Q-PCR analysis), high
concentration of salt (170 mM NaCl), mechanical injury (shearing),
ABA hormone (100 µM), cold (4°C) and drought (30%PEG6000).
Plant was harvested after stress treatments at intervals of definite
time, then immediately frozen in liquid nitrogen and stored at –80°C
for later use.
RNA isolation and cDNA synthesis
Total RNAs were isolated using TRIzol reagent (Invitrogen) according
to the manufacturer's recommendations. Total RNA was treated with
RNase-free DNaseI (Promega) for 20 min at 37°C, DNase I was
degraded at 65°C for 10 min. The integrity of the RNA was verified
after separation by electrophoresis on a 0.8% agarose gel
containing 0.5% (v/v) ethidium bromide. First-strand cDNA was
synthesized from 500 ng of total RNA using Reverse Transcription
system (Promega) with an oligo-dT15 primer. The reaction solution
was used as templates for reverse transcriptase polymerase chain
reaction (RT-PCR).
TmGSK1 gene cloning and sequence analysis
PCR was performed using the following primers for TmGSK1:
T1-forward (5'- GTTGGTGTGGTGCGTCCTTC-3') and T1-reverse
(5'- TGGGGGCCTCGATCCATGAAC -3') designed from the con-
served sequences of the known plant GSKs. Polymerase chain
reaction (PCR) was initiated with hot start method using the single
strand cDNA template and Ex-Taq polymerase (Takara) on Biometra
T-GRADIENT Themoblock. The PCR reaction was carried out for 35
amplification cycles (94°C for 30 s, 61°C for 45 s and 72°C for 90 s).
The RT-PCR product was cloned into pGEM-T vector (Promega)
and sequenced. Bioinformatic analyses were carried out using
DNASTAR software (DNASTAR, Inc., Madison, WI, USA).
DNA extraction and Southern analysis
Genomic DNA was isolated from green leaves of two-weeks-old
einkorn wheat according to the method described in molecular
cloning laboratory manual (Sambrook and Russell, 2001). For
Southern hybridization, genomic DNA was digested separately with
EcoRΙ and Hind III for 16 h and subjected to electrophoresis in 0.8%
(w/v) agarose for 16 h at 4 V cm -1 (20 µg per lane). After
depurination of DNA within the gel in 0.25 M HCl for 30 min at 25°C,
the DNA was transferred to a Hybond-N+ nylon membrane
(Amersham Bio- sciences, Little Chalfont, UK) in 20×SSC and
hybridized with the probe, which was labeled with [α- 32 P] dCTP by
Random Primer DNA Labeling kit(Takara), for 16 h at 65°C. The
blots were washed once in 0.1% SDS, 2×SSC for 15 min at 65°C
and twice in 0.1% SDS, 0.1×SSC for 15 min at 65°C
(high-stringency conditions), and exposed to X-ray film (Kodak).
Expression analysis by quantitative real-time RT-PCR
The expression of mRNA for TmGSK1 was examined by
quantitative real-time RT-PCR under several different abiotic
stresses on the Rotor-Gene 3000 system (Corbett Robotics).
RT-PCR was performed using the all-in-one qPCR kit
(Genecopoeia), following the manufacturers protocol. Primers were
designed by primer express 2.0 software and synthesized by Beijing
Sunbiotech Co., Ltd. A 189 bp product for TmGSK1 cDNA was
amplified using the following primers: T-forward 5'
GTTTGGTCTGCTGGCTGTGTTCTT 3' and T-reverse 5'
GTGCCATGGGTGAGCTTTGATTT 3'. A 118 bp product of einkorn
wheat housekeeping gene β-actin was amplified as an internal
control using the following primers: ACT-f 5'
TGGCACCCGAGGAGCACCCTG 3' and ACT-r 5' GCGACGTAC
ATGGCAGGAACA 3'. The thermal cycling profile consisted of initial
denaturation at 95°C for 3 min and 40 cycles at 95°C for 15 s, 60°C
for 15 s and 72°C for 30 s. The resultant amplification was purified
and sequenced, and showed 100% homology to target gene. To
confirm amplification specificity, the amplified fragments were
analyzed by 1.5% agarose gel electrophoresis containing ethidium
bromide. The specificity of PCR amplification was determined by
constructing a melting curve after the polymerase chain reaction
amplification, and negative controls containing RNase-free water
instead of sample was run to confirm that the samples were not
cross contaminated. Quantitation of relative expression was
determined by the 2 −∆(∆CT) method (Livak and Schmittgen, 2001).
Each sample was run in triplicate.
RESULTS AND DISCUSSION
cDNA clone and sequence analysis of the TmGSK1
protein
The TmGSK1 cDNA contained 1,543 bp with an open

Xian-Guang et al. 4067
Supplemental material 1. Alignment of sequence of GSKs predicted protein from T. monococcum L., T. aestivum, Z. mays,N. tabacum, M.
sativa and A. thaliana. The black shading indicates identical amino acids. Amino acid sequences were aligned with the MegAlign program
(DNAstar software (DNASTAR, Inc., Madison, WI, USA)) using the CLUSTALW method.
reading frame of 1,068 bp (start codon at 79 bp and stop
codon at 1146 bp) encoding a deduced protein of 355
amino acids (GenBank accession No. DQ443471). The
theoretical isoelectric point (PI) of the putative amino acid
sequence is 8.34 and molecular weight (MW) is 40.3 kDa.
The deduced amino acid sequences of TmGSK1 and
GSKs from T. aestivum (AF525086), Z. mays (AY722707),
Trifolium repens (X99100), N. tabacum (X77763),
Medicago sativa (X68409) and Arabidopsis thaliana
(BT000132) are aligned (Supplemental material 1). The
putative amino acid sequence of TmGSK1 showed high
homology to GSKs from T. aestivum (94.2%), Z. mays
(95.5%), T. repens (90.5%), N. tabacum (83.4%), M.
sativa (84.4%) and A. thaliana (73.2%). Phylogenetic
analysis of TmGSK1 is shown in Supplemental material 2.
Southern analysis of TmGSK1
Southern analysis of genomic DNA digested separately
with two different restriction enzymes, EcoR I and HindIII,
was performed to estimate the copy number of TmGSK1
in the einkorn wheat genome. As shown in Figure 1, the
banding pattern was produced with the TmGSK1
full-length probe (1,543 bp) and a high-stringency wash.
Only one hybridizing band in each digestion was
observed, indicating that the TmGSK1 sequence was
present as a single copy in the einkorn wheat genome.
Quantitative real-time RT-PCR of the TmGSK1 gene in
response to various abiotic stresses
The quantitative real-time RT-PCR analyses of TmGSK1
during various abiotic stresses are shown in Figure 2.
Constitutively expressed β-actin gene was used as an
internal control. TmGSK1 expression was upregulated in
these abiotic stresses. Accumulation of TmGSK1 mRNA
increased step by step under salt stress (Figure 2a). Its
expression was up to 1.5-folds when compared to the con-

4068 Afr. J. Biotechnol.
Supplemental material 2. The phylogenetic tree of predicted protein sequence of GSKs from T. monococcum L.,T. aestivum, Z.
mays, N. tabacum, M. sativa and A. thaliana.
trol when its was stressed 6 h with NaCl and reached its
maximum within 12 h after treatment; under wounding
treatment, the expression pattern of TmGSK1 was in-
duced earlier than that under NaCl treatment (Figure 2b).
Injury inducing its expression increased rapidly, up to
2-folds when it was injured just one hour. For abscissic
acid (ABA) treatment, TmGSK1 transcription was induced
similar to that under wounding. Its expression increased
3-folds after treated with ABA for 1 h (Figure 2c). For
drought treatment, it was 2.0 and 2.6 folds increase after
treated with PEG6000 at 6 and 12 h (Figure 2d); low
temperature (4°C) can induce its high expression too
(Figure 2e). Expression pattern analysis indicates the
possible involvement of TmGSK1 in salt, wounding, ABA,
drought and cold stress signal transduction pathways in
einkorn wheat.
To understand a mechanism of abiotic stress signal
transduction in plant, it is important to identify the
components involved in the pathway. In this study, we
Figure 1. Southern-blot analysis of TmGSK1. Left: Agrose
gel electrophoresis of the genomic DNA which was
completely digested with EcoR I (E) and Hind III (H); Right:
hybridization using the full-length sequence of AbGSK1 as
probe.
isolated the glycogen synthase kinase 3 (GSK3)/
SHAGGY-like kinases from T. monococcum L. Previously,
it has been shown that GSK3 are involved in a variety of
biological processes. Present evidence indicates that
plant GSKs are also involved in different processes such
as hormone signaling, NaCl stress and other abiotic
stresses. In accordance with these previous observations,
we found in the present study that TmGSK1 was also
involved in the response of abiotic stresses such as salt
stress, mechanical injury, abscissic acid hormone, cold
and drought stress. Interestingly, as shown in Figure 2,
the plants accumulate more TmGSK1 mRNA response to
all of these stresses. These means such stresses are
inter- connected and may induce similar cellular signal
transduction pathways. Moreover, these signal pathways
may cross-talk on TmGSK1 or TmGSK1 may integrate all
of these signal pathways in einkorn wheat. Stress signal
activate the glycogen synthase kinase through different
ways and substrates are phosphorylated and activity is

a
Stress time (h)
b
Stress time (h)
c
Stress time (h)
Figure 2. The relative mRNA level of TmGSK1 under salt stress treatment (a);
wounding treatment (b); ABA treatment (c); drought treatment (d); cold treatment
(e). The quantitative real-time RT-PCR analysis of TmGSK1 gene expression in T.
monococcum L in response to salt stress (1% NaCl), mechanical injury, ABA
hormone, drought and cold treatment (4°C) using constitutively expressed β-actin
gene as an internal control.
Xian-Guang et al. 4069

4070 Afr. J. Biotechnol.
Figure 2. Contd.
e
d
Stress time (h)
Stress time (h)
regulated. Then, stress responsive genes express and
start-up stress responsive mechanisms to re-establish
cellular homeostasis and protect and repair damaged
proteins and membranes. Therefore, TmGSK1 was
shown to be a positive regulator commonly involved in the
tolerance to salt, mechanical injury, ABA hormone, cold
and drought in einkorn wheat. However, most
mechanisms in this model are still unknown, and further
investigation should be carried out to indicate the wheat
plant abiotic stress responsive signal transduction in
details.
Acknowledgments
This work was supported by the National Nature Science
Foundation of China (NO.31000165, 31171182), the Key
Project of Chinese Ministry of Education (NO.209076) and
Foundation of science and technology research projects
of Henan Province in China (No. 092300410099,
102300413213).
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African Journal of Biotechnology Vol. 10(20), pp. 4072-4080, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2419
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Some quality traits and neurotoxin β-N-oxalyl-L-α,βdiaminopropionic
acid (β-ODAP) contents of Lathyrus
sp. cultivated in Turkey
Ugur Basaran 1 *, Ozlem Onal Asci 2 , Hanife Mut 1 , Zeki Acar 3 and Ilknur Ayan 3
1 Bozok University, Faculty of Agriculture, Department of Field Crops, Sivas Street 66200, Yozgat-Turkey.
2 Ordu University, Faculty of Agriculture, Department of Field Crops, Cumhuriyet Street, 52200, Ordu-Turkey.
3 Ondokuz Mayis University, Faculty of Agriculture, Department of Field Crops, 55139, Samsun-Turkey.
Accepted 25 March, 2011
In this study, 52 landraces accessions belonging to the species of Lathyrus sativus and Lathyrus
clymenum collected from different regions of Turkey and one released variety of Lathyrus sativus were
evaluated for some quality traits like seed coat color, 1000 seed weight, crude protein and neurotoxin β-
N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) content. Among the investigated landraces, high
variation was determined for all the investigated traits, which was attributed to both genetic as well as
environmental factors. Protein content was as between 24.07 to 30.90% and β-ODAP content was as
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
content seem to be promising material for Lathyrus breeding. Furthermore, many investigated
landraces showed lower β-ODAP and higher protein contents compared with the released variety. Also,
the local consumption of L. sativus landraces as a food or feed was determined during the collection
process.
Key words: Lathyrus, landrace, protein, β-ODAP, Turkey.
INTRODUCTION
Lathyrus sativus L. (grass pea, in Turkish “murdumuk,
culban, fasil, feslek”) is a traditional crop use for both
animal consumption as forage and grain and for human
consumption as a pulse. L. sativus has many advantages
including drought and flooding tolerance, disease
resistance, high yield potential and favorable nutritional
composition of seeds. It can easily grow in different soil
types and at high altitudes (Tivari and Campbell, 1996a,
b). L. sativus grows with as little as 250 mm of annual
precipitation and it is typically the last surviving plant in
drought times (Tekele-Haimanot et al., 1990; White et al.,
2002).
Besides the obvious advantages, L. sativus seeds
contain a major anti-nutritional compound namely β-
ODAP (Zhao et al., 1999; Wang et al., 2000). Heavy and
prolonged (3 to 4 month) consumption of L. sativus seeds
*Corresponding author. E-mail: ugurb@omu.edu.tr. Tel: (+90
354) 212 70 01. Fax: (+90 354) 212 27 89.
can cause a disease known as ‘lathyrism’ or ‘neurolathyrism’,
a disease causing paralysis of the limbs
(Spencer et al., 1986; Yan et al., 2006).
Until recently, relatively little effort has been made
towards the improvement of this hardy pulse crop due to
its toxicity. To counter the effects of climate change on
agricultural productivity, L. sativus is gaining interest in
southern Australian Mediterranean-type environments
(Hanbury et al., 1999), North America (Campbell et al.,
1994) and Southern Europe (Crino et al., 2004; Tavoletti
et al., 2005; Polignano et al., 2009). Also, many studies
have been carried out to bring grass pea in areas under
monoculture cereal cultivation in Europe (Vaz Patto et al.,
2006).
In order to reintroduce L. sativus in crop rotations under
marginal environments, breeding programs are in progress
worldwide to improve genotypes combining high
yield, high protein content and low or zeroneurotoxin
content (ODAP) (Bozzini, 1997; Siddique et al., 1998;
Hanbury et al., 2000).
Lathyrus cultivation was not well documented in

Basaran et al. 4073
Figure 1. Distribution of the collection area in Turkey. 02, Adiyaman; 15, Burdur; 16, Bursa; 18, Cankiri; 20, Denizli; 23, Elazig; 43,
Kutahya, 44, Malatya; 48, Mugla; 50, Nevsehir; 55, Samsun; 64, Usak.
Turkey. However, some authors reported that, L. sativus,
Lathyrus cicera, Lathyrus clymenum and Lathyrus ochrus
were cultivated in Turkey (Genc and Sahin, 2001; Cetin,
2006) and L. sativus was widely cultivated in the country
in the past (Davis, 1970). In addition, Lathyrus hirsutus
had been cultivated in the east and inner parts of Turkey
until 1960s (Tosun, 1974). Lathyrus cultivation dramatically
decreased from the 1960s. Today, based on actual
data, only L. sativus is cultivated in 18.000 ha for the
seed in Turkey (TUIK, 2008). This crop is mainly used as
stock-feed; however, it is rarely used for human
cunsumption in some parts of the country (Basaran et al.,
2010).
There is only one released variety of L. sativus,
‘Gürbüz 2001’ in Turkey. This means, approximately all
the cultivated genotypes are landraces; hence, they are
promising materials for breeding programs. Most of these
landraces were not previously screened. However,
Karadag et al. (2010) indicated that, it had a high
variability in β-ODAP levels in winter and spring-sown
grasspea lines. On the other hand, interestingly, there is
no lathyrism case documented for both humans and
animals in Turkey which may result from landraces with
low β-ODAP content. So, the screening of these landraces
regarding their β-ODAP content is very important.
For this purpose, expeditions were carried out to collect
Lathyrus landraces cultivated in Turkey and to evaluate
their quality traits including the protein and β-ODAP
contents. Also, this study provided comprehensive and
additional information about the general status of
Lathyrus cultivation in Turkey.
MATERIALS AND METHODS
In this study, a total of 52 local landrace accessions of Lathyrus (51
L. sativus and 1 L. clymenum) collected from Turkey were
examined. To collect Lathyrus accessions, initially 12 cities in which
Lathyrus are cultivated were determined based on data of the
Turkish Statistical Institute (TUIK, 2007) and Ministry of Agriculture
records. The collection program was carried out in 2007. The
collection area is given in Figure 1.
Lathyrus accessions were taken from different farmers or local
seed markets and their name and altitude of collection cites were
recorded. Three landraces of the collected L. sativus genotypes are
commercial material. In addition, the released variety ‘Gürbüz-2001’
was obtained from Turkish Central Research Institutes for Field
Crops. Seed coat color, 1000 seed weight, protein and β-ODAP
contents were determined for each sample.
Seed coat color was determined by visual observation. Total
nitrogen was determined by the Kjeldahl method and crude protein
content was estimated using a conversion factor of 6.25 (Kacar,
1972). All the data were presented as mean, mimimum, maximum
and standard deviation determined by using SPSS 13.0 statistical
package program.
Determination of β-ODAP
β-ODAP was determined using capillary zone electrophoresis
(CZE) (Zhao et al., 1999) with some modifications. CZE was
carried out using Agilent HP 3D and UV detection at 195 nm. The

4074 Afr. J. Biotechnol.
capillar was 55 cm (44 cm effective length) x 50 µm. The analyses
were performed at a constant voltage of 20 kV at 20°C in an
electrolyte of 75 mM (H3BO3) buffer at pH 7.5. The required pH of
the buffer was adjusted by adding NaOH. All chemicals were of
analytical-reagent grade. The reference standard of β-ODAP was
obtained from Dr. S.L.N.Rao (Lathyrus Technologies, Hyderabad,
India).
Sample extraction
0.5 g powder of L. sativus seeds was soaked in 50 ml ethanol-water
(30:70, v/v) solution and was shaken for 2 h (in ice). After
centrifugation (3500 rpm at 15 min), the upper clear solution was
filtered with 0.45 µm filter paper. Then clear solution was diluted
with ultra distilled water (1:1) and was injected directly into the CZE
system for 40 s at 50 mbar.
RESULTS AND DISCUSSION
There was very limited information about the distribution
and density of Lathyrus cultivation in previous years. But,
according to the old farmer’s statements, Lathyrus cultivation
was more widespread in the past than now and a
dramatically decrease begun in the 1960’s. During the
collection expedition, only landraces of L. sativus and L.
clymenum species were collected. But L. cicera, L.
ochrus and L. hirsutus were not encountered; their
cultivation was reported by different authors (Tosun,
1974; Genc and Sahin, 2001; Cetin, 2006). The absence
of L. cicera and L. ochrus among the encountered species
in the study clearly indicated that, the cultivated
Lathyrus materials had been exposed to genetic erosion
in the last 50 years and a lot of genetic materials
disappeared during the period.
The collection sites and local names of Lathyrus
landraces throughout the country are presented in Table
1. Seed trade for both L. sativus and L. clymenum was
very limited and also, there was only one released variety
named ‘Gürbüz-2001’ in Turkey. So, farmers usually sow
their own seed produced in previous years.
In general, L. sativus is cultivated at high altitudes (600
to 1600 m). The common name of Lathyrus species is
“murdumuk” in Turkey. Moreover, Lathyrus is known by
many local names such as “culban, coluk, fasil feslek”
and L. clymenum is known as “kara murdumuk” (Table
1).
L. sativus is mainly cultivated for its seed and it is used
as feed (Table 2). In some cities, however, its grain has
been rarely consumed by humans as a soup, pilaf or
snack (by mixing with chickpea). L. clymenum is
cultivated only in Mugla city in small field for household
consumption and its seed is used to make soup and it is
added in the traditional food named “stuffed zucchini
flowers”.
According to farmers, L. sativus has higher seed yield
than other legumes especially in dry condition and do not
shatter its pods. So, farmers prefer to harvest the plant
for seed production than for forage. For animal feeding,
seeds are used by soaking in water or grinding and
mixing with cereals in Turkey.
Seed coat colors amongst the landraces of L. sativus
examined in the study were grey, brown, creamy and
mostly mixture of these color. Creamy seed coat color
was observed in few landraces in the mixture. However,
landrace 5501 used only for human consumption had
creamy seed coat color alone. The color variation in a
single landrace can be related to genetic variability. So,
besides inter population variation, intra population genetic
variation was as high as possible, which is a desired
feature in plant breeding. On the other hand, genotypes
with light cream color seed had low neurotoxin content
(Dahiya, 1976). It was reported that, seed coat color
could be a useful visual character to select low toxic lines
(Campbell, 1997). However, Kaul et al. (1986), did not
find any relationship between seed coat color and toxic
contents. In this study, certain relationship between seed
color and β-ODAP contents was not determined. Many
landraces with grey or brown seed coat color had low β-
ODAP than creamy seed’s landraces. Moreover, the
6406 numbered landraces, which had low toxicity, had
grey and brown seed color (Table 2).
1000 seeds weight changed from 72.2 g in landrace
numbered 2303 to 148.0 g in landrace numbered 5501
(Table 2) with mean 100.2 g (Figure 2). According to
farmers’ experience, they had not observed pod shattering
in these landraces. L. sativus showed high
variations (41 to 510 g) regarding 1000 seed weight
(Joshi, 1997; Rybinski et al., 2008). Larger-seeded types
are mostly found around the Mediterranean region, while
small-seeded types are found in Indian subcontinent. In
addition, small-seeded types have a tendency for pod
shattering (Campbell, 1997). So, in general, the cultivated
L. sativus landraces in Turkey showed similar features
with the Mediterranean genotypes.
Crude protein (CP) contents of the landraces varied
from 24.07 to 30.90% for L. sativus and it was 26.33% for
L. clymenum. CP content in the released variety was
25.81% (Table 2.) and mean CP content was 28.23% for
all the landraces (Figure 3). Variation in the seed CP
contents can be attributed more to ecological factors
other than genetic variation due to the ecological differences
of the collection areas. But, Lathyrus is mostly
cultured on poor or marginal lands with no fertilization in
Turkey. So, these hard growing conditions determined
the high CP ratios (28.23% as a mean) which indicated
that many of the collected landraces may display genetic
ability for high protein content. The seed CP ratios
obtained in this study are similar to the results of other
authors (Rotter et al., 1991; Rosa et al., 2000; Urga et al.,
2005; Karadag et al., 2009; Karadag and Yavuz, 2010)
who reported protein content for L. sativus seed between
21.49 and 31.98%. But our results were lower than the
results of Abd El Zaher et al. (2007) who reported that,
seed protein was between 29.17 and 37.33% in L.
sativus and between 36.11 and 36.72% in L. clymenum.

Table 1. Collection sities and local name of accessions of Lathyrus sp.
Basaran et al. 4075
Number Accession** City Village/district Local name Altitude (m)
1
2
LS-0201
LS-0202
Adiyaman
Dardogan/ Merkez
Büklum/ Merkez
Culban/ Coluk
Culban/ Coluk
825
787
3
4
LS-1501
LS-1502
Burdur
Kizilkaya/ Bucak
Harmanli/ Yesilova
Murdumuk
Murdumuk
787
1093
5 LS-1503 Kizilkaya/ Bucak Murdumuk 787
6 LS-1601
Dogancılar/ Harmancık Murdumuk 781
7
8
LS-1602
LS-1603
Bursa
Dogancılar/ Harmancık
Demirciler/ Harmancık
Murdumuk
Murdumuk
781
719
9 LS-1604* Commercial Murdumuk -
10 LS-1801
Yukaripelitozu/ Merkez Murdumuk 768
11 LS-1802 Cankiri Yukaripelitozu/ Merkez Murdumuk 768
12 LS-1803 Elmaci / Eldivan Murdumuk 957
13 LS-2001
Uzunpinar/ Merkez Murdumuk/Fasil 1148
14 LS-2002 Cabar/ Civril Murdumuk/Fasil 1013
15 LS-2003 Ulukent/ Tavas Murdumuk/Fasil 934
16
17
LS-2004
LS-2005
Denizli
Balkica/ Tavas
Eziler/ Guney
Murdumuk/Fasil
Murdumuk/Fasil
1150
841
18 LS-2006 Baklancakirlar/ Cal Murdumuk/Fasil 886
19 LS-2007 Alaaddin/ Acipayam Murdumuk/Fasil 887
20 LS-2008 Karahoyukavsari/ Acipayam Murdumuk/Fasil 900
21 LS-2301
Uzuntarla/ Merkez Culban 995
22 LS-2302 Elazıg Acipayam/ Merkez Culban 987
23 LS-2303* Commercial Culban -
24 LS-4301 Kutahya -/ Domonic Murdumuk 880
25 LS-4401
Yenikoy/ Darende Culban 1600
26
27
LS-4402
LS-4403
Malatya
Yenikoy/ Darende
Basdirek/ Darende
Culban
Culban
1600
1445
28 LS-4404* Commercial Culban -
29 LS-5001
Kalecik/ Kozakli Murdumuk 1120
30 LS-5002 Sadik/ Hacibektas Murdumuk 1159
31 LS-5003 Sahinler/ Gulsehir Murdumuk 880
32 LS-5004 Nevsehir Hacıhalilli/ Gulsehir Murdumuk 1000
33 LS-5005 Cullar/ Acigol Murdumuk 1086
34 LS-5006 Tatlarin/ Aciöl Murdumuk 1113
35 LS-5501 Degirmenci/ Kavak Feslek 600
36 LS-6401
Hacim/ Sivasli Murdumuk/Fasil 938
37 LS-6402 Kasbelen/ Merkez Murdumuk/Fasil 960
38 LS-6403 Kasbelen/ Merkez Murdumuk/Fasil 960
39 LS-6404 Kasbelen/ Merkez Murdumuk/Fasil 960
40 LS-6405 Inay/ Ulubey Murdumuk/Fasil 743
41 LS-6406 Inay/ Ulubey Murdumuk/Fasil 743
42 LS-6407 Inay/ Ulubey Murdumuk/Fasil 743
43
44
LS-6408
LS-6409
Usak
Kisla/ Ulubey
Kisla/ Ulubey
Murdumuk/Fasil
Murdumuk/Fasil
800
800
45 LS-6410 Kisla/ Ulubey Murdumuk/Fasil 800
46 LS-6411 Karacaahmet/ Ulubey Murdumuk/Fasil 780
47 LS-6412 Ilyasli/ Merkez Murdumuk/Fasil 770
48 LS-6413 Ilyasli/ Merkez Murdumuk/Fasil 770
49 LS-6414 Ilyasli/ Merkez Murdumuk/Fasil 770
50 LS-6415 Ilyasli/ Merkez Murdumuk/Fasil 770
51 LS-6416 Ilyasli/ Merkez Murdumuk/Fasil 770
52 LS Released variety ‘Gurbuz-2001’ -
53 LC-4801 Mugla Cumalı- /Datça Kara murdumuk ?
*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.
Table 2. Local use, morphological and chemical features of accessions of Lathyrus sp.
Accession* Local Use Seed coat color*** 1000 seed weight (g) Crude protein (%) β-ODAP (mg g -1 )
LS-0201 Feed G+B 92.0 30.90 1.90
LS-0202 Feed G+B 84.9 28.03 1.66
LS-1501 Feed+food G 110.6 30.04 2.19
LS-1502 Feed+food G+B 86.4 29.00 1.75
LS-1503 Feed+food G+B 94.4 30.09 3.24
LS-1601 Feed+food G+B+C 102.3 30.57 1.68
LS-1602 Feed+food C+B 134.0 28.00 2.32
LS-1603 Feed+food G+B 107.3 28.61 2.38
LS-1604 Feed G+B 99.6 27.61 2.62
LS-1801 Feed G+B 97.8 27.98 1.83
LS-1802 Feed G+B 122.6 27.50 2.06
LS-1803 Feed B 104.7 28.50 3.86
LS-2001 Feed G+B 76.3 29.34 2.30
LS-2002 Feed G+B 80.6 30.10 2.07
LS-2003 Feed G+B 121.5 26.96 2.02
LS-2004 Feed G+B 115.0 26.67 3.33
LS-2005 Feed G+B 77.2 27.00 3.84
LS-2006 Feed B 73.3 26.23 1.74
LS-2007 Feed G+B 90.5 27.86 2.26
LS-2008 Feed G+B 101.5 27.18 1.71
LS-2301 Feed B 106.3 29.27 1.74
LS-2302 Feed B 72.2 27.34 3.15
LS-2303 Feed G+B 89.94 27.80 2.25
LS-4301 Feed G+B+C 118.6 30.41 1.81
LS-4401 Feed G+B 80.8 30.33 1.41
LS-4402 Feed G+B 101.8 28.62 1.99
LS-4403 Feed B 103.3 28.16 2.02
LS-4404 Feed G 129.0 27.02 2.67
LS-5001 Feed G+B 106.5 28.55 2.18
LS-5002 Feed G+B 102.2 28.69 2.20
LS-5003 Feed G+B 112.2 27.99 2.00
LS-5004 Feed G+B 103.4 27.98 2.07
LS-5005 Feed G+B 140.8 26.23 2.93
LS-5006 Feed B 113.6 28.80 2.06
LS-5501 Food C 148.0 24.07 1.80
LS-6401 Feed G+B 88.3 28.77 1.54
LS-6402 Feed G+B 95.4 28.21 1.42
LS-6403 Feed G+B 107.3 27.91 1.89
LS-6404 Feed G+B 92.2 27.63 1.85
LS-6405 Feed G+B 108.9 29.19 1.72
LS-6406 Feed G+B 75.3 29.78 1.35
LS-6407 Feed G+B 107.5 28.60 2.42
LS-6408 Feed G+B 79.7 28.32 2.59
LS-6409 Feed G+B 76.8 26.60 2.36
LS-6410 Feed G+B 104.0 27.63 2.32
LS-6411 Feed G 85.4 27.78 1.91
LS-6412 Feed G+B 93.4 28.09 1.71
LS-6413 Feed G+B 97.1 27.61 2.38
LS-6414 Feed G+B 85.0 29.70 2.65
LS-6415 Feed G+B 90.3 29.51 1.69
LS-6416 Feed G+B 86.7 29.15 2.90
LS** Feed B 117.8 25.81 2.50
LC-4801 Food B 118.8 26.33 2.68
* LS, L. sativus; LC, L. Clymenum; ** released variety; *** G, grey; B, brown; C, creamy.

Frequency
14
12
10
8
6
4
2
0
60.00 80.00 100.00 120.00 140.00 160.00
1000 seed weight (g)
Figure 2. General distribution of Lathyrus landraces for 1000 seed weight.
β-ODAP levels of the samples were between 1.35 and
3.86 mg g -1 (Table 2) with a mean of 2.21 mg g -1 (Figure
4.) The highest toxic level was determined in the landrace
collected from Cankiri numbered 1803, while the lowest
level was obtained in the landrace collected from Usak
numbered 6406 (Table 2). The β-ODAP concentration in
the released variety named ‘Gurbuz- 2001’ was higher
than the mean of all the samples (2.50 mg g -1 ). Low toxic
levels generally, were in landraces belonging to L. sativus
and β-ODAP content was determined as 2.68 mg g -1 in L
clymenum seed (Table 2).
β-ODAP concentration of L. sativus seeds varied between
0.2 to 7.2 mg g -1 in dry matter (Deshpande and
Campbell, 1992). For safe consumption by humans, β-
ODAP content of seeds has to be lower than 2.2 mg g -1
(Abd El Moneim et al., 1999). In the present work, β-
ODAP was lower than 2.00 mg g -1 in 21 L. sativus
landraces (Figure 3), indicating the importance of land-
Mean = 100.2083
Std. Dev. = 17.31015
N = 53
Basaran et al. 4077
races in screening for low toxic content.
There has been no lathyrism case documented in
human or animals up till now in Turkey. Also, farmers did
not know anything about lathyrism or any toxic effect of
Lathyrus seeds on human or animals. In fact, lathyrism
risk is very low in Turkey. This is probablly due to low and
rare consumption, soaking procedure and low β-ODAP
content, because, the grain of Lathyrus sativus is rarely
used in human diet for stock feeding in the country.
Neurotoxic β-ODAP is a water-soluble amino acid that
can be leached from seed by soaking in water (Akalu et
al., 1998). On the other hand, Lahyrism is induced by
heavy and prolonged (more than three months) consumption
of the L. sativus seeds (Mehta et al., 1994).
Genotype is the most important factor determining
ODAP concentration; environment has less influence
(Hanbury et al., 1999). However, environmental factors
such as drought, zinc deficiency, iron oversupply and the

4078 Afr. J. Biotechnol.
Frequency
20
15
10
5
0
24.00
25.00 26.00 27.00 28.00 29.00 30.00 31.00
Protein (%)
Figure 3. General distribution of Lathyrus landraces for seed protein contents.
Frequency
10
8
6
4
2
0
1.00
B-ODAP (mg/g)
Mean = 2.206
Std. Dev. = 0.56494
N = 53
1.50 2.00 2.50 3.00 3.50 4.00
Figure 4. General distribution of Lathyrus landraces for seed β-ODAP contents.

presence of heavy metals in soil can considerably
increase the level of β-ODAP in seeds (Lambein et al.,
2007). So, the development of varieties for a specific
region is one of the effective solutions to avoid lathyrism.
At this point, local landraces will be usefull. Analysis of a
large number of germplasm of L. sativus showed that,
samples originated from Africa, Syria, Turkey and Cyprus
had significantly lower content in the dry seed than in the
samples originated from Bangladesh, Ethiopia, India,
Nepal and Pakistan (Abd El Moneim et al., 2001). Therefore,
the variation in this study on β-ODAP level was
attributed both to genetic factors and environmental
conditions. For this reason, there are needs for new
studies, to determine the β-ODAP levels of these landraces
in different ecological conditions.
L. sativus, which is a drought tolerant legume, is a
promising crop for the diversification of cropping systems
and sustainability of agriculture in marginal and drought
areas. Furthermore, under the risks of climate change
which is supposed to cause serious drought, the plant will
become a more important crop in the world. So, future
studies should concentrate on developing new varieties
with high yield, low β-ODAP and high protein level. The
results of this showed that, L. sativus landraces cultivated
in Turkey, are promising genetic materials especially with
their low β-ODAP contents.
ACKNOWLEDGEMENTS
The authors’ thanks go to Prof. Dr. A. Nur ONAR and Dr.
Meral KARACAN of OMU, Science and Art Faculty,
Chemistry Department, for their help in the CZE analysis.
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African Journal of Biotechnology Vol. 10(20), pp. 4081-4085, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2427
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Optimization of a plant regeneration protocol for
broccoli
Ke Huang 1,2,3*,# , Qiuyun Wu 1,2# , Juncheng Lin 3 and Jingui Zheng 3
1 Hunan provincial key laboratory for germplasm innovation and utilization of crop, Changsha, 410128, P. R. China.
2 College of Horticulture and Landscape, Hunan Agricultural University, Changsha, 410128, P. R. China.
3 Institute of Agricultural Product Quality, Fujian Agricultural and Forestry University, Fuzhou, 350002, P. R. China.
Accepted 18 March, 2011
The factors which influence the regeneration of broccoli (Brassica oleracea var. italica) were studied
using an orthogonal design. The results showed that the major factor was the explant type, followed by
naphthylacetic acid (NAA), benzylaminopurine (BAP), sucrose and AgNO3 in turn. The maximum
regeneration was on Murashige and Skoog (MS) medium + NAA 0.107 µM + BAP 17.76 µM + 2% sucrose
+ 0.8% agar. Hypocotyl proved to be the optimum explant source and its regeneration frequency
reached 100%.
Key words: Naphthylacetic acid (NAA), benzylaminopurine (BAP), AgNO3, hypocotyl, broccoli (Brassica
oleracea var. italica).
INTRODUCTION
Broccoli (Brassica oleracea L. var. italica Plenck), which
originated from Italy and is related to the cabbage and
cauliflower, is a very important vegetable crop. It is well
known for its high vitamin, calcium and sulforaphane
content (Zhang et al., 1992; Henzi et al., 2000). Plant
diseases, insects and other stressors can cause enormous
yield reductions during commercial broccoli
cultivation (Yang et al., 2002; Cao and Earle, 2003;
Viswakarma et al., 2004) but, under modern production
systems, genetic engineering can be used to add target
characteristics to broccoli cultivars. Transgenic B. oleracea
plants have been obtained using several methods
involving Agrobacterium-mediated transformation (Boulter
et al., 1990; Toriyama et al., 1991; Berthomieu and
Jouanin, 1992; Christey and Sinclair, 1992; Metz et al.,
1995; Christey et al., 1997; Higgins et al., 2006; Jocelyn
et al., 2007). The efficiency of such Agrobacterium-
*Corresponding author. E-mail: huangke@yahoo.cn Tel.:86-
731-84617028, Fax: 86-731-84617181.
Abbreviations: MS, Murashige and Skoog; BAP,
benzylaminopurine; NAA, naphthylacetic acid.
#Both are Co-authors.
mediated transformation is known to be affected by
multiple factors such as the bacterial strain and
concentration used, the plant genotype, explant type and
co-cultural conditions (van Wordragen and Dons, 1992;
Puddephat et al., 1996; Birch 1997; Cogan et al., 2002;
Chakrabarty et al., 2002; Kim and Botellam, 2002; Nigel
et al., 2002; Suri et al., 2005). Most authors have
reported difficulties with one or more of the stages in the
gene transfer and regeneration process which prevent
any of their protocols from serving as the method of
choice for B. oleracea transformation. Transformation
efficiency can however, be increased by manipulating
many factors, and the important factors are the explant
and the plant growth regulator.
There have been many reports on plant regeneration of
Brassica crops, but they studied the factors that affect
plant regeneration separately, such as explant type (Dai
et al., 2009), plant regulator concentration (Dai et al.,
2009; Ravanfar et al., 2009) and sucrose concentration.
This study reports the optimization of a system for
broccoli regeneration by orthogonal design. The
improved regeneration was achieved by systematically
optimizing the combination of factors that affect plant
regeneration using an orthogonal design (Tang and
Feng, 1997) in Catharanthus Roseus (Sun et al., 2002),
white clover (Zhang et al., 1998) and so on. Together,

4082 Afr. J. Biotechnol.
Table 1. Orthogonal design table.
Medium
no.
Level code NAA (µM) BAP (µM) AgNO3 (mM) Sucrose (%) Explant
1 0 0 0 1 Cotyledon
2 0.054 8.88 0.02 2 Cotyledon plus petiole
3 0.107 17.76 0.04 3 Petiole
4 0.161 26.64 0.06 4 Hypocotyl
Table 2. Broccoli regeneration frequency results*.
NAA
(µM)
BAP
(µM)
AgNO3
(mM)
Sucrose
(%)
Explant
Mean number of
shoots per explant
Regeneration
frequency (%)
1 1(0) 1(0) 1(0) 1(1) 1 (Cotyledon) 1.4±0.54 6±1.58 K
2 1(0) 2(8.88) 2(0.02) 2(2) 2 (Cotyledon plus petiole) 2.0±0.71 22±3.53 F
3 1(0) 3(17.76) 3(0.04) 3(3) 3 (Petiole) 1.6±0.54 9.33±1.78 IJ
4 1(0) 4(26.64) 4(0.06) 4(4) 4 (Hypocotyl) 5.8±1.48 92.22±0.84 B
5 2(0.054) 1(0) 2(0.02) 3(3) 4 (Hypocotyl) 3.6±0.89 72.89±2.28 D
6 2(0.054) 2(8.88) 1(0) 4(4) 3 (Petiole) 2.0±1.22 13.78±1.10 H
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
8 2(0.054) 4(26.64) 3(0.04) 2(2) 1 (Cotyledon) 0.0±0.00 0±0.00 L
9 3(0.107) 1(0) 3(0.04) 4(4) 2 (Cotyledon plus petiole) 2.0±1.00 9.11±1.22 IJ
10 3(0.107) 2(8.88) 4(0.06) 3(3) 1 (Cotyledon) 2.2±1.30 11.78±1.48 HI
11 3(0.107) 3(17.76) 1(0) 2(2) 4 (Hypocotyl) 6.4±1.14 100±0.00 A
12 3(0.107) 4(26.64) 2(0.02) 1(1) 3 (Petiole) 1.8±1.10 8.67±1.92 JK
13 4(0.161) 1(0) 4(0.06) 2(2) 3 (Petiole) 2.4±0.89 16.89 ±2.59 G
14 4(0.161) 2(8.88) 3(0.04) 1(1) 4 (Hypocotyl) 3.8±0.84 86.67±0.89 C
15 4(0.161) 3(17.76) 2(0.02) 4(4) 1 (cotyledon) 2.4±1.14 8.89±1.64 IJK
16 4(0.161) 4(26.64) 1(0) 3(3) 2 (Cotyledon plus petiole) 2.8±1.30 28.89±1.87 E
*The regeneration frequency was recorded 21 d after inoculated, 450 explants were used for the experiment, A-F expresses differences at the
level ofα=0.01 level.
these modifications resulted in a notable improvement in
the regeneration rate of the species.
MATERIALS AND METHODS
Broccoli (B. oleracea L. var. italica Plenck) cv Xinlv was provided by
the Jiangsu Academy of Agricultural Sciences, Nanjing. Seeds,
surface sterilized in 70% ethanol for 90 s, 0.1% HgCl2 for 12 min,
were germinated at 25°C on solid MS medium (Murashige and
Skoog, 1962) containing 2% sucrose (w/v) (pH 5.8). A 16 h
photoperiod was used.
Medium constituents and culture conditions
All media used in this experiment were of MS type with 0.8% agar
plus different concentrations of benzylaminopurine (BAP: 0, 8.88,
17.76 and 26.64 µM) and naphthylacetic acid (NAA: 0, 0.054, 0.107
and 0.161 µM). The medium with a pH adjusted to 5.8 was
dispensed into 100 ml Erlenmeyer flasks before autoclaving at
121°C and 1.2 ~ 1.3 kgcm -2 for 20 min, and silver nitrate (AgNO3: 0,
0.02, 0.04 and 0.06 mM) was added after being filter sterilized. The
medium was dispensed into 11 cm Petri dishes. The culture
conditions were 25 ± 1°C and a 16 h photoperiod of approximately
28 µEm -2 s -1 . The rooting medium was composed of MS + 0.107 µM
NAA + 2% sucrose + 0.8% agar, at pH 5.8.
Shoot and root induction
Cotyledon, cotyledon with petiole attached, petiole and hypocotyl
explants were isolated from 7 days old germinated seedlings and
cultured on regeneration medium for adventitious bud induction
(Table 1), the orthogonal combination scheme is provided in Table
2. Five replications and 90 explants per replicate was used for each
treatment, and data variance analysis by DPS was used (Tang et
al., 1997).
Explants were inoculated on differentiation regeneration media
for adventitious bud induction. Adventitious buds were then cut and
transplanted onto root induction media. The root induction medium
composed of MS + NAA (0, 0.54 and 1.07 µM) + 3% sucrose +
0.8% agar, and the regenerated plants were transplanted onto
vermiculite medium after 2 weeks of root induction.
RESULTS
By examining the combination of factors that affected
broccoli regeneration using an orthogonal design and by

Huang et al. 4083
Figure 1. The regeneration of broccoli hypocotyle from cv ‘Xinlv’ (B. oleracea var. italica cv ‘Xinlv’). A: Calli formation (7 days); B:
formation of the regenerated bud (21 days); C: further growth of a regenerated bud (42 days); D: root formation in the regenerant
(56 days); E: The transplantation of a regenerated broccoli seedling (90 days); F: flower head formation (150 days).
the use of data variance analysis by DPS (Tang et al.
1997), it was apparent that the most suitable condition for
Xinlv was: MS + NAA 0.107 µM + BAP 17.76 µM + 2%
sucrose + 0.8% agar. Hypocotyls were the best explant
source (Fexplants = 1161.897, F0.01 = 5.29) with a regeneration
frequency of 100% (Table 2 and Figure 1).
The effect of plant growth regulator on broccoli
regeneration
The plant growth regulator kind and concentration have
major effect on the broccoli callus abduction and differentiation.
The results indicated that the different NAA and
BAP level have highly significant difference on broccoli
regeneration (FNAA = 18.8723, FBAP = 8.3232, F0.01 = 5.29),
and 0.107 µM NAA, 17.76 µM BAP is the best for the
broccoli regeneration.
The effect of silver nitrate on broccoli regeneration
Broccoli regeneration is not sensentive on the silver
nitrate, the broccoli adventitious bud regenerated better
while there is no silver nitrate in the medium than the
medium with silver nitrate. So for the broccoli regeneration
medium, the silver nitrate should not be added.
The effect of sucrose concentration on broccoli
regeneration
There is highly significant difference level of different
sucrose concentration on broccoli regeneration (Fsucrose =
6.45344, F0.01 = 5.29), and the 2% sucrose is the best
concentration for broccoli regeneration.
The effect of explant type on broccoli regeneration
We selected the cotyledon plus petiole, hypocotyls,
cotyledon and petiole as the adventitious bud induction
explants. The different explant type has highly significant
difference in the broccoli adventitious bud induction
(Fexplant = 1081.924, F0.01 = 5.29).
Regenerated buds were placed on rooting medium and
that which proved optimum for rooting was MS + 1.07 µM
NAA + 3% sucrose + 0.8% agar with a rooting success
rate of 100%. All roots produced on this media were
rosettes, whilst buds failed to develop on any other media.

4084 Afr. J. Biotechnol.
DISCUSSION
The establishment of a regeneration system is a major
step in the development of transgenic technology for
Brassica vegetables. This paper defined such a system
for B. oleracea L. var. italica Plenck using selection based
on the optimum explants type, plant growth regulators,
sucrose and AgNO3 concentration which proved capable
of achieving 100% regeneration from hypocotyl. The kind
and concentration of plant growth regulators, sucrose
concentration and the explant type all affected the
regeneration of broccoli, while AgNO3 also had some
influence on the successfof regeneration percentage. The
experiment validates the conclusions reached using other
broccoli varieties in which the regeneration frequency
was also 100% and suggests that the regeneration system
is applicable to a range of broccoli varieties (Figure
1). The kind and concentration of plant growth regulators
also have a strong effect on broccoli callus induction and
differentiation (He et al., 1998; Zhang and Gong, 2001;
Zhao et al, 2002; Ravanfar et al., 2009; Dai et al., 2009;
Ravanfar et al., 2009). Significant effects of NAA and
BAP levels on regeneration (FNAA = 23.2379, FBAP =
10.0031, F0.01 = 5.29) indicate that both affect
regeneration frequencies. Ravanfar et al., (2009)
indicated that 96.67% of hypocotyl explant produced
shoot on 3 mg·L - ¹ (13.32 µM) BAP, this study has
presented 100% regeneration at 4 mg·L -1 (17.76 µM),
because there is a combination relationship among the
NAA, BAP, AgNO3 and the explants. We studied the
factors systematically, and the results of regeneration
frequency (100%) and mean number of shoots per
explant (6.4) indicated that this is the best recipe for
broccoli regeneration in this paper.
The results indicated that the medium lacking AgNO3
was optimum for broccoli regeneration (FAgNO3 = 22.1231,
F0.01 = 5.29). It may therefore be appropriate to tailor the
AgNO3 concentration in the medium according to the
plant genotype (Chi et al, 1990). The sucrose
concentration was found to significantly affect the degree
of regeneration (Fsucrose = 8.1219, F0.01 = 5.29). Several
reports have described variety-specific differences in in
vitro organogeny or embryogenic development in
response to contrasting sucrose concentrations (He et
al., 1998; Kantia and Kothari, 2002; Tang et al., 2002).
Sucrose concentration has an effect on adventitious bud
induction and the amount of cluster buds. The
differentiation ability and plant growth regulator level of
explants depend upon the position and timing of excision,
which also affect the regeneration frequency (Murata and
orton, 1987). Hypo-cotyls, cotyledons, petioles and
cotyledons in combi-nation with their petioles have been
used as explants in this study. Some of these explants
have a high regeneration frequency and are easily
infected by Agro-bacterium, making them good transform
explants. Cotyledons plus petioles are usually chosen as
explants during Brassica regeneration because, when cut
from a growth point, they have some primordial meriste-
matic cells and possess higher regeneration frequencies
(Birch, 1997; Kim and Botellam, 2002; Murata and Orton,
1987; Puddephant et al., 1996). It was apparent that
hypocotyls were the best explants for broccoli
regeneration in this study as they exhibited higher
differentiation ability and the plant growth regulator
combination was suitable for bud induction. It has been
proposed that polarity occurs during Brassica hypocotyl
regeneration but, in agreement with Li and Chen (1993;
1994), this was not observed in the present experiment
using B. oleracea L. var. italica Plenck.
Conclusions
The best regeneration recipe for the broccoli is on medium
MS + NAA 0.107 µM + BAP 17.76 µM + 2% sucrose
+ 0.8% agar, and the best explant source is hypocotyl.
Based on this menu, the regeneration frequency of
broccoli reached 100%.
ACKNOWLEDGEMENTS
This work was supported by the Natural Sciences Foundation
of China (30600415). We thank Birt Stevens for
critically reviewing the manuscript.
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African Journal of Biotechnology Vol. 10(20), pp. 4086-4092, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2455
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Evaluation of bread wheat genotypes for salinity
tolerance under saline field conditions
Munir Ahmad 1 , Muhammad Munir 1 , Iftikhar Ahmad 2 and Muhammad Yousuf 2
1 Pir Mehar Ali Shah Arid Agriculture University Murree road 46300 Rawalpindi Pakistan.
2 National Agricultural Research Center Park road 45500 Islamabad Pakistan.
Accepted 25 March, 2011
In two consecutive seasons (2007-08 and 2008-09), field experiments were conducted at Soil Salinity
Research Institute, Pindi Bhattian and Biosaline Agricultural Research Station, Pakka Aana, Pakistan.
During 2007-08, 103 wheat landrace genotypes were evaluated for salinity tolerance. During 2008-09, 47
selected genotypes were evaluated at the same locations. Combined analysis of both locations during
both years revealed that genotypes differed significantly for plant height, dry biomass m -2 , fertile tillers
plan -1 , spike length, grain spike -1 , 1000 seed weight and yield m -2 . Grain yield had strong positive
correlation with plant height, dry biomass, spike length, spikelets spike -1 , grain spike -1 and 1000 grain
weight. Due to this positive correlation, yield can be used as a selection criterion under saline field
conditions. Accessions 10807 (Pak), 11299 (Pak), 11917 (Iran) and cultivars Pavon (Pak) performed better
during both years. These genotypes could be effectively used as new sources of salt tolerance.
Key words: Genetic divergence, bread wheat, field evaluation, salinity tolerance.
INTRODUCTION
Salinity is a major abiotic stress limiting crop production
in many agricultural regions of the world (Katerji et al.,
2009). Over 800 million hectares of land are affected by
salt worldwide (Munns, 2005). Some of the most serious
examples of salinity occur in the arid and semiarid
regions. In Pakistan, more than 10 million hectares are
affected by salt (FAO, 2008). A low level of salinity may
not reduce grain yield, although shoot biomass is reduced,
which is reflected in a harvest index. Wheat yield
is substantially reduced as the soil salinity level rises to
100 mM NaCl (Munns et al., 2006).
Screening large population for salinity tolerance in the
field is difficult due to tremendous heterogeneity of saline
soils (Richards, 1983). However, various statistical
*Corresponding author. E-mail: zm_muneer@yahoo.com.
Abbreviations: ECe, Electrical conductivity; SAR, sodium
absorption ratio; SSRI, soil salinity research institute; BARS,
Biosaline Agricultural Research Station; RSCw, residual sodium
carbonate.
techniques have been used to tackle similar problems,
e.g. by blocking, particularly the use of small blocks
(Bartlett, 1978). These have reduced error variations
and increased detection of varietal differences, and can
be adapted to cope with the high heterogeneity of saline
fields. Most of the experiments are carried out under
controlled conditions where plants are not exposed to
those conditions that prevail in salt affected field
conditions such as spatial and temporal heterogeneity of
soil chemical and physical properties (Munns and James,
2003). Therefore, salt tolerance of genotypes under field
conditions needs to be evaluated particularly as a
function of yield that is considered as a foremost target of
the plant breeder (Yamaguchi and Blumwald, 2005).
Kingsbury and Epstein (1984) evaluated 5000
accessions of bread wheat in 50% seawater and identified
29 accessions that produced seed. Jafari-Shabestari
et al. (1995) evaluated 400 Iranian wheat genotypes in
irrigated field conditions in California and identified
numerous accessions that were consistently high for
grain yield in both low and high salinity treatments.
Ahmad et al. (2005) studied six wheat varieties in salt
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).
Season 2007-2008 2008-2009
Parameter Unit Site 1 Site 2 Site 1 Site 2
pH – 8.1 8.3 8.2 8.4
ECe dS m -1 16.9 13.4 17.8 14.6
SAR – 1.72 14.32 5.49 16.45
CaCO3 % 1.42 1.23 1.39 1.21
OM % 0.70 0.30 0.69 0.29
N % 0.03 0.04 0.03 0.02
Extractable P mg kg -1 4.12 3.47 3.75 2.10
Extractable K mg kg -1 33.5 54.6 35.4 55.5
Sand % 42.7 12.5 - -
Silt % 37.2 34.2 - -
Clay % 20.1 53.3 - -
Textural class – Sandy loam Clayey - -
Table 2. Analysis of variance of 7 traits studied in 103 wheat genotypes at SSRI and BARS during 2007-08.
Source of variation
Plant height
(cm)
Total bio
mass m -2 (g)
Sum of squares (Percentage of total)
Spike length
(cm)
Spikelets
spike -1
Grains
spike -1
Ahmad et al. 4087
Yield m -2
(g)
1000 seed
weight (g)
Location 22.87** 33.56** 0.17 0.31 10.62* 38.55** 0.38
Replication 0.34 0.21 2.95** 1.22* 1.79** 0.72* 0.73
Block (loc* rep) 9.66** 5.07** 5.33** 5.00 4.61* 4.60** 3.38
Genotypes 35.94** 23.70** 40.89** 33.10** 34.64** 17.88** 39.10**
Loc*genotype 14.48** 17.86** 26.62** 26.38* 25.82** 19.30** 32.54**
Error 16.71 19.60 24.04 33.99 22.52 18.95 23.87
Total sum of squares 141624.11 7974152.40 841.77 2484.76 33713.58 806159.07 1950.14
CV 14.62 53.91 12.52 15.17 22.82 51.62 13.38
*Significant at 5% probability level; **Significant at 1% probability level.
produced greater yield than salt susceptible due to higher
dry weight of shoot and spike and better grain development.
El-Hendawy et al. (2009) evaluated wheat genotypes
and reported that grain weight plant -1 , number of grains
plant -1 and number of fertile spikes plant -1 are good
screening criteria under field conditions.
The objectives of the present study were to evaluate
the genetic diversity in 103 wheat landraces/cultivars for
salinity tolerance under saline field conditions and to
identify the parents for breeding salt tolerant cultivars.
MATERIALS AND METHODS
Seeds of 103 wheat landraces/cultivars (Table 2) including 4 check
cultivars namely: SARC III, SARC IV, SARC V and SARC VII, were
obtained from the gene bank of Plant Genetic Resources Program,
National Agricultural Research Centre, Islamabad; Saline
Agriculture Research Center (SARC), University of Agriculture,
Faisalabad, and Ayyub Agricultural Research Institute, Faisalabad,
Pakistan. The landraces/cultivars included 64 accessions from
Pakistan, 20 from Iran, 7 from Syria, 4 from Egypt and 8 from Italy.
The seeds were sown at Soil Salinity Research Institute (SSRI),
Pindi Bhattian and Biosaline Agricultural Research Station (BARS),
Pakka Aana (Pakistan) during the growing seasons 2007-08.
Incomplete block design was followed with 2 replications keeping
row to row distance of 30 cm and row length of 4 m. The
experiment consisted of 12 small blocks. ECw (electrical conductivity
of water) used for irrigation was 2.0 to 2.4 dSm -1 at SSRI and 3.4 to
3.7 dSm -1 at BARS, while RSCw (residual sodium carbonate) was
1.0 to 1.5 at SSRI and 2.0 to 2.5 meq L -1 at BARS. Soil analysis of
both locations is given in the Table 1. Data were recorded from 10
randomly selected plants from each replication for biomass m -2 ,
plant height, spike length, number of spikelets spike -1 , number of
grains spike -1 , 1000 gain weight and grain yield m -2 . During 2008-
2009, 47 salt tolerant accessions selected from both locations
during 2007-2008 on the basis of yield m -2 and germination and
hydroponic testing at 200 mM NaCl stress (data not given) were
sown at the both earlier mentioned locations following the same
procedure of 2007-2008.
The data were subjected to analysis of variance using the GLM
procedure in SAS (SAS Institute, 2003). The means obtained were
separated by standard error of differences of means. The
correlation coefficients among all the traits were computed following
Kown and Torrie (1964). All the traits showed strong positive cor-

4088 Afr. J. Biotechnol.
Table 3. Salt tolerance categories of 103 wheat genotypes on the basis of average yield of 2 locations.
Salt tolerant
category
Grain yield
(g/m -2 )
Number of
accessions
Accessions (country of collection)
Tolerant 95 (g/m -2 ) and above 5 10795 (Pakistan), 10851 (Pakistan) 11299 (Pakistan), 10807 (Pakistan)
and 11302 (Pakistan)
Moderately
tolerant
Moderately
susceptible
67 - 95 (g/m -2 ) 27 Sakha-92 (Egypt), 11464 (Syria), Maroon (Iran), 13193 (Pakistan),
Roushan (Iran), 11465 (Syria), 11462 (Syria), 11244 (Pakistan), 11186
(Pakistan), 10798 (Pakistan), 10803 (Pakistan), 11386
(Pakistan),11384 (Pakistan), 10862 (Pakistan), 11406 (Pakistan),
11478 (USA), 10834 (Pakistan), Pavon (Pakistan), 10833
(Pakistan),10800 (Pakistan), 11526 (Pakistan), 11193 (Pakistan),
11387 (Pakistan), 11419 (Pakistan), 11272 (Pakistan), SARC-VII
(Pakistan), 11248 (Pakistan)
39 - 67(g/m -2 ) 47 12114 (Pakistan), 11195 (Pakistan), 10783 (Pakistan), Tabasi (Iran),
12119 (Pakistan), Arvand (Iran), ADL (Iran), 10853 (Pakistan), 10849
(Pakistan), SARC IV (Pakistan), 11467 (Syria), 11418 (Pakistan), Giza-
163 (Egypt), Karaj-II (Iran), 11133 (Pakistan), 11414 (Pakistan), 11415
(Pakistan), 11374 (Italy), 12118 (Iran), 11171 (Pakistan), 11457 (Syria),
13184 (Pakistan), Falat (Iran), 10854 (Pakistan), Moghan-1 (Iran),
10809 (Pakistan), 12117 (Pakistan), 11240 (Pakistan), Khazar-1 (Iran),
10777 (Pakistan), 11454 (Syria), 10784 (Pakistan), Chenab (Iran),
11399 (Pakistan), 11407 (Pakistan), Kayeh (Iran), Sardari (Iran), Chods
(Iran), 11458 (Syria), 4098785 (Pakistan), 11409 (Pakistan), 10824
(Pakistan), 11416 (Pakistan), 11373 (Italy), SARC III (Pakistan),
4098795 (Pakistan), 11388 (Pakistan)
Susceptible 39 (g/m -2 ) and below 24 India (Iran), 11378 (Pakistan), 10812 (Pakistan), Karaj-1 (Iran), Darab
(Iran), Omid (Iran), 10788 (Pakistan), 4098815 (Pakistan), Giza-155
(Egypt), 12113 (Pakistan), 11371 (Italy), 11371 (Italy), 10811
(Pakistan), SARC-V (Pakistan), 11372 (Italy), Rasool (Iran), Hirmand
(Iran), Alborz (Iran), 11370 (Italy), 11901 (Italy), 11290 (Pakistan),
11405 (Pakistan), 11792 (Pakistan), 11369 (Italy)
relation with yield m -2 during 2007-2008, therefore, average yield of
both locations was used to classify the genotypes into four groups.
Difference between the range of average yield was divided into four
equal groups (Table 3), namely, salt tolerant, moderately salt
tolerant, moderately salt susceptible and salt susceptible.
RESULTS
Soil analysis of two salt affected soils revealed marked
differences in ECe (electrical conductivity), SAR (sodium
absorption ratio) and soil texture (Table 1). Performance
of wheat genotypes under field conditions is the combined
effect of genotype, environment and genotype ×
environment interaction. Combined analysis of both
locations during 2007-08 and 2008-09 (Tables 2 and 4)
revealed that genotypes differed significantly (P < 0.01)
for all the traits studied. During 2007-08, location effect
was significant (P < 0.05) for plant height, total biomass,
grain spike -1 and yield m -2 . During 2008-09, differences
due to location were significant (P < 0.05) for all the
seven traits studied. This may be due to differences in
ECe and SAR of the two locations (Table 1). Block
(location × replication) effect was significant (P < 0.01),
except spikelets spike -1 and 1000 seed weight during
2007-08, however, during 2008-09, it was significant only
for plant height (P < 0.05). The possible reason for non
significance may the small size of blocks. Location ×
genotype effect was significant (P < 0.01) during the both
years for all the traits, except, spikelets spike -1 where it
was significant at P < 0.05 during 2007-08.
Based on average yield of two locations, 103 wheat
genotypes were classified into four groups (Table 3),
namely: salt tolerant included five genotypes, all from
Pakistan; moderately salt tolerant included 27 genotypes,
mostly from Pakistan, Egypt, Syria and Iran; moderately
salt susceptible included 47 genotypes; and salt susceptible
included 24 genotypes. Most of the susceptible
accessions were from Pakistan, Iran and Egypt.
During 2007-08, moisture stress coupled with high
salinity rise just after sowing created high ideal saline
conditions for crop evaluation. This inhibited the germination
and plant growth at early stage at SSRI. Due to
which most of the accessions at BARS performed better
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.
Trait studied
Plant
height
(cm)
Tiller m -2
Sum of squares (Percentage of total)
Spike length
(cm)
Spikelet
s spike -1
Grains
spike -1
Yield m -2
(g)
Ahmad et al. 4089
1000 seed
weight (g)
Location 58.25** 59.89** 11.46* 28.25** 19.23** 49.26** 21.53**
Replication 0.29 0.08 0.41 0.05 0.01 0.03 0.10
Block (loc × rep) 0.53 2.20* 2.56 1.58 0.34 2.08 1.34
Genotypes 24.36** 20.40** 45.85** 31.60** 60.89** 28.52** 51.09**
Loc × genotype 10.80** 10.10** 25.72** 19.41** 14.74** 13.23** 16.50**
Error 5.77 7.33 14.00 19.11 4.78 6.88 9.43
Total sum of squares 49321.63 2218209.40 256.70 1048.07 12989.14 3294445.11 1050.27
CV 8.53 27.28 9.14 12.29 12.08 39.23 11.74
*Significant at 5% probability level; **Significant at 1% probability level.
Table 5. Correlation coefficient between various traits and yield in wheat genotypes at Pindi Bhattian
(PB) and Pakka Aana (PA) during 2007-08 and 2008-09.
Plant height (cm)
Trait studied Yield (g m -2 ) 2007-08 Yield (g m -2 ) 2008-09
Biomass (g m -2 ) (2007-8)
Fertile tillers plant -1 (2008-09)
Spike length (cm
Spikelets spike -1
Grains spike -1
1000 seed weight (g)
**Significant at 1% probability level.
reduction was manifested in plant height, fertile tillers
plant-1, spike length, spikelets spike-1, grains spike-1,
1000 grain weight and yield m-2 (Table 6). Five accessions
produced above 100 cm tall plants at SSRI but >77
cm at BARS. This showed a significant reduction in plant
height at BARS as compared to SSRI (Table 6).
Total dry biomass production at SSRI was less as
compared to BARS during 2007-08. Accessions 10795,
10807, 10851 and 11299 produced highest total biomass
at both locations. The total biomass production ranged
from 47.6 to 672.2 gm-2 at BARS. It revealed a huge
PB 0.82** PB 0.91**
PA 0.94** PA 0.77**
PB 0.93** -
PA 0.96** -
- PB 0.94**
- PA 0.95**
PB 0.80** PB 0.90**
PA 0.92** PA 0.74**
PB 0.80** PB 0.90**
PA 0.92** PA 0.74**
PB 0.80** PB 0.95**
PA 0.93** PA 0.73**
PB 0.81** PB 0.94**
PA 0.92** PA 0.76**
reduction in biomass in salt susceptible accessions as
compare to salt tolerant. During 2008-09, fertile tillers m-2
showed a considerable reduction at both locations. It
ranged from 125.4 to 409.2 at SSRI and 8 to 259 at
BARS. Accessions 10824, 11287, 11214, 11383 and
Local white were able to maintain maximum number of
fertile tillers at both locations (Table 6). Fertile tillers m-2
had a positive and highly significant correlation with grain
yield m-2 during 2008-09 at both locations (Table 5).
Information on the relationship between yield and its
components and among the components themselves can

4090 Afr. J. Biotechnol.
Table 6. Means of 7 traits studied in 47 wheat genotypes at Pindi Bhattian and Pakka Aana during 2008-09.
Accession
no.
Plant height Fertile Tiller Plant -1 Spike length Spikelets spike -1 Grains spike -1 1000 grain weight Yield m -2
PB PA PB PA PB PA PB PA PB PA PB PA PB PA
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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.
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
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
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
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
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
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
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
x Standard error of the difference between means; no., number.
be utilized to improve the efficiency of selection in
plant breeding programs. Spike length is an
important yield contributing trait which showed
reduction under saline field conditions. During
2007-08, spike length ranged from 4.85 to
13.97 cm at SSRI and from 6.1 to 11.1 cm
at BARS. During 2008-09, spike length ranged
from 6.4 to 11.3 cm at SSRI and 4.8 to
9.5 cm at BARS (Table 6).
Grains spike -1 also showed reduction at high
salinity level. At SSRI, grains spike -1 ranged from
4.8 to 48.17 during 2007-08, while at
BARS, it was from 16.5 to 46. During 2008-
09, grains spike -1 ranged from 8.3 to 45 at
SSRI, while at BARS, it was from 7.5 to
42.5. Reduction in grain spike-1 also causes
reduction in yield at high salt stress. This
reduc-tion is due to positive association
between grain spike -1 and yield. Most of the
accessions showed reduction in 1000 seed weight
at BARS during both years. The foremost target of
wheat breeder is to improve grain yield. Therefore,
the assessment of final grain yield and
growth parameters determining grain yield is a
significant aspect of breeding programs. Five
accessions: 10795, 10851, 11299, 10807 and
11302 (all from Pakistan) were identified as most
salt tolerant on the basis of average yield performance
at two locations (Table 2). Three of them:
10807, 10851 and 11299 were included in the
second phase of the experiment (2008-09).
Accession 10807 produced 128.71 and 91.0 gm -2
yield during 2007-08 and 105.6 and 84.3 gm -2
during 2008-09 at SSRI and BARS, respectively.
Accession 11299 produced 93.55 and 127.8 gm -2
yield during 2007-08, and 385.1 and 111.2 gm -2
during 2008-09 at SSRI and BARS, respectively.
DISCUSSION
Our results revealed that wheat genotypes responded
differently to salinity stress at the two
locations in terms of yield and yield components.
Similar findings were reported by (Richards et al.,
1987; Slavich et al., 1990). The decline in total dry
biomass in the sensitive accessions was most
likely due to the extra energy utilization for
osmotic accumulation (Wyn Jones and Gorham,
1993). Significant positive correlation between dry
biomass and yield m-2 (Table 5) indicated that
total dry biomass along with yield can be good
selection criteria under salinity stress.
Salts present in the soil solution reduce the
ability of plant to absorb water which slow down
plant growth and ultimately cause reduction in
yield components. This is called osmotic or water
deficit effect of salinity. Salinity stress at different
Ahmad et al. 4091
phenological stages inhibits photosynthetic activities
of the plant because it had a direct inhibitory
effect on the Calvin cycle enzymes (Ottander and
Oquist, 991). Tiller plant -1 is the most salinity
sensitive trait in wheat (El-Hendawy et al., 2005).
Therefore, to increase the yield under
stress condition, it is necessary to maintain
high plant density. Tiller formation included
tiller number and tiller biomass. Salinity reduces
tiller number by delaying and reducing tiller emergence
at the vegetative stage. After tiller emergence,
growth of tillers at all stages is inhibited by
salinity due to its damage on the essential
metabolic reaction in plants, resulting in low tiller
biomass and small tiller size (Mass and Poss,
1989). ECe >7.5 dSm -1
in soil water could eradicate
most of the secondary tillers and greatly
reduce the formation of tertiary and lateral tillers.
The yield potential of wheat is greatly dependent
on the number of tillers plant -1 that is affected in
the early life cycle. Number of tillers regulates
grain yield by its prime influence on the number of
spikes in wheat (Simons and Hunt, 1983). Our
correlation results confirmed these findings.
Spikelets spike -1 along with number of
tillers was identified as the most salt sensitive
yield components in wheat. At the time of spike
emergence, salinity suppresses the reproductive
development, spikelet initiation and ultimately

4092 Afr. J. Biotechnol.
number of spikelets (Mans and Rawson, 2004). Due to
their response to salinity and significant positive
correlation with yield, these two traits could be used as
selection criteria. These traits could be determined at
early growth stages and therefore, may be used to
screen large population. Reduction in grains spike -1 was
due to reduction in spikelets spike -1 as revealed by
positive correlation between them. The 1000 grain weight
was less affected as compare to the other yield
components because it was determined at maturity which
is the least salt sensitive stage in wheat (Frank et al.,
1997).
Grain yield had strong positive correlation with plant
height, dry biomass, spike length, spikelets spike -1 , grain
spike -1 and 1000 grain weight (Table 5). Due to this positive
correlation, yield can be used as a selection criterion
under saline field conditions. Many scientists had classified
crop species on the basis of grain yield under stress
conditions (Sadiq et al., 1994; Jafari-Shabestari et al.,
1995; Anderson et al., 1996). Reduction in yield was due
to the reduction in the yield components and high ECe at
BARS was the main cause of reduction in yield and yield
components. Wheat yield decline 10% when ECe value
goes >10 dSm -1 (Katerji et al., 2009).
Sum of square due to genotype for tillers m -2 , total
biomass m -2 , spikelets spike -1 and yield m -2 revealed
that these traits were adversely affected at both locations.
Our results showed that these traits could be effectively
used as selection criteria for screening wheat under field
conditions. Accessions 10807, 11299, 11917 and cultivars
Pavon performed better during both years. Cultivar Local
white produced better yield at both locations during 2008-
09 but check cultivar Pasban 90 performed better at SSRI
as compare to BARS. The high yield of these genotypes
was actually associated with high total biomass production
and yield components. Accession 11299 (Pak) was found
to be most salt tolerant at germination stage and seedling
stage in hydroponics under 300 and 250 mM NaCl stress,
respectively. Cultivar Pasban 90 and accession 10807
were also found to be salt tolerant at seedling stage in
hydroponics at 250 mM NaCl stress (data not shown).
These genotypes could be used as new sources of salt
tolerance.
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Pflanzenzuchtung. J. Plant Breed. 90: 249-258.
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African Journal of Biotechnology Vol. 10(20), pp. 4093-4099, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1248
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Antioxidant enzymes activities during secondary
somatic embryogenesis in Persian walnut (Juglans
regia L.)
M. Jariteh 1 *, H. Ebrahimzadeh 1 , V. Niknam 1 , K. Vahdati 2 and R. Amiri 3
1 Plant Physiology Laboratory, Department of Plant Sciences, School of Biology, College of Science, University of
Tehran, P.O.BOX: 14155-6455, Tehran, Iran.
2 Department of Horticulture, College of Abouraihan, University of Tehran, Tehran, Iran.
3 Department of Agronomy and Crop Breeding, College of Abouraihan, University of Tehran, Tehran, Iran.
Accepted 18 March, 2011
Somatic embryogenesis was achieved from immature cotyledon explants of Persian walnut (Juglans
regia L.) cave. "Chandler" on DKW medium. Secondary somatic embryogenesis, the process by which
adventitious embryos are formed from primary somatic embryos, is frequent during somatic
embryogenesis in Persian walnut. It has certain advantages compared with primary somatic
embryogenesis such as high multiplication rate, independence to the explant source and repeatability.
Proteins and activities of antioxidant enzymes including catalase (CAT), peroxidase (POX), superoxide
dismutase (SOD) and polyphenol oxidase (PPO) were evaluated in two types of secondary embryogenic
calli and secondary somatic embryos in Persian walnut. In dark brown calli from which their secondary
somatic embryos were separated, CAT, POX and POX had the highest activities while the activity of
SOD and also protein content showed the lowest level. High SOD activity, protein content and low
activities of CAT, POX and PPO were determined in secondary somatic embryos and light brown calli
containing secondary somatic embryos. According to the obtained results, some proteins and
antioxidant enzymes have been regarded as markers for secondary somatic embryogenesis.
Key words: Persian walnut, secondary somatic embryogenesis, antioxidant enzymes.
INTRODUCTION
Somatic embryogenesis and particularly the secondary
somatic embryogenesis provide an efficient regeneration
system for vegetative propagation and genetic transformation
of walnut (Mc Granahan et al., 1990). Secondary
somatic embryogenesis has been reported in many tree
species and is the basis of embryo cloning (Merkle et al.,
1995; Daigny et al., 1996; Benelli et al., 2001; Agarwal et
al., 2004). Secondary or repetitive somatic embryogenesis,
the process by which adventitious embryos are
*Corresponding author. E-mail: maryam.jariteh@gmail.com.
Fax: (+98) 21 66492992.
Abbreviations: CAT, Catalase; POX, peroxidase; PPO,
polyphenoloxidase; SOD, superoxide dismutase; DBC, dark
brown callus; LBC, light brown callus; SSE, secondary somatic
embryo; DKW, Driver and Kuniyuki Walnut medium.
derived from primary somatic embryos, is frequent during
somatic embryogenesis in walnut. Secondary embryos
are formed on the cotyledons and most frequently at the
root tips of somatic embryos. If these adventitious
embryos removed when their cotyledons began to
expand, they often develop into normal plants. Failure to
remove adventitious embryos at this stage of development
led to the formation of more somatic embryos,
abnormal growth or callus formation. Somatic embryo
tissues often turn into dark brown masses after several
months in culture. These brown tissues frequently
produce globular somatic embryos which could be
removed and used to propagate the embryogenic lines
(Tulecke and Mc Granahan, 1985). Martinelli and
Gribaudo, (2001) called the secondary embryogenesis as
re-initiation of embryogenic calli from somatic embryos.
The secondary somatic embryos of walnut can be
efficiently multiplied by repetitive or recurrent embryo-

4094 Afr. J. Biotechnol.
genesis on hormone-free DKW medium (Deng and
Cornu, 1992).
As an experimental system, secondary somatic embryogenesis
has certain advantages compared with
primary somatic embryogenesis such as high multiplication
rate, independence to the explant source and
repeatability (Karami et al., 2008). Furthermore,
embryogenicity can be maintained for a long period of
time by repeated cycles of secondary embryogenesis
(Raemakers et al., 1995). On the other hand, it is thought
that adventitious embryogenesis including somatic and
anderogenetic ones might be induced by different kinds
of stress treatments (Kamada et al., 1994). There is a
variety of data that link somatic embryogenesis, DNA
methylation and oxidative stress response. Also, an
increasing number of publications link reactive oxygen
species (ROS) and somatic embryogenesis (Zavattieri et
al., 2010).
Some antioxidant enzymes have been introduced as
indicators for somatic embryogenesis. According to
Kairong et al. (1999) the differentiation and development
of embryogenic cells in the somatic embryogenesis of
Lycium barbarum L. are regulated by three antioxidant
enzymes including CAT, POX and SOD. Somatic
embryogenesis from somatic tissues of walnut, rather
than zygotic embryos needs to be further investigated
(Vahdati et al., 2006). Thus, study of antioxidant enzymes
activities during secondary somatic embryogenesis can
help to improve primary somatic embryogenesis and
reduce problems in recalcitrant explants. For this purpose,
two different types of secondary embryogenic calli,
light and dark brown calli containing secondary somatic
embryos at different stages of development and
separated secondary somatic embryos were compared.
Proteins and activities of some antioxidant enzymes
including CAT, POX, SOD and PPO were studied during
secondary somatic embryogenesis in Persian walnut
(Juglans regia L.) cv. "Chandler".
MATERIALS AND METHODS
Plant material
Ten fruits of walnut cultivar "Chandler" were harvested 9 weeks
after pollination and stored at 4°C for 5 days. They were surface
sterilized and dissected immediately. Immature cotyledons were
cultured on DKW solid medium (Driver and Kuniyuki, 1984)
containing 0.01 mg/l IBA, 2 mg/l Kin, 1 mg/l BA and 250 mg/l
glutamine (Tulecke and Mc Granahan, 1985). Cultures were grown
in the dark at room temperature.
The repetitive somatic embryogenic line which initiated from
immature cotyledons of walnut cultivar "Chandler" was used in the
present study. This line has been maintained by subculturing every
two weeks at 25°C in the dark for over 2 years. Somatic embryos
were subcultured on basal DKW medium solidified with 0.21%
Gelrite (w/v). Dark brown calli with secondary somatic embryos at
different developmental stages on their surfaces (DBC+SSE), dark
brown calli without secondary somatic embryo (DBC-SSE),
secondary somatic embryos at different developmental stages
which were separated from the surface of dark brown calli (SSE)
and light brown calli with secondary somatic embryos at different
developmental stages (LBC+SSE) were used for all the analyses.
Protein extraction
For estimation of total protein content and enzymes activity, plant
materials were homogenized at 4°C with a mortar in 1M Tris-HCl
(pH 6.8) and 2.5 % (w/v) polyvinylpolypyrolidone (PVPP) to avoid
phenol oxidative effects. The homogenates were centrifuged at
13000 g for 30 min two times at 4°C using a Heraeus 400R
microfuge. Supernatants were kept at -70°C and used for protein
determination and enzyme assay. An UV-visible spectrophotometer
(UV-160, Shimadzu, Tokyo, Japan) were used for the determination
of the absorbance.
Protein quantification
Protein concentration was measured according to Bradford (1976),
using bovine serum albumin (BSA) as standard. Five milliliters of
the Bradford reagent and 100 µl of the each protein extract were
mixed and then, reaction mixtures were incubated at room
temperature for 20 min. The absorbance values were measured at
595 nm.
Antioxidant enzymes assays
Peroxidase (POX; E.C. 1.11.1.7) activity was measured according
to the method of Abeles and Biles (1991). The assay mixture
consisted of 4 ml of 0.2 M acetate buffer (pH 4.8), 0.4 ml H2O2 (3
%), 0.2 ml 20 mM benzidine and 0.2 ml enzyme extract. The
increase of absorbance was recorded at 530 nm. The POX activity
was defined as 1 µM of benzidine oxidated per min per mg protein
(Unit mg -1 (protein)).
Superoxide dismutase (SOD; E.C. 1.15.1.1) activity was
estimated by measuring its ability to inhibit the photochemical
reduction of nitroblue tetrazolium (NBT) at 560 nm as described by
Giannopolitis and Ries (1977) in a reaction mixture consisted of 0.1
ml enzyme extract, 50 mM sodium phosphate buffer (pH 7.5), 13
mM L-methionine, 75 µM NBT, 0.1 mM EDTA and 75 µM riboflavin.
The reaction mixture was irradiated for 14 min and absorbance was
recorded at 560 nm against the non-irradiated blank. One unit of
SOD activity was defined as the amount of enzyme which caused
50% inhibition in NBT reduction.
Catalase (CAT; E.C. 1.11.1.6) activity was assayed from the rate
of H2O2 decomposition as measured by decrease of absorbance at
240 nm (Aebi, 1974). The reaction mixture contained 0.625 ml of 50
mM sodium phosphate buffer (pH 7.0), 0.075 ml H2O2 (3%) and
0.05 ml enzyme extract.
Polyphenol oxidase (PPO; E.C. 1.14.18.1) activity was estimated
following the method of Raymond et al. (1993) at 40°C. The
reaction mixture contained 2.5 ml of 0.2 M sodium phosphate buffer
(pH 6.8), 0.2 cm 3 pyrogallol 20 mM and 0.02 cm 3 enzyme extract.
The increase in absorbance was recorded at 430 nm. The PPO
activity was defined as 1µM of pyrogallol oxidated per min per mg
protein (Unit mg -1 (protein)).
Gel electrophoresis
For determination of protein patterns, discontinuous sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
was performed according to Laemmli (1970), using 12%
acrylamide. For detection of proteins, gels were stained with 0.03%
comassie brilliant blue G250. For isoenzyme assay, native PAGE
was carried out according to a modified method of Davis (1964)

with a 12% (SOD) and 10% (POX, CAT and PPO) acrylamide gels
at 4°C.
SOD bands were visualized using the activity staining procedure
described by Wendel and Weeden (1989). Gels were incubated in
0.2 M Tris-HCl (pH 8.0) containing 4% riboflavin, 4% EDTA and
20% NBT for 40 min in the dark at room temperature and then,
exposed to white light until white bands appeared in violet
background. For SOD isoform identification, assays were performed
in the presence of selective inhibitors. Application of KCN (3 mM)
inhibits only Cu/Zn-SOD; H2O2 (5 mM) inhibits both Cu/Zn-SOD and
Fe-SOD. Mn-SOD was not inhibited by KCN or H2O2 (Lee et al.,
2001).
CAT activity was detected using the method of Woodbury et al.
(1971). The gels were incubated in 5 mM H2O2. After 10 min, the
gels were washed with distilled water and stained with a reaction
mixture containing 2% (w/v) ferricyanide (K3Fe(CN)6) and 2% ferric
chloride till yellow bands appeared on dark green background.
Electrophoresis pattern of POX was obtained by staining of the
gels with benzidine according to Van Loon and Geelen, (1971). The
gels were immersed in 0.2 M acetate buffer (pH 4.8) containing 3%
H2O2 and 4% benzidine in 50% methanol in the dark at room
temperature till the brown bands appeared.
PPO bands were visualized using the method of Van Loon and
Geelen, (1971). The gels were incubated in 0.2 M potassium
phosphate buffer (pH 6.8) containing 0.5% 3,4- dihydroxy L-
phenylalanine (L-DOPA) and 3.5% calcium chloride in the dark at
room temperature till brown bands appeared.
Statistical analysis
The data determined in triplicate were analyzed by analysis of
variance (ANOVA) using SAS Software (SAS Institute, Inc., 2002).
Significance of mean differences was determined by the Duncan
multiple range test (DMRT) at P < 0.05.
RESULTS
Tissue culture
Most of the immature cotyledon explants of Chandler formed
callus but only low percentage of them were
embryogenic and the number of somatic embryo per
callus ranged from 1 to 4 (data not shown). The embryos
loosely attached without a vascular connection to the
maternal tissues. They were removed from the
embryogenic calli and cultured on basal medium. When
walnut somatic embryos especially at the primary stages
of development including globular and heart shape were
cultured on basal DKW medium, two distinct responses
were observed; (a) development, maturation and
conversion to whole plant; and (b) secondary callus
induction and repetitive somatic embryogenesis.
In some cases, mature embryos produced new individual
embryos on their cotyledons or root tips directly.
Cotyledon-derived primary embryogenic calli were compact,
hard, yellowish and grow slowly with a few number
of somatic embryos on the surface but secondary calli
were fast growing, very friable and light brown at early
stages of formation and gradually became dark brown
while produced a large number of embryos (Figure 1).
Biochemical assays
Ahmad et al. 4095
The highest and lowest content of proteins was determined
in SSE and DBC-SSE, respectively. Protein
content in LBC+SSE was significantly higher than that in
DBC+SSE. The highest activity of CAT was measured in
DBC-SSE and the lowest activity was detected in
LBC+SSE. There was no significant difference in activity
of CAT between DBC+SSE and SSE. The highest activity
of POX was detected in DBC-SSE and POX activity
decreased in the DBC+SSE, SSE and LBC+SSE,
respectively. PPO showed the highest activity in DBC-
SSE while DBC+SSE had lower activity of PPO. The
lowest and highest activity of PPO was detected in SSE
and LBC+SSE, respectively. The lowest activity of SOD
was detected in DBC-SSE and no significant difference
was determined in SOD activity among other samples
(Table 1). According to the SDS-PAGE protein pattern
(Figure 2), the strongest and weakest protein bands were
detected in SSE and DBC-SSE, respectively. Among the
proteins detected, the most abundant were those with
molecular masses of 83, 53, 45, 40, 36, 30, 29 and 28
kDa, respectively. The 83, 53, 45 and 40-kDa proteins
were present in all samples strongly except DBC-SSE.
Also, the 30, 29 and 28-kDa proteins of light brown
secondary somatic embryos (LBC+SSE) and SSE were
expressed more than the corresponding fraction of dark
brown callus with or without embryos (DBC+SSE and
DBC-SSE).
According to non-denaturing PAGE, two CAT isoforms
were detected in the DBC+SSE. CAT1 which was observed
in SSE and LBC+SSE was stronger than CAT2
and was absent in DBC-SSE. In DBC-SSE only CAT2
was detected and this isoform was absent in SSE and
LBC+SSE (Figure 3a). Thus, CAT 2 could be regarded as
a marker for dark grown calli and embryos.
Five POX isoforms were determined in all samples
examined. According to the activity staining, POX1 band
was stronger than four other bands. This band in
DBC+SSE was very strong compared with LBC+SSE and
SSE. POX2 and POX3 isoforms were similar in all the
samples. POX4 was present only in DBC+SSE. POX5
was weak in DBC-SSE and LBC+SSE in comparison with
DBC+SSE and SSE (Figure 3b). Thus, POX4 could be
regarded as a marker for dark grown calli containing
embryos (DBC+SSE).
Five bands with PPO activity were detected on gels by
native PAGE. PPO1 and PPO3 bands in DBC-SSE were
stronger than the others. PPO2 was present only in
DBC+SSE and DBC-SSE while PPO4 was present only
in SSE and LBC+SSE. PPO5 band was common in all
samples and is very strong in SSE and LBC+SSE in
comparison with other samples (Figure 3c). Thus, PPO4
could be regarded as a specific marker for light grown
calli and secondary somatic embryos.
Six bands of SOD were detected in the samples. The
identities of the major SOD activity bands were tested by

4096 Afr. J. Biotechnol.
Figure 1. Secondary somatic embryogenesis in Persian walnut. (a) Primary embryogenic callus
derived from immature cotyledons of Chandler cultivar with two somatic embryos; 1, arrowed = a
well developed cotyledonary embryo; 2, arrowed = heart shape embryo (bar = 8 mm); (b) direct
secondary somatic embryogenesis; arrowed = two torpedo embryos on the surface of the primary
embryo's cotyledons (bar = 4 mm); (c) light brown secondary embryogenic callus with several
secondary somatic embryos; 1, arrowed = globular secondary embryo, 2, arrowed = precotyledonary
embryo (bar = 4 mm); (d) dark brown secondary embryogenic callus with several secondary somatic
embryos; 1, arrowed = torpedo embryo; 2, arrowed = precotyledonary embryo (bar = 5 mm).
Table 1. Protein content (mg g -1 (F.W.)) and activity of antioxidant enzymes (U mg -1 (protein)) in different stages of
secondary somatic embryogenesis.
Parameter Total protein CAT POX PPO SOD
DBC+SSE 2.52 ± 0.10c 144.16 ± 4.56 b 28.52 ± 1.30b 0.28 ± 0.01b 1.017 ± 0.001a
DBC-SSE 0.55 ± 0.02d 345.26 ± 10.73a 135.51 ± 2.15a 0.85 ± 0.04a 0.980 ± 0.001b
SSE 5.50 ± 0.28a 125.30 ± 4.30b 18.23 ± 1.10c 0.11 ± 0.01c 1.012 ± 0.001a
LBC+SSE 4.26 ± 0.04b 77.26 ± 5.17c 9.24 ± 0.37d 0.13 ± 0.01c 1.015 ± 0.004a
DBC+SSE, Dark brown calli with secondary somatic embryo; DBC-SSE, dark brown calli without secondary somatic embryo;
SSE, secondary somatic embryo; LBC+SSE, light brown calli with secondary somatic embryo. Means ± SE; values marked
with different letters are significantly different according to DMRT at P < 0.05.
preincubating the gels with well-characterized SOD
inhibitors: KCN is an inhibitor of Cu/Zn-SOD, whereas,
H2O2 inhibits both Cu/Zn-SOD and Fe-SOD. The upper
band (Figure 3d), in all samples, represented a
mitochondrial Mn-SOD. Five bands with higher mobility
represented the cytosolic Cu/Zn-SOD isoenzymes. The
band representing a chloroplastic Fe-SOD was not presented.
No significant qualitative differences in the
pattern of SOD isoforms were observed between tissues.
DISCUSSION
In vitro culture conditions represent an unusual combination
of stress factors that plant cells encounter
(oxidative stress as a result of wounding at excision of
the explant tissue, PGRs, low or high salt concentration
in solution, low or high light intensities). The stress not
only promotes dedifferentiation but also induce somatic
embryo formation. The drastic changes in the cellular

Figure 2. SDS-PAGE pattern of proteins during
secondary somatic embryogenesis in Persian walnut;
dark brown calli with secondary somatic embryo
(DBC+SSE) (1), dark brown calli without secondary
somatic embryo (DBC-SSE) (2), secondary somatic
embryos (SSE) (3), light brown calli with secondary
somatic embryo (LBC+SSE) (4) and molecular mass
marker (M). Equal amounts of protein (40 µg) were
loaded in each lane. Arrows indicate the affected and
embryo- specific bands.
environment of the in vitro culture induced by a ‘stressor’
in the culture medium or in the physical environment of
the culture, are responsible for reprogramming of gene
expression (Zavattieri et al., 2010). However, secondary
somatic embryogenesis from a dark brown calli in
absence of any growth regulator might be induced by oxidative
stress. The effect of stress during culture may help
to improve induction of somatic embryogenesis in
recalcitrant somatic tissues of Persian walnut.
Somatic embryos are formed from immature cotyledons
of walnut cultivar and this result is in agreement with
Tulecke and Mc Granahan (1985) and Vahdati et al.
(2006). According to Tulecke and Mc Granahan (1985)
somatic embryo tissues in walnut cultivars often turn into
dark brown masses after several months in culture and
they frequently produce globular somatic embryos which
could be removed and used to propagate the embryogenic
lines. It should be mentioned that, all of the
developmental stages of embryos including globular,
heart shape, torpedo and cotyledonary can be observed
on the surface of secondary embrygenic calli. Although,
all related articles have confirmed the secondary
embryogenesis in walnut cultivars but there is no report
Ahmad et al. 4097
on the alterations of antioxidant enzymes and proteins
during somatic embryogenesis.
Protein content in SSE which contained embryos at
different developmental stages was the highest, but DBC-
SSE which lacks any embryos had the lowest level of
protein. The difference in protein content was confirmed
by gel electrophoresis profile. These results are in good
agreement to Junaid et al. (2007), who reported that
protein content in matured and proliferated somatic
embryos were significantly higher than that in secondary
embryogenic calli of Catharanthus roseus L. They
showed that the protein content gradually increased with
advancing stages of somatic embryogenesis. It has been
reported that, the degradation of cell membranes and
browning coincident with the loss of chlorophyll and
browning is associated with cell disorganization
(Laukkanen, et al., 2000). Among the protein bands,
some were stronger than the others. Proteins with molecular
weight of about 83, 53, 45, 40-kDa were expected
to be involved in somatic embryogenesis. Sung and
Okimoto (1983) in the studies on carrot have reported
two somatic embryo-specific proteins (77 and 43 kDa).
Similar studies performed by Chen and Luthe (1987) on
rice revealed the presence of two major proteins of 54
and 24 kDa, which were regarded as somatic embryosspecific.
According to Pakusch et al. (1991) a 24 kDa
band may represent subunits of methyltransferase, an
enzyme associated with lignin synthesis. The production
of lignin compounds in callus of plants species may be as
a result of stress reactions because several stresses are
known to induce the synthesis of phenylpropanoids.
Activities of CAT, POX and PPO in DBC-SSE were
significantly higher than those in the other samples but
the lowest activity of SOD was detected in this tissue.
According to these results, the differentiation and
development of embryogenic tissues during secondary
somatic embryogenesis of Persian walnut could be
regulated by antioxidant enzymes (CAT and POX and
PPO). The study analyses showed that activities of CAT,
POX and PPO rapidly increased along with the seconddary
somatic embryogenesis. According to Kairong et al.
(1999) the acquisition of competence, induction and
development of somatic embryos in L. barbarum L. were
associated with the gradual increase in SOD activity.
POX and CAT activities were high in callus and rapidly
decreased in the early days of the differentiation culture
(Kairong et al., 1999). Laukkanen et al. (1999, 2000)
reported that, tissue browning in Scot pine is associated
with increased POX and PPO activities, as well as cell
disorganization and eventually cell death. From the
zymograms of CAT, it can be concluded that CAT1 was
somatic embryo-specific and CAT2 was present only in
dark brown calli and not in light brown ones. These
differences might be related to tissue browning and thus,
CAT could be an indicator for oxidative stress. Among
five POX isoforms, POX1 band in DBC+SSE was very
strong compared with LBC+SSE and SSE. POX4 band

4098 Afr. J. Biotechnol.
Figure 3. Activity staining for SOD; (a) CAT (b) PPO (c) and SOD (d) during secondary somatic embryogenesis in Persian
walnut: dark brown calli with secondary somatic embryo (DBC+SSE) (1), dark brown calli without secondary somatic embryo
(DBC-SSE) (2), secondary somatic embryos (SSE) (3), and light brown calli with secondary somatic embryo (LBC+SSE) (4).
Equal amounts of protein (40 µg) were loaded in each lane.
was present only in DBC+SSE. Thus, POX4 could be
related to tissue browning in these tissues. PPO1 and
PPO3 bands were stronger in DBC-SSE and PPO2
which was only present in DBC+SSE could be related to
tissue browning, while PPO4 was detected only in SSE
and LBC+SSE and PPO5 could be related to somatic
embryo development (Figure 3c). The contribution of
individual SOD isoforms to total SOD activity was
determined by performing SOD assays directly on protein
extracts separated in native gels. The upper band (Figure
3d), in all samples, represented a mitochondrial Mn-SOD.
Five bands with higher mobility represented the cytosolic
Cu/Zn-SOD isoenzymes. The band representing a
chloroplastic Fe-SOD was not detected in samples. The
levels of superoxide dismutase activity were lower in
browning tissues than that in non-browning tissues in
culture condition in Scot pine (Laukkanen et al., 2000)
and these results are in agreement with the latter
research. In Virginia pine, tissue browning decreases the
efficiency of in vitro regeneration through somatic
embryogenesis (Tang and Newton, 2004). It was reported
that, in non-browning callus cultures, PPO activity
declined while in browning calli, PPO activity continued to
increase. It is believed that the increased PPO activity in
browning calli results from wounding or oxidative
damage. Numerous experiments have recently been
carried out on several plant species with the objective of
explaining the role of oxidative stress in plant
morphogenesis (Gupta and Datta, 2003, 2004; Libik et
al., 2005). It is well known that somatic embryogenesis is
one of the most useful approaches for understanding
plant development. Higher levels of intracellular H2O2
induce and promote embryo-genesis of L. barbarum L.
callus (Kairong et al., 1999). However, the relationship
between the ROS and the callus differentiation and
regeneration has not been well understood till now (Tian
et al., 2003).
In summary, according to the present research, the
differentiation and development of embryogenic tissues
during secondary somatic embryogenesis of Persian
walnut could be regulated by protein and antioxidant
enzymes.

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African Journal of Biotechnology Vol. 10(20), pp. 4100-4105, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2673
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Sugar beet genotype effect on potential of bioethanol
production using Saccharomyces cerevisiae
fermentation
Parviz Mehdikhani 1 *, Hrachya Hovsepyan 2 and Mahmood Rezazadeh Bari 3
1 Agricultural Research Center of West Azerbaijan, Iran.
2 Agricultural Research Center of Yerevan, Armenia.
3 Agricultural Sciences College of Urmia University, Iran.
Accepted 6 April, 2011
In order to study the effect of genotype on sugar beet ethanol production potential, ten beet varieties
including diploid, triploid and tetraploid, multigerm, monogerm sugar beet and fodder beet were planted
in randomized complete block design (RCBD) with three replications in Khoy Agricultural Research
Center for two years (2008 to 2009). Some morphological and physiological traits were recorded. The
fresh root and raw sugar of each treatment were fermented to ethanol using Saccharomyces cerevisiae.
Results showed that, genotype had a remarkable effect on the ethanol production potential. Significant
difference (at the 1% level) in ethanol was observed among the beet varieties in both ethanol production
methods. In addition, more significant differences in most morphological and qualitative traits among
the varieties studied were observed. Variation on ethanol production were intensively related to the
chemical composition of root, especially sugar content, potassium impurity, syrup purity and some
characteristics such as root dry matter and root length. Bioethanol production was enhanced by
increasing the sugar content and root yield in sugar beet. Sugar beet varieties contained less root yield
and more sugar content had higher potential for ethanol production than the fodder beet varieties.
Key words: Bioethanol, Saccharomyces cerevisiae , sugar beet, raw sugar
INTRODUCTION
Oil crises occurrence in the 1970 decade due to consumption
increase over production, and the extraordinary
increase of oil price, persuaded industrial countries to
have different approach on the energy issue. Reduction
of fossil fuel resources, commencement of environmental
concerns and improvement of social living standards
under different conventions and protocols like Kyoto
protocol, led to direct human use of clean fuels
(Miyamoto, 1997). Industry leading manufacturers in the
world and producers attempt to replace petrochemical
materials with agricultural products. Nowadays, bioenergy
forms about 8% of the total annual energy used in
the United States (Anonymous c , 2010). Governments and
*Corresponding author. E-mail: Mehdikhani20@gmail.com. Tel:
+98 461 223 3158 or +98 914 161 3755. Fax: +98 461 223
3159.
industries make use of various initiatives associated with
bio-energy production, increase of production efficiency
and decrease of production cost (National research
council, 2000). Ethanol or ethyl alcohol, unlike past
decades that was only used in few industries, is
considered as one of the important and strategic
commodities in many countries and yet the production
volume of this substance is increasing day by day. More
than 80% of the existing ethanol in the world is used as
fuel consumption and its by-products, and 20% of the
remaining is used for traditional applications in industries
such as medical, health and cosmetics, paint and resin
(Anonymous a, 2010). Brazil and United States are the
world's leading producer of ethanol. In 2008, United
States produced 9 billion U.S. liquid gallons of ethanol
fuel and 10.6 billion U.S. liquid gallons of ethanol fuel in
2009. This represents around 55% of the world's total
ethanol production (Anonymous a , 2010). Ethanol also can
be considered as an agricultural sector stabilizer, for

example, if ethanol conversions factories are enable to
produce ethanol from sugar beet, potato and maize, we
will not be faced with difficulty of production increase of
these products in different periods (Parvin, 2010).
Currently, more than 90% of the existing ethanol in the
world is produced as bio-ethanol and 7% synthetically. In
fact, until the late 1980 a noticeable proportion of ethanol
in the world was artificially produced from petrochemicals,
but after this decade new investment has not
been done in this case. It is obvious that each country
based on the dominant culture and climate conditions can
make use of some agricultural products for producing
ethanol. For an example, more than 20% of the nation’s
total corn supply is consumed for ethanol production in
United States (Anonymous a , 2010), while ethanol is
produced in Iran, countries like South America, Brazil,
India, Pakistan and Thailand and some European
countries from sugar beet and cane molasses. Meanwhile,
countries such as Russia, Canada, China,
Australia and some European countries use wheat for
ethanol production (Venturi and Venturi, 2003).
Sugar beet (Beta Vulgaris L.) is a sucrose-rich product
that has many industrial applications in the today world.
Beets are planted from northern latitudes of 30 to 60° and
from Cairo in Egypt, to Helsinki in Finland and generally,
in Europe, Africa and North America and from the height
of zero to over 2000 meters above sea level (Khajehpour,
2006). Beet has a relatively wide consistency range with
an extensive resistance to water stress, salinity and cold.
Among agricultural products, sugar beet is a plant that
has allocated itself a special place in supplying a part of
energy required for human, in particular, in the third world
countries that do not have access to other energy
sources and it is considered as one of the key of economic,
agricultural and national components (Cook and
Scott, 1998).
Today, sugar beet provides 40% of world sugar trade
within less than 200 years progress (USDA, 2008). Sugar
beet cultivation in 41 countries is estimated as 8.1 million
hectares. In the United States, every year about 0.5
million hectares of lands are planted with sugar beet,
Germany with more than 0.4 million hectares and Italy
and England, each with more than 0.2 million hectares
are placed in next ranks (USDA, 2008). Different researches
have been done on ethanol production from sugar
beet. Theurer et al. (1987) evaluate different varieties of
sugar and fodder beets in different weather conditions
with respect to ethanol production potential. They stated
that the amount of ethanol produced from sugar beet is
more than fodder beet. Gibbons and Westby (1987) did a
study with the aim of optimizing ethanol production from
different sizes of fodder beet slices and achieved 4.83%
of ethanol.
Venturi and Venturi (2003) and Koga (2008) compared
agricultural products of temperate regions and concluded
that, energy consumption efficiency of sugar beet for
producing ethanol is much higher than those of wheat,
Mehdikhani et al. 4101
corn and oil seeds. A research conducted in Turkey
about ethanol production from sugar beet varieties
showed that this country has enough potential to supply
energy required for domestic consumptions and independence
from imports of fossil fuels (Icoz et al., 2009).
With respect to pure carbohydrates, sugar beet has
very high harvest index, because either the aerial part or
underground part of sugar beet can be used to ferment
and produce ethanol. On the other hand, sugar beet
compared with other agricultural products, can be
harvested in a three-month period and this provides a
good chance for ethanol production (Chatin et al, 2004).
In a research conducted in the UK, bio-ethanol values
produced from different crops were studied and it was
announced that 2688 and 5250 L of bio-ethanol were
produced from every hectare of wheat and sugar beet
farms, respectively (Anonymous b , 2010). In another study
in India, 0.38 and 0.126 L of ethanol were produced from
every kg of sugar and fresh sugar beet root, respectively
(Blazek, 2007).
Srivastava et al. (2008) observed significant differences
between different varieties with respect to bio-ethanol
production and announced that the varieties with more
root yield and higher sugar percentage are more
promising for ethanol production. Kosaric et al. (1983)
compared two types of yeast including Saccharomyces
cerevisiae and Saccharomyces diastaticus for bio-ethanol
production from fresh sugar beet root and announced
that the yeast of S. cerevisiae had more efficiency.
Undoubtedly, ethanol production from renewable materials
and biomass mostly depends on physico-chemical
properties of the materials, pre-treatment manner,
efficiency of zymogenic microorganisms and fermentation
conditions such as initial concentration of sugar, PH,
temperature, microbial density and fermentation time
(Camire and Camire, 1994; linko et al., 1983; Zhan et al.,
2003). Increase of fermentable biomass amount is an
issue that must be studied by different varieties with
respect to fermentation efficiency.
Plant breeding strategies can be implemented to
improve biomass yield, biomass quality, biomass
conversion efficiency, resistance to diseases and pests,
sugar content and other characteristics associated with
ethanol production. This study investigated the ethanol
production potential in some beet genotypes and its
relation with some characteristics such as root yield and
sugar content.
MATERIALS AND METHODS
The quality and quantity of the ethanol produced from sugar beet is
strongly dependent on variety. In order to evaluate some characteristics
of sugar beet varieties that affect Bioethanol production,
this experiment was carried out with ten beet varieties in
randomized complete block design (RCBD) with three replications
for two years (2008 to 2009) in Khoy Agricultural Research Station,
Iran. Ten beet varieties including 8 sugar beets and 2 fodder beets
were the most appropriate varieties with respect to plantation

4102 Afr. J. Biotechnol.
season and region. Some traits including green biomass, fresh and
dry weight of root, leaves and petiole, root yield, sugar content,
white sugar content, sugar yield, molasses, nitrogen, sodium and
potassium impurities, syrup purity, leaf area index (LAI), length of
root and green cover percentage were measured and recorded. At
the end of the growth season, all roots and crowns were harvested
and then samples were taken for recording some characteristics in
the laboratory. Fresh sugar beet root and raw sugar were used for
the ethanol production in the laboratory.
Preparing of yeast culture
The special yeast (S. cerevisiae) was maintained on malt agar
medium [contains yeast extract (3 g); malt extract (3 g); peptone (5
g); glucose(10 g); agar (20 g); all dissolved in 1 L of distilled water
and adjusted to pH 5.6] accordingly (Zayed and Foley, 1987).
Inoculum was prepared from 1 ml of yeast with 100 ml of the earlier
stated medium. After maintaining for 30 h in an incubator at 35°C,
the culture contained approximately 5 × 10 8 cells per ml.
Production of ethanol from fresh sugar beet root
After washing the roots of 20 kg of each treatment, using cutting
machine, the roots were cut into slices with a thickness of 1.5 mm
and a length of 5 to 7 cm (Mesbahi, 2003). Then, the slices were
kept in warm water (60°C) for 2 h with agitator timer till their syrup
leaked into the water (Jones et al., 1981). After filtration of the total
solution, the remaining syrup was separated from pulp by pressure.
The sap was boiled for 2 h in a vacuum till its concentration
reached the sufficient limit and also, its microbial contamination was
destroyed. Then the sap was fermented under 35°C and at pH 5.5
for 72 h (Srivastava et al., 2008) using a special strain of S.
cerevisiae Persian type culture collection (PTCC) 5269 obtained
from the Persian type culture collection of yeast cultures, Tehran,
Iran. After fermentation, the obtained solution was filtered and then
purified with a distiller apparatus.
Production of raw sugar from sugar beet
For the production of raw sugar, 20 kg beet root from each
treatment after washing with water was sliced to the dimensions of
1.5 mm in thickness and 5 to 7 cm in length (Mesbahi, 2003). In
whole, the current syrup in the slices after rinsing with pure water of
almost the same volume and temperature of 75°C was exited by a
press machine. In order to refine the obtained solution, lime and
CO2 were added, respectively. In this stage, the pH increased to
about 11 (Clarke, 1988). The solution was maintained in a closed
container for at least an hour with a gentle agitation at 85°C to
some of the impurities deposit. Then the solution was transferred to
a pre-provided apparatus with special tubes passing steam until it
was concentrated up to 70% at 120°C (Mesbahi, 2003). Some
sugar powder was added to the produced syrup in vacuum at
temperature below 100°C and it was kept at the same temperature
till sugar crystals were formed and enlarged. After cooling the
mixture, it was centrifuged at 1800 rpm for 5 min and the produced
sugar was weighed and kept after drying. The remaining syrup was
weighed as molasses (Mesbahi, 2003).
Production of ethanol from raw sugar
The sugar produced from each treatment with 25% concentration
was dissolved in distilled water at 40°C. The obtained solution was
fermented for 72 h under the earlier mentioned temperatures and
pH for all the treatments by using suitable population density of S.
cerevisiae. After filtering the produced solution, it was purified up to
94% by using distiller apparatus (Koga, 2008).
Statistical analysis
Ethanol concentration was determined by specific gravity method
(AOAC, 1990). Ethanol was analyzed using HPLC system with an
Aminex HPX-87H column (Bio-Rad, Hercules, CA) to compare the
results with earlier method. The mobile phase was 5 mm H2SO4
pumped at a flow rate of 0.6 ml/min. Data acquisition and analysis
were performed using the SHIMADZU EZ START 7.1.1 software.
The results reported were based on specific gravity method.
Analysis of variance (ANOVA), least significant difference (LSD)
and comparison of means were done using ASA (SAS, 1995).
RESULTS AND DISCUSSION
In the experiment of the ethanol production from fresh
sugar beet root, the total amount of distilled alcohol was
recorded for each treatment with 94% alcohol. Sugar
beet roots had around 10 to 18% sugar for different
varieties. So the theoretical yield of the produced ethanol
was almost 15% for fresh sugar beet root and around
50% for sugar. An important characteristic of the ethanol
production process is its feedstock quality, which makes
it susceptible to contamination by non-S. cerevisiae
yeasts. The most important aspect of the fuel-ethanol fermentation
is ethanol yield or more generally the industrial
yield. It depends on the fermentative capacity of the yeast
population (Grote and Rogers, 1985) and their resistance
to stress conditions (Bai et al., 2008).
Analysis of variance
Analysis of variance for sugar beet physiological and
quali-tative characteristics for two years showed that,
there was a significant difference among the different
varieties with respect to ethanol production potential from
fresh root and raw sugar. Varieties also showed significant
differences in some recorded traits including root
yield, sugar content, sugar yield, nitrogen, potassium and
sodium impurities, syrup purity and molasses yield. This
difference was completely normal because the various
types of beet including N, E and Z types and/or sugar and
fodder beet and also diploid, triploid and tetraploid were
applied in this study. These differences resulted in
different ethanol yields of the varieties.
Combined analysis of variance for morphological traits
for the two years showed that root length, leaf number,
leaf area index (LAI), dry weight of roots and crowns had
significant differences (p < 0.01).
Comparison of means
Comparison of some traits of beet varieties showed that

Table 1. Comparison of some traits of beet varieties
Mehdikhani et al. 4103
Variety Root Yield Sugar Yield White Sugar Yield Sugar Content K NA N
9597 57.33 c 9.68 bc 8.59 b 16.9 a 3.89 b 1.88 bc 2.23 abc
7233 57.86 c 9.90 bc 8.63 b 17.12 a 4.48 b 2.11 bc 2.34 abc
Shirin 58.14 c 10.37 bc 9.27 b 17.85 a 4.00 b 1.68 c b 2.42 abc
BR1 61.68 bc 10.14 bc 8.65 b 16.43 a 4.81 b 2.21 bc 2.93 ab
7112 71.35 b 11.40 b 9.59 b 16.03 a 4.42 b 3.05 ab 2.76 ab
IR2 87.49 a 14.51 a 12.42 a 16.58 a 4.56 b 2.54 abc 2.60 ab
37RT 65.67 bc 10.78 b 9.23 b 16.45 a 4.42 b 2.62 abc 2.37 abc
19669 68.13 b 10.05 bc 8.85 b 14.75 a 3.78 b 1.69 c 1.90 bc
Fd1 66.60 bc 8.03 cd 5.75 c 11.59 b 7.21 a 2.20 bc 1.17 c
Fd2 69.40 b 6.53 d 4.05 c 9.9 b 8.20 a 3.52 a 3.42 a
Numbers followed by similar letters are not significantly different at 5% level of probability.
Table 2: Comparison of beet varieties regarding ethanol production potential
Variety Ethanol from Root (L Ton–1 ) Ethanol from Root (L ha–1 ) Ethanol from Raw Sugar (L ha–1 )
9597 107.10 a 6194 bc 4725 b
7233 108.40 a 6334 bc 4750 b
Shirin 113.20 a 6637 b 5100 b
BR1 104.80 a 6489 bc 4756 b
7112 102.40 a 7294 b 5275 b
IR2 105.60 a 9285 a 6829 a
37RT 105.70 a 6901 b 5077 b
19669 93.64 a 6436 bc 4868 b
Fd1 74.57 b 4974 cd 3164 c
Fd2 63.25 b 4395 d 2228 c
Numbers followed by similar letters are not significantly different at 5% level of probability.
in the bioethanol production from fresh root of beet,
cultivar of IR2 had the highest root yield with 87.49 ton/h
and the highest ethanol yield with 9285 L/h (Tables 1 and
2). Also, IR2 with 12.42 ton/h of white sugar yield had
higher yield than the rest of the varieties, while fodder
beet varieties (Fd1 and Fd2) with 5.75 and 4.05 tone/h of
white sugar yield, respectively, had the lowest yield and
these varieties had the lowest bioethanol yield (Table 1).
The fodder varieties had impurities amounts of nitrogen,
potassium and sodium. Hence, the amount of molasses
production in these varieties also was more than the rest
(data not shown).
Theurer et al. (1987) evaluated sugar beet and fodder
beet varieties, and declared that sugar beet with 8640 L/h
of ethanol production, compared with 6380 L/h fodder
beet, had more potential for ethanol production.
Ethanol production from raw sugar, IR2 with 6829 L/h
had the highest value, while the fodder beet varieties with
3164 and 2228 L/h had the lowest ethanol yield (Table 2).
According to the qualitative analysis of raw sugar
obtained from the different varieties, it can be stated that,
the quality of sugar in different varieties had no difference
and so ethanol production was related to quantity of the
produced sugar for each variety. The amounts of
potassium and nitrogen impurities of the fodder varieties
were comparable with those of other varieties. Significant
difference between the two years of the tests was not
observed in terms of our studied traits. Regional weather
data also showed that, no large changes in climate
parameters were observed during the years 2008 and
2009 (data not shown).
Correlations of some sugar beet traits with ethanol
production
Correlation between some sugar beet traits with ethanol
production from the fresh root indicated that, ethanol yield
had high positive correlation with the sugar yield, white
sugar yield, sugar content, white sugar content, syrup
purity and dry weight of root. Also, it had significant
negative correlation with molasses sugar (Table 3).
The correlation between some sugar beet traits with
ethanol production from raw sugar is shown in Table 3.
The results indicated that, ethanol yield had high positive
correlation with sugar yield, white sugar yield, sugar

4104 Afr. J. Biotechnol.
Table 3. Correlation of some beet traits with ethanol production from fresh root and raw sugar.
Ethanol from Fresh Root (L/Ton) Ethanol from Raw Sugar (L/H)
Root Yield - 0.575 **
Sugar Yield 0.727 ** 0.969 **
White Sugar Yield 0.838 ** 0.986 **
Sugar Content 0.921 ** 0.828 **
White Sugar Content 0.985 ** 0.830 **
Potassium Impurity 0.266 -0.475 *
Sodium Impurity 0.340 -0.218
Nitrogen Impurity 0.112 0.070
Syrup Purity 0.829 ** 0.655 **
Molasses Sugar -0.628 ** -0.342
Root Length -0.002 0.584 **
Number of leaves -0.128 0.174
Leaf Area Index 0.007 -0.102
Fresh Weight of Crown 0.038 -0.093
Dry Weight of Crown -0.037 0.427 *
Dry Weight of Root 0.369 * 0.582 **
**& * represent correlations with significant levels of 0.01 and 0.05, respectively.
content, white sugar content, root yield, syrup purity, root
length, dry weight of root and crown. Also, it had
significant negative correlation with potassium impurity.
Sodium and nitrogen impurities, number of leaves and
leaf area index (LAI) had no significant correlation with
ethanol production.
In this study, S. cerevisiae was used for the fermentation
and the used sugar type was sucrose that can be
converted to bioethanol. Hence, cellulose tissue in fresh
beet root cannot have great role in ethanol production
(Singh et al., 1995). Therefore, the high correlation
between ethanol yield and white sugar in this study
seems to be reasonable. Variations of 32 to 43% for
ethanol yields were observed among the 10 beet
varieties. The effect of genotype on ethanol production is
related to both the chemical composition and morphological
properties of the beet root samples, with a
stronger effect observed for chemical composition such
as sugar content and impurities. Ethanol production
increased as sugar content increased, whereas the
ethanol production decreased as nitrogen and potassium
impurities increased. Further research is needed to test a
broad number of varieties across a wide range of growing
conditions to further evaluate the effects on ethanol
fermentation yields. Both sugar content and root yield
were strongly correlated with ethanol yield based on
regression analysis using single chemical composition.
The relationship between sugar content and theoretical
percentage of ethanol yield indicated that, sugar content
had significant effect on ethanol yield (Table 3).
Zhan et al. (2003) studied a number of varieties of grain
and fodder sorghum and found that the chemical composition
and physical properties of grain are very important
factors on ethanol production potential.
Conclusions
Bioethanol production from sugar beet via fermentation
technology is a promising fuel alternative. In order to
produce ethanol from sugar beet and by-products via
fermentation, it is important to know the correlations
between some morphological and physiological traits with
ethanol production. The novelty of this study compared
with other investigations is to produce ethanol from fresh
sugar beet root. It was observed that, several varieties
had different ethanol production potentials highly
correlated with root quantitative and qualitative traits. In
fact, this study highlighted breeding of sugar beet
varieties particularly for ethanol production. Based on the
analysis of the experimental data, ethanol production
from fresh root has more efficiency than fermentation of
raw sugar. Among all of the investigated varieties, sugar
beet varieties produced more ethanol per hectare than
fodder beet. Sugar beet varieties had more root yield and
sugar content than fodder beet and these two
characteristics play basic role in ethanol production. The
adapted sugar beet hybrids showed better promise than
fodder beet as a fuel crop in the USA, since sugar beet
produces an equal or greater quantity of fermentable
sugar, it has less bulk to transport and more extractable
sugar per unit mass (Theurer et al., 1987).
The results obtained from this research showed that, a
limited number of beet genotypes had significant effects
on the potential of ethanol production. A significant difference
(p < 0.01) was observed in ethanol yield among
varieties in both methods of ethanol production. Also,
highly significant differences were observed in the
morphological and physiological traits among the varieties.
Genotype variation effects were found on ethanol

yield, that is, highly related to the chemical composition of
roots, especially sugar content, potassium impurity, crude
syrup purity and some morphological characteristics such
as root length and root dry matter. In addition, ethanol
production increased as sugar content and root yield
increased.
When sugar beet was compared with other sugar
crops, its irrigation requirement was less and so, it was
suitable in our agricultural system. It needs more ethanol
from beet genotypes and support from private sugar
industry from all over the world. This subject may create
a new vista.
ACKNOWLEDGMENT
The authors would like to acknowledge all personnel of
biotechnology research institute in Urmia University
especially Dr. Sabzi for preparation of all apparatus and
services. The authors gratefully acknowledge also Dr.
Taleghani and Dr. Mahmoodi, directors of sugar beet
seed institute for the analysis of sugar beet pulps in their
institute lab and Dr. moghaddamnia for kindly reviewing.
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African Journal of Biotechnology Vol. 10(20), pp. 4106-4113, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2211
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Modeling and experiment to threshing unit of stripper
combine
Xu Lizhang* and Li Yaoming
Key Laboratory of Modern Agricultural Equipment and Technology, Ministry of Education and Jiangsu Province, Institute
of Agricultural Engineering, Jiangsu University, Zhenjiang 212013, China.
Accepted 7 April, 2011
The aim of this study was to establish the mathematical models to axial threshing unit of stripper
combine harvester. The exponential distribution model of the un-threshed grain Tu ( z ) , free grain Tf ( z )
and cumulative gain F( z ) along the axial threshing drum z ∈ [0, L]
was obtained. The loss of
separation L f was the free grain which had reached the end of the threshing unit but still not
separated. On the designed testing equipment, experiments of threshing performances were conducted
with the different feed rates and drum rotator speeds for the rice stripped mixtures. Experimental
results showed that the distribution of the cumulative separated gain was in agreement with the
simulation. The results were very useful for practical analysis of grain separation and damage as well
as for threshing unit design and process optimization.
Key words: Stripping, combine harvester, threshing unit, model, experiment.
INTRODUCTION
The stripper combine is combined with a stripper header.
And the basic concept of the stripper header is that a
rearwards rotating rotor fitted in the front of the header is
fitted with 6 to 8 rows of stripping fingers that strip grain
from the crop as the combine moves the head forwards
while it spins backwards (Price,1993; Jiang et al., 2001).
The speed of the rotor can be varied according to crop
conditions. Those stripped by stripping fingers was called
the stripped mixtures, which include the free grain, the
un-threshed grain and some straws etc. For this reason,
characteristic of the stripped mixtures which was fed into
the threshing unit of the stripper combine through the
feeder house was quite different from the material fed into
the threshing unit of conventional combine harvester.
A scientific model which can describe the threshing
process is extremely important to study threshing unit.
Based on the view of mechanics, Trollope (1982) deduced
a set of differential equations which describe the
threshing process, but it is difficult to apply.
*Corresponding author. E-mail: lzxu1979@gmail.com. Tel: +86-
0511-88797214-219. Fax: +86-0511-88780175.
On the assumption of the rule of threshing process, which
meets the qualification of exponential distribution, Huynh
(1982) established a mathematical model, however, it is
too simple and experiential, and it has great differences
with the actual situation. Taking into account the
parameters of threshing process, and properties of gain,
a rather nice probability model of the threshing cylinder
with rasp bar was published (Wan et al., 1990; Zhou et
al., 1998; Yin, 1999).
Miu (2002 a, b) presented a more detailed model of the
axial threshing unit of the conventional combine harvester,
but it is not completely suitable for the threshing unit
of stripper combine. Specific objectives of the work reported
in this study were to: (1) establish a mathematical
model for describing the threshing process; (2) perform
experimental studies on the designed equipment for
threshing performance; (3) simulation by computer on the
mathematical model.
Model development
As shown in Figure 1, a threshing separation unit is in
common use in the stripper combine harvester. The sign

l
rotor
concave
L
straw export
Lizhang and Yaoming 4107
o z
feeding
Figure 1. Schematic diagram of axial threshing unit.
“l ” denotes the breadth of the entrance of the unit and
“L” denotes the length of the threshing platen. Taking the
point of intersection between the axial line of the platen
and the side face of the entrance as the origin, the central
axis of platen is defined as the z axes and the direction of
axial movement of the grain is set for the plus direction of
the z axes.
Assume that the feed rate of the mixture is constant;
the mass of the mixture fed into the threshing unit per
second is 1, then:
z
z ) = ( k 1 + k )
(1)
l
q s ( 2
Where, qs (z ) is the mass of grain fed into the threshing
unit per second over the interval [0,z], z ∈ [ 0,
l]
; k1 is the
percentage of the free grain, which has been threshed
but not separated, in the total mass of the mixture and
k2 is the percentage of the un-threshed grain, which has
not been threshed, in the total mass of the mixture.
Experimental results shows that the mixture in threshing
space includes the free grain (60 to 70%), the unthreshed
grain (15 to 25%) and the straw (about 10 to
20%). Therefore, the threshing process of the rice
stripped mixtures includes two parts. One, the unthreshed
grain are threshed, the other is the free grain
separated form the concave during screwing with the
rotator.
Development of the model is based on the following
assumptions: (1) the probability of grain threshed and
separated during the threshing space is equal in the
plane through the arbitrary point of the z axis; (2) within
the region from z to z+ ∆ z , the probability of threshing for
the un-threshed grain is in direct proportion to the amount
of the un-threshed grain. And the proportion coefficient,
named as 1 u , is related to the parameters, such as the
rotator speed, the cylinder-concave clearance, the structure
of concave and the properties of the grain etc; (3)
within the region z to z+ ∆ z , the probability of separating
form the concave for the free grain is in direct proportion
to the amount of the free grain. And the proportion
coefficient, named as u 2 , is also related to the parameters
such as the rotator speed, the cylinder-concave
clearance, the structure of concave and the properties of
the grain etc.
According to assumptions (1) and (2), if ∆z ->0, then:
[ 1 P(
z)
]
p1( z)
u1
− 1
= (2)
Where, 1( ) z p is the probability of grain threshing at a z
point and 1( ) z P is the mass of the threshed grain within
the region from 0 to z, So:
∫
P ( z)
p ( s)
ds
(3)
1
= z
0
1
It can be obtained as follows:
p ( z)
1
−u1z
= u1e
(4)

4108 Afr. J. Biotechnol.
Similarly, it is known:
p ( z)
2
u
e
−u2
z
= 2
(5)
Where, 2 ( ) z p is the probability of free grain separating
out of the concave at z point; At z ∈[ 0,
l]
is the mass of
the un-threshed grain is equal to subtraction of the
threshed mass from the fed-in mass:
T
u1
( z)
z
z z −u
s z
1
−u1z
= k2
− k2
u1e
ds = k2
e
l l ∫ (6)
l
0
Where, 1( ) z Tu is the mass of the un-threshed grain at
z ∈ [ 0,
l]
.
Similarly, when z ∈ [ l,
L]
, the equation can be expressed
as follows:
T
u2
( z)
z
−u1
( s−l
)
−u1z
= k 2 − Tu1(
l)
∫ u1e
ds = k 2e
(7)
0
Where, 2 ( ) z Tu is the mass of the un-threshed grain;
z ∈ [ l,
L]
.
If z= L, then:
L
t
−u1L
= Tu
2 ( L)
= k2e
(8)
Where, L t is the loss of the un-threshed.
According to the probability theory, the probability of
grain separation is equal to the convolution of the
probability of the un-threshed grain threshing and the
probability of the free grain through which the concave
occurs (Miu, 2002b). As z ∈ [ 0,
l]
:
z z uu z z
f ( z) = k p( z) ∗ p ( z) + k p( z) = k ( e − e ) + k ue
1 2 −u2 z −uz 1 −u2
z
1 2 1 2 1 2 2 1 2
l l u1 −u2
l l
Where, f ( ) is the probability of grain separation;
z ∈ [ 0,
l]
.
Integrate:
F(
z)
1
1 z
k z
[ u1(
1−e
u −u
l
) −u
( 1−e
z
)] + k1
( 1−e
l
)
(9)
2
−u2z
−u1z
−u2z
= (10)
1
2
2
Where, 1( ) z F is the cumulative mass of the separating
grain; z ∈ [ 0,
l]
.
Similarly, when [ l,
L]
z ∈ :
k2
−u2z
−u1
z
−u2z
F2(
z)
= [ u1(
1−e
) −u2(
1−e
)] + k1(
1−e
) (11)
u −u
1
2
Where, 2( ) z F is the cumulative mass of the separated
grain; z ∈ [ l,
L]
.
The grain of the rice stripped mixtures which fed into the
threshing unit finally is divided into three parts: the unthreshed
grain, the free grain and the separated grain.
Hence:
⎧
⎪
⎨
⎪
⎩
z
Tu1(
z)
+ Tf
1(
z)
+ F1(
z)
= ( k1
+ k2)
l
T ( z)
+ T ( z)
+ F ( z)
= k + k
u2
f 2
2
1
2
( z∈[
0,
l])
( z∈[
l,
L])
(12)
Where, T f ( ) , ( ) are the free grain corresponding
1 z
T f 2 z
to different positions z over the rotor length.
Hence:
⎧
⎪T
⎪
⎨
⎪T
⎪⎩
f 1
f 2
z
( z)
= [ k
l
( z)
= k
2
2
u1
u −u
u1
( e
u − u
1
1
2
2
( e
−u2z
−u2z
−e
− e
−u1z
−u1z
) + k e
) + k e
−u2z
1
−u2z
1
]
(13)
The free grain which has reached the end of the
threshing unit but still not separated is called the loss of
separation:
L
u
1 −u2L
−u1L
−u2L
f = Tf
2 ( L)
= k2
( e − e ) + k1e
(14)
u1
−u2
Where, L f is the loss of separation.
MATERIALS AND METHODS
Experimental equipment
The testing equipment of axial threshing unit includes rice stripped
mixtures conveyor, feeding unit, threshing unit, data acquisition and
control system etc., as shown in Figure 2. The rice stripped
mixtures conveyor, feeding unit and threshing unit are driven by
different electromotor whose rotator speed are controllable. The
structural parameters such as the cylinder-concave clearance,
height and screwing angle of the oriented board on the cover

Lizhang and Yaoming 4109
Figure 2. Schematic on testing equipment of axial threshing unit. 1, stripped mixture conveyor; 2, intakes; 3, feeding chain; 4, swath
deflector; 5, threshing drum cover; 6, threshing drum; 7, separation concave; 8, frame; 9, straw outtake; 10, grain box; 11, valve handle;
12 straw boxes.
Figure 3. Distribution of spike tooth on threshing drum.
threshing element are adjustable. The sensor is installed to inspect
the rice stripped mixtures conveyor speed, while sensors of rotate
speed and torque are installed on the axis of feeding chain wheel
and threshing drum. The side boards consist of transparent glass,
so it is convenient to observe movements of grain in the test device
(Xu, 2003).
In order to keep the environment of field, structure and motion
parameters suitable, the testing are similar to the 4LGT-150 stripper
combine harvester in marketing. Variety: late japonica rice; thousand
grain weight: 29.4 g; mass percentage of the free grain in the rice
stripped mixtures: 65%; mass percentage of the un-threshed grain
in the rice stripped mixtures: 20%; mass percentage of the straw in
the rice stripped mixtures: 15%; moisture content of mixture: 37.2%;
moisture content of grain: 24.3%; moisture content of straw: 61.2%;
rotor radius: 297mm; entrance breadth ( l ):480 mm; whole rotor
length ( L ):1580 mm; threshing element: spike tooth (distribution of
spike tooth is shown in Figure 3); number of tooth bar: 6; cylinderconcave
clearance: 8 mm; concave style: grid concave; concave
enveloping angle: 220°; conveyor speed: 1 m/s; feeding chain wheel
rotate speed: 466 r/min.
Experimental methods
The whole length of threshing rotor was divided into 10 testing
zones, the entrance breadth 5 equally uniforms and the rest 5
equally divvied, as shown in Figure 4. The testing arrangements

4110 Afr. J. Biotechnol.
Figure 4. Distribution of the testing zones.
Table 1. Experimental result of the threshing unit.
Serial
number
Feed rate /kg.s -
1
Rotator rotate speed
/r.min -1
Separation loss
rate/%
Un-threshed loss
rate/%
Total loss
rate/%
1 1.5 750 0.154 0.304 0.458
2 1.5 850 0.158 0.226 0.384
3 1.5 950 0.161 0.172 0.333
4 1.8 750 0.206 0.544 0.75
5 1.8 850 0.212 0.376 0.588
6 1.8 950 0.223 0.208 0.431
7 2.0 750 0.328 0.51 0.838
8 2.0 850 0.344 0.448 0.792
9 2.0 950 0.338 0.244 0.582
were carried out on three feed rates and three rotate speeds, as
listed in Table 1. Each group repeats 3 times.
Before the test begin, the rice stripped mixtures of certain mass
are uniformly placed on the conveyer 30 m long. In the process of
test, the rice stripped mixtures were fed into the threshing space, in
which the un-threshed grain is threshed and the free grain is
separated through feeding chain. Grains from the concave grate
drop into the testing zone of the grain box. The ejection from the
threshing unit falls into the straw box with the straw, from which the
un-threshed loss and the separation loss can be obtained.
s
Separation loss rate s 100%
T
L = × (15)
W
Where, T s is the mass of the un- separation grain and W is the
mass of the total grain including un- separation grain and unthreshed
grain.
Un-threshed loss rate:
Tu
L t = × 100%
(16)
W
Where, T u is the mass of the un-threshed grain.
Total loss rate:
Lto = Lt + Ls
(17)
RESULTS AND DISCUSSION
Experimental analysis
The experimental results on the rice stripped mixtures to
the threshing unit are listed in Table 1 with three different
feed rate and three levels of the rotate speeds of the
threshing drum.
It is known from the table that: (1) at the same feed
rate, rotate speed of threshing drum has an obvious
effect to the loss rate of un-threshed and the higher the
rotate speed is, the smaller the un-threshed loss rate. But
it is unobvious to the loss rate of separation; (2) on the
condition that other parameters do not change, the feed

Cumulative mass percent /%
A
B
Figure 5. Axial distribution of grain and straw.
rate of the rice stripped mixtures into the threshing unit
has a remarkable effect on the un-threshed loss rate and
separation loss rate and the larger the feed rate is, the
bigger both the loss rate increase accordingly.
As feed rate is 1.8 kg/s and rotate speed of threshing
drum is 850 r/min, the axial distribution of the grain in
testing zone is shown in Figure 5. It can be deduced from
the figure that: (1) as shown in Figure 5a, the grain
separated from the concave grate increases rapidly in
width of the feeding entrance and reaches maximum at
the center point of the entrance, then reduces fast. The
curve of grain falls slowly in the rest length, which tends
to zero by the end of the threshing drum axis. It means
that the loss of grain is little. The distribution of the straw
separated from the concave grate is average along the
whole length; (2) it is known from Figure 5b that, about
70% of the grain has been separated during width of the
feeding entrance (1/3 whole length). If no measure is
adopted, the load to cleaning must be uneven, so that the
cleaning performances become worse.
Simulation analysis
Lizhang and Yaoming 4111
As feed rate is 1.8 kg/s and rotate speed of threshing
drum is 850 r/min according to the test, it can be
observed that the un-threshed loss rate is 0.376% and
the separation loss rate is 0.212%, then solving equation
(8) and (14), it is obtained as follows:
u 1 =2.5151; 2
u =6.994. (18)
Where, l =480 mm; L =1580 mm; k 1 =65% and
k 2 =20%.
Using equations (1), (6), (7), (10), (11) and (13),
simulations are conducted by computer, as shown in
Figure 6. It is known from the simulation that: (1) the
cumulative mass of the separated grain increased rapidly
as exponential curve during the entrance of the mixture,
after which, curve of the mass went gentle gradually. In

4112 Afr. J. Biotechnol.
Figure 6. Variation of the indices of threshing process.
the whole drum, nearly 70% of the grain has been
separated in the anterior part of one third of the whole
length, which is in agreement with the experimental
results, as shown in Figure 5b; (2) at first, the mass of the
free grain in the threshing space increases continuously
and it reaches maximum at the end of the feeding
entrance, then it decreases gradually. By the end of the
threshing drum, the value of the free grain mass is called
the loss of separation; (3) comparatively, the mass of the
un-threshed grain in the threshing space changes slowly,
it also increases firstly and then reduces, which only
accounts for 20% in the rice stripped mixtures. So, it
seems small comparatively. By the end of the threshing
drum, the value of the un-threshed grain mass is called
the loss of un-threshed.
Conclusion
In this study, a mathematical model of threshing unit with
axial feeding of the stripper combine is established with
the method of probability and simulations are also
conducted. The simulations show the variation of the
indices of threshing process along threshing drum axis,
including un-threshed gain, free gain and cumulative
separated gain. On the self-designed testing equipment,
experiments on threshing performance with different feed
rate and drum rotator speed are conducted for the rice
stripped mixtures. Experimental results show that, the
distribution of the cumulative separated gain is in
agreement with the simulation. It is known from the test
that about 70% of grain is separated in the width of
feeding entrance, so the measures must be taken to
make the cleaning loads even. It is helpful for a better
understanding of threshing processes in axial units and
significant for the design of the threshing unit of the
stripper combine.
ACKNOWLEDGEMENTS
This work was supported by a grant from the National
Natural Science Foundation of China (No. 50475123),
high-tech key laboratory of agricultural equipment &
intelligentization of Jiangsu province (No. BM2009703 )
and A Project Funded by the Priority Academic Program
Development of Jiangsu Higher Education Institutions
(PAPD). The authors would like to thank the reviewers for
their helpful suggestions on the manuscript.
REFERENCES
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model. Trans. ASAE, 25(1): 65-73.
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African Journal of Biotechnology Vol. 10(20), pp. 4114-4120, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2368
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Yield responses of forage sorghums to salinity and
irrigation frequency
Siti Aishah 1 , H. A. R. Saberi 1 *, R. A. Halim 1 and A. R. Zaharah 2
1 Department of Crop Science, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
2 Department of Land Management, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
Accepted 18 March, 2011
Water stress restricts crop yield in both the arid and semi-arid zones of the world. In particular, water
stress is associated with low availability of water, as well as osmotic effects associated with salinity.
The response of forage sorghum [Sorghum bicolor (L.) Moench] varieties to salinity and irrigation
frequency were studied from December 2007 to December 2008. Speedfeed and KFS4 were grown
under salinity levels of 0, 5, 10 and 15 dS m -1 and irrigated when the leaf water potential reached -1.0
(control), -1.5 and -2.0 MPa. The irrigation frequency was found to affect growth and yield of the forage
sorghums. When irrigation was delayed in leaf water potential of -1.0 to -2.0 MPa, the yield and yield
components were found to decrease. The maximum dry forage yields were 45.1, 38.9 and 38.5 g plant -1
for frequent, intermediate and infrequent irrigation regimes, respectively. Increased salinity significantly
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
every one unit increase in salinity, the forage yield decreased by 5.2 units and for every one unit
increase in water stress (irrigation frequency), the forage yield decreased by 3.6 units. The variety
Speedfeed had higher total dry mass than KFS4 under well-watered conditions but KFS4 performed
better than Speedfeed under water stress. For both varieties, infrequent watering reduced dry matter
and biomass accumulation, but increased water use efficiency (WUEs) (6.88).
Key words: Salinity, irrigation frequency, dry matter, water use efficiency (WUEs), forage sorghum.
INTRODUCTION
Salinity is one of the most important environmental
factors limiting crop production of marginal agricultural
soils in many parts of the world (Qureshi et al., 2007).
Salts in soil and water can reduce water availability and
this can lead to stressful conditions. Water stress restricts
crop yields, particularly in the arid and semi-arid zones.
However, cultivation under salinity and water stress
conditions is normally practiced for food supply in developing
countries (Munns, 2002). Sorghum has greater salt
and drought tolerances than other summer forages. Only
warm season annual grasses provide substantial forage
yield in a short period of time. There are a number of
potential forage sorghum varieties which may be appro-
*Corresponding author. E-mail: alireza_sa70@yahoo.com.
Abbreviations: LWP, Leaf water potential; FC, field capacity;
PWP, permanent wilting point.
priate for various salinity levels of seawater, but the levels
of salt tolerance among most of the grown forage sorghum
varieties and cultivars have not been adequately
characterized (Qadir and Oster, 2004). The general effect
of salinity is the reduction in growth (Ghoulam et al.,
2002) which resulted from ion toxicity, osmotic stress,
mineral deficiencies, physiological and biochemical
perturbations, as well as the combinations of these
stresses (Munns, 2002; Hasegawa et al., 2000). Due to
low availability of soil moisture from osmotic effects associated
with salinity, water stress occurs during crop
establishment and development. However, where the
growing season is long, tillering varieties of forage
sorghum are able to recover to a certain extent from
water deficits in the early growth periods by forming additional
head-bearing tillers. The resulting yield reduction
can be partly offset by additional head-bearing tillers
(FAO, 2001). Despite significant contributions, earlier
investigations on sorghum were focused primarily on
grain sorghum. Hence, studies are still needed for forage

sorghum to improve the understanding of the effects of
salt and water stresses for different varieties. Many
factors are still needed to be considered when addressing
the suitability of irrigation water with respect to
salinity. Therefore, the objective of this experiment was to
determine the growth responses of two forage sorghum
varieties to different levels of salinity and irrigation
frequency.
MATERIALS AND METHODS
The factorial experiment was conducted under rain shelter at the
University Putra Malaysia (02°N 59.476´ 101°E 2.867´, 51 m
altitude), from December 2007 to December 2008. The climatic
conditions recorded under the rain shelter were 30°C mean temperature,
90% humidity, 4.5 mm evaporation and 72.5% light at 12
am. Two selected (Fouman et al., 2003) salt tolerant varieties
namely Speedfeed and KFS4, of forage sorghum [Sorghum bicolor
(L.) Moench] were subjected to the salinity levels of 0, 5, 10 and 15
dS m -1 of NaCl concentrations, and irrigated when the leaf water
potential (LWP) reached -1.0 (control), -1.5 and -2.0 MPa. KFS4 is
an open pollinated variety from Iran, while Speedfeed is a hybrid
variety originally from Australia.
The treatments were arranged in a randomized complete block
design with three replications. Polybags (40 × 45 cm) were filled
with a mixture of top soil, peat moss and sand at the ratio of 3:2:1
(v/v), respectively. The soil mixture had a pH of 5.4. During mixing,
60 g of CaCO3, 10 g of complete fertilizer (15% N, P2O5, K2O), 1 g
of triple super phosphate (45% P2O5) and 2.4 g of urea (46% N)
were added to each polybag. Soil field capacity (FC) and
permanent wilting point (PWP) were measured based on pressure
plat and pressure membrane procedures outlined by Richards
(1947), before and after the end of the experiment. Soil moisture
was determined by gravimetric method (Muhammad et al., 2008).
The plants were irrigated with non-saline water for seedling
establishment and with saline water starting from 2 weeks after
germination according to the treatments. Plants were harvested at
pre flowering stage and washed with deionized water. Leaves,
stems and roots were separately weighed and dried at 75°C for 48
h for dry mass determination. Forage weight was calculated based
on cumulative shoot dry weight. Irrigation treatments were applied
based on leaf water potential (-1, -1.5 and -2 MPa) using the
pressure chamber (Santa, Barbara, CA, USA). When leaf water
potential reached the considered potential, the soil samples were
taken. The amount of water required for the respective irrigation
treatment was calculated using the following equation (Muhammad
et al., 2008):
V = SMD × A
Where, V = volume of water to be applied (litre); A = polybag area
= חr 2 .
SMD = ( FC θ
θ
- i ) D Bd /100
θ
SMD = soil moisture deficit; FC = gravimetric soil moisture
content at field capacity (%);
θ i = soil moisture content before irrigation (%); D = rooting depth
(cm); Bd = bulk density (1.5 g cm -3 in this soil).
Water use efficiency (WUE) was determined by dividing the total
dry forage by the volume of irrigation water applied in each
treatment during the experimental period. The data were subjected
Aishah et al. 4115
to analysis of variance (ANOVA) using the Statistical Analysis
System (SAS) procedure. Treatment means were compared using
least significant differences (LSD) at the 5% (P ≤ 0.05) probability
level. Regression analysis was used to determine the relationship
among variables and salinity levels.
RESULTS AND DISCUSSION
The irrigation frequency and salinity affect growth and
yield of forage sorghums. The above ground part which is
important in forage sorghum was highly affected by the
main factor, while the total dry weight was significantly (P
< 0.01) influenced by the interaction of salinity, irrigation
and variety (Table 1).
The total dry mass of both varieties declined as salinity
level increased and water regime decreased (Figure 1).
Salinity may affect the growth by creating an external
osmotic potential that prevents water uptake or due to the
toxic effects of Na + and Cl - ions on the plant, it affects the
uniformity of plant density with negative effect on yield
(Okcu et al., 2005). Our result is also in agreement with
Munns (2002). Between varieties tested, the interaction
effect was more significantly pronounced on the
Speedfeed. This was shown by 41.5% decreased in its
biomass under normal irrigation with increasing salinity to
15 dS m -1 (Figure 1B) as compared to 30.5% for the
KFS4 (Figure 1A). At other levels of irrigation (irrigation at
-1.5 and -2 Mpa), from 0 to 15 dS m -1 , the Speedfeed
biomass decreased by 23.8 and 24.1%, respectively.
Whereas for the KFS4, the greatest biomass differences
between irrigation regimes in respect to salinity, were
found only at the highest salinity level. This response
would indicate that the KFS4 variety is more tolerant to
stress conditions than the Speedfeed. Furthermore,
based on varietal characteristic, their total biomass is not
significantly different from each other (Table 1).
Although irrigation and salinity had highly significant
effects on dry forage yield and root growth, an interaction
effect was only found between irrigation and variety.
Irrespective of variety, the dry forage decreased linearly
with increasing salinity (Figure 2A). A similar result was
reported by Hester et al., (2001) who indicated that the
crop yield performance decreased markedly with the
increase in the concentration of salt. A linear reduction
due to salinity was also found on root mass (Figure 2B).
Based on varietal responses (Table 2), Speedfeed was
more sensitive to water stress as indicated by a
significant reduction in its dry root and dry forage. The
impact on Speedfeed however was greatly shown only
when the irrigation schedule changed from -1 to -1.5 Mpa
and as a result, the forage yield decreased by 22.2% and
roots declined by 29.4%. However, further delay in
irrigation did not show any significant reduction in both
parameters. For KFS4, irrigation schedule did not affect
its forage yield and root growth. This characteristic may
contribute favourably to the persistence of this species in
saline soils under natural condition. In irrigated agriculture,

4116 Afr. J. Biotechnol.
Table 1. Effects of varieties, irrigation and salinity on dry matter yield and root-to-shoot ratio.
Source / Treatment
Dry root
(g plant -1 )
Dry forage
(g plant -1 )
Total dry weight (g
plant -1 )
Root to
shoot ratio
Variety
KFS4 7.47a z 42.25a 49.72a 0.176b
Speedfeed 7.96a 39.41b 47.42a 0.201a
LSD 0.59 2.66 3.23 0.021
Irrigation frequency (MPa)
at LWP -1.0 8.60a 45.12a 53.729a 0.731a
at LWP -1.5 7.41b 38.88b 46.298b 0.611b
at LWP -2.0 7.13b 38.48b 45.624b 0.631b
LSD 0.76 3.42 4.164 0.024
Salinity (dS m -1 )
0 8.93 a 45.73 a 54.669 a 0.657b
5 8.97 a 43.61a 52.592 a 0.736a
10 7.27 b 39.79b 47.073 b 0.653a
15 5.68 c 34.17c 39.867 c 0.591b
LSD 0.91 4.08 4.963 0.029
F value
V*I 5.91** 4.37* 15.66** 75.29**
V*S 0.07 ns 0.03 ns 1.38 ns 17.84**
I*S 0.27 ns 0.29 ns 0.75 ns 15.04**
V*I*S 0.75 ns 0.70 ns 3.08** 22.42**
Error and CV
Error (MS) 1.57 31.43 46.43 0.001
CV (%) 16.25 13.73 14.03 6.08
z Means with same letter within a column are not significantly different at 5% level by LSD. V1 = KFS4, V2 =
Speedfeed; S1, S2, S3 and S4 = salinity 0, 5, 10 and 15 dS m -1 , respectively; I1, I2 and I3 = irrigation
frequency when the LWP reach -1, -1.5 and -2MPa, r
salt would normally be leached from the surface at
sowing, and in dry-land agriculture, the crop is normally
planted after rain (Serraj and Sinclair, 2002).
Unlike the dry forage yield and roots, root to shoot ratio
of both forage varieties responded differently to salinity
and irrigation. The root shoot ratio at normal irrigation
exhibited by KFS4 (Figure 3A) seems to be opposite to
Speedfeed (Figure 3B). The root shoot ratio of KFS4
irrigated at normal frequency was lower than plants under
water stress conditions at all levels of salinity. This
phenomenon is a normal plant growth reaction towards
water stress conditions. On the other hand, the root-toshoot
ratio of well-watered Speedfeed was higher than
that under infrequent irrigation. The higher root-to-shoot
ratio of KSF4 under water stress would probably explain
the better performance of KFS4 under water stress
conditions.
Table 3 shows that infrequent irrigations gave higher
WUE’s when compared to frequently irrigated treatments.
In this study, the WUEs obtained for the infrequently
watered plants were higher than earlier reports (Gulzar et
al., 2003; Mustafa and Abdel Magid, 1982). Although
larger volumes of water were used in this investigation as
compared to the two previous reports, the higher WUEs
obtained here might have been due to the reduction of
irrigation by infrequent watering. Irrespective of variety,
the frequently watered plants accumulated greater dry
matter which eventually had produced high dry forage
yield than other irrigation frequencies (Table 3).
The results derived for the irrigation study showed that
despite the possibility of greater surface evaporation with
the light frequent irrigations, sorghum varieties and other
indicators of plant water stress were found to be
improved with low frequent irrigation. Pearson correlation
matrix reveals that most of the studied characters are
significantly and positively correlated, on the other hand,
root to shoot ratio is negatively correlated with all the
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
(average of two years).
forage, dry stem, dry leaf and dry root (Table 4). Similar
scenario is observed for the forage dry weight with yield
components.
The regression reveals that the forage yield is
significantly and negatively correlated with salinity and
irrigation frequency (F < 0.05). The relationship between
yield, salinity and irrigation frequency was studied, and
Aishah et al. 4117
the regression equation was: Y=-3.277 -5.207X1-3.586X2,
where R 2 = 0.90 (n = 72). For every one unit increase in
salinity (X1), the forage yield would be decreased by 5.2
units and for every one unit increase in water stress (X2),
the forage yield would be decreased by 3.6 units.
Ultimately, when irrigation was delayed from -1 to -1.5
and -2MPa, the forage yields were decreased significantly,

4118 Afr. J. Biotechnol.
Figure 2. Effects of salinity on forage (A) and root dry weight (B) of forage sorghum.
Table 2. Interaction of variety and irrigation on dry root and dry forage mass.
Irrigation schedule
Dry root (g plant -1 ) Dry forage (g plant -1 V1 V2
)
V1 V2
I1 7.97 a 9.98 a z 43.78 a 46.46 a
I2 7.22 a 7.04 b 41.62 a 36.14 b
I3 7.22 a 6.85 b 41.34 a 35.63 b
LSD (%) 1.231 2.131 4.176 7.961
z Means with same letter within a column are not significantly different at 5% level by LSD; V1 = KFS4, V2 =
Speedfeed; I1, I2 and I3 = irrigation frequency when the LWP reach -1, -1.5 and -2MPa, respectively.
regardless of variety from 45.1 to 38.9 and 38.5 g plant –1
for frequent, intermediate and infrequent irrigation
regimes, respectively. The reduction was found to be
mostly between the frequently and the less and least
frequently watered plants which decreased (Table 3).
This finding suggests that in semi-arid environments
(where water saving is very important), if the aim is to get
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
irrigation frequency.
Table 3. Effect of irrigation frequency on yield, total amount of water applied and water use efficiency (WUE).
Irrigation
schedule
Days after treatment Dry forage
4 14 22 31 36 41 48 56
Amount of water applied (liter/plant)
yield
(g plant- 1 )
Total
water
used (liter)
Aishah et al. 4119
WUE (g
plant- 1
liter- 1 )
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
I2 0.5 1 0 1.19 0 1.42 0 1.98 38.88 b 6.10 b 6.35 ab
I3 0.5 1 0 0 2.02 0 0 2.12 38.48 b 5.64 c 6.88 a
LSD (5%) 3.42 0.61 0.60
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
not significantly different at 5% level by LSD.

4120 Afr. J. Biotechnol.
Table 4. Correlation coefficients between plant weight parameters.
Parameter DR DL DS DF TDW R:SH
DR 1 0.74** 0.86** 0.87** 0.92** -0.28*
DL 1 0.76** 0.87** 0.86** 0.16 ns
DS 1 0.98** 0.97** -0.48**
DF 1 0.99** -0.32**
TDW 1 -0.32**
R:SH 1
**Correlation is significant at the 0.01 level (2-tailed test); DR = dry root, DL = dry leaf, DS = dry
stem, DF = dry forage, TDW = total dry weight, R:SH = root to shoot ratio.
and infrequently. This contradicted the finding by Saeed
and El-Nadi (2004) who recommended using light
frequency to get high WUEs.
Conclusion
Under salinity and water stress conditions, the KFS4
variety has a better vegetative growth performance as
compared to Speedfeed. Infrequent irrigation had
reduced biomass accumulation; the reduction was higher
when low irrigation frequency was coupled with salinity.
Irrigation may be intensified in saline soils to mitigate the
effect of salinity on plant growth. However, there is a
critical level of salinity after which irrigation cannot
mitigate the effect of salinity. The critical level of salinity
for KFS4 was 15 dS m -1 , while for Speedfeed, it was 10
dS m -1 . WUE of forage sorghum could be increased by
infrequent irrigation, and new fields could be well irrigated
by saving water. The results obtained in this study would
serve as a useful guide for managing forage sorghums in
saline and water stressed field conditions.
REFERENCES
FAO STAT (2001). Land and Water division, FAO AGLw, Water, News,
Contacts Agriculture 21 http://www.fao.arg/ag/agl/. Accessed 10
June 2009.
Fouman A, Majidi Heravan E, Nakano Y (2003). Evaluation forage
sorghum varieties for salt tolerance. Proceedings of 7th International
Conference on Development of Dry Lands. 14-17 September 2003,
Tehran (IRAN).
Ghoulam C, Foursy A, Fares K (2002). Effects of salt stress on growth,
inorganic ions and proline accumulation in relation to osmotic
adjustment in five sugar beet cultivars. Environ. Exp. Bot. 47: 9-50.
Gulzar S, Khan MA, Ungar IA ( 2003). Salt tolerance of a coastal salt
marsh grass. Commun. Soil Sci. Plant Anal. 34: 2595-2605.
Hasegawa PM, Bressan RA, Zhu JK, Bohnert H J (2000). Plant cellular
and molecular response to high salinity. Annu. Rev. Plant Physiol.
51: 463-499.
Hester MW, Mendelssohn IA, Mckee KL (2001). Species and population
variation to salinity stress in Panicum hemitomon, Spartina patens
and Spartina alterniflora: Morphological and physiological
constraints. Environ. Exp. Bot. 46: 277-297.
Muhammad A, Haji Kh, Ahmad H, Muhammad A, Ejaz A, Muhammad A
(2008). Effec of available soil moisture depletion levels and topping
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Res. 46(3): 229-243.
Munns R (2002). Comparative physiology of salt and water stress. Plant
Cell Environ. 25: 239-250.
Mustafa MA, Abdel Magid EA (1982). Effects of irrigation interval, urea
and gypsum on N, P and K uptake by forage sorghum on highly
saline-sodic clay. Exp. Agr. 18(2): 177-182.
Okcu G, Kaya MD, Atak M (2005). Effects of salt and drought stresses
on germination and seedling growth of pea (Pisum sativum L.). Turk
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Richards LA (1947). Pressure-membrane apparatus-construction and
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Saeed AM, El-Nadi AH (2004). Forage sorghum yield and water use
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333-341.

African Journal of Biotechnology Vol. 10(20), pp. 4121-4126, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2406
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Isolation and characterization of a novel sulfuroxidizing
chemolithoautotroph Halothiobacillus from
Pb polluted paddy soil
Jiyan Shi 1 , Huirong Lin 1, 2 *, Xiaofeng Yuan 3 and Yidong Zhao 4
1 Institute of Environmental Science and Technology, Zhejiang University, Hangzhou, 310029, China.
2 Department of Environmental Science and Engineering, Tan Kah Kee College, Xiamen University, Zhangzhou, 363105,
China.
3 Life Science Department, Zhejiang Chinese Medical University, Hangzhou, 310053, China.
4 Institute of High Energy Physics, Chinese Academy of Science, Beijing Synchrotron Radiation Facility, Beijing, 100049,
China.
Accepted 7 April, 2011
A new mesophilic and chemolithoautotrophic sulfur oxidizing bacterium (SOB) strain HT1 was isolated
from a rice rhizosphere soil polluted by Pb using thiosulfate as electron donor at pH 7.0. The 16S rRNA
gene sequence showed that the new isolate was a sulfur oxidizing obligate chemolithotroph belonging
to Gammaproteobacteria, Halothiobacillus and utilizing different reduced sulfur compounds (sulfide,
elemental sulfur, thiosulfate and sulfite) as chemolithotrophic substrates. Strain HT1 was able to use
CO2 as a carbon source responsible for the reduction of nitrate to nitrite, which represented a halophilic
SOB capable of growth within a broad salinity range of 0 to 3 M NaCl and a heavy-metals-tolerant SOB.
HT1 was Gram negative, motile and was proposed as the type strain of a novel species of sulfur
oxidizing bacteria. The soxB gene could not be detected in strain HT1 during thiosulfate oxidation. The
metabolism pathway of HT1 was ‘S4 intermediate’ (S4I) pathway. Sulfur globules accumulated in HT1
were mainly S8.
Keywords: Halothiobacillus, heavy-metals-tolerant, soxB gene, 16S rRNA gene, sulfur globule.
INTRODUCTION
Sulfur oxidizing bacteria play an important role in mineral
cycling in environments. The species of the genus
Thiobacillus include a wide diversity of Gram negative,
rod sulfur oxidizing bacteria which obtain energy from
oxidation of reduced sulfur. They fall into α, β and γ
subclasses of the Proteobacteria and could be classified
as Acidithiobacillus, Halothiobacillus and Thermithiobacillus
(Kelly and Wood, 2000). Biological reduced sulfur species
such as aqueous (hydro) sulfide as well as insoluble
metal sulfides, polysulfides, elemental sulfur, sulfite, thiosulfate
and polythionates are finally oxidized to sulfate, or
more precisely to sulfuric acid. During these processes,
protons are produced. The oxidation of reduced sulfur to
* Corresponding author. E-mail: linhuirong@yahoo.com.cn. Tel:
+86-571-86971424. Fax: +86-571-86971898.
sulfuric acid is of great importance for biohydrometallurgical
technologies. Contrary to its significant role
in the global sulfur cycle and its biotechnological
importance, the microbial fundamentals of sulfur oxidation
are incompletely understood.
Sulfur oxidizing bacterium (SOB) exhibits a wide range
of metabolic flexibility, particularly with respect to processes
involving respiration and energy transduction.
Previous studies concluded that there were at least two
metabolic pathways of SOB during the thiosulfate
oxidation (Friedrich et al., 2001). Some SOB was
involved in the toxicity of heavy metal ion and possesses
unique metabolic and ecophysiological features with
extraordinary properties. Novel SOB with extraordinary
properties was reported (Sorokin et al., 2006; Ghosh and
Roy, 2007). Wood and Kelly (1991) found the first halophilic
SOB capable of growing at very high salt
concentrations (4 M NaCl). Some SOB that is capable of

4122 Afr. J. Biotechnol.
anaerobic growth with sulfur compounds and nitrogen
oxides as electron acceptors play an important role in
mineral cycle.
Herein, we isolated a novel SOB from a Pb polluted
paddy soil. The nearly complete 16 s rRNA gene sequences
and physiological characteristics of the new isolate
were analyzed. Functional soxB gene was detected in
order to investigate its metabolic pathway. Sulfur K-edge
x-ray absorption near edge structure spectroscopy
(XANES) was used to identify the speciation of sulfur in
the cells of HT1.
MATERIALS AND METHODS
Rhizosphere soil of rice was used as the inoculums for enrichment
cultures. Selected physical and chemical properties of the soil used
are: organic matter, 2.26%; pH, 5.63; Pb concentration, 680.09
mgkg -1 ; Zn concentration, 68.96 mgkg -1 ; Cu concentration, 21.10
mgkg -1 ; S concentration, 247 mgkg -1 . Mineral medium used for
enrichment, isolation and cultivation of SOB contained (per liter of
deionised water, added 15 agar for solid medium): Na2HPO4 1.2 g,
KH2PO4 1.8 g, MgSO4·7H2O 0.1 g, (NH4)2SO4 0.1 g, CaCl2 0.03 g,
FeCl3 0.02 g and MnSO4 0.02 g. Ten grams of Na2S2O3 was added
as model sulfur source (Graff and Stubner, 2003). Enrichment of
SOB was conducted with liquid medium on a rotary shaker at 200
rpm at 28°C. For isolation and purification, solid medium was prepared.
0.2 ml of aliquots of the enrichment cultures were transferred
onto the solid medium. The plates were incubated in an incubator at
28°C. Colonies formed on agar plates of the mineral medium were
transferred at least three times to be pure.
DNA of the new isolate -was obtained for amplification and
sequencing of the 16S rRNA gene. The 16S rRNA gene was
amplified with primers BSF8/20: 5'-AGAGT TTGAT CCTGG
CTCAG-3' and BSR1541/20: 5'-AAGGA GGTGA TCCAG CCGCA-
3'. PCR was run using 50 µl reaction volumes and reactant
concentrations in each 50 µl reaction which were 10 to 15 ng of
DNA template, 25 pM of each primer, 2.5 mM deoxynucleotide
triphosphates (dNTPs, Proega, USA), 10×PCR buffer (applied
Biosystems, USA), 0.1 mM MgCl2 solution (Sigma) and 1 U of Taq
polymerase (Applied Biosystems, USA) in nuclease-free water. The
reaction was carried out as follows: 4 min initial denaturation at
94°C, followed by 30 cycles of denaturation at 94°C for 50 s,
annealing at 57°C for 50 s and DNA extension at 72°C for 40 s.
Cycling was completed by a final elongation step of 72°C for 10
min. The PCR product were purified and sequenced by the
Invitrogen Corporation (USA) in China. The nearly complete 16S
rRNA gene sequences of the new isolate were compared using the
BLAST program. A phylogenetic tree was constructed with sequences
aligned with the CLUSTAL X 1.83 and the Paup v.4.0b.8.a
program.
Physiological characteristics were analyzed as described by
Dong and Cai (2001). Cells were suspended in sterile double
distilled water and adhered on copper mesh. Then, the cells were
dyed with 1% uranygl acetate for about 15 s. Transmission electron
microscope (H-7650, HITACHI) was used to observe the morphology.
Different reduced sulfur compounds (thiosulfate, thiocyanate,
elemental sulfur, sulfide, sulfite and tetrathionate) were used to
determine the utilization of substrates as energy sources. The
sulfite solution was prepared in 50 mM EDTA to prevent
autooxidation (Sievert et al., 2000). Nitrogen (nitrate, nitrite) was
used to test as an electron acceptor in the absence of oxygen with
KNO3 and KNO2. Effects of salinity (NaCl) and heavy metal
tolerance (Pb, Zn and Cu) on bacterial growth were examined in
Luria-Bertani medium containing varying concentrations.
PCR amplifications of soxB gene fragments with extracted DNA
were performed as a two-step PCR using the primer sets and
protocols as described by Petri et al., (2001). Sulfur species in the
cell were analyzed by XANES as described by Prange et al., (1999)
at Beijing synchrotron radiation facility, institute of high energy
physics of China. Spectra were recorded at 4B7A beam line and
scanned in the region between 2420 and 2520 eV.
RESULTS AND DISCUSSION
A new isolate using thiosulfate as sulfur was obtained and
named as strain HT1. Colonies grown on mineral medium
with thiosulfate as sulfur source were smooth and whitelight
yellow (1 to 3 mm). The cells of strain HT1 appeared
singly or in pairs and motile with flagellums (Figure 1).
The 16S rRNA genes were amplified using general
bacterial primers. A phylogenetic tree was obtained as
shown in Figure 2. Comparison of the nearly complete
16S rRNA genes showed that strain HT1 was closely
related to Halothiobacillus and belonged to the γ subclass
of the Proteobacteria. Its lineage is Bacteria,
Proteobacteria, Gammaproteobacteria, Chromatiales,
Halothiobacillaceae and Halothiobacillus. The GenBank
accession number for the nearly complete 16S rRNA
gene sequence of Halothiobacillus HT1 is GU013549.
Table 1 shows the physiological characteristics of HT1
when compared with known Halothiobacillus. Reduced
inorganic sulfur compounds sulfide, polysulfides, elemental
sulfur, sulfite and thiosulfate were oxidized as electron
donors for energy by HT1. No growth was observed on a
mineral medium with thiocyanate as energy source.
Under aerobic conditions, the bacterium was able to
grow with nitrate as electron acceptor. However, no
growth was observed with nitrite. It was concluded that
strain HT1 is a nitrate to nitrite reducer and can serve as
a nitrite provider. The isolate grew autotrophically on
thiosulfate and can use carbon dioxide as carbon source.
HT1 was capable of adapting to high salt (3 M) and could
use CO2 as a carbon source, suggesting that it possessed
carboxysomes that contain enzymes involved in
carbon fixation (Tsai et al., 2007).
HT1 could grow in 621 mg.kg -1 of Pb 2+ , 192 mgkg -1 of
Cu 2+ and 325 mgkg -1 of Zn 2+ , respectively, showing
relatively high tolerance of heavy metals. Possible toxicity
and mechanisms of resistance to Cu have been studied
in Sulfolobus solfataricus and Sulfolobus metallicus
(Ettema et al., 2006; Remonsellez et al., 2006). Microbial
sulfur oxidation is of great importance in influencing the
transformation of heavy metals. This useful application
requires heavy-metals-tolerant SOB which can grow in
heavy metals polluted environment. They may possess
genetic determinants for metal resistance encoding
bacterial metallothioneins and heavy metal-transporting
ATPases to exist in toxic heavy metal ions and contribute
to sulfur oxidation (Auernik et al., 2008).
As a result of the phylogenetic and physiological diversity
of sulfur oxidizing bacteria, several different enzymatic

Figure 1. Morphology of cells of strain HT1 grown in LB medium. (a) In pair; (b) in
pair with flagellums; (c) single.
Shi et al. 4123

4124 Afr. J. Biotechnol.
10
65
Escherichia coli MC4100
100
60
100
Acidithiobacillus thiooxidans FJ172634
Acidithiobacillus ferrooxidans FJ157225
Thiomicrospira thyasirae NR_024854
87
Thiomicrospira halophila DQ390450
Thiomicrospirasp AB301715
Thiofaba tepidiphila AB304258
99
100
97
89
97
63
71
Halothiobacillus sp. EF397577
79
HT1
Halothiobacillus sp. EF397570
Halothiobacillus sp. AY096035
Halothiobacillus neapolitanus AB308268
Thiobacillus halophilus THU58020
Thioalkalibacter halophilus EU124668
Thiobacillus neapolitanus AF173169
Halothiobacillus neapolitanus EU871645
Figure 2. Phylogenetic tree derived from 16S rDNA sequence data of strain selected and other related species.
The tree was rooted with Escherichia coli as an out group.
systems and pathways are involved in the dissimilatory
oxidation of thiosulfate. At least two major pathways have
been proposed for different SOB: (1) the sulfur oxidation
pathway (PSO) and (2) the S4 intermediate pathway
involving polythionates (S4I) (Friedrich et al., 2001). The
S4I pathway includes the formation and oxidation of
polythionate or S and sulfite from thiosulfate. In the PSO
pathway, thiosulfate is oxidized directly to sulfate and
does not accumulate intermediate products such as
polythionate, S and sulfite. Thiosulfate oxidation is carried
out by a thiosulfate-oxidizing multi-enzyme system in
which one enzyme is coded by soxB (Anandham et al.,
2008). SoxB contains a prosthetic manganese cluster in
the reaction center and is essential for thiosulfate

Table 1. Comparative characteristics of strain HT1 and typical Halothiobacillus.
Characteristic HT1 Halothiobacillus *
Gram stain test - -
Oxidase / /
Catalase - /
Indole + /
Methyl red test - /
Amylohydrolysis - -
Oxidative fermentation of glucose Ferment /
Nitrate reduction + +
Nitrite reduction - -
Denitrification - -
Gelatin liquefaction - /
H2S + /
Cu tolerance 621 mgkg -1 /
Pb tolerance 192 mgkg -1 /
Zn tolerance 325 mgkg -1 /
NaCl tolerance 175.5 gkg -1 0-234 g.kg -1
Sulfur sources
sulfide + +
S + +
sulfite + +
thiosulfate + +
tetrathionate + +
thiocyanate - -
“/” Denotes this index does not test or exist; “*” denotes reference control
oxidation. HT1 is a sulfur oxidizing obligate chemolithotroph
capable of using thiosulfate as substrate. In
order to detect the pathway of HT1, different primers
were conducted on it to detect the existence of soxB.
PCR-based analysis showed that no specific bands were
obtained in HT1. Our results were consistent with soxB
gene analysis with Halothiobacillus hydrothermalis conducted
by Petri et al., (2001), suggesting that HT1 oxidize
thosulfate via the S4I path way which is enzymatically
different from the PSO path-way.
It was reported that many SOB took up and stored
sulfur globules in the reduced forms (usually as elemental
sulfur). Different bacterial groups stored various forms of
globules (Prange et al., 1999). Sulfur in the globules of
Beggiatoa alba and Thiomargarita namibiensis consisted
of S8, whereas Acidithiobacillus ferrrooxidans stored
polythionates as globules. Purple and green sulfur
bacteria also consisted mainly of polymeric sulfur chains
while the sulfur chains were found in the globules of
Thermoanaerobacter sulfurigignens and
Thermoanaerobacterium thermosulfurigenes. As an
essential macronutrient for microorganisms, plants and
animals, sulfur (S) exists in soils in a great variety of
organic and inorganic species with oxidation states
ranging from -2 to +6. Reference compounds were used
to explore different sulfur oxidation states and chemical
structures. For analysis, the fitting and plotting package
Shi et al. 4125
WinXAS version 3.1 was used. The spectra were
normalized by fitting first and second order polynomial
functions to the pre- and post-edge regions respectively.
Sulfur K-edge XANES analysis showed that sulfur
globules in HT1 fit well with cyclooctasulfur (S8) which
was consistent with the results of Engel et al., (2007)
(Figure 3). This could be either a biosignature of bacteria
that preferentially take up and oxidize cyclooctasulfur (S8)
or a sign that the polymeric form of sulfur is preferred,
thus leaving cyclooctasulfur to accumulate.
HT1 was a heavy-metals-tolerant SOB which might
play an important role in biogeochemical cycle. Microorganisms
are considered to be important contributors to
the biogeochemical cycle of trace elements through
several microbe mediated processes. Most sulfur transformations
are fundamentally controlled by biosphere
processes, especially by the specialized metabolisms of
microorganisms. Biological oxidation was the principal
pathway for mineralization of organic S. Sulfur oxidation
by metal resistant SOB contributed to the formation of
SO4 2- , leading to the transformation of sulfur.
ACKNOWLEDGEMENTS
This work wassupported by Zhejiang Provincial Natural
Science Foundation of China (R5110031), the National

4126 Afr. J. Biotechnol.
Absorption
2.460 2.465 2.470 2.475 2.480 2.485 2.490
Energy (KeV)
Figure 3. Sulfur K-edge XANES spectra of HT1 cells.
Natural Science Foundation of China (40601086,
20777066), and the Fundamental Research Funds for the
Central Universities (2010QNA6025).
REFERENCES
Anandham R, Indiragandhi P, Madhaiyan M, Ryu KY, Jee HJ, Sa TM
(2008). Chemolithoautotrophic oxidation of thiosulfate and
phylogenetic distribution of sulfur oxidation gene (soxB) in
rhizobacteria isolated from crop plants. Res. Microbiol. 159: 579-589.
Auernik KS, Maezato Y, Blum PH, Kelly RM (2008). The genome
sequence of the metal-mobilizing, extremely thermoacidophilic
archaeon Metallosphaera sedula provides insights into bioleachingassociated
metabolism. Appl. Environ. Microb. 74: 682-692.
Engel AS, Lichtenberg H, Prange A, Hormes J (2007). Speciation of
sulfur from filamentous microbial mats from sulfidic cave springs
using X-ray absorption near-edge spectroscopy. FEMS Microbiol.
Lett. 269: 54-62.
Ettema TJG, Brinkman AB, Lamers PP, Kornet NG, de Vos WM, van der
Oost J (2006). Molecular characterization of a conserved archaeal
copper resistance (cop) gene cluster and its copper-responsive
regulator in Sulfolobus solfataricus P2. Microbiol. 152: 1969-1979.
Friedrich CG, Rother D, Bardischewsky F, Quentmeier A, Fischer J
(2001). Oxidation of reduced inorganic sulfur compounds by bacteria:
Emergence of a common mechanism? Appl. Environ. Microb. 67:
2873-2882.
Ghosh W, Roy P (2007). Chemolithoautotrophic oxidation of thiosulfate,
tetrathionate and thiocyanate by a novel rhizobacterium belonging to
the genus Paracoccus. FEMS Microbiol. Lett. 270: 124-131.
Graff A, Stubner S (2003). Isolation and molecular characterization of
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Kelly DP, Wood AP (2000). Reclassification of some species of
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nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov. Int.
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HG, Steudel R, Dahl C, Hormes J (1999). In situ analysis of sulfur in
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near edge spectroscopy. BBA-Gen Subjects, 1428: 446-454.
Remonsellez, F, Orell A, Jerez CA (2006). Copper tolerance of the
thermoacidophilic archaeon Sulfolobus metallicus: possible role of
polyphosphate metabolism. Microbiology, 152: 59-66.
Sievert SM, Heidorn T, Kuever J (2000). Halothiobacillus kellyi sp. nov.,
a mesophilic, obligately chemolithoautotrophic, sulfur-oxidizing
bacterium isolated from a shallow-water hydrothermal vent in the
Aegean Sea, and emended description of the genus Halothiobacillus.
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Muyzer G (2006). Thiomicrospira halophila sp nov., a moderately
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usual Manual, Science Press. Beijing.

African Journal of Biotechnology Vol. 10(20), pp. 4127-4132, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2452
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Biomonitoring of some heavy metal contaminations
from a steel plant by above ground plants tissue
M. Ataabadi, M. Hoodaji* and P. Najafi
Islamic Azad University, Khorasgan Branch, Isfahan, Iran.
Accepted 21 March, 2011
Soil and plants growing in the vicinity of industrial areas display increased concentrations of heavy
metals and give an indication of the environmental quality. The contamination source for aluminum,
iron, nickel and lead in the Botanical garden of Mobarakeh Steel Company was recognized by analyzing
the leaves and topsoil of two evergreen species: Quercus brantii and Ligustrum vulgare. Availability of
the studied elements for plants was investigated by measuring their total and extractable concentration
inthe soils. For ensuring air borne source of these metals, plant tissues were washed with distilled
water. Plant available heavy metals of the soils were few in comparison with the total values which were
related to the high pH value and the CaCO3 percentage in the soils around each plant. On the other
hand, significant differences were obtained between the washed and unwashed leaves in both species
indicating metal contaminations from the atmosphere. Significant differences were detected between
the contaminated and background site samples in both plants for Al, Fe and Ni. In addition, the leaves
of L. vulgare accumulated more elements partially than Quercus brantii. Difference in the accumulation
potentials of the plants were related to the diversity of the physical and chemical properties of the
leaves and the type of elements. The enrichment ratio of plants (Ccontaminated/Cbackground) was calculated
and it indicated that, the Fe in both plants was moderately enhanced by anthropogenic activities.
Key words: Industrial contamination, Quercus brantii, Ligustrum vulgare, enrichment factor.
INTRODUCTION
Human activities cause the slow extermination of plant
and animal species in nature through toxic pollution due
to industrial and technological advancement in recent
decades (Ives and Cardinale, 2004). Many heavy metals
emitted mostly from anthropogenic sources, have now
exceeded or equaled their natural emissions (Biney et al.,
1994) and have been posing a serious threat to the
ecosystems (Wen Kuang et al., 2006).
Soil and plants growing in the nearby zone of industrial
areas display increased concentration of heavy metals,
serving in many cases as biomonitors of pollution loads.
Plants take up large quantities of pollutants and translocate
them into vegetative and generative organs at
various rates (Kovács et al., 1993), which make clear the
quality of the environment. Calzoni et al. (2007) in their
studies on the ability of Rosa rugosa for biomonitoring of
*Corresponding author. E-mail: M_Hoodaji@Khuisf.ac.ir Tel:
0311-5354061, 09131172651. Fax: 0311-5354061.
heavy metal indicated that, leaf accumulation was due to
atmospheric deposition rather than to soil uptake (Calzoni
et al., 2007). Chemical foliar analysis has also been employed
to study the impact and extent of air pollutants
(Djingova et al., 1999; Ericsson et al., 1995; Hüttl and
Fink, 1991) and the pollutant accumulation capacity of
different plants (Bicchiega et al., 1994; Somsak et al.,
2000). Leaves of higher plants have been used for biomonitoring
heavy metals since the 1950s (Al-Shayeb et
al., 1995). Pine needles for the determination of airborne
pollutants are also a suitable technique for monitoring
purposes (Holoubek et al., 2000).
Parts of the deposited particles are not removed by
rainfall and become irreversibly adsorbed or incorporated
into the hydrophobic wax layer of the foliage (Rossini and
Raitio, 2003). Therefore, cleaning has an effect on the
foliar concentrations of Al, As, At, Br, Cd, Cr, Fe, Ni, Pb,
V, T and other heavy metals (Rossini and Raitio, 2003).
Aksoy and Öztürk (1996), for example, used date palm
leaves to monitor the distribution of airborne Pb, Cd, Zn
and Cu in the city of Antalya in Turkey. A comparison of

4128 Afr. J. Biotechnol.
Table 1. Mean values of soil properties.
Site pH O.C (%) CEC (meq/100 g) CaCO3 (%) Texture
Contaminated
Q. brantii 8.1 0.3 11.2 68 Sandy loam
L. vulgare 8.3 0.2 10.5 70 Sandy loam
Background
Q. brantii 8.1 1.3 29.1 26 Clay loam
L. vulgare 8 1.2 28.5 23 Clay loam
O.C, organic carbon; CEC, cation exchange capacity.
washed and unwashed samples showed that, leaf
analyses gave a reasonably reliable measure of the total
aerial fallout of heavy metals in the studied area.
Plants contamination in most cases arises from
atmospheric particles accumulation through their foliage
and leaves and the degree of contamination depend on
be smoothness of leaves, wind speed and on the value of
rainfall. Ward et al. (1977) expressed that plant washing
after sampling, decreased the element contents to about
10 to 30% in comparison with unwashed plants (Ward et
al., 1977).
Hoodaji and Jalalian (2003 a,b) showed that, soil and
plant near the Mobarakeh Steel Company were slightly
contaminated with some heavy metals, such as Fe, Zn
and Mn; for example they also reported that, topsoils (0
to 5 cm) and shoot of rice, wheat and bean were enriched
in Fe (Hoodaji and Jalalian, 2003a, 2003b). The plant
contamination was assessed using the enrichment factor.
The enrichment factor (EF) was based on the
standardization of a tested element against a reference
one (Reinmann et al., 2001). The aim of this study was to
assess the leaves of Quercus brantii and Ligustrum
vulgare as possible biomonitors of heavy metal contamination
in the vicinity of industrial area.
MATERIALS AND METHODS
Site description
Mobarakeh Steel Company (MSC) is located at 65 km south west of
Isfahan, near the city of Mobarakeh, Isfahan Province, Iran. It is
Iran's largest steel maker and one of the largest industrial
complexes operating in Iran and is one of the main contamination
sources in this region. This study was done in the Botanical garden
of Mobarakeh Steel Company located in 32°34'15’N and 51°25' 21,
E southwest of Isfahan, Iran. This region has an arid climate with a
mean annual rainfall of 140 mm and wind direction of SW-NE. The
region’s type of soil, according to information obtained from profile
description was Typic Haplocalcids. The garden was approximately
3 ha in area, with 9 plots and 3 replications. The background site
was located at 50 km away in a village with low traffic in the Bagh
Bahadoran region. Minimum and maximum annual temperatures of
this location were 7.6 and 24.33°C, respectively. Relative humidity
was 40% and mean wind speed was 1.77 km/h.
Two species, Q. brantii and L. vulgare were selected. These
species are widely distributed in landscape and can survive under a
wide temperature range and grow in almost any type of soil. They
have different behaviors to metal contaminants due to various
morphological and physico-chemical characteristics of their leaves.
L. vulgare is a shrub with oval to lance-shaped sub-shiny dark
green leaves, thick cuticle and moderate epicuticular waxy layer,
whilst Q. brantii is a tree with spiny shape of the lightly lobed leaves
and thin cuticle.
Sampling, preparation and analysis of plant samples
The sampling of both species was performed in August 2007 from
the studied and background sites. Leaf sampling was done with
wind direction (SW to NE) and from approximate height of 190 and
140 cm for Quercus and Ligustrum, respectively, along 9 plots in 3
replications. After transferring the samples to a laboratory, leaves
were divided in to two parts. One part was washed with de-ionized
water to clean dust and any deposited substances on the leaves
and another part was not cleaned. At first, all samples were air
dried and then oven dried at 70°C for 48 h to constant mass, milled
and sieved through a 35 mesh screen. 1 g milled powder of each
samples were digested with 10 ml 2N HCl (Chapman and Pratt,
1961).
Sampling, preparation and analysis of soil samples
Soil samples were also collected randomly from a depth of 0 to 30
cm around each plant. Soil samples were air dried, ground and
passed through sieve (2 mm).The main soil chemical properties
were determined by laboratory analysis. Organic carbon was
determined by a modified wet oxidation (Nelson and Sommers,
1982). Soil pH was measured using potentiometric titration of the
soil extract. Cation exchange capacity (CEC) was measured by
ammonium acetate extraction (Rhoads, 1982). Calcium carbonate
was determined by back titration (FAO, 1974). Soil texture was
measured by the Hydrometer method (Gee and Bauder, 1986). The
total metal concentrations (Al, Fe, Ni and Pb) were determined after
digestion with 10 ml HNO3 70% (Pyatt, 1999; Soon and Abbund,
1993). The plant available heavy metals in soils were extracted with
diethylen triamine penta acetic acid (DTPA) solution (0.005 M
DTPA + 0.01 M CaCl2 + 0.1 M TEA at pH = 7.3) (Lindsay and
Norvell, 1978). Finally, heavy metal concentrations in plant and soil
samples were determined by ICP- AES (GBC Integra XL model).
Some chemical and physical properties of soils are given in Table
1.
To identify the possible origin of metals in the plants, an
enrichment factor of plant (EFplant), was calculated with Equation 1
for each metal as follows:

Table 2. Total and plant available mean metal concentration (mg kg -1 ) ± Standard error in topsoil around each plant.
Ataabadi et al. 4129
Location/species
Contaminated site
Al
Q. brantii
Fe Ni Pb Al
L. vulgare
Fe Ni Pb
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
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
Available percent* 0.005 0.04 5.07 6.62 0.002 0.06 3.98 4.31
Background site
Total 19250 ± 1000 30000 ± 680 57.2 ± 1 15.9 ± 1 18750 ± 788 30400 ±1500 44.8 ± 5.5 21.5 ± 2
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
Available percent* 0.01 0.06 2.3 19.8 0.01 0.1 2.71 13.5
*Available percent: (plant available / total) × 100.
M
EF = (1)
plant
plant
M control
Where, Mplant and Mcontrol are the concentrations of the
examined elements of the plant in the contaminated and
background sites, respectively (mg kg -1 ) (Reinmann et al.,
2001).
Data analysis was done by a statistical package SPSS
14.0. To evaluate the effect of washing treatment on
element concentration, the statistical significance (p <
0.05) of the differences between unwashed and washed
samples was determined using t-test.
RESULTS AND DISCUSSION
Relationship between soil properties and
heavy metal uptake by plant
The total and plant available heavy metal values
in the soils are given in Table 2. The total metal
concentrations were high, but only a little fraction
of them were available for the plants. These
results can be explained with the consideration of
the chemical properties of the soils in Table 1.
Total soil heavy metal concentration is commonly
used to indicate the degree of
contamination (Karaca, 2004), although, DTPAextractable
concentration provides a more
suitable chemical evaluation of the amount of
metals available for plant uptake (Lindsay and
Norvell, 1978; Pretuzzelli, 1989; Zufiaurre et al.,
1998). Loading and accumulation of heavy metals
in the soil depend on different factors such as
chemical form of elements, pH, organic matter
content, texture and cation exchange capacity
(CEC) of the soil (Logan and Chaney, 1983). With
increasing pH, organic matter content, CEC and
clay percentage and availability of the metals
reduced. In addition to the existence of carbonate,
sulfate, phosphate and sulfide forms of elements
in soil causes an increase in the metal precipitation
and consequently decrease their availability
for plant (Shuman, 1985 and Forstner,
1985).Therefore, in calcareous soils (CaCO3 >
60%) mobility and uptake of heavy metals were
very low. Kabata-Pendias and Pendias (1999)
also expressed that, uptake and disuptake of
elements depend on plant species, growth stage
and composition of soil solution, especially Ca
(Kabata-Pendias and Pendias, 1999).
Consequently, in the contaminated site, the
existence of more than 60% CaCO3 with pH >8
led to decrease in mobility and availability of these
elements in the soils.
Consideration of the available percents of the
metals also confirmed the results, therefore, it can
be concluded that the source of these elements in
plants cannot be the soil.
Concentration of elements in plants
Leaf samples from contaminated site (MSC) had
significantly higher concentrations of Al, Fe and Ni
than the background site for both species. This
result showed the direct effect of atmospheric
contamination in industrial regions. The effects of
industrial activities on heavy metal pollution have
been reported by many authors (El-Hassan et al.,
2002; Rossini and Mingorance, 2006). Comparison

4130 Afr. J. Biotechnol.
Table 3. The mean values of EFplant.
Plant species Al Fe Ni Pb
Q. brantii 1.5 2.6 1.9 0.9
L. vulgare 1.5 2.9 1.9 1.5
Table 4. Elemental concentrations ± standard error in leaves of Q. brantii compared by t-test.
Element
Site
Al
Unwashed Washed
T-test
Fe
Unwashed Washed
T-test
Ni
Unwashed Washed
T-test
Pb
Unwashed Washed
T-test
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 *
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 *
T-test * ns ** * * * ns ns
Significance: *p < 0.05, **p < 0.01. ns: not significant.
Table 5. Elemental concentrations ± Standard error in leaves of L. vulgare compared by t-test.
Site
Al
Unwashed Washed
T-test
Fe
Unwashed Washed
T-test
Ni
Unwashed Washed
T-test
Pb
Unwashed Washed
T-test
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 *
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 *
T-test * * ** * ns * ns ns
*p < 0.05; **p < 0.01; ns, not significant.
of the normal content of Fe in plants (100 to 500
mg kg -1 of dry weight) (Pais and Benton, 1997)
with the detected concentrations, showed that the
contamination of Fe in the study area was
considerable.
Enrichment factor of plant
EFplant showed the different ratio of metal bioaccumulation
in each part of the plant in relation
to the control site. EF > 2 indicates sample
enrichment (Mingorance et al., 2007). 1

Investigation of atmospheric source of elements with
water washing treatment
The ability to distinguish airborne and soil borne
contamination was assessed by washing the leaves.
Washing with water was an effective procedure for the
removal of Pb, Fe, Al, Cr and V from the leaves of
ornamental species (Rossini Oliva and Raitio, 2003)
General comparison between the element concentrations
before and after washing in the contaminated site
indicated significant differences in both species except for
Al in Ligustrum vulgare (Table 4 and 5). In the
contaminated site, after washing, element contents of
leaves decreased about 29-64 and 28-74 percents for
Quercus and Ligustrum, respectively. Therefore, it can be
concluded that metal concentrations in the plants in the
studied area were due to atmospheric dust from industrial
activities. Removal efficiency of the studied elements
after washing with water were much higher in comparison
with Ward et al ΄s results (1977), which reported
reduction in element content about 10-30 percents with
washing (Ward et al., 1977). Reduction in element
content after washing with water is different by physical
and chemical characters of pollutants, plant species and
subsequently properties of leaves (Rea et al., 2000,
(Rossini Oliva and Raitio, 2003)
Conclusions
The findings of this study showed that, both
species were suitable biomonitors for Al, Fe and Ni
contamination in the studied area. L. vulgare acted better
than the Q. brantii in some cases which confirmed that
the accu-mulative ability of plant tissues depend on their
surface properties (Tables 4 and 5). Both species were
mode-rately enriched in Fe due to the EFplant > 2.
Considerable differences between the total and DTPAextractable
heavy metals in soils due to high pH value
and CaCO3 percentage disproved the probability of plant
contamination through the soil and fortified the theory of
atmospheric source of these elements in the industrial
area. On the otherhand, the use of water-washing
procedure which led to significant differences in the
heavy metals content of the leaves in both plants in the
contaminated site, demon-strated the atmospheric source
of the metals.
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African Journal of Biotechnology Vol. 10(20), pp. 4133-4137, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1388
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Isolation, purification and identification of bacteria from
the shoes worn by children
Hui Li, Changqing Zhao, Jin Zhou, Huanhuan Shao and Wuyong Chen*
National Engineering Laboratory for Clean Technology of Leather Manufacture, Institute of Life Sciences, Sichuan
University, Chengdu 610065, China.
Accepted 4 April, 2011
13 strains of bacteria were isolated from 12 shoes that were worn by children aged 6 to 12 for more than
half a year. Through morphological observation, physiological and biochemical measurements, as well
as 16SrRNA sequence analysis, the bacteria were identified as follows: Bacillus licheniformis, Bacillus
subtilis (5 subspecies), Bacillus spore (3 subspecies), Bacillus anthracis, Staphylococcus aureus,
Bacillus amyloliquefaciens and Bacillus thuringiensis. The results may contribute to the selection of
efficient antimicrobial agents for children's shoes and insole.
Key words: Children’s shoes, bacteria, isolation, identification.
INTRODUCTION
Shoes are necessary for everyone, especially as they are
worn by all in almost 2/3 of humans’ life time. They are
vulnerable to contamination by microorganisms when
worn. Some published results show that a great amount
of bacteria and fungi attach themselves to the feet and
socks of humans, with the density of 5,000 to 100,000
colonies/cm 2 , and more severely with a density of
10,000,000 to 1,000,000,000 colonies/cm 2 (Xu, 2006). It
is these microorganisms that react with the sweat of
humans and cause an undesired odor. However, various
microorganisms could grow and breed largely in the
micro-environment of inner shoes and would rightly
cause incomparable beriberi by reacting with human’s
sweat. On the other hand, there was little information
about these bacteria and fungi in shoes, although the
pathogens which these microorganisms belong to have
not been reported.
Since children are usually engaged in many physical
activities, they could secrete more sweat than adults for
their vigorous metabolism. As a result, more bacteria
would breed and lead to more serious foot diseases (Cai,
2005). In the long-term result, a series of diseases could
be induced, which would severely do harm to children's
physical and mental health ultimately (Dorman et al., 2000).
*Corresponding author. E-mail: wuyong.chen@163.com.
Tel/Fax: + (86)28 8540 4462.
Shamez had successfully isolated a strain of
Staphylococcus from children’s shoes (Shamez and
Mehdi, 2005), but up till now, no report has been made
on the isolation of other bacteria from children’s shoes. If
the specific pathogenic microorganisms could be isolated
and identified from the shoes, some effective antimicrobial
agents for the different species of bacteria
could be selected for materials used in making shoes to
protect children's feet from being infected by the microorganisms.
For this purpose, the bacteria from children’s
shoes were isolated and identified with morphological
observation, physiological and biochemical tests, extraction
of genomic DNA, PCR amplification and analysis of
16SrRNA sequence.
MATERIALS AND METHODS
12 pairs of shoes that were worn by children aged 6 to 12 for more
than half a year, were selected as the shoes’ sample. Beef extract
peptone medium was used for the isolation of bacteria (Ausubel et
al., 2005). LB medium, TE buffer (pH 8.0), CTAB/NaCl solution (pH
8.0) and dNTPs mixture were selected for 16SrRNA sequence
analysis (Joseph et al., 2001).
Isolation of bacteria
The leather insole, plastic insole and leather lining from the 12 pairs
of shoes were cut into pieces. Then, the pieces were put in a sterile
conical flask with glass beads and 100 ml sterile water was added
under sterile conditions. After the pieces were soaked in sterile

4134 Afr. J. Biotechnol.
water for 1 h, the conical flask was shook in a vibrator (CHZ-82,
Jintan Fuhua Instrument Co., China) at 200 rpm for 3 h to disperse
the cells of germs. When the cells were singly dispersed, 1 ml
sample of the cell suspension and 9 ml sterile water were added
together into a test tube and mixed fully. Then 1 ml mixture suspension
was taken into 9 ml sterile water to obtain the 10 -2 dilution.
According to the same procedure, different concentrations of
bacteria (10 -3 , 10 -4 , 10 -5 , 10 -6 and 10 -7 ) were obtained (Lu et al.,
2006). For isolation of the aerobic bacteria, 0.05 mg/ml nystatin was
added to sterile beef extract peptone medium to inhibit the growth
of molds and yeasts (Papamanoli et al., 2003). When the nystatin
and medium were mixed uniformly, 15 ml of the mixture was poured
into culture plates. After the medium was solidified by cooling, 0.2
ml diluents of 10 -5 , 10 -6 and 10 -7 for each concentration were drawn
into plates by a sterile pipette. Each concentration of germ
suspension was inoculated for 3 plates, and then the plates were
coated by a sterile glass rod uniformly. Lastly, the culture plates
were stored in a biochemical incubator (SPX-80BS-II, Shanghai
Medical Equipment Manufacture Co., China) at 37°C for 2 days.
For isolation of the anaerobic bacteria, 15 ml of beef extract
peptone medium was poured into culture plates. After the medium
was solidified by cooling, 0.2 ml diluents of 10 -4 , 10 -5 and 10 -6 for
each concentration were drawn into plates by a sterile pipette. Each
concentration of germ suspension was inoculated for 3 plates and
then plates were coated by a sterile glass rod uniformly. Lastly, the
culture plates were sealed by sterile Vaseline and placed in a
vacuum dryer. After been vacuumed, the plates were stored in a
biochemical incubator at 37°C for 2 days to enable the anaerobic
bacteria grow largely on the plates.
Purification of the isolates
The morphology of colonies was observed with optical microscope
(XZE-H, Chongqing Optical Instrument Co., China). According to
different morphology of the colonies, the single colony of
predominant bacteria was picked and inoculated to the medium
slant, and in total, there were 13 strains of bacteria identified
preliminarily. Then purified colonies were obtained by repeated
streaking of the single colony on fresh agar plates and their
morphology was recorded as the basis for classification in detail
(Christine, 2002). In the experiment, there were no strict anaerobe
bacteria on the vacuum dryer flat.
Morphological identification
After it was cultured at 37°C for 24 h, the strains were picked from
the slants and were stained with gram as well as spore (Kandler et
al., 1986). Then, the cell and spore of the strains were observed
with optical microscope. At the same time, the common standard
strains of Escherichia coli and Bacillus were selected as references
(Kim et al., 2000).
Physiological and biochemical appraisal
There were a total of 10 trials based on the identification of a
common bacterial system for appraising physiological and
biochemical characteristics of the bacteria (Joseph et al., 2001).
The common standard strains such as E. coli and Bacillus were
also used as references.
Extraction of genomic DNA, amplification and analysis of
16SrRNA
According to the manufacturer’s instructions, the extraction of the
total genomic DNA was performed using DNA extraction kit
(Promega, USA) and PCR amplification was performed. A portion of
the bacterial 16SrRNA gene was amplified using the primers, 27F
(5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-TACGGCT
ACCTTGTTACGACTT-3') (Mora et al., 1998). The reaction mixture
was set up on ice and it included: 10 × Taq buffer (with Mg 2 + ) (5 μl),
dNTP (3.5 μl), 1.5 μl each for forward and reverse primer, Taq DNA
polymerase (0.5 μl), template DNA (2 μl) and an addition of ddH2O
up to 36 μl as the final volume. The PCR program was denaturized
at 94°C for 5 min, and at 94°C for 30 s, while it was annealed at
57°C for 40 s and extended at 72°C for 90 s. These steps cycled a
total of 32, while the program was finally extended at 72°C for 10
min (Mora et al., 1998). Subsequently, the PCR product was
separated by 1% agar-gel electrophoresis, and the band of expected
size was cut-off and purified with a purification kit (Promega,
USA). The expected bands were sequenced by Huada Genomic
Company (Beijing, China).
Sequence identification was initially estimated using the BLAST
facility of the National Center for Biotechnology Information. All
available subsets of 16SrRNA gene sequences were selected,
analyzed and aligned with CLUSTALX 1.8 (Thompson et al., 1997).
RESULTS AND DISCUSSION
Morphology, physiological and biochemical
identification
According to the different morphology of the colony
(Figure 1), there were 13 strains of bacteria distinctly
labeled from 1 to 13#, respectively. The morphological
characteristics of the bacteria are shown in Table 1, and
the physiological and biochemical characteristics of the
bacteria are shown in Table 2. The colony characteristics
of the isolates could thus be helpful for their genetics
identification.
Identification of the sequences of 16SrRNA
According to Table 3, the similarities of the 13 strains of
16SrRNA sequence reached up to 98% when compared
with the standard strains in the GenBank (Kolbert and
Persing, 1999). Combining it with the morphological and
physiological characteristics (Buchanan et al., 1984;
Breed et al., 1994), these strains were identified as:
Bacillus licheniformis CICC 10037 (1#); Bacillus subtilis
64-3 (2#); Bacillus spore CO64 (3#); B. subtilis SRS-15
(4#); B. subtilis W-9 (5#); B. subtilis I527 (6#); B. subtilis
I3 (7#); Bacillus anthracis (8#); Staphylococcus aureus
(9#); 1Bacillus amyloliquefaciens SDG-3 (10#); B. spore
DC3158 (11#); B. spore LY (12#) and Bacillus
thuringiensis 61436 (13#).
Among the strains isolated from the children's shoes, a
strain of S. aureus accounted for 7.70%, while 12 strains
of Bacillus accounted for 92.30%. Published researches
show that most Bacillus can secrete amylase, lipase and
protease to strongly decompose carbon pollutants, complex
polysaccharides and proteins, and they also play an
important role in decomposing water-soluble organic
matter such as sweat, leading to foot odor (Wan, 2001;

Li et al. 4135
Figure 1. Morphology of the bacteria strains. The morphology of the 13 strains of the bacteria were observed with optical
microscope (labeled from 1 to 13#), respectively; the size of bacteria observed with optical microscope were magnified by
1000 times.
Table 1. Morphological characteristics of bacteria.
S/N Gram Shape Spore Colonies color Morphology Transparency Edge
1 G + Short rod Had a spore Yellowish Round Opaque Irregular
2 G + Short rod Had a spore White Irregular Opaque Irregular
3 G + Short rod Had a spore White Round Sub transparent Regular
4 G + rod Had a spore Yellowish Round Opaque Irregular
5 G + Long rod Had a spore White Round Opaque Irregular
6 G + Short rod Had a spore Yellowish Round Opaque Regular
7 G + Long rod Had a spore Yellowish Round Opaque Regular
8 G + Long rod Had a spore Yellowish Irregular Opaque Irregular
9 G + Spherical No spore Yellowish Round Opaque Regular
10 G + Rod Had a spore Yellowish Round Opaque Irregular
11 G + Rod Had a spore White Round Transparent Regular
12 G + Rod Had a spore White Round Opaque Irregular
13 G + Rod Had a spore Yellowish Round Opaque Irregular
Bruce et al., 1988). B. anthracis could spread widely in
dust pollution and easily cause skin anthrax (Quan et al.,
2005). Also, S, aureus is the most common infection
pathogen of the purulent, and it can produce toxin and
cause serious harm, as aggressive bacteria, to the skin of
people’s feet (Proctor et al., 1995). In this study, 13
strains of bacteria from children’s shoes were identified.
The results of the identification will be helpful in screening
efficient antibacterial agents for shoes and insole to
protect children's feet effectively. Also, the results of the
strain’s identification will provide more useful information
for the medical care of children’s feet.
CONCLUSION
13 strains of bacteria were isolated from the children’s
shoes and were identified as B. licheniformis, Bacillus
subtilis (5 subspecies), B. spore (3 subspecies), B.
amyloliquefaciens, S. aureus, Bacillus amyloliquefaciens
and B. thuringiensis. This is the first report on the
bacteria isolated and identified from children’s shoes in
this area. Based on the results, some efficient antibacterial
agents could be chosen against the pathogenic
microorganisms for children's shoes and insole.
ACKNOWLEDGEMENTS
The authors wish to thank the Ministry of Science and
Technology, China for co-operating in the Science and
Technology project between Romania and the People’s
Republic of China (Item No. 2009DFA42850). Also, the
authors would also like to thank the Agency of Science and

4136 Afr. J. Biotechnol.
Table 2. Characteristics of the physiology and biochemistry of bacteria.
S/N 1 2 3 4 5 6 7 8 9 10 11 12 13
Amylolysis + + + + + + + + − + + + +
Gelatinolytic + + − + + + + + + + + − +
Methyl red test − − − − − + + − + − − − +
Indole test − − − − − − − + − − − − +
Vp + + − + + + + + + − − − +
Urea test − − + − − − − − + − + + −
Sugar fermentation + + + + + + + + + + + + +
Glucose fermentation + + + + + + + + + + + + +
Mannitol fermentation + + + + + + + + + + + + +
Catalase test + + + + + + + + + + + + +
+ Indicates positive reaction results; - indicates negative reaction results.
Table 3. Identification of the sequences of 16S Rrna.
S/N Sequence number Strain number Species number Similarity (%)
1 AY871102.1 CICC10037 B. licheniformis 99.29
2 EF472462.1 64-3 B. subtilis 99.14
3 DQ643066.1 CO64 B. spore 99.89
4 GU056808.1 SRS-15 B. subtilis 99.92
5 EU815066.1 W-9 B. subtilis 99.86
6 GQ199597.1 I527 B. subtilis 98.21
7 GQ199595.1 I3 B. subtilis 100
8 AY138383.1 2000031664 B. anthracis 99.47
9 AB114634.1 A9784 S. aureus 99.31
10 FG436406.1 SDG-31 B. amyloliquefaciens 99.98
11 GU121479.1 DC3158 B. spore 99.49
12 AY787805.1 LY B. spore 98.54
13 FJ932761.1 61436 B. thuringiensis 99.48
Technology of Sichuan Province for financially supporting
this study (Item No. 2009HH0004).
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African Journal of Biotechnology Vol. 10(20), pp. 4138-4146, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1410
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Analyzing the two-dimensional plot of the interannual
climate variability for detection of the climate change in
the Large Karoun River Basin, Iran
Narges Zohrabi 1 *, Ali Reza Massah Bavani 2 , Hossein Sedghi 3 and Abdol RasolTelvari 4
1 Department of irrigation, Science and Research Branch, Islamic Azad University, Khouzestan -Iran.
2 Department of Irrigation and Drainage Engineering, College of Abouraihan, University of Tehran, Iran.
3 Department of Water Engineering, Science and Research Branch, Islamic Azad University, Tehran, Iran.
4 Department of Civil Engineering, Islamic Azad University, Ahwaz, Iran.
Accepted 20 January, 2011
In most studies on climate change, the first problem often faced by the researcher is detecting the
climate change of the study area during the past periods and attributing it to the greenhouse gases. In
this study, an attempt has been made to introduce a method for detecting the climate change during the
past periods in regional scale and attributing it to greenhouse gases with regard to climate processes
in a region. For this purpose, at first it is necessary to calculate the interannual variability range of the
region climatic variables, resulting from the interaction between the climate systems of the ‘earth’
(atmosphere, biosphere, etc.). Hence, long-term statistics (1000 years) of the temperature and
precipitation, resulting from control run (fix greenhouse gases) of AOGCM models (HadCM3 and
CGCM3), were used for Large Karoun River Basin. Then, based on the two-variant normal distribution,
the interannual climate variability range of the study area was plotted as two-dimensional temperatureprecipitation
graphs. Next, the annual temperature and precipitation anomaly values of the observation
stations in different regions of Large Karoun River Basin were compared with the region interannual
variability range for detecting the climate change of the study area during the past and attributing it to
greenhouse gases. The results show increase in temperature and decrease in precipitation trends,
denoting the fact that the temperature variable has been influenced by the climate change. So, in all
regions of the Large Karoun River Basin, the final years of the period (1971 to 2009) have almost been
located outside the interannual climate variability range, indicating the effect of climate change on the
climatic variables of the said years.
Key words: Interannual climate variability, detecting the climate change, AOGCM models, Large Karoun River
Basin.
INTRODUCTION
Different factors can unbalance the stationary time series
of a region’s climatic variables, and a recognition of this
fact can greatly contribute to the climate assessment of
that specific region in future periods. Part of these factors
relate to the interactions between the components of the
Earth’s climate system (atmosphere, hydrosphere,
cryosphere, land surface and biosphere), such as Atlantic
Multidecadal Oscillation (AMO), Pacific Decadal
*Corresponding author. E-mail:
n.zohrabi@khouzestan.srbiau.ac.ir.
Oscillation (PDO), El Niño/Southern Oscillation (ENSO)
and El Nino which can cause internal variability in time
series of climatic variables (Wang and Schimel, 2003;
Hegerl et al., 2007). Various studies have been
accomplished so far in order to detect such variability.
Kazadi and Kaoru (1996) in their research on interannual
variability, during long term periods of the climate (30
years from 1960 to 1992) in Zaire river basin (Africa),
more attentively dealt with understanding the ENSO
phenomenon, its relationship with it and its effect on
internal climate variability and showed that solar annual
cycles determine the seasonal changes of climatic

variables over a region. Timilsena et al. (2009) investtigated
the influence of interannual/interdecadal climate
variability on the Colorado river basin. In this research,
the relationship between individual impact of ENSO, PDO
and AMO and its combined effect on stream-flow was
determined using the non parametric ‘rank sum’ test. The
results indicated an increase in stream-flow during
ElNiNo and a decrease during LaNiNo phenomena,
respectively in the Colorado river basin. Peel and
McMahon (2006) investigated the recent changes in
internal variability of temperature and precipitation on a
global scale. For this purpose, they used empirical mode
decomposition (EMD) (Huang et al. 1998) for quantifying
the proportion of variation in the annual temperatureprecipitation
variability. They reported that the annual
variability of temperature and precipitation have marginally
decreased in the USA, Canada, Europe, Central
Asia, China, Japan and Australia since 1970.
Another part of the factors causing non-stationary
status in climatic variables in a region may be external
factors related to the changes in solar radiation and
changes in the earth orbital and volcanic activity.
Although changes in solar radiation or in the earth orbital
may occur in a several thousand years scale, the
temperature of the region tends to get cold for a few
months by volcanic eruption. Overall changes due to
natural external factors and internal climatic variability
within the system are called natural climate variability.
Another important factor affecting the stationary status of
the climatic variables in a region and thereby resulting in
different trends in such variables is increasing the
greenhouse gases volume in the atmosphere due to
anthropogenic activity (Wang and Schimel, 2003; Hegerl
et al., 2007; Baede et al., 2001). Many scientists believe
that the primary cause for higher earth's surface temperature
in recent decades has been the increased
concentration of the greenhouse gases (Pagano and
Garen, 2005; Garfin et al., 2008; Milly et al., 2008). To
investigate the effect of the said factors on climatic
variables of a region, the trend tests are usually used for
examining the observation records. Khliq et al. (2009)
documented guidelines of different statistical trend tests
in time series for detecting the hydrological trends on
local scales. Moreover, studies by Chen and Grasby
(2009) demonstrated the impacts of the natural
fluctuations of the quasi-cyclic components on the Mann-
Kendall and Thiel-Sen tests, which are the most common
methods used in data analysis and detection of trend in
time series. Although, from the results of this study and
other similar previous researches, this study provides a
better insight for appropriate temporal trend analyses of
the hydroclimate and associate climate data time series,
inference can be drawn that the main method used in
researches for detecting the climate change during the
past, has been analyzing the trends in climatic variables
using non-parametric tests and one-dimensional test in
most cases. The reason for the fame of such tests, proba-
Zohrabi et al. 4139
bly lie in the fact that for the application of nonparametric
methods, no assumptions on parent
distribution of the time series are necessary (Khliq et al.,
2009). It should be noted that although an increase in
greenhouse gases in the atmosphere can create a trend
(especially in temperature), the opposite case is not true,
that is, the existence of a trend in the climate data of a
region can not necessarily be attributed to the increase in
greenhouse gases. In other words, after proving the
existence of a trend in the past climate data of a region,
the relationship between the trend and the increase in
greenhouse gases should be proved. In this case, it can
be said that the climatic variables of the region in the past
have been affected by the climate change. Therefore, in
the climate impact assessment studies, the system
behavior which is a result of alteration by natural
fluctuations or human-induced climate change needs to
be separately investigated (Sridhar and Nayak, 2010).
Braganza et al. (2004) showed the influence of the
external natural forces (solar radiation and volcanic activities)
and the forces resulting from anthropogenic
activities on the climate change observed throughout the
twentieth century, in the course of considering 5 simple
indices of the surface temperature including: the globalmean
temperature (GM), mean land-ocean temperature
contrast (LO), mean magnitude of the annual cycle in
temperature over land (AC), the meridional temperature
gradient in the northern hemisphere mid-latitudes (MTG)
and the northern/southern hemisphere temperature
contrast (NS). For attributing the changes that occurred in
the temperature indices, they used simple and multiple
linear regression between observation records of the
temperature indices and the simulated data derived from
the control run (under the influence of natural forces and
fix greenhouse gases) of 5 models coupled in the
atmosphere-ocean. The linear trends observed in all the
indices except for the hemispheric temperature contrast
(NS) are significantly larger than the values that resulted
from the simulation of such models.
In this research, the detection and attribution of internal
variability to the greenhouse gases and surveying the uncertainty
of different models has been perfectly performed,
but only in one dimension of “temperature”, while
nothing has been done about the “precipitation” variable.
More detailed researches in the field of detection and
attribution of variability which occurred in greenhouse
gases can be found in the paper presented by the
“International ad hoc detection and attribution group”
(Barnett et al. 2005). The mentioned paper, while
reviewing the best part of the studies regarding the
detection of internal variability and attributing them to the
greenhouse gases, shows that the main goal of detection
and attribution studies during the past several years has
been a comparison of the observed changes in climate,
primarily during the past century, with the data simulations
by the Coupled General Circulation Model
(CGCM) that have been forced by estimates of historical

4140 Afr. J. Biotechnol.
Figure 1. Location of the Large Karoun River Basin in Iran and some of its important
installations.
changes in anthropogenic and (natural external forces)
most frequently in “temperature” on a global-scale. Also,
it is indicated that according to the results of the studies
carried out in different parts of the globe, the temperature
variable has been influenced by the climate change.
Accordingly, although in a study conducted in the UK
by Hulme and Brown (1998), the internal climate
variability range and its attribution to greenhouse gases is
well recognized, but the uncertainty of the simulation
models in internal climate variability calculations has
been ignored. This is why in climate change studies there
are various uncertainty resources in different stages
required by simulation of the climatic variables by
Atmosphere-Ocean General Circulation Mode (AOGCM)
model on regional scales (Jones and Page, 2001)
It can be seen therefore in most previous researches
that first, the natural and internal climate variability have
been investigated often in one-dimensional state using
trend tests in order to detect the climate change that is
not attributed to the greenhouse gases. Secondly, the
uncertainty of the simulation models of the internal
climate variability has not been considered. Thus, the
present study intends to propose a new method for twodimensional
detection of the climate change during the
past periods, considering the uncertainty and the way it is
attributed to greenhouse gases. Meanwhile, considering
the fact that this is contrary to numerous studies in Iran,
concerning the trend detection in climate variables time
series, no works have been carried out regarding the
occurred variability’s attribution to the greenhouse gases
and uncertainty analysis.
Description of the study area
The Large Karoun River Basin is located in the southwestern
part of Iran, and due to the fact that it
encompasses the country's largest water potential, this
basin houses the most and the greatest water projects in
Iran. The basin area of 67,257 km 2 encompasses about
5% of the total area of Iran. 67% of the basin area is
mountainous and the remaining 33% is made of high
plains. Its two main tributaries, including Karoun and Dez
rivers, flow over Khouzestan Plain after journeying separately
through anfractuous routes in mountains and join
together at Band Ghir located 50 km off north of Ahwaz
city. As such, the Karoun River eventually terminates at
the Persian Golf (Figure 1).
A brief description of the study area is thus presented
and details can be found in Jamab (1999). Climatic

Mean Monthly precipitation(m m /m onth)
200
150
100
50
0
Jan Feb Mar Apr Mar Jun Jul Aug Sep Oct Nov Dec
P Khoram abad P Shahrekord P Ahwaz P Pol shalo
P Talezang P Gotvand T Khoramabad T Shahrekord
T Ahwaz T Talezang T T Gotvand
Gotvang Gotvan
T Pol Shalo
Figure 2. Mean monthly precipitation (P) and mean monthly temperature in different regions of the Large
Karoun basin.
variation in Large Karoun River Basin is numerous and
the types of dry desert, semi dry, semi wet, wet, very wet
Mediterranean and cold humid climates are found in it.
Precipitation regime in the basin is Mediterranean, that is,
the dry season exactly matches the summer and the
rainfall season coincides with winter. About 96% of
rainfall occurs between October and May which varies
from 1800 mm /year in the heights of 150 mm/year in
plain areas (Jamab, 1999).
Fundamentally, Figures 2 and 3 show the precipitation
and temperature conditions in different regions of the
Large Karoun River Basin. As it is clear from the figures,
a wide range of precipitation data have been recorded in
basin stations, indicating various climates dominating the
Large Karoun River Basin. This is due to the large extent
and considerable heights of the basin which makes the
investigation of the precipitation variable and climate
conditions analysis dependant on the division of the basin
into its constituting micro-climates. The classification of
the micro-climates has been performed based on the
precipitation pattern in relation with the region
temperature, vegetation and height of different regions
and lesser attention has been paid to the precipitation
volume. This means that in classification of microclimates,
some parameters like altitude, temperature and
vegetation of land are more influential than precipitation
volume. For example, as it can be seen from Figure 3,
Zohrabi et al. 4141
despite the identical volume of the rainfall in pluviometery
stations of Ahwaz and Shahrkurd, these two regions have
different climates due to their different height and
temperature. Also, Sepid-dasht and Yasouj despite their
identical volume of precipitation are located in two
regions with different climates. So, the Large Karoun
River Basin has been divided into four distinct climatic
regions (Figure 1 and Table 1).
Each region will be discussed as a separate uniform subbasin
(Table 1) as follows:
(1) Mountainous region of Karoun River Basin.
(2) Mountainous region of Dez River Basin.
(3) Middle part of the Large Karoun River Basin (from
installed stations at an input of Karoun1 and Dez dams
reservoirs to Ahwaz).
(4) Southern regions of the Large Karoun River Basin
(from Ahwaz to the Large Karoun estuary (Arab, 2009).
MATERIALS AND METHODS
Data
Considering the climatic classification, data verification and
suitability of the statistical period, precipitation data of more than 50
years (1956 to 2009) and temperature data of more than 30 years
(1971 to 2009), four main stations were selected in this basin
40
35
30
25
20
15
10
5
0
-5
Mean Monthly T em perature( o C/m onth)

Row
4142 Afr. J. Biotechnol.
Mean annual precipitation (mm)
1000
900
800
700
600
500
400
300
200
100
0
Shahrekord
Figure 3. Mean annual precipitation (P) during 1956 to 2009 in different regions of the Large Karoun basin.
Table 1. Geographical characteristics of the selected river stations.
Different regions of Large
Karoun River Basin
Station
name
Long Lat
Elevation
masl
Drainage
area (km 2 )
Time period
P T
1 Mountainous region of Karoun River Basin Pol Shalo 50.08 31.45 700 23400 1956- 2008 1971-2009
2 Mountainous region of Dez River Basin Tale Zang 48.46 32.49 480 16213 1956- 2008 1971-2009
3 Middle basin of Large Karoun River Basin Gotvand 48.49 32.15 75 32425 1956- 2008 1958-2009
4 Southern region of Large Karoun River Basin Ahwaz 48.41 31.2 20 60737 1957- 2008 1957-2009
(Table 1). The aforementioned data were acquired from “Iran
Meteorological Organization” and “Ministry of Energy” [Iran Water
Resource Management Company and Khouzestan Water and
Power Authority (KWPA)].
According to this classification, Pol-e Shaloo (located at Karoun3
dam), Tale Zang (located upstream of Dez dam), Gotvand and
Ahwaz stations are considered as the representative of the
mountainous region of Karoun river basin, the mountainous region
of Dez river basin, the middle part of the Large Karoun River Basin
and the southern region of the Large Karoun River Basin,
respectively (Figure 1 and Table 1).
Simulation of climatic variables during the past periods using
HadCm3 and CGCM3 models
One of the methods for simulating internal climate variability within
a region is to explore climatic variables time series in long-term
records (that is, more than several hundred years). Due to the lack
of long-term observation records around the world, the alternative
method will be the use of a three-dimensional coupled ‘atmosphereocean
general circulation model’ or AOGCM (Wilby and Harris,
2006; Mitchell, 2003). These models are based on the laws of
physics represented by mathematical relationships which are
resolved in a three-dimensional network on the earth surface. In
order to simulate the earth’s climate, the main climatic processes
including atmosphere, cryosphere, biosphere and hydrosphere,
were simulated in independent sub-models. So far, different general
Ahwaz
Pol Shalo
Talezang
Gotvand
Station
Sepiddasht
Barz
Armand
circulation models have been developed by different research
institutes including UKMO-HadCM3, CCCma-CGCM3, GFDL_CM2,
CM2.1, NCAR_PCM, CCSM3 and CSIRO-MK3 (cited in IPCC-AR4,
2007). In this research, to consider the uncertainty of the model, the
outputs of two CGCM3 and HadCM3 models with spatial resolution
of roughly 3.75 degrees lat/lon and 2.5/3.75 lat/lon, respectively,
were used. In a specific type of simulation by this model, the
amount of greenhouse gases over simulation time is kept at an
observed level in the year 1850. In this type of simulation, known as
the control run, the climatic variables are often simulated over a
1000-year period. Obviously, due to the fixed amount of
greenhouse gases in such simulations, the time series of the
variable under study is only affected by internal forces of the
climate system and thus determines the internal variability of the
climate system. If it is assumed that no changes will occur in the
external factors including solar radiations and volcanic activities
over future periods (that is, the next one hundred years), one can
conclude that such simulation reflects natural climate variability
(Oldenborgh et al., 2005; Lambert et al., 2005).
Calculation of interannual climate variability
In this study, in order to separate climate changes caused by
greenhouse gases from interannual variability, the variability range
of the temperature and precipitation are first examined. On the
other hand, the recorded statistical periods’ length available at
gauging stations is not adequate for calculating the interannual
Marghak
Yasoj

climate variability of the study area. Also, the observation records of
the recent decades have been probably affected by radiation force
resulting from increasing greenhouse gases, thereby making
separation of interannual variability from climate changes quite
difficult. Thus, in order to show the interannual variability across a
region, long-term records of 1000 years generated by the control
run of AOGCM models are used (Ruosteenoja et al., 2002). To
analyze the interannual variability range of two “temperature” and
“precipitation” variables of the study area, first, their annual
anomaly time series with respect to the average base period is
calculated. [By definition, temperature anomaly is the temperature
difference from a base temperature, while precipitation anomaly is
the precipitation ratio difference from a base precipitation]. The
world meteorological organization (WMO) has suggested that in
order to harmonize the selection of the 30 years base period in
different climate change studies and the possibility of comparing
them, the 1961 to 1990 base period should be considered. On the
other hand, as the organization recommends, where data recorded
on the station of the study area for this period is not available, the
period of 1971 to 2000 will be replaced (IPCC-TGCIA, 1999).
So, because the statistics of the temperature variable of the Large
Karoun River Basin upstream do not cover the 1961 to 1990 period,
the period of 1971 to 2000 is considered as the base period. Then,
it is assumed that the time series of these two variables follow twovariant
normal distribution. With this assumption, the relationship
governing the two-variant normal distribution of the temperature
and precipitation anomaly will be realized as follows (Von Storch
and Zwiers, 2002):
1
1
2
2
⎧ ′ ′ ′ ′
T
⎨
⎩
T
− 2ρ
σ
R
σ
2 2
2
− ρ σ T
T R σ R
+
R
⎫ 2
⎬ = χ
⎭
Where, T ′ and R′ are temperature and precipitation anomalies,
σ T σ and R are standard deviation of temperature and
precipitation and ρ �represents the correlation between the
2
χ temperature and precipitation anomalies. The amount of is
2
χ determined by square distribution of with 2 degrees of freedom
which is extracted from the respective tables at 95% confidence
2
χ level (df = 2, 95% → = 5.99). By plotting Equation (1) for 95%
confidence level, elliptic circuits whose internal area indicates the
interannual climate variability range for temperature and
precipitation are obtained and its external area represents the
variability related to other factors (like greenhouse gases).
RESULTS
Approximation of interannual climate variability in
the study area
Figure 4 shows the interannual climate variability range
with regard to the calculated 95% confidence level in the
study area. In this figure, the horizontal axis represents
temperature anomaly, while the vertical axis shows
precipitation anomaly with respect to the base period
average of 1971 to 2000. As it can be seen from this
figure, most of the points showing annual anomalies are
depicted inside the ellipse or too close to its perimeter. It
means that the interannual climate variability range of the
(1)
Zohrabi et al. 4143
region has been plotted quite well by the ellipses. Overall,
due to low skewness, the depicted figure indicates the
assumed two-variant normal distribution of temperature
and precipitation as acceptable for plotting the
interannual climate variability range of the region under
study. On the other hand, the excellent overlap obtained
from each of the two models shows low uncertainty of the
model selection. Therefore, it can be concluded with 95%
probability that the combined interannual temperatureprecipitation
variability anomaly values of the Large
Karoun River Basin are less than 1.5°C and 75%,
respectively.
After the determination of the interannual climate variability
range within the study area, for evaluating the
significance of climate change in the past half century (its
attribution to greenhouse gases), the values of these
variability were compared with the observed annual
anomalies of temperature and precipitation for each
region of the Large Karoun River Basin. Figure 5 shows
the two-variant trend of temperature-precipitation anomalies
during the past half century with respect to
average (1971 to 2000) and the interannual climate
variability range in different regions of the Large Karoun
River Basin.
Since the inside area of each ellipse represents the
combined interannual temperature-precipitation variability
range within the study area, a point outside this range is
indicative of the temperature and precipitation changes
being dependant on factors other than interannual
climate variability. Frequency time evolution of annual
climate in the Large Karoun River Basin over the last
years (1971 to 2009) can be traced from A to Z. As the
points, A and Z, are considered as the start and ending
years of the recent century, respectively, the results of
the two-dimensional trend of temperature-precipitation
anomalies for the past half-century with respect to the
average (1971 to 2000 period) of four regions specified in
Figure 5 shows an increase in temperature and a
decrease in precipitation. Approximately, in all regions of
the Large Karoun River Basin, especially the most
southern part of it, the “temperature” found throughout
the ending years of this period lies outside the interannual
climate variability range, indicating the significance
of climate change (that is, its relationship with the
increase of greenhouse gases) in these years. The
droughts that occurred in the ending years of the period
confirm these results. Meanwhile, water years of 1999 to
2000 and 2000 to 2001 and also 2007 to 2008 and 2008
to 2009 have been defined as ‘dry years’. A review of
annual precipitation changes in the representative
northern stations of Karoun and Dez river basins, namely
Pol-e Shallo and Taleh Zang stations shows more than
50% decrease in precipitation than under normal
conditions. As such, higher precipitation in these regions
of the basin compared with that of downstream regions,
considerably reduces the basin yield, especially on
upstream Dez and Karoun 3 dams at Pol-e Shallo and
Taleh Zang stations (Arab, 2009). The recent trend towards

4144 Afr. J. Biotechnol.
Figure 4. Interannual temperature-precipitation variability, with respect to the average period of 1971
to 2000 for Large Karoun River Basin.
Figure 5. Two-dimensional trend of annual temperature–precipitation anomalies for the past half
century with respect to the average period of 1971 to 2000 for the Large Karoun River Basin,
together with interannual climate variability range. a: Mountainous region of Karoun river (Pole
Shalo station); b: Mountainous region of Dez river (Talezang station); c: Middle basin of Large
Karoun River Basin (Gotvand station); d: Southern region of the Large Karoun River Basin
(Ahwaz station). Black points: observed data Grey points: Ten last years.

Figure 5. Continued
warming and drying, evidenced in Figure 5 is reinforced
and, in the context of the past half century of Large
Karoun River Basin climate variability, it becomes more
significant. Therefore, detecting the range of inter-annual
climatic variability of a region helps in identifying the
years when extreme events such as drought and flood
occur.
DISCUSSION
In this study, an attempt has been made to present a
method of detecting past climate change in a regional
scale and attributing it to greenhouse gases with regard
to the climate process in a region. For this purpose,
initially the variability range of climate variables related to
the regional interannual variability was determined using
control run data of two AOGCM models. The two models
Zohrabi et al. 4145
data were used to study the uncertainty of two AOGCM
models in interannual variability of the study area. As
such, it was assumed that the probability function of the
temperature and precipitation was a two-variant normal
distribution. With this assumption, the interannual climate
variability range of the region was determined. The
results of this study showed that: Firstly, the assumption
of normality of temperature-precipitation probability function
was acceptable due to low skewness of the plotted
range. Secondly, the uncertainty between AOGCM
models in estimating the natural climate variability range
is low.
The comparison between the two-dimensional observed
temperature-precipitation trend of the region and the
interannual variability range showed that the climatic
variable trend in the past half century in Large
Karoun river basin had led to increased temperature and
decreased precipitation, denoting the fact that the

4146 Afr. J. Biotechnol.
temperature variable has been influenced by the climate
change. Khordadi et al. (2008) studies also show that
regional climate in Iran is becoming dryer and warmer,
and probably this will continue in the future (Montazeri
and Fahmi, 2003). Also, the occurrence of low-flow and
drought crisis in the second half of the century has been
more frequent, incurring considerable damages because
of decreased quality water resources on regional and
national scales. Meanwhile, the impacts of greenhouse
gases are clearly noticeable in the ending years of the
observation period. In general, the proposed method in
this paper can be used as an alternative to conventional
stochastic methods in which only the data trend tests
(such as Mann-Kendall test) for detecting and attributing
climate change in the past periods are used.
ACKNOWLEDGEMENTS
The authors wish to thank organizations and individuals
who supported this work. Also, they wish to thank, in a
special way, the Ministry of Energy, Khouzestan Water
and Power Authority (KWPA) and Iran Water Resource
Management Company for the provision of necessary
data sets and for their valuable support during this study.
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African Journal of Biotechnology Vol. 10(20), pp. 4147-4156, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1905
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Screening and optimization of extracellular lipases by
Acinetobacter species isolated from oil-contaminated
soil in South Korea
Periasamy Anbu # *, Myoung-Ju Noh # , Da-Hye Kim, Jun-Seok Seo, Byung-Ki Hur* and Kyeong
Ho Min
Department of Biological Engineering, College of Engineering, Inha University, Incheon 402-751, Republic of Korea.
Accepted 6 December, 2010
A total of 53 strains of bacteria were isolated from oil contaminated soil collected in South Korea. The
isolated bacteria were screened using spirit blue agar and Rhodamine-B agar media. Two of the
isolated strains exhibited a greater clear zone than the others, indicating higher lipase activity.
Therefore, these two strains (BK43 and BK44) were selected and identified based on their
morphological and physiological characteristics. The 16S rRNA gene sequencing was also
implemented. Phylogenetic analyses based on the results of 16S rRNA gene sequencing revealed that
BK43 and BK44 were close in identity to Acinetobacter junii. The optimum pH and temperature for
lipase production by BK43 were found to be 6.0 at 30°C, after 24 h of incubation, while BK44 were found
to be 6.0 at 25°C, after 12 h of incubation. In addition, increased enzymatic production was obtained
when the organisms were cultured in medium supplemented with 1% sucrose as the carbon source.
Among the different lipase inducers tested, both strains utilized Tween 80 and produced a great level of
extracellular lipase. Overall, the results of the present study demonstrate that the genus Acinetobacter
is good for extracellular lipase production under acidic conditions.
Key words: Acinetobacter, lipase, optimization, screening.
INTRODUCTION
Lipases are triacylglycerol acylhydrolases (EC 3.1.1.3)
that catalyze the hydrolysis of triacylglycerol to glycerol
and fatty acids (Sharma et al., 2001). Lipases are ubiquitous
in nature and produced by animals, plants and
microorganisms. Currently, microbial lipases are receiving
a great deal of attention due to their potential for use
in industrial processes. Most commercial extracellular
products are obtained from microorganisms and have the
ability to catalyze a wide variety of reactions in aqueous
and non-aqueous phases (Saxena et al., 2003). Microbial
lipases have also received more attention due to their
*Corresponding author. E-mail: anbu25@yahoo.com,
biosys@inha.ac.kr. Tel: +82-32-860-7512. Fax: +82-32-872-
4046.
#These authors contributed equally to this work.
selectivity, stability and substrate specificity (Treichel et
al., 2010). Particularly, extracellular bacterial lipases are
of commercial importance in the food, detergent, cosmetic
and pharmaceutical industries, as well as
inprocesses involving organic synthesis and fat/oil degradation
(Jaeger et al., 1994).
Extracellular lipase production by bacteria are influenced
by the composition of the growth medium, cultivation
conditions and many physico-chemical (pH and temperature)
and nutritional factors (carbon, nitrogen and lipid
sources) (Jaeger et al., 1994). Most bacterial lipases are
generally induced in medium that contains the proper
fatty acids and oils (Joseph et al., 2006; Immanuel et al.,
2008; Kiran et al., 2008). However, a few investigators
have reported that the activity of lipase produced by
Pseudomonas aeruginosa EF2 and Acinetobacter
calcoaceticus was repressed by the presence of a lipid
source in the medium (Gilbert et al., 1991; Mahler et al.,
2000). In addition, Lin et al. (2006) have found that

4148 Afr. J. Biotechnol.
vitamins influenced lipase production by the edible Basidiomycetes,
Antrodia cinnamomea.
A variety of extracellular lipases of bacterial origin with
different properties and specificities have been described
and characterized. Extracellular lipase was isolated from
many different bacterial species, including Bacillus
(Ertugrul et al., 2007) and Pseudomonas (Kiran et al.,
2008; Wang et al., 2009). Additionally, many studies have
been con-ducted to evaluate lipase production under
alkaline condi-tions (Chen et al., 1998; Kasana et al.,
2008; Kiran et al., 2008; Wang et al., 2009) but few
studies have focused on acidic lipases (Bradoo et al.,
1999; Liu et al., 2007).
Lipase production by Acinetobacter radioresistens
under alkaline conditions in the presence of n-hexadecane
was evaluated (Chen et al., 1998). In recent
years, most studies conducted to improve lipase production
have focused on their production by one important
genus, Acinetobacter (Chen et al., 1998; Dharmsthiti et
al., 1998). Acinetobacter strains have been isolated from
a variety of sources, including soil (Bompensieri et al.,
1996) and water (Blaise and Armstrong, 1973). However,
industries are still seeking strains of bacteria that produce
a high yield of potent lipase with excellent properties
using cost-effective methods. Therefore, the present
study was conducted to isolate novel lipase producing
bacteria, after having the isolated strains identified and
the culture conditions for optimal production of extracellular
lipase determined.
MATERIALS AND METHODS
Materials
Gum arabic, p-nitrophenyl palmitate, sodium deoxycholate and
tributyrin were purchased from Sigma (USA). Tween 80 was
obtained from Duchefa, Biochemica (The Netherlands). All other
chemicals used in this study were of analytical grade.
Isolation and screening of lipase-producing microorganisms
Fifty three strains of bacteria were isolated from oil contaminated
soil collected in South Korea using serial dilution. The isolates were
maintained on LB agar plates at 4°C and stored at -80°C in glycerol.
The isolated bacteria were screened for the production of lipase
using spirit blue agar containing lipase reagent as Marshall (1992)
method. The lipolytic activities of all isolates were then compared by
measuring the width of the areas of clearing or areas of deep blue
color around the colonies. In addition, the same strains were
screened using the Rhodamine-B method (Kouker and Jaeger,
1987). The bacterial strains were inoculated on the agar plates,
after which the lipolytic activity was determined by the formation of
an orange fluorescent zone around the fungal colonies that was
visible upon UV irradiation (350 nm).
The liquid culture medium (Tryptic soy broth) used for lipase
production contained the following (g/L): Pancreatic casein, 17;
enzymatic digest soybean, 3; NaCl, 5; dipotassium phosphate, 2.5;
glucose, 2.5; pH 7.5. To produce the lipase, an Erlenmeyer flask
(250 ml) containing 50 ml of medium was inoculated with an aliquot
of approximately 1% of the preculture prepared in LB broth (g/L):
Tryptone, 10; yeast extract, 5; NaCl, 10; pH 7.0. The inoculated
flasks were then incubated at room temperature with constant
shaking at 180 rpm. Then, the cell-free supernatant was recovered
by centrifugation (10,000 rpm, 10 min at 4°C) and the clear supernatant
was used to determine the lipase activity. The growth of the
microorganisms was then determined by measuring the absorption
at 600 nm.
Identification of the microorganisms
The lipase producing bacteria was identified by morphological and
biochemical characterizations. A gram (positive/negative) stain
reaction was conducted using the Biomerieux system according to
the manufacturer’s instructions. The biochemical tests were conducted
using the API 20NE system according to the manufacturer’s
protocols (Biomerieux). The identification was further confirmed by
the 16S rRNA gene sequencing method. Briefly, genomic DNA was
extracted using a genomic purification kit (Promega, USA). The
DNA was then amplified by PCR using the following universal 16S
rRNA gene primers, 8-27F: 5’-AGAGTTTGATCCTGGCTCAG-3’
and 1472R: 5’-TACGGYTACCTTGTTACGACTT-3’. PCR was
conducted by subjecting a reaction mixture to initial denaturation at
94°C for 5 min, followed by 35 cycles of 94°C for 45 s, 55°C for 1
min, 72°C for 1 min and a final extension step at 72°C for 10 min.
Phylogenetic analysis
The 16S rRNA gene sequence was compared with sequences
available in the nucleotide database using the BLAST algorithm at
the NCBI. Phylogenetic tree was constructed using the neighbor
joining method (http://www.phylogeny.fr).
Fatty acid analysis
To analyze total cellular fatty acid content, the bacterial cells were
cultured at 30°C for 24 h, after which the fatty acid profiles were
determined based on the method described by Lepage and Roy
(1984) using a gas chromatograph (Hewlett Packard 6890, USA)
equipped with a flame-ionized detector (FID) and a DB23 (30 m ×
0.25 mm × 0.26 µm, Agilent Technologies, USA) capillary column.
Lipase assay
Lipase activity was assayed quantitatively using 4-nitrophenyl
palmitate as the substrate according to the method described by
Winkler and Stuckmann (1979). Briefly, 10 ml of isopropanol
containing 30 mg of 4-nitrophenyl palmitate (pNPP) was mixed with
90 ml of 0.05 M phosphate buffer (pH 8.0) containing 207 mg of
sodium deoxycholate and 100 mg of gum arabic. A total of 2.4 ml of
freshly prepared substrate solution was then pre-warmed at 37°C
and mixed with 0.1 ml of enzyme solution. After incubation at 37°C
for 15 min, the absorbance was measured at 410 nm against an
enzyme free control. One enzyme unit was defined as the amount
of enzyme that liberated 1 µmol of 4-nitrophenol per minute under
the assay conditions.
Optimization of lipase production
Initially, the lipase production was determined after culture in tryptic
soy broth from 6 to 48 h at 28°C. The lipase production was then
evaluated at different pHs ranging from 5.0 to 10.0 and at tempera-

Table 1. Screening isolated bacterial strains for extracellular lipase.
Isolates No.
Sprit blue
agar (mm)
Rhodamine-B agar
(mm)
Isolate No.
Sprit blue
agar (mm)
Anbu et al. 4149
Rhodamine-B agar
(mm)
BK01 - ++ BK28 - -
BK02 ++ - BK29 + -
BK03 ++ + BK30 ++ +
BK04 - - BK31 + +++
BK05 - ++ BK32 ++ +
BK06 + ++ BK33 - +
BK07 ++ ++ BK34 - ++
BK08 - + BK35 - -
BK09 - ++ BK36 ++ +++
BK10 + + BK37 - -
BK11 - - BK38 ++ +
BK12 - - BK39 - ++
BK13 - - BK40 - -
BK14 - + BK41 ++ +
BK15 - - BK42 + -
BK16 - - BK43 +++ +++
BK17 - + BK44 +++ +++
BK18 ++ + BK45 - -
BK19 - - BK46 - -
BK 20 ++ - BK47 - -
BK21 - - BK48 + -
BK22 + - BK49 ++ +
BK23 + - BK50 - -
BK24 + - BK51 + -
BK25 ++ + BK52 ++ ++
BK26 - ++ BK53 - +
BK27 ++ ++
+++, High activity (above 50 mm); ++, moderate activity (above 25 to below 50 mm); +, low activity (below 25 mm); -, no activity.
tures of 20, 25, 30, 37 and 45°C. The pH of the medium was
adjusted prior to autoclaving. Further, the changes in lipase
production in response to the following carbon sources (1%) were
evaluated: glucose (control), fructose, xylose, maltose, lactose,
sucrose, mannitol and starch. The carbon sources were sterilized
separately and then aseptically added to the autoclaved medium.
The following substances were used as lipase inducers (1%): olive
oil, sesame oil, soybean oil, tributyrin and Tween 80. Medium that
contained no lipase inducer was used as a control.
RESULTS AND DISCUSSION
Isolation, screening and identification of lipase
producing bacteria
A total of 53 distinct morphological bacterial strains were
isolated from oil-contaminated soil in South Korea. The
isolated strains were screened for extracellular lipase
using spirit blue agar and Rhodamine-B agar media. Two
of the isolates produced a larger clear zone than the
others, indicating higher lipase activity. These two strains
that produced blue color around bacterial colonies, were
grown on spirit blue agar medium due to the hydrolysis of
tributyrin and Tween 80. Furthermore, a fluorescent zone
on Rhodamine-B agar medium was observed under UV
irradiation (350 nm), indicating that the two strains were
able to hydrolyze olive oil (Table 1). The earlier results
confirmed that the two strains were potent to produce
lipase and also indicate that lipolytic bacteria are widespread
in the oil-contaminated environments. The selected
strains (BK43 and BK44) were then identified based
on morphological, physiological and biochemical characterizations.
The physiological and biochemical characteristics
are presented in Table 2. The results showed that
both strains are gram negative, aerobic and coccoid rod
shape. A biochemical test was conducted using an API
identification kit. Both strains were oxidase negative and
capable of assimilating caprate and malate (Table 2),
which indicates that strains BK43 and BK44 were closely
related to Acinetobacter junii. Taken together, these
characteristics indicated that both strains belong to the
genus Acinetobacter.

4150 Afr. J. Biotechnol.
Table 2. The morphological, biochemical and physiological characteristics of strains BK43 and
BK44.
Tests Reactions BK43 BK44
Gram staining - Negative Negative
Shape - Coccoid rod Coccoid rod
NO3 Nitrate/nitrite reduction - -
TRP indole production - -
GLU Acid production from glucose - -
ADH arginine dihydrolase - -
URE Urease - -
ESC β-glucosidase - -
GEL Protease (gelatin hydrolysis) - -
PNPG β-galactosidase - -
GLU assimilation of glucose - -
ARA assimilation of arabinose - -
MNE assimilation of mannose - -
MAN assimilation of mannitol - -
NAG assimilation of N-acetyl-glucosamine - -
MAL assimilation of maltose - -
GNT assimilation of gluconate - -
CAP assimilation of caprate + +
ADI assimilation of adipate - -
MLT assimilation of malate + +
CIT assimilation of citrate - -
PAC assimilation of phenyl-acetate - -
OX cytochrome oxidase - -
+; Positive result, -; negative result.
Sequencing and phylogenetic analysis
The identities of the bacteria were further confirmed by
16S rRNA sequencing. Approximately 1381 and 1383 bp
sequences were obtained from BK43 and BK44,
respectively, and then aligned with other 16S rRNA sequences
available in the GenBank database. A phylogenetic
tree was then constructed using the neighbor-
joining method by only culturable Acinetobacter species
(Figure 1). The phylogenetic analysis indicated that the
sequences of strain BK43 and BK44 were highly homologous
(99 and 100%, respectively) with the sequence of
A. junii (NCBI accession No. AM184300) followed by
Acinetobacter sp. (NCBI accession No. AM412159). The
16S rRNA sequences of strains BK43 and BK44 were
deposited in GenBank under accession numbers
GQ202270 and GQ202271, respectively. Based on these
results, BK43 and BK44 are Acinetobacter sp.
Fatty acid analysis
The fatty acid profiles of the Acinetobacter species
isolated in this study are presented in Table 3. Although
the patterns of fatty acid compositions of both strains
were similar, the fatty acid contents differed. The fatty
acid profiles were more similar to A. junii (Yoon et al.,
2004) than to other Acinetobacter species such as
Acinetobacter antiviralis (Lee et al., 2009) and A.
radioresistens (Nishimura et al., 1988). The major fatty
acids produced by the strains were C16:0, C16:1 (n-7)
and C18:1 (n-9). However, the C18:1 (n-9) accumulated
more than 50% in both strains. These results further
suggest that the isolated strains BK43 and BK44 are
Acinetobacter sp. The identified bacterial strains BK43
and BK44 were used for further studies to optimize their
extracellular lipase production.
Production of extracellular lipase
Most extracellular bacterial lipases are influenced by
nutritional and physiological factors such as pH, temperature
and carbon sources (Immanuel et al., 2008; Kiran
et al., 2008; Wang et al., 2009). The lipase activity of both
isolates was evaluated from 6 to 48 h. The maximum
lipase production was observed after 12 h of incubation
for BK44 and after 24 h for BK43 (Figure 2). Furthermore,
the enzyme activity was gradually decreased after 12 and
24 h, respectively. Therefore, the optimum incubation

Figure 1. Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the
relationships between strains BK43, BK44 and related strains. Bar, 0.005 changes per nucleotide
position.
periods for each strain were maintained throughout the
studies. The results of the present study are similar to
those of several other studies in which the optimum incubation
period for lipase production was found to be 12 to
24 h (Chen et al., 1998; Dharmsthiti et al., 1998).
However, some bacterial species required more than 2
days for the maximum lipase activity to occur (Joseph et
al., 2006; Kiran et al., 2008; Wang et al., 2009).
Anbu et al. 4151
Theresults of the present study indicate that the
organisms isolated here can be used to produce large
quantities of lipase within a short period of time.
Both strains were able to produce a high level of lipase
at pH 6.0. However, the enzyme activity decreased
rapidly at alkaline pHs. Figure 3A shows that both strains
prefer an acidic pH. These findings differ from the results
of other studies in that the optimum lipase production

4152 Afr. J. Biotechnol.
Lipase activity (U/m l)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Table 3. Fatty acid profiles of Acinetobacter species.
Fatty acids Contents in BK43 (%) Contents in BK44 (%)
C10:0 1.504 1.864
C12:0 1.602 1.497
C14:0 trace trace
C15:1 3.6 3.09
C16:0 19.2 20.81
C16:1 (n-9) trace 1.506
C16:1 (n-7) 16.31 16.86
C17:0 trace trace
C17:1 trace trace
C18:0 trace trace
C18:1(n-9) 54.2 51.524
Trace: fatty acids present in amounts less than 1%.
BK43
BK44
6 12 24 36 48
Incubation periods (h)
Figure 2. Effect of different incubation periods on lipase production. Each point represents the
mean ± SEM of three independent experiments.
occurred at neutral pH (Joseph et al., 2006) or alkaline
pH (Chen et al., 1998; Kiran et al., 2008; Wang et al.,
2009), but similar at pH 6.0 (Ertugrul et al., 2007). Most
bacterial species are able to produce greater amounts of
lipase at pH 6.5 to 7.0 (Dharmsthiti et al., 1998; Gao et al.,
2004; Joseph et al., 2006). Most reports, available regarding
the production of lipases by fungi, involved studies
conducted under acidic conditions (Cihangir and Sarikaya,
2004; Mhetras et al., 2009).
Among different temperatures tested (20 to 37°C), the
highest production by BK43 was obtained at 30°C, while
the highest production by BK44 was obtained at 25°C
(Figure 3B). In the case of BK44, approximately 25% of
the enzyme activity was lost when the temperature increased
from 25 to 30°C. In addition, the enzyme
production by both strains has decreased dramatically at
37°C, which indicates that these strains are unable to
grow and produce lipase at higher temperatures. Many
species of Acinetobacter have been reported to be
psychrophilic and psychrotrophic (Kasana et al., 2008;

Figure 3. Effect of different pHs (A) and temperatures (B) on lipase production. Each point
represents the mean ± SEM of three independent experiments.
Park et al., 2009), however, the strains isolated here
failed to grow and produce lipase at low temperatures
even after 1 week of incubation. The optimum temperature
for lipase production was 25 to 30°C, which agrees
with the production of lipase by many microorganisms,
including A. radioresistens and A. calcoaceticus LP009
(Chen et al., 1998; Dharmsthiti et al., 1998; Kiran et al.,
2008).
A
B
Anbu et al. 4153
Several studies have shown that lipase production was
influenced by different carbon sources present in media
(Mahler et al., 2000; Immanuel et al., 2008). In general,
the required specific carbon source and its concentration
differ among organisms. Therefore, in this study, various
carbon sources (1%) were added to the culture medium
and their effects on lipase production, by both strains,
were evaluated. The greatest increase in lipase production

4154 Afr. J. Biotechnol.
B
A
Figure 4. Effect of various carbon sources (A) and lipase inducers (B) on lipase production.
Each point represents the mean ± SEM of three independent experiments.
by both strains was observed in response to supplementation
of the culture medium with sucrose as the
carbon source, followed by lactose in the case of BK43
and xylose in the case of BK44 (Figure 4A) compared
with glucose (control). Sucrose has been reported to
have similar effects on lipase production by Bacillus
pumilus SG2 (Sangeetha et al., 2008). The other carbon
sources evaluated in this study, such as fructose, starch

and mannitol, led to a decrease in lipase production by
BK44 of more than 50%. These results suggest that the
above carbon sources act as inhibitors of lipase production
and indicate that the carbon source has the potential
to increase the lipase production significantly.
Lipase inducers were evaluated as shown in Figure 4B,
lipase production by both strains increased dramatically
(2-fold) when 1% Tween 80 was added to the media.
However, a moderate level of lipase production by BK43
and BK44 was obtained when soybean and sesame oil,
respectively, were used as the lipase inducers (Figure
4B). These findings agree with the results of other studies
in which other lipids induced lipase production (Gilbert et
al., 1991; Joseph et al., 2006; Immanuel et al., 2008;
Kiran et al., 2008). Similarly, Dharmsthiti et al. (1998) and
Gao et al. (2004) reported that lipase production by A.
calcoaceticus LP009 and Serratia marcescens ECU1010
was induced by 1 and 0.5% Tween 80, respectively.
Conversely, Joseph et al. (2006) reported that Tween 20
and 80 induced a poor level of lipase production by
Staphylococcus epidermis. In the present study, other
lipase inducer such as tributyrin resulted in very low
enzyme production by both strains.
The results of the present study provide useful information
for the optimization of culture conditions such as
carbon sources and lipase inducers, and physicochemical
properties such as pH and temperature to
provide the best lipase production by Acinetobacter
species. These results clearly demonstrated that lipaseproducing
bacteria are widespread in oil contaminated
soil. The optimized growth conditions developed in this
study can be used for a large scale in industrial purposes.
Furthermore, the majority of studies conducted to date
have evaluated lipase production under alkaline conditions;
therefore, the results of the present study will be
useful for development of methods of lipase production
under acidic conditions. To our knowledge, this is the first
report on the highest lipase production in Acinetobacter
species under acidic conditions. The final optimized medium
resulted to about 5.9 fold (BK43) and 5.7 fold
(BK44) more lipase production when compared with
lipase obtained in the original medium. More studies will
be conducted to purify and characterize the acidic lipase
produced by the two strains isolated here under optimized
culture and physico-chemical conditions.
ACKNOWLEDGEMENT
This work was supported by Inha University Grant, Inha
University, Republic of Korea.
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African Journal of Biotechnology Vol. 10(20), pp. 4157-4165, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2085
ISSN 1684–5315 © 2011 Academic Journal
Full Length Research Paper
Antifungal activity of selected plant leaves crude
extracts against a pepper anthracnose fungus,
Colletotrichum capsici (Sydow) butler and bisby
(Ascomycota: Phyllachorales)
Lucy Johnny*, Umi Kalsom Yusuf and R. Nulit
Department of Biology, Faculty of Science, University Putra Malaysia, 43400 UPM
Serdang, Selangor Darul Ehsan, Malaysia.
Accepted 8 April, 2011
The antifungal activities of the leaves extract of 15 selected medicinal plants; Alpinia galanga (L.) Willd.,
Alstonia spatulata Blume., Annona muricata L., Blechnum orientale L., Blumea balsamifera L., Centella
asiatica L., Dicranopteris linearis (Burm. f.) Underw., Dillenia suffruticosa (Griff ex Hook.f. and
Thomson) Martelli, Litsea garciae Vidal., Melastoma malabathricum L., Momordica charantia L.,
Nephrolepis biserrata (Sw.), Pangium edule Reinw., Piper betle L. and Polygonum minus Huds., were
evaluated on the plant pathogenic fungus, Colletotrichum capsici which was isolated from chilli. The
antifungal assay was carried out in potato dextrose media in five different treatments, which were
distilled water as the negative control, crude extract of leaves in methanol, chloroform, acetone and
Kocide 101 as the positive control. They were carried out in three replicates. The two-way analysis of
variance (ANOVA) was carried out on all the data to justify the difference between critical difference
(CD) of mean (P = 0.05) and coefficient of variation (CV %) in terms of mean percent reduction in colony
diameter, sporulation and minimum inhibitory concentration (MICs) of C. capsici to take statistical
decisions. Crude extract of P. betle in all the solvents was found to be the most effective and exhibited
the highest antifungal activities. Crude extract of P. betle in methanol inhibited 85.25% of radial growth
of C. capsici followed by 78.53% leaves crude extract in chloroform and 73.58% leaves crude extract in
acetone at the concentration of 10 µg/ml (p < 0.05). The exact concentrations that had definite potential
to fully restrict the growth (100% inhibition) of C. capsici (MIC) by P. betle was 12.50 µg/ml in methanol,
17.50 µg/ml in chloroform and 15.00 mg/ml in acetone. The sporulation assay also revealed that, P. betle
leaves crude extracts showed the highest inhibition of spore germination rate of C. capsici overall at
the concentration of 10 µg/ml; with 80.93% inhibition by leaves crude extracts in methanol, 74.09% by
leaves crude extracts in chloroform and 72.91% by leaves crude extracts in acetone. Concentration of
plant leaves crude extracts that inhibited 50% or more of the radial growth and sporulation was
considered as effective (LC ≥ 50). As a conclusion, the leaf crude extracts that exhibited effectiveness
by showing more than 50% inhibition against C. capsici should be considered for further evaluation. P.
betle leaf crude extracts was the most effective in inhibiting the fungus respectively and thus, exhibited
the highest potential as a new leading biofungicide in the agriculture field.
Key words: Plant leaves crude extracts, antifungal activities, Colletotrichum capsici.
INTRODUCTION
Pepper fruit anthracnose which is caused by Colleto-
*Corresponding author. E-mail: lucyjohnny13784@yahoo.com.
Tel: +6017-3434717. Fax: +603-86567454.
trichum species is one of the most serious disease which
leads to serious yield loss and quality deterioration in
many Asian countries and in tropical areas (Oanh et al.,
2004; Sang et al., 2007). The most destructive disease of
pepper anthracnose is caused by Colletotrichum capsici
(Sydow) Butler and Bisby (1931) (Ascomycota:

4158 Afr. J. Biotechnol.
Phyllachorales) (Amusa et al., 2004). It is well known for
infection on leaves, stems, mummification of unripe green
pepper fruits, pre-mature fruit drop and fruit rot (Agrios,
1988; Marvel, 2003). It also has been reported that, pre-
and post-harvest fruit losses of up to 50% was caused by
this fungi (Boali, 1991). Generally, Colletotrichum
diseases can be controlled by a wide range of chemicals
such as copper compounds, dithiocarbamates, benzimidazole
and trizole compounds; other fungicides such as
chlorothalonil, imazalil and prochloraz are also effective
against Colletotrichum (Waller, 1992). Although, the use
of systemic fungicides simplifies the management strategy,
not many systemic fungicides are practically in use on
chilli. This limits the choice of systemic fungicides on
chilli, thus, there is a strong need to find alternative
systemic fungicides to the existing chemical carbendazim,
which is the only systemic fungicide currently
used in chilli fields (Gopinath et al., 2006). Furthermore,
there is also the raise of concerns for problems of
fungicide insensitivity, residues on edible produce and for
tree crops; efficiency of spraying has increased in
importance (Bailey and Jeger, 1992). One approach
might be the testing of plants traditionally used for their
antifungal activities as potential sources for drug development.
Hence, this study provided broader options in agriculture
by evaluating the antifungal activity of plant’s
leaves crude extracts from selected medicinal plants as
Alpinia galanga (L.) Willd., Alstonia spatulata Blume.,
Annona muricata L., Blechnum orientale L., Blumea
balsamifera L., Centella asiatica L., Dicranopteris linearis
(Burm. f.) Underw., Dillenia suffruticosa (Griff ex Hook.f.
and Thomson) Martelli, Litsea garciae Vidal., Melastoma
malabathricum L., Momordica charantia L., Nephrolepis
biserrata (Sw.)., Pangium edule Reinw., Piper betle L.,
and Polygonum minus Huds., in order to test their
antifungal potential against phytopathogenic fungi C.
capsici. These plants were selected for extraction due to
their well-known medicinal properties in traditional uses
by local ethnics in Malaysia. The collected plant samples
were identified at Herbarium, Department of Biology,
Universiti Putra Malaysia. Leave samples were extracted
in methanol, chloroform and acetone. Then, C. capsici
was treated with different concentration of plant crude
extract and antifungal activities were determined by
measuring the percentage inhibition of radial growth,
sporulation rate and minimum inhibition concentration
(MIC).
MATERIALS AND METHODS
Plant collection and extraction
The leaves of 15 plants (A. galanga, A. spatulata, A. muricata, B.
orientale, B. balsamifera., C. asiatica, D. linearis, D. suffruticosa, L.
garciae, M. malabathricum, M. charantia, N. biserrata, P. edule, P.
betle, and P. minus) were collected locally from the nearby areas of
Sarikei, Sarawak (Table 1).
The leaf samples were air dried and weighed. Leaves samples
were then, ground using mortar and pestle into coarse powder.
Leaves of the plants were extracted in polar solvent (methanol),
semi-polar solvent (chloroform), and non-polar solvent (acetone) by
following cold percolation method (Valsaraj et al., 1997). Leaves
sample were then, soaked in three different solvents; methanol,
chloroform and acetone, at room temperature for 72 h. The
obtained extract was then filtered through Whatman (no.1) filter
paper extracts solution and was transferred into 250 ml round
bottom flasks which were previously weighed. Then, the extracts
solution was evaporated using Buchi Rotavapor R-210, Switzerland
to concentrate the extracts. Concentrated extracts were allowed to
dry in fume cupboard, weighed again and were kept in 4°C for
bioassays evaluation. Their volume was made up to obtain respective
concentrations.
Source of isolate
The culture of C. capsici from Capsicum annuum L. was obtained
from the Faculty of Agriculture, Universiti Putra Malaysia. Pure
cultures were maintained on potato dextrose agar (PDA) and slants
(Gupta, 2004).
Antifungal assays
Agar-dilution assay
The agar dilution assay was carried out according to Alam (2004)
with a slight modification. 39 g of potato dextrose agar (PDA)
powder was boiled until the agar completely dissolved in 1 L of
distilled water. The solution was then transferred to the blue cap
bottle and sterilized using autoclave at 121°C for 15 min. 19 ml of
the sterilized PDA and 1 ml of plant extract were mixed and plated
on the sterilized Petri dishes (8.5 mm in diameter). 10 mm diameter
of mycelia discs were inoculated at the centre of the medium. The
antifungal assay was divided into five different treatments as crude
extract of leaves in methanol, chloroform and acetone, commercial
fungicide Kocide 101 as positive control and a negative control.
Colony growth was determined on the basis of linear dimensions.
Assay was carried out in three replicates. The percent reduction
(Rr) or stimulation (Rs) of colony diameter by each extract was
determined using the following formula (Nduagu et al., 2008):
Rr =
Rr =
(R1 – R2) x 100
R1
(R2 – R1) x 100
R2
Where, Rr = percent reduction in colony diameter; Rs = percent
stimulation in colony diameter; R1 = colony diameter on the
untreated medium (control); and R2 = colony diameter on the
treated medium.
Minimum inhibitory concentration (MIC) assay
Minimum inhibitory concentration (MIC) was determined using agardilution
method (Yazdani et al., 2007). 1 ml of various crude
extracts concentrations (0.001, 0.005. 0.050, 0.500, 5.0, 12.5, 15.0,
17.5 and 20.0 µg/ml) were prepared in the laboratory universal
bottle containing 9 ml of PDA and were sterilized. The mixture of
PDA and extracts were poured into Petri dish under sterile
condition. Then, 2 µl of adjusted spore suspension was added to

Table 1. Details about plants used, their family, English and local names.
Plant used Family English name Local name
A. galanga (L.) Willd. Zingiberaceae Greater galangal or blue
ginger
A. spatulata Blume. Apocynaceae Hard milkwood or siamese
balsa
A.muricata L. Annonaceae Brazilian pawpaw, soursop,
prickly custard apple
Johnny et al. 4159
Lengkuas (Malay), Engkuas/Lankwas/Puar
(Iban)
Pulai puteh/Rejang (Iban), Pulai basong (Malay)
Hampun kapal (Kadazan), Durin mekah (Iban),
Durian belanda (Malay)
B. orientale L. Blechnaceae Oriental blechnum, Paku ikan/Paku Lipan/Paku ular/Paku ulat
centipede fern
(Malay)
B.balsamifera L. Asteraceae Sambong Susuoh (Bidayuh), Urok bung (Kayan), Dun
supiro (Kiput), Keymabo (Selakau), Sembong
(Malay)
C.asiatica L. Mackinlayaceae Indian pennywort Pegaga (Malay)
D. linearis (Burm. f.)
Underw.
D. suffruticosa (Griff ex
Hook.f. and Thomson)
Martelli
Gleicheniaceae Uluhe, staghorn fern, false
staghorn, resam
Bengkawang (Iban), Resam (Malay)
Dilleniaceae Simpoh ayer Buan (Iban), Abuan (Semai), Bu’ua (Bidayuh),
Simpoh air (Malay)
L. garciae Vidal. Lauraceae Bagnolo, wuru lilin Madang enkala/Pedar (Iban), Pong Labon
(Sabah), Ta’ang (Bidayuh)
M.malabathricum L. Melastomaceae Malabar melastome,
Singapore rhododendron,
senduduk
Sekenduduk/kenduduk/seduduk (Iban),
Senduduk (Malay)
M. charantia L. Cucurbitaceae Bitter melon Peria (Malay)
N. bisserrata (Sw.) Polypodiaceae Giant sword fern Paku larat (Iban), Paku uban (Malay)
P. edule Reinw. Salicaceae football fruit, kepayang,
kluwak
Kepayang (Iban, Malay)
P. betle L. Piperaceae Betel leaf Sireh (Malay, Iban)
P.minus Huds. Polygonaceae Pygmy smartweed Kesum (Malay)
each Petri dish plate. The agar without any plant crude extract
served as the control. Assay was carried out in three replicates. The
minimum inhibitory concentration (MIC) was regarded as the lowest
concentration of the extract that did not show any visible growth
(100% inhibition) after 14 days of incubation (compared with
control). The minimum inhibitory concentration (MIC) was expressed
in µg/ml.
Sporulation assay
The sporulation assay was carried out according to Nduagu et al.
(2008) with a slight modification. The rate of sporulation was
determined by adding 10 ml sterile distilled water to each seven
days old plate that were obtained from agar dilution assay and
gently scraping with a sterile glass rod to dislodge the spores. The
spore suspensions obtained were filtered through sterile cheese
cloth into a sterile 50 ml glass beaker and homogenized by manual
shaking. The spores were then counted using a haemocytometer.
Assay was carried out in three replicates. The percent sporulation
reduction (Sr) or stimulation (Ss) by each extract was
determined using the following formula (Nduagu et al., 2008):
Sr =
(R1 – R2) x 100
R1
(R2 – R1) x 100
Ss =
R2
Where, Sr = percent reduction in sporulation; Ss = percent
stimulation in sporulation; S1 = sporulation on the untreated
medium (control); and S2 = sporulation on the treated medium.
Statistical analysis
The two-way analysis of variance (ANOVA) was carried out on all
the data to justify the difference between critical difference (CD) of
mean (P = 0.05) and coefficient of variation (CV %) in terms of
mean percent reduction in colony diameter, sporulation and MICs of
C. capsici to take statistical decisions (Snedecor and Cochran,
1989). Results with p < 0.05 were considered to be statistically
significant. If the result was significant, CD test was adapted to find
which of the concentrations were same or different in their percent
reduction in colony diameter, sporulation and MICs. For the
calculation of CV, standard deviation was converted into a relative
measure of dispersion for the purpose of comparison. If CV was
greater, it was said that, the treatment was more variable and less
stable in terms of action and vice versa. CD and CV were
calculated using the following formula: CD = Standard error
difference X table value for error degrees of freedom at 5% level
and CV = Standard deviation / mean X 100.

4160 Afr. J. Biotechnol.
RESULTS
Inhibition of radial growth of C. capsici by plant crude
extracts
Table 1 shows that only 5 out of the15 plants screened
showed more than 50% inhibition of radial growth. These
plants were P. betle, A. galanga, C. asiatica, M. charantia
and P. minus. Crude extracts of P. betle in all the
solvents exhibited significant reduction in colony radial
growth against C. capsici in all the concentrations (Table
2). Furthermore, these studies revealed that, the percent
inhibition of radial growth against C. Capsici increased as
the concentration of plant crude extract increased.
Methanol crude extract of P. betle exhibited the highest
antifungal activity with 72.30 to 85.18% inhibition against
C. capsici. This was followed by methanol crude extract
of A. galangal and C. asiatica which exhibited 68.77 to
74.60% and 57.60 to 71.87% inhibition, respectively.
Chloroform crude extracts of P. betle also showed the
highest inhibition (72.36 to 78.53%) against radial growth
of C. capsici followed by A. galangal (50.72 to 63.57%),
M. charantia (57.46 to 61.31%) and C. asiatica (56.58 to
50.61%) (Table 2). While in B. balsamifera and P. edule
less than 50% antifungal activity was exhibited (46.61to
49.82% inhibition), respectively.
Acetone crude extracts of P. betle, A. galanga, M.
charantia, C. asiatica, B. balsamifera and P. edule were
found to be effective against C. capsici which showed
more than 50% inhibition of radial growth with different
concentration (Table 2). P. betle exhibited 75.02%
inhibition in 10.00 µg/ml, 75.38% inhibition in 1.00 µg/ml,
73.73% inhibition in 0.10 µg/ml and 70.36% inhibition in
0.01 µg/ml against C. capsici. This was followed by
acetone crude extract of B. balsamifera which exhibited
74.56% inhibition in 10.00 µg/ml, 74.56% inhibition in
1.00 µg/ml, 70.82% inhibition in 0.10 µg/ml and 65.80%
inhibition in 0.01 µg/ml against C. capsici. Acetone crude
extract of M. charantia exhibited 57.24% inhibition in
10.00 µg/ml, 54.94% inhibition in 1.00 µg/ml, 51.44%
inhibition in 0.10 µg/ml and 51.53% inhibition in 0.01
µg/ml against C. capsici. Acetone crude extract of D.
suffruticosa exhibited effective antifungal activities only in
10.00 µg/ml with 51.33%.
MIC of C. capsici by plant crude extracts
Plant crude solvent extracts with the lowest concentration
that did not show any visible growth (100% inhibition) of
C. capsici after 14 days of incubation (compare to
control) were determined as MIC. Among the plants
screened, P. betle exhibited the lowest MIC value against
C. capsici in 12.5 µg/ml in methanol crude extract. This
was followed by methanol crude extract of A. galanga
(15.00 µg/ml), C. asiatica (17.5 µg/ml) and finally, by M.
charantia and B. balsamifera (20 µg/ml), respectively
(Table 3). In chloroform crude extracts, both P. betle and
A. galanga exhibited the lowest MIC value against C.
capsici in 17.5 µg/ml, followed by C. asiatica and M.
charantia (20.00 µg/ml). The lowest MIC value was
observed in P. betle (15.00 µg/ml), B. balsamifera (17.50
µg/ml) and M. charantia and P. minus (20.00 µg/ml) in
acetone crude extracts (Table 3).
Inhibition/stimulation of sporulation of C. capsici by
plant crude extracts
Inhibition of sporulation of C. capsici by leaf crude
extracts in methanol, chloroform and acetone of the 15
medicinal plants species are summarized in Table 4.
Plant crude solvent extracts with concentration that
inhibited more than 50% of the normal sporulation were
considered as effective (Begum et al., 2007). Among the
plants screened, five species showed 50% and more of
antifungal activity against; C. capsici A. galanga, C.
asiatica, B. balsamifera, M. charantia, P. betle and P.
minus. P. betle exhibited the highest antifungal activity in
all the chosen four treatments of solvent extracts. P. betle
leaf crude extract of 10.00 µg/ml exhibited the highest
percent of inhibition against C. capsici between 72.91
and 80.93%, respectively (Table 4).
DISCUSSION
The findings from this study revealed that, leaf crude
extracts of P.betle exhibited the highest antifungal
activities overall in inhibiting the mycelial growth of C.
capsici among the 15 medicinal plants. The methanol
crude extract of P. betle in 10.00 µg/ml exhibited the
highest inhibition overall with 85.25% inhibition (P <
0.05). Compared with the positive control, commercial C.
capsici fungicide (Kocide 101), the percentage of
inhibition by methanol crude extract of P. betle in 10.00
µg/ml exhibited almost the same percentage of Kocide
101 (87.24%) of the respective concentration. At the
lowest concentration of acetone crude extract which was
0.01 µg/ml, P. betle still effectively inhibited the mycelial
growth with 70.36% inhibition. This showed that the
inhibitory action of the P. betle crude extracts was
recorded even at very low dose, which is a clear indication
that the crude extract contained active components
that have antifungal properties.
The methanol crude extract of P. betle in 10.00 µg/ml
exhibited the highest inhibition in sporulation of C. capsici
overall with 80.93% inhibition (P < 0.05). The positive
control, commercial C. capsici fungicide (Kocide 101),
exhibited 91.73% inhibition of the respective concentration.
At the lowest concentration of acetone crude
extract which was 0.01 µg/ml, P.betle still effectively
inhibited the sporulation with 68.96% inhibition. This
showed that the inhibitory action of the P. betle crude

Johnny et al. 4161
Table 2. Mean ± S.E of inhibiton of radial growth (mm) of C. capsici by leaf extracts in methanol, chloroform and acetone with
various concentrations.
Leaf extract in methanol
Mean ± S.E of inhibiton of radial growth (mm)
0.01 (µg/ml) 0.10 (µg/ml) 1.00 (µg/ml) 10.00 (µg/ml)
A. galanga L. 55.22 ± 1.08* 56.99 ± 0.59* 59.31 ± 1.28* 62.78 ± 1.51*
A. spatulata Blume. 19.13 ± 0.92 19.89 ± 1.04 22.06 ± 0.56 23.64 ± 0.91
A. muricata L. 30.25 ± 1.23 31.86 ± 1.21 35.60 ± 1.20 39.96 ± 1.54
B. orientale L. NI NI 2.68 ± 1.07 4.02 ± 1.13
B. balsamifera L. 25.86 ± 0.73 27.24 ± 1.19 30.37 ±1.22 34.33 ±1.46
C. asiatica L. 54.78 ± 1.48* 46.90 ± 1.80* 55.26 ± 1.58* 59.07 ± 1.44*
D. linearis 4.44 ± 1.35 5.84 ± 0.77 7.85 ± 0.90 9.78 ± 0.85
D. suffruticosa 30.57 ± 0.59 31.08 ± 0.77 33.77 ± 0.78 36.71 ± 0.76
L.garciae Vidal. 13.59 ± 0.84 15.83 ± 1.22 29.34 ± 0.66 31.02 ± 1.41
M. malabathricum L. 30.71 ± 2.52 30.27 ± 1.24 33.41 ± 1.08 34.51 ± 2.16
M.charantia L. 42.17 ± 1.32* 42.42 ± 2.81* 46.25 ± 1.59* 48.32 ± 1.57*
N.bisserrata (Sw.) NI NI 2.89 ± 0.11 3.67 ± 1.13
P.edule Reinw. NI NI 1.68 ± 0.43 4.26 ± 1.17
P. betle L. 61.22 ± 0.53* 65.50 ± 1.10* 66.59 ± 0.89* 71.87 ± 0.78*
P. minus Huds. 39.10 ± 1.72 40.20 ± 1.42 40.37 ± 2.03 46.92 ± 1.11*
Leaf extracts in chloroform
A. galanga (L.) Willd. 42.64 ± 1.30* 46.40 ± 0.72* 48.97 ± 1.36* 53.38 ± 1.05*
A. spatulata Blume. 24.96 ± 1.13 24.74 ± 1.12 27.44 ± 0.72 27.40 ± 0.73
A. muricata L. 8.39 ± 0.84 10.61 ± 1.16 12.65 ± 1.19 18.01 ± 0.92
B. orientale L. NI NI 1.77 ± 1.01 5.33 ± 1.13
B. balsamifera L. 35.50 ± 0.43 39.17 ± 1.35 44.87 ± 1.10* 46.26 ± 1.34*
C. asiatica L. 38.69 ± 1.87 40.82 ± 1.54 42.61 ± 1.01* 47.41 ±1.82*
D. linearis (Burm. f.) Underw. 32.05 ± 1.02 34.23 ± 0.62 36.19 ± 1.13 37.89 ± 0.62
D. suffruticosa 21.41 ± 0.50 22.10 ± 0.93 23.98 ± 1.26 29.90 ± 2.20
L. garciae Vidal. 1.94 ± 1.70 5.84 ± 1.26 8.85 ± 1.99 9.68 ± 1.53
M. malabathricum L. 25.58 ± 1.55 23.56 ± 0.69 27.27 ± 1.13 41.12 ± 1.55
M. charantia L. 48.12 ± 1.53* 50.24 ± 2.12* 51.85 ± 1.94* 50.09 ± 1.56*
N. bisserrata (Sw.) NI NI 3.27 ± 0.15 5.60 ± 0.62
P. edule Reinw. 36.93 ± 1.43 37.73 ± 0.70 41.24 ± 1.17 44.01 ± 1.40*
P.betle L. 61.27 ± 0.75* 63.90 ± 1.72* 65.37 ± 0.90* 66.20 ± 1.15*
P. minus Huds. 20.23 ± 2.27 25.62 ± 1.51 29.43 ± 0.52 31.57 ± 1.38
Leaf extracts in acetone
A. galanga L. 19.70 ± 0.95 21.68 ± 0.66 23.17 ± 1.55 26.94 ± 1.26
A. spatulata Blume. 28.87 ± 0.97 31.06 ± 0.84 34.65 ± 0.43 37.27 ± 1.06
A. muricata L. 16.00 ± 0.85 17.56 ± 1.04 28.75 ± 1.01 33.55 ± 1.25
B. orientale L. NI 3.77 ± 1.32 2.83 ± 1.15 4.70 ± 1.47
B. balsamifera L. 55.86 ± 0.36* 60.06 ± 1.00* 62.55 ± 1.15* 62.44 ± 1.13*
C. asiatica L. 14.47 ± 0.84 24.16 ± 1.24 28.48 ± 1.23 35.28 ± 1.59
D. linearis 21.74 ± 0.99 23.28 ± 1.26 28.25 ± 1.21 34.80 ± 0.91
D. suffruticosa 39.14 ± 0.49 39.14 ± 1.42 38.78 ± 1.38 43.23 ± 1.50*
L. garciae Vidal. NI NI 24.48 ± 1.38 25.48 ± 1.26
M. malabathricum L. 22.74 ± 1.25 31.07 ± 1.63 28.98 ± 1.89 37.72 ± 0.84
M. charantia L. 43.16 ± 0.72* 43.23 ± 1.25* 46.45 ± 0.94* 47.78 ± 1.28*
N. bisserrata (Sw.) NI NI 2.05 ± 0.92 3.18 ± 1.00
P. edule Reinw. 8.12 ± 1.28 36.91 ± 2.03 36.92 ± 1.27 35.36 ± 0.75
P. betle L. 59.57 ± 1.39* 62.17 ± 1.18* 63.77 ± 0.55* 63.56 ± 0.77*
P. minus Huds. 25.34 ± 1.88 27.65 ± 1.73 28.08 ± 1.30 42.64 ± 0.78
Positive control (Kocide 101) 61.60 ± 0.64 66.08 ± 0.90 72.55 ± 0.71 73.54 ± 1.06
Negative control (distiiled water) 84.66 ± 0.24 84.34 ± 0.57 85.00 ± 0.00 84.30 ± 0.65
Each value represented the mean (3 replicates) ± standard error; NI = no inhibition; * represent crude extracts that effectively inhibited growth
(P < 0.05).

4162 Afr. J. Biotechnol.
Table 3. Mean ± S.E of inhibiton of radial growth (mm) of C. capsici
by leaf extracts in methanol, chloroform, and acetone with various
concentrations.
Leaf extracts in methanol Minimum inhibition
concentration (µg/ml)
A. galanga L. 15.00
A. spatulata Blume. >20.00
A. muricata L. >20.00
B. orientale L. >20.00
B.balsamifera L. 20.00
C. asiatica L. 17.50
D. linearis >20.00
D.suffruticosa >20.00
L. garciae Vidal. >20.00
M. malabathricum L. >20.00
M. charantia L. 20.00
N.. bisserrata (Sw.) >20.00
P.edule Reinw. >20.00
P.betle L. 12.50
P.minus Huds. >20.00
Leaf extracts in chloroform
A.galanga L. 17.50
A.spatulata Blume. >20.00
A. muricata L. >20.00
B.orientale L. >20.00
B. balsamifera L. 20.00
C. asiatica L. 20.00
D.linearis >20.00
D. suffruticosa >20.00
L.garciae Vidal. >20.00
M. malabathricum L. >20.00
M.charantia L. >20.00
N.bisserrata (Sw.) >20.00
P. edule Reinw. >20.00
P.betle L. 17.50
P.minus Huds. >20.00
Leaf extracts in acetone
A.galanga L. 20.00
A. spatulata Blume. >20.00
A.muricata L. >20.00
B.orientale L. >20.00
B.balsamifera L. 17.50
C. asiatica L. 20.00
D.linearis >20.00
D. suffruticosa >20.00
L.garciae Vidal. >20.00
M.malabathricum L. >20.00
M.charantia L. >20.00
N. bisserrata (Sw.) >20.00
P. edule Reinw. >20.00
P.betle L. 15.00

Table 3. continued
P.minus Huds. >20.00
Positive control (Kocide 101) 12.50
Negative control (distilled water) NI
NI = No Inhibition
Johnny et al. 4163
Table 4. Mean ± S.E of inhibiton of sporulation (x10 5 ) of C. capsici by leaf extracts in methanol, chloroform, and acetone with
various concentrations.
Leaf extracts in methanol
Mean ± S.E of inhibition/stimulation of sporulation (x10 5 )
0.01 (µg/ml) 0.10 (µg/ml) 1.00 (µg/ml) 10.00 (µg/ml)
A.galanga (L.) Willd. 2.40 ± 0.07* 2.62 ± 0.06* 2.57 ± 0.12* 2.73 ± 0.10*
A. spatulata Blume. 0.84 ± 0.02 0.86 ± 0.03 1.11 ± 0.02 1.09 ± 0.03
A. muricata L. 0.94 ± 0.04 0.91 ± 0.03 1.35 ± 0.06 1.59 ± 0.06
B.orientale L. NI NI NI NI
B.balsamifera L. 1.29 ± 0.08 1.28 ± 0.10 1.43 ± 0.04 1.62 ± 0.08
C.asiatica L. 1.49 ± 0.13 1.57 ± 0.14 2.03 ± 0.10* 2.41 ± 0.09*
D.linearis (Burm. F.) Underw. 0.22 ± 0.04 0.17 ± 0.03 0.32 ± 0.05 0.48 ± 0.04
D. suffruticosa 1.08 ± 0.08 1.30 ± 0.03 1.58 ± 0.13 1.61 ± 0.06
L.garciae Vidal. 0.40 ± 0.02 0.54 ± 0.02 0.93 ± 0.03 0.95 ± 0.04
M. malabathricum L. 1.46 ± 0.03 1.39 ± 0.05 1.59 ± 0.11 1.54 ± 0.12
M.charantia L. 1.65 ± 0.11 1.70 ± 0.14 2.00 ± 0.10* 2.01 ± 0.04*
N.bisserrata (Sw.) NI NI NI NI
P.edule Reinw. NI NI NI NI
P.betle L. 2.78 ± 0.09* 2.92 ± 0.06* 2.95 ± 0.03* 3.20 ± 0.07*
P. minus Huds. 1.81 ± 0.08 1.73 ± 0.03 1.77 ± 0.07 2.06 ± 0.05*
Leaf extracts in chloroform
A. galanga (L.) Willd. 1.91 ± 0.05 1.96 ± 0.15* 2.25 ± 0.09* 2.49 ± 0.06*
A. spatulata Blume. 1.23 ± 0.02 1.17 ± 0.03 1.19 ± 0.03 1.29 ± 0.03
A.muricata L. 0.12 ± 0.03 0.11 ± 0.04 0.45 ± 0.04 0.55 ± 0.04
B. orientale L. NI NI NI NI
B. balsamifera L. 1.53 ± 0.06 1.63 ± 0.13 1.92 ± 0.08 2.13 ± 0.07*
C.asiatica L. 1.50 ± 0.09 1.52 ± 0.15 1.82 ± 0.10 2.00 ± 0.06*
D. linearis (Burm. f.) Underw. 1.16 ± 0.02 1.60 ± 0.03 1.58 ± 0.04 1.57 ± 0.04
D. suffruticosa 1.00 ± 0.04 1.06 ± 0.04 1.07 ± 0.03 1.11 ± 0.03
L. garciae Vidal. 0.06 ± 0.02 0.07 ± 0.02 0.21 ± 0.03 0.30 ± 0.02
M. malabathricum L. 1.09 ± 0.07 1.05 ± 0.11 1.26 ± 0.08 1.76 ± 0.08
M. charantia L. 1.74 ± 0.12 1.88 ± 0.05 2.02 ± 0.05* 2.11 ± 0.04*
N.bisserrata (Sw.) NI NI 0.20 ± 0.03 0.20 ± 0.03
P.edule Reinw. 1.39 ± 0.06 1.58 ± 0.05 1.58 ± 0.04 1.83 ± 0.04
P.betle L. 2.71 ± 0.06* 2.84 ± 0.04* 2.86 ± 0.13* 2.93 ± 0.04*
P.minus Huds. 1.03 ± 0.09 1.25 ± 0.04 1.30 ± 0.09 1.43 ± 0.05
Leaf extracts in acetone
A.galanga (L.) Willd. 0.98 ± 0.08 1.01 ± 0.04 1.01 ± 0.07 1.21 ± 0.10
A. spatulata Blume. 1.31 ± 0.03 1.33 ± 0.03 1.49 ± 0.03 1.47 ± 0.03
A. muricata L. 0.60 ± 0.05 0.53 ± 0.04 1.06 ± 0.04 1.09 ± 0.04
B. orientale L. NI NI NI 0.10 ± 0.03
B. balsamifera L. 1.91 ± 0.07 2.48 ± 0.15* 2.73 ± 0.10* 2.80 ± 0.10*
C. asiatica L. 0.66 ± 0.10 0.89 ± 0.08 1.43 ± 0.06 1.81 ± 0.09
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.
Table 4. Continued
D. suffruticosa 1.70 ± 0.04 1.67 ± 0.12 1.64 ± 0.03 1.77 ± 0.09
L. garciae Vidal. NI NI 0.83 ± 0.04 0.89 ± 0.04
M. malabathricum L. 1.06 ± 0.04 1.40 ± 0.05 1.41 ± 0.05 1.62 ± 0.10
M.charantia L. 1.66 ± 0.13 1.85 ± 0.06 1.93 ± 0.04* 1.99 ± 0.06*
N. bisserrata (Sw.) NI NI NI 0.07 ± 0.01
P. edule Reinw. NI 1.17 ± 0.04 1.38 ± 0.05 1.42 ± 0.03
P.betle L. 2.71 ± 0.07* 2.82 ± 0.02* 2.83 ± 0.12* 2.88 ± 0.07*
P. minus Huds. 1.14 ± 0.06 1.26 ± 0.13 1.33 ± 0.03 2.05 ± 0.04*
Positive control (Kocide 101) 0.76 ± 0.09 0.79 ± 0.05 0.55 ± 0.04 0.33 ± 0.02
Negative control (distilled water) 3.93 ± 0.02 3.95 ± 0.01 3.97 ± 0.01 3.95 ± 0.02
Each value represented the mean (3 replicates) ± standard error; NI = No Inhibition; * represent crude extracts that effectively inhibited
growth (P < 0.05).
extracts was recorded even at very low dose, which is a
clear indication that the crude extract contained active
components that had antifungal properties. Phangthip et
al. (2005) found that Piper betle leaves have antimicrobial
activity due to the essential oils of the plant which
contained phenolic compounds such as cavicol,
cavibetol, carvacrol, eugenol and allilpyrocatechol. These
compounds are assumed could inhibit bacteria and fungi.
Begum et al. (2007) had found the extract of P.betle to
have a wide spectrum of antifungal activity. It has also
been reported that the leaves of P. betle possess various
medicinal properties such as antioxidant, antibacterial,
digestive, stimulant, antifungal and nematocidal properties
(Phangthip et al., 2006).
Johann et al. (2007) stated that the chemistry of Piper
species has been widely investigated and phytochemical
investigations from all parts of the world have led to the
isolation of a number of physiologically active compounds
such as alkaloids/amides, propenylphenols, lignans,
neolignans, terpenes, steroids, kawapyrones, piperolides,
chalcones, di-hydrochalcones, flavones and flavanones
which exhibited high antimicrobial and antifungal
properties. According to Lee et al. (2004) most Piper
chemistry has been conducted to find potential pharmaceuticals
or pesticides and over 90% of the literature
focuses on compounds that are cytotoxic, antifungal,
antitumor, fragrant or otherwise useful to humans.
The antimicrobial properties of different species of the
genus Piper have also been studied (Johann et al.,
2007). In a screening for medicinal plants with
antimicrobial activity in Colombia, the methanolic extract
of the leaf of Piper lanceafolium showed activity against
Candida albicans, Klebsiella pneumoniae, Enterococcus
faecalis, Mycobacterium phlei, Bacillus subtilis and
Staphylococcus aureus. Piper nigrum (black pepper) is
known to have antifungal activity due to lactones,
terpenoids, alkaloids and saponins. 4,5-Dimethoxy-2,3-
(methylenedioxy)-l-allylbenzene, a natural isolate of Piper
hispidum and Piper aduncum, also has strong
antimicrobial activity. This natural product and three other
related compounds, [4-(5’-hydroxy-5’-nonanyl)-1,2
(methylenedioxy) benzene, 4-(5’-non-4’-enyl)-1,2-
(methylenedioxy) benzene and 6-methoxy-2,3-
(methylenedioxy)-4-allylphenol], were synthesized from
piperonal and screened for their biological activity. These
four compounds showed high levels of antifungal and
antibacterial activity against several fungi and bacteria.
In conclusion, the leaves crude extracts that exhibited
good potential and showed effectiveness as fungicides of
C. capsici should be drawn with an in-depth study of
testing the phytoextracts for their potentiality under in vivo
condition.
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African Journal of Biotechnology Vol. 10(20), pp. 4166-4174, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2250
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Isolation, identification and application in lignin
degradation of an ascomycete GHJ-4
Huiju Gao, Yanwen Wang, Wenting Zhang, Weile Wang and Zhimei Mu*
College of Forestry, Shandong Agricultural University, Shandong, China.
Accepted 24 March, 2011
This study was undertaken to isolate an ascomycete producing ligninolytic enzyme and characterize its
lignin degradation capability. Among 20 isolates, GHJ-4 was isolated from decayed wood of Salix
matsudana Koidz in Mount Tai, China, by different indicator compounds assay. The taxonomy of the
fungi was Paraconiothyrium variabile Damm, Verkley and Crous, which had been confirmed by both
morphological and 5.8S rDNA/ITS analyses. The capability of utilizing several lignin model compounds
and decoloration of aromatic dyes by GHJ-4 strain revealed its ligninolytic potentiality. After incubation
for 40 days, the weight loss of the wood was 20.91% and lignin loss was 22.99%, which indicated that,
GHJ-4 strain had higher degradation ability for lignin. To our best of knowledge, this study represented
the first report that P. variabile could produce ligninolytic enzyme and degrade lignin.
Key words: Ligninolytic enzyme, ascomycete, identification, lignin degradation.
INTRODUCTION
Biofuel plays an essential role in replacing petroleumbased
fuels in current worldwide energy situation (Yuan
et al., 2008). Among all the feedstocks, lignocellulosic
biomass, which could be acquired from biomass crop and
agricultural residuals, might be the best choice in the long
term (Smeets and Faaij, 2007). However, the key
obstacle for transitioning lignocellulosic feedstock was
the complicated structure of the plant cell wall,
particularly the presence of lignin. It was highlighted that,
microbial degradation of lignin had potential advantages
over the prevailing chemical degradation, due to energy
and environmental concerns (Keller et al., 2003). However,
degrading lignocellulosic biomass by current
available microorganism is still far to meet the industrial
*Corresponding author. E-mail: ghj@sdau.edu.cn. Tel: (86)538-
8249561. Fax: (86)538-8249164.
Abbreviations: LiP, Lignin peroxidise; MnP, manganese
peroxidase; PDA, potato dextrose agar; SNA, synthetic nutrient
agar medium; OA, oatmeal; CMA, cornmeal; MEA, malt extract;
PDA, potato dextrose; ITS, internal transcribed spacer; PCR,
polymerase chain reaction; BLAST, Basic Local Alignment
Search Tool; NJ, Neighbors joining; RBBR, Remazol Brilliant
Blue R; LiP, lignin peroxidise; MnP, manganese peroxidise.
demands (Shary et al., 2007; Afrida et al., 2009). Thereby,
isolation of new strains for degradation of lignin is still
essential.
Extracellular oxidoreductases, including lignin peroxidase
(LiP), manganese peroxidase (MnP) and laccase
produced by wood-decomposing microorganism, are
directly involved in the degradation of lignin in their
natural lignocellulosic substrates and various xenobiotic
compounds including dyes (Abrahão et al., 2008;
Thurston, 1994; Call and Mücke, 1997; Leonowicz et al.,
2001). Some wood-degrading fungi contain all three
classes of lignin-modifying enzymes, while the others
contain only one or two of these enzymes (Hatakka,
1994; Dhouib et al., 2005). The production of ligninolytic
enzymes is observed as a colourless halo around
microbial growth (Dhouib et al., 2005). So, the lignin
degradation capability of microbes is initially screened
indirectly on solid media containing different indicator
compounds.
Although, wood decay fungi are primarily basidiomycetes,
other microorganisms are also involved in the
lignocellulosic decaying processes (Nilsson et al., 1989).
Wood biodegradation by certain ascomycetes was first
described in detail and designated as “soft rot” by Savory
(1954). Nilsson et al. (1989) demonstrated that, some
higher ascomycetes, particularly Daldinia concentrica,

degraded Aspen wood with the same intensity as
Trametes versicolor, a basidiomycete typically classified
as white-rot fungus. The ascomycete, Chrysonilia sitophila
could degrade rice hull and Pinus radiata bark products
and also produce ligninolytic and cellulolytic enzymes
(Ferraz et al., 1991). Whereafter, different Penicillium
strains was described as potential degraders or was able
to degrade compounds with related lignin structures
(Rodriguez et al., 1994). The family Xylariaceae had
been reported to cause a special type of extensive woodrot,
which could produce laccase and low amounts of
peroxidase as potential ligninolytic enzymes along with
different polysaccharide-cleaving hydrolases as well as
esterase (Liers et al., 2006).
Ascomycetous fungi with coniothyrium-like anamorphs
are common colonizers of woody host plants (Damm et
al., 2008). Nilsson (1973) found that, Coniothyrium
cerealis could degrade wood. A new woody plants hosted
genus Paraconiothyrium was established by Verkley et
al. (2004), which included the old Coniothyrium minitans
and Coniothyrium sporulosum. Later species were frequently
isolated from wood and leaves of Prunus,
Actinidia, Laurus and Dendrobium. Recently, two
additional new species, Paraconiothyrium africanum and
Paraconiothyrium variabile, were identified based on their
DNA sequence data and unique morphological characteristics
(Damm et al., 2008).
In this study, an ascomycete producing ligninolytic
enzyme, forming coniothyrium-like anamorphs, was
isolated from decayed wood of Salix matsudana Koidz in
Mount Tai. It was a new strain with lignin-transformation
capability. The fungus was morphologically and phylogenetically
identified and its ability for lignin degradation
was characterized.
MATERIALS AND METHODS
Isolation of ligninolytic enzyme producing fungi GHJ-4 strain
Different indicator compounds were added to potato dextrose agar
(PDA) in order to detect ligninolytic enzyme production. These
compounds included 0.04% Remazol Brilliant Blue R (RBBR),
0.04% guaiacol and 0.02% tannic acid (Dhouib et al., 2005). The
plates were incubated at 28°C for at least a week under dark.
Positive strains were subcultured when clear positive reactions
were visible.
Morphology
To enhance sporulation, autoclaved wood sawdust was placed onto
the surface of synthetic nutrient agar (SNA) medium (Kwaśna and
Bateman, 2007) and incubated at 25°C for 1 to 4 weeks under
natural light. Measurements, photographs of characteristic structures
and vertical sections through conidiomata were made
according to Damm et al. (2007). Cultural characteristics and radial
growth rates were determined on oatmeal (OA), cornmeal (CMA),
3% malt extract (MEA) and PDA agars (Damm et al., 2008). Plates
were incubated in a growth chamber at 25°C under natural light.
Growth characteristics were studied on MEA plates incubated in the
Gao et al. 4167
dark at temperatures ranging from 5 to 35°C, with 5°C intervals.
DNA isolation, amplification and analyses
Genomic DNA of GHJ-4 strain was extracted from fungal mycelium
grown on PDA plates following previously described protocols
(Pryor and Gilbertson, 2000). The internal transcribed spacer (ITS)
regions of 5.8S rDNA gene was amplified and sequenced using
primer ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3') and ITS4 (5'-
TCCTCCGCTTATTGATATGC-3'). Polymerase chain reaction (PCR)
was performed by denaturation for 5 min at 95°C followed by 35
cycles of 1 min at 94°C for 1 min at 56°C and 1 min at 72°C, with a
final extension of 10 min at 72°C. The PCR products with the
expected size (about 600 bp) were cut from gel and purified by DNA
gel extraction kit (TaKaRa, Japan), cloned into pEASY-T3 vector
(TransGen, China) and transformed into Escherichia coli competent
cells DH5α. Sequences were performed with an AB13730XL DNA
autosequencer (Applied Biosystems). Sequence homolog analysis
was performed by Basic Local Alignment Search Tool (BLAST)
(http://www.ncbi.nlm.nih.gov). Fungi identification was based on
5.8S rDNA gene sequence similarity to BLAST hits. Neighbors
joining (NJ) phylogenetic trees were generated based on partially
sequenced 5.8S rDNA and closely related sequences. In the NJ
analyses, the Kimura distance calculation was used. A bootstrap
analysis (1000 replicates using NJ option) was performed to
determine the confidence levels of the nodes.
Cultivations in liquid media
GHJ-4 strain was cultivated on a basic media (Media V) for
production of laccase on 200 rpm at 28°C. Media V contained 15.0
g sucrose and 200.0 g potato extract per liter. Subsequently, it was
cultivated in four types of rich media to improve laccase production.
Media I contained (g/l): 3.0 soluble starch, 2.0 yeast extract, 0.17
KH2PO4, 0.44 MgSO4, 0.37 CaCl2, 0.5 Tween-80 ; media II contained
(g/l): 200.0 potato extract, 25.0 glucose, 3.0 yeast extract,
3.0 KH2PO4, 2.5 MgSO4, 0.1 vitamin B1, pH 6.0; Media III contained
(g/l): 24.0 soluble starch, 24.0 glucose, 10 bran, 3.2 KH2PO4, 0.2
MgSO4, 0.006 CuSO4, 3.2 NH4Cl, pH6.5; Media IV contained (g/l):
30 bran, 2.0 peptone, 3.0 beef extract, 3.0 KH2PO4, 0.5 MgSO4,
0.02 Vitamin B1, 0.01 CaCl2, 0.1 NaCl.
LiP and MnP production by GHJ-4 strain was followed for 14
days. The fungus was inoculated into 250 ml Erlenmeyer flasks
containing 50 ml medium which involve 30.0 g bran, 58.0 g soluble
starch, 3.5 g beef extract, 1.0 g NaNO3, 3.0 g KH2PO4 and 0.1 g
MnSO4.
Laccase activity assay
Laccase activity was measured spectrophotometrically with guaiacol
as substrate. A modified method of Chakraborty et al. (2000)
was used. An aliquot of enzyme solution was incubated in 4.0 ml of
50 mM sodium acetate buffer (pH 4.5) containing 1.0 mM guaiacol
at 30°C. The changes in absorbance due to oxidation of guaiacol in
the reaction mixture was monitored at 465 nm (ε465=12,100 M -1 cm -
1 ). One unit of enzyme activity was defined as the amount of
enzyme that oxidized 1 µmol guaiacol per minute under the
aforementioned condition.
Lignin peroxidase activity assay
The lignin peroxidase activity was evaluated by UV spectrometry of
the veratraldehyde produced ( TM 310 =9,300 M −1 cm −1 ) during veratryl
alcohol oxidation (Moldes et al., 2003). The reactive mixture was

4168 Afr. J. Biotechnol.
composed of 2.5 ml sodium tartrate buffer (100 mM, pH 3.0), 1.0 ml
veratryl alcohol (10 mM), 100 µl hydrogen peroxide (10 mM) and
400 µl enzyme extract. The reaction was started by adding
hydrogen peroxide and the veratraldehyde was determined at 310
nm. One enzyme unit was defined as 1 µmol product formed per
minute under the assay conditions.
Manganese peroxidase activity assay
Manganese peroxidase activity was assayed following the method
of Moldes et al. (2003). The reaction mixture contained 3.4 ml
sodium malonate (50 mM pH 4.5), 0.1 ml MnSO4 (15 mM) and 400
µl enzyme extract. The reaction was started by adding 0.1 ml H2O2
(10 mM) and the produced Mn 3+ complexes was determined at 240
nm (ε240=6,500 M -1 cm -1 ). One activity unit was defined as the
amount of enzyme that oxidized 1 µmol MnSO4 per minute and the
activities were expressed in U ml -1 .
Characterization of ligninolytic potential
GHJ-4 strain was incubated in 250 ml Erlenmeyer flasks containing
25 ml minimal salts medium, which involves 2.0 g NH4Cl, 0.5 g
MgSO4, 1.0 g KH2PO4, 0.007 g CuSO4, 0.2 g Na2HPO4, 0.035 g
MnSO4, 0.007 g FeSO4 per liter and lignin (Sigma-Aldrich) and
guaiacol as sole carbon source. The flasks were inoculated with 2
pieces (1.0 cm diameter) of 7-day precultured agar plates and the
cultures maintained under stationary at 28°C for 15 days. Growth of
the fungus and indication of the lignin degradation capability was
measured based on the dry weight of the mycelia after filtration,
followed by drying at 80°C to constant weight.
Detection of decolourization ability
For dye decolourization studies, GHJ-4 strain was incubated on
PDA plate supplemented with 0.02% RBBR, aniline blue and
phenol red at 28°C (Rodriguez et al., 1996). Decolourization was
observed after 15 days.
Wood biodegradation by GHJ-4 strain
Wood chips (2.0×2.0×0.1 cm) were obtained from Populus bonatii
Levl (10-year old). Wood biodegradation was performed under
solid-state fermentation at 28°C for 40 days. Inoculum was
prepared as follows: 3 pieces (1.0 cm diameter) of 7-day precultured
agar plates were inoculated into a 250 ml Erlenmeyer flask
containing 5% Wood chips, 15% bran and 80% nutrient solution
that was composed of 2.0 g NH4Cl, 0.5 g MgSO4, 1.0 g KH2PO4,
0.007 g CuSO4, 0.2 g Na2HPO4, 0.035 g MnSO4, 0.007 g FeSO4
and 1,000 ml of water. After biodegradation, bio-treated wood chips
were washed to remove the mycelium grown on wood surfaces and
then dried to constant weight at 105°C. Initial and final dry weights
were used to determine weight losses. A set of uninoculated
sterilized wood chips served as a control wood sample.
Decayed and non-decayed wood samples were milled in a knife
mill to pass through a 0.5 mm screen. Klason insoluble and soluble
lignins in benzene/ethanol extracted milled wood samples were
determined by acid hydrolysis as described by Ferraz et al. (2000).
RESULTS
Isolation of GHJ-4 strain
Reactions with three different indicators, guaiacol,
Remazol Brilliant Blue R (RBBR) and tannic acid, were
tested with collected samples. The positive reactions with
guaiacol, RBBR and tannic acid correlated well with each
other. Altogether, 20 fungal strains showing positive
reactions on indicator plates were isolated from decayed
wood of S. matsudana Koidz in Mount Tai. Fungal strain
GHJ-4 was selected for further studies, as the oxidative
polymerization of guaiacol to form reddish brown zones in
the medium was higher than other isolated strains. Its
lignin degradation capability was further confirmed by
other indicators (Figure 1).
Phylogenetic analysis
An expected 589 bp DNA fragment of the 5.8S rDNA/ITS
region was obtained from the GHJ-4 strain by PCR amplification.
The sequence was deposited in the GenBank
database (Accession no. GQ331986). BLAST analysis of
the amplificons indicated that, it had the highest
similarities (over 99%) sequences generated from
Paraconiothyrium or Microdiplodia strains. To clarify the
phylogenetic position of GHJ-4, a phylogenetic tree was
constructed based on the 5.8S rDNA sequences (Figure
2). The result revealed that GHJ-4 strain, P. variabile and
Paraconiothyrium brasiliense formed one cluster, which
implied they were closely related.
Taxonomy
The strain GHJ-4, obtained from wood of S. matsudana
Koidz, could be assigned to P. variabile based on the
DNA sequence data and its morphology (Damm et al.,
2008). Its conidiomata were pycnidia and the shape of
the conidiogenous cells was more variable. Conidia were
smaller than those of most other Paraconiothyrium
species and grew more slowly than P. brasiliense.
Conidiomata pycnidial, produced on wood sawdust on
SNA in 1 to 4 weeks was solitary, subglobose, 1 to 3
ostioles, black, superficial or semi-immersed, 200 to 600
µm diameter, wall consisting of 6 to 8 cell-layers (30 to 45
µm) of thick-walled dark brown textura angularis, becoming
hyaline and thin-walled towards the inside of the
pycnidium, that was surrounded by brown hyphal
appendages. Conidiophore, lining the inner conidiomatal
cavity, was hyaline, branched with conspicuous umbilici,
3 to 10 × 2 to 5 µm. Conidiogenous cells varied in shape,
conical to subulate or subcylindrical, broadly or elongated
ampulliform, phialidic with periclinical wall thickening or
with one or more percurrent proliferations near the apex.
Conidia was pale brown or light green, subcylindrical to
ellipsoidal, both ends obtuse, 1-celled, smooth walled, 3
to 4.5 × 1.5 to 2.5 µm. Vegetative hyphae were 2 to 5 µm
wide, hyaline to pale brown, septate and smooth. On
PDA medium, parts of the hyaline vegetative hyphae was
transformed to very dark-walled hyphal pieces, which
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,
decolourize of RBBR; C, yellow zones of tannic acid; D, decolourize of aniline blue; E, decolourize of phenol red.
Figure 2. 5.8S rDNA/ITS region phylogenetic tree of GHJ-4 strain obtained by the
neighbor-joining method based on the two-parameter Kimura correction of
evolutionary distances. Numbers at the nodes are the bootstrap confidence values
obtained after 1000 replicates. Camarosporium leucadendri was used as the outgroup.
Gao et al. 4169

4170 Afr. J. Biotechnol.
5 µm D
A B C
E F
Figure 3. Morphology of GHJ-4 strain. A, Dark walled hyphae; B, conidia oozing from pycnidia; C, longitudinal section through a
pycnidium; D, pycnidial wall with conidiophorese; E, conidiophores; F, conidia.
brown material on the outer wall surface (Figure 3).
Cultural characteristics
Colonies on OA reached 53.2 mm after 7 days and 85
mm after 14 days (25°C, in diffuse daylight). It was flat,
with an even to slightly ruffled colourless and glabrous
margin, mycelium whitish, aerial mycelium absent or
consisting of sparse, scattered white to grayish tufts.
Colonies on MEA reached 42.6 mm diameter in 7 days
and 68.5 mm in 14 days, with an even to slightly ruffled,
glabrous and colourless margin, colony surface almost
entirely covered by a dense mat of woolly aerial
mycelium, which was pale olivaceous-grey, in the centre
olivaceous-black and near the margin paler to almost
pure white. Colonies on PDA reached 54.7 mm diameter
in 7 days, 82.8 mm diameter in 14 days. It was flat, with
an even, whitish margin, most of the colony surface
covered by felty floccose aerial mycelium, in the centre
becoming olivaceous-grayish. Colonies on CMA reached
85 mm diameter in 14 days (46 mm in 7 days), as on
PDA. It was flat, white, in the centre becoming olivaceousgrayish
(Figure 4).
Growth characteristics
The growth temperature range of GHJ-4 was 5 to 35°C
and the optimum growth temperature was approximately
25°C.
Production of ligninolytic enzyme by GHJ-4 strain
Laccase production by GHJ-4 strain was studied in liquid
cultures and proved to be highly dependent on the
medium. The production level of laccase in liquid cultures
was quite low in basic media. Laccase production was
enhanced by the addition of bran. Among the five media
tested in this study, media IV had the highest laccase
production (Figure 5). The production of laccase activity
occurred on day 2 and reached its maximum (484.72
U/ml) on day 10 and then declined gradually.
Furthermore, GHJ-4 also produced LiP and MnP. LiP
activity first appeared on the 2 nd day (0.31 U/ml) and
reached its highest level of activity (7.28 U/ml) on the 12 th
day, after which the activity started to decrease (Figure
6). As regards MnP activity, it began on the 4 th day (0.73
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)
and MEA (D) (25°C, natural light).
-1)
Enzyme activity (U ml -1 )
500
400
300
200
100
0
0 2 4 6 8 10 12
Time(day)
MediaⅠ Media Ⅱ Media Ⅲ
Media Ⅳ Media Ⅴ
Figure 5. Time course of extracellular laccase activity of GHJ-4 on different media.
Gao et al. 4171

4172 Afr. J. Biotechnol.
Enzyme activity(U ml
-1 )
cultivation (3.81 U/ml) (Figure 7).
8
7
6
5
4
3
2
1
0
0 2 4 6 8 10 12 14
Time(day)
Figure 6. Time course for LiP activity produced by GHJ-4.
Lignin model compounds utilization capability
The ability of GHJ-4 strain to utilize guaiacol or lignin as
the sole carbon and energy source is shown in Table 1.
Results showed that lignin and guaiacol, particularly
lignin, could be utilized by GHJ-4 strain. So, GHJ-4 was
able to utilize some lignin model substrates.
Dye decolourization ability of GHJ-4
The polymeric dye RBBR, an anthracene derivative, was
used as a starting material in dye production and represented
an important class of often toxic and recalcitrant
organopollutants. Results showed that, GHJ-4 could
readily decolorize RBBR (Figure 1). We had also found
the decolourization of another two polymeric dyes, aniline
blue and phenol red, by GHJ-4 in solid culture (Figure 1).
Biodegradation capacity for wood
The decay capacity of GHJ-4 strain for wood blocks was
examined. Results showed that, GHJ-4 had 20.91%
losses of wood weight and approximately 22.99% lignin
loss after 40 days incubation period. So, GHJ-4 strain
could be considered as an efficient wood degrader.
DISCUSSION
P. variabile was frequently isolated from different plant
species worldwide. Riccioni et al. (2007) isolated it from
wood of Actinidia chinensis and Actinidia deliciosa in
association with trunk or vine disorders and from necrotic
wood under pruning cut surfaces. There was also a
report on the isolation of this species from leaves of
Laurus nobilis (Göre and Bucak, 2007) and branches of
Prunus persica and Prunus salicina with dieback symptoms
or pruning debris (Damm et al., 2008). However,
there is no isolation of P. variabile having been reported
in China. Our present study represents the first report of
isolation of this species. This indicated its broad host
spectrum, including several distantly related host plants
and a wide geographical distribution.
The basidiomycetous white-rot fungi and related litterdecomposing
fungi are the most efficient lignin-degrading
organisms in nature (Fackler et al., 2006; Ohkuma et al.,
2001). However, the degradation of lignocellulose by
ascomycetes is an important route for carbon cycling in
plant litter and soils (Nilsson and Daniel, 1989; Liers et

Enzyme activity(U ml
-1 )
6
5
4
3
2
1
0
0 2 4 6 8 10 12 14
Time(day)
Figure 7. Time course for MnP activity produced by GHJ-4.
Table 1. Growth of GHJ-4 on different medium.
Carbon source
3 days
Dry weight of mycelial (mg)
6 days 9 days 12 days 15 days
Lignin 2.2 4.9 10.0 16.9 25.5
Guaiacol 0.5 1.2 6.4 8.8 12.3
al., 2006). Our results showed that, the newly isolated
ascomycete GHJ-4 could produce extracellular oxidoreductases
including laccase, lignin peroxidase (LiP) and
manganese peroxidase (MnP), which is the first record of
P. variabile producing ligninolytic enzyme. Furthermore,
laccase production by GHJ-4 was largely affected by
culture conditions, particularly bran, which was in
agreement with the reported results (Lee et al., 1999;
Schlosser et al., 1997; Dong et al., 2005). Chen et al.
(1982) had clearly demonstrated that, part of lignin
degradation proceeds via low-molecular-weight aromatic
acids and the dye serves as a substrate for fungal enzymes
possibly related to lignin degradation (Glenn and
Gold, 1983). Our results showed that, the newly isolated
P. variabile GHJ-4 strain was capable of utilizing several
lignin model compounds and decoloration of aromatic
dyes, which provided additional evidence of degrading
lignin of GHJ-4. In addition, this finding proved that GHJ-
4 was able to degrade natural lignin.
In conclusion, our results showed that this new isolated
P. variabile GHJ-4 had a certain lignin degradation
capacity and provided a new microorganism resource for
Gao et al. 4173
lignin degradation. The ability of this fungi strain could be
of interest for lignocellulosic biomass degradation as well
as treatments of pulp and paper mill effluent. However, it
remains to be studied concerning the role of every
ligninolytic enzyme of GHJ-4 strain on lignin biodegradation
and how lignin is degraded by it. Furthermore,
it would be more informative to compare the ligninolytic
capabilities of this fungus with other lignin-degrading
ascomycetes and basidiomycetes and further research
concerning the role of ascomycetes on lignin biodegradation
should be carried out using P. variabile GHJ-4
and other microorganisms of this class.
ACKNOWLEDGEMENT
This work was funded by Natural Science Foundation of
Shandong province, China (Grant No. Q2008D08).
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African Journal of Biotechnology Vol. 10(20), pp. 4175-4182, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2285
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Improved taxol production in Nodulisporium sylviforme
derived from inactivated protoplast fusion
Kai Zhao 1,2 , Lixin Sun 3 , Xi Ma 1 , Xiuliang Li 1 , Xin Wang 1 , Wenxiang Ping 1 and Dongpo Zhou 1 *
1 Key Laboratory of Microbiology, College of Life Science, Heilongjiang University, 74 Xuefu Road, Harbin 150080, China.
2 Biotechnology Research Center, Heilongjiang Academy of Agricultural Sciences, 368 Xuefu Road, Harbin 150086,
China.
3 Hospital of Heilongjiang University, Harbin 15008.
Accepted 6 April, 2011
Inactivated protoplast fusion by UV irradiation and UV+LiCl mutation was conducted using
Nodulisporium sylviforme strain UV40-19 and UL50-6 to breed a high taxol-producing fungus. Qualitative
and quantitative analysis of taxol production was confirmed using thin-layer chromatography, high
performance liquid chromatography and mass spectrometry. The protoplasts of UV40-19 and UL50-6 were
fully inactivated by heating at 54°C for 5 min and by UV irradiation (30 w UV light and vertical distance 30
cm) for 85 s. The highest fusion rate (14.31 ± 1.13%) between UV40-19 and UL50-6 was obtained under the
conditions of 35% PEG, 90 s fusion time and the addition of 0.01 mol/l CaCl2. One high taxol
production strain HDF-68 was obtained. The taxol production was up to 468.62 ± 37.49 �g/l, which was
increased by 24.51 and 19.35% compared with the parental strain UV40-19 and UL50-6, respectively. This
study provided a good basis for the application of this technique to the breeding of the strains
with high taxol output.
Key words: Taxol, endophytic fungi, protoplast preparation, protoplast fusion.
INTRODUCTION
Taxol is a diterpenoid with anticancer activities. It was first
isolated from the bark of Taxus brevifolia Nutt by Wani et
al. (1971) and is still mainly extracted from the bark of
yews. The action of taxol is to inhibit the depolymerization
of microtubulin and disturb the function of microtubes thus,
affect the formulation of spindle and inhibit tumor cell
mitosis (Zhou et al., 2003). Recently, great effort has been
made to develop alternative means of taxol production,
including complete chemical synthesis (Holton et al.,
1994a, b; Nicolaou et al., 1994), semi-synthesis
(Comnercon et al., 1995; Holton et al., 1995) and the
Taxus spp. plant cell culture (Christem et al., 1991; Arteca
and Wickremesinhe, 1993; Ketchum et al., 1995;
Furmanowa and Syklowska-Baranek, 2000; Wang at al.,
2001).
Using microbe fermentation to produce taxol is a
*Corresponding author. E-mail: zhoudp2003@yahoo.
com.cn. Tel: +86 0451 86609016. Fax: +86 0451
86609016.
very prospective method for obtaining a large
amount of taxol. Several endophytic fungi that produce
taxol have been isolated (Stierle et al., 1993; Strobel et al.,
1996; Zhou et al., 2009). Since 1993, the authors have
isolated five endophytic fungal species that can produce
taxol by screening samples from the inner bark
(phloem-cambium) and xylem of Taxus cuspidata Sieb. et
Zucc. These fungi are Nodulisporium sylviforme (Zhou et
al., 2001), Pleurocytospora taxi (Sun et al., 2003),
Alternaria taxi (Ge et al., 2004), Botrytis (Zhao et al.,
2008a) and Aspergillus niger var. taxi (Zhao et al., 2009).
However, the method is still at the experimental
stage due to the low yield of the isolated and bred
strain, which makes industrial production difficult.
Therefore, the bottleneck lies in the breeding of high
yield strain (Zhou et al., 2003). Two high taxol-
production mutants UV40-19 and UL50-6, were screened
from N. sylviforme HQD33 by UV irradiation and UV+LiCl
mutating in our lab to induce mutagenesis. The
common mutagenesis may have little effect on taxol
production. Alternatively, protoplast fusion can be used
to produce high yield stable strain and is widely applied to

4176 Afr. J. Biotechnol.
fungal breeding (Gokhale, 1992; Muralidhar and Panda,
2000). Up to date, no report on the breeding of high
taxol-production strain by inactivated protoplast fusion
is available. In this study, we report the production
of a high taxol-producing strain by inactivated
protoplast fusion of two parental strains.
MATERIALS AND METHODS
Strains
The spores from N. sylviforme HQD33 (a taxol-producing endo-
phytic fungus isolated from T. cuspidata in China, CCTCC M 202049)
with a taxol output of 51.06 to 125.70 µg/l were subjected to
a series of mutagenesis screening (UV, EMS, 60 Co and NTG).
A mutagenesis-derived strain NCEU-1 with a taxol output of
314.07 μg/l was used as the primary starting strain. UV 40-19
with taxol output of 376.38 µg/l and UL50-6 with taxol output
of 392.63 µg/l were the mutants from strain NCEU-1
protoplasts which underwent UV mutagenesis and the
combined mutagenesis by UV and LiCl, respectively (Zhao
and Zhou, 2004). UV40-19 and UL50-6 were used as parental strains in
this study for inactivated protoplast fusion.
Media
Potato dextrose agar (PDA) medium and potato dextrose liquid
medium (Shen et al., 1999) were used as the growing medium. For
the regeneration of protoplasts, solid regeneration medium (PDA
medium containing 0.7 mol/l NaCl) and semi-solid regeneration
medium (reducing the content of agar in solid regeneration medium
to 6 to 8 g/l) were used as hypertonic media. S-7 medium (Stierle
et al., 1993) with the addition of tyrosine, linoltic acid and
phenylalanine at a final concentration of 1.5 to 5.0 mg/l was
used as the fermentation medium.
Lytic enzyme solution
Lytic enzyme solution was prepared by dissolving 30 mg/ml
lywallzyme (Institute of Microbiology of Guangdong, Guangzhou,
China), 20 mg/ml snailase (Beijing BioTech Co., China) and 10
mg/ml lysozyme (Institute of Biochemistry, Academy of China) in 0.7
mol/l NaCl, followed by clarifying the solution by centrifuged at 4000
r/min at 4°C for 15 min. The supernatants were collected by filtration
and the pH was adjusted to 5.5 to 6.0.
Osmotic stabilizer and fusogen
NaCl at 0.7 mol/l was used as the osmotic stabilizer. Polyethylene
glycol (PEG, MW 6000) was dissolved in the osmotic stabilizer to
yield final concentration of 25, 30, 35and 40% (w/v), respectively.
The PEG solutions containing 0.01 mol/l CaCl2 and 0.05 mol/l
glycine were used as fusogen.
Preparation and regeneration of protoplasts
Preparation and regeneration of protoplasts were carried out
as previously described (Zhao and Zhou, 2004).
Inactivation and fusion of protoplasts
Protoplasts of strain UV40-19 were inactivated by heating at 54°C for
5 min. Protoplasts of strain UL50-6 were inactivated by UV irradiation
(30 w UV light and vertical distance 30 cm) for 85 s. Two
inactivated protoplast suspensions were selected
randomly with a concentration of 1.0×10 6 protoplasts/ml. 1
ml of each was mixed and centrifuged at 3000 r/min for 10
min. The protoplasts were collected and suspended in 0.2 ml
osmotic stabilizer, then 1.8 ml different concentrations of
PEG preheated at 30°C was added to the suspensions. The
mixed liquid was treated at a 30°C water bath for different
periods, then 5 ml osmotic stabilizer at 4°C were added to
stop the fusion, followed by washing and centrifugation to
discard the fusion agent. The washed protoplasts were
resuspended into osmotic stabilizers and regenerated by
double-layer culture for 3 to 5 days at 28°C (Zhao et al., 2008b).
The fusion rate was calculated based on the regeneration of
syzygies and inactivated parents.
The fusion time was set at 60 s with PEG concentrations at
25, 30, 35 and 40%. Then, the fusion time was set at 30, 60,
90 and 120 s under the optimal PEG concentration. In
addition, the effect of Ca 2+ (0.01 mol/l CaCl2) in the fusion
reagents on the fusion rate of the protoplasts was estimated.
The fusion rate of the protoplasts was calculated as follows
(Zhao et al., 2008b):
a= ((b-c)/d) ×100%
Where, a, refers to the fusion rate of the protoplasts; b refers
to the number of colonies on the regeneration plate; c refers
to the number of colonies on the inactivated parent
regeneration plate; d refers to the number of parent colonies
on regeneration plate.
Screening of fusants with high taxol yield
The obtained fusants with good mycelia growth and spore-
production abilities were successively transferred and cultured.
They were transferred onto plates containing 135 μg/ml Nystatin and
cultured at 28°C for 3 days (Zhao et al., 2005). Single colony
growing fast on the plates with large diameter was selected as
resistant strains through primary screening which were used for the
screening of high-output strains. Other colonies were discarded.
The fusants obtained by primary screening were activated on PDA
slope culture at 28°C, then transferred into 50 ml PDA liquid medium
in 250 ml flask and cultured at 28°C for 3 days. The products were
inoculated into modified S-7 culture at the concentration of 3% (v/v)
and fermented at 28°C and 150 r/min for 12 days. At the end of
fermentation, the filtrate and mycelium were collected. The filtrate
was extracted twice using acetic ether, one hour each time and
supernatants were collected. The mycelium was whetted fully and
extracted using 30 ml acetic ether for 1 h. The organic phase was
combined and then, distilled to remove the organic solvent.
Purification by column chromatography
The silica gel used for 60 to 100 um column chromatography
was dipped in CHCl3 overnight and packed (15 × 260 mm) by
gravity settling. The impurities were washed out from silica
gel thoroughly using CHCl 3. The sample was dissolved into
the CHCl3 and loaded. The unabsorbed impurities were
washed out using CHCl3. Thereafter, methanol: CHCl3 (3:97,
v/v) was used for elution and the peaks were collected. All
the process was carried out at room temperature.
Thin-layer chromatography (TLC) analysis
The chloroform-methanol (7:1, v/v) was used as developer

Regeneration rate of protoplasts (%)
90
80
70
60
50
40
30
20
10
0
1 2 3 4 5 6 7 8 9 10 11 12
Heat inactivation time (min)
Figure 1. Effect of heat inactivation time and temperature on
regeneration rate of strain UV40-19 protoplasts.
and taxol standard (Sigma Ltd) was used as the control. 1%
vanillin-concentrated sulfuric acid was used as
chromatography agent. After TLC developing,
chromategenic agent was sprayed and dried at 90 to 105°C
for chromatogenic reaction.
High performance liquid chromatography (HPLC) analysis
Using taxol standard as control, the corresponding TLC area of 1
cm 2 of the sample was scratched and dissolved using 1 ml methanol
and eluted using ultra sonication. The eluate was filtered. A Waters
Millennium32 HPLC workstation equipped with a photodiode array
detector was used for quantitative analysis. An aliquot of taxol
extract (10 μl) was lyophilized and dissolved in 1% methanol and
was injected onto a 250 × 4.6 mm Taxsil-3 C18 reverse phase
column. The mobile phase was a mixture of methanol-water (60:40)
and the flow-rate was set at 1.0 ml/min. Taxol in the eluent was
detected by measuring the absorbance at 227 nm where taxol has
the maximum absorbance. Qualification was achieved using the
standard curve generated from the taxol standard over a
concentration range of 0.05 to 1.00 mg/ml at which the peak area
showed linear relationships with the absorbance (r = 0.9988).
Mass spectrometry analysis
The HPLC peak with the same retention time as the standard
was collected and dried. The structure of the extracted taxol was
confirmed with a waters triple quadrupole tandem LC-MS system
(Waters, MA, USA). The HPLC portion was run isocratically with
acetonitrile: water (49:51) as mobile the phase. The sample was
loaded onto a 250 × 4.6 mm Taxsil-3 C18 reverse phase column
(Metachem, Co. Ltd) and separated at a flow rate of 0.8 ml/min with
the column temperature at 35°C. The MS scanning ranged from 100
to 1000 m/z and the shell gas (N2) and assistant gas (N2) were 65
international units (IU) and 20 IU, respectively. The discharge
current was 5 µA. The evaporator and capillary temperatures were
465 and 180°C, respectively.
Measurement of hereditary stability of fusants
Fusants with high taxol output were cultured for ten gene-
Zhao et al. 4177
44°C
52°C
54°C
rations. Strains with good hereditary stability and high yield
of taxol were stored.
RESULTS
Preparation, inactivation and fusion of protoplasts
The yield of N. sylviforme UV40-19 and UL50-6 protoplasts
was up to 1.26 × 10 and 1.82 × 10 7 /ml, respectively.
Regeneration tests of inactivated protoplasts of both
strains showed that no colony grew on the regeneration
medium (Figures 1 and 2), demonstrating that the
inactivation process was successful.
The protoplast fusion rate at different PEG
concentration and different fusion time was shown in
Tables 1 and 2, respectively. It can be seen that both PEG
concentration and fusion time had great effects on the
protoplast fusion. The optimal PEG concentration was
35% and the optimal fusion time was 90 s.
The addition of Ca 2+ into the fusion agent can
improve the fusion rate of the protoplasts signifi-
cantly. Figure 3 showed that under conditions of 90
s and 35% PEG, the addition of 0.01 mol/l CaCl2
increased the fusion rate of the protoplasts, from
10.92 ± 0.76 to 14.31 ± 1.13%.
Screening of fusants with high taxol output
Isolated fusants were transferred onto the PDA medium
and incubated at 28°C for 2 to 3 days. There were great
differences on the morphological characters and growth
rate among fusants. The fusants growing faster were
selected out and employed to fermentation and hereditary
stability tests. From thirty five fusants, one high yield
fusant HDF-68 was obtained. Morphological comparisons
between parents and HDF-68 were shown in Table 3.

4178 Afr. J. Biotechnol.
Regeneration rate of protoplasts (%)
45
40
35
30
25
20
15
10
5
0
30 40 50 60 70 80 90 100
UV irradiation time
Figure 2. Effect of UV irradiation time on regeneration rate of strain UL50-6 protoplasts.
Table 1. Effect of PEG concentration on fusion rate.
PFG concentration (%, w/v) Fusion rate (×10 -2 )*
25 1.53 ± 0.11
30 4.92 ± 0.21
35 9.61 ± 0.52
40 6.44 ± 0.48
*Data were shown as Means ± SD and mean values were based
on five replicates.
Clearly, the morphological characters of fusant HDF-68
were not identical to either of the parents. It displayed
mixed characters of both parents’ strains.
Taxol production in the fusant HDF-68
TLC analysis showed that the fermentation extractions
from the fusant HDF-68 appeared one blue dot located at
the same place as the standard taxol Rf. This test
indicated that the fusant HDF-68 can produce taxol or
taxol-like compounds. Extracted from fermented fungal
culture, the taxol sample showed the characteristic peaks
as the standard taxol in the HPLC chromatogram (Figure
4a and b). After the addition of the taxol standard into the
samples, the peaks from the samples increased,
supporting that the purified fermentation product from the
fusant HDF-68 is taxol. Based on taxol standard curve,
the taxol content produced in the sample was 468.62 ±
37.49 �g/l. The taxol output of the fusant HDF-68 was
higher than their parent strains N. sylviforme UV40-19 and
UL50-6 by 24.51 and 19.35%, respectively (p < 0.01).
Waters LC-MS analysis confirmed that purified product
Table 2. Effect of fusion time on fusion rate.
Fusion time (s) Fusion rate (×10 -2 )*
30 4.08 ± 0.26
60 6.18 ± 0.28
90 10.92 ± 0.76
120 8.27 ± 0.41
*Data were shown as Means ± SD and mean values
were based on five replicates.
is taxol with the expected molecular ion mass (M+H) + (m/z
854.92) (Figure 5).
Stability of strain HDF-68
After ten generations of successive subculturing, the
colony morphological characteristics of strain HDF-68
were identical to those of the original fusant strain and the
taxol output of successive strain of the fusant HDF-68
was almost the same as the original fusant strain (Table
4). The results showed that strain HDF-68 remained
stable after being cultured for ten generations.
DISCUSSION
Effects of PEG concentration and fusion time on
fusion rate
PEG is the most commonly used fusogen and its con-
centration has a significant effect on the rate of protoplast
fusion (Gokhale, 1992). Previous studies showed that, in

Fusion rate (%)
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
1
0
(%)
A B
Figure 3. Effect of Ca 2+ on fusion rate. (A) With 0.01 mol/l Ca 2+ ; (B) without Ca 2+ .
Table 3. Morphological characters of parents and fusant HDF-68.
Strain UV40-19 UL50-6 HDF-68
Colony morph
Colony diameter
(PDA, 5 days)
Colonies loose, hoary,
reverse light yellow, margin
irregular, mycelium effuse
Colonies dense, hoary,
reverse light brown, margin
regular, mycelium effuse
5.8-6.7 cm 5.4-6.2 cm 6.1-6.2 cm
Zhao et al. 4179
Colonies dense, hoary,
reverse light yellow,
margin irregular,
mycelium effuse
Spore volume* 33.68 ± 7.58 �m 3 29.45 ± 12.77 �m 3 36.95 ± 6.36 �m 3
*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
shown as Means ± SD and mean values were based on ten replicates.
case of fungi, 25 to 40% PEG had the optimal effect on
improving fusion rate (Zhou and Ping, 1990). In this study,
35% PEG was the optimal concentration for protoplast
fusion. Compared with the concentration of fusogen,
fusion time has less influence on the fusion rate
(Wesseling, 1982; Zhou and Ping, 1990). Only short
period (about 1 min) of exposure to PEG was enough to
induce protoplast fusion and with the prolonged fusion
time, the fusion frequency did not increase accordingly
(Hopwood and Wright, 1979; Baltz and Matsushima,
1981). The results of this study showed that, over the
range of 30 to 90 s, with the increasing of fusion time, the
fusion rate increased. The optimal fusion time for N.
sylviforme was 90 s. Further prolonging the fusion time
will reduce the fusion rate. The optimal fusion time varies
with the diffe- rent species.
Production of HDF-68 using inactivated protoplast
fusion
In this study, the inactivation rates of both parents were
100%, so all the colonies growing on the regeneration
medium should be fusants, which made the selection
procedure of fusants easier. The regeneration rate in this
study was up to 14.31 ± 1.13%. This is because heat

4180 Afr. J. Biotechnol.
Figure 4. HPLC chromatograms of taxol extracted from strain HDF-68. Arrows indicate the taxol-specific peaks. A, Taxol
sample extracted from strain HDF-68; B, taxol molecule standard.
treatment and UV-irradiation affected the different
processes of the protoplasts. For example, UV-irradiation
could enhance crossing over between the two genomes,
leading to high complementation ability (Gokhale, 1992).
Although, a lot of fusants were obtained, many were
unstable. This may be because the protoplasts derived
from mycelium of N. sylviforme contain several nuclei,
which usually leads to multinucleate protoplasts (Gokhale,
1992).
Through protoplast fusion with inactivated parents, we
have succeeded in obtaining a high yield fusant HDF-68
that could produce 468.62 �g/l taxol and the fusant HDF-
68 exhibited characteristics of both parents and genetic
stability after subculturing. These results suggest that
intraspecific chromosomal recombination between N.
sylviforme UV40-19 and UL50-6 might have occurred since
the parents were inactivated.
Up to date, there is no report on using inactivated
protoplast fusion to produce a strain with high taxol
output. This is the first successful application of the
methods in improving taxol output of endophytic fungi.
Although, the yield of taxol obtained in this study is
insufficient for industrial production, we proved that the
inactivated protoplast fusion is an effective method in
fungus breeding and provided a good basis for the
application of this technique to the breeding of the
strains with high taxol output. Further improvement of
strain HDF-68 and the optimization of fermentation
conditions and culture medium might lead to higher
production of taxol.
ACKNOWLEDGEMENTS
This study was supported by National Natural
Science Foundation of China (30970090), China
Postdoctoral Science Foundation (20090450136),
Innovation Foundation of Harbin (2010RFQXS043),
Program for New Century Excellent Talents in
Heilongjiang Provincial University, Research
Program Heilongjiang Education Bureau (11551377),
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) +
Table 4. Properties of HDF-68 after 10 generations.
Heilongjiang University (Hdtd2010-17) and
Outstanding Youth Science Fund of Heilongjiang
University.
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African Journal of Biotechnology Vol. 10(20), pp. 4183-4189, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2286
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Production of ethanol from mango (Mangifera indica L.)
peel by Saccharomyces cerevisiae CFTRI101
Lebaka Veeranjaneya Reddy 1 *, Obulam Vijaya Sarathi Reddy 2 and Young-Jung Wee 3 *
1 Department of Microbiology, Yogi Vemana University, Kadapa (A.P.) 516 003, India.
2 Department of Biochemistry, Sri Venkateswara University, Tirupati 517 502, India.
3 Department of Food Science and Technology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, Korea.
Accepted 4 April, 2011
Mango fruit processing industries generate two types of waste, including solid waste (peel and stones)
and liquid waste (juice and wash water). Utilization of this waste is both a necessity and challenge. This
work was aimed to investigate the suitability of dried mango peel for ethanol production. The mango peel
contained good amount of reducing sugars up to 40% (w/v). Direct fermentation of mango peel extract
gave only 5.13% (w/v) of ethanol. The rate of the fermentation was very slow. Nutrients such as yeast
extract, peptone and wheat bran extract were tested for the supplementation of mango peel medium and
it was observed that the nutrient supplementation increased the ethanol production significantly up to
7.14% (w/v). The suitability of wheat bran extract (WBE) based medium, which is cheap and abundantly
available for mango peel fermentation was also discussed.
Key words: Mango peel, ethanol fermentation, nutrient supplementation, wheat bran extract.
INTRODUCTION
The excessive consumption of fossil fuels, particularly in
large urban areas, has greatly contributed to generation
of high levels of pollution. There is a need for environmentally
sustainable energy sources to find a viable and
long-term substitute for liquid petroleum. As a step to
solve this problem, the use or addition of biofuels to
gasoline, which reduces emission of carbon monoxide
and unburned hydrocarbons that form smog, has widely
been enforced in recent years (Wyman, 1994). In this
regard, India reforms are taken by blending 10 to 15%
ethanol in its gasoline usages. Converting a renewable
non-fossil carbon, such as organic wastes and biomass
consisting of all growing organic matter (plants, grasses,
fruit wastes and algae) to fuel would assure a continual
energy supply (Wyman, 1996).
The economics of ethanol production by fermentation
*Corresponding author. E-mail: lvereddy@yahoo.com;
yjwee@ynu.ac.kr. Tel: +91 92909 75611; +82 53 810 2951.
Fax: +82 53 810 4662.
are significantly influenced by the cost of the raw materials,
which accounts for more than half of the production costs
(Classen et al., 1999). To achieve a lower production
cost, the supply of cheap raw material is thus a necessity.
Production of value added products from agro-industrial
and food processing wastes is now a focusing area, as it
reduces pollution in the environment in addition to energy
generation. The annual availability of these wastes
amounts to 1.05 billion tons (Anonymous, 2004). The
major part of this is mostly discarded and it is the main
source for increasing the pollution in environment on
occasions and also, the discarding process become a
very expensive step due to high transportation costs.
Majority of fruit and vegetable wastes available from their
processing industries are seasonal and they do not
decompose rapidly. The mechanical drying of these
wastes (mango peel, citrus peel, pineapple peel and
tomato processing wastes) gave opportunity to store the
substrate all over the year. The yeast Saccharomyces
cerevisiae and facultative bacterium Zymomonas mobilis
are better candidates for industrial alcohol production. Z.
mobilis possesses advantages over S. cerevisiae with

4184 Afr. J. Biotechnol.
respect to enthanol productivity and tolerance. However,
ethanol is produced commercially by yeast because it
ferments glucose to ethanol as a virtually sole product
and it is known for its high ethanol tolerance, rapid
fermentation rates and insensitivity to temperature and
substrate concentration (Linden and Hahn-Hägerdal,
1989).
Mango is processed to a maximum extent, thereby producing
high quality of solid and liquid wastes. Solid
wastes, stones, stalks, trimmings and fibrous materials
are obtained during the preparation of raw material. This
contributes about 40 to 50% of total fruit waste out of
which, 5 to 10% is pulp waste and 15 to 20% is kernel
(Anonymous, 2004; Madhukara et al., 1993; Maini et al.,
2000; Pandey et al., 2000). Liquid waste is the waste
material that comes out of a factory after washing of
fruits, packaging, blanching, cooling and plant and machinery
clean up and so on. Utilization of this mango waste
is both a necessity and a challenge. If a factory is
processing five tons of Totapuri mangoes per hour, about
six tons of peel would be available as waste per day of 8
h work. Approximately, 0.4 to 0.6 million tons of mango
peel is generated annually in India (Anonymous, 2004).
This waste is either used as cattle feed or dumped in
open areas, where it adds to environmental pollution. The
use of mango peel as a source of pectin and fibre
production also has been reported (Pandia et al., 2004).
Grohmann et al. (1994; 1995; 1996; 1998) previously
reported ethanol production from orange peel. Ethanol
production from banana (Manikandan et al., 2008) and
pineapple peels (Ban-koffi and Han, 1990) were also
investigated. Mango peel is difficult to decompose, as it
takes a very long time, because of its complex composition.
Suitability of mango peel for biogas production was
investigated by Madhukara et al. (1993). However,
ethanol fermentation from fruit and vegetable wastes like
mango peel appears to give better returns. The presence
of high amount of reducing sugars in dried and fresh
mango peel prompted us to make an attempt to utilize it
as a raw material for ethanol production and development
of cheap medium. As far as we know, this is the first
report of its kind on ethanol production from mango peel.
MATERIALS AND METHODS
Strain and medium
Non-amylolytic and ethanol-producing yeast strain S. cerevisiae
CFTRI 101 was used throughout the experiments; was obtained
from CFTRI, Mysore, India. The culture was maintained on MPYD
(malt extract 0.3%, peptone 0.5%, yeast extract 0.3% and dextrose
2%) agar (1.5%) slants at 4°C. The inoculum was prepared by
inoculating the slant culture into 25 ml of the sterile MPYD liquid
medium in 100 ml conical flask and growing it on a rotary shaker
(100 rpm) for 48 h. 10% (v/v) inoculum (3 × 10 4 cells ml -1 ) was
inoculated into 100 ml sterile mango peel extract broth in 250 ml
conical flask and was incubated up to 5 days under stationary
conditions. All the stated experiments were conducted at pH 5.0
and 30°C.
Mango peel
Mango peel was procured from local mango pulp industry (Vinsari
Fruit Pulp Industries Ltd., Renigunta, Tirupati, India). It was dried
and milled to a particle size of 40 BS (British Standard) mesh in an
apex mill.
Extraction of sugars from mango peel
Mango powder (100 g) was mixed with water (1:3) and left overnight.
The liquid containing sugars was extracted with the help of
cheesecloth by squeezing. This acted as control. In the case of
enzymatic digestion with 1% (v/v) pectinase, Trizyme 50 (Triton
Chemicals, Mysore, India) was used for improved results. The
extraction medium pH was 5.0 and the temperature was 37°C. The
extract was suitably diluted to obtain the desired concentration of
sugars (15-17%, w/v), and was supplemented with various nutrients
(yeast extract 1%, peptone 1.5%, ammonium phosphate 2% and
wheat bran 3%) in order to study the effect of nutrients on fermentation.
The unsupplemented medium acted as the control. In the
case of wheat bran extract supplementation experiments, mango
peel sugars were extracted into the wheat bran extract solution
instead of water.
Preparations of wheat bran extract (WBE)
Wheat bran obtained from the local market was used in preparation
of the wheat bran extract. Wheat bran (30 g) was boiled with 500 ml
of water for 10 min. After cooling, it was filtered and equal volume
(500 ml) of the extract was collected by washing the residue and
made to 1 L (Shamala and Sreekantiah, 1988).
Analytical methods
Mango peel analysis for the determination of moisture, non-reducing
sugars, protein, total soluble solids, cellulose and lignin was
carried out according to the methods of Ranganna (1986).
Reducing sugar concentration was estimated by Shaffer and
Somogyi (1933) method. Ethanol and other metabolites were determined
by gas chromatography coupled with flame ionization detector
(Antony, 1984). Final cell biomass was estimated by weighing the
dried yeast cells after fermentation. All data are shown as the
average values and standard deviations from three independent
experiments, unless otherwise stated.
RESULTS
Extraction of sugars from dried mango peel
From the aqueous extraction, low amount of sugars were
obtained; only 20% (w/v). Mango peel treated with crude
pectinase yielded higher levels of solubilisation and
reducing sugars (30 ± 5%, w/v) (Tables 1 and 2). The
optimum incubation period for solubilisation of the maximum

Table 1. Composition of fresh and dried mango peel used in this
study.
Content Fresh mango
peel
sugars was found to be 24 h (Figure 1). The results also
indicated a relatively low inhibition of hydrolytic enzymes
(amylases and cellulases) by the sugars released from
Dried mango
peel
Moisture 70 ± 5 10 ± 1.2
Total solids 25.6 ± 4.6 70.5 ± 2.7
Reducing sugars 7.0 ± 1.8 30 ± 2.5
Non-reducing sugars 5.9 ± 0.4 4.3 ± 0.5
Protein 3.5 ± 0.5 4.0 ± 0.8
Cellulose and lignin 25.2 ± 2.0 23 ± 1.2
Table 2. Effect of pectinase enzyme (1%, v/v) on sugar extraction
from dried mango peel.
Time (h)
Reducing sugar (%, w/v)
Pectinase non-treated Pectinase treated
5 5.8 ± 0.5 10 ± 0.63
10 10 ± 0.65 15.6 ± 2.3
15 15.5 ± 1.5 21 ± 1.6
20 18 ± 1.3 24.3 ± 1.3
25 20 ± 1.5 30 ± 5.5
Figure 1. Total percentage of water soluble solids and sugars in the
dried mango peel extract after pectinase (1%, v/v) treatment. –●–,
total water soluble solids; –■–, soluble sugars.
Reddy et al. 4185
the mango peel. Another significant observation made
during this study was decrease in the initial pH from 5 to
4.5 at the end.

4186 Afr. J. Biotechnol.
Effect of pH, temperature and nutrients on the dried
mango peel fermentation
The levels of reducing sugars were adjusted to 15% (w/v)
with the dilution and the required nutrients were supplemented
for fermentation. The direct fermentation of
mango peel extract gave 5.14% (w/v) ethanol. The
results of optimizing the culture conditions such as pH
and temperature indicated that, the changes in pH and
temperature could affect the final ethanol concentration.
The final ethanol concentration at different pH and temperature
experiments (Figure 2a, b) showed that 30°C
and pH 4.5 were optimum for ethanol production from
mango peel extract.
The supplementation of nutrients significantly increased
the ethanol concentration and fermentation rate. Yeast
extract alone and combination with peptone supple-
Figure 2. Effect of (a) pH and (b) temperature on ethanol
fermentation from the dried mango peel extract.
mentation attributed to the ethanol formation very rapidly
and formed 7.0 and 7.14% (w/v) ethanol, respectively,
instead of 5.14% (w/v) from unsupplemented media at
the end of the fermentation. The data on fermentation of
mango peel extract with nutrient supplementation are
presented in Table 3. The mango peel extract with wheat
bran extract medium significantly increased the yeast
growth and the ethanol formation when compared with
the peel extract alone (Table 3). The wheat bran extract
increased the ethanol concentration from 51.4 to 67.5 g l -
1 (Figure 3). We also tried the fresh mango peel, which
contained low levels of reducing sugars (7 to 10%, w/v)
because of its high amount of water (90%, v/v). Experiments
with the fresh mango peel extract without supplementation
of nutrients was also done, but it gave low
ethanol productivity (3%, w/v).
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 .
Supplement
Ethanol
concentration
(g L -1 )
24 h 48 h 72 h
Theoretical
ethanol
yield (%)
Volumetric
productivity
(g L -1 h -1 )
Ethanol
concentration
(g L -1 )
Theoretical
ethanol
yield (%)
Volumetric
productivity
(g L -1 h -1 )
Ethanol
concentration
(g L -1 )
Theoretical
ethanol yield
(%)
Reddy et al. 4187
Volumetric
productivity
(g L -1 h -1 )
Control 23 30.7 0.95 45.6 60.0 0.93 51.3 68.4 0.71 5.2
Peptone 30 40 1.25 55.4 73.3 1.15 68.8 91.7 0.95 6.3
Yeast extract 33.2 44.3 1.38 60.5 80.7 1.26 70.0 93.7 0.92 6.6
Ammonium
phosphate
25.4 33.6 1.05 46.7 62.3 0.97 53.2 70.9 0.73 5.1
Peptone +
yeast extract
34.5 46.0 1.43 62.2 82.9 1.29 71.4 95.2 0.99 6.8
Wheat bran
extract
30.8 41.1 1.28 53.9 71.9 1.12 67.5 90 0.93 6.2
a The values presented in the table are mean values of three independent experiments.
the supplemented medium had high cell count
at the end of the fermentation (data not shown).
The stated results were supported by the increase
of final biomass in the nutrient supplemented
mango peel extract medium (Table 2).
DISCUSSION
The dried mango peel contained high amount of
reducing sugars (up to 45%, w/v) and the results
are in accordance with those of previous reports
(Anonymous, 2004; Madhukara et al., 1993). In
the case of aqueous extraction, the sugar content
was very low. The reason for the low content
could be due to the presence of pectin, which held
the sugar molecules and could not be released
with simple water extraction. The presence of
other enzymes like amylase and cellulase in the
crude pectinase, may aid to increase sugar concentration
by hydrolyzing the respectable substances
(Grohmann et al., 1995). The significant
pH drop during the enzymatic hydrolysis of mango
peel is undoubtedly caused by the release of Dgalacturonic
acid from pectin. The pKa value of Dgalacturonic
acid is 3.51 (Filippov et al., 1978) and
the pH values of peeled hydrolyzates appeared be
to stabilized in the range of 3.3 and 3.5. The low
yields of ethanol from dried mango peel increase
the cost of production. Increase in the ethanol
production up to 7 to 7.5% (w/v) as in general
industrial output from molasses can economize
the process. The development of cheap medium
for fruit waste fermentation to ethanol also
required low-cost ethanol production.
To improve the concentration of ethanol, the fermentation
medium was supplemented with variety
of nutrients like yeast extract, peptone and
Biomass
(g L -1 )
ammonium phosphate to overcome the nutritional
deficiency. The result presented in Table 3 clearly
indicates that, in the case of supplementation of
nutrients, not only the rate of ethanol synthesis
but also the final ethanol concentration increased
significantly. The combination of yeast extract and
peptone gave the maximum improvement in rate
of ethanol synthesis as well as final concentration
in the medium. The ethanol production as well as
viable cell count was significantly increased up to
50 and 20%, respectively, in the mango peel
extract with yeast extract and peptone supplementation.
However, the ammonium phosphate
supplemented one did not stimulate the ethanol
production. Similar results were also obtained in
the case of ethanol fermentation from orange peel
by genetically modified Escherichia coli, which
can utilize the glucose, galactose and galcturonic
acid for ethanol product (Grohmann et al., 1994,

4188 Afr. J. Biotechnol.
Figure 3. Effect of wheat bran extract on the conversion of mango peel to
ethanol fermentation. –▲–, mango peel extract medium supplemented with
wheat bran extract; –■–, control (unsupplemented medium).
1996). It is possible to produce high ethanol
concentrations by extending exponential growth phase of
yeast to longer periods and soluble sugar concen-tration
as in the case of beer production (Kirsop, 1978; Casey
and Magnus, 1984). It is expected that the nutrient
supplementation would overcome nutritional deficiencies
of yeast and allow them to stay longer in growth phase. In
order to verify whether the amount of nutrients and their
mode of feeding influenced the alcoholic fermentation by
S. cerevisiae, experiments were conducted in batch fermentation
with various amounts of nutrients and different
feeding strategies.
Compared with initial total supplementation, exponential
feeding strategy improved the performance of the
fermentation process and the ethanol tolerance of the
yeast. In a recent study reported by Reddy and Reddy
(2005), the nutrients and polyphenols rich horse gram
flour improved the ethanol formation in very high gravity
fermentation. The suitability of wheat bran extract as a
medium for ethanol production from mango peel investigations
successfully replaced the costly medium components
and developed a novel wheat bran extract (WBE)
medium which could provide a cheap source of amino
acids and other nutrients (Shamala and Sreekantiah,
1988). Reddy and Basappa (1996) also successfully
replaced the nutrients with wheat bran extract in the
direct fermentation of starch to ethanol by Endomycopsis
fibuligera and Z. mobilis. However, supplementation with
yeast extract and peptone was superior to that of wheat
bran extract. This showed that wheat bran extract has
limited amount of nutrients when compared with yeast
extract or peptone. Further optimization studies on the
supplementation of WBE are to be made to make the
process economically viable. The initial fermentability of
the unsupplemented peel extracts by S. cerevisiae was
extremely poor because of insufficient growth nutrients in
the peel medium. The fermentation of mango peel extract
was stimulated by supplementation with low amounts of
yeast extract and peptone. The wheat bran extract
though stimulated not only the rate of fermentation and
also the final concentration of ethanol, but it was not as
good as the yeast extract and peptone. However, higher
concentrations of WBE is needed to be supplemented to
make the mango peel extract fermentation process more
economical since the supplementation with yeast extract
and peptone is obviously more expensive. Given the
promise of the proposed WBE based medium for ethanol
fermentation, it should be tested beyond the bench scale.
Generally, the production concentration in commercial
ethanol production plants is between 7.5 and 10% (w/v).
Based on previous reports, the ethanol production
concentration was 4.02% (w/v) in citrus peel waste, 3.5%
(w/v) in grape fruit peel and 4.2% (w/v) in pineapple peel
(Ban-koffi and Han, 1990; Nishio et al., 1980; Wilkins et
al., 2007a, b).
In this study, mango peel was proved as one of the
novel and potential raw material for ethanol production.
Ethanol production from mango peel requires supplementation
of nutrients because of its low nutrient availability.
Supplementation of yeast extract, peptone and
optimization of fermentation conditions enhanced the
fermentation rate and final ethanol concentration. WBE
supplementation showed comparable improvement in all
terms with the very expensive, yeast extract and peptone.
Further optimization studies on peel hydrolysis using
commercial enzymes and optimization of WBE supple-

mentation will make the process economically viable and
it should be tested beyond the bench scale.
ACKNOWLEDGMENTS
We profusely thank Dr T.N Bhavanishankar, plant manager,
Bacardi-Martine India Limited (Nanjangud, Karnataka
State) for his support in the GC–FID analysis. Special
thanks to Dr S. C. Basappa, former deputy director and
scientist, Central Food Technological Research Institute
(CFTRI), Mysore, for his encouragement and critical
comments on the manuscript.
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African Journal of Biotechnology Vol. 10(20), pp. 4190-4196, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2298
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Secretory expression of Rhizopus oryzae α-amylase in
Kluyveromyces lactis
Song Li, Wei Shen, Xianzhong Chen, Guiyang Shi and Zhengxiang Wang*
Research Center of Bioresource and Bioenergy, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi
214122, China.
Accepted 17 March, 2011
Kluyveromyces lactis is a non-conventional yeast species extensively used in the expression of
heterologous genes. In this study, a genetically modified K. lactis with high-level expression of αamylase
from Rhizopus oryzae was obtained, which could successfully hydrolyze and use starch for
growth very well. Shake flask fermentation indicated that, the recombinant yeast was able to produce
the α-amylase at considerable secretion levels using a variety of carbon sources. The highest level of
amylase expression was 22.4 U/ml when cultivated at 30°C and pH 7.0 in the presence of galactose.
Moreover, it was shown that the recombinant yeast, which could efficiently degrade starch, yielded a
final biomass of 12.25 g/l with enzyme activity of 11 U/ml in the culture medium using 20 g soluble
starch/l as the sole carbon source.
Key words: Kluyveromyces lactis, α-amylase, secretion, starch, fermentation.
INTRODUCTION
Yeasts are eukaryotic micro-organisms that have been
used for centuries in traditional fermented food (Romanos
et al., 1992). They have recently become attractive host
organisms for the production of foreign proteins because
of developments in recombinant DNA technology (Romanos
et al., 1992; Dominguez et al., 1998). Compared with the
extensively used Escherichia coli expression system, the
yeast expression system has important advantages in the
expression of proteins from eukaryotic micro-organisms;
these advantages stem from its effective posttranslational
modification system and ability to produce
foreign proteins in soluble and correctly folded form (van
Ooyen et al., 2006).
Kluyveromyces lactis is a Crabtree-negative, nonconventional
yeast that is able to grow solely on lactose
as the carbon source (Dominguez et al., 1998). For decades,
K. lactis has been used in the production of lowlactose
milk because of its lactase-producing ability,
which is necessary for lactose degradation (Freitas et al.,
2008). Its GRAS (generally regarded as safe) status, efficient
secretion capacity and low catabolite repression
*Corresponding author. E-mail: zxwang@jiangnan.edu.cn.
Tel/Fax: +86-510-85918121.
makes it an attractive alternative expression system to
traditional baker’s yeast in the production of certain
proteins (Dominguez et al., 1998; Schaffrath and Breunig,
2000). As an expression host, K. lactis is best known for
its use in the production of bovine chymosin on an
industrial scale, widely recognised as a major biotechnological
achievement (van Ooyen et al., 2006). The yeast
was successfully used in the production of β-galactosidase
in the food industry (Dagbagli and Goksungur,
2008) and was able to achieve high-level expression of
human serum albumin (Lodi et al., 2005) and human
interleukin-1β (Fleer et al., 1991) in pharmaceutical
research. In addition, a large variety of proteins from
different sources such as bacteria, fungi, plants and
mammals (van Ooyen et al., 2006), have been produced
by K. lactis, including α-amylase from Bacillus
amyloliquefaciens (Bartkeviciute and Sasnauskas, 2003),
mouse (Tokunaga et al., 1997) and wheat (Russell et al.,
1993).
The aim of this study was to enhance the expression
and secretion of the Rhizopus oryzae α-amylase using K.
lactis. The amylase gene was integrated into the K. lactis
genome and expressed under the control of the K. lactis
lactase (LAC4; β-galactosidase) promoter and
Saccharomyces cerevisiae α-mating factor pre-pro
sequence. Different cultivation conditions, including tem-

perature, pH and carbon sources were investigated to
study their effects on the production of the R. oryzae αamylase
in K. lactis. Specifically, the starch fermentation
characteristics of the recombinant K. lactis strain were
also observed.
MATERIALS AND METHODS
Strains, plasmids and culture media
E. coli JM109 was used for the propagation of plasmids. The host
strain K. lactis and the expression plasmids pKLAC1 were
purchased from New England BioLabs Company. Recombinant
plasmid pMD-RoAmy containing the R. oryzae α-amylase gene
coding sequence was constructed in previous work (Li et al., 2011)
LB medium (5 g yeast extract, 10 g peptone, 10 g NaCl per litre)
was used for cultivation of E. coli. YPD medium (10 g yeast extract,
20 g peptone, 20 g dextrose per litre) and YCBA agar plate (10 g
yeast carbon base, 15 g agar powder per litre, 5 mM acetamide)
were prepared for the pre-cultivation of K. lactis and selection of
yeast recombinants, respectively. YPDS agar plate (YPD medium
supplemented with 5 g soluble starch, 15 g agar powder per litre)
was used for halo assays of α-amylase activity. YPX medium (10 g
yeast extract, 20 g peptone, 20 g carbon source per litre) was used
for the expression of α-amylase.
Construction and transformation of expression plasmid
The mature RoAmy coding sequence (RA) was amplified by the
polymerase chain reaction (PCR) from pMD-RoAmy with primers:
5'-ATTGTCGACGTGCCTGTCATCAA-3' and 5'-GTAGCGGCCGCG
ATAAGCTTGCACAAACGAAC-3'. The amplification yielded a copy
of the gene flanked by SalI and NotI sites provided by the two
primers (underlined), respectively. The PCR product was gelpurified,
digested with the two indicated enzymes and inserted at
the corresponding site of pKLAC1 vector; this procedure yielded a
recombinant expression plasmid pKLRA, in which the α-amylase
gene was under the control of the K. lactis lactase (LAC4; βgalactosidase)
promoter and the S. cerevisiae α-mating factor prepro
sequence.
The secretion cassette pKLRA was linearized with SacII and
introduced into K. lactis cells by electroporation transformation
method (Delorme, 1989). The electroporated cells were spread on
YCBA agar plates. After incubation, transformants appeared on the
plates at 30°C for 3 to 4 days and were selected for further study.
α-Amylase halo assay
For halo assay, the transformed yeast cells were patched onto
YPDS agar plates and incubated for 48 h at 30°C. The halo-forming
ability of transformants was detected by staining with weak iodine
solution (20 g KI and 2 g I2 per litre).
Expression of α-amylase
The selected K. lactis transformants with different halo-forming abilities
were cultured in shake flasks to screen the RoAmy expression
levels. The recombinant strains were inoculated into 10 ml YPD
medium and grown in a shaking incubator with 200 rpm at 30°C
overnight; 0.2 ml of the overnight culture was transferred into 50 ml
fresh medium and cultivated to an OD600=6.0 to 10.0. Then, the precultures
were inoculated into 50 ml YPX medium to a final optical
Li et al. 4191
density (600 nm) of 0.2. In all the shake flask cultivation processes,
250 ml baffled flasks were used.
Analytical techniques
The α-amylase activity was determined as follows: 1 ml 1% (w/v)
soluble starch was mixed with 0.25 ml citric acid–Na2HPO4 buffer
(0.2 M, pH 5.0) and incubated at 55°C for 5 min, followed by
addition of 0.1 ml enzyme solution. It was further incubated for 5
min. The reducing sugar formed was determined according to the
method described previously (Miller, 1959), using maltose as a
standard. One unit of α-amylase activity was defined as the amount
of enzyme that released 1 mg reducing sugar per minute under the
mentioned conditions.
The yeast growth was measured in terms of dry weights, which
were determined by drying 3 ml samples to constant weight at
65°C. The starch concentration was quantified according to the method
described by Xiao et al. (2006).
RESULTS
Expression of α-amylase in K. lactis
More than one hundred transformants appeared on YCBA
agar plates. Eleven colonies were randomly selected and
replicated to YCBA agar plates for further purification.
Then, the purified clones were patched onto YPDS agar
plates and grown at 30°C for 3 to 4 days. After staining
with weak iodine solution, clear zones were formed
around clones no. 1, 2, 5 and 11, as shown in Figure 1. It
was seen that clones no. 1 and 11 shown larger zones on
the plate assay, but the α-amylase activities observed in
shake flask fermentation were almost the same (data not
shown).
Effects of culture conditions on α-amylase
expression in K. lactis
To investigate the effects of initial pH on α-amylase
expression, this study tested the YPD media with initial
pH values ranging from 4.0 to 8.0. The highest obtained
level of amylase expression, calculated from the triplicate
fermentations, ranged from 1.4 to 11.2 U/ml, as shown in
Figure 2a. To study the effects of temperature on αamylase
expression, the temperature of the shake flask
incubator was set from 20 to 35°C and the initial pH value
of the medium was adjusted to 7.0. Among the cultivation
temperatures observed, the highest amylase expression
level (11.8 U/ml) was achieved at 30°C (Figure 2b).
α-Amylase expression with different carbon sources
The carbon source for amylase expression in the shake
flask culture was screened and the initial amount of
different carbon source was kept at 20 g/l. Figure 3
shows that, amylase was successfully secreted in all the

4192 Afr. J. Biotechnol.
Figure 1. Test of the amylase activity of selected clones of the
K. lactis transformats. The clones’ labelled no. 3, 4, 6 to 10
and the colony labelled “C” of non-transformed control host K.
lactis cells show no clearance zones indicating the absence of
amylase activity.
tested culture media, of which the galactose-containing
culture provided the highest level of amylase secretion at
22.4 U/ml- approximately two times higher than that
obtained in other carbon source-containing cultures.
However, the highest biomass (15.75 g/l) was achieved
using glycerol as the carbon source. Interestingly, at the
end of the shake flask fermentation, the starch- and
maltose-containing cultures yielded similar biomass,
which was slightly higher than that observed in the
glucose-containing culture. Samples from the galactosecontaining
culture media of recombinant K. lactis were
analyzed by SDS-PAGE for the presence of the αamylase,
as shown in Figure 4, both glycosylated and
non-glycosylated α-amylases were secreted into the
culture medium.
Starch fermentation characteristics of the
recombinant K. lactis strain
The characterisation of the growth of the recombinant K.
lactis strain and the K. lactis host strain as well as their
starch hydrolysis and fermentation, were observed using
soluble starch as the sole carbon source. Figure 5a
shows that, the native K. lactis strain could not attain
continuous growth in starch, whereas the recombinant
strain, which has the α-amylase secretion cassette,
displayed a high growth rate in starch. The effects of
initial starch concentrations ranging from 10 to 40 g/l
were examined to study the growth of recombinant yeast.
Because of the α-amylase secreted by the recombinant
yeast, the starch in the cultures was efficiently and com-
Figure 2. (A) Effects of pH and (B) temperatures on αamylase
production in YPD culture medium. Samples
were harvested from 48 h fermentation cultures. All data
are mean values of three independent experiments; error
bars indicate standard deviation.
pletely degraded during fermentation from 12 to 24 h
(Figure 5b). The maximum cell density increased with
rising initial starch concentrations from 10 to 30 g/l;
although, the 40 g starch/l in the medium was completely
degraded in the first 24 h of fermentation, the maximum
cell density observed at 84 h was slightly lower than that
obtained at 20 and 30 g starch/l concentrations. In
addition, the maximum yeast growth rate during the first
12 h was achieved with 10 g starch/l and the yeast exhibited
better growth with 20 g starch/l during the 24 to 60 h
cultivation time. Further details are given in Table 1.
DISCUSSION
It is difficult to accurately compare the expression levels
of various α-amylases in K. lactis, because of variations
in the starch origin and α-amylase activity definition, as
well as in the details of the enzyme assays. However, the

Figure 4. SDS-PAGE analyses of α-amylase
produced in galactose-containing culture medium by
recombinant K. lactis. Proteins were concentrated 5fold
by 10% (w/v) trichloroacetic acid precipitation,
before loading (15 µl). Protein bands were stained
with Coomassie brilliant blue R-250. Lane 1 to 5,
samples from 0, 6, 12, 24 and 48 h fermentation
cultures, respectively; lane M, molecular mass
marker.
highest yield of the R. oryzae α-amylase obtained in the
galactose-containing culture medium by shake-flask
fermentation was 22.4 U/ml (approximately 20 mg/l),
Figure 3. Effects of carbon sources on α-amylase secretion and
biomass production. Samples were harvested from 48 h fermentation
cultures. All data are mean values of three independent experiments;
error bars indicate standard deviation.
Li et al. 4193
which was much higher than the expression level of
mouse α-amylase (0.527 U/ml) (Tokunaga et al., 1997) or
Schwanniomyces occidentalis α-amylase (30 mU/ml)
(Strasser et al., 1989) obtained in K. lactis. To the best of
this study’s knowledge, among various α-amylases expressed
in K. lactis, the expression level of the R. oryzae
α-amylase obtained in this study is one of the highest
reported? In the previous work of these authors, the R.
oryzae α-amylase expression levels observed in E. coli
and S. cerevisiae were only approximately 0.11 and 1.3
U/ml, respectively. In addition, the α-amylase expression
level obtained by recombinant Pichia pastoris was up to
45 U/ml in shake-flask fermentation, however, the
application of recombinant α-amylase in food industry
could be limited due to the characteristics of the host
stain (not a GRAS status strain) and slight residual
methanol in the final fermentation broth. Thus, it was
conclude that K. lactis could be more promising for the
expression of the R. oryzae α-amylase at food status.
Among physical parameters, cultivation temperature
and pH of growth media are important for organism
growth and enzyme secretion (Gupta et al., 2003). Similar
to the temperatures used in many studies (Rocha et
al., 1996; Mincheva et al., 2002), 28 to 30°C yielded the
highest α-amylase activity and biomass. Attempts to
express amylase at an initial pH of 4.0 resulted in an
absence of enzyme activity and the amylase activity
produced at pH 8.0 was only half of the highest amylase
expression level at pH 7.0 (Figure 2). This result indicates
that pH control is crucial for R. oryzae α-amylase
expression in K. lactis.
The expression and secretion of R. oryzae α-amylase

4194 Afr. J. Biotechnol.
Figure 5. Time courses in the fermentation of starch using
recombinant K. lactis and host strain. Biomass produced
(A) and starch hydrolyzed (B) by recombinant K. lactis with
initial starch concentrations of 10 g/l (●), 20 g/l (▲), 30 g/l
(▼) and 40 g/l ( ) and host strain with 20 g starch/l (■). All
data are mean values of three independent experiments;
error bars indicate standard deviation.
in K. lactis was under the control of the LAC4 promoter,
which is one of the most frequently used promoters for
high-level heterologous gene expression in K. lactis and
induced by the presence of lactose or galactose in the
growth medium, but not fully repressed in the absence of
the inducer (van Ooyen et al., 2006). A number of carbon
sources were tested for the growth and α-amylase
expression of recombinant K. lactis. Galactose generated
the highest α-amylase expression probably because the
LAC4 promoter was induced by galactose present in the
culture medium (Rubio-Texeira, 2006). By contrast, αamylase
expressed in lactose-containing cultures were
almost the same as that observed in other carbon source
cultures, indicating that lactose failed to induce the LAC4
promoter for the enhancement of α-amylase expression.
From another point of view, the LAC4 promoter was not
repressed by the presence of a variety of studied carbon
sources. However, considering the price of galactose,
other carbon sources, such as glucose, glycerol, lactose
and starch, are more suitable for α-amylase production.
Most of the wild-type yeast strains cannot directly utilise
starches because of their inability to produce starchdecomposing
enzymes (Jamai et al., 2007). Numerous
studies have demonstrated that, both α-amylase and
glucoamylase are required for efficient starch hydrolysis
in industrial ethanol production using S. cerevisiae strains
(Knox et al., 2003; Kosugi et al., 2009). In previous study,
R. oryzae α-amylase was secreted through an S.
cerevisiae strain. However, the recombinant strain in the
starch-containing culture medium showed extremely low
α-amylase activity (approximately 0.06 U/ml), a level insufficient
for efficient starch degradation without exogenous
glucoamylase. In this study, the host K. lactis strain
used could not grow in the medium with starch as the
sole carbon source, whereas the recombinant K. lactis
strain obtained was able to hydrolyse soluble starch
efficiently because of its ability to express α-amylase at
high concentrations in the starch-containing culture (11.2
U/ml), as shown in Figure 5 and Table 1.
The maltose uptake system of S. cerevisiae has been
extensively studied and is a highly specific strain-dependent.
For example, Weusthuis et al. (1993) observed a
linear decrease in cell yield with increasing amounts of
maltose in the medium feed for S. cerevisiae CBS 8066
strain, whereas Batistote et al. (2006) reported that, S.
cerevisiae VIN7 wine strain yielded a higher final biomass
in the presence of maltose rather than glucose under
certain conditions. Similar results were found for both S.
cerevisiae 70 and 254 strains, as described by Zastrow et
al. (2000). These findings are similar with those observed
in the recombinant K. lactis strain obtained in this study.
Specifically, the recombinant K. lactis produced higher
final cell density in the presence of soluble starch rather
than glucose, probably because of its ability to produce
R. oryzae α-amylase. Unlike bacterial α-amylase, fungal
α-amylase can be used to produce high-maltose syrup
(Doyle et al., 1989), in which the maltose concentration is
up to 50% (w/w) and the glucose concentration is usually
around 10% (w/w). In addition, considerable amounts of
maltotriose, which also yielded higher final biomass than
in the presence of glucose (Zastrow et al., 2000), can be
released during starch hydrolysis (Doyle et al., 1989). In
previous study, soluble starch hydrolysed with R. oryzae
α-amylase yielded a final concentration of glucose and
maltose at 12 and 67% (w/w), respectively and a large
amount of maltotriose was still released at the initial
hydrolysis stage of the gelatinised starch (data not
shown). Overall, it can be conclude that the ability of R.
oryzae α-amylase to produce high levels of maltose and
maltotriose accounts for the higher cell density obtained
by the recombinant K. lactis in the presence of soluble

starch.
Conclusion
Table 1. Soluble starch fermentation characteristics of recombinant K. lactis strain.
Parameter
Biomass (g l -1 h -1 ) a
Starch (g l -1 h -1 ) b
Starch concentration (g/l)
Time (h) 10 20 30 40
0-12 0.064 0.064 0.063 0.601
12-24 0.289 0.343 0.261 0.262
24-36 0.031 0.333 0.292 0.281
36-84 0.146 0.271 0.563 0.261
0-6 0.717 0.852 0.683 0.883
6-12 - c 1.667 1.517 1.602
12-15 - 4.001 2.933
15-18 - 3.967
18-21 -
a Biomass production rate, gram of dry weight per litre per hour; b starch degradation rate,
gram of soluble starch per litre per hour; c the calculation of starch degradation rate in its
running out time is not accurate and the data are not shown. All data are mean values of
three independent experiments.
In conclusion, because of the high-level expression and
secretion of R. oryzae α-amylase in the culture medium,
the recombinant K. lactis was able to efficiently grow
using soluble starch as the sole carbon source and
attained a relatively high cell density probably because of
the considerable amounts of maltose and maltotriose
released by R. oryzae α-amylase. These findings indicate
that, the production of heterologous proteins or other
specific products from K. lactis using starchy materials as
the sole carbon source may be achieved through the coexpression
of the genes of interest and R. oryzae αamylase
gene.
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African Journal of Biotechnology Vol. 10(20), pp. 4197-4206, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2139
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Volatile compounds of maari, a fermented product from
baobab (Adansonia digitata L.) seeds
Charles Parkouda 1 *, Brehima Diawara 1 , Samuel Lowor 2 , Charles Diako 3 , Firibu Kwesi Saalia 4 ,
Nana T Annan 3 , Jan S Jensen 5 , Kwaku Tano-Debrah 4 and Mogens Jakobsen 6 .
1 Département Technologie Alimentaire/IRSAT/CNRST 03 BP 7047 Ouagadougou 03, Burkina Faso.
2 Cocoa Research Institute of Ghana, Physiology/Biochemistry Division P.O. Box 8, Akim-Tafo, Ghana.
3 Council for Scientific and Industrial Research, Food Research Institute, P.O. Box M-20, Accra, Ghana.
4 Department of Nutrition and Food Science, University of Ghana, Legon, Accra, Ghana.
5 University of Copenhagen, Faculty of Life Science, Forest and Landscape, DK-2970, Hørsholm, Denmark.
6 University of Copenhagen, Faculty of Life Science, Centre for Advance Food Studies, Department of Food Science,
Rolighedsvej 30 DK 1958 Frederiksberg C, Denmark.
Accepted 1 April, 2011
The volatile compounds associated with baobab seeds fermentation for Maari production were
extracted and analysed by Likens-Nickerson simultaneous steam distillation-extraction method and gas
chromatography-mass spectrometry (GC-MS), respectively. Furthermore, the titratable acidity, tannin
content and proximate composition were evaluated. A total of 96 compounds were identified with
esters, acids, alcohols and ketones being quantitatively the major groups. Fermentation led to an
increase in the concentration of total volatile compounds from 121.6 in unfermented cooked seeds to
809.1 mg kg -1 in the fermented product. Drying resulted in a significant loss of up to 80.7% of the total
volatiles.
Key words: Volatile compounds, maari, baobab, fermentation.
INTRODUCTION
Several fermented food condiments are commonly found
in several African and Asian countries (Parkouda et al.,
2009). In these countries, they are extensively used due
to their pleasant taste and particular odour as well as
their important nutritional qualities (Azokpota et al., 2008;
Ouoba et al., 2005; Owens et al., 1997). Volatile compounds
are one of the most valuable traits contributing to
their quality and sensorial attributes (Beaumont, 2002).
Maari is one of these fermented food condiments
obtained by processing and spontaneous fermentation of
seeds from the baobab tree (Adansonia digitata L.) in
Burkina Faso. The process is laborious, energy- and
time-consuming and procedures are based on traditional
knowledge and experience of each processor. Contrary
to similar fermented condiments produced from African
locust beans (Soumbala, afitin, iru, sonru and netetu), the
*Corresponding author. E-mail: cparkouda@yahoo.fr.
baobab cooked seeds were not dehulled before the
fermentation; this can lead to a difference in the chemical
content and the profile of volatile compounds. Maari is
especially used as flavouring agent for sauces, soups
and other dishes in some African countries (Chadare et
al., 2008; Parkouda et al., 2010). Traditional maari is
characterized by a specific strong odour which is the
main criterion used by consumers to appreciate the
quality of the condiment. Similar products like afitin,
chungkuk-jang, iru, natto, sonru, soumbala and thua-nao
have been extensively studied and a diversity of volatile
compounds reported (Azokpota et al., 2008;
Leejeerajumnean et al., 2001; Ouoba et al., 2005;
Tanaka et al., 1998). The flavour of these condiments
has mainly been attributed to various volatile compounds
produced through the metabolic activities of microorganisms
during fermentation or the processing conditions
(Azokpota et al., 2008; Leejeerajumnean et al.,
2001; Ouoba et al., 2005). Differences in the traditional
processes for fermented condiments occur among ethnic

4198 Afr. J. Biotechnol.
tribes and these differences presumably influence the
quality of the final products. The identification of volatile
compounds is one of the steps which could give information
for a future selection of starter culture to be used
in controlled fermentation (Azokpota et al., 2010). In
afitin, iru and sonru, compounds belonging to pyrazines,
aldehydes, ketones, esters, alcohols, acids, alkanes,
alkenes, benzenes, phenols, sulphurs and furans groups
were identified (Azokpota et al., 2008). In addition to
these compounds, Ouoba et al. (2005) found amines and
pyridines groups in soumbala. However, volatile compounds
that characterize maari have not yet been
reported. The aim of the present study was to investigate
the volatile compounds associated with spontaneous
fermentation of baobab seeds for maari production using
Likens-Nickerson simultaneous distillation-extraction
method and gas chromatography mass spectrometry
(GC-MS).
MATERIALS AND METHODS
Maari processing and sampling
Maari samples were obtained by spontaneous fermentation of
baobab seeds by three producers (V, C and B) from Pousghuin (a
village in Zorgho, Burkina Faso) according to their own procedure.
Briefly, maari production was done as follows: The baobab seeds
were cleaned and boiled for about 36 h. After the 24 h of boiling, an
ash lye solution was added as softening agent. After the boiling
period, the seeds were drained and transferred into a basket and
left to ferment spontaneously (first fermentation) for 72 h at room
temperature (30 to 35°C). The fermenting mash was pounded and
moulded with further addition of the alkaline ash lye solution. It was
left to undergo a second spontaneous fermentation for about 24 h
at room temperature before drying, including an initial steam
cooking step. The final product obtained was characterized by dark
brownish colour, stickiness and strong smell. Maari is sometime
slightly roasted to give a specific trait to the product. For each of the
three producers, duplicate unfermented boiled seeds, fermenting
mash just before steam cooking and the final dried maari were
collected from the producers and stored at -20°C before analysis.
Additional commercial dried maari ready for consumption was also
collected at Toulfé (a village in Burkina Faso) for comparison. The
analyses were performed in triplicate.
Proximate analysis
Moisture, protein, lipid and ash analyses were determined according
to AOAC official methods (AOAC, 2005). The tannins content
of the samples were estimated by the vanillin-HCl colorimetric
method as described by Addy et al. (1995). The tannins were
extracted with methanol (Merck, Darmstadt, Germany) and made to
react with the vanillin reagent (Merck, Darmstadt, Germany), to
produce a colour reaction, which was measured spectrophotometrically
at 500 nm. The calibration curve was established with
(+)-catechin (Merck, Darmstadt, Germany). For titratable acidity
(TA), 5 g of sample was mixed with 50 ml of distilled water and
mixed in a stomacher (Stomacher 400 Lab Blender, London,
England) at normal speed for 4 min. After filtration through a
Whatman paper filter, the TA was evaluated by titrating the filtrates
with 0.1 N sodium hydroxide (Merck, Darmstadt, Germany) using
phenolphthalein as the indicator (Jideani and Okeke, 1991).
Extraction and identification of the volatile compounds
Extraction of volatile compounds
The simultaneous distillation and extraction method described by
Nickerson and Likens (1966) was used for the investigation of the
volatile compounds of the different types of maari. The volatile
compounds of the samples were extracted using a microscale
steam distillation low density solvent extraction device (micro-SDE;
Chrompack, Middelburg, the Netherlands) as described by Annan
et al. (2003) and modified as follows: a known quantity of pounded
samples (according to their moisture content) was mixed in 400 ml
distilled water to obtain 2.5% slurries (on dry weight basis) of
samples (w/v). A 1 ml internal standard solution (2-methyl-1propanol)
was added to the sample slurry in a 1000 ml Erlenmeyer
flask. About 6 ml of a mixture of pentane and diethyl ether (1:1) was
placed in a 9 ml pear-shaped solvent flask. Both flasks were
connected to the distillation apparatus and the solutions were
brought to boil. Extraction of volatile compounds was carried out for
30 min, from the beginning of condensation of vapours on the walls
of the condenser. The organic solvent phase was collected and
stored at -20°C to freeze out any water present. The solvent extract
was poured off, dried over 2 g of anhydrous Na2SO4 and concentrated
to 100 mg by gently blowing N2 gas over the surface. The
concentrated extract was analyzed for volatile compounds using
gas chromatography and mass spectrometry (GC-MS). Extractions
were performed in triplicate.
Separation and identification of the volatile compounds by GC-
MS
Separation and identification of volatile compounds in the
concentrated extract was performed on a Varian CP 3900 GC/MS
equipped with Varian CP-8400 Autosampler (GC-MS; Varian, NA,
USA). Two microlitres of extract were injected (split ratio, 1:20) into
Varian factor four capillary column (VF-5ms) (30 m x 0.25 µm x
0.39 mm film thickness) using the temperature program: 10 min at
40°C, raised at 6°C min -1 to 240°C and then, maintained for 30 min
at 240°C. The carrier gas was helium with a constant flow rate of 1
ml min -1 .
The MS detector conditions were as follows; transfer line
temperature, 250°C; manifold temperature, 80°C; ion trap
temperature, 20°C; ionisation mode, EI auto; mass range, 25 to 550
m/z; multiplier delay, 1.77 min.
Identification of volatile compounds was determined in the total
ion mode scanning a mass to charge ratio (m/z) range between 25
and 550. Further identification was obtained by probability-based
matching with mass spectra in the NIST 05 mass spectral library
(Varian). Only compounds showing high quality index (degree of
agreement between mass spectrum of sample and mass spectrum
in database ≥ 90) were retained. Concentrations of volatile compounds
were estimated by comparing the relative peak areas of the
compounds with that of the 2-methyl-1-propanol internal standard
and reported in mg kg -1 based on the concentration of the internal
standard.
Statistical analysis
Differences between mean values were determined by analysis of
variance and Student-Newman-Keuls test using SAS statistical
software package (SAS, release, 8.1, Cary, N.C., USA).

RESULTS
As seen from Table 1, during fermentation, protein content
decreased from 156 ± 4.1 g kg -1 to values ranging
from 147 ± 0.7 to 134 ± 0.5 g kg -1 dry weight basis, while
lipid content increased from 122 ± 5.3 g kg -1 to values
ranging from 151 ± 3.8 to 181 ± 1.5 g kg -1 . Ash content
increased from 45 ± 0.4 up to 88 ± 3.6 g kg -1 . The
titratable acidity of unfermented baobab seeds had a
value of 0.17% OAE and increased to 1.87% in the
fermented product. The tannins content decreased from
0.29 mg g -1 CE to values between 0.25 and 0.09 mg g- 1
after fermentation. Changes resulting from the
fermentation were statistically significant (P < 0.05).
The results in Table 2 show that, a variety of volatile
compounds characterized the studied samples and a
total of 96 volatile compounds were found. They belonged
to 10 groups of compounds including acids (6),
alcohols (13), aldehydes (6), alkanes (2), alkenes (2),
esters (23), ketones (22), pyrazines (7), sulphur compounds
(4) and others (11). Esters, ketones, alcohols
groups constituted the groups with the largest number of
volatile compounds.
No volatile compounds belonging to the aldehydes,
alkenes and pyrazines group were found in the unfermented
cooked seeds (UCS). Fermentation led to an
increase in the concentration of total volatile compounds
from 121.6 mg kg -1 in an unfermented cooked seeds
(UCS) up to 809.1 mg kg -1 in the fermented product
(ZV1). In the fermented samples, the total volatile compounds
were 809.1, 582.9 and 636.8 mg kg -1 ,
respectively, for ZV1, ZC1 and ZB1; after the drying
process, they were 291.3, 204.03 and 123 mg kg -1 for
ZV1, ZC1 and ZB1, respectively. Drying process resulted
in an important loss up to 80.7% of the total volatiles
including principally esters, alcohols and acids groups.
The amount of the volatile compounds in the dried commercials
maari sample (DTA) was 352.9 mg kg -1 .
Qualitatively, only few volatile compounds including 2nonadecanone,
γ-dodecalactone, 2-decanone, 3-ethyl-2pentanone,
14-methyl pentadecanoate, di-tert-Butyl
dicarbonate, ethyl acetate, Z,Z,Z-4,6,9-nonadecatriene,
nonadecane, benzeneacetaldehyde and 2,4-dimethyl-3pentanol
were common in all dried fermented samples
from the three producers, showing a specificity of each
sample. Table 3 shows that, the relative amounts of the
compound groups varied widely among samples and
ranged from 0.0 to 65.4%. The major compounds group
found in unfermented cooked seeds was sulphur compounds
(26% of its total amount of volatiles). No
aldehydes, alkynes, furans, phenols and pyrazines compounds
were found in the unfermented cooked seeds. In
the fermented products, the major compounds found
generally were alcohols, aldehydes, alkanes, esters and
ketones groups. The sample ZB1 showed the highest
relative amount of esters (65.4%), ZV1 for ketones
(37.3%) and ZC1 for alcohols (26%), which were the
Parkouda et al. 4199
common components groups among all the samples. The
commercial samples DTA stands out significantly as the
sample richest in the content in pyrazines group (20%).
DISCUSSION
Production of maari resulted in a decrease of carbohydrates.
Decrease in carbohydrates has been reported
in previous studies on similar products like bikalga and
daddawa (Parkouda et al., 2008; Ibrahim and Antai,
1986). The decrease is likely to be explained by cooking
and loss of carbohydrate in the cooking water and by the
fermentation where carbohydrates are metabolised by
the microorganisms responsible for fermentation
(Parkouda et al., 2008). The formation of some volatile
aroma compounds belonging to ketones and aldehydes
groups which are generally formed via Maillard reactions
between saccharides and peptides may also play a role
(Ledauphin et al., 2003). The increase of the titratable
acidity observed is likely due to the accumulation of acid
from the hydrolytic activities of the amylolytic enzymes
(Schaffner and Beuchat, 1986) as well as the role of
microbial metabolism such as lactic acid bacteria and
Bacillus spp. during fermentation. The observed decrease
in tannins during fermentation is in line with previous
studies on baobab seeds (Addy et al., 1995; Nnam and
Obiakor, 2003). Hydrolysis of the polyphenolic compounds
to simpler substances by the enzyme polyphenol
oxidase or the breakdown of the tannin complexes for
example, tannin-protein, tannic acid-starch and tanniniron
complexes to release free nutrients could explain this
decrease (Obizoba and Atti, 1991). Chemical content,
including titratable acidity and tannins is expected to
contribute to the sensorial quality and volatile compounds
profiles of the different samples. For instance, free fatty
acids are known to contribute positively to the production
of characteristic flavours in food but high levels of fatty
acids may easily cause rancidity (Nawar, 1985).
In maari, as other fermented condiments, volatile aroma
compounds are important for quality and sensorial
attributes. A number of volatile compounds are present in
the unfermented baobab cooked seeds; others are
produced during fermentation. The volatile compounds
found in the unfermented cooked seeds can be explained
by a complex sequence of chemical reactions, for example,
strecker degradation, taking place as a function of
moisture, cooking temperature and duration (Rodríguez-
Bernaldo De Quirós et al., 2000). Esters group found in
the unfermented cooked seeds were also reported
previously in unfermented cooked Africa locust bean
(Azokpota et al., 2008; Ouoba et al., 2005). In contrast,
no ester was reported in unfermented cooked Africa
locust bean during controlled production (Azokpota et al.,
2010). The latter result can be explained by the severe
heat treatments applied to the locust beans, leading to
complete loss of esters due to thermal degradation

4200 Afr. J. Biotechnol.
Table 1. Chemical composition † of Unfermented and Fermented baobab seeds*.
Sample
Compound
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)
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
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
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
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
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.
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.
Table 2. Volatile compounds of cooked unfermented baobab seeds (UCS) and various samples of maari.
RT
Mean concentration (mg kg -1 Acid (6)
Volatile compound UCS
of product dry weight basis)
ZV 1 ZV2 ZC1 ZC 2 ZB 1 ZB 2 DTA
12.14 2,2-dimethyl propanoic acid 1.6 b - - - - 7.5 a - -
14.67 2-methyl-Propanoic acid - - - - 13 a 0.7 b - -
19.99 2-(aminooxy) pentanoic acid 1.3 b - - - - 4 a - -
22.7 Benzoic acid - - - - - 1.0 a 0.7 a -
38.78 Palmitic acid 2.9 f - 8.1 d 30.6 a 5.3 e 18.5 b - 14.7 c
41.58 Oleic acid - 44.3 a 2.7 e 6.5 d - 14.4 b 0.9 f 8.7 c
Total acids 5.83 44.34 10.78 37 18.35 46.18 1.6 23.39
Alcohol (13)
13.88 1-isopropoxy-2-methyl-2-Propanol 1 d 5.5 a - 4.4 b - 2.5 c - -
14.91 3,3-Dimethyl-2-pentanol 0.2 b - - - - 6.8 a 6.4 a -
18 2-methyl-3-hexanol 0.8 d 9.2 a 4.5 b - 1.5 c - - 1.1 c,d
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
22.22 2,4,4-trimethyl-1-pentanol 0.5 d 75 a 11.5 c - 19.7 b - - -
22.37 1,5-hexadien-3-ol 12 c - - 61.2 a - 48.1 b 13.7 c -
22.43 5-methyl-4-octanol - 12.5 a - 3.9 b - - - -
23.53 2-dodecanol 0.2 b - - - - 0.6 a - -
25.07 2-pentadecyn-1-ol - - - 31.6 a 1.1 b - - -
26.42 2,5-dimethyl-1,5-hexadiene-3,4-diol - - - - - 9.3 a 3.5 b -
26.43 2-(hydroxymethyl)-2-methyl-1,3-propanediol 1.8 a - - - - 1.7 a - -
30.93 1-hexadecanol - - - - - 1.2 a 1.0 b -

Table 2 cont.
Parkouda et al. 4201
45.11 2-hexyl-1-octanol 1.0 c 7.2 a - - - 1.5 b 0.8 c -
Total alcohols 21.04 195.19 34.88 151.28 36.53 75.67 29.62 12.45
Aldehyde (6)
6.69 3-Bromopropionaldehyde Ethylene acetal - - - - - 30.6 b 15.6 c 44.1 a
13.26 Heptanal - - - 4.8 a - - - 1.3 b
17.58 Octanal - - 1.6 d 21.3 a - 3.7 c 0.2 e 4.3 b
19.01 Benzeneacetaldehyde - - 22.6 a - 8.3 c 2.8 d 1.8 e 9.7 b
26.41 (E,E)-2,4-nonadienal - - 2.9 b 56.5 a 3.6 b - - -
26.42 2,4-Decadienal - - - 15.4 a - 1.2 c - 2.9 b
Total aldehydes 0 0 27.1 97.95 11.86 38.3 17.61 62.35
Alkane (2)
45.52 Nonadecane - - 1.1 c 16.1 a 1.4 b 1 c,d 1 d -
47.68 Tetratetracontane 0.9 d - - 8.3 a 0.8 e 1.7 b 0.6 f 1.3 c
Total alkanes 0.9 0 1.11 24.35 2.22 2.69 1.51 1.3
Alkenes(2)
20.85 Z-1,9-hexadecadiene - - - 5.3 a 4.2 b - 1.8 c 5.1 a
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
Total alkenes 0 0 9.7 10.08 5.06 0 3.7 6.55
Ester (23)
7.7 Methoxyacetic acid, pentyl ester 1.2 b - - - - - - 5.2 a
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
14.29 2-butanol, 2-nitrosoacetate 1.3 a - - - 0.8 b - - -
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
18.79 Vinyl butyrate 5.9 b - - 11.8 a 2.2 d 2.7 c 2.3 c 2.1 c,d
24.87 2-phenylethyl acetate - - 1.1 c 15.2 a 1.3 b - - -
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
38.11 14-methyl pentadecanoate - - 4.7 c 14.3 b 17.1 a - 2.2 d -
38.12 Methyl hexadecanoate 0.3 d 4.7 c - - - 12 b - 13.3 a
38.89 L-(+)-ascorbic acid 2,6-dihexadecanoate - 116.9 a - - - 64.1 b - -
39.22 Ethyl hexadecanoate 0.9 f 26 b 14.6 d 9.7 d 7.9 e 109.5 a - 15.3 c
40.36 Methyl 7,10-octadecadienoate - - - - - 4 a - 1.3 b
40.72 Propyl hexadecanoate - - 1.5 b - - 7.8 a - -
40.78 Methyl 9,12-octadecadienoate - 7 c 1.9 f 5.2 d 2.5 e 18.1 a - 7.7 b
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.
Table 2. cont.
41.36 9,12-octadecadienoate - - - - - 52.5 - -
41.83 Ethyl (Z,Z) 9,12-octadecadienoate - - 5.3 c - 2.3 d 33.1 a - 6.4 b
41.84 Ethyl linoleate 0.5 b 17.8 a - - - 0.2 c - -
41.87 Ethyl (E)-9-octadecenoate - - - 6.7 b - - - 11.1 a
41.9 Ethyl oleate 0.6 f 8 c 8.2 b 4.6 d 3 e 68.4 a - 8 b,c
42.21 Butyl hexadecanoate - 7.8 a - - - 0.7 b - -
44.06 Isopropyl linoleate - 8.9 a - - - 2.5 b - -
44.39 Decyl oleate - 11.8 a - - - 2.3 b - -
Total esters 20.47 267.46 47.79 121.98 48.13 416.31 11.51 84.19
Ketone (22)
8.23 3,4-dimethyl-2-pentanone 9.1 b - - - - 10.5 b 10.3 b 15.8 a
8.24 3-methyl-2,4-pentanedione - - 16.9 a - 15.3 a - - -
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
9.77 4-methyl-2-hexanone 0.4 e - 4.2 b 26.1 a 3.5 c 2 d - 3.5 c
14.31 3-methyl-2-hexanone 2.2 b 5.3 a - - 1.2 c 2.1 b - -
14.81 3-hydroxy-2-butanone 2.8 a - - - - - 2.6 b 3 a
15.94 2-chloro-3-methyl-1-phenyl-1-butanone - 26.7 a - - 7.8 b - - -
15.95 2-chloro-acetophenone - - 17.9 b - 0.6 c - 16.5 b 21.1 a
16.01 2,2-dihydroxy-1-phenyl-ethanone - - - - 1.7 b - - 7.4 a
18.78 3-Hydroxybutanone - 74.1 a - - - - - 12.3 b
20.48 2,5-dimethyl-4-hydroxy-3-hexanone 1.3 d 19 a - 8.2 b 4 c 1.2 d - -
23.24 2-decanone - 27.9 a 5.5 c 14.6 b 1.9 e 2.6 e 1 f 4.1 d
25.46 4,4,6-trimethyl-2-Cyclohexen-1-one - - - - 0.8 b - - 3.4 a
25.75 2-undecanone - - 3.3 a - - - 2.7 b -
26.88 3,5-dimethyl-2-octanone - 78.9 a 16.5 b 9.4 c 1 e 1.5 d - -
26.89 3-methyl-2-heptanone - - - - 2.4 a - 0.9 b -
28.05 2-dodecanone - 16.9 a - 2.6 b - 0.6 c - -
29.12 1-cyclododecyl-ethanone - 14.9 a - - - 0.4 c 1 b -
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 -
30.21 2-tridecanone - - 6.7 a - - 1.7 b 1.4 b -
33.91 Gamma. dodecalactone - 16.7 a 8.3 b - 1.6 c 0.8 d 0.6 d -
34.16 2-nonadecanone - - 5.6 b 11.1 a 1.2 d 1 d 0.9 d 4.1 c
Total ketones 20.12 302.1 91.55 101.49 46.91 32.65 39.67 78.91
Pyrazine (7)
17.65 Trimethyl-pyrazine - - - - - - - 2.4

Table 2 cont.
Parkouda et al. 4203
19.38 1-(2-tetrahydrofurylmethyl) pyrazine - - - - - 1.7 - -
20.32 Tetramethyl-pyrazine - - - - - - 56.9
21.79 2-isobutyl-3-methylpyrazine - - - - - 0.4 - -
22.32 2,3,5-trimethyl-6-ethylpyrazine - - - - - - - 9.9
25.36 2-(2-methylpropyl)-3,5,6-trimethylpyrazine - - - - - - - 1.4
27.59 2,6-Bis(2-methylpropyl)pyrazine - - - - - 2.2 a 2 a -
Total pyrazines 0 0 0 0 0 4.27 1.98 70.61
Sulphur compounds (4)
2.66 Thioacetic acid 29.3 b 52.3 a - - - - -
4.52 Disulfide, dimethyl - - - - 3.6 b 4.1 a - -
12.93 Diacetyl sulphide 0.8 b - - - - 4.3 a 0.5 c -
16.08 Dimethyl trisulfide 1.5 d - - 7.4 b 7.8 a 3.1 c 3.9 c 1.7 d
Others (11)
Total sulphur compounds 31.63 0 52.28 7.44 11.48 11.56 4.44 1.71
4.02 Tetrahydro-thiazole 20.3 a - - - - - 9.4 b -
11.79 d-Threo-O-ethylthreonine 1.1 b - - 5.5 a - - 0.8 b 1.6 b
16.92 Phenol - - 7.6 b - 9.3 a - - -
17 2-pentyl-furan - - - 4.5 a - 1.2 b - -
17.13 O-(3-methylbutyl)-hydroxylamine - - - - - 1.2 a - 1.4 a
18.19 1-undecyne - - 5.5 b 21.3 a 5.1 b 0.1 c - -
25.91 Indole - - 1.8 b - 9.1 a - - 1.5 c
26.25 2-methoxy-4-vinylphenol - - - - - 1.2 a - 1.5 a
35.73 Benzamide - - 1.2 b - - 1.9 a 1.2 a,b 1.6 a,b
35.93 O-decyl-hydroxylamine 0.2 b - - - - - - 1.5 a
37.88
7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-
Diene-2,8-dione
- - - - - 3.6 a - 2.3 a
Total others 21.64 0 16.14 31.32 23.49 9.15 11.35 11.41
Total all compounds (96) 121.63 809.09 291.33 582.89 204.03 636.78 122.99 352.87
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 <
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)
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,
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
Faso; RT, retention time.

4204 Afr. J. Biotechnol.
Table 3. Relative amounts of volatile compound groups identified in baobab unfermented cooked seeds and maari samples.
Aroma compound
group
Relative amount of the compound groups (%) a
UCS b ZV1 ZV2 ZC1 ZC2 ZB1 ZB2 DTA
Acids 4.8 5.5 3.7 6.3 9.0 7.3 1.3 6.6
Alcohols 17.3 24.1 12.0 26.0 17.9 11.9 24.1 3.5
Aldehydes 0.0 0.0 9.3 16.8 5.8 6.0 14.3 17.7
Alkanes 0.7 0.0 0.4 4.2 1.1 0.4 1.2 0.4
Alkenes 0.0 0.0 3.3 1.7 2.5 0.0 3.0 1.9
Esters 16.8 33.1 16.4 20.9 23.6 65.4 9.4 23.9
Ketones 16.5 37.3 31.4 17.4 23.0 5.1 32.3 22.4
Pyrazines 0.0 0.0 0.0 0.0 0.0 0.7 1.6 20.0
Sulfur Compounds 26.0 0.0 17.9 1.3 5.6 1.8 3.6 0.5
Others 17.8 0.0 5.5 5.4 11.5 1.4 9.2 3.2
a % Concentration of volatile compounds relative to the total concentration of volatile compounds identified; b unfermented cooked
baobab seeds– group not detected; ZV1 and ZV2, maari samples from the producer V, collected respectively at the end of the
fermentation (before the drying process) 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, 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 Faso.
(Rodríguez-Bernaldo De Quirós et al., 2000).
This study showed that, the fermentation of the baobab
seeds for maari production led to an increase of volatile
compounds. Increase of volatile compounds in other
fermented condiments during fermentation was also
reported in previous studies (Azokpota et al., 2008;
Ouoba et al., 2005; Owens et al., 1997). The formation of
volatile compounds during fermentation observed is
obviously due to the microorganisms involved in
fermentation as well as the processing factors such as
boiling or roasting (Azokpota et al., 2010; Dakwa et al.,
2005; Ouoba et al., 2005; Yuliana and Garcia, 2009).
Indeed, difference between the volatile compounds
profiles of soumbala samples was reported to rely on the
variable ability of the species of microorganism to
degrade the proteins, lipids and carbohydrates of African
locust beans (Ouoba et al., 2005). Comparing volatile
compounds produced during spontaneous and controlled
fermentation of afitin, iru and sonru, a very pronounced
increase of the concentration of total volatile compounds
was reported during the use of Bacillus subtilis as starter
culture (Azokpota et al., 2010). This increase was
explained by the use of pure culture (Azokpota et al.,
2010; Leejeerajumnean et al., 2001). Investigating on
processing factors, Dakwa et al. (2005) found pretreatment
of soybeans by either boiling or roasting before
fermentation to generate different volatile compounds
profiles of soy-dawadawa.
The amount of the total volatile compounds decreased
during the drying process resulting in a loss of up to
80.7% of the total aroma volatile compounds. A previous
study on Bacillus fermented soybeans also reported a
loss of aroma compounds during drying
(Leejeerajumnean et al., 2001). This loss is attributable to
most of the compounds simply evaporating during the
open-air drying process. On the other hand, other volatile
components like 2-undecanone, 2-chloro-acetophenone
and 3-methyl-2-heptanone appeared in the dried maari
and a few volatile compounds like dimethyl trisulfide were
higher in the dried maari than in the fermented non-dried
maari. These changes could probably be related to
variable factors such as enzymatic activities, Maillard
reaction, strecker degradation or microbial metabolism
that could continue to occur during the drying process.
Overall, the volatile compounds reported in this study
are highly variable and confirmed the non-standard
production practices among producers. The present
results support earlier studies that show the diversity of
aroma volatile compounds in fermented food condiments
such as Beninese afitin, iru and sonru, Burkinabe soumbala,
Ghanaian soy-dawadawa, Japanese natto, Korean
chungkuk-jang and Thai thua-nao (Azokpota et al., 2010;
Azokpota et al., 2008; Dakwa et al., 2005;
Leejeerajumnean et al., 2001; Ouoba et al., 2005;
Tanaka et al., 1998). For soumbala, Ouoba et al. (2005)
revealed the presence of pyrazines, aldehydes, ketones,
esters, alcohols, acids, alkanes, alkenes, benzenes, phenols,
sulphurs, furans pyridines and amines. Amines and
pyridines were not found in afitin, iru and sonru (Azokpota
et al., 2008). In addition to the chemical groups found in
soumbala, (excluding pyridines) in this study, the
presence of alkynes compounds was reported. In natto
and thua-nao, ketones, acids and pyrazines were reported
to be the major volatile compounds (Owens et al.,
1997). Ester, mainly formed by esterification of carboxylic
acids and alcohols was reported to determine the
characteristic pleasant aromatic notes (Klesk and Qian,
2003; Rodríguez-Bernaldo De Quirós et al., 2000).
According to this study, maari shows a low level of
pyrazines contrary to what has been reported for the

African locust bean fermented condiments such as afitin,
iru, sonru and soumbala (Azokpota et al., 2008; Ouoba et
al., 2005). Similar to our results, low presence of
pyrazines was also recorded in soy-dawadawa obtained
by boiled soybeans compared with roasted soybeans
(Dakwa et al., 2005). The formation of pyrazines is
generally associated with heating (Owens et al., 1997).
Shu (1998) revealed that, pyrazines are characterized by
their extremely high volatility and reactivity. Due to the
physical proprieties of baobab seeds, the seeds are
intensively cooked for about 36 h prior to the fermentation;
this may be explained as the difference observed
in the pyrazines content in the present study. The
presence of arginine was also reported to decrease the
yield of the pyrazines production (Hwang et al., 1995). In
others studies, arginine content was recorded to be
higher in baobab seeds (2.21 mg g -1 ) than in African
locust bean (0 mg g -1 ), (Konlani et al., 1999; Glew et al.,
1997) which could also explain the low content of
pyrazines in fermented baobab seeds.
Diversity of the volatile components from each typical
sample was observed. This difference may be explained
by the specificity of each typical product giving specific
compounds which are presumably more critical for the
sensory characteristics as perceived (and probably
expected) by consumers in tasting the product. In
previous studies, the use of pure culture of microorganisms
led to uniform final fermented product
preferred by a panel of consumers (Ouoba et al., 2005;
Azokpota et al., 2010). This study is one of the steps
which could give information for a future selection of
starter culture to be used in controlled fermentation.
ACKNOWLEDGEMENTS
This work was supported by the Danida (Danish
International Development Agency) funded project titled
“Improving food potential in West African parkland trees”.
Special thanks to the women who helped us with the
maari processing during the study.
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African Journal of Biotechnology Vol. 10(20), pp. 4207-4211, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2476
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Antioxidant activities of Rosmarinus officinalis L.
essential oil obtained by hydro-distillation and solvent
free microwave extraction
Okoh O. O. 1 , Sadimenko A. P. 1 and Afolayan A. J. 2 *
1 Department of Chemistry, University of Fort Hare Alice, 5700, South Africa.
2 Department of Botany, University of Fort Hare Alice, 5700, South Africa.
Accepted March 3, 2011
The essential oils of Rosmarinus officinalis L. growing in a rural area within the Nkonkobe Municipality
of the Eastern Cape, South Africa, were extracted using the solvent free microwave extractor (SFME)
and hydro-distillation (HD) methods. The antioxidant and free radical scavenging activity of the
obtained oils were tested by means of 1,1-diphenyl-2-picrylhydrazyl radical (DPPH + ) assay and βcarotene
bleaching test. In the DPPH + assay, while the free radical scavenging activity of the oil
obtained by SFME method showed percentage inhibitions of 48.80, 61.60 and 67.00%, the HD oil
showed inhibitions of 52.20, 55.00 and 65.30% at 0.33, 0.5 and 1.00 mg/ml, respectively. In the βcarotene
bleaching assay, the percentage inhibition increased with increasing concentration of both
oils, with a high antioxidant activity of the oil obtained through the SFME than through the HD method.
The significance of this observation is discussed with respect to the properties of essential oils
obtained using different methods.
Key words: Essential oil, antioxidant, hydrodistillation, solvent free microwave extraction; Rosmarinus
officinalis.
INTRODUCTION
Plants, especially herbs and spices, have many phytochemicals
which are potential sources of natural antioxidants.
These include phenolic diterpenes, flavonoids,
tannins and phenolic acids (Dawidowicz et al., 2006).
Some antioxidants have been widely used as food
additives to protect food against oxidative degradation by
free radicals. Spices which are used in different types of
food to improve flavors are well known for their
antioxidant properties (Madsen and Bertelsen, 1995).
Recently, there has been increasing interest in the use of
natural antioxidants such as tocopherols, flavonoids and
extracts from rosemary (Rosmarinus officinalis L.) for
food preservation (Hras et al., 2000; Bruni et al., 2004;
Williams et al., 2004; Fruitos and Hernandez-Herrero,
2005). According to these authors, these natural
*Corresponding author. E-mail: aafolayan@ufh.ac.za. Fax:
+27866282295.
antioxidants do not present health problems that may
arise from the use of synthetic antioxidants such as
butylated hydroxy anisole (BHA), butylated hydroxy
toluene (BHT) and propyl gallate (PG) which have side
effects (Amarowicz et al., 2000; Aruoma et al., 1992).
Antioxidants are compounds that neutralize chemically
active products of metabolism, such as free radicals
which can damage the body. It has been documented
that plant phenols, with their potential to act as
antioxidants, play a major role in the prevention of
cancer, cardiovascular and neurogenerative diseases
which are believed to be caused by oxidative stress
(Losso et al., 2007).
R. officinalis L. is a perennial herb with ever-green
needle like leaves that belongs to the Lamiaceae family.
The Lamiaceae is a large family, rich in aromatic species
that are used as culinary herbs, in folk medicines and
fragrances, and many members of this family possess
essential oils that are secreted by glandular trichomes
(Marin et al., 2006). Previous studies have shown that rose-

4208 Afr. J. Biotechnol.
mary essential oil have antimicrobial, antioxidant, anticarcinogenic,
cognition-improving and certain glucose
level lowering properties which make it useful as a
natural animal feed additive (Fahim et al., 1999;
Debersac et al., 2001; Fu et al., 2007).
Whilst the general antioxidant potential of R. officinalis
essential oil have been reported before (Gachkar et al.,
2007), there is no information on the possible effect of the
method of extraction of the essential oils on the
antioxidant property of this herb. The biological and
phytochemical properties of essential oils extracted
through different methods have been found to depend on
the extraction method (Okoh et al., 2010). In this paper,
we report the effects of solvent free microwave extraction
and hydro-distillation extraction methods on the
antioxidative properties of the essential oils of R.
officinalis. The effects of these two extraction methods on
the chemical compositions and antimicrobial activities of
the oils have been previously reported in our laboratory
(Okoh et al., 2010).
MATERIALS AND METHODS
All solvents and reagents used in these experiments such as
methanol, chloroform and ethanol were of analytical grade and
together with 2,2-diphenyl-2-picylhydrazyl hydrate (DPPH), 3,5-ditert-butyl-4-hydroxytoluene
(BHT) -tocopherol, gallic acid (GA),
rutin, β-carotene, linoleic acid and Tween 40, were all purchased
from Sigma Aldrich (Pty) Ltd- Johannesburg, South Africa.
Collection of plant material and extraction of the essential oil
Collection, processing of the plant materials and extraction of the
essential oils using SFME and HD methods were in accordance
with our previous report (Okoh et al., 2010). Briefly, two samples of
fresh leaves (250 g each) of R. officinalis were collected in
February, 2009 from the vicinity of the University of Fort Hare, Alice
campus in the Eastern Cape Province of South Africa (latitudes
30°00-34°15S and longitudes 22°45-30°15E). The plant was
identified by Prof Don Grierson of the Botany Department,
University of Fort Hare, Alice and a voucher specimen (Okoh/10)
was deposited at the University herbarium.
SFME was carried out with a Milestone DryDIST (2004)
apparatus. The multimode reactor has a twin magnetron (2 × 800
W, 2450 MHz) with a maximum delivered power of 500 W in 5 W
increments. A rotating microwave diffuser ensures homogeneous
microwave distribution throughout the plasma coated PTFE cavity.
The temperature was monitored by an external IR sensor. Constant
conditions of temperature and water were guaranteed by the reflux
of condensed water, which was achieved by a circulating cooling
system at 5°C. Two hundred and fifty grams (250 g) of the plant
leaves were placed into the reactor without addition of water or any
solvent. The exhaustive extraction of the essential oil was obtained
in 40 min.
For the HD, 250 g of the plant leaves were hydrodistilled for 3 h
in an all-glass Clevenger apparatus in accordance with the
description of the British Pharmacopoeia (1980). Heat was supplied
to the heating mantle (50°C) and the essential oil was extracted
with 4 L of water for 3 h (until no more essential oil was released).
The oils collected from both extraction methods were analyzed
immediately after each collection using GC-MS. The compositions
of these oils have been previously reported (Okoh et al., 2010).
Antioxidant activity
DPPH radical scavenging activity
The method of Liyana-Pathiranan and Shahidi (2005) was used for
the determination of scavenging activity of DPPH free radical in the
essential oils. A solution of 0.135 mM DPPH in methanol was
prepared and 1.0 ml of this solution was mixed with 1.0 ml of oil
prepared in methanol containing 0.025 to 0.5 mg of the essential oil
and standard drugs (BHT and rutin). The reaction mixture was
vortexed thoroughly and left in the dark at room temperature for 30
min. The absorbance of the mixture was measured spectro-photometrically
at 517 nm. The ability of the plant essential oils to
scavenge DPPH radical was calculated by the following equation:
DPPH radical scavenging activity = {(Abscontrol – Abssample)} / (Abs
control} × 100
where, Abscontrol is the absorbance of DPPH radical + methanol,
Abssample is the absorbance of DPPH radical + sample oil /standard.
β- carotene bleaching assay
This method evaluates the capacity of the oil to reduce the
oxidative loss of β-carotene in a β-carotene linoleic acid emulsion
(Taga et al., 1984). β-Carotene (10 mg) was dissolved in 10 ml of
chloroform (CHCl3). An aliquot (0.2 ml) of this solution was added
into a boiling flask containing 20 mg of linoleic acid and 200 mg of
Tween 40. The chloroform was removed using a rotary evaporator
at 40°C for 5 min. Distilled water (50 ml) was slowly added to the
residue with vigorous agitation, to form an emulsion. The emulsion
was added to a tube containing 0.2 ml of essential oil. The
absorbance was immediately measured at 470 nm and the test
emulsion was incubated in a water bath at 50°C for 5 min, after
which the absorbance was measured again. BHT was used as the
positive control. In the negative control, the essential oils were
substituted with an equal volume of ethanol. The antioxidant activity
(%) of the oil was evaluated in terms of the bleaching of the βcarotene
using the following formula:
% inhibition = {(At-Ct) / (CO-Ct)} × 100
where, At and Ct are the absorbances measured for the oil and
control, respectively, after incubation for 5 min, and CO is the
absorbance values for the control measured at zero time during the
incubation.
The oil concentration providing 50% antioxidant activity (EC50)
was calculated by plotting antioxidant percentage against oil
concentration.
RESULTS AND DISCUSSION
The principle of antioxidant activity is based on the
availability of electrons to neutralize free radicals. In this
study, the antioxidant activity of R. officinalis oil was
evaluated by two complementary tests: scavenging of
DPPH + free radicals and the β-carotene bleaching test.
The results are as shown in Figures 1 and 2, respectively.
The free radical scavenging activity of essential oil of R.
officinalis obtained by SFME revealed percentage
inhibitions of 48.80 , 61.60 and 67.00%, while that of the
HD oil showed percentage inhibitions of 52.20, 55.00 and

Inhibition of beta carotene (%)
Inhibition of DPPH + (%)
65.30% both at concentrations of 0.33 , 0.5 and 1.0
mg/ml, respectively. These results show that activity
increases as the concentrations of the oils were
increased, at least within the limit of the test concentrations
of the oils (Figure 1). The SFME essential oil
showed a slightly higher DPPH-radical scavenging
activity (IC50 = 0.34 mg/ml) than the hydrodistilled
essential oil (IC50 = 0.46 mg/ml), whereas for BHT, IC50
Figure 1. Free radical-scavenging activity of R. officinalis
essential oil evaluated by the 1,1-diphenyl-2-picrylhydrazyl
(DPPH).
Figure 2. Antioxidant activity of R. officinalis L. essential oil
determined by β-carotene bleaching test.
Okoh et al. 4209
was 0.22 mg/ml at concentration of 0.33 mg/ml.
The result of lipid peroxidation inhibitory activity of the
essentials oils, assessed by the β-carotene bleaching test
are shown in Figure 2. β-Carotene usually undergoes
rapid discoloration in the absence of an antioxidant. This
is because the oxidation of β-carotene and linoleic acid
generates free radicals (Jayaprakasha et al., 2001). The
linoleic acid free radical formed upon the abstraction of a

4210 Afr. J. Biotechnol.
hydrogen atom from one of its diallylic methylene groups
attacks the highly unsaturated β-carotene molecule,
hence β-carotene is oxidized, losing its orange color
which is then monitored spectrophotometrically (Shon et
al., 2003). The results obtained from this assay are
similar to the data obtained from DPPH test. The
percentage inhibitions were 48.62, 68.06 and 86.79% for
hydrodistilled oil; and 35.87, 76.29 and 91.19% for
solvent free microwave extracted oil at concentrations of
0.33, 0.5 and 1.0 mg/ml, respectively. Although, both oils
prevented the bleaching of β-carotene, the SFME
extracted oil had a slightly higher activity than the HD
extracted oil. These results are also consistent with the
results obtained from DPPH test. The concentrations
providing 50% inhibition were 0.338, 0.282, and 0.22
mg/ml for HD, SFME and BHT, respectively. It was noted
that the antioxidant activities of the tested samples were
dependent on their concentrations. The IC50 of SFME was
however lower than that of HD essential oil.
In plant essential oils, oxygenated monoterpenes and
monoterpene hydrocarbons are mainly responsible for
the antioxidant potential (Ruberto and Baratta, 2000), and
in our previous findings (Okoh et al., 2010) on the
chemical constituents component of this study, we
reported that oxygenated monoterpenes and monoterpene
hydrocarbons were the main components of R.
officinalis essential oil.
Many reports on the investigations of the activity of R.
officinalis have shown that there are biologically active
compounds in R. officinalis essential oil that exhibit
cytotoxic, antioxidant, anti-carcinogenic and cognitionenhancing
properties (Okamura et al., 1994; Frankel et
al., 1996; Thorsen and Hildebrandt, 2003). These
compounds that have the potential to influence glucose
level in diabetic patients, modify rumen microbial
fermentation and enhance bone desorption, but do not
enhance immune response (Oluwatuyi et al. 2004; Wang
et al., 2008). Essential oils, despite their wide uses and
fragrances, constitute effective alternatives to synthetic
compounds produced by chemical industry without
showing the same side effects as the latter (Faixova and
Faix, 2008). In this study, both SFME and HD extracted
oil exhibited remarkable antioxidant activities. In general,
the essential oil obtained from SFME showed greater
activity than the one obtained from HD.
It is very difficult to attribute the antioxidant effect of a
total essential oil to one or a few active principles
because an essential oil always contains a mixture of
different chemical compounds. In addition to the major
compounds, minor molecules may make significant
contributions to the oil activity. Therefore, the antioxidant
property of the oils from this herb might be the combined
activities of the various major and minor components of
the oils.
In conclusion, the results of this study showed some
differences in the antioxidant activities and chemical
composition of the essential oils obtained by SFME and
HD extraction methods. The SFME extracted oil showed
a higher activity than that obtained by HD. This is probably
due to the higher proportions of oxygenated compounds
in SFME extracted oils. According to Okoh et al. (2010),
higher amounts of oxygenated compounds with lower
amounts of monoterpene hydrocarbons were present in
the essential oils of this plant isolated by SFME in
comparison with the oil obtained by traditional hydrodistillation.
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African Journal of Biotechnology Vol. 10(20), pp. 4212-4216, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2656
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Effects of bagging on sugar metabolism and the activity
of sugar metabolism related enzymes during fruit
development of Qingzhong loquat
Zhaojun Ni 1 , Zhen Zhang 1 , Zhihong Gao 1 *, Linping Gu 2 and Leifang Huang 2
1 College of Horticulture, Nanjing Agricultural University, Nanjing, Jiangsu, People’s Republic of China.
2 Service Centre of Agriculture and Forestry, Suzhou, Jiangsu, People’s Republic of China.
Accepted 17 March, 2011
To investigate the effects of bagging on sugar metabolism and the activity of sugar metabolism related
enzymes in Qingzhong loquat fruit development, the contents of sucrose, glucose and soluble solids as
well as the activities of sugar metabolism related enzymes were evaluated. The content of sucrose,
glucose and soluble solids increased, while the content of fructose, sorbitol and titratable acidity
decreased in ripe fruit in response to bagging. In addition, the activities of acid invertase (AI) and
neutral invertase (NI) in the bagged fruit were lower than that in the non-bagged fruit, and the activities
of sucrose synthase (SS) and sucrose-phosphate synthase (SPS) in the bagged fruit were higher than
that in the non-bagged fruit. The activities of SDH (sorbitol dehydrogenase) and SOX (sorbitol oxidase)
in the bagged fruit were lower than those in the non-bagged fruit, but there was no significant
difference, whereas the activities of SS and SPS in the bagged fruit were significantly higher than that
in the non-bagged fruit, suggesting that bagging mainly increased the products of photosynthesis by
enhancing the activities of SS and SPS.
Key words: Loquat, bagging, sugar metabolism.
INTRODUCTION
Bagging is an effective method of improving fruit quality
in fruit production and has been widely used to improve
fruit appearance, decrease pesticide residues and
increase commercial value. Moreover, bagged fruit is
preferred by the consumers. Yang et al. (2009) proved
that bagging could modify the microenvironment during
fruit development, decreasing the rate of fruit drop and
reducing the content of organic acid in longan fruit. Kim et
al. (2008) reported that the appearance of bagged
peaches improved, while the content of soluble solids,
anthocyanin and chlorophyll increased in white coloured
*Corresponding author. E-mail: gaozhihong@njau.edu.cn. Tel:
+86 2584395724. Fax: +86 2584395266.
Abbreviations: AI, Acid invertase; NI, neutral invertase; SS,
sucrose synthase; SPS, sucrose-phosphate synthase; SDH,
sorbitol dehydrogenase; SOX, sorbitol oxidase.
bags. Lin et al. (2008) demonstrated that bagged pear
fruits were more attractive with less russet and visible
dots than non-bagged fruit, while the residues of
chlorpyrifos carbendazim and cyhalothrin were also greatly
reduced. Consumers also showed preference for bagged
fruit.
Loquat (Eriobotrya japonica Lindl.) originated in China,
is distributed in Zhejiang, Fujian and Jiangsu Province
(Qiu and Zhang, 1996). ‘Qingzhong’ loquat is a local
cultivar from Suzhou, Jiangsu Province. The fruit has
good flavour and storage ability. Xu et al. (2008, 2010)
and Feng et al. (2009) studied the effects of bagging on
‘Baiyu’ loquat and ‘Ninghaibai’ loquat fruit quality and
antioxidative capacity. However, the effects of bagging on
sugar metabolism during fruit development were not
explored. The purpose of this study was to determine the
effects of bagging on sugar metabolism and the activities
of related enzymes during loquat fruit development, and
establish a basis for the improvement of this cultivation
technique.

MATERIALS AND METHODS
Materials
Thirty-year-old ‘Qingzhong’ loquat trees grown at Suzhou, Jiangsu
Province were used in this project. The fruits were bagged (white,
single-layer sulphuric acid paper) about 40 days after initial fruit set,
with bagging 80 fruits per plant. Ten fruits were sampled every 15
days until harvest. Sampling was repeated three times. Flesh was
separated from the fresh fruit for each treatment and frozen at -
70°C for further study.
Sugar content
Dried fruit (0.1 to 0.2 g) was powdered in a chilled mortar with 5 ml
of distilled water. The homogenate was centrifuged at 5000 ×g for
10 min. The supernatant was poured into a Sephadex G-25 column
and re-extracted once. The supernatants were combined and the
volume was increased to 25 ml. The contents of sucrose, glucose
and fructose were determined by the anthrone colorimetric method.
Sampling was repeated three times.
Frozen fruit was powdered in a mortar chilled with liquid nitrogen.
Two grams of fruit were added to 7 ml 70% ethanol and mixed into
a homogenate for about 20 min in 35°C water. Then, the
homogenate was centrifuged at 10, 000 ×g for 5 min. The
supernatant was poured into a Sephadex G-25 and re-extracted
once. The supernatants were combined and the volume increased
to 25 ml. A 2 ml sample of supernatant was evaporated to dryness
and dissolved with 1 ml of distilled water. The content of sorbitol
was quantified by HPLC (high performance liquid chromatography,
Agilent 1100). A 15 µl aliquot was passed through a CA column (10
µm diameter). Flowing water was used for the mobile phase. The
temperature of the column was 85°C. Sampling was repeated three
times.
Enzyme activity
Extraction and determination of acid invertase (AI), neutral
invertase (NI), sucrose synthase (SS) and sucrose-phosphate
synthase (SPS) were determined by the method described by
Nielsen et al. (1991). Frozen fruit was powdered in a mortar chilled
with liquid nitrogen. Fruit (1 g) was added to 5 ml of pre-cooled
extraction buffer (0.2 M Tris-Hcl buffer, pH = 8.8) containing 25 mM
MgCl2 and 15 mM NaN3, and made into a homogenate in an icebath.
Then, the homogenate was centrifuged at 10,000 ×g for 15
min. The supernatant was poured into a Sephadex G-10 and reextracted
once. The supernatants were combined and the volume
was increased to 10 ml, which was used as the enzyme extract.
Sampling was repeated three times.
The activity of sorbitol dehydrogenase (SDH) was determined by
the following method: Enzyme solution (150 l) and 1.05 ml of 25
mM NAD + were combined, to which 1.05 ml of 150 mM D-sorbitol
was added. The absorbance was determined at 340 nm. In the
enzyme-free reaction system, 150 l of 0.2 M Tris-HCl (pH = 8.8)
was used as the control instead of enzyme solution. The treatment
was repeated three times. The units (OD • mL –1 • min –1 ) of SDH
activity was the change of absorbance of 1 ml reaction solution per
minute. The activity of SOX (sorbitol oxidase) was determined by
the method of Yamaki (1980) and Yamaki and Ishikawa (1986).
Statistical analysis
Statistical analysis was carried out using analysis of variance and
the significance was determined using SPSS 16.0 (Statistical
package for the social science) with LSD (least significant
difference) values at P < 0.05.
RESULTS
Effects of bagging on the content of sugar
Ni et al. 4213
The glucose content in ripe fruit increased in response to
bagging (P < 0.05), while the levels of sorbitol and titratable
acid decreased (Table 1). Soluble solid to acidity ratio
increased in bagged fruit. The contents of sucrose,
fructose and glucose in the bagged and non-bagged fruit
increased with fruit development (Figure 1 A, B and C).
The content of fructose in the bagged fruit was slightly
lower than that in the non-bagged fruit before 70 days
after initial fruit set and decreased by 2.01 mg·g -1 in ripe
fruit when compared with control. The amount of sucrose
and glucose in the bagged fruit were slightly lower than
those in the non-bagged fruit before 85 days after initial
fruit set, after which the amounts were higher for bagged
than non-bagged fruit. The contents of sucrose and
glucose were higher in bagged than in non-bagged fruits
by 1.30 mg·g -1 and 2.75 mg·g -1 in ripe fruit, respectively.
The accumulation of sorbitol in the bagged fruit was
very similar to that in non-bagged fruit during fruit development,
decreasing at first and then increased during
development. The content of sorbitol in the bagged fruit
was lower than that found in the non-bagged fruit (Figure
1D).
Effects of bagging on the activities of sucrose
metabolism related enzymes
Invertase belongs to an enzyme class in which the
regulation of sucrose conversion into fructose and
glucose in the plant is mediated by NI and AI (Ni et al.,
2009). The activities of NI and AI in the bagged fruit were
quite similar during fruit development (Figure 2A and B).
The activities of NI and AI in the non-bagged fruit initially
increased faster and then more slowly until the fruit
ripened. The activity of AI was slightly higher in the
bagged fruit than in the non-bagged fruit when fruit
ripening approached.
SS is an enzyme that plays a key role in sucrose
decomposition in the course of sucrose metabolism and
SPS is a key enzyme that regulates sucrose synthesis in
the plant (Zheng et al., 2006). The activity of SS in the
bagged fruit increased during fruit development and was
higher than that in the non-bagged fruit (Figure 2C). The
change in SS and SPS activities in the bagged and nonbagged
fruit was almost the same before 85 days after
the initial fruit set. After this date, the activities of SS and
SPS in the bagged fruit increased more rapidly (Figure
2D).
Effects of bagging on the activities of sorbitol
metabolism related enzymes
SDH is an enzyme that catalyses the conversion of sorbitol

4214 Afr. J. Biotechnol.
Table 1. Effects of bagging on the contents of sugar, sucrose, fructose, glucose, sorbitol, soluble solids and titratable acidity in fruit of
Qingzhong loquat.
Treatment
Content
of sugar
(mg·g – 1 )
Content of
sucrose
(mg·g – 1 )
Content of
fructose
(mg·g – 1 )
Content of
glucose
(mg·g – 1 )
Content of
sorbitol
(mg·g – 1 )
Soluble
solid
(%)
Titratable
acidity
(%)
Solid/acid
ratio
Control 60.04 b 21.62 a 24.46 a 7.55 b 6.41 a 14.35 a 0.45 a 31.89
Bagged 57.95 a 22.92 a 22.45 a 10.3 a 2.28 b 15.13 a 0.35 b 43.23
The different letters indicate significant difference at 0.05 level; sugar = sucrose + fructose + glucose + sorbitol. Solid/acid ratio represents the
ratio of soluble solid to titratable acidity.
Content of sucrose(mg.g-1)
Content of glucose(mg.g-1)
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Days after initial fruit set
C
A
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Days after initial fruit set
Content of fructose(mg.g-1)
Content of sorbitol(mg.g-1)
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©
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Days after initial fruit set
D
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�¤�
Days after initial fruit set
Figure 1. Effects of bagging on the content of (A) sucrose, (B) fructose, (C) glucose and (D) sorbitol in fruit
of Qingzhong loquat.
to fructose and SOX is an enzyme that catalyses the
conversion of sorbitol to glucose (Liang et al., 2004). The
activities of SDH and SOX in the bagged and nonbagged
fruit were quite similar during fruit development
and decreased before 85 days after initial fruit set before
increasing slowly until the fruit ripened (Figure 3A and B).
The correlation between sugar accumulation and
activities of sucrose and sorbitol-metabolizing
enzymes during fruit development
Sucrose content displayed a very significant positive
correlation with the activities of SS and SPS during
bagged fruit development, while glucose content also
positively correlated very significantly with the activity of
sucrose-metabolizing enzymes. Sorbitol content also
exhibited a significant positive correlation with the activity
of SDH. The content of sucrose, fructose, glucose and
the activity of sucrose-metabolizing enzymes during nonbagged
fruit development showed a very significant
positive correlation, suggesting that bagging affects the
accumulation of fructose and the activity of sucrosemetabolizing
enzymes (Table 2).
DISCUSSION
The results demonstrated that average fruit weight in the
bagged fruit was lower than that in non-bagged fruit. The

Activity of neutral invertase
(mg.g-1FW.h-1)
Activity of sucrose synthase
(mg.g-1 FW.h-1)
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A
control � bagged fruit
�
control � bagged fruit
�
��� � � � � ���
���
Days after initial fruit set
��� � � � � ���
���
Days after initial fruit set
Activity of acid invertase
(mg.g-1FW.h-1)
Acticity of sucrose phosphate
synthase
(mg/g.h FW)
C D
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B
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Days after initial fruit set
��� � � � � � �
���
Days after initial fruit set
Figure 2. Effects of bagging on the activities of (A) neutral invertase, (B) acid invertase, (C) sucrose synthase and (D)
sucrose-phosphate synthase in fruit of Qingzhong loquat.
Activity of sorbitol dehydrogenase
(OD.ml-1.min-1FW)
�����
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�����
�
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�
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Days after initial fruit set
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A B
Activity of sorbitol oxidase
(mg.g-1FW.h-1)
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Days after initial fruit set
Ni et al. 4215
Figure 3. Effects of bagging on the activities of (A) sorbitol dehydrogenase and (B) sorbitol oxidase in fruit of Qingzhong loquat.
trend in sucrose, fructose, glucose and sorbitol accumulation
were quite similar but the rates of accumulation
were not synchronous in the bagged and non-bagged
fruit during fruit development. The accumulation of
sucrose and glucose in the bagged fruit increased, while
the accumulation of fructose and sorbitol decreased. The
content of soluble solids in the bagged fruit increased and
the content of titratable acidity decreased when compared
with the non-bagged fruit. These results coincide with
what Xu et al. (2008, 2010) report on the ‘Baiyu’ loquat
treated with a white single-layer paper bag.
This study found that the content of sorbitol in
‘Qingzhong’ loquat mature leaf was 24.52 mg·g -1 , 3.83
times that of the mature fruit (data was not shown in the
results), which revealed that the content of sorbitol in the
mature fruit was much lower than that in the mature leaf.

4216 Afr. J. Biotechnol.
Table 2. The correlation between sugar accumulation and activities of sucrose-metabolizing enzyme and
sorbitol-metabolizing enzyme during fruit development.
Treatment Sugar content NI AI SS SPS SDH SOX
Sucrose 0.996** 0.990** 0.991** 0.986** — —
Control
Fructose
Glucose
0.988**
0.994**
0.978**
0.989**
0.977**
0.983**
0.970**
0.981**
—
—
—
—
Sorbitol — — — — 0.836 0.76
Bagging
Sucrose 0.981* 0.984* 0.994** 0.999** — —
Fructose 0.957* 0.958* 0.972* 0.984* — —
Glucose 0.993** 0.993** 0.997** 0.994** — —
Sorbitol — — — — 0.968* 0.558
**Correlation is significant at the 0.01 level (2-tailed); *correlation is significant at the 0.05 level (2-tailed); — correlation is
negative. AI, acid invertase; NI, neutral invertase; SS, sucrose synthase; SPS, sucrose-phosphate synthase; SDH,
sorbitol dehydrogenase; SOX, sorbitol oxidase.
However, the accumulation of sorbitol in bagged fruit was
lower than that of the non-bagged fruit. Ruan et al. (1997)
reported that the difference in sugar content in fruit was
independent of export from a source leaf but the transport
capacity of sugar was different in different kinds of fruit
trees, which suggested that the content of sorbitol
entering the fruit did not depend on the output capacity of
the leaf but on the gathering ability of the fruit. A possible
reason why the content of sorbitol in the bagged fruit was
lower than that in the non-bagged fruit is that bagging
might affect the ability to transport sorbitol from the
leaves to the fruits. Bagging influenced the activities of
sucrose and sorbitol metabolism related enzymes. The
activities of NI and AI were lower in bagged fruit than
those in the non-bagged fruit and were significantly
different before 85 days after initial fruit set. The activity
of NI was similar in the bagged and non-bagged fruit,
whereas the activity of AI was higher in the bagged than
in the non-bagged fruit until the fruit ripened. The
activities of SS and SPS were higher in the bagged than
in the non-bagged fruit, which suggested that bagging
mainly increased the activities of sucrose metabolism
related enzymes.
Sugar introduced into the fruit from the leaf depends on
the sink strength of the fruit to a large extent. An important
physiological marker that determines sink strength is
the activities of sucrose metabolism related enzymes
(Vizzotto et al., 1996). As photosynthesis in fruit decreased
after bagging (Xu et al., 2010), the activities of sugar
related enzymes may be required to increase the sink
strength of the fruit to obtain more products of photosynthesis.
This study suggests that bagging mainly
offsets the loss induced by lack of sunlight by increasing
the activities of SS and SPS.
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CM (2008). Effects of bagging on fruit quality and antioxidant capacity
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African Journal of Biotechnology Vol. 10(20), pp. 4217-4225, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2181
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Anti-inflammatory evaluation of immature fruit and seed
aqueous extracts from several populations of Tunisian
Citrullus colocynthis Schrad
Belsem Marzouk 1¥ *, Zohra Marzouk 2¥ , Ehsen Haloui 2 , Manel Turki 3 , Abderrahman Bouraoui 3 ,
Mahjoub Aouni 1 and Nadia Fenina 2
1 Laboratoire des Maladies Transmissibles et Substances Biologiquement Actives, Faculté de Pharmacie de Monastir,
Rue AVICENNE 5000 Monastir, Tunisie.
2 Unité de Pharmaco-économie et Développement des Médicaments, Laboratoires de Biologie Végétale et Laboratoire
de Pharmacologie, Faculté de Pharmacie de Monastir, Tunisie.
3 Unité de recherche URSAM, Laboratoire de Pharmacologie, Faculté de Pharmacie, Monastir, Tunisie.
Accepted 21 February, 2011
Plant extracts are some of the most attractive sources of new drugs and have shown promising results
for the treatment of inflammation and immune-related diseases, including rheumatoid arthritis. Citrullus
colocynthis Schrad. (Cucurbitaceae) endemic in Tunisia, is widely used in folk medicine to treat many
inflammation disorders. The aim of this study is to quantify the alkaloid and the flavonoid levels of
different populations of C. colocynthis fruit and seed aqueous extracts at immature state. After acute
toxicity assay, these extracts were screened for anti-inflammatory activity using the carrageenaninduced
paw edema assay in rats. Alkaloid and flavonoid levels vary among the population. The best
anti-inflammatory activities were obtained with immature fruits from south Tunisia. Therefore, C.
colocynthis Schrad. could be a potential useful product suitable for further evaluation for inflammatory
diseases.
Key words: Citrullus colocynthis Schrad., alkaloids, flavonoids, toxicity, anti-inflammatory, Tunisia.
INTRODUCTION
Since ancient times, several diseases have been treated
by administration of plant extracts. Interest in ethnopharmacy
as a source of active compounds has increased
worldwide, particularly for the development of antiinflammatory
drugs. Plants with analgesic and antiinflammatory
activities have become more interesting
because some of these plants are part of the arsenal of
*Corresponding author. E-mail: belsemmarzouk@yahoo.fr. Tel:
216 73 450 389.
Abbreviations: TOF, Total oligomeric flavonoids; TLC, thin
layer chromatography; ASL, acetyl salicylate of lysine.
¥ These authors contributed equally.
modern medicine and many people are aware of
problems associated with the over-prescription and
misuse of usual drugs.
Citrullus colocynthis Schrad. (Cucurbitaceae), growing
in Tunisia (Pottier-Alapetite, 1981), is widely used in
Tunisian folk medicine for treating many diseases such
as various contagious diseases, hypertension and
rheumatism (Le Flock, 1983; Boukef, 1986). Anti-inflammatory
traditional healers seem not to pay attention to the
fruit’s degree of maturity and literature rarely mention if
seeds are present in preparations involving ground
fruit/pulp. Common preparations use juice, fresh or dried
(often ground) fruit material. Extracts (maceration or
boiling) are prepared either in water or in aqueous
mixtures (honey, milk and water/olive oil at various ratios).
Methods of administration are by ingestion or massage
(Le Flock, 1983; Boukef, 1986; Bellakhdar, 1999; El-

4218 Afr. J. Biotechnol.
Ghadi and Bshana, 1988).
Some studies have demonstrated the medicinal effect
of C. colocynthis Schrad. as anti-tumour (Tannin-Spitz et
al., 2007), immunostimulant (Bendjeddou et al., 2003),
anti-microbial (Marzouk et al., 2009, 2010a) and antioxidant
(Marzouk et al., 2010b), and against hepatic diseases
(Gebhardt, 2003), hyperglycaemia (Al-Gaithi et al., 2004)
and hair loss (Roy et al., 2007).
However, plants in the environment are exposed to a
range of abiotic stresses like osmotic, salinity, temperature
and heavy metal toxicity which affects their growth
and other metabolic processes such as alkaloid and
flavonoid productions. These secondary metabolites were
synthesized by plants to defend themselves against the
harmful action of external agents (Heller et al., 1993;
Waller and Nowacki, 1978). Thus, alkaloid and flavonoid
contents depend on the geographical distribution.
In a previously published paper, C. colocynthis
immature fruits and seeds were demonstrated as the
most efficient analgesic and anti-inflammatory parts
(Marzouk et al., 2010c). The current study measured in
vivo toxicity and anti-inflammatory activity of seven
populations of C. colocynthis Schrad. using mice and rat
as models. Preparation and tests were carried out on the
reconstituted lyophilized aqueous extracts.
MATERIALS AND METHODS
Plant materials
C. colocynthis Schrad. plants were collected in August (2007) from
seven stations (Table 1 and Figure 1). The identification was
performed according to the flora of Tunisia (Pottier-Alapetite, 1981)
and voucher specimens were deposited in the biological laboratory
of the Faculty of Pharmacy of Monastir.
Extraction protocol
The extraction was performed on fresh immature fruits or seeds.
Each plant part was ground with a mixer and added to distilled
water. The mixture was allowed to reflux for 30 min, after which the
solution was allowed to cool (4 h at 4°C). The mixture was then
filtered using filter paper (Whatman no.1) under the vacuum of a
water pump. The filtrate obtained was lyophilized, yielding the
lyophilized aqueous extract. Yields are given in Table 2.
Quantitative alkaloid screening
Alkaloids were quantified according to the volumetric procedure of
the Official Method of European Pharmacopoeia. Briefly, to 3 g of
each dried plant parts, ammonium hydroxide (5 ml), ethanol (10 ml)
and diethyl ether (30 ml) were successively added. After 4 h of
maceration, the mixture was lixiviated by 150 ml of ether and 50 ml
of chloroform. The obtained solution was reduced to 50 ml by a
rotavapory (Heidolph) and then treated with sulphuric acid (0.5 N, 3
x 20 ml). Bases were removed from the extract through precipitation
with an excess of ammonium hydroxide (3 ml). The supernatant
was then extracted with chloroform (3 x 10 ml). The precipitate
containing alkaloids was separated by means of filtration and
treated again with chloroform which was evaporated under low
pressure. The presence of alkaloids was verified by Dragendorff’s
reagent confirmed with Bouchardat’s and Meyer’s reagent (Treas
and Evans, 1984). To the residue, 20 ml of sulphuric acid was
added. The excess sulphuric acid was titrated with sodium
hydroxide (0.02 N) using the phenolphthalein indicator. The
percentage of total alkaloids was then calculated according to the
following formula:
Alkaloid level (%) = ((VH2SO4-V H2SO4ex) x 5.788 / W) x 100
Where, VH2SO4 = 20 ml, V H2SO4ex = volume of excess sulphuric acid,
W = 3 g and 1 ml of sulphuric acid correspond to 5.788 mg of
alkaloids.
Quantitative oligomeric flavonoid screening
In order to obtain total oligomeric flavonoids (TOF), the powdered
seeds were macerated separately for 24 h in a mixture of
acetone/water (2:1). Each extract was then filtered and the acetone
was evaporated under low pressure. Tannins were removed from
the aqueous phase through precipitation with NaCl for 24 h at 5°C
and the supernatant was then extracted with ethyl acetate,
concentrated and precipitated with excess chloroform. The
precipitate containing TOF was separated by means of filtration
(Paris and Moyse, 1976). The presence of flavonoids was verified
by the reaction of ‘cyanidine’ in the presence of hydrochloric acid
and magnesium. After release of hydrogen, an orange colour with
red purple indicates the presence of the flavonoids. The
appearance of the spots corresponding to the flavonoids on the
plates of thin layer chromatography (TLC) is done by pulverization
using a solution of 2% AlCl3 in methanol. The plates of TLC are
then observed under UV with 366 nm; the spots corresponding to
the flavonoids have a yellow fluorescence at intense yellow
fluorescence (Paris and Moyse, 1976). The percentage of total
oligomer flavonoids was then calculated according to the following
formula:
Total oligomer flavonoids level (%) = (weight of total oligomer
flavonoids / powdered material weight) x 100
Animals
Male adult Wistar rats weighing 160 to 180 g and Swiss albino mice
(weighing 18 to 25 g) of both sex were obtained from Pasteur
institute (Tunis, Tunisia). They were housed in polypropylene cages
and were left for 2 days for acclimatization to animal room
maintained under controlled condition (a 12 h light – dark cycle at
22 ± 2°C) on standard pellet diet and water ad libitum. Before the
day of assay, only the Wistar rats were fasted overnight with free
access to water. Housing conditions and in vivo experiments
approved according to the guidelines established by the European
Union on Animal Care (CFE Council (86/609)) were used. The rats
were used for the anti-inflammatory evaluation of the aqueous
extracts, while the mice were used for the analgesic investigation
and for the acute toxicity testing. Animals were divided into drugtreated
‘test’ and saline-treated ‘control’ groups of six or eight
animals per group.
Acute toxicity
For acute toxicity, mice were divided into groups of eight animals
each. One group served as a control and received 0.9% NaCl alone
(10 ml/kg) given intraperitoneally (i.p.), while the remaining groups
were treated with increasing doses of the aqueous extract; 50, 100,

Table 1. Sites and voucher specimens of C. colocynthis Schrad. populations.
Population Site Voucher specimens
Hammamet Hammamet Sud (36°25 N, 10°34 W) C.C-07.07
Mahdia Chorban (35°20 N, 10°32 W) C.C-06.07
Kasserine Mozgam (35°11 N, 8°46 W) C.C-05.07
Sbeitla Sbitla (35°14 N, 9°07 W) C.C-04.07
Sidi-Bouzid Jelma (35°16 N, 9°25 W) C.C-03.07
Sfax Skhira (34°10 N, 10°04 W) C.C-02.07
Medenine Sidi Makhlouf (33°33 N, 10°27 W) C.C-01.07
N: North; W: west.
Figure 1. Geographical map of Tunisia.
250, 500, 750, 1000, 1500, 2000, 3000 and 4000 mg/kg (i.p.),
respectively.
The mortality rate within a 48 h period was determined and the
LD50 was estimated according to the method described by Miller
and Tainter (1944). According to the results of acute toxicity test,
doses were chosen for pharmacological evaluations. After the last
observation, the mice were killed and the liver, lungs, heart, spleen
and kidneys were withdrawn, weighed and stored for next
evaluations.
Anti-inflammatory activity
The anti-inflammatory activity was assessed on the basis of
Marzouk et al. 4219
inhibition of paw edema induced by the injection of carrageenan (an
edematogenic agent) into the subplantar region of the right hind
paw of the rat (Winter et al., 1962). Male Wistar rats were divided
into different groups of eight animals. The control group received
2.5 ml/kg of saline, the standard group received the reference drug
(acetyl salicylate of lysine (ASL), 300 mg/Kg) and the test groups
received different population extracts of C. colocynthis at a dose of
1 and 4 mg/kg. Thirteen minutes after intraperitoneal administration
of different substances, 0.05 ml of 1% of carrageenan suspension
was injected into all animals in the right hind paw.
The paw volume, up to tibiotarsal articulation, was measured
using a plethysmometer. The measures were determined at 0 h (V0:
before edematogenic agent injection) and 1, 2, 3, 4, 5, 6 and 24 h
intervals later (VT). The difference between VT (1, 2, 3, 4, 5, 6 and

4220 Afr. J. Biotechnol.
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.
Population of Citrullus colocynthis Schrad.
Parameter Hammamet Mahdia Kasserine Sbeitla Sidi-Bouzid Sfax Medenine
Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits Seeds Fruits
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
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
Flavonoid levels 0.24 - 0.18 - 0.18 - 0.20 - 0.17 - 0.13 - 0.10 -
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
24 h) and V0 was taken as the edema value. The
percentage of inhibition was calculated according to the
following formula:
Percentage inhibition = ((VT-V0) control – (VT-V0) treated group) x
100 / (VT-V0) control
Statistical analysis
Data obtained from animal experiments were expressed as
mean ± S.E.M. and as percentage. Results were statistically
evaluated by ANOVA and using student’s t-test.
P ≤ 0.05 were considered significant.
RESULTS
Extraction yields
After all population (Table 1 and Figure 1)
extractions, experimental results (Table 2) reveal
that seeds and fruits from Kasserine, Sfax and
Medenine have significantly more higher extraction
yields than the other population plant parts.
Hammamet population showed the lowest yield.
Quantitative alkaloid and oligomeric flavonoid
screening
Alkaloid presence was reported in all C.
colocynthis organs (immature seeds and fruits
whose alkaloids are quantified in this study)
except the roots (Marzouk et al., 2009). Results
(Table 2) were dependent upon the population.
Sfax (3.90 and 2.86%, respectively, for seeds and
fruits) and Medenine (3.87 and 2.90% correspondingly,
for seeds and fruits) populations
contained more alkaloids, but Hammamet organs
presented the lowest amounts of it. Differences
were also noted between plant parts: seeds
showed better percentage (from 2.01 to 3.90%)
than fruits (from 1.55 to 2.90%). Flavonoids which
are only detected in seeds (Marzouk et al., 2009)
were present just at low levels (from 0.10 to
0.24%, Table 2). Hammamet was the richest
population in these components.
Toxicity studies
Swiss-albino mice were observed for 48 h and
morbidity and/or mortality were recorded, for each
group at the end of observation period. Due to
death index, the LD50 of all extracts were
determined (Table 2). This value is in relation with
plant parts and also with origin states. The LD50 of
seeds ranged from 438.62 mg/kg (Sfax
population) to 2298.48 mg/kg (Sidi-Bouzid
population). For the C. colocynthis fruits, Sfax
population had the lowest LD50 (385.54 mg/kg)
and that of Sbeitla was evaluated as the less toxic
(LD50 = 799.64 mg/kg). Except Kasserine
population, immature seeds were less toxic than
immature fruits. C. colocynthis seeds and fruits
from Sfax seem to be the most toxic plant parts of
all. Regarding the LD50, the quantitative alkaloid
and oligomeric flavonoid screening, this toxicity
could be, probably, attributed to the alkaloid
levels.
Anti-inflammatory effect
In carrageenan-induced rat paw edema, all extracts
produced a significant reduction of the edema
throughout the entire period of observation
(Tables 3 and 4). The intraperitoneal administration
of the aqueous extracts of seeds and fruits
significantly reduced the paw edema induced by
the noxious agent. This inhibition differs with the
plant part aqueous extract and its origin state. For
this second assessment of the anti-inflammatory
activity, the obtained results demonstrated that
the reduction of the paw edema vary in a dose
dependent fashion with its maximum at 4 mg/kg.
On the contrary, at 1 mg/kg, only a weak to
moderate activities were noted. In terms of plant
parts, immature fruits showed better activity than
immature seeds. Experimental results, at 4 mg/kg,
reveal that the anti-inflammatory effect seems to
remain moderate with Mahdia seeds and
Hammamet fruits which showed a very weak
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.
Seed population Dose (mg/Kg)
Mean swelling thickness (10-2) ± S.E.M. (% inhibition)
1 h 2 h 3 h 4 h 5 h 6 h 24 h
Marzouk et al. 4221
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
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
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
Hammamet 1
(C1)
4
(C2)
Mahdia 1
(C2)
4
(C2)
Kasserine 1
(C2)
4
(C2)
Sbeitla 1
(C1)
4
(C2)
Sidi-Bouzid 1
(C3)
4
(C2)
Sfax 1
(C1)
4
(C2)
14.00±1.41 ns
(6.67)
15.75±0.96***
(52.27)
33.00±0.82 ns
(0.00)
27.00±1.15***
(18.18)
23.75±1.71***
(28.03)
12.75±0.96***
(61.36)
20.00±1.63***
(39.39)
18.00±0.82***
(45.45)
14.00±1.41 ns
(6.67)
14.50±1.29***
(56.06)
15.00±2.45 ns
(0.00)
14.75±1.50***
(55.30)
30.50±1.91*
(11.59)
22.50±1.29***
(53.13)
40.25±1.26***
(16.15)
35.25±0.96***
(26.56)
31.00±1.41***
(35.42)
18.50±1.29***
(61.45)
25.00±1.15***
(47.91)
22.00±2.16***
(54.17)
16.75±2.52***
(30.21)
20.00±2.16***
(58.33)
31.50±2.38 ns
(8.69)
19.75±0.96***
(58.85)
44.25±2.75***
(22.37)
20.50±0.58***
(66.66)
33.00±0.82***
(46.34)
32.00±1.41***
(47.97)
29.00±2.16***
(52.85)
23.50±1.29***
(61.79)
28.50±1.29***
(53.66)
25.75±1.71***
(58.13)
33.25±2.16***
(32.14)
24.50±1.29***
(60.16)
44.25±2.75***
(22.37)
23.50±2.52***
(61.79)
46.75±3.10***
(23.98)
19.00±1.41***
(75.08)
31.00±2.31***
(59.34)
26.75±1.26***
(64.92)
32.50±2.52***
(57.38)
24.00±0.82***
(68.52)
35..00±0.82***
(54.10)
26.75±1.71***
(64.92)
31.75±1.29***
(44.78)
28.00±0.82***
(63.28)
47.00±3.16***
(23.58)
28.00±0.82***
(63.28)
36.00±2.16***
(46.27)
22.75±0.96***
(76.05)
30.50±0.58***
(67.89)
29.75±0.96***
(68.68)
34.50±1.91***
(63.68)
29.00±0.82***
(69.47)
30.50±1.73***
(67.89)
25.75±1.71***
(72.89)
34.50±1.29***
(43.69)
24.25±2.06***
(74.47)
53.00±1.83***
(20.89)
28.75±0.96***
(69.74)
32.00±2.16***
(54.93)
21.75±0.96***
(79.53)
27.00±0.82***
(74.59)
25.75±0.96***
(75.76)
28.25±2.06***
(73.41)
19.00±1.41***
(82.12)
30.00±1.63***
(71.76)
18.25±0.96***
(82.82)
29.50±3.41***
(54.26)
23.00±2.16***
(78.35)
49.50±1.91***
(25.00)
20.50±1.29***
(80.71)
18.25±1.71***
(54.94)
14.00±0.82***
(81.99)
20.50±0.58***
(73.63)
17.75±0.96***
(77.17)
24.50±1.29***
(68.49)
13.75±0.50***
(82.31)
24.50±1.29***
(68.49)
12.75±0.96***
(83.60)
14.50±1.29***
(55.38)
16.00±0.82***
(79.42)
28.00±0.82***
(30.86)
8.75±1.71***
(88.75)

4222 Afr. J. Biotechnol.
Table 3. Contd.
Seed population Dose (mg/Kg)
Medenine
1
(C3)
4
(C3)
ASL 300
(C1)
Mean swelling thickness (10-2) ± S.E.M. (% inhibition)
1 h 2 h 3 h 4 h 5 h 6 h 24 h
14.50±2.89 ns
19.25±2.21** 33.25±2.21*** 32.50±2.38*** 39.00±3.91*** 29.25±3.86*** 14.25±0.96***
(3.33)
(19.79) (32.14) (43.48) (34.45) (54.65) (56.15)
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***
(88.33) (91.66) (92.86) (97.40) (95.80) (98.06) (97.69)
7.25±0.96***
(51.66)
14.25±1.71***
(58.69)
17.50±2.38***
(69.30)
15.75±2.22***
(74.40)
19.50±2.08***
(70.89)
19.50±1.29***
(72.53)
23.75±1.71***
(41.36)
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.
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.
Table 4. Effects of different populations of C. colocynthis Schrad. fruit aqueous extracts and reference drug on carrageenan-induced paw edema.
Fruit population Dose (mg/kg)
Mean swelling thickness (10-2) ± S.E.M. (% inhibition)
1 h 2 h 3 h 4 h 5 h 6 h 24 h
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
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
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
Hammamet 1
15.00±2.45
(C1)
ns
31.75±1.26* 46.50±2.08*** 47.00±3.16*** 53.00±1.83*** 53.25±3.20*** 18.00±0.82***
(0.00) (7.97) (18.42) (23.58) (20.90) (30.28) (30.86)
4
(C2)
Mahdia 1
(C2)
4
(C2)
Kasserine 1
(C1)
4
(C2)
Sbeitla 1
(C2)
4
(C2)
24.00±0.82***
(27.27)
18.75±0.96 ***
(43.18)
17.00±0.82***
(48.48)
10.50±1.71***
(30.00)
17.75±0.96***
(46.21)
23.50±1.73***
(28.79)
19.25±0.96***
(41.67)
27.00±0.82***
(43.75)
24.75±1.26***
(48.44)
22.75±1.71***
(52.60)
23.25±1.41***
(32.61)
24.25±0.96***
(49.48)
26.50±1.29***
(44.79)
22.50±1.29***
(53.13)
31.00±0.58***
(49.59)
31.25±0.96***
(49.19)
27.5±0.58***
(55.28)
33.25±2.08***
(41.67)
29.75±1.71***
(51.63)
33.25±2.21***
(45.93)
22.75±2.06***
(63.01)
23.00±0.82***
(69.84)
30.50±1.29***
(60.00)
27.75±1.71***
(63.61)
27.00±0.82***
(56.10)
24.00±1.41***
(68.52)
32.50±2.38***
(57.38)
25.25±1.71***
(66.89)
23.75±0.96***
(75.00)
32.50±2.08***
(65.79)
22.50±0.58***
(76.32)
36.00±2.16***
(46.27)
23.00±1.86***
(75.79)
30.50±1.73***
(67.89)
22.00±2.16***
(76.84)
15.75±0.96***
(85.18)
25.50±2.08***
(76.00)
20.50±0.58***
(80.71)
32.00±2.06***
(54.93)
17.50±1.29***
(83.53)
30.00±1.63***
(71.76)
16.00±0.82***
(84.94)
11.25±0.96***
(85.83)
17.25±0.96***
(77.81)
14.25±0.96***
(81.67)
13.00±1.15***
(67.90)
12.75±0.50***
(83.60)
24.50±1.29***
(68.49)
11.25±0.96***
(85.53)

Table 4. Contd.
Sidi-Bouzid 1
(C3)
4
(C2)
Sfax 1
(C2)
4
(C2)
Medenine 1
(C3)
4
(C3)
ASL 300
(C1)
14.50±2.89 ns
(3.34)
18.25±1.71***
(44.96)
33.00±2.08 ns
(0.00)
14.50±3.11***
(56.06)
13.25±2.22
(11.67)
1.50±0.57***
(90.00)
7.25±0.96***
(51.66)
21.75±1.50***
(9.38)
22.50±1.29***
(53.13)
40.25±1.26***
(16.14)
16.75±0.96***
(65.10)
16.75±3.30**
(30.21)
1.75±0.96***
(92.71)
14.25±1.71***
(58.69)
42.75±2.16***
(12.76)
26.00±0.82***
(57.72)
43.75±1.26***
(43.50)
20.75±1.71***
(66.26)
29.00±2.16***
(40.82)
2.75±0.50***
(94.39)
17.50±2.38***
(69.30)
37.50±2.38***
(34.78)
25.75±1.71***
(66.23)
32.50±2.52***
(57.38)
15.75±1.50***
(79.34)
31.75±3.86***
(44.78)
1.75±0.96***
(97.65)
15.75±2.22***
(74.40)
39.00±1.29***
(34.45)
21.50±2.08***
(77.37)
34.50±1.92***
(63.68)
17.50±0.58***
(81.58)
34.50±1.29***
(42.02)
2.75±0.96***
(95.38)
19.50±2.08***
(70.89)
29.25±2.08***
(54.65)
21.00±2.54***
(80.24)
27.00±0.82***
(74.59)
14.75±1.26***
(86.12)
21.75±3.95***
(66.28)
0.75±0.96***
(98.84)
19.50±1.29***
(72.53)
Marzouk et al. 4223
17.75±1.71***
(45.38)
5.75±0.25***
(92.60)
24.50±1.29***
(68.49)
5.50±0.50***
(92.93)
16.75±2.22***
(48.46)
0.50±0.58***
(98.46)
23.75±1.71***
(41.36)
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
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.
significantly at the second one, the other populations
throughout the experiment, showed an
important effect. This difference may be correlated
to the process of synergism or antagonism
between all extract components (Marzouk et al.,
2009). With all extracts, 3 h after carrageenan
injection, the anti-inflammatory activity instigated
unambiguous increase to attain the maximum
level at 6 and 24 h. The inhibition percentages range
at 6 and 24 h respectively, are from 75.76 to
98.06% and from 77.17 to 97.69% for seeds and
from 80.24 to 98.84% and 81.67 to 98.46% for
fruits. South Tunisia aqueous extracts (at 4
mg/kg) were strongly more active than the other
population (1 to 6 h) and seem to have close
values after 24 h after the carrageenan injection.
The highest activity was found for immature fruit
extract from Medenine. Standard drug decreased
paw edema by a maximum of 74.39% after 4 h.
DISCUSSION
This is the first study evaluating the in vivo acute
toxicity and the anti-inflammatory activities of
extracts from different population of Tunisian C.
colocynthis immature fruits and seeds.
The study clarified complexes of biodiversity
pictures in terms of plant parts and plant geographical
distribution. Results previously obtained
(Marzouk et al., 2010c) showed the importance of
immature seeds and fruits. Fruits and seeds
differed, as made obvious by their extraction
yields, analgesic and anti-inflammatory activeties.
The present investigation showed, in addition, that
for a given plant part, the intensity of the pharmacological
activity can change from a population to
another. The trend for all the populations to have
higher activity for immature fruit aqueous extracts
and immature seeds points towards various
active, potential or antagonistic compounds present
in various concentrations according to the plant

4224 Afr. J. Biotechnol.
population, especially flavonoids and alkaloids. These
two phytochemical key families are quantified and the
results indicated that the geographical distribution of C.
colocynthis Schrad. influenced second metabolite levels
and subsequently their toxicity and biological activities.
Based on the LD50 calculation, the acute administration
doses of all population aqueous extracts were estimated
(1 and 4 mg/kg). Carrageenan has been widely used as a
noxious agent which is able to induce experimental
inflammation for the screening of compounds possessing
anti-inflammatory activity. This phlogistic agent, when
injected locally into the rat paw, produced a severe
inflammatory reaction, which was discernible within 30
min (John and Nodine, 1999). The development of
edema induced by carrageenan is a biphasic event: The
early phase (90 to 180 min) of the inflammation is due to
the release of histamine, serotonin and similar substances.
The later phase (270 to 360 min) is associated
with the activation of kinin-like substances and the
release of prostaglandins, proteases and lysosome
(Olajide et al., 1999). All population extracts inhibited hind
paw edema and showed a dose-dependent anti-inflammatory
activity but the results were different for each
population plant parts depending on the early/later
phases. All tested extracts inhibited both the phases of
the carrageenan-induced edema by reducing the release
of histamine and serotonin and also the kinin-like substances
and prostaglandins. Pharmacological properties
of all C. colocynthis Schrad. populations may be attributed
to a possible molecular mechanism by effectively
decreasing the production of the pro-inflammatory
cytokines like IL-6 and IL-1β and the expression of COX-
2, and simultaneously elevating the level of anti-inflammatory
cytokine IL-4 in the carrageenan-injected rat paw
tissues (Moulin and Coquerel, 2002).
Differences in the LD50 and the anti-inflammatory
efficacy are related to the plant population compositions.
Alkaloids and flavonoids found in C. colocynthis plant part
were dependent upon the population. At any rate, these
results indicate that the anti-inflammatory activities could
not be imputed to one family of phytochemicals only (or
its absence). Alkaloids are commonly found to have antiinflammatory
properties (Moulin and Coquerel, 2002).
Flavonoids (detected in all the seed extracts) are known
to have the same activities (Borgi et al., 2008). However,
alkaloids and flavonoids cannot be only responsible for
the pharmacological effect. Steroids and iridoids which
are present in this plant (Marzouk et al., 2009) may
contribute to better performance as an anti-inflammatory
agent (Bames and Adcock, 2009). Differences found
between the seven populations tested, in the alkaloid and
flavonoid levels, in the toxicity and in the pharmacological
activities, may be attributed to the climatic conditions and
soils.
But, despite the complexity of the chemistry of the
inorganic and organic nature, it is possible to find some
general trends like genetics and breeders which are more
and more interested in the link between genetic factors
and phenotypic variation of qualitative and quantitative
composition.
With these anti-inflammatory property, C. colocynthis
Schrad., the Tunisian population, can be considered an
effective agent to treat inflammation diseases. Immature
seeds and fruits demonstrated a high activity at very low
aqueous extract doses (1 and 4 mg/kg). The study
corroborated the pharmacological effects of this species,
justified and supported scientifically, the use of C.
colocynthis as an anti-inflammatory agent to treat pain
and rheumatoid arthritis. Additional studies are ongoing
to confirm this C. colocynthis Schrad. properties with
other models. Further, attempts are made to isolate and
define the active anti-inflammatory fractions and its
components.
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African Journal of Biotechnology Vol. 10(20), pp. 4226-4233, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB07.500
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Effects of artemether on the plasma and urine
concentrations of some electrolytes in rats
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.
Odeleye 4 and B.O. Akanji 5
1 Department of Physiological Sciences, Obafemi Awolowo University, Ile-Ife, Nigeria.
2 Department of Chemical Pathology, Obafemi Awolowo University, Ile-Ife, Nigeria.
3 Department of Pharmacology, Obafemi Awolowo University, Ile-Ife, Nigeria.
4 Department of Pharmacognosy, Obafemi Awolowo University, Ile-Ife, Nigeria.
5 Department of Chemical Pathology, Obafemi Awolowo University Teaching Hospital Complex, Ile-Ife, Nigeria.
Accepted 24 March, 2011
This study was carried out to determine the changes in the urine levels of sodium (Na + ), potassium (K + ),
and calcium (Ca 2+ ) of rats during a week of intramuscular administration of artemether (12.5 to 50.0
mg/kg/day), another one week thereafter and their concentrations in the plasma at the end of the study.
At 12.5 and 25.0 mg/kg of artemether, urine Na + concentration was significantly increased throughout
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
significantly different from the control. At 12.5 mg/kg of the drug, urine K + concentration was
significantly increased throughout the study (p < 0.05). Artemether caused no significant changes in
urine Ca 2+ concentration in the control rats as well as those that received 12.5 and 25.0 mg/kg of
artemether. Progressive and significant reductions in the urine concentrations of all the electrolytes at
50.0 mg/kg of artemether were observed. Their concentrations in the plasma were also significantly
reduced at this dose of the drug. A dose-dependent degeneration of the renal tissue of all the
experimental rats was also observed. We concluded that high doses of artemether caused progressive
degeneration of the renal tissue of rats, inability of the damaged kidneys to concentrate urine, which
manifested as excessive water loss and electrolyte depletion.
Key words: Artemether, electrolytes in plasma, urine concentrations, rats.
INTRODUCTION
Artemether, one of the derivatives of artemisinin, is
reputed for its efficacy in the treatment of malaria,
including those resulting from infection by chloroquineresistant
strains of plasmodium (Qinghaosu antimalarial
coordinating research group, 1979; China cooperative
research group on Qinghaosu and its derivatives as antimalarials,
1982). Though in combination with other antimalarials,
it is gaining ground as one of the first lines of
treatment of cerebral malaria caused by chloroquineresistant
plasmodium (Van Vugt et al., 1999; Nosten et
al., 2000). It has been reported that intramuscular administration
of multiple doses of the drug to dogs, rats
and rhesus monkeys produced neurotoxic effects such as
*Corresponding author. E-mail: rufakom@yahoo.co.uk.
gait disturbances, loss of spinal and pain reflexes (Petras
et al., 1997; Sumalee et al., 1997; Nontprasert et al.,
1998; 2000; Xiao et al., 2002). High doses of artemether
were also reported to have caused neuronal necrosis in
the region of the brainstem of rats (Raymond et al., 1998;
Xiao et al., 2002). Anorexia and a dose-dependent
reduction in body weight have also been reported at
these high doses (Qigui et al., 1998). We have studied
the changes in some of the visceral functions of Wistar
rats following one week of intramuscular administration of
12.5 to 50.0 mg/kg of artemether (Akomolafe et al.,
2006). We reported a pattern of anorexia which
manifested as a significant reduction in the food and
water intake of all the treated rats. This was accompanied
by significant increases in their urine output. These
effects persisted until even one week after the stoppage
of drug administration in those rats that received 50.0

mg/kg of the drug, whereas those that received lower
doses had only their food intake restored during this
period (Akomolafe et al., 2006). We concluded that the
significant increase in urine output without a corresponding
increase in the water intake of the rats could
exacerbate dehydration and lead to a deleterious effect
on the ionic balance of the body fluid of the rats
(Akomolafe et al., 2006). We also postulated that high
doses of artemether could cause impaired renal function
of the treated rats and that the significant increase in
urine output could be due to other effects of the drug on
thirst, anti-diuretic hormone output and the osmotic
pressure of their blood (Akomolafe et al., 2006). The
plasma levels of some electrolytes namely sodium (Na + ),
potassium (K + ), and calcium (Ca 2+ ) are very important for
the proper functioning of the neuromuscular and
cardiovascular systems (Guyton and Hall, 2001; Ganong,
2003). Excessive loss of these ions form the body
through urine stool or sweat could have serious
deleterious effects on these two systems likewise their
excessive retention (Guyton and Hall, 2001; Ganong,
2003). There is a dearth of information on the influence of
artemether on the plasma and urine levels of these
important electrolytes of the body fluid in laboratory
animals, especially rats. This study was carried out to
determine the effects of artemether on the plasma and
urine levels of Na + , K + , and Ca 2+ of rats with a view to
shedding more light on its toxicity in their body fluid and
renal tissue.
MATERIALS AND METHODS
Experimental animals
Eighty adult Wistar rats (200 to 250 g) were used for this study. The
rats were obtained from the Animal Holding of the Department of
Physiological Sciences, Obafemi Awolowo University, Ile-Ife,
Nigeria. They were kept in the laboratory under natural light/dark
cycle and were fed on normal mouse cubes (Ladokun feeds,
Ibadan, Nigeria) and water. The rats were divided into four groups
labeled I, II, III and IV. Each of the groups consisted of twenty rats,
ten males and ten females. Each of the rats was housed in a
separate metabolic cage (Ohaus R Model; Ohaus, Pine Brook, NJ,
USA) with access to food and water ad libitum. The rats were
acclimatized for 2 weeks before the commencement of the
experiments.
Drug administration
Injectable form of artemether (80 mg/ml) manufactured by Kunming
Pharmaceutical Factory, Kunming, People’s Republic of China) was
dispensed in 1 ml ampoules for intramuscular injection.
Dose regimens
Each rat in Group I that weighed 250 g was given 0.16 ml of normal
saline (equivalent to the volume of the drug that was administered
to each 250 g rat that received 50.0 mg/kg/day) for 1 week. This
group served as the control. Each of the rats in Groups II, III and IV
Akomolafe et al. 4227
received 12.5, 25.0 and 50.0 mg/kg/day of artemether respectively
via the intramuscular route for 1 week.
Urine samples were collected into clean specimen bottles for 24
h on the day before the commencement of drug administration and
this was taken as the Day 0 urine for each of the rats. This
procedure was repeated for Days 3, 7, 11 and 14 of the study, that
is, one week of drug administration and another one week later.
The concentrations of Na + , K + and Ca 2+ in the samples were
measured. The urine concentrations of Na + and K + were determined
by Flame Photometry using Coming 410C Flame Photometer. The
Ca 2+ level was measured using Cresolphthalein Complexone
method (Burtis and Ashwood, 2001).
On Day 14, the rats were sacrificed under chloroform anaesthesia.
A midline incision was made with a surgical blade to expose
the abdominal organs. Blood was collected from the hearts of the
rats by cardiac puncture and delivered into lithium heparinized
specimen bottle. A new syringe was used for the collection of blood
from each rat; the blood was immediately centrifuged at 3000
revolutions per minute for 20 min. The plasma was thereafter
separated into a specimen bottle in readiness for analysis. The
concentrations of Na + , K + , and Ca 2+- in the samples were
determined using the same methods that were used in the analysis
of urine.
The rats kidneys were dissected out and kept inside 10%
formalin until their sections were cut and stained with eosin and
hematoxylin for histological studies. Photomicrographs of the
tissues were taken using Lect3 Dialux Microscope (Bright Field) at
×40 magnification.
Statistical analysis
The results were expressed as mean ± SEM and subjected to oneway
analysis of variance (ANOVA). Significant differences were
further tested by the Duncan’s Multiple Range and Student Neuman
Keuls tests. Student’s t-test was used to compare the urine
concentration of the electrolyte for each day with the Day 0 value
for each group. Differences with probability values of p < 0.05 were
considered significant.
RESULTS
Effect of artemether on urine concentration of
electrolytes
Sodium (Na + ): Throughout the study, there was no
significant change in the concentration of Na + in the urine
of the control rats (Figure 1). At 12.5 and 25.0 mg/kg,
urine Na + concentration was significantly higher than the
Day 0 value and the control rats throughout 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 significantly different from
these values. At 50.0 mg/kg, the urine Na + concentration
decreased gradually and significantly throughout the
study (p < 0.05).
Potassium (K + ): The K + concentration in the urine of the
control rats did not vary significantly throughout the study
(Figure 2). At 12.5 mg/kg, K + concentration in the urine
was significantly higher than that of the control rats and
that of the Day 0 value throughout the study (p < 0.05).
At 25.0 mg/kg, there was no significant change in K +

4228 Afr. J. Biotechnol.
Urinary potassium concentration (mmol/L)
Urinary concentration of sodium (mmol/L)
100
90
80
70
60
50
40
30
20
10
*
*
NS
12.5 mg/Kg
25.0 mg/Kg
50.0 mg/Kg
a
*
a * a
a
* *
0
0 3 7 11 14
Figure 1. Variation in the urine concentration of sodium due to a week of intramuscular administration of
artemether to rats. Each point is mean ± SEM (n = 20). *, Significantly different from the Day 0 value (p <
0.05); a, significantly different from control (NS) (p < 0.05).
600
500
400
300
200
100
0
(mmol/L)
*
*
a
a
a
Day
0 3 7 11 14
Day
a
NS
12.5 mg/Kg
25.0 mg/Kg
50.0 mg/Kg
Figure 2. Variation in the urine concentration of potassium due to a week of intramuscular administration of
artemether to rats. Each point is mean ± SEM. (n = 20). *, Significantly different from the Day 0 value (p

DISCUSSION
Urinary concentration of calcium (mmol/L)
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
(mmol/L)
*
a a
0
0 3 7 11 14
*
Day
*
NS
Akomolafe et al. 4229
12.5 mg/Kg
25.0 mg/Kg
50.0 mg/Kg
Figure 3. Variation in the urine concentration of calcium due to a week of intramuscular administration of
artemether to rats. Each point is mean ± SE (n = 20). *, Significantly different from the Day 0 value (p <
0.05). a, significantly different from control (NS) (p < 0.05)
This study showed a dose-dependent degeneration of the
renal tissue of the rats under the influence of high doses
of artemether (Plates 1 to 4). Chemical compounds which
furnish oxygen based radicals are reputed for their cell
membrane lipid peroxidation properties. Lipid
peroxidation refers to the oxidative degradation of lipids.
It is the process whereby free radicals take electrons
away from lipids in cell membranes, resulting in cell
damage. The endoperoxide bridge in artemether molecule
produces free radicals which are responsible for the lipid
peroxidation properties of the drug (Goodman and
Gilman, 1985; Meshnick, et al., 1991, 1993; Maeno et al.,
1993). The degenerative changes observed in the renal
tissue of the treated rats could be attributed to the
destruction of the membrane of the renal tubular cells of
the rats by the free radicals produced by the drug.
Analysis of the urine of the rats for electrolytes revealed
a progressive and significant reduction in the concentration
of the electrolytes at 50.0 mg/kg of artemether.
This indicated that the rats that received this dose of
artemether produced more dilute urine with each passing
day of the study, despite the anorexia induced by the
drug. Inability of the kidney to concentrate or dilute the
urine is one of the features of damaged nephrons (Leaf
and Cotran, 1980; Neinman, and Lorenz, 1989; Guyton
and Hall, 2001; Dunn, 2003; Ganong, 2003). Rapid
tubular flow occurs in the remaining nephrons of the
diseased kidneys. The renal tubules lose their ability to
concentrate or dilute the urine (Guyton and Hall, 2001).
This study indicated that high doses of artemether are
toxic to the renal tissue of the rats. We have earlier
reported a significant increase in the volume of the urine
produced by rats that received 50.0 mg/kg of artemether,
during treatment and even one week post-treatment,
without a corresponding increase in their food and water
intake for the same period (Akomolafe et. al., 2006). The
excessive fluid loss of rats that received this dose of
artemether could lead to dehydration and a severe
depletion of the major electrolytes of their body fluid. The
photomicrograph of the kidney of rats that were given this
dose of artemether showed a very high degree of tissue
degeneration. The continuous significant decreases in
the urine concentration of the electrolytes could not have
been due to increased reabsorption of the electrolytes by
the renal tubules, but rather to the artemether-induced
anorexia and the excessive water loss that usually
accompanies increased electrolyte loss in urine (osmotic
diuresis).
Throughout the study, there was no significant change
in the concentration of Na + in the urine of the control rats
(Figure 1). This is an indication that the kidney function of
the rats was not impaired, as evidenced by the photomicrograph
of their kidneys (Plate 1). At 12.5 and 25.0
mg/kg, urine Na + concentration was significantly higher
than the Day 0 and control values throughout 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 significantly different from
these values. Our earlier report indicated that rats that
*
a

4230 Afr. J. Biotechnol.
M
S
G
B
Plate 1: Photomicrograph of the kidney of rats that received normal saline i.m. for 7 days (Control 1 rats). Mag x 400.
The kidney tissue shows no pathological changes, as reflected by the Bowman’s capsule (B), the glomerulus (G), renal
tubules (T) and other cells of the renal tissue. The glomerulus (G) and the capsular space (S) are distinct and intact.
The tubules retain their normal shapes and the tubular cells have well defined boundaries, and nuclei. The mesangial
cells (M) are normal in size shape and number.
.
T
C
Plate 2. Photomicrograph of the kidney of rats that received 12.5 mg/kg of artemether i.m. for 7 days.
Mag×400. The glomerulus (G) has expanded to the extent of almost closing the capsular space(S). The tubular
cells (C) are densely stained. The tubules (T) have started losing their integrity as reflected in the distortion of
their shape.
received 25.0 mg/kg of artemether had significantly
increased urine output which lasted for even a week after
treatment, without a corresponding increase in food and
water intake, most especially during treatment (Akomolafe
et al., 2006).
The significant increase in urine Na + concentration at
25.0 mg/kg of artemether is an evidence of dehydration
induced by the drug. The integrity of the nephrons of the
rats had been severely compromised as revealed by
Plate 3. The significant increase in urine Na +
concentration at this dose of the drug could also be a
reflection of excessive sodium loss by the damaged
T
G
kidneys of the rats.
The K + concentration in the urine of the control rats did
not vary significantly throughout the study (Figure 2). At
12.5 mg/kg, K + concentration in the urine was significantly
higher than the control and Day 0 values throughout the
study (p < 0.05). At 25.0 mg/kg, there was no significant
change in K + concentration throughout the study.
Potassium, once absorbed by the intestinal tract, is
partially removed from the plasma by glomerular filtration
and is then nearly completely reabsorbed in the proximal
tubule. Unlike sodium, it is effectively re-excreted by the
distal tubules There is no threshold level for K + ( Guyton
S

.
Plate 3. Photomicrograph of the kidney of rats that received 25.0 mg/kg of artemether, i.m. for 7 days.
Mag×400. The Bowman’s capsule (B) started breaking down. The glomerulus (G) has undergone
degeneration. The capsular space (C) has been completely obliterated. The tubular cells (S) are still
densely stained. The tubules (T) have lost much of their integrity as their boundaries are no more well
defined. The intertubular space (P) are almost completely closed due to inflammation of the tubules.
T
C
B
T
C
G
B
Akomolafe et al. 4231
Plate 4.Photomicrograph of the kidney of rats that received 50.0 mg/kg of artemether i.m. for 7 days. Mag×400. The
Bowman capsule (B) is almost non-existent due to degeneration. The densely stained tubular cells (C) are still
observable, though the tubular intergrity (T) has been grossly distorted as a result of inflammation and degenerative
changes.
and 25.0mg/kg of artemether in this study could be
attributed to the differences in the renal handling of the
two electrolytes. Potassium is mainly an intracellular
electrolyte. The damaged renal tissue could also be a
source for additional K + in the body fluid of the rats.
Artemether caused no significant change in urine Ca 2+
concentration in the control rats as well as those that
received 12.5 and 25.0 mg/kg of artemether, throughout
the study (Figure 3). It could be due to the fact that
artemether does not have any appreciable effects on
renal Ca2 + excretion at these doses of the drug.
However, this needs further verification.
The plasma Na + concentration was significantly increased
at 25.0 mg/kg of artemether only (p < 0.05) (Figure
4). This is in conformity with the observed significant
elevation of the urine concentration of this electrolyte in
rats that received this dose of artemether. The
significantly increased Na + concentration in the plasma of
rats that received 25.0 mg/kg of the drug could be due to
the same reasons given for the elevated urine level of the
electrolyte. The reductions observed at 12.5 and 50.0
mg/kg of the drug were not significant. The plasma K +
concentration increased significantly at all the doses of
artemether studied, except at 50.0 mg/kg where it was
significantly reduced (Figure 5). The significant increase
in the plasma concentrations of this electrolyte at lower
doses of artemether could be as a result of the druginduced
renal tissue damage at these doses, while the

4232 Afr. J. Biotechnol.
Potassium (mmol/L)
7
6
5
4
3
2
1
0
Sodium (mmol/L)
(mmol/L)
142
140
138
136
134
132
130
128
(mmol/L)
NS 12.5 25.0 50.0
Dose (mg/kg)
Figure 4. Effect of artemether on the plasma sodium concentration of rats. *Significantly different
from control (p < 0.05). The plasma Na + concentration was significantly increased at 25.0 mg/kg of
artemether only (p < 0.05). The reduction observed at 12.5 and 50.0 mg/kg of the drug was not
significant.
*
NS 12.5 25.0 50.0
Dose (mg/kg)
Figure 5. Effect of artemether on the plasma potassium concentration of rats. *Significantly different from control (p <
0.05). The plasma K + concentration increased significantly at all the doses of artemether studied, except at 50.0 mg/kg
where it was significantly reduced.
significant decrease at 50.0 mg/kg could be attributed to
the earlier reported sustained significant reduction in food
consumption, increased urine output, as well as much
degeneration of the nephrons induced by artemether.
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 of artemether (Figure 6). This is an indication
that, at these doses of artemether, calcium excretion was
not adversely affected by the renal tubular damage
induced by the drug. However, a significant decrease in
*
*
the plasma level of this electrolyte was observed at 50.0
mg/kg of the drug.
Conclusion
High doses of artemether caused progressive degeneration
of the renal tissue, inability of the nephrons to
concentrate urine, excessive water loss and electrolyte
depletion in rats.
*

Calcium (mmol/L)
2.5
2
1.5
1
0.5
0
NS 12.5 25.0 50.0
Dose (mg/ ) kg)
Akomolafe et al. 4233
Figure 6. Effect of artemether on the plasma calcium concentration of rats. *Significantly different from control (p < 0.05).
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
of artemether. At 50.0 mg/kg, plasma Ca 2+ decreased significantly.
ACKNOWLEDGMENTS
We are grateful to Obafemi Awolowo University, Ile-Ife for
financing this study and to Mr. J.A. Ibeh, of the
Department of Anatomy and Cell Biology, and Messrs T.
A. Ogundoyin, F. A. Abidoye, C. O. Ola, R. T. Olatoye,
and A. E. Adebiyi of the Department of Physiological
Sciences for their technical assistance in the process of
carrying out this research work
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*

African Journal of Biotechnology Vol. 10(20), pp. 4234-4241, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2174
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Expression of biological active VHH camelid single
domain antibody in transgenic tobacco
Behnaz Korouzhdehy 1 , Mehdi Dadmehr 1 *, Issa Piri 1 , Fatemeh Rahbarizadeh 2 and Mahmood
Solouki 3
1 Department of Agriculture, University of Payame Noor, Tehran, Iran.
2 Department of Medical Biotechnology, University of Tarbiat Modares, Tehran, Iran.
3 Department of Agriculture, University of Zabol, Zabol, Iran.
Accepted 1 April, 2011
Functional VHH single domain antibody lacking light chains occur naturally in Camelidae. The single
domain nature of VHH gives rise to several unique features when compared to antigen-binding
derivatives of conventional antibodies. The level of expression in Escherichia coli was found to be too
low for therapeutic purposes. Therefore, there is a need to examine other production systems such as
plants. Several plants are the facile and economic bioreactor for large-scale production of industrial
and pharmaceutical agents like proteins and antibodies. Here, we have selected tobacco as the host
plant because of large scale production capability and many other advantages such as greater safety
and lower production costs when compared to animal-based systems. In this study, we have subcloned
VHH gene into pBI121 using phasmid pCANTAB5E. The new construct was used to transform the
Agrobacterium strains C58GV3101 and LBA4404. Agrobacterium strain C58GV3101 showed a higher
virulence on leaf disks of Nicotiana tabacum (NC25). Transgenic tobacco plants were then developed by
introducing VHH gene under the control of CaMV 35S promoter. The presence of the VHH antibody gene
in the plant genome was verified by PCR analysis and Southern hybridization experiments. Northern
blot analysis showed that the genes coding for the VHH could be expressed in tobacco plants. Three
lines of transgenic plant that expresses high levels of mRNA were screened in a further analysis. The
expression of VHH was then observed in transgenic plants by ELISA using the specific antibody
developed, the results showed three to five folds higher than non-transgenic tobaccos.
Key words: VHH antibody fragment, subcloning, antibodies, transgenic tobacco, bioreactor.
INTRODUCTION
Antibodies have been used as diagnostic or therapeutic
agents in vivo as well as ex vivo for the last two decades.
Their clinical applications are evident in the treatment of
diseases such as cancer, transplantation, autoimmunity
and cardiovascular disorders. They have been specifically
used in cancer therapy because of their high
specificity for tumour antigens and low cross-reactivity
with normal cells. VHHs are heavy chain variable domains
derived from immunoglobulins (IgG) naturally devoid of
light chains such as those derived from Camelidae in pre-
*Corresponding author. E-mail: mehdidadmehr@yahoo.com.
vious studies (Hamers-Casterman et al., l993) Furthermore,
their H chain is devoid of the CH1 domain due to
an unconventional splicing event during mRNA maturation.
Therefore, the antigen-binding fragment of the heavychain
antibody consists of a single domain referred to as
VHH that replaces a four-domain Fab fragment in the
immunoglobulin structure (Muyldermans et al., 1994).
VHH molecules are about 10 times smaller than IgG
molecules. They are single polypeptides and very stable,
resisting extreme pH and temperature conditions.
Moreover, they are resistant to the action of proteases
which is not the case for conventional antibodies.
Furthermore, in vitro expression of VHH’s produces high
yield, properly folded functional VHHs. In addition,
antibodies generated in Camelids will recognize epitopes

than those recognised by antibodies generated in vitro
through the use of antibody libraries or through immunization
of mammals other than Camelids (Ghahroudi et
al., 1997). As such, anti-MUC1 VHHs may interact more
efficiently with its target than conventional antibodies,
thereby blocking its interaction with the target ligands
more efficiently. Since VHH’s are known to bind into
unusual epitopes such as cavities or grooves (Spinelli et
al., 2000), the affinity of such VHH's may be more
suitable for therapeutic treatment. Besides the
advantages of easy cloning (single gene) and selection
from in vivo matured library, it has other technological,
physiochemical and functional advantages, such as close
homology to human VH fragments, high expression yield,
highly soluble and the generation of antigen-specific high
affinity binders (Nguyen et al., 2001). Recently, Camelid
single domain antibodies have been used to target MUC1
antigens in breast cancer (Taylor-Papadimitriou et al.,
2002). These fragments have been expressed in both
Escherichia coli and P. pastoris (Rahbarizadeh et al.,
2006). However, these expression systems have several
limitations that hinder maximum output of biologically
active and safe therapeutic agents. Some of these
limitations that have been reported are: (1) formation of
inclusion bodies in bacteria, (2) formation of non-native
proteins having different biological activities in yeast, (3)
low transgene expression levels, (4) transgene induced
instability of certain cell lines in mammalian cell cultures
and (5) contamination of animal-based products with
human pathogens (Koprowski et al., 2005). Such
shortcomings invite alternative methods of production to
ensure the safety and economical benefits of recombinant
therapeutic proteins (Carmer et al., 1999). Plantbased
systems are increasingly used for the production
of recombinant proteins including antibodies (During et
al., 1990; Peeters et al., 2001; Torres et al., 1999;
Schillberg et al., 2005). Plant-based systems have
several advantages over the other production systems,
such as the ability to carry out necessary posttranslational
modifications not available in bacterial
systems, as well as greater safety and lower production
costs when compared to animal-based systems. Plantbased
technology has been recently reviewed, with full
description of commonly used plants (Hellwig et al.,
2004). Processing of transgenic crops would require
relatively little capital investment, making the commercial
production of biopharmaceuticals an exciting prospect. It
has been estimated that the cost of producing
recombinant proteins in plants could be 10 to 50 fold
lower than that for producing the same protein in E. coli
or mammalian cells (Stoger et al., 2000, 2002). Several
proteins, enzymes and antibodies have been produced in
plants and used in clinical trials, with a prospect of
commercial exploitation (Ma et al., 2003). In this report,
which is the first of its kind, we described the cloning and
expression of VHH single domain antibody in tobacco
plants as a first step in the development of a plant based
Korouzhdehy et al. 4235
vaccine that could produce VHH and a low-cost biomass
scale-up was performed with this bioreactor.
MATERIALS AND METHODS
Construction of the expression vector pBI-VHH
The phasmid vector pCANTAB5E containing the VHH coding
sequence that was partially optimized for expression in competent
E. coli TG1 was obtained from the Department of Medical
Biotechnology, Tarbiat Modares University (AY544575). The VHH
encoding region was amplified from pCANTAB5E phasmid DNA
using the primers back: 5´-
GGAAATTCGAGCTCTTAGTGAGATGGTGAC-3´ and forward:
5´TCTAGAGGA TCCTAAACAATGGTCCTGCTACAGTCA-3´. Each
PCR mixture was prepared in a final volume of 50 µl containing 50
ng pCANTAB5E phasmid DNA template, 50 pM forward primer, 50
pM of the corresponding backward primer, 40 pM dNTPs, 25 mM
MgCl2, 1.25 U Taq DNA polymerase (Cinnagen) and 5 µl 10 × PCR
buffer II (Cinnagen). Hot start PCR was performed at 95°C for 5
min, followed by 35 cycles of denaturation at 94°C for 30 s,
annealing at 52°C for 45 s and elongation at 72°C for 1 min. The
reaction was completed by a final extension time at 72°C for 10
min. The amplified variable regions were purified by electrophoresis
on a 1.5% agarose gel and subsequently extracted with an
AccuePrep Gel Extraction Kit (Bioneer) according to the
manufacturer’s instructions.
Amplified DNA was cloned into T/A vector (pTZ57R) using the
InsT/A clone PCR product cloning kit from Fermentas. pTZ57R
plasmid was cut with BamH I and Sac I (Roche Applied Science).
After another electrophoresis on a 1.5% agarose gel, the variable
regions were excised and extracted with the AccuPrep Gel
Extraction Kit (Bioneer). The fragment of interest was ligated with
the corresponding cut vector pBI 121 in a 10 µl volume containing
50 ng vector DNA, threefold molar excess of the PCR product and
5 U of T4 DNA ligase (Fermentas) for 16 h at 14°C. E. coli TG1
cells (50 µl) were transformed with 2 µl of the ligation product via
CaCl2 method. One milliliter of prewarmed SOC medium
(Sambrook et al., 2001) was immediately added, and the cells were
grown at 37°C for 1 h shaking with 250 rpm. Cells were plated on
LB agar medium containing kanamycin (25 µg/ml) and incubated
overnight at 30°C.
Colonies were picked for plasmid extraction using the Accuprep
Plasmid Extraction Kit (Bioneer). Agrobacterium tumefaciens strains
C58GV3101 and LBA4404 have been previously described
(Hoekema et al., 1983; Koncz et al., 1986). The engineered pBI 121
plasmids were used to transform the both earlier mentioned
Agrobacteriums using a freeze and thawing standard protocol
(Sambrook et al., 2001).
Transformation
Axenic plants of tobacco (Nicotiana tabaccum var.NC25) were
maintained under in vitro growth conditions at 26°C and 16 h
photoperiod in the laboratory. For tobacco transformation,
Agrobacterium cells were used for transformation. Leaf explants of
in vitro-grown were inoculated with Agrobacterium in a YEP liquid
medium for 10 min. For effective transformation, the explants were
placed in a co-culture medium containing 2.0 mg/l BAP and 0.01
mg/l NAA. Two days later, the explants were transferred to the
regeneration medium (MS medium, supplemented with 2.0 mg/l
BAP, 0.01 mg/l NAA, 200 mg/l cefatoxim and 100 mg/l kanamycin).
The explants were transferred to the fresh medium at 2 weeks
intervals. As a control, non-inoculated explants were cultured in the
same medium without hormones and antibiotics. The induced

4236 Afr. J. Biotechnol.
shoots were then dissected from the explants and transferred to
free hormone MS rooting medium containing cefatoxim (200 mg/l)
for Agrobacterium elimination and kanamycin (100 mg) as a
selective antibiotic. Also, specific modified MS media were
developed and used for co-culture, regeneration and rooting steps
by Dadmehr (Unpublished data). When the roots were induced on
the regenerated shoots, plantlets were transplanted to moist
vermiculite for acclimatization. When the shoots had began to grow,
the plantlets were transplanted to moist soil and the plants were
grown to maturity.
PCR analysis and Southern blot hybridization
Genomic DNA from 200 mg each of non-transgenic plants as
negative control and all putative kanamycin resistant plants was
extracted from transgenic plants using the CTAB method (Rogers et
al., 1994). Transformed and control plant genomic DNA was used
as a template to detect the VHH gene by polymerase chain reaction
(PCR) under the conditions that were described before and with
specific primers. The 327 bp amplified DNA fragments were
analyzed by electrophoresis on a 1.5% agarose gel stained with
EtBr and observed under UV illumination. The genomic DNA also
was digested with SacI to confirm the copy number of inserted T-
DNAs for the VHH derived transformants. The digested DNA was
electrophoresed on a 1.0% agarose gel and then transferred to
positively charged nylon membrane (Roche Co., Germany) using a
Turboblotter system (Schleicher and Schuell). Dig-labeled probes
were generated by PCR Dig Labeling Mix (Roche Co., Germany)
with the specific primer set for the VHH gene. DNA gel blots were
hybridized at 50°C in a DIG easy hybridization buffer. After
hybridization overnight, the membrane was washed in an SSC
buffer series and then detected using the Dig Detection Kit following
the manufacturer’s instructions (Roche Co., Germany).
Northern blot analysis
Total RNA from the leaves of transformed plants was isolated using
the RNAgents ® Total RNA isolation system (Promega Co., USA)
according to the manufacturer’s instructions. Samples of 30 µg of
total RNA were denatured with formaldehyde and formamide and
fractionated in a 3.0% agarose gel using a 3-(N-morpholino)
propane sulfonic acid (MOPS) buffer. The RNA was blotted to a
positively charged nylon membrane, and fixed by ultraviolet
irradiation. The membranes were hybridized with Dig-labeled
probes using a template containing most of the coding region for
VHH. After hybridization for 10 h at 68°C, the blots were washed in
an SSC buffer series and then detected by means of the Dig
Detection Kit according to manufacturer’s instructions (Roche Co.,
Germany).
Quantification of VHH levels in transgenic tobaccos
Antibodies from leaves were obtained by homogenization in a
blender with liquid nitrogen, and the resulting powder was
resuspended in cold extraction buffer containing PBS, pH 7.2, 10
mM EDTA, 0.03% Triton X-100, 5 mM mercaptoethanol and 1 mM
PMSF (1 g of fresh weight/ml). The extract was filtered and
centrifuged for 20 min at 12,000 ×g and the resulting supernatant
was used for the VHH protein expression analysis. ELISA plates
were coated with 100 µl of coating buffer containing the sample
proteins from the transgenic plants overnight at 4°C. After standing
overnight, the plates were washed and blocked 2 h at RT with 5%
skim milk in PBS-Tween 20. The plates were then washed three
times with 0.05% Tween 20 in PBS and 100 µl of MUC1 synthetic
peptide TSA-P1-24 as the primary antibody diluted at 1:1000 in
PBS-Tween 20% was added to each well and allowed to react for 2
h at RT. The plates were again washed three times, and treated
with 100 µl of anti-(camel) rabbit antibodies conjugated with
horseradish peroxidase as the second-antibody, diluted at 1:2000 in
PBS-Tween 20 for 2 h at RT. Finally, the plates were again washed
four times, and 200 µl of a freshly prepared solution of
ophenylenediamine dihydrochloride and H2O2 was added.
Reactions were stopped by adding 2.5 M H2SO4. The absorbance
was measured in a Multiskan ELISA Reader (Labsystems, Helsinki,
Finland) using a 492-nm filter.
RESULTS
Construction and transformation of vectors
Single domain antibody gene of the pCANTAB5E was
subcloned into the expression vector pBI121. The
transfer of this gene is schematically shown in Figure 1.
The isolation and PCR amplification of the gene encoding
the VHH antibody was performed using pCANTAB5E
phasmid DNA as template. Targeting the restriction sites
BamH1 and Sac1 in pTZ57R plasmid facilitated
subcloning. New construct (plasmid) was transformed
into TG1 E. coli and the colonies were appeared on the
kanamycin containing plate. After the plasmid extraction,
both pTZ57R and pBI121 plasmids were cut by BamH1
and Sac1 restriction enzymes. Electrophoresis showed
327 bp bands from pTZ57R and 12000 and 1900 bp GUS
removed from pBI121. The amplified 327 and 12000 bp
bands were gel-purified from agarose. During the ligation,
VHH gene was inserted into pBI121 plasmid and was
called PBI-VHH (Figure 1). PBI-VHH plasmid was
transferred to Agrobacterium strains C58GV3101 and
LBA4404.
Effects of two strains of Agrobacterium on the
efficiency of transformation in tobacco
To improve the efficiency of transformation, this study
examined the effects of two Agrobacterium strains on
shoot formation from leaf disks. 168 and 162 leaf disks
were infected by Agrobacterium strains C58GV3101 and
LBA4404, respectively with a copy of pBI-VHH. All of
these were transferred to plates with media containing
cefatoxim. The frequency of regenerated shoots from leaf
disks that were infected with C58GV3101 was higher.
Fourteen (14) transformants were obtained from leaf
disks infected with the Agrobacterium strain LBA4404
after 3 months in culture. In this experiment, the
transformation efficiency was at least 8.3%, a ratio of
about 7.8-fold of C58GV3101 (Table 1). These results
indicated that kind of Agrobacterium strains affects the
efficiency of transformation in N. tabacum. When the leaf
explants were inoculated with Agrobacterium immediately
after excision, shoots on the cut edges of the explants
were observed in the presence of 100 mg/l kanamycin
and 200 mg/l cefatoxim after two weeks. The putatively

Figure 1. Schematic representation of pBI-VHH construction. VHH gene inserted
between CaMV 35S promoter and NOS terminator.
Table 1. Effect of two Agrobacterium strains on transformation frequency.
Number of experiment Strain
EX1
EX2
EX3
Total
transformed shoots were excised when they were about
1 cm tall and transferred to a shoot elongation medium
containing cefatoxim and kanamycin.
Consequently, shoots were transferred to rooting media
and after hardenization to the greenhouse. The genes
that had been stably integrated into the plant genome
were transcripted under the control of CaMV35S,
resulting in the expression of the VHH gene.
Number of leaf
disk
Number of
regeneration shoot
Korouzhdehy et al. 4237
Number of
transgenic shoot
C58GV3101 68 26 7
LBA4404 52 10 3
C58GV3101 24 3 1
LBA4404 30 0 0
C58GV3101 76 28 6
LBA4404 80 14 0
C58GV3101 168 57 14 (8.3%)
LBA4404 162 24 3 (1.85%)
Expression of VHH antibody fragment in plants and
molecular analysis
More than 160 kanamycin-resistant putative transformants
were obtained (Table 1) and carried out further
analysis of VHH gene expressions on selected transgenic
plants. Untransformed tobacco plants that had been
regenerated from leaf discs without kanamycin selection,
were used as negative controls. PCR analysis was

4238 Afr. J. Biotechnol.
carried out as the first method to confirm the transgenic
nature of the regenerated plants. The presence of VHH
DNA in the genomic DNA isolated from regenerated
tobacco was confirmed. Transformants were detected by
PCR amplification of inserted VHH with specific primers.
The expected 327 bp VHH bands were found in the
transformants (Figure 2). No DNA product was detected
in untransformed control plant DNA. The same fragment
was amplified using plasmid pBI 121 as our positive
control. Southern blot analysis of PCR positive transgenic
lines (T1) was done to verify site-specific integration and
to establish copy number. In order to determine the T-
DNA copy number in each transformant, the DNA was
digested with SacI, which cuts the T-DNA between VHH
Figure 2. Agarose gel electrophoresis of VHH amplification
products from transgenic plants. Lane M: 100 bp ladder; lane WT:
wild type tobacco; lanes 1 to 7: transgenic tobacco plants lines 2,
3, 6, 7, 12, 18 and 19.
Figure 3. Southern blot analysis of transgenic tobacco lines 1, 9 and
22 for the presence of VHH gene. Lane WT: genomic DNA from nontransgenic
tobacco; Lanes 1 to 5: genomic DNA from transgenic
tobacco lines 2, 6, 7, 9, 12 and 18.
Figure 4. Expression of VHH mRNA extracted from leaf tissue.
Lane WT: non-transgenic tobacco; Lanes 1 to 4: transgenic
tobacco lines 2, 6, 7 and 12.
and the nos terminator. The probe then hybridizes to one
T-DNA end fragment from each single-copy insertion,
thus permitting the copy number of the VHH gene to be
established. The transgene copy number as estimated by
Southern hybridization ranged from one to two copies
(Figure 3).
Detection of the VHH transcripts
The transformants were analyzed by hybridizing RNA
samples with a labeled probe encompassing the coding
region of the VHH gene. Figure 4 shows the results of an
experiment, where selected transformants harboring the

Figure 5. VHH expression levels in transgenic tobacco lines. Production of VHH in tobaccos
of four independent transgenic plants (T2, T6, T7 and T12) and one wild type plant (WT) was
analyzed by ELISA. Mean values and standard deviations of three independent analysis are
represented. A reaction buffer (B) was used as a blank control.
pBI 121constructs and a non-transgenic control were
probed. A VHH transcript was found in the four lines of
transgenic tobacco (nos. 2, 6, 7 and 12), and the mRNA
levels in individual transgenic lines were all quite different
(Figure 4). A high level of a VHH transcript was found in
the 6 and 7 transgenic lines. The signal was variable
among different transformants, due to random integration
of the foreign gene into the host tobacco genome. The
size of VHH specific transcript was confirmed by RNA
Century TM Size Markers (Ambion, Inc., Austin, TX USA)
and supplied manuals. Five micrograms of RNA Century
Markers was electrophored along with RNA transcripts
from transformants and cutted for staining with EtBr. The
transcripts from the pBI 121 transformants were ≈ 327 bp
in length by comparison with stained RNA Century
Markers, which is consistent with the expected size.
Detection of VHH protein expression in transgenic
plants
Based on northern analysis, the transformed plants were
further checked for recombinant protein expression by
ELISA analysis using a monoclonal specific antibody. As
a result, the expression of recombinant VHH protein was
confirmed by an ELISA assay. The results demonstrated
that concentrated extracts from all selected plants were
positive by ELISA (Figure 5). Expression level of VHH
varied significantly among the four transformed plants.
Plant derived protein in the transformed plants ranged
from 0.9 to 2.5 ng/50 µg of total soluble protein.
Transgenic plant line 6 showed the highest level of
Korouzhdehy et al. 4239
expression, accounting for 0.005% of the total soluble
protein.
DISCUSSION
Expression of the first recombinant antibody in tobacco
plant opened the field of the expression of recombinant
antibodies as a real alternative to eliminate many
constraints of monoclonal antibody production in
bioreactors (Hiatt et al., 1989). Expression of antibodies
in plants is being studied for their potential uses in
biotechnology. Camelidae are known to produce
immunoglobulin (Igs) devoid of light chains and constant
heavy-chain domains (CH1). Antigen-specific fragments
of these heavy-chains IgGs (VHH) are of great interest in
biotechnology applications (Muyldermans et al., 2001).
VHH single domain antibody fragments against MUC1
antigen have been produced, but exclusively in bacteria
and yeast. We have described here for the first time, the
expression of a MUC1 specific VHH antibody in plants.
Selecting the most appropriate vector for use in plant
transformation can be a complex task and various factors
need to be considered. Here, we have used pBI-VHH
plasmid which carries origins of replication from spa and
colE1 to facilitate maintenance in Agrobacterium and E.
coli, respectively. The presence of unique multiple
cloning sites (MCSs) for introducing target genes into the
T-DNA region and a bacterially expressed marker gene
(kanamaycin), permits selection and maintenance of the
vector in E. coli and Agrobacterium. To facilitate immunohistochemical,
biochemical and bioassay investigation,

4240 Afr. J. Biotechnol.
large amount of VHH is required; therefore, we used the
CaMV 35S promoter which is a very strong constitutive
promoter, causing high levels of gene expression in dicot
plants. Prior to that, we used the T/A cloning vector
(PTZ57R) to take advantage of inserted MCS (multi
cloning site) which is beneficial for cloning. M13 primers
around the MCS that facilitate the gene sequencing were
also used. The resulting PCR product ligated into a linear
vector with a 3´ terminal 'T' or 'U' at both ends. Tobacco
as a laboratory bench model was our target plant
because of its large scale production and as compared to
other plants represents a potentially safer production
bioreactor for human proteins from the standpoint of
containment.
This study established an efficient protocol for plant
regeneration and the Agrobacterium mediated
transformation of tobacco. The effects of different
Agrobacterium strains C58GV3101 and LBA 4404 on
transformation rate of tobacco was investigated. The
results indicate that the presence of pBI-VHH plasmid
with kanamycin resistance in C58GV3101 increased the
level of T-DNA transfer into the tobacco cells. This may
be due to the higher sensitivity of C58GV3101 when
compared to LBA4404. Therefore, using this
Agrobacterium strain may provide a better opportunity to
use tobacco for genetic engineering. In an effort to study
the safety and reproducibility of VHH single domain
antibody production in plant-based systems, a transgenic
tobacco plant expressing this antibody was grown as a
leaf disk culture on selective medium. Two weeks later,
the plant cell cultures were initiated in selective medium
and examined for VHH antibody production. Expression
of the VHH appeared to remain constant throughout the
growth periods, with no effect on the growth rate.
Transient and stable plant system used here to express
VHH gene, has the same advantages and limitations that
have been already described in literature for similar
cases. Various methods have been recently applied to
produce and select functional antibodies. The transgene
copy number as estimated by Southern hybridization
ranged from one to two copies (Figure 3). According to
the same experiment (Olhoft et al., 2001), we can
conclude that multiple integrations of the T-DNA are due
to the nature of the binary plasmid. Usually, the
correlation between transcript level and T-DNA copy
number is known in the transgenic plant. However, we
guess that differences in the amount of specific transcript
for foreign gene could be due to the promoter used or to
a position effect, rather than to the number of transgene
copies. One of the most commonly used methods to
estimate the expression level of the recombinant antibody
produced in transgenic plants is the enzyme-linked
immunosorbent assay. In this work, we reported the
validation of an enzyme (alkaline phosphatase)-linked
immunosorbent assay to determine a plant-derived
antibody directed against the MUC1 concentrations up to
2.5 ng//50 µg of total protein. The binding between MUC1
synthetic peptide and the plant-derived antibody in this
assay was not affected by the impurities of the samples,
demonstrating that our assay is free from interference in
the range of concentrations. This VHH appears to be an
intrinsically stable molecule, able to accumulate in the
plant cell cytosol and to maintain its functionality. At
present, the expression levels of a foreign antibody in
transformed plants have been shown in relatively small
amounts (0.01 to 0.1% of the total soluble protein). The
highest expression level for the cholera toxin B subunit in
transgenic tobacco chloroplasts, up to 4.1% of the total
soluble protein has been reported (Daniell et al., 2001).
The use of different promoters (Arakawa et al., 2001;
Chong et al., 1997), the use of plant-derived leader
sequences and signal peptides (Kong et al., 2001) and
mainly the modification of codon usage of the foreign
protein (Mason et al., 1980) could improve expression
levels in plants. More studies are required to use the
different subcloning procedures by different plant vectors,
and optimize the expression and production of VHH gene
in plants and the extraction/purification procedures that
have substantial impacts on the final outcome
ACKNOWLEDGEMENTS
The authors thanks Dr. Reza Sheikhnejad (ProNAi
Therapeutics Inc., Kalamazoo, Michigan, USA) for his
valuable advice and support in this study.
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African Journal of Biotechnology Vol. 10(20), pp. 4242-4248, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2343
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Aloe arborescens aqueous gel extract alters the
activities of key hepatic enzymes and blood
concentration of triglycerides, glucose and insulin in
alloxan-induced diabetic rats
Motetelo Alfred Mogale 1 , Sogolo Lucky Lebelo 2 , Leshweni Jeremia Shai 3 * and Jacobus
Nicholaas Eloff 4
1 Department of Physiology, Faculty of Medicine, University of Limpopo, P. O. Box 130, Medunsa 0204, South Africa.
2 Department of Life and Consumer Sciences, University of South Africa, Private Bar X6, Florida 1710, South Africa.
3 Department of Biomedical Sciences, Tshwane University of Technology, Acardia campus, Pretoria, South Africa.
4 Phytomedicine Programme, University of Pretoria, Onderstepoort, South Africa.
Accepted 22 February, 2011
The present study investigated the antidiabetic activity and the possible mechanisms of action of
aqueous extract of Aloe arborescens leaf gel (AALGEt) on normal and alloxan-induced diabetic rats.
Diabetes was induced in 12 h fasted rats by intraperitoneal injection of 140 mg/kg body weight of
alloxan. Blood glucose levels, body weight and water intake were determined on day 7, 14 and 21 of
AALGEt treatment. Plasma insulin and triglycerides levels, as well as activities of hepatic glucokinase
and glucose 6-phosphatase (G6Pase) were determined at the end of the study. Blood glucose levels,
plasma triglyceride and insulin levels, as well as the activity of hepatic G6Pase were significantly
increased in diabetic rats. With the exception of hepatic glucokinase activity, daily oral administration
of AALGEt to diabetic rats significantly reversed the effects induced by alloxan. The activities of
glucokinase and glucose-6-phosphatase as well as plasma insulin levels in AALGEt-treated normal rats
were comparable with those observed in untreated normal rats. The results suggest that AALGEt
ameliorates physiological parameters altered by the diabetic state. These effects may be mediated in
part, through the protection of pancreatic beta cells from further damage by alloxan.
Keywords: Aloe arborescens, alloxan, diabetic rats, antidiabetic activity, hepatic enzymes.
INTRODUCTION
Ethnomedical reports suggest that more than 1200 plants
are used to control diabetes mellitus in traditional medicinal
systems of different cultures, worldwide (Marles and
Farnsworth, 1995; Grover et al., 2002). The hypoglycemic
*Corresponding author. E-mail: shailj@tut.ac.za. Tel: +2712 382
6342. Fax: +2712 382 6342.
Abbreviations: AALGEt, Aqueous Aloe arborescens leaf gel
extract; FFAs, Free fatty acids; G6Pase, glucose 6phosphatase;
HDL, high density lipoprotein; HSL, hormone
sensitive lipase; LPL, lipoprotein lipase; SANBI, South African
National Biodiversity Institute.
effects of a large number of these plants have been
evaluated and confirmed in animal models of diabetes
(Day and Bailey, 2006; Frode and Medeiros, 2006) as
well as in clinical studies (Jayawardena et al., 2005; Day
and Bailey, 2006). However, many of these plants still
need to be validated and characterized in terms of
toxicity, active principles and hypoglycemic mechanism of
action.
Aloe arborescens Miller., also known as Krantz aloe, or
Kidachi aloe, is a multi-stemmed succulent shrub with a
height of about 3 m. It belongs to the Asphodelaceae
family (van Wyk and Smith, 2004; Smith et al., 2008). It is
indigenous to southern Africa but it is reported to be
cultivated all over the world (Smith et al., 2008). Some
black communities in the Northwest Province (Odi district)

of South Africa use the leaf components (gel and juice) of
A. arborescens as antidiabetic remedies (Mogale,
personal communication). The hypoglycemic activity of A.
arborescens leaf components as evaluated and confirmed
in experimental animal models of diabetes (Beppu et al.,
1993; 2006) are attributed to the presence of the
polysaccharides, arboran A and B in the leaves of this
plant (Hikino et al., 1986; Beppu et al., 2006). Katerere
and Eloff (2005) also discussed the use of Aloes in
diabetes. However, the mechanism whereby A.
arborescens leaf components exert their hypoglycaemic
effect remains unknown.
An antidiabetic agent may be effective in reducing
blood glucose concentration through: 1) stimulating
insulin secretion from pancreatic beta-cells; 2) enhancing
glucose uptake by fat and muscle cells; 3) altering the
activity of some enzymes (e.g. α-glucosidase, α-amylase,
glucokinase and glucose-6-phosphatase) that are
involved in glucose metabolism and 4) slowing down the
absorption of sugars from the gut (Tanira, 1994; Klover
and Mooney, 2004; Cheng and Fantus, 2005). The
present study was undertaken to investigate the effects of
aqueous A. arborescens leaf gel extract on fasting blood
glucose levels, insulin secretion and activities of selected
hepatic enzymes in normal and alloxan-induced diabetic
rats.
MATERIALS AND METHODS
Plant material and preparation of AALGEt
Leaves of A. arborescens were collected from a private garden in
the North West province of the Republic of South Africa and
confirmed as those of A. arborescens (Miller) by the South African
National Biodiversity Institute (SANBI) (Genspec no. 2206- 12).
Leaf gels were dried and milled into a fine powder after which
AALGEt was prepared by homogenizing 25 g of the powder in 250
ml of water, followed by centrifugation at 3000 xg for 20 min. The
resulting supernatants were pooled and stored at 4°C until use.
Animals and induction of diabetes
Male albino Wistar rats weighing 220 to 280 g were obtained from
the animal facility of the University of Cape Town, South Africa. All
animals were kept in individual cages in an environmentally
controlled room with a 12 h light/12 h dark cycle. The animals had
free access to water and standard rat diet. The study was approved
by the Ethics Committee of the University of Limpopo, South Africa
(Animal Ethics Approval number AEC09/06). Diabetes mellitus was
induced in 12 h fasted animals by intraperitoneal injection of alloxan
monohydrate (Sigma, St. Louis, MO., USA) dissolved in sterile
normal saline at a dose of 140 mg/kg body weight. Since alloxan is
capable of producing fatal hypoglycaemia as a result of massive
pancreatic insulin release (Szudelski, 2001; Dhandapani et al.,
2002), rats were treated with 20% glucose solution intraperitoneally,
8 h after alloxan treatment. The rats were then kept for the next 24
h in their cages with 5% glucose in dispenser bottles to prevent
hypoglycaemia (Dhandapani et al., 2002). Diabetes was confirmed
in rats by measuring fasting blood glucose, 72 h post alloxan
treatment. Rats with marked hyperglycaemia (blood glucose level
above 11.0 mM) were selected for use in the study. The highest
Mogale et al. 4243
blood glucose concentration measured in a diabetic rat was 25 mM.
Experimental procedure
The experiment was carried out using four groups of eight rats
each: Group I (untreated normal rats), group II (treated normal rats),
group III (untreated diabetic rats) and group IV (treated diabetic
rats). Experimental animals (groups II and IV) received a daily dose
of 300 mg/kg body weight of AALGEt incorporated in their drinking
water for 3 weeks. Control animals received water without AALGEt
during the same period. Fasting blood glucose levels were
determined by means of MediSense ’s Optimum Xceed Diabetes
Monitoring system and blood glucose test strips before treatment
(day 0) and on day 1 (4 h), day 7, day 14 and day 21 after initiation
of treatment. Body weights and water intake of all groups of rats
were assessed on the same days that blood glucose levels were
measured.
On day 22, blood was withdrawn from the heart of each rat under
general anaesthesia. The collected blood was heparinised and
centrifuged for 20 min at 3000 xg and used for the mea-surement of
plasma glucose, plasma triglycerides and plasma insulin. Following
blood collection, rats were euthanized using sodium thiopentone
(200 mg/kg). Liver tissue was removed from each animal, washed
with saline and stored at -70°C until used for the assay of
glucokinase and glucose-6-phosphatase activities.
Determination of plasma glucose, triglycerides and insulin
levels
Plasma glucose and plasma triglyceride levels were measured
using commercially available kits based, respectively on the glucose
oxidase and the glycerol blanked methods. Both methods
were adapted to the Beckman Coulter®’s UniCell DXC 800
Synchron® Clinical System. Plasma insulin levels were determined
by the enzyme linked immunosorbant assay (ELISA) adapted to the
Beckman Coulter® Ireland Inc’s UniCell DXI 800 Access®
Immunoassay System.
Measurement of hepatic glucokinase activity
About 500 mg liver tissues from each rat was homogenized in 5 ml
ice-cold homogenization buffer containing 100 mM KCl and 20 mM
HEPES (pH 7.9). The homogenates were centrifuged at 3000 xg for
20 min at 4°C. Glucokinase activity in the supernatants was
measured by the pH sensitive colorimetric assay published by
Chapman and Wong (2002) in a reaction mixture containing 20 mM
HEPES pH 7.9, 5 mM ATP, 5 mM MgCl2, 5 mM D-glucose and 0.2
mM Cresol Red. Absorbance measurements were taken at 570 nm
after incubation at 37°C for 30 min. The glucokinase activity in the
supernatants was expressed as units/mg protein. One unit of
glucokinase is defined as the amount of protons liberated/minute at
37°C under the specified assay conditions. Protein concentration in
supernatants was assayed by the method of Lowry et al. (1951)
using bovine serum albumin as a standard.
Assay of hepatic glucose-6-phosphatase activity
Liver microsomal fractions were prepared according to the ultracentrifugation
method published by Pari and Satheesh (2006).
Briefly, rat liver (1.0 g) was homogenized in 5 ml ice-cold 5 mM
HEPES (pH 7.4) containing 0.25 M sucrose. The homogenate was
centrifuged at 10 000 ×g for 20 min, at 4°C after which the
supernatant was centrifuged again at 105 000 ×g for 60 min. The
microsomal pellets obtained were re-suspended in same buffer and

4244 Afr. J. Biotechnol.
Table 1. Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on blood glucose
levels of normal and alloxan-induced diabetic rats.
Rat groups
Average blood glucose (mmol/L)
Day 0 Day 7 Day 14 Day 21
Normal control rats 4.1 ±0.5 5.9 ±0.6 4.7±0.9 4.8 ±0.7
Normal + AALGEt 4.3 ±1.3 5.2 ±0.8 4.6 ±0.5 5.1 ±0.7
Diabetic control rats 1.5 ±1.3 2.2 ±8.2 >27.8** >27.8**
Diabetics + AALGEt 20.1 ±4.2 1.2 ±5.6 ¥ 9.4 ±4.3 ¥¥ 7.2 ±6.8 ¥¥
Each value represents ± SD, n = 8. Mean values were significantly different from those of normal control
rats. *p < 0.01, **p < 0.001. Mean values were significantly different as compared to diabetic control
rats, ¥ p < 0.01, ¥¥ p < 0.001.
their glucose-6-phosphatase enzyme activity was assayed according
to the method developed by Baginski et al. (1974). In this
method, glucose-6-phosphate is converted by glucose-6-phosphatase
(G6Pase) in liver microsomal fractions into glucose and
inorganic phosphate. The inorganic phosphate liberated is allowed
to react with ammonium molybdate in the presence of ascorbic acid
(Fiske and Subbarow, 1925). The amount of phosphate liberated
per unit time determined as the blue phosphomolybdenum complex
at 700 nm is a measure of G6Pase. G6Pase activity was expressed
as units/mg of protein. One unit of glucose-6-phosphatase activity
was defined as the amount of Pi liberated/min at 37°C under the
specified assay conditions.
Water consumption and body weight determination
Daily water consumption was determined by calculating the difference
between amount of water (200 ml) supplied and the volume
of water remaining after 24 h. Body weight was determined
gravimetrically.
Statistical analysis
Data expressed as mean ± SD were analyzed using the Sigma Stat
statistical program (version 8.0). Comparisons were made between
normal and alloxan-induced diabetic rats as well as between
treated and untreated alloxan-induced diabetic rats by means of
unpaired Student’s t-test and their significance were established by
analysis of variance analysis (ANOVA). Differences of p < 0.05
were considered statistically significant.
RESULTS
There was no significant difference in the fasting blood
glucose levels of normal rats treated with AALGEt when
compared with that of normal untreated rats (Table 1).
However, a steady increase in the blood glucose levels of
diabetic control rats was observed throughout the entire
experiment. The increase in the blood glucose levels of
untreated diabetic rats was significantly higher on day 7
(p < 0.01), day 14 (p < 0.001) and day 21 (p < 0.001)
when compared with normal control rats. Treatment of
diabetic rats with AALGEt for 3 weeks significantly
reduced their blood glucose levels when compared with
those of diabetic control rats on day 7 (p < 0.01), day 14
(p < 0.001) and day 21 (p < 0.001).
At the end of the experimental period, plasma glucose,
triglyceride and insulin were measured. Plasma glucose
and triglyceride levels in untreated diabetic animals were
significantly higher (p < 0.001 and p < 0.05, respectively)
(Figure 1A and B).
Treatment of diabetic rats with AALGEt for 3 weeks
significantly reduced plasma glucose (p < 0.01) by 66.7%
and plasma triglycerides by 25% when compared with
untreated diabetic rats. Oral administration of AALGEt to
diabetic rats for 3 weeks significantly increased plasma
insulin levels when compared to diabetic controls (Figure
1C).
Changes in body weight and water intake of normal
control, treated normal rats, diabetic controls and
AALGEt treated diabetic rats on days 1, 14 and 21 of
treatment are shown in Figure 2A and B. The changes in
the activities of hepatic glucokinase (GK) and G6Pase in
normal and diabetic control are shown in Table 2. Alloxan
treatment caused a decrease in the hepatic GK activity
and a significant increase (p < 0.05) in the activity of
hepatic G6Pase of diabetic rats when compared to
normal control rats (Table 2). Furthermore, oral administration
of AALGEt to normal and diabetic rats did not
cause any significant difference in the activity of hepatic
GK. On the other hand, oral administration of AALGEt for
3 weeks resulted in a significant decrease (p < 0.05) in
hepatic G6Pase activity.
A significant decrease in body weight was observed in
untreated diabetic rats when compared with normal
control rats on day 14 (p < 0.01) and day 21 (p < 0.001)
(Figure 2A). There was a significant increase in the water
intake of diabetic rats than in normal control rats on days
7, 14 and 21 (p < 0.05, p < 0.01 and p < 0.001, respecttively)
(Figure 2B). Treatment of diabetic rats with
AALGEt did not significantly attenuate the observed
weight loss. However, treatment of diabetic rats with
AALGEt significantly reduced the water intake to lower
levels than untreated diabetic rats (p < 0.01 on day 14
and p < 0.001 on day 21).

Plasma glucose (mM/L)
50Normal controls
AALGEt treated normals
2.0
Diabetic controls
AALGEt treated diabetics 1.8 B
40
30
20
10
0
A
Rat groups
**
¥¥
Plasma triglyceride (mmol/L)
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
B
Rat groups
Plasma insulin (umol/L)
250
250
200
150
100
50
0
Mogale et al. 4245
Rat groups
Figure 1. Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on plasma glucose (A), plasma triglyceride (B) and
plasma insulin (C) levels of normal and alloxan-induced diabetic rats. Mean values were significantly different as compared to normal
Figure 1: Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on plasma glucose (A)
plasma triglyceride (B) and plasma insulin (C) levels of normal and alloxan-induced diabetic rats. Mean
values were significantly different compared to normal control rats, * P < 0.05, ** P < 0.01. Mean values
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,
P < 0.01.
¥ P < 0.05, ¥¥ p < 0.01.
DISCUSSION
mice.
Alloxan induces diabetes by damaging the insulin secreting
cells of the pancreas leading to hypoinsulinemia and
hyperglycaemia (Szudelski, 2001). In agreement with
these known effects of alloxan, the blood glucose levels
of untreated alloxan-induced diabetic rats were significantly
increased throughout the current study when
compared to those of normal control rats. Continuous
treatment of alloxan-induced diabetic rats with AALGEt
(300 mg/kg body weight) for a period of 3 weeks resulted
in a significant decrease in the blood glucose levels of
treated diabetic rats when compared to diabetic controls
but no such effect was observed in normal treated rats.
Thus, unlike the use of insulin or sulfonylurea drugs,
which cause severe hypoglycaemia when taken in excessive
doses (Bastaki, 2005; Cheng and Fantus, 2005),
continuous use of AALGEt or its accidental overdose will
not result in hypoglycaemic shock. Furthermore, results
of the current study suggest that the blood glucose
lowering effect of AALGEt can last for a longer period of
time. These observations concur with the findings of
Beppu et al. (1993, 2006) in alloxan-induced diabetic
*
¥
Continuous treatment of alloxan-induced diabetic rats
with AALGEt (300 mg/kg body weight) for 3 weeks also
significantly increased (but did not normalize) the plasma
insulin levels of diabetic rats as compared to diabetic
controls. However, AALGEt did not significantly alter
plasma insulin levels of normal rats. Thus, like the aloe
vera leaf gel extract (Reynolds and Dweck,1999),
AALGEt might exert its blood glucose lowering effects
through stimulation of insulin secretion from pancreatic
beta cells. However, the fact that AALGEt did not
significantly alter the plasma insulin levels of normal rats
but significantly increased plasma insulin levels of alloxan
induced-diabetic rats towards the normal value, suggest
that, AALGEt may either regenerate damaged pancreatic
beta cells or protect them from further damage by alloxan.
Since alloxan is known to exert its diabetogenic
effect through generation of oxygen free radicals, this
hypothesis is in agreement with the findings of Beppu et
al. (2003) and Chacko et al. (2008), who reported that A.
arborescens components possess free radical scavenging
effects that may prevent pancreatic islet beta-cell
destruction by free radicals generated by alloxan. Thus,
C
**
C
¥

4246 Afr. J. Biotechnol.
Water intake (ml/rat/day)
Figure 2. Effects of 3 weeks treatment with AALGEt (300 mg/kg body weight) on body weight (A) and water intake (B) of
normal and alloxan-induced diabetic rats. Mean values were significantly different compared to normal control rats, * p <
0.05, *** p < 0.001. Mean values were significantly different compared to diabetic control rats, ¥ p < 0.05, ¥¥ p < 0.01, ¥¥¥ p <
0.001.
Table 2. Effects of treatment with AALGEt (300 mg/kg body weight) on the activities of hepatic glucokinase
and glucose-6-phosphatase
Rat groups Glucokinase (U/g protein) Glucose 6- phosphatase (U/mg protein)
Normal control rats 298.01 ± 11.4 0.198 ± 0.05
Normal + AALGEt 304.56 ± 8.34 0.213 ± 0.06
Diabetic control rats 282.82 ± 6.18 0.521 ± 0.02*
Diabetics + AALGEt 288.13 ± 9.81 0.282 ± 0.06 ¥
Each value represents ± SD, n = 8. Mean value was significantly different from those of normal control rats. *p <
0.05. Mean value was significantly different as compared to diabetic control rats, ¥ p < 0.05.
in this respect, AALGEt probably act like plant ex-tracts of
Gymnema sylverstre (Chattopadhyay, 1998) and Ipomea
batatas L. (Kusano and Abe, 2000) which are reported to
repair pancreatic islet cells damaged by either alloxan or
streptozotocin (Bnouham et al., 2006).
Uncontrolled diabetes mellitus is often associated with
abnormal plasma lipid levels, in particular, elevated
plasma triglyceride levels and reduced plasma HDLcholesterol
levels (Sniderman et al., 2002; Mooradian,
2009). Hypertriglyceridemia represents an independent
risk factor for the development of coronary heart disease
250
200
150
100
50
0
in people with type 2 diabetes (Ooi and Ooi, 1998;
Sniderman et al., 2002). We have observed in this study,
that continuous treatment of alloxan-induced diabetic rats
with AALGEt (300 mg/kg body weight) for a period of 3
weeks significantly reduced their plasma triglyceride
levels when compared with diabetic control rats. Under
normal circumstances, insulin inhibits the enzyme hormone
sensitive lipase (HSL), which mobilizes free fatty
acids (FFAs) from adipose tissues (Kwiterovich, 2000;
Smith et al., 2005). FFAs released by the action of HSL
are the major substrates for the hepatic triglyceride

synthesis (Kwiterovich, 2000). Insulin also activates the
enzyme lipoprotein lipase (LPL), which clears triglyceride
rich proteins from blood plasma (Kwiterovich, 2000;
Smith et al., 2005). However, in a diabetic state, HSL is
not inhibited and LPL is not activated due to insulin
deficiency (Kwiterovich, 2000; Mlinar et al., 2007). Thus,
the effect of AALGEt on plasma triglyceride levels could
be attributed to its effects on plasma insulin levels.
Hepatic glycogenolysis and gluconeogenesis are major
causes of fasting hyperglycemia seen in both type 1 and
type 2 diabetes mellitus (Smith et al., 2005). Inhibition of
enzymes involved in gluconeogenesis and/or glycolgenolysis
therefore constitutes an alternative approach to
suppress hepatic glucose production and lower fasting
plasma glucose (Agius, 2007). G6Pase catalyses the
final reaction in hepatic glucose production by both gluconeogenesis
and glycogenolysis, and has been proposed
as a potential target for antihyperglycaemic drugs for
type-2 diabetes (Pari and Satheesh, 2006; Agius, 2007).
A significant increase in the activity of the hepatic
G6Pase of untreated alloxan-induced diabetic rats as
compared to normal rats was observed in the current
study. Treatment of diabetic rats with AALGEt (300 mg/kg
body weight) for 3 weeks resulted in a significant
reduction of GPAase activity when compared to diabetic
controls. Thus, AALGEt could contain substances that act
like the oral hypoglycaemic agent, metformin (Bastaki,
2005; Cheng and Fantus, 2005) and plant extracts of
Boerhaavia diffusa L. (Pari and Satheesh, 2004) and
Pterocarpus marssupium (Pari and Satheesh, 2006)
which have been shown to lower blood sugar levels by
inhibiting hepatic production of glucose.
Glucokinase is a rate limiting enzyme involved in the
hepatic storage and utilization of glucose (Smith et al.,
2005). Following a carbohydrate rich meal, hepatic GK
clears a significant amount of glucose from the blood
circulation and facilitates its conversion into glycogen and
fatty acids (Smith et al., 2005; Agius, 2007). Thus,
hepatic GK play a significant role in the prevention of
postprandial hyperglycemia. The activity of hepatic GK is
reported to be reduced in alloxan-induced experimental
diabetes (Zhang et al., 2007) and some antidiabetic plant
extracts are reported to exert their hypoglycemic activity,
in part by increasing the activity of this enzyme (Pari and
Satheesh, 2006). In agreement with the known effects of
alloxan on the activity of GK (Zhang et al., 2007), we
have observed in this study that the hepatic GK activity
was significantly reduced when compared to normal
controls. However, oral administration of AALGEt (300
mg/kg body weight) to both normal and alloxan-induced
diabetic rats for 3 weeks did not significantly alter the
activity of hepatic GK in these animals.
Uncontrolled diabetes mellitus is also associated with
body weight loss, polyurea, polydypsia and polyphagia
(Bastaki, 2005). Weight loss in diabetes mellitus results
from a combination of dehydration (caused by frequent
urination), increased breakdown of muscle proteins (for
Mogale et al. 4247
provision of gluconeogenic amino acids) and enhanced
mobilization of fat stores (provision of FFAs to be used as
fuel) (Bastaki, 2005; Smith et al., 2005). These events
are directly or indirectly related to insulin deficiency or
lack of insulin actions (Smith et al., 2005). We have
observed a significant decrease in the body weight of
untreated alloxan-induced diabetic rats as compared to
normal control rats. The loss of weight in alloxan-induced
diabetic rats was accompanied by increased water intake
(polydypsia) by these rats, suggesting that dehydration
was a contributing factor towards weight loss. Continuous
treatment of alloxan-induced diabetic rats with AALGEt
for 3 weeks significantly prevented body weight loss.
However, the mean body weight of AALGEt treated rats
was still significantly reduced when compared to that of
the normal control rats. The marked improvement in body
weight loss of treated diabetic rats as compared to
untreated alloxan induced diabetic rats could also be
attributed to the effect of AALGEt on plasma insulin levels
and hence on improved glucose metabolism in these
rats.
The findings of this study suggest that oral administration
of an aqueous extract of the leaf gel of A.
arborescens (AALGEt) (300 mg/kg body weight) for 3
weeks brings about significant beneficial effects in
various physiological/biochemical parameters altered by
the diabetic state. Although, the exact chemical compound(s)
responsible for the antidiabetic activity of
AALGEt are currently unknown, AALGEt appears to exert
its blood glucose lowering effect in alloxan-induced
diabetic rats by protecting insulin secreting pancreatic
beta cell from further damage by alloxan. Further studies,
in particular, histological studies of pancreatic beta cells
of AALGEt treated alloxan-induced diabetic rats are
needed in order to confirm this hypothesis. Furthermore,
the isolation and identification of active compounds
AALGEt will go a long way in promoting the use and
acceptance of A. arborescens leaf as an antidiabetic
remedy. Although, lower doses may also have led to a
positive effect, the dosage that was used in these experiments
(equivalent to 2.4 g/day per 80 kg human) were
probably higher than the dosage used by traditional
healers. In general, these results strongly support the
traditional use and preparation of AALGEt for treating
diabetes in humans.
ACKNOWLEDGEMENTS
The authors would like to thank Mrs A. Freitas for her
assistance in handling and maintaining the animals. The
study was funded by the Department of Physiology,
University of Limpopo, South Africa. The authors would
also like to thank Prof Leon Hay, Head of the Department,
for making funds available for this study. L.J. Shai
is a recipient of NRF funding under the Thutuka-REDIBA
programme.

4248 Afr. J. Biotechnol.
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African Journal of Biotechnology Vol. 10(20), pp. 4249-4255, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2158
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Increased vascular endothelial growth factor (VEGF)
expression in rats with spinal cord injury by
transplantation of bone marrow stromal cells
Deshui Yu 1,3 , Libo Liu 2 , Xiaodong Zhi 3 , Yang Cao 3 and Gang lv 1 *
1 Department of Orthopaedics, the First Affiliated Hospital, China Medical University, Shenyang 110001, People’s
Republic of China.
2 Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang 110001, People’s
Republic of China.
3 Department of Orthopaedics, The First Affiliated Hospital, Liaoning Medical University, Jinzhou 121001, People’s
Republic of China.
Accepted 24 March, 2011
Vascular endothelial growth factor (VEGF), a well known angiogenic factor, has been shown to have
direct and/or indirect influence on spinal cord injury (SCI). The purpose of this study is to observe
VEGF expression changes in rats with SCI by bone marrow stromal cells (BMSCs) treatment. The mRNA
expression of VEGF in rats was analyzed by semi-quantitative RT- PCR and the protein expression level
was quantified by means of western blot and immunohistochemistry technology. It was found that
VEGF was significantly up-regulated in BMSCs treatment group in comparison to the sham group and
DMEM group on mRNA level. On protein level, VEGF was also highly increased in BMSCs treatment
group, which reached the highest level after 14 days treatment. The present study suggests a potential
local role for VEGF as mediators of SCI which might provide a certain reasonable clue to clarify
molecular mechanisms of BMSCs treatment in SCI.
Key words: Vascular endothelial growth factor (VEGF), spinal cord injury, bone marrow stromal cells, reverse
transcription-polymerase chain reaction (RT-PCR), western blot, immunohistochemistry.
INTRODUCTION
Each year, many people worldwide suffer from spinal
cord injury (SCI). These injuries cause death of neural
cells, severance and demyelination of descending and
ascending axons, and consequently, loss of motor and
sensory function. Endogenous repair efforts fail to repair
*Corresponding author. Email: ganglv514@yahoo.com.cn. Tel:
+86-0416-467-3886. Fax: +86-0416-467-3528.
Abbreviations: SCI, Spinal cord injury; BMSCs, bone marrow
stromal cells; DMEM, Dulbecco's modified Eagle's medium;
FBS, fetal bovine serum; RT, room temperature; DEPC,
diethylpyrocarbonate; VEGF, vascular endothelial growth factor;
PCR, polymerase chain reaction; IDV, integrated densities
value; EDTA, ethylenediaminetetraacetic acid; DAB,
diaminobenzidine; COX-2, cyclooxygenase-2.
the spinal cord and, as a result, the functional impairments
are permanent (Wrigley et al., 2009; Wollaars et al.,
2007). Potential treatments for SCI are being tested in
the clinic but so far, none of these have emerged as one
that reverses the devastating functional consequences of
SCI, however, several studied demonstrated that bone
marrow stromal cells (BMSCs) may also have therapeutic
promise for SCI (Shields et al., 2010; Bunge., 2008).
Survival, migration and differentiation of BMSCs after
transplantation have been widely researched in recent
years (Shields et al., 2010; Bunge, 2008; Someya et al.,
2008). The underlying molecular mechanisms of repairing
injured spinal cord and improving functional recovery with
BMSCs have not, however, been largely understood.
Better understanding of the molecular mechanism of
BMSCs treatment for SCI may contribute significantly to
the development of BMSCs reagents as well as help in

4250 Afr. J. Biotechnol.
SCI control. Consequently, it is of vital importance to
define the molecular mechanisms of BMSCs treatment
for successful treatment of SCI.
Angiogenesis plays an important role in increasing
blood flow at the lesioned site, where hypoxia occurs
after SCI and is an essential component of nerve regeneration
across a gap of injured spinal cord (Carmeliet et
al., 2002). Similarly, neo-angiogenesis is also essential
for trauma repair, which requires nutrients and oxygen to
overcome hypoxia (Carmeliet et al., 2002). It has been
reported that SCI may induce angiogenesis (Casella et
al., 2002), however, under normal circumstances after
SCI, the angiogenesis of the injured spinal cord is
insufficient to support the spinal cord plasticity required
for functional recovery. Recent data from the medical
literature have also shown a positive correlation between
the density of blood vessels and functional improvement
following SCI (Blight., 1991; Widenfalk et al., 2003).
These findings suggest that stimulating angiogenesis
may improve recovery of function after SCI. BMSCs have
the ability to stimulate angiogenesis, which had been
reported in recent medical literature (Tang et al., 2006;
Chen et al., 2003; Kayo et al., 2005). BMSC may
promote angiogenesis through VEGF expression in the
spinal cord, according to the results of the present study,
which may contributes to shed more light on the
molecular mechanism of BMSCs treatment in SCI.
In this study, we investigated whether BMSCs transplantation
could induce the expression of VEGF in the
injured spinal cord and provided a certain reasonable
clue that shed more light on the molecular mechanisms
of BMSCs treatment in SCI.
MATERIALS AND METHODS
The adult male Wistar rats (12 weeks, 250 to 300 g) were
purchased from the Center for Experimental Animals of China
Medical University. All animal experiments were carried out in
accordance with the National Institute of Health Guide for the Care
and Use of Laboratory Animals.
Culture and identification of MSCs
BMSCs were isolated from rat bone marrow and cultured as
described by previous literature (Yano et al., 2005). Briefly, fresh
whole bone marrow was harvested aseptically from tibias and
femurs by 5 ml syringe needle. The marrow was extruded with 5 ml
of Dulbecco's modified Eagle's medium (DMEM)-low glucose
medium. Bone marrow was mechanically dissociated to obtain a
homogeneous cell suspension. The cells were placed in a 25 cm 2
plastic flask for cell culture with 5 ml DMEM-low glucose medium
containing 10% fetal bovine serum (FBS). The cells were incubated
at 37°C in 95% humidity and 5% CO2 for 24 h. At this time, nonadherent
cells were removed by replacing the medium. The culture
medium was replaced three times a week. After the cultures have
reached 80 ~ 90% confluency, they were lifted by incubation in a
solution containing 0.25% trypsin for 5 min at 37°C for passage.
Cultured BMSCs were indentified with antibodies against CD34 and
CD44 according to previous literature (Yano et al., 2005).
Spinal cord injury model preparation and intramedullary spinal
cord injection
The adult male Wistar rats (12 weeks, 250 to 300 g) were
anesthetized with pentobarbital and laminectomies were performed
at the T8 to T10 level. Spinal cords were injured using the weight
drop technique according to Allen’s method (Allen, 1914) with a
slight modification. A plastic impounder (2-mm diameter) was
placed gently on the exposed dura and a 10-giron weight was
dropped from a height of 10 cm onto the impounder. The weight
and impounder were immediately removed after impact and
paravertebral muscle and skin were closed. Rats were randomly
divided into three groups: Sham group, DMEM group and BMSCs
group.
Thirty minutes after injury, rats received four injections of a
suspension of BMSCs (passage 3 ~ 4 cells, 75000 in 3µl of DMEM
each) into peri-lesion area of the exposed spinal cord (BMSCs
group). Two injections were applied 1 mm rostral and two injections
1 mm caudal to the lesion epicenter, and one injection at each side
0.5 mm lateral to the cord midline. The microneedler was carefully
inserted from the dorsal surface 1.75, 1.25 and 0.75 mm deep into
the spinal cord. Each rat received a total of 3.0 × 10 5 BMSCs. In
DMEM group, the rats received four injections of an equal volume
of DMEM alone at the same position and depth. Sham controls
underwent the same operations but without spinal cord insult. Rats
used in this study were treated strictly according to the NIH Guide
for Care and Use of Laboratory Animals.
RNA preparation and semi-quantitative RT-PCR
Total RNA was isolated from frozen spinal tissue using TRIzol
reagent (Invitrogen Corp., Carlsbad, CA) according to the
manufacturer’s protocol and as described in the online supplement.
In short, the spinal tissue was ground with mortar and pestle,
cooled by liquid nitrogen of the ground tissue, and 100 mg was
incubated with 1 ml TRIzol for 5 min at room temperature (RT). Cell
debris was removed by centrifugation (12,000 ×g at 4°C for 10 min)
and 0.4 ml chloroform was added. After vortexing, the mix was
incubated for 5 min at RT. The phases were separated by
centrifugation (12,000 ×g at 4°C for 15 min) and the aqueous phase
was transferred to a new tube. 0.6× volume of isopropyl alcohol and
a 0.1× volume of 3 M sodium acetate were added to this aqueous
phase and incubated for 10 min at 4°C. The precipitated RNA was
pelleted by centrifugation (12,000 ×g at 4°C for 15 min) and after
the removal of the supernatant, the RNA pellet was washed twice
with 70% ethanol. After drying, the RNA was resuspended in 30 µl
diethylpyrocarbonate (DEPC)-treated water. The quality and
quantity of the RNA was verified by the presence of two discrete
electropherogram peaks corresponding to the 28S and 18S rRNA
at a ratio approaching 2:1. Using mRNA as template, singlestranded
cDNAs were generated by Superscript II reverse
transcriptase (Invitrogen) according to the manufacturer’s
directions. The vascular endothelial growth factor (VEGF) primer
sequences were as follows: Sense prime: 5′-GAGTATATCT
TCAAGCCGTCCTGT-3′; anti-sense prime: 5′- ATCTGCATAG
TGACGTTGCTCTC -3′. GAPDH (Applied Biosystems) served as
the internal control. The polymerase chain reaction (PCR)
conditions were 94°C for 3 min, followed by 30 cycles of DNA
amplification (45 s at 94°C, 1 min at 61°C and 1 min 30 s at 72°C)
and 8 min incubation at 72°C. PCR products were separated by
electrophoresis at a constant voltage (2 V/cm) in a 1.2% (w/v)
agarose gel. Images were captured using a Gel Print 2000i/VGA
(Bio Image), and the integrated densities value (IDV) was analyzed
with computerized image analysis system (Motic Images Advanced
3.2). All DNA manipulations were performed as described by
Sambrook and Russell, (2001).

Western blot
Yu et al. 4251
Figure 1. Bone marrow stromal cells, culture and detect. A, Third passage of bone marrow mesenchymal stem cells was
observed by inverted phase contrast microscope; B, the expression of CD44 in bone marrow stromal cells was observed
by phase contrast the inverted microscope
In order to detect protein expression level of VEGF, western blot
was performed as described in the online supplement. The spinal
cord tissues were obtained from the peri-lesion region at days 3, 7
and 14 post-surgery. Protein homogenates of spinal cord samples
were prepared by rapid homogenization in 10 volumes of lysis
buffer (2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM
ethylene glycol tetraacetic acid (EGTA), 0.4% NaF, 20 mM Tris-
HCl, pH7.5). Tissue homogenate were centrifuged at 17,000 xg for
1 h at 4°C and the protein concentration in the supernatant was
determined by the Coomassie (G250) binding method. Equal
amounts of protein (20 µg) from each sample were loaded and
separated into a 4 to 7.5% gradient sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing
conditions. Electroblotting proteins were transferred onto nitrocellulose
membranes (Santa Cruz Biotechnology, Inc). After
blocking with 5% nonfat dry milk overnight at 4°C, membranes were
incubated for 2 h at room temperature in agitation with the following
antibodies: rabbit polyclonal anti-VEGF (dilution 1:400; Santa Cruz
Biotechnology, Inc), and rabbit polyclonal anti-β-actin (dilution
1:400; Santa Cruz Biotechnology, Inc). Secondary horseradish
peroxidase conjugated rabbit anti-goat/goat anti-rabbit antibodies
(Santa Cruz Biotechnology, Inc) were used at 1:5000 dilution for 2 h
at room temperature in agitation. Immunoreactive bands were
visualized using the enhanced chemiluminescence (ECL kit, Santa
Cruz Biotechnology, Inc) and scanned using Chemi Imager 5500
V2.03 software. The integrated densities value (IDV) was analyzed
with computerized image analysis system (Fluor Chen 2.0) and
normalized with that of β-actin.
Immunohistochemistry
To detect expression and localization of VEGF in spinal cord tissue,
immunohistochemistry was performed. The cryostat sections were
thawed, air-dried and then blocked with goat serum for 30 min at
room temperature, and then incubated with rabbit polyclonal anti-
VEGF antibody (dilution 1:150, Santa Cruz Biotechnology, USA) at
4°C over night. Following reaction with primary antibodies, the
sections were stained using the ABC Kit (Santa Cruz Biotechnology,
USA), and the color was developed with diaminobenzidine
(DAB). Negative controls were conducted by exchange of primary
antibody for phosphate buffered saline (PBS).
Statistics analysis
To calculate the statistical differences among the sham, DMEM and
BMSCs groups, the statistical package SPSS13.0 (SPSS Incorporated,
USA) was used for all analysis. One-way analysis of
variance (ANOVA) followed by Bonferroni’s post hoc test were
utilized to determine the significant difference among multiple
groups. Student’s t test was used to determine the significance of
differences between the groups. All values were expressed as
mean ±SD. In general, p values less than 0.05 were considered
statistically significant.
RESULTS
BMSC cell culture and detection
Most cells suspended in culture medium, non-adherent
cells were removed by replacing the medium. Adherent
cells began to proliferate and form cell colony at about 72
h later. Cells surrounded the cell colony center to
distribute toward circumference. Cell morphology was
homogeneous, which appeared spindle-shaped with
serial sub-cultivation (Figure 1A). Most cultured adherent
passage 3 cells expressed CD44 (Figure 1B). In contrast,
a majority of adherent passage 3 cells were negative for
CD34.
Semi-quantitative RT-PCR analysis of VEGF
expression
In order to detect the mRNA expression of VEGF in rat
with SCI by BMSC treatment, reverse transcription PCR
was conducted. As shown in Figure 2, VEGF mRNA
levels in BMSCs group were significantly increased at 1,

4252 Afr. J. Biotechnol.
1 3 5
Day
Figure 2. The mRNA expression of VEGF at different time points in rats from each group
after transplantation. A, The expression of VEGF in different group by RT-PCR; M: marker; 1:
sham operation group; 2, 4, 6: DMEM group at transplantation after 1, 3, 5 days,
respectively; 3, 5, 7: BMSCs group at transplantation after 1, 3, 5 days, respectively. B: The
integrated density value of vascular endothelial growth factor mRNA at different time points in
rats from different group after transplantation. Different mark represent the significant
difference at p < 0.05
3 and 5 days after transplantation of BMSC as compared
to DMEM group (P < 0.05), moreover, DMEM group were
significantly higher than sham group (P < 0.05) in
different time. These results showed that transplantation
of BMSC could up-regulate VEGF mRNA expression.
Western blot analysis of VEGF expression
The western blot was performed to detect protein expression
of VEGF. The result showed that VEGF expression
was at a low level in the normal group rats. The
expression level of VEGF protein in the DMEM group rats
increased and peaked at 3 days (P < 0.01), then decreased
quickly with the extension of time, approaching the
expression levels of the sham group rats at 7 and 14
days (P < 0.05) after spinal cord injury. In the BMSCs
group rats, the up-regulation of VEGF protein expression
was observed at 7 and 14 days after SCI as compared
with that in DMEM group rats (P < 0.05), which were
time-dependent and reached the highest level at 14 days
after spinal cord injury (Figure 3).
Immunohistochemistry analysis of VEGF expression
In order to further confirm VEGF protein expression,
immunohistochemistry was conducted. The results
showed that there were no obvious differences (P < 0.05)
in VEGF expression between BMSCs and DMEM groups
rats at 3 day after transplantation (Table 1). The expression
level of VEGF in BMSCs group rats were increased

3 7 14
Day
Figure 3. The expression of VEGF at different time points in rats from each group after
transplantation in protein level. A: The expression of VEGF in different group by western blot; 1:
sham operation group; 2, 4, 6: DMEM group at transplantation after 3, 7, 14 days, respectively; 3,
5, 7: BMSCs group at transplantation after 3, 7, 14 days, respectively. B: The integrated density
value of vascular endothelial growth factor protein at different time points in rats from different
group after transplantation. Different mark represent the significant difference at p < 0.05.
Table 1. The mean optical density of VEGF at different time points in rat in all groups after the transplantation by
immunohistochemical staining (n = 6,x ± s). Different mark represent the significant difference at p < 0.05.
Time point after the
transplantation (day)
3 7 14
Sham group 0.1800±0.0061 0.1811±0.0089 0.1823±0.0063
DMEM group 0.3153±0.0098 ##
0.2381±0.0095 ## 0.1954±0.0038
BMSCs group 0.3150±0.0097 0.2520±0.0052* 0.2281±0.0079**
significantly at 7 and 14 days (P < 0.05) after
transplantation when compared with that in DMEM group
(Figure 4 and Table 1).
DISCUSSION
VEGF is essential for developmental angiogenesis and
Yu et al. 4253
plays important roles in adult animals to control vascular
permeability and homeostasis, blood pressure, and
pathological angiogenesis associated with wound healing
(Ku et al., 1993; Shibuya, 2006; Lee et al., 2007). Based
on its major role as an angiogenic factor and autocrine
growth factor for many diseases, therapeutic angiogenesis
drugs have been developed for clinical use by
improvement of VEGF expression. Previous studies

4254 Afr. J. Biotechnol.
Figure 4. The expression of VEGF by immunohistochemical staining at 7 days after transplantation. A, Sham operation group; B,
DMEM group; C, BMSCs group.
had shown that administration of VEGF could significantly
increase the microvascular density in central nervous
system (Blight., 1991; Widenfalk et al., 2003). In this
study, we found that BMSCs treatment not only increased
the level of VEGF expression, but also extended the time
of VEGF expression in SCI, which would strengthen the
biological effects of VEGF in promoting angiogenesis of
SCI, and this seems to be compatible with the reported
results that BMSCs implantation could induce
angiogenesis in a rat model of chronic hind limb ischemia
(Chen et al., 2003), acute myocardial infarction and
stroke (Rubén et al., 2006). The present study suggests
that VEGF probably plays a pivotal role in the treatment
of SCI by BMSC.
BMSCs are mesenchymal elements normally providing
structural and functional support for hemopoiesis
(Pittenger et al., 1999), which have the potential to differentiate
into other kinds of cells such as osteoblasts,
adipocytes, and chondrocytes (Pittenger et al., 1999;
Prockop, 1997), and produce a variety of neurotrophic
factors, cytokines, cell adhesion molecules, and growth
factor such as VEGF, thereby providing the pathway for
regenerating axons. BMSCs transplanted into a rat model
of chronic hind limb ischemia (Al-Khaldi et al., 2003),
acute myocardial infarction (Tang et al., 2006) and
ischemic brain (Chen et al., 2003) have provided their
beneficial effects by enhancing angiogenesis. The
angiogenesis stimulated by the BMSCs transplantation is
considered to at least in part, result from angiogenic
factors secreted by BMSCs and/or stimulating endogenous
parenchymal cells (Chen et al., 2003). VEGF is
one of the most potent angiogenic factors, capable of promoting
proliferation, migration and survival of endothelial
cells and plays the most important role in the initiation of
new blood vessel formation (Kayo et al., 2005; Breen.,
2007; Leung et al., 1989), therefore, VEGF may play
important role in the treatment of SCI by BMSC.
In conclusion, we have demonstrated that VEGF
expression was significantly up-regulated in BMSC group
in comparison to the normal group and DMEM group. The
current study provides a new approach for studying the
molecular mechanism underlying BMSCs treatment in
SCI. However, this molecular mechanism is largely
unknown; elucidation of these questions will depend on
further experiments. The next proposal of our study will
be to analyze whether VEGF expression could be accompanied
by cyclooxygenase-2 (COX-2), a protein relevant
in regulating pro-inflammatory processes, which have
been demonstrated to play a relevant role in VEGF
regulation (Siner et al., 2007). These data have to be
confirmed in future experiments, which would provide
further valuable clues to clarify molecular mechanisms of
BMSCs treatment in SCI.
ACKNOWLEDGEMENTS
We thank Dr. Yanfeng Wang for his assistance with
histological preparations. This work was supported by
Special Fund for Scientific Research of Doctor-degree
Subjects in Colleges and Universities of Ministry of Education
(20060159019) and Natural Science Foundation of
Liaoning Province (20052096).
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African Journal of Biotechnology Vol. 10(20), pp. 4256-4258, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2225
ISSN 1684–5315 © 2011 Academic Journals
Short Communication
In-ovo evaluation of the antiviral activity of methanolic
root-bark extract of the African Baobab (Adansonia
digitata Lin)
Sulaiman, Lanre K. 1,2 , Oladele, Omolade A. 2 *, Shittu, Ismaila A. 1 , Emikpe, Benjamin O. 3 ,
Oladokun, Agnes T. 1 and Meseko, Clement A. 1
1 Viral Research Department, National Veterinary Research Institute, Vom, Plateau State, Nigeria.
2 Department of Veterinary Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria.
3 Department of Veterinary Pathology, University of Ibadan, Ibadan, Oyo State, Nigeria.
Accepted 18 March, 2011
Application of ethnoveterinary medicine in the control of poultry diseases is being embraced in many
parts of the world for more profitable production. This study investigated the antiviral property of the
root-bark extract of the African Baobab tree (Adansonia digitata Lin) against Newcastle disease virus.
One hundred and seventy five specific antibody negative embryonated chicken eggs and a field strain
of Newcastle disease virus were used to test for the antiviral activity of the methanolic root bark extract
of the tree. Following a 2-h exposure of the virus to eight graded concentrations of the extract, it was
incubated at 37°C and observed 12-hourly for mortality. Dead embryos were spot tested for
haemagglutinating activity. The 100EID50 concentration of the virus and the highest concentration of the
extract were inoculated as positive and negative controls, respectively. All eggs inoculated with the
virus alone as well as 5 and 2 mg/ml extract/virus suspensions, died by 72 h post inoculation, while no
mortality was observed amongst those inoculated with 250 and 200 mg/ml virus/extract suspensions as
well as those inoculated with the pure extract. Mortalities of 40, 40 and 20%, due to viral activity were
recorded for 25, 50 and 100 mg/ml suspensions, respectively. This study showed that methanolic rootbark
extract of A. digitata has antiviral activity against Newcastle disease virus in ovo, particularly when
used at dose rates of 200 and 250 mg/ml.
Key words: Ethnoveterinary, African Baobab, antiviral activity, Newcastle disease virus.
INTRODUCTION
A major constraint to profitable broiler production is
disease outbreaks. The tropical environment provides
optimum conditions like high environmental temperature
and relative humidity as well as poor biosecurity for
disease agents to thrive. These are major challenges
being faced by the poultry industry in Nigeria. In the face
of persistent disease outbreaks and in spite of the use of
*Corresponding author. E-mail: laprecieux@yahoo.com or
lade.oladele@gmail.com. Tel: 234-806-113-8531.
Abbreviation: DMSO, Dimethylsulfoxide; EID50, embryo
infective dose 50; HA, haemagglutination; ND, Newcastle
disease; NVRI, Nigerian Veterinary Research Institute; SAN,
specific antibody negative.
conventional chemicals as disinfectants and drugs, it has
become imperative to seek means of achieving effective
biosecurity and control measures which ethnoveterinary
preparations could offer (Gueye, 1999; Musa et al.,
2008).
The Baobab tree (Adansonia digitata Lin) is indigenous
in many African countries (Wickens, 1982; Sidibe and
Williams, 2002). Many parts of the plant, especially
leaves, fruit pulp, seeds and bark fibers, have been used
traditionally for medicinal and nutritional purposes (Sidibe
and Williams, 2002; Chadare et al., 2009) and some
commercial enterprises produce standardized preparations
derived from its parts. The medicinal applications
include treatment of intestinal and skin disorders and
various uses as anti-inflammatory, anti-pyretic and
analgesic agents (Ramadan et al., 1994; Palombo, 2006;

Ajose, 2007; Karumi et al., 2008). In addition, antibacterial,
antiviral and anti-trypanosomal activities of the
plant extracts have been reported (Anani et al., 2000;
Hudson et al., 2000; Atawodi et al., 2003; Vimalanathan
and Hudson, 2009). Most of the reports were on the
efficacy of the leaves, fruit pulp, seeds and bark fibers of
this plant with very little information on the antiviral
properties associated with the root-bark extracts.
However, the root bark is commonly used in traditional
African Medicine in the treatment of fever (SCUC, 2006;
Wickens, 2008). This study was therefore carried out to
evaluate the antiviral property of the methanolic root bark
extracts of the African Baobab tree (A. digitata Lin)
against Newcastle disease (ND) virus.
MATERIALS AND METHODS
Extract preparation
Root-bark of A. digitata was harvested, cut into small pieces and
air-dried on a clean surface for 10 days. Two kilograms of the rootbark
was soaked (completely immersed) in 5 L of methanol in a
covered container for 3 days (Wickens, 2008; Egunyomi et al.,
2010). The methanol was decanted and fresh methanol was added
to the root-bark. The procedure was repeated twice and the extract
was poured into a small glass container and refrigerated.
Antiviral activity test
Preparation of virus: Extract inoculum
The virus used was a field strain of ND virus from the Virology
Research Laboratory of the National Veterinary Research Institute
(NVRI), Vom, Nigeria. The virus was propagated and titrated in 9
day-old specific antibody negative (SAN) embryonated chicken
eggs and the 100EID50 was calculated. The extract was diluted to
the desired concentrations of 250, 200, 100, 50, 25, 10, 5 and 2
mg/ml. These extract concentrations were substituted for the virus
buffer diluent at the end point dilution of the virus to achieve the
100EID50. The virus/extract suspensions were kept at 4°C for 2 h to
allow reaction to occur.
Egg inoculation
The embryonated chicken eggs were labeled according to the
extract concentrations used. A set of plastic egg trays were
thoroughly cleaned with Virkon ®, the eggs were swabbed with
70% alcohol in cotton wool and transferred into the cleaned trays.
The swabbed eggs were placed in the micro-safety cabinet where
they were punched and immediately inoculated with the
extract/virus suspension via the allantoic route. Each egg was
inoculated with 0.1 ml of the inoculum and 5 eggs were inoculated
with each con-centration of the extract/virus suspension, while
inoculating the 250 mg/ml extract only (without virus) and the
100EID50 concentration of the virus (without extract- virus control)
as negative and positive controls, respectively. The eggs were
sealed with molten wax and incubated at 37°C. The inoculated
eggs were monitored for embryonic death 12-hourly for up to 96 h
post inoculation. Dead embryos were removed and chilled in the
refrigerator for 3 h after which they were opened to check for
haemagglutinating (HA) activity by spot testing.
Spot haemagglutination test
Sulaiman et al. 4257
Dead embryos that had been chilled were brought out of the
refrigerator and kept at room temperature for about 30 min. The
eggs were swabbed and placed in the biosafety cabinet. The shell
of each egg was opened to reveal the air space and a pipette was
used to dispense a drop of 1% washed chicken red blood cells on a
white tile. A wire loop was thoroughly flamed and used to pick a
drop of the allantoic fluid which was mixed with the drop of blood.
The tile was gently rocked and observed for visible agglutination,
indicating viral activity (Thayer and Beard, 1998; Murakawa et al.,
2003). This was done for every egg and the observations were
recorded.
RESULTS AND DISCUSSION
All eggs inoculated with the virus control, 5 and 2 mg/ml
of the extract/virus suspension died between 48 and 72 h
of inoculation and showed positive haemagglutination by
spot-testing. However, no mortality was observed amongst
the embryonated eggs inoculated with the extract only,
250 and 200 mg/ml of extract/virus suspension up till 96 h
post inoculation. There were varying percentages of
embryo mortality amongst the 100, 50, 25 and 10 mg/ml
extract/virus suspension groups (Table 1). The haemagglutination
test was carried out to associate embryo
mortality with viral activity. The results of mortalities and
HA spot-testing are shown in Table 1. Only the group of
eggs inoculated with 5 mg/ml extract/virus suspension
had a dead embryo at 24 h post inoculation.
This study has evaluated the potentials of the methanolic
root-bark extract of A. digitata Lin as an antiviral
agent against ND virus using multiplication of the ND
virus in embryonated egg as an indicator for antiviral
property. The results of the virus propagation showed
that 250 and 200 mg/ml concentrations of the extract
completely inhibited the growth of ND virus in
embryonated chicken eggs, indicating that the methanolic
root bark extract of A. digitata at these doses are
effective against the virus when exposed in-ovo.
The results of this study corroborrates the findings of
Vimalanathan and Hudson (2009) that the methanolic,
water and dimethylsulfoxide (DMSO) extract of the leaf,
stem and pulp of A. digitata has both antiviral effect,
especially against influenza virus and anti-inflammatory
effects. The antiviral property observed might be ascribed
to the presence of various potentially bioactive ingredients
(Chadare et al., 2009), including triterpenoids,
flavonoids and phenolic compounds, but at present, the
antiviral activity observed in this study could not be
ascribed to specific compounds.
At extract concentrations of 100, 50 and 25 mg/ml,
there were 20, 40 and 40% mortalities due to viral activity
when compared with 0% at concentrations of 250 and
200 mg/ml. This indicates inhibitory rather than virucidal
effect of the extract on the ND virus at these doses.
Whereas, at concentrations of 2 to 10 mg/ml, as well as
at 0 mg/ml (virus control), the study recorded total or near

4258 Afr. J. Biotechnol.
Table 1. Embryo mortality and haemagglutination (HA) test result of embryonated chicken eggs inoculated with graded doses
of A. digitata extract/ND virus suspension.
Virus
Mortality HA Test Mortality due to
(mg/ml) 24 h pi 48 h pi 72 h pi 96 h pi +ve -ve viral activity (%)
250 0/5 0/5 0/5 0/5 0 5 0
200 0/5 0/5 0/5 0/5 0 5 0
100 0/5 0/5 1/5 0/4 1 4 20
50 0/5 1/5 3/4 1/1 2 3 40
25 0/5 0/5 2/5 0/3 2 3 40
10 0/5 0/5 3/5 2/2 5 0 100
5 1/5 0/4 4/4 - 4 1 80
2 0/5 0/5 5/5 - 5 0 100
Vc 0/5 0/5 5/5 - 5 0 100
Uic 0/5 0/5 0/5 0/5 0 5 0
Ec 0/5 0/5 0/5 0/5 0 5 0
Vc = Virus control; Uic = uninoculated control; Ec = egg control; pi = post inoculation.
total mortalities due to viral activity, indicating the ineffectiveness
of the extract at these doses. The dead
embryos in the 50 and 5 mg/ml extract concentrations
that were negative for haemagglutinating activity must be
a result of nonspecific deaths from manipulations during
inoculation (Villegas, 1998).
In conclusion, this work has shown that the methanolic
root-bark extract of A. digitata Lin has direct antiviral
activity against ND virus and could therefore be useful in
the control of the disease in poultry birds.
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Int. J. Derm. 46(suppl 1): 48-55.
Anani K, Hudson JB, de Souza C, Akpagana K, Towers GHN, Arnason
JT, Gbeassor M (2000). Investigation of Medicinal Plants of Togo for
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Atawodi SE, Bulus T, Ibrahim S, Ameh DA, Nok AJ, Mamman N,
Galadima M (2003). In Vitro trypanocidal effect of methanolic extract
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Chadare FJ, Linnemann AR, Hounhouigan JD, Nout, MJR, Van Boekel
MAJ (2009). Baobab Food Products: A Review on their Composition
and Nutritional Value. Crit. Rev. Food Sci. Nutr. 49: 254-274.
Egunyomi A, Gbadamosi IT, Osiname KO (2010). Comparative
effectiveness of ethnobotanical mosquito repellents used in Ibadan,
Nigeria. J. Appl. Biosci. 36: 2382-2388.
Gueye EF (1999). Ethnoveterinary medicine against poultry diseases in
African villages. World Poult. Sci. J. 55: 187-198.
Hudson JB, Anani K, Lee MK, De Souza C, Arnason JT, Gbeassor M
(2000). Further Investigations on the Antiviral Properties of Medicinal
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(2008). Ethnoveterinary remedies used for the management of
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Palombo EA (2006). Phytochemicals from traditional medicinal plants
used in the treatment of diarrhoea: Modes of action and effects on
intestinal function. Phytother. Res. 20: 717-724.
Ramadan A, Harraz FM, El-Mougy SA (1994). Anti-inflammatory,
analgesic and antipyretic effects of the fruit pulp of Adansonia
digitata. Fitoterapia, 65: 418-422.
SCUC (2006). Baobab manual, Field manual for extension workers and
farmers. University of Southampton, Southampton, UK.
Sidibe M, Williams JT (2002). Baobab. Adansonia digitata. International
Centre for Underutilized Crops. University of Southampton p. 105
(ISBN 0854327649).
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Glisson JR, Jackwood MW, Pearson JE, Reed WM (eds). A
laboratory manual for the isolation and identification of avian
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Square, PA. USA pp. 255-266
Villegas P (1998). Titration of biological suspensions. In: In: Swayne E,
Glisson JR, Jackwood MW, Pearson JE, Reed WM (eds). A
laboratory manual for the isolation and identification of avian
pathogens. American Association of Avian Pathologists. Kennett
Square, PA. USA, pp. 248-254.
Vimalanathan S, Hudson JB (2009). Multiple inflammatory and antiviral
activities in Adansonia digitata (Baobab) leaves, fruits and seeds. J.
Med. Plan. Res. 3(8): 576-582.
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and Australia. Springer.

African Journal of Biotechnology Vol. 10(20), pp. 4259-4264, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2064
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Effect of gamma radiation on different stages of Indian
meal moth Plodia interpunctella Hübner (Lepidoptera:
Pyralidae)
Hosseinzadeh Abbas 1 , Shayesteh Nouraddin 2 , Zolfagharieh Hamid Reza 3 , Bernousi Iraj 5 ,
Babaei Mohammad 3 , Zareshahi Hasan 4 , Ahari Mostafavi Hossein 3 and Fatollahi Hadi 3
1 Department of Entomology, Agricultural Faculty, Urmia University, Urmia, Iran.
2 Department of Plant Medicine, Mahabad Branch, Islamic Azad University, Mahabad, Iran.
3 Agricultural, Medical and Industrial Research School, Karaj, Iran.
4 Radiation Application Research School, Yazd, Iran.
5 Department of Agronomy and Plant Breeding, Agricultural faculty, Urmia University, Urmia-Iran.
Accepted 4 March, 2011
Indian meal moth Plodia interpunctella Hübner is one of the most important stored products pests in
the world. In this research, the effect of gamma irradiation was studied on different developmental
stages of this pest and the doses required to prevent each of these developmental stages was
investigated. From the results, required dose to prevent larval emergence from irradiated 1 to 24 h eggs
was 400 Gray (Gy), and 400 Gy was required to prevent pupae from 15 days old larvae. Also, the dose of
radiation required to prevent adult emergence from irradiated 5 days old pupa was 650 Gy. According to
the results, dose of 650 Gy is adequate to control all immature stages of this pest. In addition, the effect
of gamma ray was studied on developmental stage period of each irradiated existence stage till adult
eclosion. The results revealed that there was a dose-dependent increase in the developmental periods,
and the growth index of the adults was significantly decreased with increasing dose of radiation
administered to the eggs, larvae and pupae too. It is concluded that irradiation can be used as a safe
method to control stored pests.
Key words: Gamma irradiation, prevention dose, developmental period, growth index, Plodia interpunctella.
INTRODUCTION
Iran is the largest producer of pistachios and dates in the
world and according to the Food and Agriculture Organization
(FAO), approximately 10% of stored products in
Iran are lost due to insect infestation (Zolfagharieh,
2002). The Indian meal moth Plodia interpunctella
Hübner is a cosmopolitan pest whose larvae can infest a
variety of stored grain, nuts, pulses, meals, dried fruits
and processed foods (Simmons and Nelson, 1975).
Methyl bromide (MB) is a widely used fumigant for the
control of insect infestation in many agricultural commo-
*Corresponding author. E-mail: abas1354@yahoo.com or
abmelis@gmail.com.
Abbreviations: MB, Methyl bromide; PTM, potato tuber moth.
dities. As methyl bromide has the potential to destroy the
ozone layer in the environment, alternative methods had
to be sought to replace its use in insect control
(Gochangco et al., 2002). It has been reported that
irradiation could offer an important and attractive alternative
for the export commodity fumigation uses of MB
and for eliminating the problem of chemical residues
(Ahmed, 1991; Marcotte, 1993). Brower and Tilton (1983)
reported that irradiation is an approved method of direct
control for stored product insect in wheat and wheat flour
in many countries, and indications were that it would
soon be approved for all grain, grain products and other
dry food commodities. Commodities are exposed to
gamma rays from the radioactive isotopes cesium 137 or
cobalt 60, electron beams, or X rays converted from an
electron beam generator. The mode of action of ionizing
irradiation as a quarantine treatment involves breaking

4260 Afr. J. Biotechnol.
chemical bonds. When this happens to DNA, normal
development or reproduction of the organism may be
prevented. Decades of research conducted worldwide on
radiation disinfestation of food and agricultural products
have shown that this method is effective. The advantages
of irradiation processing include no undesirable residues
in the foods treated, no resistance developed by pest
insects and few significant changes in the physicchemical
properties or the nutritive value of the treated
products (Ahmed, 2001; Lapidot et al., 1991; Boshra and
Mikhaiel, 2006). Unlike other disinfestations techniques,
irradiation does not need to kill the pest immediately to
provide quarantine security; therefore, live (but nonviable
or sterile) insects may occur with the exported commodity
(Follett and Griffin, 2006).
In the present work, we have tested gamma radiation
sensitivity of different life stages of P. interpunctella
Hübner. The study was also designed to assess the
effect of gamma irradiation on different stages of P.
interpunctella.
MATERIALS AND METHODS
Insect culture
P. interpunctella Hübner used in all experiments were derived from
a laboratory culture initially established for larvae collection from
infested pistachios in Urmia Province, Northwest Iran. The larvae
were reared on artificial diet containing bran (800 g), dry yeast
powder (160 g), honey (300 g) and glycerol (200 ml / 1000 g) dry
ingredient. The diet was decontaminated at 60°C for 6 h.
Throughout the experiments, insect cultures were maintained under
controlled laboratory conditions of 28 ± 1°C and 65% ± 5% relative
humidity (RH) with a 14:10 h light : dark photoperiod. Larvae were
placed in 500 ml glass containers half full of rearing medium.
Fifteen virgin pairs of males and females (0 to24 h old) were
selected for the experiments. These adults were placed in funnels
capped with screen lids. Funnels were placed over radiology film
for 24 h and oviposited eggs on the film were collected every 12 h.
Irradiation of eggs
Twenty eggs (1 to 2 days old) were placed in glass Petri dishes and
irradiated in a calibrated 60 Co irradiator (Issledovatel type PX-30)
with the activity of 4.5 kCi and dose rate of 0.65 Gy/s at the Iran
Nuclear Research Center for Agriculture and Medicine. Ferric
dosimetry system was used for the facility.
Radiation was applied at nine dose levels ranging from 0 to 400
Gy to the eggs. Controls were subjected to the same conditions as
the irradiated eggs in that, they were transferred to the irradiator
and subsequently removed without exposure to radiation. After
irradiation, eggs were returned immediately to the laboratory and
then placed in Petri dishes that had a central well made by ringing a
central area (2 cm diameter) with glue and allowing the glue to dry.
Rearing medium was placed in the Petri dish around the central
well to provide food for any hatched larvae, and the apparatus was
placed at 28 ± 1°C and 65 ± 5% RH. The Petri dishes were
examined under a binocular microscope, and the number of
hatched eggs was counted daily until no further hatched egg was
observed. The rearing medium containing the small larvae was then
transferred to Petri dishes containing food. Pupation and adult
emergence of these larvae were recorded. Four replicate batches
of eggs were used for each dose level and the control.
Irradiation of 15 days old larvae
Fifteen days old larvae were selected for uniformity in size before
irradiation. Larvae were irradiated in the same irradiator and dose
ranges from 0 to 400 Gy. Immediately after treatment, irradiated
and control larvae were transferred to Petri dishes containing
rearing medium. The Petri dishes were subsequently transferred to
the rearing conditions. Pupation, adult emergence and mortality of
these larvae were recorded every 2 days. Mortality of larvae was
determined by the brown color with no observable movement. The
adults that developed from irradiated larvae were removed and
counted daily. Four replicate (20 larvae for each replicate) were
used for each dose level and control.
Developmental periods and longevity of the adults derived
from irradiated eggs, larvae and pupae
The developmental periods, from egg to adult of P. interpunctella
Hübner that developed from irradiated eggs, larvae and pupae were
recorded. The longevity of the adults was also determined. The
growth index (percentage adult eclosion/total developmental period)
was calculated for the irradiated eggs, larvae and pupae.
Irradiation of pupae
Five-day old pupae were placed in Petri dishes and irradiated at six
dose levels of 0, 150, 250, 350, 450, 550 and 650 Gy. The number
of adults that emerged from irradiated pupae was recorded. Four
replicates (20 pupae in each) were used for each dose level.
Statistical analysis
Data from the experiments were subjected to analysis of variance
(ANOVA) using the Statistical Package for the Social Sciences
(SPSS) for windows 11.5. Data were transformed using arcsine √x
before ANOVA. Means were separated at the 5% significance level
by the Tukey test.
RESULTS AND DISCUSSION
Effects of gamma radiation on eggs
The data obtained from experiments dealing with the irradiation
of eggs are summarized in Table 1. A significant
reduction in hatched eggs was observed and it was
correlated with the radiation doses (F = 596.522; df = 8; P
< 0.05). Percentage larval emergence from irradiated
eggs was 93.75% in the untreated controls but was
reduced to 2.81% at a dose of 350 Gy, and all irradiated
eggs were completely sterile at 400 Gy. The percentage
of emergent larvae that survived to the pupa stage also
decreased with increasing radiation doses (F = 769.212;
df = 8; P < 0.05). At doses of 250 and 300 Gy, pupal
development was 10.31 and 4.37%, respectively.
The percentage of pupation that survived to the adult
stage was also decreased with increasing radiation doses
(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).
Dose (Gy) Egg hatch (%) ± SE Pupation (%) ± SE Adult emergence (%) ±SE
0 93.75±1.61 a 75.00±1.02 a 61.25±0.72 a
50 78.12±1.20 b 65.00±1.02 b 60.00±1.02 a
100 62.5±1.02 c 52.50±1.44 c 48.75±1.25 b
150 43.13±1.20 d 35.00±1.02 d 31.87±1.20 c
200 31.87±1.20 e 20.62±1.20 e 16.25±0.72 d
250 21.25±0.72 f 10.31±0.79 f 8.75±.72 e
300
350
400
11.87±1.20 g
2.81±0.79 h
0 i
4.37±1.20 g
Means within each column followed by the same latter are not significantly different (P > 0.05).
0 h
0 h
1.87±1.20 f
Table 2. Pupation, adult emergence and mortality (%) of the P. interpunctella irradiated as
larvae (15 days old).
Dose (Gy) Pupation(%)±SE Adult emergence (%) ±SE Mortality(%) ±SE
0 85.62±1.57 a 60.00±1.77 a 11.87±1.20 g
150 76.25±0.72 b 45.94±0.60 b 17.81±1.07 f
200 51.87±1.20 c 21.25±0.72 c 35.31±1.07 e
250 37.50±1.77 d 8.12±1.20 d 51.87±1.20 d
300 20.31±1.02 e 0 e 61.87±1.20 c
350 5.00±1.02 f 0 e 81.87±1.20 b
400 0 g 0 e 100 a
Means within each column followed by the same latter are not significantly different (P > 0.05).
emergence from irradiated eggs was 61.25% in the
untreated controls but was reduced to 1.87% at a dose of
300 Gy and no adult emerged at doses of 350 Gy and
above.
Effects of gamma radiation on 15 days old larvae
The effects of gamma radiation on irradiated larvae are
shown in Table 2. The data showed that the percentage
of irradiated larvae that survived to the pupal stage were
decreased with increasing radiation doses (F = 1077.006;
df = 6; P < 0.05). The percentages of pupae that developed
from irradiated larvae were 5% at 350 Gy when
compared with 85.62% in the control. The percentage of
pupation that survived to the adult stage were also
decreased with increasing radiation doses (F = 998.931;
df = 6; P < 0.05). Statistical analysis of data indicated that
irradiation significantly affected mortality percentages of
larvae (F = 1038.098; df = 6; P < 0.05). Mortality of
irradiated larvae began 4 days after the treatment and
continued through the time of observation. Irradiation at
400 Gy caused 100% mortality.
0 f
0 f
Abbas et al. 4261
Effects of gamma radiation on the developmental
period and longevity of the adults derived from
irradiated eggs, larvae and pupae
There was a dose-dependent increase in the
developmental time of P. interpunctella Hübner adults
developed from irradiated eggs (F = 21.876; df = 7; P <
0.05), larvae (F = 42.931; df = 7; P < 0.01) and pupae (F
= 21.650; df = 7; P < 0.05) (Table 3). The mean developmental
time from egg to adult was 44.20 days in the
controls, which rose to 52.50 days for insects derived
from eggs irradiated with a dose of 350 Gy. The
development of the irradiated larvae was also extended
from 23.40 days in the controls to 36.40 days for insects
exposed to a dose of 350 Gy. The mean developmental
time from pupa to adult was 3.70 days in the controls,
which rose to 6.90 days at a dose of 350 Gy. The growth
index of the adults was significantly decreased with
increasing doses of radiation administered to the eggs (F

4262 Afr. J. Biotechnol.
Table 3. Mean (± SE) developmental period of P. interpunctella adults irradiated as eggs, larvae and pupae a .
Dose
(Gy)
Developmental period from eggs to adults (days) Growth index b
Eggs treated Larvae treated Pupae treated Eggs treated Larvae treated Pupae treated
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
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
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
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
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
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
300 49.40±0.37 b - 6.90±0.23 a 0.03 f - 5.37 g
350 52.50±0.40 a - - - - 5.16 g
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
eclosion/total developmental period.
Adult emergence (%)
100
90
80
70
60
50
40
30
20
10
0
92.5a
66.87b
35c
23.75d
12.5e
Control 150 250 350 450 550 650
Dose (Gy)
Figure 1. Adult emergence of the P. interpunctella irradiated as pupae (5 days old).
= 1141.967; df = 7; P < 0.05), larvae (F = 456.928; df = 7;
P

obtain complete sterility for Ephestia cautella (Walker)
and P. interpunctella eggs respectively (Ozyardimci et al.,
2006). In our study, a dose of 350 Gy completely
prevented adult emergence from irradiated eggs. Ayvaz
and Tuncbilek (2006) found that no Ephestia kuehniella
Zeller late-instar larvae received an irradiation dose of
250 Gy as emerged adults.
When the effects of six gamma radiation doses ranging
from 50 to 1000 Gy were investigated against all life
stages of E. cautella, the development of adults from
treated eggs and larvae was prevented by 300 and 200
Gy, respectively (Cogburn et al., 1973). In the present
study, a dose of 400 Gy completely prevented larval
development and the larvae did not reach pupation. The
minimum dose required to prevent adult emergence from
P. iterpunctella exposed as larvae was 300 Gy, while
doses required to cause death were 450 Gy and above
(Azelmat et al., 2005). A dose of 250 Gy was sufficient to
give complete mortality for last-instar larvae of E.
kuehniella Zeller (Ayvaz and Tuncbilek, 2006). Our
results showed that, when the prevention of adult
emergence is used as a standard for measuring the
effectiveness of radiation against P. interpunctella larvae,
a dose of 300 Gy is required. It was also found that the
ultimate development of adults from 11-days-old P.
interpunctella larvae was completely prevented when
these larvae were exposed to 149 Gy (Johnson and Vail,
1988). Ayvaz et al. (2006) reported that younger larvae
were more sensitive to radiation than the older ones, and
that the dose required to prevent adult emergence was
lower (200 Gy) than for mature larvae (250 Gy). Cogburn
et al. (1973) reported that from the larvae of Cadra
cautella Walker treated with 20 krad (200 Gy), no adults
were produced. Mansour (2002) also reported that
200 Gy doses were enough to prevent adult emergence
from irradiated mature larvae of codling moth Cydia
pomonella (L.). Ayvaz and Tunçbilek (2006) found that a
dose of 200 Gy applied to the young larvae of E.
kuehniella completely prevented female adult emergence
(all emerged moths were males).
There was a dose-dependent delay in the developmental
period of the P. iterpunctella eggs, larvae and
pupae. The longevity of the treated larvae increased
when compared with controls that continued development
to adult emergence. Hallman (2000) noted that insects
may live for considerable periods of time after irradiation.
Makee and Saour (2003) assumed that unbalanced
hormonal system led to prolonged developmental time in
the F1 larvae of the potato tuber moth (PTM),
Phthorimaea operculella Zeller. Hosseinzadeh et al.
(2010) found that there was a dose-dependent increase
in the developmental time of Oryzaephilus surinamensis
L. adults developed from irradiated eggs, larvae and
pupae. Increasing post-treatment longevity for irradiated
P. interpunctella, the false codling moth, Cryptophlebia
leucotreta Meyrick, the Mediterranean flour moth, E.
kuehniella, and the red flour beetle Tribolium castaneum
Abbas et al. 4263
Herbst have been reported previously (Azelmat et al.,
2005; Johnson and Vail, 1988; Bloem et al., 2003; Mehta
et al., 1990; Ayvaz and Tuncbilek, 2006). Typically, irradiated
nymphs or larvae will have a prolonged nymphal
or larval stage and may live longer than non-irradiated
control insects (Hasan and Khan, 1998).
When the pupae of P. interpunctella were irradiated,
the percentage of adult emergence was decreased by
doses up to 650 Gy. When male and female pupae of
Ephestia calidella (Guenee) were irradiated with doses of
200 to 800 Gy, the percentage adult emergence was
decreased in accordance with increasing gamma
radiation doses (Boshra and Mikhaiel, 2006). This work
also showed that a 1000 Gy dose prevented the emergence
of both sexes.
Hallman (2000) reported that some stored-product
moths, especially the Angoumois grain moth, Sitotraga
cerealella (Oliver) and the Indian meal moth P.
interpunctella (Hübner), were among the most tolerant
arthropods to radiation. In comparison with dipteran
species, Lepidoptera require much higher levels of
ionizing radiation to obtain full sterility (LaChance and
Graham, 1984). The main cause of this difference is
thought to be as a result of the different kinetic organization
of chromosomes in these two groups of insects.
The Diptera possess typical monocentric chromosomes
with kinetic activity restricted to the centromere, whereas
lepidopteran chromosomes are holokinetic (Gassner and
Klemetson, 1974).
The results of this study support the assumption that a
dose of 400 Gy is required in order to prevent hatching of
eggs and 300 Gy is required to prevent the larvae from
reaching adulthood. It is recommended that dose of 650
Gy should be used to control population growth of P.
interpunctella when targeting pupae are within stored
products. Delayed developmental periods were observed
after irradiation of the eggs, larvae and pupae. Irradiation
could be used as a disinfestation treatment in stored
products as an alternative to chemical fumigants. Phostoxin
and methyl bromide are the most common fumigants
for insect disinfestation in storage. Fumigants leave
residues in treated products, and due to the ozonedepleting
properties, methyl bromide was banned in
many countries. Therefore, irradiation is a safe alternative
to fumigation.
Acknowledgments
The authors wish to express their deep appreciation to
Urmia University and Karaj Nuclear Research Center for
Agriculture and Medicine. The authors also thank Dr.
Aramideh for assistances analyzing data.
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4264 Afr. J. Biotechnol.
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African Journal of Biotechnology Vol. 10(20), pp. 4265-4268, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1903
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Body composition of freshwater Wallago attu in relation
to body size, condition factor and sex from southern
Punjab, Pakistan
Muhammad Yousaf*, Abdus Salam and Muhammad Naeem
Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan 60800, Pakistan.
Accepted 3 February, 2011
Wallago attu is one of the large freshwater catfish found in Pakistan. The rapid growth and high
nutritional quality encouraged investigation into the aquaculture potential of this excellent food fish. It
was observed that body size had a positive influence on percent ash, percent fat and percent protein
contents (wet weight) but there was no significant effect on percent water content. The condition factor
had a positive correlation with fat, ash and protein contents (%wet weight) but no influence on percent
water content. There was no significant influence of sex on body composition of W. attu. If it is
impossible to find out the water content, then the body constituents can be estimated from the wet
body weight, total length and condition factor of this species. As the variations in body composition are
related to these variables, so the equations of each constituent were estimated. The predictive
equations can be used to estimate the body composition with a fair level of accuracy.
Key words: Wallago attu, body size, condition factor, body composition, sex.
INTRODUCTION
Wallago attu is one of the large freshwater catfish found
in Pakistan, India, Sri Lanka, Nepal, Bangladesh, Burma,
Thailand, Vietnam, Kampuchea, Malay Peninsula,
Afghanistan, Sumatra and Java (Talwar and Jhingran,
1991; Giri et al., 2002). The rapid growth (Goswami and
Devraj, 1992) and high nutritional quality of its flesh
(Lilabati and Viswanath, 1996) encourage investigation
into the aquaculture potential of this excellent food fish.
Taking into consideration the various health risks, fish’s
mineral and body composition and their health status
were assessed in order to establish the safety level of the
table sized species prior consumption (Fawole et al.,
2007). Body composition illustrates the nutritional quality
of food because analysis of biochemical composition
including protein, fat and ash is very important in
assessing food value (Kamal et al., 2007). So, biochemical
evaluation is necessary to ensure the nutritional
value as well as eating quality fish (Azam et al., 2004).
However, the value of these body constituents vary
*Corresponding author: E-mail: mahyousaf@hotmail.com.
significantly from one species and one individual fish to
another depending on age, sex, feeding season,
sampling time, activity and environmental condition
(Weatherley and Gill, 1987; Jobling, 1994; Tang et al.,
2009). The aim of the present study is to examine
changes in the proximate composition in relation to body
weight, length, condition factor and sex. Predictive
equations are developed to describe these relationships
in wild W. attu.
MATERIALS AND METHODS
Seventy-eight, wild W. attu of different body sizes, ranging from
16.7-50.2 cm total length and 14.54-648.82 g body weight, were
obtained from different localities of Indus River Southern Punjab
using a cast net and were transported live to the Institute of Pure
and Applied Biology in plastic containers. On arrival at the
laboratory, fresh fish were washed with tap water several times to
remove adhering blood and slime. They were anaesthetized with
Tricaine Methanesulfonate (MS 222), weighed to nearest 0.01 g on
an electronic digital balance (MP-3000 Chyo, Japan) and their
length measured to the nearest 0.1 cm on wooden measuring tray.
These fish were placed in a pre-weighed aluminum foil tray in an
electric oven (Memmert ® 8540) at 70°C until a constant weight

4266 Afr. J. Biotechnol.
Table 1. Wet body weight (g) versus body constituents of Wallago attu.
Body constituents (%) r a b S.E. (b) t value when b = 0
Water contents 0.010 76.112 -0.0001 0.002 -0.05
Ash contents (wet weight) 0.120 3.224 0.001 0.0003 1.667
Fat contents (wet weight) 0.448*** 3.570 0.003 0.001 4.143
Protein contents (wet weight) 0.265** 15.035 0.003 0.001 2.462
Statistical parameters of various relationships, correlation coefficient (r), intercept (a), regression coefficient (b),
standard error of b (S.E.) and n = 78 in each case.
Significance level: **P

Table 3.Condition factor (K) versus body constituents of Wallago attu.
Body constituent (%) r a b S.E. (b) t value when b = 0
Water contents 0.009 74.601 1.189 15.424 0.077
Ash contents (wet weight) 0.108 3.027 0.679 0.720 0.943
Fat contents (wet weight) 0.434*** 1.146 6.743 1.605 4.203
Protein contents (wet weight) 0.117 14.166 3.410 3.324 1.026
Yousaf et al. 4267
Statistical parameters of various relationships, correlation coefficient (r), intercept (a), regression coefficient (b), standard
error of b (S.E.) and n = 78 in each case.
Significance level: ***P

4268 Afr. J. Biotechnol.
375.
Kamal D, Khan AN, Rahman MA, Ahamed F (2007). Biochemical
composition of some small indigenous fresh water fishes from the
river Mouri, Khulna, Bangladesh. Pak. J. Biol. Sci. 10(9): 1559-1561.
LeCren ED (1951). The length-weight relationship and seasonal cycle in
gonad weight and condition in the perch (Perca fluviatilis). J. Anim.
Ecol. 20: 201-219.
Lilabati H, Viswanath W (1996). Nutritional quality of freshwater catfish
(Wallago attu) available in India. Food Chem. 57: 197-199.
Love RM (1980). The chemical biology of fishes. Vol II. Academic
press, London.
McComish TS, Anderson RO, Goff FG (1974). Estimation of Bluegill
Lepomis macrochirus proximate composition with regression models.
J. Fish. Res. Bd. Can. 31: 1250-1254.
Memid D, Celikkale MS, Ercan E (2006). Effects of different diets on
growth performance and body composition of Russian sturgeon
(Acipenser gueldenstaedtii, Brandt & Ratzenburg, 1833). J. Appl.
Ichthyol. 22(1): 287-290.
Salam A, Davies PMC (1994). Body composition of northern pike (Esox
lucius L) in relation to body size and condition factor. Fish. Res. 19:
193-204.
Salam A, Ali M, Anas M (2001). Body composition of Oreochromis
nilotica in relation to body size and condition factor. Pak. J. Res. Sci.
12(1): 19-23.
Talwar PK, Jhingran AG (1991). Inland fishes of India and adjacent
countries. Volume 2. A.A. Balkema, Rotterdam.
Tang H, Chen L, Xiao C, Wu T (2009). Fatty acid profiles of muscle
from large yellow croaker (Pseudosciaena crocea R.) of different age.
J. Zhejiang Univ. Sci. B. 10(2): 154-158.
Weatherley AH, Gill HS (1987). The biology of fish growth. Academic
Press. London. pp. 1-443.
Wootton RJ (1990). Ecology of Teleost Fishes. Chapman and Hall,
London
Wootton RJ (1998). Ecology of Teleosts fishes 2 nd Dordrecht: Kluwer.
Zar JH (1996). Biostatistical Analysis. Prentice-Hall. New Jersey.

African Journal of Biotechnology Vol. 10(20), pp. 4269-4275, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB09.786
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Rhizopus stolonifer exhibits dimorphism
C. O. Omoifo
Department of Crop Science, Ambrose Alli University, Ekpoma, Edo State, Nigeria. E-mail: coomoifo@yahoo.com.
Accepted 4 March, 2011
This study showed that multiple morphologies could be induced from sporangiospores of Rhizopus
stolonifer in minimal medium. These included moniliform hyphae, septate hyphae and terminal budding
yeast cells. When considered along the normal coenocytic filamentous growth form, the study showed
that R. stolonifer could be polymorphic.
Key words: Rhizopus stolonifer, polymorphic, multiple morphologies.
INTRODUCTION
Rhizopus stolonifer, a Zygomycete has a filamentous
growth habit. Its filaments are coenocytic, that is, they are
non-septate. It is the only fungus yet known to produce
rhizoids which penetrate the substratum in order to obtain
nutrients. The rhizoids also serve as support. Opposite
the rhizoids, a sporangiophore juts into the atmosphere
and this terminates in a club-shaped collumelum
enclosed within the sporangial wall. Between the collumelum
and wall are numerous asexual reproductive
structures known as sporangiospores. This organism is
also characterized by the presence of stolons, which
connect rhizoid joints.
Other members of the Zygomycetes, especially species
of the genus Mucor, have been shown to undergo fungal
dimorphism (Bartnicki-Garcia and Nickerson, 1962a, b, c,
d; Friedenthal et al., 1974; Schulz et al., 1974; Ruiz-
Hereira, 1985; McIntyre et al., 2002; Lubberhusen et al.,
2003). In such phase transitions, sporangiospores convert
to yeast like cells, with a central globose mother cell
which multilaterally produces daughter buds by blastic
action. This occurs under CO2 tension or high hexose
concentration.
However, terminal budding yeast cells have been
induced from sporangiospores of Mucor circinelloides
Tieghem in minimal medium (Omoifo, 2005). When the
external medium was subjected to K + variation, the 1.0 g/l
K + supplementation was found to be critical for protoplast
formation, a transient morphology, but Na + was required
for yeast induction. Thus, a treatment of 1.0 g/l K + , 0.10
g/l Na + gave optimum yeast induction; even though thallic
growth forms, including holoblastic-, holothallic- and
enterothallic conidia were also induced, uracil supplementation
drastically diminished the presence of these
thallic forms (Omoifo, 2006a). This suggested that these
ions impacted on the structural modification led to the
induction of yeast cells from sporangiospores. A follow-up
study showed that the incorporation of myoinositol and
zinc into the minimal medium of growth of the
microorganism enhanced yeast induction by more than
63%. These studies further demonstrated that environmental
factors influence mould-yeast interconversion.
In the study of Bartnicki-Garcia and Nickerson (1962b),
several members of the Mucurales were tested for their
ability to undergo mould-yeast interconversion, but R.
stolonifer was found to be incapable of it. Their study was
conducted in solid cultures of complex nature (yeast
extract-peptone-glucose, YPG) and incubated under 30%
CO2 pressure. This claim in minimal medial composition
that is known to induce terminal budding yeast cells from
M. circinelloides Tieghem was validated or otherwise
disproved (Omoifo, 2005, 2006a, b).
It was also found that Mucor species could only convert
to multipolar budding yeastlike cells under CO2 atmosphere
when there is fermentable carbon substrate like
galactose, fructose, mannose, glucose and disaccharide
like sucrose; when it was grown with xylose as substrate,
intact sporangiospores remained in the broth after 72 h
(Bartnicki-Garcia and Nickerson, 1962b).
On the other hand, McIntyre et al. (2002) cultivated M.
circinelloides in xylose substrate-Vogel’s minimal medium
which was incubated in CO2 atmosphere and the
morphological expression was multipolar budding yeast
like cells. Lubberhussen et al. (2003) used similar
medium (Vogel’s) with xylose or glucose as substrate for
the cultivation of M. circinelloides and obtained filamenttous
growth in cultures sparge under the atmosphere.
Since preliminary study showed that R. stolonifer could
utilize glucose for growth, this study examined the effect

4270 Afr. J. Biotechnol.
of the pentose sugar, xylose, on its habit.
Reported here is the induction of several morphological
forms of R. stolonifer in minimal medium.
MATERIALS AND METHODS
Fungal strain
The strain of R. stolonifer used in this study was a gift from Miss
Elsa Thomas, the Project Assistant to Dr. G. S. Prasad of Microbial
Type Culture Collection 1, Institute of Microbial Technology, Sector
39A, Chandigarh, India. It was purified through three different
inoculations and subculturing and maintained as malt extract (MEA,
31.2 g/L) solid cultures which exhibited filamentous growth habit. A
fresh culture was prepared after seven days.
Inoculum preparation for growth
Inoculum was obtained by pouring sterile deionized distilled water
over aerobic growth and a sterile glass rod was gently passed over
the surface so as to dislodge the spores. The suspension was not
subjected to extreme preparation as was done in the method of
Omoifo (1996, 2003, 2006a).
This was to ensure that some mycelia fragments were retained in
the inoculum. This showed to be so on examination under the
microscope (x400 magnification). But generally, spore count which
was taken with Neubauer Haemocytometer. (BSS No. 784
Hawksley, London Vol. 1/4000) was adjusted to 10 x 10 5 spores per
ml in sterile deionized distilled water, with the aid of tally counter.
Reagent and culture media
This has been described (Omoifo, 1996, 2006a) and subsequently
modified. Briefly, media were prepared per litre: Xylose (Hymedia),
10 g; (NH4)2SO4 (Merck), 5 g; K2HPO4 (Qualigens), 5 g; KH2PO4
(Merck), 5 g; MgSO4 (Qualigens), 2 g; FeS04 (Hymedia), 0.1 g;
NaCl (Qualigens), 0.1 g; CuSO4 (Analytical Rasayan), 0.06 g;
MnSO4 (Qualigens), 0.065 g; ZnSO4 (BDH), 0.06 g; CaCl2 (Sigma)
uracil (Hymedia), 100 mg; myoinositol (Sigma), 0.25 g.
The media where prepared in 2000 ml beaker. Weights of buffer
components 0.2 M Na2HPO4: 0.1 M citrate were obtained using
H54Ar meter balance and added to the beaker. The pH was
adjusted to 4.5 with 2 N NaOH or 1 N HCl using a control dynamics
pH meter model APX 175 E/C in the 2000 ml beaker before
dispensing 80 ml of broth in each duplicate of 250 ml Erlenmeyer
flasks for the test. The solution in each flask was made up to 100 ml
with glass distilled deionized water and sterilized at 121°C per 15
min.
Inoculation, growth conditions and sample collection
A 1 ml spore suspension was drawn and inoculated into each broth
flask using Eppendorf micropipette. Each culture flask was then
shaken for 30 s and thereafter incubated at 28 ± 1°C (laboratory
temperature). Culture flasks were examined after 72 and 168 h of
growth.
The culture flasks were returned for further incubation. Slides
were prepared for viewing by putting one or two drops of
lactophenol on a glass slide with the aid of wire loop, one drop from
the culture flask properly shaken was then added and covered with
a glass cover slip and viewed under a binocular microscope at x
1000 magnification using the microscope Olympus BX51 attached
with camera model No. C-3250. The observed morphologies were
recorded.
RESULTS
Visual examination showed the emergence of mycelia on
the surface of growth medium. This appeared white to
grey on broth surface but aerial mycelia were grey.
The culture was cloudy and sediments were formed by
day 3. After one week, aerial mycelia became profuse
and thick sediment was formed at the bottom of the
culture. Microscopic examination revealed that, aerial
mycelia originated from broth level mycelia made up of
rhizoids and stolons. Figure 1a showed a section of a
long sporangiophore, which is vertically opposite the
rhizoids that penetrate into the broth as well as a side
(horizontal) branch, which is a section of the stolon
interconnecting two rhizoid joints. Shown in Figure 1b is
globose sporangium enclosing numerous sporangiospores;
within it also is the collumelum which is the terminal
end of the characteristically long sporangiophore.
Figure 1c showed collapsed sporangia that had released
their content of spores. Sporangiospores are grey to dark
brown in colour, have striated walls and vary in shape
(Figure 1d).
Three different types of hyphae were formed within the
broth. These were tan to light brown coenocytic hyphae
(Figure 2c), dark brown septate hyphae (Figure 2s) and
moniliform hyphae which has narrow diameter and simple
septa-like partitions which have no cell wall material
deposition (Figure 2m).
On the other hand, yeast cells which were profusely
produced, were terminal budding and assumed varying
shapes. They could be globose, subglobose, variations of
cylindrical and allantoidal cells (Figure 2a and y). Yeast
formation also occurred by moniliform budding whereby
the growing tip enlarges; a constriction occurs behind the
expanded apex which assumes the yeast shape. It could
be globose, subglobose or ellipsoidal. This eventually
detaches from the hyphae into the medium where it
continues to proliferate by terminal budding (Figure 3a
and b).
DISCUSSION
Several growth forms were observed in submerged
cultures in this study. These included coenocytic hyphae,
septate hyphae, moniliform hyphae and terminal budding
yeast cells, which was preponderant. Therefore, R.
stolonifer is polymorphic in minimal medium.
The study of Bartnicki-Garcia and Nickerson (1962b)
showed that R. stolonifer could not convert to yeast-like
morphology under carbon dioxide pressure, unlike the
Mucor rouxii used in their study. In the aforementioned
study, it was shown that M. rouxii proliferate by multipolar
budding, whereby a yeast mother cell gave rise to
numerous multipolar daughter buds by blastic action.

Figure 1. Substrate-level and aerial mycelia of Rhizopus stolonifer showing (a) part of
stolon; (b) part of sporangiophore; (c) rhizoids; (d) sporangium; (e) collapsed sporangia
and (f) striated sporangiospores.
This is in contrast with the results obtained in the
present study whereby R. stolonifer exhibited different
growth forms in minimal medium. A weight proportion for
each morphological type in submerged culture was not
determined. Subjective assessment however showed that
the terminal budding yeast morphology was the dominant
form.
Although, nuclei were not visualized in this study, it
should be pointed out that dimorphic M. rouxii produces
yeast like cells which are multipolar budding and the
mother cell and daughter buds, each contain numerous
nuclei, as revealed by Giemsa staining (Bartnicki-Garcia
and Nickerson, 1962b) or 6-diamidino 2-phenylidole
(DAPI) staining (McIntyre et al., 2002; Lubberhussen et
Omoifo 4271
al., 2003), whereas terminal budding yeast cell like that of
Saccharomyces cerevisiae, harbours one nucleus per
cell. Thus, after conversion to the yeast cell from sporangiospore
or after intermediary stage as in moniliform
hypha, proliferation of R. stolonifer was in the manner
similar to the budding pattern of yeast cells of S.
cerevisiae.
It does not appear that formation of septa by
Zygomycetous species in minimal medium has been
previously reported. In the study of McIntyre et al. (2002),
the use of calcofluor white revealed the presence of
septa in filaments formed by M. circinelloides. But in that
case, it was due to the formation of arthrospores that
occurred after the growth and ceased (Lubberhussen et

4272 Afr. J. Biotechnol.
Figure 2. Submerged mycelia of R. stolonifer showing (c) coenocytic hyphae; (m) moniliform
hyphae; (s) septate hypha; (c) terminal budding yeast cells and (a) subglobose, ellipsoidal and
allantoid yeast cells, which may be 1 to 3 celled.
al., 2003). In the present study, arthrospores were not
observed. Septation is a characteristic of the higher fungi
where it compartmentalizes cellular components. Since
septate hyphae were observed in submerged culture in
this study, it thus means that R. stolonifer could exhibit
characteristics of the higher fungi. As this study showed,
it also applies to the formation of terminal budding yeast
cells.
Formation of terminal budding yeast cell took two
different lines. These include induction from (a)
sporangiospore and (b) moniliform hypha. The process of
induction of terminal budding yeast cells from
sporangiospores has been previously observed (Omoifo,
2003, 2005).
Swollen and then lysed sporangiospore, released
nucleates into medium of growth. These gave rise to
protoplasts which subsequently became yeast cells,
thereafter, proliferation by terminal bud formation
occurred. Omoifo (2006a, b) demonstrated the effect of
myoinositol and zinc on the induction and subsequent
proliferation of terminal budding yeast cells from sporangiospores
of M. circinelloides Tieghem in a system thought
to be of high ionic strength that possibly led to the
establishment of a pH-transmembrane gradient; such
would stimulate second messengers generation and
signals transduction within the living cell (Dawes, 1986;
Mitchell, 1978).
It was hypothesized that, activity at the molecular level
activated mitogenic factors that led to yeast form
induction (Omoifo, 2005). Thereafter, it could assume
that terminal yeast budding observed here is in the
manner of proliferation of S. cerevisiae, that is, daughter
bud production by blastic action whereby the inner wall of
the mother cell extends into the new bud, and this entails
the extension of the existing cell wall (Marchant and
Smith, 1968; Sentandreu and Northcote, 1969).
As shown in this study, formation of an intermediary
structure, similar to that elaborated for the initial stages of
formation of sporogenous hyphae was involved in
moniliform yeast induction (Omoifo, 2005). It was shown
that sporogenous hyphae emerges from sporangiospore
as coenocytic thin walled vegetative filament which
possesses thin lumen that becomes compartmentalized;
with the centripetal growth of the plasma membrane thus
forming septa-like partition (without the involvement of
cell wall). A morphologically distinct conidium is produced
at the distal end of this filament, and this holoblastically
produced catenate conidia (Omoifo, 2005). In the present
study, the growing tip of moniliform filament formed yeast
mother cell instead, and this on detachment becomes
terminal budding. This method of primary bud formation
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
bud; (b) mature bud near detachment. The moniliform hypha may have simple septa-like partition, (i).
Phialophora dermatitidis whereby there is bud eruption
from hyphal wall; bud wall is continuous with inner hyphal
wall, whereas the outer wall forms a collar (Grove et al.,
1973). In contrast too, is the study of Hardcastle and
Szaniszlo (1974) that showed that moniliform hyphae
emerged from induced yeast cells of Cladosporium
Omoifo 4273
werneckii but these produced lateral conidia, which on
release did not exhibit any form of budding.
In the present study, terminal budding yeast cells were
induced from sporangiospores in two different ways, viz
(a) sporangiospore to nucleates to protoplasts to yeast
cells; (b) sporangiospore to moniliform hypha to yeast

4274 Afr. J. Biotechnol.
cell. This study confirmed earlier reports which showed
that terminal budding yeast cells can be induced from
sporangiospores (Omoifo, 1996, 1997, 2003, 2006a, b;
Omoifo et al., 2006). For biochemical and molecular
studies to be conducted successfully, pure cultures of the
morphotypes or anamorphs of any particular fungus is
required. The achievement of this objective has been
demonstrated in several studies (Omoifo, 2003, 2005,
2006a, b). This is further confirmed in the foregoing
report.
ACKNOWLEDGEMENTS
This work was done at the MTCC-1, Institute of Microbial
Technology, Chandigarh, India during the Postdoctoral
fellowship programme supported by the Third World
Academy of Sciences, Triestie, Italy and the Council for
Scientific and Industrial Research, India. To both
institutions, I express my sincere gratitude.
REFERENCES
Bartnicki-Garcia S, Nickerson WJ (1962a). Induction of yeast-like
development in Mucor by carbon dioxide. J. Bacteriol. 84: 829- 840.
Bartnicki-Garcia S, Nickerson WJ (1962b). Nutrition, growth and
morphogenesis of Mucor rouxii. J. Bacteriol. 84: 841-858.
Bartnicki-Garcia S, Nickerson WJ (1962c). Isolation, composition and
structure of cell walls of filamentous and yeast-like forms of Mucor
rouxii. Biochim. Biophys. Acta. 58: 102-119.
Bartnicki-Garcia S, Nickerson WJ (1962d). Assimilation of carbon
dioxide and morphogenesis of Mucor rouxii. 64: 584-551.
Dawes EA (1986). Microbial Energetics: Tertiary Level Biology
Chapman and Hall, New York, p. 189.
Friedenthal JW Epstein A, Passon S (1974). Effect of potassium
cyanide, glucose and anaerobiosis on morphogenesis of Mucor
rouxii. J. Gen. Microbiol. 82: 15-24.
Grove SN, Oujezdsky KB, Szaniszlo PJ (1973). Budding in the
Dimorphic Fungus Phialophora dermatitidis. J. Bacteriol. 115(1): 323-
329.
Hardcastle RV, Szaniszlo PJ (1974). Characterization of dimorphism in
Cladosporium werneckii. J. Bacteriol. 119: 294-302.
Lubberhusen TL, Nielsen T, McIntyre M (2003). Characterization of the
Mucor circinelloides life cycle by on-line image analysis. J. Appl.
Microbiol. 95: 1152-1160.
Marchant R, Smith DG (1968). Bud formation in Saccharomyces
cerevisiae and a comparison with the mechanism of cell division in
other yeasts. J. Gen. Microbiol. 53: 163-169.
McIntyre M, Breum J, Arnau J, Nielsen J (2002). Growth physiology and
dimorphism of Mucor circinelloides (synracemosus) during
submerged batch cultivation. Appl. Microbiol. Biotechnol. 58: 495-
502.
Mitchell P (1978). Translocation through natural membranes. Adv.
Enzymol. 29: 33-87.
Omoifo CO (1996). Modeling sporangiospore-yeast transformation of
Dimorphonyces strain. Hind. Antibiot. Bull. 38: 12-31.
Omoifo CO (1997). Auxotrophic requirement for sproangiospore -yeast
transformation of Dimorphomyces diastaticus Strain C12. Hind.
Antibiot. Bull. 39: 11-15.
Omoifo CO (2003). Sequential sporangiosopore-yeast transformation
hypothesis. Afr. Scientist. 4: 191-273.
Omoifo CO (2005). Development of Yeast Cells from Sporangiospores.
Idehuan Publishing Company, Nigeria, p. 402.
Omoifo CO (2006a). Effect of K+, Na+ and uracil on sporangiosporeyeast
transformation of Mucor circinelloides Tieghe. Afr. J.
Biotechnol. 5(9): 715-722.
Omoifo CO (2006b). Effect of myoinositol and zinc on sporangiosporeyeast
transformation of Mucor circinelloides Tieghe Afr. J. Biotechnol.
5(9): 723-730.
Omoifo CO, Aruna MB, Omamor IB (2006). Effect of myoinositol on
sporangiospore-yeast transformation of Mucor circinelloides Tieghem
cultivated in synthetic broth. Hind. Antibiot. Bull. 47-48: 24-30.
Ruiz-Hereira J (1985). Dimorphism in Mucor species with emphasis on
M. Rouxii and M. Baciliformis. In: Szaniszlo PJ (ed). Fungal
Dimorphism With Emphasis on Fungi Pathogenic for Humans, New
York. pp. 361-385.
Schulz BE, Kraepelin G, Hinkelmann W (1974). Factors affecting
dimorphism in Mycophyta (Mucorales): a correlation with the
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Gen. Microbiol. 55: 393-398.

African Journal of Biotechnology Vol. 10(20), pp. 4276-4288, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB09.787
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Effect of extracellular calcium chloride on
sporangiospore-yeast transformation of Rhizopus
stolonifer
C. O. Omoifo
Department of Crop Science, Ambrose Alli University, Ekpoma, Nigeria. E-mail: coomoifo@yahoo.com.
Accepted 4 March, 2011
Previous studies showed that zinc and myoinositol, which are intracellular, serve as components of
signalling elements in many eukaryotes, and participate in stimulating the induction and proliferation of
yeast cells from sporangiospores of fungi. It was thought that a transmembrane-pH-gradient, similar to
what is obtained in bacterial mitochondrion or proton-substrate symport in yeasts, permitted
influx/efflux of materials into the cell that triggered the induction and subsequent proliferation of yeast
cells. To examine this model further, this study evaluated the ability of sporangiospores of Rhizopus
stolonifer to undergo morphogenetic transformation in the presence of different levels of extracellular
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
and proliferation to varying extent. Ca 2+ at 0.50 and1.8 mM, which was outstanding, was compared with
broth treatment at 0.25 mM ZnSO4: pH 4.5 which gave optimum biomass in a previous study. Medium
pH was further varied: pH 4.2, 4.5 and 5.0. The results showed that there was interaction between the
level of divalent cations and pH. At all levels tested, Ca 2+ was better stimulatory than Zn 2+ . Direct
induced yeast cell count, which eliminated possible impact of protoplasts and calcium phosphate
precipitate on optical density measurements, made these results more valid. Treatment at 0.50 mM Ca 2+ :
pH 5.0 gave the most profound result. This was therefore chosen for further biochemical work.
Key words: Rhizopus stolonifer, synthetic broth, dimorphism, extracellular Ca 2+ , sporangiospores, induced
yeast cells.
INTRODUCTION
Dimorphism is the conversion of a microorganism from
one growth habit to another, an effect caused by changes
in environmental conditions (Romano, 1966). In a report
presented over a decade ago, it was shown that
sporangiospores of a mucoraceous strain is transformed
to terminal budding yeast cells (Omoifo, 1996). This was
opposite the multipolar budding yeast-like morphology
that has been reported for many mucoraceous species
including Mucor rouxii (Bartnicki-Garcia, 1968; Bartnicki-
Garcia and Nickerson, 1962a, b, c, d; Lubberhusen et al.,
2003; McIntyre et al., 2002), Mycotypha (Hall and
Kolankaya, 1974; Schulz et al., 1974; Price et al., 1973).
The preceding report showed that several distinct
morphologies can be induced from the fungus, Rhizopus
stolonifer. These include yeast cells, coenocytic hyphae,
moniliform hyphae and septate hyphae. This is in
contrast to the multipolar budding yeast-like cells that
have been reported for other members of the
Zygomycetes. It is also in sharp contrast to the study of
Bartnicki-Garcia and Nickerson (1962b) whereby it was
shown that R. stolonifer was incapable of morphogenetic
interconversion.
Conversion of sporangiospores to terminal budding
yeast cells require structural modifications that involved
transient morphologies, including growth spheres and
cytosolic nucleates that are released when cell wall is
lysed, and protoplasts till the yeast is formed. This
becomes the stable form, which is proliferated by terminal
budding. These various forms have been shown in
several publications (Omoifo, 1996, 1997, 2003; Omoifo
and Omamor, 2005; Omoifo et al., 2006) including the
research monograph “development of yeast cells from
sporangiospores” (Omoifo, 2005).
In an attempt to explain the cryptic biophysical changes
that occurred leading to the formation of yeast, a system
model known as the sequential sporangiospore-yeast

transformation (SSYT) hypothesis was formulated
(Omoifo, 2003). This was based on electrochemical
gradient which permits transport processes and energy
metabolism in mitochondria or proton-substrate symport
through plasma membranes (Alderman and Hofer, 1981;
Albert et al., 1994; Dawes, 1986; Mitchell, 1967; Slayman
and Slayman, 1974; Voet and Voet, 1995; West and
Mitchell, 1972; 1973). It holds that the multiple conversions
are possible because the individual entities are
bathed in a milieu whereby a transmembrane-pHgradient
is set up and this encourages influx/efflux of
materials with consequent fundamental biochemical and
biophysical changes in cytosolic components and cell
wall integrity. It was shown that monovalent elements like
H + , K + and Na + , play important roles in the induction of
terminal budding yeast cells from sporangiospores of
muco-raceous strains. H + was involved in the
maintenance of the transmembrane-pH-gradient; K + was
absolutely necessary for the formation of protoplasts from
spora-ngiospores of Mucor circinelloides Tieghem
(Omoifo, 2005). On the other hand, accumulation of Na + ,
which is antiported with K + (Tonomura, 1986) was
necessary for the protoplasts to convert to the yeast form
(Omoifo, 2005). The effect of K + was confirmed in another
study where protoplast formation peaked at a high level
of K + 1.0 g/l, and yeast induction was optimal at 0.10 g/l
Na + . In a combinational manner under various
treatments, the interaction between 1.0 g/l K+ and 0.1 g/l
Na + had the greatest impact on yeast induction,
notwithstanding the occurrence of other anamorphic
subtypes including enterothallic and holothallic conidia,
which were scanty. However, these anamorphs were
eliminated when 100 mM uracil was incorporated into the
medium of growth (Omoifo, 2006a).
Subsequent study showed the effect of intracellular
signalling precursors on yeast induction from sporrangiospores.
Although, myoinositol and zinc were not the
primary initiators, they individually enhanced yeast
induction from sporangiospores (Omoifo and Aruna,
2006; Omoifo et al., 2006). When the cyclic hexitol and
divalent ion were used in combination, induced yeast
sigmoid growth pattern ensued and biomass was upped
by 63 to 65% in comparison with control cultures
(Omoifo, 2006b). Calcium is a primary element in signal
transduction in eukaryotic cells where it’s binding to
proteins triggers changes in conformation as well as
altering local electrostatic fields, thereby controlling
cellular processes (Kretsinger, 1979; Albert et al., 1994;
Berridge, 1987; Berridge et al., 2000; Voet and Voet,
1995; Clapham, 2007). Since zinc which plays key role in
catalytic and structural component of many proteins
(Eide, 2006) has been shown to enhance yeast induction
from sporangiospores, perhaps calcium, which functions
as an intracellular messenger (Albert et al., 1994) and
further involved in cytosolic Ca 2+ homeostasis (Forster
and Kane, 2000; Ton and Rao, 2004), would also
promote yeast induction from sporangiospores.
Omoifo 4277
The objective of this study was to evaluate the effect of
CaCl2 on the ability of R. stolonifer to convert to terminal
budding yeast cells. Since exogenous culture pH
influence Ca 2+ uptake through Ca 2+ channels in plasma
membrane (Youatt, 1990), the effect of pH on the
stimulatory ability was also evaluated.
MATERIALS AND METHODS
Chemicals
Reagent grade chemicals were obtained from BDH Laboratory
supplies (Poole, UK) except Bacto peptone (Sigma), myoinositol
(Sigma) and yeast extract (Oxoid).
Fungal strain
R. stolonifer used in this study was isolated at the Microbial Type
Culture Collection Laboratory 1, Institute of Microbial Technology,
Chandigarh India as reported in the preceding report. It was
maintained on glucose-yeast extract-peptone agar (GYPA: 10-0.3-
5.0-15g/l) slants. Fresh culture was prepared on Petri dish unto
which 20 ml of agar was poured and used after 7days of growth.
Inoculum preparation for growth studies
A 7 day old culture of R. stolonifer was flushed with sterile deionized
distilled water and a sterile glass rod was gently passed over
the fungal growth so as to dislodge the spores. The suspension,
poured into centrifuge bottles, was washed with 3 changes of sterile
deionized distilled water in an MSE 18 centrifuge at 5000 rpm for 7
min at 25°C. Spore count was taken with a Neubauer hemocytometer
and was made up to 10 6 /ml in sterile deionized distilled
water.
Reagents and culture media
Procedure for preparing the base medium has been described
(Omoifo, 1996) and has been used in subsequent studies (Omoifo,
2003, 2006a, b; Omoifo and Aruna, 2005; Omoifo et al., 2006). It
contained per litre: 10 g glucose, 5 g (NH4)2SO4, 5 g K2HPO4, 5 g
KH2PO4, 0.10 g NaCl, 0.10 g FeSO4, 2.0 g MgSO4, 0.060 g CuSO4,
0.060 g ZnSO4, 0.065 g MnCl2, 100 mg uracil and 2 Mm
myoinositol. Where the effect of Zn 2+ was to be tested, zinc
sulphate was not added to the basal preparation. The initial culture
was buffered at Ph 4.5 with 0.2 M Na2HPO4: 0.1 M citrate. When Ph
level was the test factor, culture was then buffered with equivalent
weights of the buffer species. Since levels of calcium were to be
tested, these were prepared separately. Each of the duplicate
flasks of the synthetic broth was incorporated with the various
concentrations of calcium chloride at the following levels: 0.0, 0.20,
0.25, 0.50, 1.0, 1.5 and 1.8 Mm. The Ph was adjusted with 2 N
NaOH or 1 N HCl using a Cole-Parmer Ph Tester model 59000.
The solution in each flask was made up to 100 ml with glass
distilled deionized water and sterilized at 121°C for 15 min.
Inoculation, growth conditions and sample collection
A 1 ml spore suspension was inoculated into each broth flask using
Eppendorf micropipette in an operation done in a laminar flow
chamber model CRC HSB-60-180. Before each inoculation, the

4278 Afr. J. Biotechnol.
Table 1. Treatments and biomass estimates with their respective standard errors, and form of growth
during the cultivation of R. stolonifer in synthetic broth for 120 h at Ph 4.5 and temperature 28°C.
Treatment: Calcium chloride (Mm) Mean biomass at 620 nm Form of growth
Control 0.1399 ± 0.0075 Yeast cell
0.2 0.1361 ± 0.0062 Yeast cell
0.25 0.1462 ± 0.0107 Yeast cell
0.5 0.1666 ± 0.0095 Yeast cell
1.0 0.1381 ± 0.0153 Yeast cell
1.5 0.1360 ± 0.0086 Yeast cell
1.8 0.1854 ± 0.0418, turbidity Yeast cell
spores were kept in suspension by shaking for 30 s and the broths
were similarly shaken after each inoculation before transferring to a
laboratory side bench for incubation at 28 ± 1°C. At 24 h intervals,
the culture flasks were brought to the chamber and 10 ml were
withdrawn from each culture flask with sterile pipettes, one for each
flask, into factory-sterilized plastic sample tubes. The culture flasks
were thereafter returned for further incubation. The samples were
kept at -18°C until further analysis. Slides were prepared for
viewing by putting one or two drops of lactophenol-in-cotton blue on
a glass slide and with the aid of wire loop, one drop from the culture
flask properly shaken was then added and covered with a glass
cover slip and viewed under a binocular microscope at x1000
magnification using the microscope Olympus CHB14. The observed
morphologies were recorded.
Biomass determination
Culture samples were thawed up to room temperature before
biomass determination. The absorbance was obtained at 620 nm,
using a Camspec M105 spectrophotometer (Cambridge, UK).
Statistical analysis
Results were subjected to a 2-way analysis of variance (ANOVA)
test for the single factor, or a split-plot format for combined factors
and considered significant if p < 0.05 and comparison between
means was performed using the Genstat 5 package.
RESULTS AND DISCUSSION
The effect of various levels of Ca 2+ on morphological
expression of R. stolonifer cultivated in still cultures is
shown in Table 1. Yeast cell was the predominant
morphology. In other dimorphic fungi, Ceratocystyis ulmi
(Paranjape et al., 1990) and Ophiostoma ulmi (Gadd and
Brunton, 1992), exogenous Ca 2+ stimulated germ tube
formation in media designated for yeast production, while
it led to a reduction of mycelia production in
Aureobasidium pullulans (Madi et al., 1997). Madi and
colleagues attributed this difference to the use of
‘restrictive medium’ in the former, as compared to their
use of what they termed ‘general medium’ for the growth
of A. pullulans. The preceding report illustrates the fact
that multiple morphologies could be induced from
sporangiospores in buffered synthetic broth, but this
could be streamlined to obtain specific morphology,
depending on the environmental factor incorporated into
the medium of growth. For example, multipolar budding
yeast like cell was obtained in complex nitrogen sourced
medium incubated at elevated temperature (Omoifo,
1996, 2005) and terminal budding yeast cells from simple
nitrogen sourced-medium (Omoifo, 1996), or myoinositolincorporated
medium (Omoifo, 1997, 2003, 2005; Omoifo
et al., 2006).
In the present study, proliferation of terminal budding
yeast cells induced from sporangiospores of R. stolonifer
occurred at all concentration levels tested. Growth profile
did not describe a sigmoid pattern (Figure 1). Mean
biomass showed differences in growth between cultures
where control broth apparently had higher values than
0.2, 1.0 and 1.5 mM Ca 2+ (Table 1). Analysis of variance
of O.D. readings showed that only time had significant
impact on yeast induction and growth (Table 2). However,
on separation of means, LSD 0.04889 p < 0.05, the
aforementioned concentration levels were not distinct
except for 0.5 and 1.8 mM Ca 2+ , which were in separate
subsets (Table 3).
In this study, control culture or cultures with low level
Ca 2+ made had no difference with yeast proliferation,
except when there was high level challenge, 0.5 or 1.8
mM Ca 2+ . But the magnitude of yeast cells formed in the
1.5 mM Ca 2+ supplemented broth compares with that of
control although protoplasts were part of entities in the
latter culture. This study decided to subject the Ca 2+
levels with the higher O.D. values to different pH
treatments.
The effect of extracellular pH levels on sporangiosporeyeast
transformation had earlier been shown (Omoifo,
1996). In synthetic broth, pH 4.5 was found to be most
ideal for conversion of sporangiospores to yeast cells
(Omoifo, 1996). A subsequent report showed that
buffered synthetic broth pH 4.5, supplemented with 0.25
mM ZnSO4 further enhanced conversion of sporangiospore
of M. circinelloides to yeast (Omoifo, 2006a).
In the present study, Zn 2+ was contrasted with Ca 2+ under
the optimum conditions previously determined but also
varied this with a ≤0.5 unit pH differences in either
direction. Cumulative mean values showed that the
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
of calcium chloride for 120 h at pH 4.5 and temperature of 20°C, ambient.
Omoifo 4279

4280 Afr. J. Biotechnol.
Table 2. Analysis of variance of optical density readings (620 nm) of induced yeast
cells of R. stolonifer.
Source of variation DF SS MS F-pr.
Concentration 6 0.043517 0.007253 0.316
Time 4 0.068225 0.017056 0.029*
Concentration x time 24 0.187666 0.007819 0.192
Residual 105 0.638335 0.006079
Total 139 0.937743
*Significant at p < 0.05 probability level; DF, degree of freedom; SS, sum of square, MS,
mean sum; F-pr, frequency of probability.
Table 3. Homogenous subsets of mean biomass estimates
obtained during sporangiospore-yeast transformation of R.
stolonifer cultivated in synthetic broth for 120 h with pH 4.5
at 28°C, ambient.
Treatment : Calcium chloride (mM) Mean
Subset 1
1.8 0.1854
Subset 2
0.5 0.1676
Subset 3
Control 0.1399
0.2 0.1361
0.25 0.1462
1.0 0.1381
1.5 0.1360
Means separation based on L.S.D. 0.04889 at p < 0.05.
supplementation but was the least with the 0.25 mM Zn 2+
challenge which was barely lower than 1.8 mM Ca 2+
insult (Table 4). This indicated that Ca 2+ ion at 0.5 mM
concentration was more effective in promoting yeast
induction/proliferation.
To know the contribution of the various factors
incorporated into the growth medium, a split-plot analysis
of variance of growth data was done. It was shown that
the main factors including ionic concentration, pH and
time, as well as their interactions made significant impact
(p < 0.1) on growth of R. stolonifer as yeast cells (Table
5). The specific contribution of pH levels to yeast proliferation
is shown in Figure 2. The pH influenced yeast
proliferation. At 0.5 mM Ca 2+ concentration where
optimum growth occurred, maximum growth was at pH
4.5, whereas it was at pH 5.0 when the 1.8 mM Ca 2+
concentration (which had lower optimum values than the
0.5 mM Ca 2+ concentration) was used. While the least
biomass was recorded at 0.25 mM Zn 2+ concentration,
maximum growth with this ion also occurred at pH 5.0.
But at pH4.2 where the least growth occurred for all the
ionic concentrations, the 0.25 mM Zn 2+ had higher growth
value than the 1.8 mM Ca 2+ concentration. While growth
was compromised at pH 4.2 in the 1.8 mM Ca 2+ culture,
the pressure was relieved as the external pH rise. This
indicated that at the cytosolic level, there was interaction
between the type of ion and pH on the one hand, and the
ionic concentration and pH level on the other. A
comparison of biomass of the Ca 2+ challenge at all the pH
levels tested suggested that yeast induction and proliferation
is more sensitive to higher level Ca 2+ insult.
Since there is interest in conditions where determinable
growth phases (lag, log and stationary) occurred, a timecourse
pattern should illustrate this. Induced proliferating
yeast cells of R. stolonifer assumed sigmoid curve with
0.25 mM Zn 2+ supplementation at the lower pH values,
whereas at pH 5.0 where the biomass was higher (Figure
2), there appeared to be a resurgence of growth after the
initial rapid log growth and wind-down 96 h from
commencement of experiment (Figure 3a). The occurrence
of sigmoid growth of yeast cells induced from
sporangiospores of M. circinelloides cultivated in 0.25
mM Zn 2+ supplemented pH 4.5 minimal cultures has been
previously reported (Omoifo, 2006a). In that study, other

Optical density at 620
Table 4. Mean biomass of induced yeast cells from sporangiospores of R.
stolonifer cultivated in buffered synthetic broth at different levels of
exogenously applied Ca 2+ and Zn 2+ ions.
Parameter
Optical density at 620 nm
0.25 mM ZnSO4 0.50 mM CaCl2 1.80 mM CaCl2
Mean 0.3795 0.8252 0.3941
S.E. 0.003 0.010 0.006
Fold-increment 2.2 2.1
Table 5. Analysis of variance of growth data of R. stolonifer cultivated in buffered synthetic
broth at different pH levels and exogenously applied ions.
Source of variation d.f. Sum of square F-value
Concentration 2 7.693008

4282 Afr. J. Biotechnol.
Figure 3. Time-course pattern in the growth of induced yeast
cells of R. stolonifer at different combinations of ions and pH
levels.
overcame inherent repression and assumed proliferation,
and hence the resurgence of growth prior to termination
of experiments. In contrast, in the 0.5 mM Ca 2+ challenge,
a sigmoid growth pattern was described in the buffered
pH 5.0 culture. However, a resurgence of growth occurred
after sigmoid pattern when the cultures were buffered
at the lower levels of pH (Figure 3b). Perhaps similar
explanation for this phenomenon stated earlier is here
forth attributable. Growth profiles at the 1.8 mM Ca 2+
concentration showed greater variability from the sigmoid
pattern (Figure 3c). Growth had extended lag: 96 h at pH
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
cultivated in buffered synthetic broth at different levels of exogenously applied Ca 2+
and Zn 2+ ions.
Parameter
Unicellular count (x10 6 )
0.25 mM ZnSO4 0.50 mM CaCl2 1.80 mM CaCl2
Mean 8.99 87.31 53.56
Standard error 0.149 1.629 1.64
Fold-increment 9.71 1.63
Comparing tables 4 and 6, the difference that exists between 0.25 mM ZnSO4 and 0.5
mM CaCl2 on one hand and 0.5 mM CaCl2 and 1.8 mM CaCl2 on the other are also
reflected in the direct cell count data, although the magnitude vary. As the CaCl2 level
increases, growth of induced yeast cells is suppressed.
where a resurgence of growth also occurred after 96 h.
The higher profile description at pH 5.0 is a reflection of
the magnitude of growth shown in Figure 2 and shows
similarity with the pattern in the 0.25 mM Zn 2+ supplementation
(Figure 3a) although its magnitude was greater
(Figure 2). Microscopic examination revealed that
morphological expression in the pH 5.0 buffered cultures
was protoplast and yeast cell, except when the ionic
concentration was 0.5 mM Ca 2+ , which was of mainly
terminal budding yeast cell.
In all experiments, except with control and 0.25 mM
Zn 2+ supplementation, turbidity appeared to occur probably
resulting from precipitation of calcium phosphate
and this appeared to increase with Ca 2+ levels. Similar
observation was made in the study which showed the
effect of exogenous Ca 2+ on the growth of Aureobasidium
pullulans (Madi et al., 1997). In order to validate the
findings of this study, especially where sigmoid curves
were obtained for optical density readings, direct cell
count was do. This would eliminate any discrepancy in
O.D. readings that could arise from calcium phosphate
precipitation.
Unicellular population size in the 0.5 mM Ca 2+ culture
was 9.7-fold that of the 0.25 mM Zn 2+ , while it was 1.6fold
the magnitude of the 1.8 mM Ca 2+ broth (Table 6). In
contrast, when biomass estimation was done through
measurement of O.D., the value of 0.5 mM Ca 2+ broth
was 2.2-fold that of 0.25 mM Zn 2+ and 2.1-fold the
magnitude in 1.8 mM Ca 2+ culture. Earlier experiment had
shown that protoplast, cytosolic units that is, nucleates in
earlier studies, are released when cell wall lysed, and
thallic subtypes and conidia were induced in Zn 2+
supplemented broths (Omoifo, 2006a). However, thalli
were absent in the presence of 100 mM uracil (Omoifo,
2006b). In the present study, protoplasts and yeast cells
contributed to the O.D. readings in the Zn 2+ supplemented
medium, whereas only yeast cells were herein
counted. Calcium phosphate precipitated only in Ca 2+
supplemented media. Thus, the contributors to the O.D.
readings included the induced cellular entities and
calcium phosphate precipitate. Since the value differential
between the 1.8 and 0.5 mM Ca 2+ estimate was larger, as
Omoifo 4283
reflected in the greater than 2-fold increase when O.D.
was used in biomass assessment in contrast to the use
of direct unicellular count (less than 2-fold increase), it
was difficult to assume that calcium phosphate precipitation
was the sole cause of the reduction in the biomass
estimate in the 1.8 mM Ca 2+ supplemented medium.
Perhaps, the high Ca 2+ concentration suppressed yeast
cell induction and proliferation.
This was probably confirmed at pH 5.0 where yeast
count in the 1.8 mM Ca 2+ supplementation was significantly
less than that of 0.5 mM Ca 2+ (Figure 4).
Furthermore, the 1.8 mM Ca 2+ medium contained terminal
budding yeast cells, unbudded yeast cells as well as
protoplasts, whereas 0.5 mM Ca 2+ broth contained
terminal budding yeast cells only. Apparently, excess
Ca 2+ influenced the ability of protoplasts to convert to
yeast form on the one hand, and of unicellular yeast form
to undergo mitogenesis and or proliferation on the other.
Similar trend, higher values for 0.5 mM Ca 2+ as against
1.8 mM Ca 2+ , occurred at pH 4.2 or 4.5 and further
supports the results obtained when optical density was
used for biomass estimation (Figure 2). These results
illustrate the effect of interaction between pH level and
exogenous Ca 2+ supply.
To further test all such interactions, an analysis of
variance of induced yeast count data was done (Table 7).
There was significant (p < 0.001) difference for the
induction and proliferation because of treatments, indicating
the existence of variability in the transformation
process.
The existence of variation in the treatments is in agreement
with such findings when optical density was the
measure of biomass, as shown earlier. This also agrees
with the finding of Omoifo (2006a) when M. circinelloides
Tieghem was the test microorganism and Zn 2+ and
myoinositol were incorporated into the growth medium. In
the present study, the mean performances of treatments
are presented in Table 8.
Of the treatments, the least induced yeast cell count
was obtained in the 0.25 mM ZnSO4: pH 4.2 medium and
progressively increased with pH until the optimum for this
ion in 0.25 mM ZnSO4: pH 5.0 broth. The 0.5 mM Ca 2+

4284 Afr. J. Biotechnol.
Ind uced yeast cell count (1 x 10 6 )
0.25 mM ZnSO4
0.5 mM CaCl2
1.8 mM CaCl2
Figure 4. Microscopic cell count of induced yeast cells of R. stolonifer in Ca 2+ and Zn 2+ incorporated buffered
synthetic broth as affected by different levels of pH.
Table 7. Treatment and induced cell estimates with their respective standard errors and form
of growth during the cultivation of R. stolonifer in synthetic broth for 120 h at temp28°C.
Treatment Mean cell count (x10 6 ) Form of growth*
0.25 mM ZnSO4: pH4.2 4.94±2.04 Y,P
0.25 mM ZnSO4: pH4.5 9.68±4.47 Y,P
0.25 mM ZnSO4: pH5.0 12.42±4.83 Y,P
0.50 mM CaCl2: pH4.2 56.60±29.87 Y, P
0.50 mM CaCl2: pH4.5 71.02±33.99 Y
0.50 mM CaCl2: pH5.0 134.08±62.67 Y
1.80 mM CaCl2: pH4.2 24.24±14.21 Y, P
1.80 mM CaCl2: pH4.5 41.38±19.61 Y, P
1.80 mM CaCl2: pH5.0 80.15±35.84 Y, P
*Y, yeast cell; P, protoplast.
concentration on average had higher growth value. The
buffered pH 4.2 culture had mean count of 56.60 ± 29.87
and this increased as the medium pH until the maximum
at 134.08 ± 62.27 at pH 5.0. Comparatively, 0.5 mM
CaCl2 supported higher level of growth than 1.8 mM
CaCl2. The increase in growth value also followed the
same trend as those at the other levels. Therefore, the
results of treatment means confirmed the influence of
extracellular pH on the induction from, and subsequent
proliferation of yeasts from sporangios pores of R.
stolonifer.
Figure 5 shows yeast proliferation profiles in the different

Table 8. Analysis of variance of induced yeast cell count of R. stolonifer cultivated in
buffered synthetic broth with different pH levels and exogenously applied ions.
Source of variation Degree of freedom Sum of square F-value
Concentration 2 184113.5

4286 Afr. J. Biotechnol.
Figure 5. Time-course pattern in induced yeast cell count of R.
stolonifer at different combinations of ions and pH levels. Growth
pattern was sigmoid in the buffered pH 4.5 synthetic broths
when Ca2+ was exogenously supplied, and at pH 4.2and 5.0,
growth was still at the logarithmic phase when the experiments
were terminated. Growth magnitude was reduced at pH 4.2.
Table 9. Paired t-test on total unicellular count in media incorporated with Ca 2+ or
Zn 2+
Ionic concentration Mean Variance N
0.5 mM Ca 2+ 9.31567E7 1.05997E16 60
0.25 mM Zn 2+ 8.10667E6 6.52549E13 60
t = 6.87839, p = 4.34432E-9. At the 0.05 level, the two means are significantly different

Log10 (number of induced yeast cells)
Log10 (number of induced yeast cells)
Time (h)
Time (h)
0.5 mM CaCl2 pH 4.2
0.5 mM CaCl2 pH 4.5
0.5 mM CaCl2 pH 5.0
1.8 mM CaCl2 pH 4.2
1.8 mM CaCl2 pH 4.5
1.8 mM CaCl2 pH 5.0
Figure 6. Log transformed induced yeast cell count of R. stolonifer in cultures where sigmoid growth was exhibited
Table 10. Lag phase and specific growth rate in cultures where sigmoid growth pattern was
exhibited.
Extracellular concentration 0.5 mM Ca 2+ 1.8 mM Ca 2+
Extracellular pH 4.2 4.5 5.0 4.2 4.5 5.0
Lag phase (h) 24 24 24 48 48 48
µ 0.025 0.026 0.029 0.037 0.039 0.031
The longer lag possibly means that growth was repressed at that concentration/pH level resulting in lack
of cell number increase. Conceivably, the eventual increase in cell number may then have been due to
a minority of cells in the medium not initially suppressed, which could continue to grow and divide. In
this situation, the lag would represent the time required for the subpopulation of cells not repressed to
overgrow the arrested population. Alternatively, the cells could overcome the repressive effect of Ca 2+,
by genetic adaptation and subsequently proliferate, thus achieving higher specific growth rate.
Omoifo 4287

4288 Afr. J. Biotechnol.
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African Journal of Biotechnology Vol. 10(20), pp. 4289-4295, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1198
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Genetic variation and bottleneck in Japanese quail
(Coturnix japonica) strains using twelve microsatellite
markers
Hossein Emrani 1 *, Cyrus Amirinia 1 and Mohammad Ali Radjaee Arbabe 2
1 Department of Biotechnology, Animal Science Research Institution of Iran (ASRI), Karaj, Iran.
2 Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran.
Accepted 28 January, 2011
The genetic structure of four strains of Japanese quail (Pharach, Panda, Tuxedo and Golden) was
investigated by 12 microsatellite markers in Iran. Whole blood samples were collected from 200
individuals belonging to four strains and genomic DNA was extracted by salting out procedure. The 12
microsatellite markers were amplified through polymerase chain reaction (PCR). The results indicated
that the average heterozygosity between strains ranged from 0.4343 to 0.7902. The Chi-square and
likelihood ratio test performed to examine strains for Hardy-Weinberg equilibrium showed some highly
significant deviations from deficiency. FIS value, which indicates the degree of departure from random
mating, was particularly high in the four strains when compared to that of other breeds, indicating
heterozygosity deficiency. Maximum Nei’s genetic distance was observed between Tuxedo and Golden
strains; whereas the minimum Nei’s genetic distance was observed between Pharach and Tuxedo
strains. Tuxedo strain revealed bottleneck event under three models of microsatellite evolution for sign,
standardized differences and Wilcoxon sign rank tests. The power of the microsatellite marker as a
useful tool for evaluating genetic variation within and between Japanese quail strains was also noted.
Key word: Bottleneck, genetic distance, genetic variation, microsatellite, Japanese quail.
INTRODUCTION
The Japanese quail, originally domesticated around the
11th century as a pet song bird (Howes, 1964; Crawford,
1990), is valued for its eggs and meat. It is also a
valuable laboratory species because of its small body
size, rapid generation interval and high prolificacy (Mills
et al., 1997). Japanese quail is phylogenetically closely
related to the chicken (Stock and Bunch, 1982). Both
species have a karyotype of 2n = 78 chromosomes and a
similar genome length of 1.2 × 10 9 bp, consisting of
morphologically distinct macrochromosomes (1–8 and
the ZW sex chromosomes) and cytologically indistinguishable
microchromosomes (Shibusawa et al., 2001).
*Corresponding author. E-mail: h_emrani@asri.ir. Tel: +98-261-
4430010-14 (465). Fax: +98-261-4413258.
Abbreviations: PIC, Polymorphism information content; PCR,
polymerase chain reaction; HWE, Hardy-Weinberg equilibrium.
Thus, the Japanese quail has been recommended as a
model species for poultry (Baumgartner, 1994; Mills et
al., 1997; Kayang et al., 2004). Recently, 100 microsatellite
markers were developed for Japanese quail
(Kayang et al., 2000, 2002) and used to build the first
microsatellite linkage map, which spans 576 cM and
contains 58 loci assigned to 12 linkage groups (Kayang
et al., 2004). Microsatellites are valuable genetic markers
due to their dense distribution in the genome, great
variation, co-dominant inheritance and easy genotyping.
They were extensively used in parentage testing, linkage
analyses, population genetics and bottleneck studies
(Goldstein and Pollock, 1997).
Genetic diversity in populations and the evolutionary
forces that affect it are central to both evolutionary
(Wright, 1931) and conservation biology (Frankham,
1995b). When populations undergo temporary large
reductions in size of so-called population bottlenecks (Nei
et al., 1975), they lose genetic diversity through random
drift. Identifying populations that have experienced a

4290 Afr. J. Biotechnol.
severe reduction in size (a bottleneck) is important
because bottlenecks can increase demographic stochasticity,
rates of inbreeding, loss of genetic variation and
fixation of mildly deleterious alleles, thereby reducing
evolutionary potential and increasing the probability of
population extinction (Frankel and Soule, 1981;
Frankham, 1995a, b; Rall et al., 1988; Bryant et al., 1986;
Goodnight, 1987; Luikart et al., 1998). In this study, the
bottleneck and genetic diversity within and among four
strains of Japanese quail was analyzed (Panda, Pharach,
Golden and Tuxedo) by 12 microsatellite markers as
recom-mended by Kayang et al. (2002).
MATERIALS AND METHODS
Sampling and DNA extraction
Japanese quail strains, reared at Bonab Research Center,
Northwest of Iran, were used as the experimental animals. These
strains were reared for 17 generations and were phenotypically
selected for high weight gain. Any new individual has not been
introduced to the strains.
Whole blood samples were randomly collected from 200
individual belonging to four strains: 70 individuals from Pharach
strain, 40 individuals from Panda strain, 50 individuals from Tuxedo
strain and 40 individuals from Golden strain. About 200 µl of blood
per individual was collected in 0.5 mM EDTA (pH 8). Genomic DNA
was extracted by the salting out procedure (Miller et al., 1988).
Microsatellite primers
12 microsatellites with high polymorphism information content (PIC)
value, indicating the informative of the markers, recommended by
Kayang et al. (2002) were used in this study. The primers were
encoded by GUJ0001, GUJ0021, GUJ0023, GUJ0034, GUJ0041,
GUJ0049, GUJ0052, GUJ0055, GUJ0059, GUJ0070, GUJ0097
and GUJ0099 characters. Primers were synthesized by TIBMO-
LBIOL Company, Germany.
PCR amplification
Genomic DNA was amplified by PCR containing 50 ng of template
DNA, 2.5 µl of 10 x buffer (10 Mm Tris, 50 mM Kcl, 0.1% gelatin,
pH 8.4), 1.5 to 2.0 mM MgCl2 (as optimized for each marker), 200
µM of each dNTPs , 1 µl of 5 pmol/µl each for forward and reverse
primers and 1 u of Taq DNA polymerase; ddH2O was added to the
volume of 25 µl. Reactions were carried out on a thermal cycler
(Biometra) using an initial 2.5 min denaturation at 95°C, followed by
30 cycles of denaturation at 95°C for 1 min, annealing at 50 to 62°C
for 30 s, extension at 72°C for 30 s, and a final extension step at
72°C for 5 min. The PCR products were electrophoresed on 8%
denatured urea-polyacrylamide gel and bands visualized by rapid
silver staining method (Sanguinetti et al., 1994). The gel was
photographed using Gel-Doc XR (BioRad). Patterns of the different
genotypes for each microsatellite locus were analyzed using Gel-
Pro analyzer, version 3.1 for windows TM, which determines the
allele's sizes in each animal.
Statistical analysis
Allelic frequencies were estimated using genotype counting. Hardy-
Weinberg equilibrium (HWE) based on likelihood ratio were tested
for different locus-population combinations by POPGENE software
Version 1.31 (Yeh et al., 1999). Nei’s (1972) standard genetic
distance and Reynolds genetic distance (Reynolds et al., 1983)
matrices were calculated by tools for population genetic analysis
(TFPGA) software version 1.3 (Miller, 1997) and dendrograms were
constructed using unweighted pair-group method with arithmetic
mean (UPGMA) by TFPGA software (Miller, 1997) with 1000
bootstrap replications. Polymorphism criteria such as PIC
(Botestein et al., 1980) and the number of observed and effective
alleles (Hedrick, 1999) were also estimated by HET software
version 1.8 (Ott, 2001) and POPGENE software packages, respectively
with assumption of Hardy-Weinberg equilibrium. Average
expected heterozygosity was calculated by POPGENE software
(Yeh et al., 1999).
To accomplish the goal of finding evidence of fluctuations in
population sizes, bottleneck software package was used (Cornuet
and Luikart, 1996) to detect deviations from mutation drift
equilibrium. This method consisted three excess heterozygosity
tests developed by Cornuet and Luikart (1996): (i) sign test, (ii)
standardized differences test and (iii) a Wilcoxon sign-rank test. The
probability distribution was established using 1000 simulations
under three models: infinite allele model (IAM), stepwise mutation
model (SMM) and two-phase model of mutation (TPM) by using
Bottleneck v1.2.02 Software (http://www.ensam.inra.fr/URLB).
RESULTS
All loci were polymorphic in the four Japanese quail
strains, except GUJ0001 and GUJ0041, which were
monomorphic in the Panda and Tuxedo, respectively.
The number of alleles per locus varied from 9 (GUJ0059)
to 3 (GUJ0001, GUJ0021 and GUJ0041). New alleles
were found in several loci that have not been previously
reported in the same loci for other Japanese quail by
Kayang et al. (2002). Significant deviation from HWE in
all population-locus combinations were observed except
for GUJ0041 in Pharach strain, GUJ0023, GUJ0099,
GUJ0021, GUJ0034, GUJ0041 and GUJ0097 in Panda
strain and GUJ0034, GUJ0055, GUJ0049 and GUJ0070
in Golden strain (P < 0.05). Effective number of alleles,
PIC, observed heterozygosity and expected heterozygosity
are given in Table 1. All the expected heterozygosities
were larger than the observed heterozygosities,
except for GUJ0097 in Tuxedo.
Nei’s (1972) standard genetic distance (Da), corrected
for bias, due to sampling of individual, and Reynolds
genetic distance (D Reynolds) matrices (Reynolds et al.,
1983) are reported in Table 2.
The expected heterozygosity (HExp) ranged from 0.841
(GUJ0070) in the Pharach strain to 0.486 (GUJ0052) in
the Golden strain. Polymorphism criteria such as PIC
values and number of alleles indicate high polymorphism
at some of the loci. The PIC is a good standard for
evaluating genetic markers. The highest and the lowest
PIC values belonged to GUJ0059 in Golden (0.815) and
GUJ0041 in Panda strain (0.427), respectively. Effective
number of alleles is a reciprocal of gene homozygosity
(Hartl and Clark, 1989). The highest and the least
effective numbers of alleles were in GUJ0059 locus
(6.04) in Pharach strain and in GUJ0041 locus (1.91) in

Table 1. Statistical results of 12 loci of quail strains.
Microsatellite
primer
GUJ0001
GUJ0021
GUJ0034
GUJ0041
GUJ0049
GUJ0059
GUJ0070
GUJ0097
Genetic parameter of
microsatellite loci
Sample size (n)
Pharach 70 Panda 40
Tuxedo
50
Emrani et al. 4291
Golden 40
NO 5 1 3 4
Ne 3.37 1.00 2.95 3.07
PIC 0.641 0.000 0.586 0.613
HObs 0.230 0.000 0.000 0.000
HExp 0.717 0.000 0.676 0.687
FIS 0.672 0 1 1
NO 6 3 5 5
Ne 4.48 2.57 3.66 3.58
PIC 0.751 0.535 0.680 0.677
HObs 0.526 0.666 0.428 0.055
HExp 0.798 0.666 0.727 0.730
FIS 0.322 -0.091 0.410 0.922
NO 5 5 5 7
Ne 3.10 4.57 3.20 4.78
PIC 0.627 0.745 0.636 0.761
HObs 0.200 0.750 0.250 0.571
HExp 0.690 0.883 0.709 0.805
FIS 0.705 0.040 0.636 0.277
NO 6 3 1 6
Ne 2.65 1.91 1.00 2.75
PIC 0.590 0.427 0.000 0.568
HObs 0.321 0.250 0.000 0.250
HExp 0.629 0.491 0.000 0.646
FIS 0.484 0.475 0 0.601
NO 6 6 5 7
Ne 4.00 4.26 3.50 4.00
PIC 0.715 0.735 0.668 0.715
HObs 0.600 0.666 0.400 0.583
HExp 0.769 0.787 0.733 0.766
FIS 0.200 0.129 0.441 0.222
NO 5 4 6 8
Ne 3.27 3.57 4.96 6.03
PIC 0.650 0.762 0.767 0.815
HObs 0.333 0.000 0.250 0.071
HExp 0.709 0.757 0.815 0.849
FIS 0.520 1 0.687 0.914
NO 7 5 6 7
Ne 6.04 4.00 4.88 4.57
PIC 0.813 0.707 0.765 0.750
HObs 0.300 0.333 0.600 0.500
HExp 0.841 0.782 0.815 0.797
FIS 0.640 0.555 0.245 0.360
NO 6 6 6 6
Ne 4.84 3.66 4.76 3.52
PIC 0.762 0.690 0.758 0.685
HObs 0.500 0.625 0.846 0.647
HExp 0.807 0.7500 0.805 0.727
FIS 0.369 0.139 - 0.071 0.096

4292 Afr. J. Biotechnol.
Table 1. Contd.
GUJ0055
GUJ0052
GUJ0023
GUJ0099
NO 5 4 4 4
Ne 4.458 3.446 3.188 3.173
PIC 0.740 0.658 0.637 0.489
HObs 0.45 0.555 0.307 0.411
HExp 0.783 0.730 0.699 0.547
FIS 0.399 - 0.217 0.552 0.237
NO 5 4 5 4
Ne 3.913 3.677 4.083 1.933
PIC 0.701 0.685 0.712 0.563
HObs 0.166 0.666 0.642 0.166
HExp 0.750 0.755 0.768 0.486
FIS 0.864 - 0.077 0.148 0.589
NO 5 4 4 4
Ne 3.161 2.976 3.881 2.919
PIC 0.647 0.616 0.694 0.597
HObs 0.414 0.375 0.500 0.235
HExp 0.689 0.685 0.755 0.667
FIS 0.357 0.435 0.326 0.687
NO 5 4 3 4
Ne 4.304 3.521 2.945 3.085
PIC 0.729 0.662 0.586 0.625
HObs 0.607 0.666 0.143 0.388
HExp 0.774 0.736 0.672 0.685
FIS 0.209 - 0.014 0.785 0.234
NO, observed number of alleles; Ne, effective number of alleles; PIC, polymorphism information content; HObs , observed
heterozygosity; HExp, expected heterozygosity; FIS , is the inbreeding coefficient.
Table 2. Distance matrices estimated by Da (above diagonal) and DReynolds (below diagonal)
distance using 1000 bootstrap replications.
Strain Pharach Panda Tuxedo Golden
Pharach *** 0.307 0.247 0.571
Panda 0.050 *** 0.402 0.743
Tuxedo 0.044 0.071 *** 0.794
Golden 0.112 0.142 0.128 ***
Panda strain, respectively (Table 1). FIS of the inbreeding
coefficient, measure the relative heterozygote deficit and
non-random mating in samples. Its value ranges between
-1 (all individuals heterozygote), 0 (random association of
alleles) and 1 (all individuals homozygote). If inbreeding
is avoided, F = 0; negative F indices are usually from
selection in favor of the heterozygotes, whereas positive
values indicate that the considered population has an
inbreeding system of mating (Liu et al., 2008). The mean
values of FIS estimates obtained 0.45, 0.3, 0.47 and 0.46
for Pharach, Panda, Tuxedo and Golden strains, respectively
indicating the high level of inbreeding in the four
strains. This could be as a result of a bottleneck effect.
Bottlenecks influence the distribution of genetic variation
within and among populations, thus the genetic effects of
reductions in population size requires evaluation. To characterize
it, sign, standardized differences and Wilcoxon
sign rank tests were utilized. The values of average
heterozygosity (He) and their probabilities (H > He) in the
sign test, under three models of microsatellite evolution
(IAM, SMM and TPM) were calculated and used to
measure the expected number of loci with heterozygosity
excess (Table 3). If the probability values for each model
were less than 0.05, then null hypothesis was rejected,
indicating that the bottleneck event occurred in this
model. For example, the expected number of loci with
heterozygosity excess in Pharach strain was 7.10 and
7.15 for TPM and SMM, under null hypothesis in the sign
test, respectively (Table 3). The probability values were,
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
and TPM.
Parameter IAM TPM SMM
Sign test: number of loci with heterozygosity excess (probability)
Pharach Expected = 7.01(0.0151) * 7.10 (0.0749) 7.15(0.0792)
Panda Expected = 6.37(0.0024) * 6.46(0.0028) * 6.59 (0.1179)
Tuxedo Expected = 6.32(0.0023) * 6.49(0.0030) * 6.54(0.0032) *
Golden Expected = 7.03(0.0690) 7.11 (0.2095) 7.07 (0.3649)
Standardized differences test (Ti values and probability)
Pharach 3.272 (0.00053) * 2.077 (0.0189) * 0.131 (0.4480)
Panda 4.204 (0.00001) * 3.435 (0.0003) * 2.497 (0.0062) *
Tuxedo 4.998(0.00001) * 4.490(0.0000) * 3.729 (0.0001*)
Golden 2.666 (0.0038) * 1.461 (0.0720) -0.441 (0.3295)
Wilcox sign-rank test (probability of heterozygosity excess)
Pharach 0.03418* 0.04248* 0.2334
Panda 0.00049* 0.00049* 0.00342*
Tuxedo 0.00049* 0.00049* 0.00049*
Golden 0.00122* 0.05225 0.67725
*Rejection of null hypothesis/bottleneck.
hypothesis was accepted when using sign test. These
results indicate that, due to mutation-drift equilibrium, the
Pharach strain has not undergone a recent genetic bottleneck.
However, the expected number of loci with
heterozygosity excess was 7.01 for IAM with probability
of 0.0151 and thus rejected the null hypothesis, indicating
bottleneck under this model.
The standardized difference test for Pharach strain
provided the Ti (probability) statistics equal to 3.272
(0.00053), 2.077 (0.0189) and 0.131 (0.4480) for the IAM,
TPM and SMM models, respectively. The probability
values were less than 0.05 for IAM and TPM, thus
hypothesis of mutation-drift equilibrium was accepted
under SMM only. Using the Wilcoxon rank test, the
probability values were 0.03418 (IAM), 0.04248 (TPM)
and 0.2334 (SMM) under the three models, indicating
that the null hypothesis was accepted under SMM model.
Among the used tests, Wilcoxon rank test and standard
difference test showed bottleneck event in golden strain
under IAM. However other P values tests were not
significant, demonstrating that the null hypothesis of
mutation–drift equilibrium was fulfilled in this strain. The
results of all the three tests together showed that the
bottleneck event did not occur in this strain. Golden strain
is a wild strain that was collected from its natural niche
and suffered few selective breeding programs. Results
revealed bottleneck event in Tuxedo strain under three
models for sign, standardized differences and Wilcoxon
sign rank tests.
DISCUSSION
Emrani et al. 4293
The Hardy-Weinberg equilibrium test showed several
deviant loci in the four strains. There were many causes
of disequilibrium such as small population, selection at or
near the genomic locus, non-random mating, genetic drift
and inbreeding. In addition, the possible occurrence of
null alleles could have led to false observation of homozygotes
which could have accounted for more deviations
from HWE. The mean observed heterozygosity for these
four strains were 0.387, 0.462, 0.364 and 0.325 for
Pharach, Panda, Tuxedo and Golden, respectively. In all
the four strains, the heterozygosity observed was lower
than that reported for wild Japanese quail (0.66) (Chang
et al., 2005). The average PIC of the 12 microsatellite loci
in the four strains was close to the results of Kayang et
al. (2002). Based on the classification of Botstein et al.
(1980), PIC > 0.5 is highly informative, 0.25 < PIC < 0.5 is
middle informative and PIC < 0.25 is slightly informative.
In this study, all loci were highly informative (PIC > 0.5),
except GUJOO41 (PIC = 0.427) in Panda strain. Reynolds
distance were used to estimate pairwise genetic
distance between the breed. This measure is recommended
by Eding and laval (1999) for population with
short divergence time. Maximum Nei’s genetic distance
was observed between Tuxedo and Golden strains;
whereas the minimum Nei’s genetic distance was
observed between Pharach and Tuxedo strains. Both
cluster resulted from Nei’s standard genetic distance and

4294 Afr. J. Biotechnol.
a
b
Figure 1. UPGMA showing the genetic relationships among strains using
Nei’s standard genetic distance (a) and Reynolds genetic distance (b).
Reynolds genetic distance revealed two branches. One
branch included Pharach, Tuxedo and Panda and
another branch was consisted of Golden. Pharach and
Tuxedo grouped closely (Figure 1). It was expected to
find high genetic distance between Golden and other
strains. Golden is the most prolific type among the
Japanese quail strains. The mean values of FIS among
the four strains were higher than that of the population
studied by Kim et al. (2007), indicating a certain level of
heterozygote deficiency. Significant heterozygote deficiencies
have been reported in chicken breeds (Qu et al.,
2006; Liu et al., 2008). The heterozygote deficiency (FIS >
0) might be attributed to a number of factors, namely,
sample relatedness, linkage with loci under selection
(genetic hitchhiking), population heterogeneity, null
alleles (non-amplifying alleles) and inbreeding
(Shahsavarani and Rahimi-mianji, 2010). Null alleles
were most unlikely segregated at all the loci. The most
plausible explanation for the heterozygote deficiency was
inbreeding in the four strains. These four closed strains
especially Tuxedo strain were under selective breeding
program for 17 generations. When a breeder conducts a
selective breeding program, his primary objective is to
alter, not conserve gene and genotypic frequencies in
order to improve the population. Inbreeding and genetic
drift are inevitable during a selective breeding program,
because each act of selection creates a bottleneck event
that accelerate the accumulation of inbreeding and
magnify genetic drift. There are some techniques that can
be used to moderate inbreeding and finally bottleneck so
that it does not counteract selection. Managing a
population to minimize the effect of bottleneck event can

e accomplished only by managing the number of
effective population (Ne). Bottleneck reduces the Ne and
make it difficult, if not impossible, to achieve genetic
goals. Unfortunately, it is difficult to maintain a constant
Ne generation after generation because Ne can decline
due to diseases, selective breeding program, etc (Tave,
1999). One way to maintain a constant Ne generation
after generation can be pedigree mating. The weak
performance of some economic traits (meat and number
of eggs) in these strains, especially Tuxedo can be as a
result of bottleneck event. Breeding strategies should
therefore be designed to amplify the population size and
simultaneously avoid inbreeding in these four strains. The
information generated in this study will greatly aid in the
establishment of effective breeding for these four strains
and may further be utilized for studying differentiation and
relationships among different quail strains.
ACKNOWLEDGEMENTS
This work was financially supported by the Animal Science
Research Institute of Iran. The authors would like to
acknowledge Dr. Ali Akbar Masoudi for his valuable input
and assistance in the preparation, analysis and
interpretation of this study.
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African Journal of Biotechnology Vol. 10(20), pp. 3978-3985, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2334
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Characterization of a chestnut FLORICAULA/LEAFY
homologous gene
Tao Liu 1 *, Yun-qian Hu 2 and Xiao-xian Li 2
1 Faculty of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming, Yunnan, 650201, China.
2 Kunming Institute of Botany, the Chinese Academy of Sciences, Kunming, Yunnan, 650204, China.
Accepted 24 March, 2011
The FLORICAULA/LEAFY (FLO/LFY) homologues’ genes are necessary for normal flower development
and play a key role in diverse angiosperm species. In this paper, an orthologue of FLORICAULA/LEAFY,
CmLFY (chestnut FLO/LFY), was isolated from the inflorescence of chestnut trees. Its expression was
detected in various tissues. Furthermore, the flowering effectiveness of the gene was assessed with
transgenic Arabidopsis. CmLFY protein showed a high degree of similarity to PEAFLO (78%), which is a
homologue of FLO/LFY from pea. RT-PCR analysis showed that, CmLFY expressed at high levels in
inflorescences, but not in young leaves, fruits or stems. The transgenic Arabidopsis with
over-expressed CmLFY showed accelerated flowering, which supports that CmLFY encodes a functional
orthologue of the FLORICAULA/LEAFY genes of angiosperms despite its sequence divergence. These
results suggest that CmLFY is involved in inflorescence development in chestnut.
Key words: Chestnut, homologue, FLORICAULA/LEAFY.
INTRODUCTION
Two developmental programmes can be distinguished
during the life cycle of flowering plants. During the initial
vegetative phase, the apical meristem produces leaves
and lateral shoot. After floral induction, the plant enters
into a reproductive phase and the apical meristem chan-
ges its developmental pattern and initiates the production
of flowers. In Antirrhinum and Arabidopsis this transition
from vegetative to reproductive development pattern
requires the establishment of floral meristem identity in
the lateral meristems of the inflorescence, a process
which has been demonstrated to be controlled by the
homo- logous genes FLORICAULA (FLO) and LEAFY
(LFY), respectively (Ma, 1998; Pidkowich et al., 1999).
LFY is a transcriptional regulation gene thought to play a
primary role in determining flower meristem identity.
FLORICAULA (FLO), a LFY homologue in snapdragon,
has been shown to have almost the same role. Loss func-
* Corresponding author. E-mail: beixue2007@gmail.com. Tel:
+86-871 5227721.
Abbreviations: PCR, Polymerase chain reaction; RT-PCR,
reverse transcriptase-PCR; RACE, rapid amplified cDNA ends.
tion mutants of these genes result in the conversion of
flowers into indeterminate secondary shoots (Coen et al.,
1990; Weigel et al., 1992). The over-expression of LFY
under a constitutive promoter in Arabidopsis caused early
flowering and converted all lateral shoots into solitary
flowers. In heterologous plants such as aspen, LFY has
been shown to have effects on the acceleration of flower-
ing and induction of ectopic flowers (Weigel and Nilsson,
1995; Rottman et al., 2000), suggesting a conservation of
LFY function across long phylogenetic distances within
angiosperms. These reports strongly suggest that, the
ability to control the expression of LFY or of its orthologs
from other plants, could make it possible to artificially
induce various plants to blossom and even skip or shorten
the juvenile phase of woody plants.
Flowering is an essential stage for fruit production and
thus, an understanding of the genetic mechanisms under-
lying the flowering event is important for efficient fruit
production. During the last decade, molecular mecha-
nisms of flowering have been studied extensively in
herbaceous ‘‘model’’ plants such as Arabidopsis and
Antirrhinum. In contrast, studies on the molecular
mechanisms of flowering in fruit trees have just begun.
Although, several flowering-related FLO/LFY orthologs
have been cloned from kiwifruit (Actinidia deliciosa)

(Walton et al., 2001), grapevine (Vitis vinifera) (Carmona
et al., 2002), and apple (Malus domestica) (Kotoda et al.,
2002), their specific role in the induction of the
characteristic features of tree reproductive development is
still being elucidated.
Trees are characterized by an extended adolescence.
This is particularly important for commercial fruit/nut tree
growers and breeders, because prolonged juvenile
periods delay harvesting and the evaluation/breeding of
new strains. Chestnut (Castanea mollissima) is one of the
most commercially valuable fruit trees, with production
once only annually. Chestnut has an extended juvenile-
vegetative phase, during which vegetative growth is
maintained. Attempts to exploit existing knowledge of the
genetic control of flower development in Arabidopsis to
engineer nut trees could have significant economic and
scientific implications for the tree fruit industry. As yet, no
studies have reported on the molecular mechanism
underlying the development of chestnut flowers. Recently,
MADS-box genes have been cloned and characterized in
chestnut by us (Liu et al., 2006). Our results suggest that
these chestnut MADS genes may be involved in floral
organ and fruit development in chestnut. In this paper, we
identified a chestnut FLORICAULA/LEAFY homologous
gene involved in floral development. We described the
isolation and characterization of CmLFY (DQ270548).
The function of CmLFY was demonstrated by transgenic
Arabidopsis, as expected for normal plants, transgenic
plants showed earlier flowering phenotype, indicating that
CmLFY may play a similar role of promoting flowering as
the FLORICAULA/LEAFY genes in flower development.
MATERIALS AND METHODS
Plant materials
The chestnut and Arabidopsis ecotype Columbia (Col) were used in
this study. Chestnut examples were collected from Kunming,
Yunnan, People’s Republic of China and were frozen in liquid
nitrogen immediately and stored at -80°C.
RNA extraction and cDNA cloning
Total RNA was extracted from the inflorescences using a modified
CTAB method (Zeng and Yang, 2002). The cDNA was prepared
using Smart cDNA library construction kit (BD Biosciences Clontech,
American). Full-length CmLFY cDNA was obtained by the 5′ and 3′
rapid amplified cDNA ends (RACE) method (Chenchik et al., 1996).
The first amplified chestnut cDNA contained about 440 bp between
primers CmS and CmA, designed from Arabidopsis thaliana LFY
(Weigel et al., 1992) and Antirrhinum majus FLO (Coen et al., 1990)
cDNA sequences. The 3′ RACE was carried out between cassette
CDSIII/3’ primer and CmS2 primer. The DNA fragment amplified
was cloned into PMD18-T vector (Takara, Japan). The 5′ RACE was
carried out between cassette CDSIII/5’ primer and CmA2 primer.
Full-length cDNAs were amplified with the sense primer LFY-ORF5
and antisense primer CDSIII/3’ primer. These clones were sequenced
completely by the dideoxy method using ABI3730 automated
sequencer (ABI Company, USA).
Phylogenetic comparisons
Liu et al. 3979
Protein sequences of different LFY homologs were retrieved from
GenBank and aligned with CLUSTAL. Phylogenetic relationships
among these genes were inferred by NJ analysis (MEGA Ver.3.1).
Plant transformation
The full length CmLFY (including start codon ATG) was amplified
between LFY-ORF5 and LFY-ORF3 (Figure 1). Then, the amplified
cDNA was blunted and KpnI and SalI linkers were ligated both ends,
cloned into a pCambia2301-101 binary vector in a sense-oriented
manner under the CaMV 35S promoter. An Agrobacterium
tumifaciens GV3101 strain (Van-Larebeke et al., 1974) was used for
the transformation of the A. thaliana Columbia ecotype by the
floral-dip method (Clough and Bent, 1998). For the selection of
transformed plants, resultant seeds were planted on a 1/2MS culture
medium containing kanamycin (50 µg mL --1 ) as selective antibiotics
and then, transferred to 22°C under LD conditions (16 h light, 8 h
dark). Transformants were identified as kanamycin-resistant when
seedlings in the medium produced green leaves and well-
established roots. Resistant transformants were transplanted to
moistened potting soil composed of vermiculite and perlite (1:1
(v/v --1 )) after two to four adult leaves had developed and their flower
phenotypes were observed under long-day conditions.
RNA analysis of transgenic plants by RT-PCR
Detection of CmLFY transcripts was performed by RT-PCR. RNA
was isolated from whole plants of Arabidopsis by a method using
CTAB described previously described. CmLFY specific transcripts
were identified using 1 µg of total RNA as a template and the
following primers: a sense primer LFY-ORF5 and an antisense
primer LFY-ORF3 for CmLFY (Table 1), giving rise to about 1300 bp
long PCR product. PCR reactions were run for 35 cycles at 58°C for
CmLFY. The PCR products were run on 1.5% (w/v --1 ) agarose gels
stained with ethidium bromide.
RESULTS
Cloning and sequence analyses of CmLFY
A comparison of Arabidopsis LFY (Weigel et al., 1992)
and Antirrhinum FLO (Coen et al., 1990) showed the
presence of several conserved regions. Two of these
regions, CmS and CmA, were used to design degenerate
oligo- nucleotide primers for RT-PCR. Using these
primers with cDNA prepared from an inflorescence of
chestnut, we obtained a PCR amplified fragment
containing both primers and other conserved sequences.
This fragment was named CmLFY (chestnut
FLORICAULA/LEAFY homologue). To obtain a full-length
CmLFY cDNA, several primers for 5’/3’ RACE were
designed. Primer sites and the nucleotide sequences of
primers used for cloning were shown in Figure 1 and
Table 1, respectively.
The coding region of CmLFY is 1,161 bp, encoding
386 amino acids. The deduced amino acid sequence of
CmLFY cDNA has 78% homology with PEAFLO, 79%
with PtFL and 59.9% with PlaraLFY. NLF, PRFLL, PEAFLO,

3980 Afr. J. Biotechnol.
Table 1. Primer sequences used in PCR cloning of CmLFY.
Primer Oligonucleotide
Primers for an internal fragment
CmS sense primer
CmA antisense primer
Cassette primers
CDSIII/5’ primer
CDSIII/3’ primer
3’ RACE primer
CmS2 primer
5’ RACE primer
CmA2 primer
CmLFY specific primers
LFY-ORF5
LFY-ORF3
5’- GGGAGCACCCGTTCATTGTGACTG -3’
5’-GA/CAGCTTG/TGTG/TGGGACATACCAGAC-3’
5’-AAGCAGTGGTATCAACGCAGAGT-3’
5’-ATTCTAGAGGCCGAGGCGGCCGACATG-3’
5’-CGGCCTTGATTACCTCTTCCATCTC -3’
5’- CACCGCGCTCCTTGGCAATGTTCTGT -3’
5’-GGTACCAAGCTAGCTTCATTGATG -3’
5’-CGGTCGACTAGAAATGCAAATTTTTCTC-3’
Figure 1. Nucleotide and deduced amino sequences (single-letter code) of CmLFY cDNA. The asterisk (*) indicates a stop
codon. Arrows above the sequences indicate the primers used for RACE-PCR.
ELF1 and CmLFY lacked a proline-rich region in
the variable region (roughly the first 40 amino acids). To
more closely determine the evolutionary relationship
between FLO/LFY-like proteins, a phylogenetic tree was
con- structed (Figure 2b). This tree showed that, the
topology of these genes seems to be concordant with the
topology of the species phylogeny and suggests that
CmLFY is an angiosperm ortholog of FLO/LFY-like genes.

a
Liu et al. 3981

3982 Afr. J. Biotechnol.
b
Expression patterns of CmLFY
Figure 2. (a) Sequence comparison of FLO/LFY-like proteins (accession numbers in
parentheses): NLY and PRFLL from P. radiata (U76757 and U92008, respectively); CmLFY
from chestnut (DQ270548); BOFH from B. oleracea (Z18362); FLO from A. majus (M55525);
LFY from A. thaliana (M91208); NFL1 and NFL2 from N. tabacum (U16172 and U16174,
respectively); ALF from P. hvbrida (AF030171); PtFL from P. balsamifera (U93196); RFL from
O. sativa (AB005620); TOFL from L.esculentum (AF197934); PEAFLO from P. sativum
(AF010190); ClLFY from C. glaziovii (AY633622); ELF1 from Eucalyptus globules (AF034806);
JunefLFY from J. effusus (AF160481) and PlaraLFY from P. hispanica (AF106842). Black
boxes indicate identical amino acids, shaded boxes indicate amino acids with similar
properties, and dots indicate gaps introduced to optimize alignment. c1 and c2, conserved
regions; v1 and v2, variable regions; (b) Protein sequence comparisons of CmLFY to other
LFY/FLO homologs. The deduced amino acid sequence of CmLFY was compared with some
published LFY homologs. Box characters represent chestnut protein.
CmLFY mRNA expression was analyzed in various
tissues by RT-PCR. Total RNA was isolated and PCR
primers specific to CmLFY were used to detect the
expression patterns in several tissues. The result showed
that CmLFY mRNA was expressed in inflorescence
tissues, but not in young leaves, fruits and stems (Figure
3). The RT-PCR of CmLFY -specific primers resulted in
amplification of a single band. Increasing the PCR cycle
did not change these expression patterns. The amplified
DNA fragments were the expected length.
Ectopic expression in Arabidopsis
To further verify the function of CmLFY, we constructed a
binary vector pCambia2301-101-CmLFY containing full-
length CmLFY coding area inserted in the sense-oriented
direction under the control of the Cauliflower 35S
promoter. The pCambia2301-101-CmLFY was introduced
into wild- type Arabidopsis plants by
Agrobacterium-mediated transformation. Fourteen
independent transgenic plants that survived on kanamycin
were identified (Table 2). Five (lines one-five) of them
flowered earlier than the wild-type plants by five-seven

Figure 3. Expression pattern of CmLFY gene revealed by RT-PCR
analysis. Lane 1: inflorescences; Lane 2: stem; Lane 3: tender leaves with
no flowers; Lane 4: tender leaves with flowers; Lane 5: fruit. rRNA is used
as RNA standard. Amplification of CmLFY was performed for 30 cycles.
Table 2. Comparison of flowering time, number of rosette leaves in T1 transgenic and wild-type Arabidopsis (Col)
plants in LD conditions.
Transgenic line Days to flowering a Rosette leaves at time of flowering b Note
1 18 4 Early flowering
2 16 3 Early flowering
3 18 4 Early flowering
4 20 5 Early flowering
5 20 4 Early flowering
6 24 7
7 24 8
8 25 9
9 22 8
10 24 8
11 25 8
12 26 9
13 26 10
14 25 8
wt c 25.5 d 9.5 d (n=10) e
v-wt f 26 d 9.5 d (n=8) e
a Days to flowering is defined as the time when flower primordial were first visible to the naked eye; b rosette leaves were
counted on the day that flower primordial were first visible; c w wild-type Arabidopsis Columbia plants; d Mean number; e
Number of plants;
f vector-transformed plants.
days and produced only four to five rosette leaves when
they flowered (Figure 4 and Table 2). Most of the T1
transgenic plants could self-pollinate and grow siliques
normally and the resultant seeds also had the ability to
germinate except for the lines 1, which is so small that it
was difficult to obtain its seeds. The early flowering were
inherited in the next generation and co- segregated with
the kanamycin-resistant genes. The expression of the
CmLFY mRNA in T1 plants was confirmed by RT-PCR, all
putative transformed lines showed the expected products
of CmLFY. No amplifi- cation was observed for the cDNA
prepared from non-transformed Arabidopsis plants and
vector-trans- formed Arabidopsis plants. The transgenic
plants are stably transformed and the siblings which
segregate without the transgene had no amplification of
CmLFY and flowered at the same time as wild-type
plants.
DISCUSSION
Liu et al. 3983
Despite the importance of understanding the regulation of
the flowering process in woody perennials for the
management and improvement of woody species, very
little is known about the underlying molecular
mechanisms. Regulation of flowering in woody perennials
shows remarkable differences in contrast to herbaceous
species, that is, long juvenile phases, bud dormancy and
the alternating vegetative and reproductive development
according to the season.
To investigate the molecular mechanism in chestnut
flowering, we cloned CmLFY, a putative homologue
of Arabidopsis LFY. A comparison (Figure 2a) of amino
acid sequences with Arabidopsis LFY (Weigel et al.,
1992), Antirrhinum FLO (Coen et al., 1990) and other
FLO/LFY homologs (Brassica oleracea. BOFH.

3984 Afr. J. Biotechnol.
Figure 4. Transgenic and wild-type Arabidopsis plants grown under LD
photoperiods for 20 days. Arabidopsis plants were grown in one-half strength of
medium for 9 days and transferred to potting soil. (a) wild-type Arabidopsis
Columbia. (b) Transgenic Arabidopsis carrying a 35S::CmLFY gene.
Anthony et al., 1993; Nicotiana tabacum. NFL. Kelly et al.,
1995; Oraza sativa. RFL. Kyozuka et al., 1998; Pinus
radiata. NLY. Mouradov et al., 1998 ; Petunia hybrida. ALF.
Souer et al., 1998; Eucalyptus globulus, ELF1, Southerton
et al., 1998 ; Lycopersicon esculentum. TOFL.
Molinero-Rosales et al., 1999; Juncus effusus. JunefLFY.
Frohlich and Parker., 2000; Platanus hispanica. PlaraLFY.
Frohlich and Parker., 2000; Populus balsamifera. PTFL.
Rottmann et al., 2000; Cedrela glaziovii. ClLFY. Marcelo
and Adriana, 2006) showed the presence of conserved
regions including two large conserved regions (c1 and c2)
and two shorter regions of lower similarity (variable
regions v1 and v2). These domains are typical markers for
transcriptional acti- vators and may be functionally
important for FLO/LFY-like proteins (Weigel et al., 1992;
Coen et al., 1990). The phylogenetic tree showed that the
topology of these genes seems to be concordant with the
topology of the species phylogeny and suggests that
CmLFY is an angiosperm ortholog of FLO/LFY-like genes
(Figure 2b).
The expression of CmLFY was only found in
inflorescences, but not in tissues such as fruits, stems and
tender leaves (Figure 3). It supports that the CmLFY is a
functional homologous gene of chestnut. In the species
such as apple, kiwifruit and grape, the expression levels
of their FLO/LFY homologs increase in the proliferating
inflorescence meristems generating inflorescence
branches, with the highest levels detected in young floral
meristems (Walton et al., 2001; Carmona et al., 2002).
Therefore, the high levels of CmLFY expression in inflo-
rescence suggests that CmLFY plays a role during chest-
nut reproductive development as it has been suggested
for most FLO/LFY-like genes studied.
To further demonstrate the function of CmLFY, we
have produced transgenic Arabidopsis plants expressing
CmLFY. They flowered earlier and had a shorter
inflorescence and reduced number of rosette leaves
compared with the controls (Figure 3). Based on the result
that several transgenic Arabidopsis with 35S::CmLFY
flowered earlier than the controls, the expression of
CmLFY should have some relationship with the early
flowering phenotypes, although, the mechanism of
flower-bud formation in chestnut might be different from
that in Arabidopsis. Of course, to be more certain that the
CmLFY gene functions similarly to LFY, LFY mutant-
rescue experiments will be required. The CmLFY gene
was not expressed strongly in transformants showing
early flowering relative to those that were wild type in
appearance, which suggests that the severity of pheno-
type in transformants does not depend solely on the level
of gene expression in this case, although the exact cause
is still unknown. Some reports are available that describe
transgenic Arabidopsis plants expressing LFY homologs
from other plant species. For example, Arabidopsis
transgenic lines expressing NEEDLY from Radiata pine
and ELF1 from Eucalyptus showed early flowering
(Mouradov et al., 1998; Southerton et al., 1998). Until now,
however, our ability to manipulate fruit/nut tree strains
through genetics has been limited by the extended
maturation period of these plants. Both taking long time to
flowering and the fact that, no characterized flowering
mutants have been described in this genus make genetic
studies in chestnut more difficult. Nevertheless, advances
in the establishment of transformation protocols for

chestnut may allow us to use reverse genetic approaches
and to define more clearly the role played by CmLFY in
the reproductive development.
The breeding of fruit trees of chestnut often requires
more than 20 years, including periods of cross pollination,
seedling selection and regional trials, to produce varieties
that meet the demands of consumers. One of the limit
factors of chestnut breeding is the long juvenile phase of
at last several years. It had been found that transgenic
approaches of LFY introducing could reduce the juvenile
phase of Populus and the transgenic poplar flowered in 5
months after regeneration (Weigel and Nilsson, 1995).
Therefore, probably we can anticipate that these techni-
ques are likely applicable to fruit trees of chestnut in future
years and believed that, the transgenic approach would
be a useful breeding strategy for reducing the time
required for generation among woody plants. However,
the efficiency of producing early flowering transgenic lines
with Arabidopsis LFY gene seems to be low in woody
plants in some case (Pena et al., 2001). This suggests
that regulatory genes such as LFY do not always function
beyond species as well as expected. Since environmental
and genetic factors controlling flower development in
chestnut have not been made clear so far, the CmLFY
gene could at least, be one of the tools available for
studying the mechanism of flower development in tree
fruits such as the chestnut.
ACKNOWLEDGMENTS
This research was financially supported by the Faculty of
Agronomy and Biotechnology (SJCX09012 to T Liu) and
the National Natural Science Foundation of China (NSFC,
30800062 to XX Li).
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African Journal of Biotechnology Vol. 10(20), pp. 4303-4312, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.2361
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
Productive performance of laying hens fed wheat-based
diets included olive pulp with or without a commercial
enzyme product
M. Zarei 1 , M. Ehsani 2 and M. Torki 2 *
1 Member of Young Researchers Club of Islamic Azad University, Aligudarz Branch, Iran.
2 Department of Animal Science, Agriculture Faculty, Razi University, Imam Avenue, Kermanshah, Iran.
Accepted 21 March, 2011
Olive pulp (OP) is the remainder of olive cake (the raw material resulting from extraction of olive oil)
after the removal of the seed fractions. It can be achieved by sieving the dry olive cake to separate most
of the seeds. To assess effects of dietary inclusion of OP supplementing with a cocktail commercial
enzyme on performance of laying hens and egg quality characteristics, one hundred and twenty 80week-old
Lohmann LSL-Lite hens, with an average laying rate of 80.3 ± 3.8% (late production phase)
and 1450 ± 14 g live body weight, were divided in 20 cages (n = 6). Hens in 5 cages (replicates) were
randomly assigned to feed on one of the 4 experimental diets. Based on a 2×2 factorial arrangement, 4
iso-caloric and iso-nitrogenous diets (ME =2720 kcal/kg and CP=150 g/kg) including OP (0.0 and 90.0
g/kg) and a commercial cocktail enzyme with mostly xylanase activity (Nutrase®, 0.0 or 0.9 g/kg) were
formulated. To determine blood biochemical parameters and differentiable count of white blood cells,
one hen per replicate was bled via wing vein on day 35 of trial. To determine egg quality parameters, all
eggs during three frequent days were collected on week 4 of trial. Collected data of feed intake (FI), egg
production (EP), egg mass (EM) and calculated feed conversion ratio (FCR), blood biochemical
parameters and egg quality traits during 6 week trial period was analyzed based on completely
randomized design. Hens fed the OP-included diet shown decreased EP compared with hens fed the
control diet during week 3 of experimental period (p ≤ 0.05). There was no significant difference
between hens fed the OP-included diet and the control diet in terms of FI and EM. In addition, the same
trend was observed in terms of enzyme effect on FI and EM. Dietary enzyme supplementation improved
FCR compared with hens fed the control diet during week 6 of the experimental period (p ≤ 0.05).
Including OP in the diets of hens decreased the blood level of triglycerides (p ≤ 0.05). Including OP in
the diets of hens increased the yolk index (p ≤ 0.05). From the results of this investigation, it can be
concluded that including OP in diets of laying hens up to 9% would not have deleterious effects on
bird's productive performance. In addition, dietary supplementation with a cocktail enzyme with mainly
xylanase activity improved FCR in hens.
Key words: Olive pulp, enzyme, laying hens, performance, egg quality characteristics, blood parameters.
INTRODUCTION
The biggest single expense in any system of poultry pro-
duction is feed, accounting for up to 70% of total
*Corresponding author. E-mail: torki@razi.ac.ir. Tel:
00989133277266.
Abbreviations: AME, Available energy; NSP, non-starch
polysaccharides; OP, olive pulp; FI, feed intake; EP, egg
production; EM, egg mass; FCR, feed conversion ratio.
production cost. In order to reduce feeding costs, attempts
have been made to use agricultural and industrial byproducts
as feed ingredients. Improved utilization of crop
residues and by-products to be used in animal feeding
deserves more attention. Examples of crop residues and
agricultural by-products in Iran are; cereal bran, citrus
pulp, tomato pulp, poultry litter and olive pulp (OP). Olive
pulp is the remainder of olive cake (the raw material
resulting from extraction of olive oil) after the removal of
the seed fractions. OP is considered as a good source of

4304 Afr. J. Biotechnol.
calcium, copper and cobalt but poor in phosphorus,
magnesium and sodium and with fair levels of manganese
and zinc (Harb, 1986). Due to low nutritive value
(low in energy, digestible proteins and minerals and high
in lignin), OP is seldom integrated into poultry feeding. In
addition, a xyloglucan, one of the non-starch polysaccharides
(NSP) which has anti-nutritive effects on
monogastrics such as poultry and pigs, from OP has
been reported by Gil-Serrano and Tejero-Mateo (1988).
Coimbra et al. (1995) also showed the occurrence of the
xylan-xyloglucan complexes in the OP cell walls. In
addition, Rosa´rio and Domingues (2002) extracted
glucuronoxylans with a xylose/glucose ratio of 7: 1 from
OP.
Although starch is the predominant carbohydrate in
cereals, some of the constituent carbohydrates are watersoluble
NSP, which are considered the major antinutritive
factors in cereals and other varieties of feed ingredients
(Bedford and Classen, 1992; Campbell and Bedford,
1992). The common NSP include β-glucans, arabinoxylans
and fructans (Classen and Bedford, 1991). The
structures of NSP differ between grains and also between
varieties of the same grain (Annison, 1990). The similarity
between the types of NSP is usually the presence of β (1
→ 4) backbones with or without the presence of β (1 →
3) side linkages (Iji, 1999). The effects of NSP upon feed
utilization and poultry productivity have been discussed in
many reports. Salih et al. (1991) reported that, NSP
reduced feed intake and decreased broiler chicken
performance. The most noticeable effect of NSP in
poultry diets is an increase in the viscosity of digesta and
the excretion of sticky droppings. This is considered to be
one of the greatest influences of NSP on broiler chicken
productivity (Smits and Annison, 1996) and it is
necessary to use exogenous enzymes to overcome these
problems.
Poultry naturally produces enzymes to aid the digestion
of feed nutrients. However, they do not have enzyme to
break down fiber completely and need exogenous enzymes
in feed to aid digestion. Enzymes are used mainly to
achieve consistency in performance and to reduce
formulation costs by easing constraints on the inclusion
level of some ingredients, such as wheat and barley.
Hence, the supplementation of microbial enzymes in
poultry diets is now common practice in many countries
where the predominant cereals are wheat or barley.
Improving poultry performance by dietary manipulation
has been the goal of nutritionists. Enzyme has been
reported to alleviate the negative effects of arabinoxylans,
present in wheat as the dominant cell-wall constituent,
by lowering gut viscosity and increasing protein,
fat and starch digestibility (Bedford and Morgan, 1996).
Moreover, an improvement in overall protein digestibility
of dietary components other than wheat, such as soybean,
has also been reported (Bedford and Morgan,
1996). Pan et al. (1998) demonstrated that, commercial
enzyme mixtures could promote metabolizable energy
particularly utilized in wheat based rations. Many years of
intensive research, both in official trials as well as in field
trials, have shown that its bacterial origin gives it some
very distinctive and valuable characte-ristics, distinguishing
it clearly from xylanase. It reaches its highest
efficiency in rations with a high inclusion of wheat and
wheat by-products. Many years of practical experience
have shown that, it also has an important effect on
improving the digestibility of other grains, such as barley,
corn, rice, rye and sorghum. Improved starch digestibility,
for instance, has been reported to account for up to 35%
of the improvement in available energy (AME) as a result
of xylanase supplementation (Carre et al., 1992).
Xylanase has been reported to alleviate the negative
effects of arabinoxylans, present in wheat as the
dominant cell-wall constituent, by lowering gut viscosity
and increasing protein, fat and starch digestibility
(Bedford and Morgan, 1996).
The objectives of this study were to investigate the
effects of dietary inclusion of OP and enzyme supplementation
on performance of the laying hens fed with
wheat-based diets.
MATERALS AND METHODS
All procedures used in this six-week experiment were approved by
the Animal Ethics Committee of Razi University and complied with
the “Guidelines for the Care and Use of Animals in Research". A
total number of one hundred and twenty 80-week-old Lohmann
LSL-Lite hens with an average egg production rate of 80.3 ± 3.8%
(late laying phase) and 1450 ± 14 g live body weight, were obtained
from a commercial supplier. After a week of adaptation, the hens
were randomly allocated to one of four experimental diets. Hens
were semi-randomly distributed between 20 cages (n=6) with
almost same average body weight and egg production throughout
the cages. The hen distribution was based on the previous production
records of birds during one month before selecting them
from their original flock and transferring to the experimental cages.
Hens in 5 cages (replicates) were assigned to feed on one the 4
experimental diets. Four iso-caloric and iso-nitrogenous diets (ME
=2720 kcal/kg and CP=150 g/kg) including OP (0.0 and 90.0 g/kg)
and a commercial cocktail enzyme with mostly xylanase activity
(Nutrase ® , 0.0 or 0.9 g/kg) were formulated (Table 1).
The approximate analysis of the OP used in this study is; dry
matter (DM=93%), crude protein (CP= 6.06%), ether extract (EE%=
7.6%), crude fiber (CF%= 48.2), ash (7.4%), calcium (Ca%=0.6)
and total phosphorous (P%= 0.07). Fatty acid composition (%) of
oils from OP used in this study is; total saturated fatty acids (∑SFA=
15.01%), total monounsaturated fatty acids (∑MUFA= 71.38%) and
total polyunsaturated fatty acids (∑PUFA= 9.10%). Nutrase® xyla is
a bacterial endo-1,4-beta-xylanase, designed for use in pig and
poultry rations with a high content of arabinoxylans. Nutrase® xyla
is produced by Bacillus subtilis, which makes it today, the only EU
registered bacterial xylanase preparation available on the market.
The hens were housed in laying cages made from galvanized
metal wire which provided approximately 430 cm 2 /hen. The cages
were located in a windowless and environmentally controlled room
with the room temperature kept at 21-23°C and the photoperiod set
at 16 h of light (incandescent lighting, 10 lux) and 8 h dark. Each
cage had a nipple waterier. Water was available ad libitum throughout
the experiment. Feed consumption was measured on a weekly
basis. To determine blood biochemical parameters and differentiable

Table 1. Ingredients and composition of the experimental diets.
Olive pulp (g /100 g) 0.0 9.0
Enzyme (g /100 g) 0.00 0.09 0.00 0.09
Feed ingredients g / 100 g diet
Wheat 62.78 62.78 58.06 58.07
Fish meal 5.00 5.00 5.00 5.00
Soybean meal 8.80 8.80 8.78 8.78
Dried tomato pomace 3.50 3.50 3.50 3.50
Oil 5.03 5.03 5.03 5.03
Olive pulp 0.00 0.00 9.00 9.00
Enzyme - Nutrease® 0.00 0.09 0.00 0.09
Dicalcium phosphate 1.64 1.64 1.64 1.64
Limestone 8.07 8.07 8.07 8.07
Common salt 0.16 0.16 0.17 0.17
Vit. and Min. Premix 1 0.25 0.25 0.25 0.25
Sand 4.38 4.29 0.10 0.00
DL-Methionine 0.15 0.15 0.15 0.15
Calculated analyses
ME (Kcal/kg) 2720 2720 2720 2720
Crude protein (%) 15 15 15 15
Ether extract (%) 6.65 6.65 6.65 6.65
Crude fiber (%) 4.37 4.37 4.37 4.37
Calcium (%) 3.67 3.67 3.67 3.67
Available P (%) 0.33 0.33 0.33 0.33
Lys (%) 0.68 0.68 0.68 0.68
Met (%) 0.38 0.38 0.38 0.38
Met and Cys (%) 0.61 0.61 0.61 0.61
1 The vitamin and mineral premix provide the following quantities per kilogram of diet:
Vitamin A, 10,000 IU (all-trans-retinal); cholecalciferol, 2,000 IU; vitamin E, 20 IU (αtocopheryl);
vitamin K3, 3.0 mg; riboflavin, 18.0 mg; niacin, 50 mg; D-calcium
pantothenic acid, 24 mg; choline chloride, 450 mg; vitamin B12, 0.02 mg; folic acid, 3.0
mg; manganese, 110 mg; zinc, 100 mg; iron, 60 mg; copper, 10 mg; iodine, 100 mg;
selenium, 0.2 mg; antioxidant, 250 mg.
count of white blood cells, one hen per replicate was bled via wing
vein on day 35 of trial. To determine egg quality parameters, all
eggs during three frequent days were used. Collected data of feed
intake (FI), egg production (EP), egg mass (EM) and calculated
feed conversion ratio (FCR) during 6 week trial period was analyzed
based on a 2×2 factorial arrangement of treatments and completely
randomized design using GLM procedure of SAS. All statements of
significance are based a probability of less than 0.05. The mean
values were compared by Duncan’s multiple range test.
RESULTS AND DISCUSSION
Effects of dietary OP inclusion and enzyme supplementation
on EP, FI, FCR, EW and EM are presented in
Tables 2 to 6, respectively. There was no significant
interaction between dietary OP inclusion and enzyme
supplementation on the measured productive performance
parameters throughout the experimental period (p
> 0.05). Egg production (%) was not significantly affected
by dietary OP inclusion (p > 0.05), except for week 3 of
Zarei et al. 4305
trial period. Hens fed with the OP-included diet shown
decreased EP compared with hens fed with the control
diet during week 3 of experimental period (p ≤ 0.05); but
the overall EP during the whole trail period (weeks 1 to 6)
was not significantly affected. There was no significant
difference between hens fed with OP-included diet and
the control diet in terms of FI (Table 3) and EM (Table 6).
In addition, the same trend was observed in terms of
enzyme effect on FI and EM. Feed conversion ratio was
not significantly affected by dietary OP inclusion (p >
0.05). Including OP in diet improved EW on weeks 4 and
6 as well as the overall trial period (weeks 1 to 6). Studies
concerning feeding with OP to monogastrics are limited
(Tortuero et al., 1989). Several research studies were
conducted to investigate the feasibility of utilizing OP in
broiler rations. The proportion of OP in its rations is
variable. There seems to be a limit between 50 and 100g/
kg (Abo Omar, 2000; Rabayaa, 2000). Diets with different
levels of fiber showed certain influence on gastro-

4306 Afr. J. Biotechnol.
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.
Treatment
Enzyme (g /100 g)
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6
0.00 67.85 75.71 79.28 84.04 86.42 83.80 79.52
0.09 70.47 72.61 79.52 85.71 86.43 88.33 80.51
Olive pulp (g /100 g)
0.00 71.42 76.42 84.28 a 85.95 87.14 85.47 81.78
9.00 66.90 71.90 74.52 b 83.81 85.71 86.66 78.25
Olive pulp Enzyme
0.00 0.00 69.52 75.71 83.81 84.76 86.66 82.85 80.55
0.00 0.09 73.33 77.14 84.76 87.14 87.62 88.09 83.01
9.00 0.00 66.19 75.71 74.76 83.33 86.19 84.76 78.49
9.00 0.09 67.61 68.09 74.28 84.28 85.24 88.57 78.01
SEM 5.83 5.36 3.84 3.32 3.16 2.26 3.09
CV 19.91 17.06 11.41 9.24 8.64 6.21 9.14
Source of variation Probability
Enzyme 0.676 0.591 0.954 0.641 0.999 0.076 0.765
Olive pulp 0.473 0.435 0.028 0.549 0.674 0.625 0.296
Enzyme × Olive pulp 0.849 0.435 0.862 0.841 0.779 0.770 0.659
a-b Means within a column (within main effects) with no common superscript differ significantly (p < 0.05); SEM= standard error of means.
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
laying hens.
Treatment
Feed intake (g/ hen/ day)
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6
Enzyme (g /100 g)
0.00 105.66 116.55 117.72 118.22 116.89 118.13 115.53
0.09 111.25 109.91 113.57 112.79 114.14 115.40 112.84
Olive pulp (g /100 g)
0.00 110.78 114.97 117.16 114.92 114.49 116.32 114.77
9.00 106.13 111.49 114.13 116.09 116.54 117.21 113.60
Olive pulp Enzyme
0.00 0.00 109.95 116.88 119.14 116.45 114.81 117.19 115.74
0.00 0.09 111.62 113.07 115.19 113.40 114.16 115.45 113.81
9.00 0.00 101.38 116.23 116.30 120.00 118.97 119.07 115.33
9.00 0.09 110.88 106.76 111.95 112.19 114.12 115.35 111.87
SEM 4.18 4.00 2.96 2.52 3.30 1.61 2.29
CV 9.11 8.34 6.05 5.16 4.71 3.26 4.75
Source of variation Probability
Enzyme 0.224 0.135 0.203 0.058 0.275 0.128 0.284
Olive pulp 0.307 0.422 0.346 0.667 0.410 0.606 0.634
Enzyme × Olive pulp 0.388 0.511 0.949 0.385 0.400 0.569 0.756
SEM, Standard error of means.

Zarei et al. 4307
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:
g egg) of laying hens.
Treatment
Enzyme (g /100 g)
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6
0.00 2.58 2.49 2.39 2.24 2.16 2.26 a 2.35
0.09 2.57 2.48 2.30 2.12 2.14 2.11 b 2.29
Olive pulp (g /100 g)
0.00 2.57 2.47 2.25 2.16 2.14 2.21 2.30
9.00 2.59 2.51 2.44 2.20 2.17 2.15 2.34
Olive pulp Enzyme
0.00 0.00 2.64 2.53 2.31 2.20 2.15 2.31 2.36
0.00 0.09 2.49 2.40 2.18 2.11 2.12 2.12 2.24
9.00 0.00 2.50 2.45 2.46 2.27 2.18 2.21 2.34
9.00 0.09 2.68 2.56 2.42 2.13 2.16 2.10 2.34
SEM 0.16 0.13 0.10 0.08 0.07 0.06 0.07
CV 14.68 12.74 10.23 9.15 8.73 6.57 7.46
Source of variation Probability
Enzyme 0.944 0.950 0.419 0.213 0.770 0.035 0.451
Olive pulp 0.907 0.776 0.094 0.629 0.717 0.380 0.596
Enzyme × Olive pulp 0.353 0.414 0.680 0.775 0.934 0.562 0.466
SEM, Standard error of means.
intestinal tract weight, length and content (Abo Omar et
al., 1994; Abo Omar, 1995). The different levels of olive
cake had no effect on carcass cuts of broilers carcass
when fed with OP at levels up to 100 g/kg (Abo Omar and
Gavoret, 1995). Rabayaa et al. (2001) who incorporated
OP in four of the experimental groups at rates of 2.5, 5,
7.5 and 10% in both starter and finisher feeds to replace
similar rates of yellow corn reported that weight gain of
chicks was the same in chicks consuming up to 7.5% of
OP. However, weight gain of chicks fed the level of 10%
OP had the lowest significant weight gain. In addition,
similar trends were observed in chicks for F and feed
conversion efficiency.
Dietary enzyme supplementation improved FCR compared
with hens fed with control diet during week 6 of the
experimental period (p ≤ 0.05). Although enzyme addition
decreased FCR during the whole trail period, this effect
was significant only on week 6 (Table 4). Enzyme
supplementation did not have significant effect on EW
(Table 5). Xylans are the principal NSP of wheat and high
levels of wheat in poultry diets can increase the viscosity
of the gut contents, which impedes the circulation and
absorption of nutrients, causing reduced feed intake, BW
gain and feed efficiency (Annison and Choct, 1991).
Chickens do not produce some enzymes, such as galactosidases
(xylanase/β-glucanase); thus, corn-soybean-
based diets without supplemented enzymes such as xylanases
and pectinases might result in gas accumulation in
the gut and diarrhea (Jaroni et al., 1999a; Wu et al.,
2005). The endosperm cell wall of wheat is mainly composed
of arabinoxylans. The addition of exogenous
enzymes to wheat-based diets increases digestibility of
nutrients in cockerels (Carré et al., 1990). It may also
provide additional dietary energy as well as short chain
fatty acids and oligosaccahrides (Iji, 1999). Such phenomena
are associated with cellular proliferation and
improved gut health (Iji, 1999). Xylanase is used
extensively in wheat-based diets to counteract the effects
of NSP in broiler (Bedford and Schulze, 1998). In addition,
xylanase has been used in combination with other
enzyme(s) (xylanase-based cocktail enzyme) in layer
diets containing wheat or wheat and barley (Brenes et al.,
1993; Pan et al., 1998; Scott et al., 1999, Francesch et
al., 1995; Oloffs et al., 1998; Salobir, 1998; Jaroni et al.,
1999a,b; Mathlouthi et al., 2002) and broiler diets
(Bedford and Schulze, 1998) to counteract the effects of
NSP; however, the results obtained with different experimental
conditions and diets have been inconclusive. The
degree of improvement obtained by adding enzymes to
the diet depends on many factors including the level of
antinutritive factor in the diet, the spectrum and concentration
of enzymes used, the type of animal and the

4308 Afr. J. Biotechnol.
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).
Treatment
Enzyme (g /100 g)
Week 1 Week 2 Week 3
Egg weight (g)
Week 4 Week 5 Week 6 Weeks 1 to 6
0.00 62.29 62.80 63.07 62.77 62.81 42.53 59.38
0.09 61.96 61.93 62.34 62.15 61.85 42.19 58.74
Olive pulp (g /100 g)
0.00 61.80 61.86 62.25 61.89 b 61.72 41.40 b 58.48 b
9.00 62.46 62.87 63.16 63.03 a 62.94 43.32 a 59.63 a
Olive pulp Enzyme
0.00 0.00 61.95 62.05 62.10 62.00 61.90 41.37 58.56
0.00 0.09 61.64 61.66 62.39 61.78 61.54 41.43 58.41
9.00 0.00 62.63 63.54 64.04 63.55 63.73 43.69 60.19
9.00 0.09 62.28 62.21 62.29 62.52 62.16 42.95 59.07
SEM 0.61 0.68 0.71 0.51 0.72 0.32 0.52
CV 2.35 2.58 2.67 1.93 2.75 1.83 2.07
Source of variation Probability
Enzyme 0.620 0.249 0.344 0.266 0.226 0.345 0.260
Olive pulp 0.326 0.176 0.240 0.050 0.130 0.045 0.049
Enzyme × Olive pulp 0.976 0.525 0.194 0.465 0.442 0.266 0.390
SEM, Standard error of means.
age of the animal (young animals tend to respond better
to enzymes than older animals), type of gut micro flora
present and the physiology of the bird. Older birds,
because of the enhanced fermentation capacity of the
micro flora in their intestines, have a greater capacity to
deal with negative viscosity effects (Choct et al., 1995).
Typically, enzymes added to layer feed appear to have
little effect on egg mass but improve feed efficiency
(Benabdeljelil and Arbaoui, 1994). Mathlouthi et al.
(2002) who determined the effects of xylanase on true
metabolizable energy values of wheat, barley and wheat
bran as well as performance of laying hens fed with
wheat-, barley- or wheat bran-based diets reported
improved egg mass of layers fed with wheat- or wheat
bran-based diets supplemented by xylanase. In addition,
xylanase supplementation improved egg production, egg
mass and feed conversion ratio of layers fed with low
energy diet. It did improve the feed conversion ratio of
layers fed with high-energy diet. Xylanase increased the
TME values for wheat and barley and did not affect the
TME value of wheat bran (Mathlouthi et al., 2002).
Senkoylu et al. (2009) who investigated the effects of
30% whole-wheat inclusion in a standard layer diet
supplemented with xylanase, on laying performance,
digestive organs and ileal mucosa development and
reported that xylanase supplementation to whole wheat
significantly improved egg production and feed
conversion rate compared with the ground wheat and
whole wheat fed groups. Pirgozliev et al. (2010) who examined
the effect of dietary xylanase on the availability of
nutrients for laying hens when fed on wheat-rye-soybased
diets and reported that, the AME and nitrogen
metabolisability coefficients of xylanase-supplemented
diets were greater than the control diet. In addition, they
reported supplementary xylanase significantly improved
the coefficients of metabolisability of indispensable, dispensable
and total amino acids. Their data suggested
that, the use of a xylanase might improve the metabolisability
of some nutrients, but that such effects might
not always benefit production parameters. Feed intake
and feed conversion ratio for egg production were not
affected by xylanase (Pirgozliev et al., 2010). Diet
supplementation with an enzyme cocktail providing 7 U/g
of α-1, 6-galactosidase and 22 U/g of β-1, 4-mannanase
significantly improved feed conversion of Lohmann
Brown-Lite laying hens (Han et al., 2010).
As it is shown in Table 7, among the blood biochemical
parameters (cholesterol, high density lipoprotein, low
density lipoprotein and triglycerides), only blood level of
triglycerides was affected by dietary OP inclusion.
Including OP in the diets of hens decreased the blood
level of triglycerides (p ≤ 0.05); however, there was no
significant difference between blood levels of cholesterol,
high density lipoprotein and low density lipoprotein in

Zarei et al. 4309
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
hens.
Treatment
Egg mass (g/ hen/ day)
Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Weeks 1 to 6
Enzyme (g /100 g)
0.00 42.17 47.60 49.99 53.01 54.29 52.43 49.92
0.09 43.59 44.92 49.64 53.33 53.42 54.72 49.94
Olive pulp (g /100 g)
0.00 43.98 47.27 52.51 53.47 53.80 52.70 50.62
9.00 41.78 45.26 47.13 52.86 53.91 54.46 49.23
Olive pulp Enzyme
0.00 0.00 42.97 47.07 52.11 53.04 53.64 50.90 49.96
0.00 0.09 45.00 47.47 52.90 53.90 53.97 54.50 51.29
9.00 0.00 41.38 48.14 47.88 52.97 54.95 53.97 49.88
9.00 0.09 42.19 42.38 46.38 52.76 52.88 54.95 48.59
SEM 3.52 3.54 2.53 2.09 2.04 1.41 2.02
CV 19.39 18.04 11.98 9.07 8.93 6.19 9.53
Source of variation Probability
Enzyme 0.708 0.483 0.895 0.883 0.691 0.142 0.992
Olive pulp 0.562 0.597 0.061 0.781 0.961 0.252 0.522
Enzyme × Olive pulp 0.872 0.420 0.674 0.806 0.584 0.391 0.545
Table 7. Effect of dietary inclusion of olive pulp (0 and 90 g/kg) and enzyme (0 and 0.9 g/kg) on serum
biochemical parameters (Cholesterol, High density lipoprotein, Low density lipoprotein and
Triglycerides) of laying hens.
Blood biochemical parameters
Treatment CHOL 1 Enzyme
(mg/dl) HDL (mg/dl) LDL (mg/dl) TG (mg/dl)
0.00 144.63 59.875 61.25 1463.9
0.09 137.88 66.125 58.37 1270.9
Olive pulp
0.00 156.75 62.62 64.00 1749.9 a
9.00 125.75 63.37 55.62 984.9 b
SEM 21.44 8.36 8.28 346.82
CV 30.36 26.55 27.67 50.73
Source of variation Probability
Enzyme 0.758 0.469 0.734 0.588
Olive pulp 0.173 0.930 0.331 0.047
Enzyme
pulp
× Olive 0.810 0.815 0.964 0.776
1-Cholesterol, triglycerides, high density lipoprotein, low density lipoprotein; a-b means within a column
(within main effects) with no common superscript differ significantly (p < 0.05). SEM= Standard error of
means.

4310 Afr. J. Biotechnol.
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
differentiable counts of white blood cells (%, heterophil, lymphocyte, monocyte, eosinophil and basophil) of laying hens.
Treatment
Enzyme (g /100 g)
Heterophil
Differentiable counts of white blood cells (%)
Lymphocyte Monocyte Eosinophil Basophil
0.00 28.50 67.37 1.00 0.37 2.75
0.09 25.12 70.87 1.37 0.00 2.62
Olive pulp (g /100 g)
0.00 23.12 72.50 1.25 0.25 2.87
9.00 30.50 65.75 1.12 0.12 2.50
SEM 3.72 3.83 0.48 0.28 1.05
CV 27.72 11.08 81.53 298.14 78.38
Source of variation Probability
Enzyme 0.381 0.378 0.453 0.204 0.907
Olive pulp 0.070 0.103 0.800 0.662 0.728
Enzyme × Olive pulp 0.317 0.216 0.453 0.662 0.563
Table 9. Effect of dietary inclusion of guar meal (0, 25 and 50 g/kg) and enzyme supplementation (0 and 0.4g/kg) on
egg quality (egg index, yolk index, Haugh unit, eggshell weight and eggshell thickness) of laying hens.
Egg quality characteristics
Treatment
Enzyme (g /100 g)
Egg index Yolk index Haugh unit Egg shell weight Egg shell thickness
0.00 74.60 41.83 70.06 6.87 40.00
0.09 74.97 42.03 70.78 6.48 37.40
Olive pulp (g /100 g)
0.00 74.23 40.56 b 68.41 6.53 38.60
9.00 75.34 43.29 a 72.44 6.82 38.80
SEM 1.38 2.39 2.90 0.30 1.78
CV 4.36 5.65 9.73 11.65 10.91
Source of variation Probability
Enzyme 0.798 0.853 0.817 0.281 0.187
Olive pulp 0.455 0.020 0.207 0.423 0.917
Enzyme × Olive pulp 0.946 0.092 0.312 0.612 0.131
a-b Means within a column (within main effects) with no common superscript differ significantly (P < 0.05); SEM, standard
error of means.
hens fed with control and the OP-included diets. There
was no significant effect of enzyme supplementation on
the analyzed blood biochemical parameters (p > 0.05).
Differentiable count of white blood cells was not
significantly affected by dietary OP inclusion and enzyme
supplementation (Table 8). There is no record in the
literature presenting the effects of diet inclusion of OP on
blood biochemical parameters of laying hens.
As it is shown in Table 9, among the egg quality cha-
racteristics (egg index, yolk index, haugh unit, eggshell
weight and eggshell thickness); only yolk index was
affected by dietary OP inclusion. Including OP in the diets
of hens increased the yolk index (p ≤ 0.05); however,
there was no significant difference between the other egg
quality traits in hens fed with control and the OP-included
diets. There was no significant effect of enzyme supplementation
on the analyzed egg quality characteristics (p >
0.05). Gunawardana et al. (2009) evaluated the effects of

Rovabio (a natural mixture of enzymes produced by the
organism Penicillium funiculosum), dietary energy and
protein on performance, egg composition, egg solids and
egg quality of commercial Leghorns in phase 2 and reported
dietary protein significantly increased feed
consumption but decreased yolk color. As dietary energy
increased, feed consumption decreased and yolk color
increased. They also found a significant interaction
among dietary protein, energy and Rovabio on egg
production, BW, egg mass, feed conversion and yolk
solids. Egg weight of hens fed with diets supplemented
with enzyme was significantly greater than that of hens
fed with diets without enzyme during weeks 3 and 4.
However, enzyme did not significantly influence average
egg weight (Gunawardana et al., 2009). In the research
study by Pirgozliev et al. (2010) the yolk color of the birds
receiving xylanase was darker than the yolk of the birds
given the control diet. In addition, birds receiving
xylanase had a significantly higher weight gain than those
fed on the unsupplemented diet. Dirty and cracked eggs
in their study were not affected by xylanase (Pirgozliev et
al., 2010). The improvement in feed conversion for
Lohmann Brown-Lite laying hens fed with enzyme
cocktail (providing 7 U/g of α-1, 6-galactosidase and 22
U/g of β-1, 4-mannanase) supplemented diet was accompanied
by an increase in albumen weight and percentage
and a decrease in the percentage of yolk and the ratio of
yolk to albumin in the eggs (Han et al., 2010).
Conclusions
In conclusion, OP can be inserted in laying hens diets up
to 9% with no adverse effect on bird's performance.
Dietary inclusion of OP would have beneficial effect on
laying hens' performance in terms of egg weight, yolk
index and blood level of triglycerides. In this investigation,
dietary supplementation with a cocktail enzyme with
mainly xylanase activity improved FCR in hens.
Acknowledgment
Appreciation is expressed to Research Sector of Islamic
Azad University, Aligudarz Branch, Iran, for financial
support of this research.
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African Journal of Biotechnology Vol. 10(20), pp. 4313-4317, 16 May, 2011
Available online at http://www.academicjournals.org/AJB
DOI: 10.5897/AJB10.1883
ISSN 1684–5315 © 2011 Academic Journals
Full Length Research Paper
An investigation on natural radioactivity from mining
industry #
E. Esmeray* and M. E. Aydin
Selcuk University, Environmental Engineering Department, Konya, Turkey.
Accepted 25 March, 2011
The environmental problem of naturally occurring radioactive materials (NORM) is omnipresent on
earth and their radioactivity may become concentrated as a result of human activities. Various
industries produce concentrated radioactivity in their by-products. Mining originating industries such
as the coal industries, petroleum extraction and processing and natural gas, mining enrichment waste,
phosphate, etc have been well known and widely investigated. The Environmental Protection Agency
(EPA) describes NORM wastes from the mining and processing of three categories of metals: Rare
earth metals, special application metals and metals produced in bulk quantities by industrial extraction
processes. Moreover, NORM has a lot of negative effects on the natural resources (water supplies,
soils, air, etc.) and living organisms (human, animals, plants, microorganisms, etc.). In this study, we
investigated NORM levels that originated from mining industry and the concentration of NORM in
drinking water supplies. NORM parameter of gross alpha and gross beta were also in this study,
seasonal changes in gross alpha and gross beta were investigated. The obtained results showed that,
natural activity concentrations of α- and β-emitting radionuclides in all water samples did not exceed
World Health Organisation (WHO) and Turkish Standards of Drinking Water (TS 266) recommended
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
observed for the gross α and gross β activities, respectively. For all samples, the gross β activities were
higher than the corresponding gross α activities.
Key words: Natural radioactivity, mining industries, gross alpha, gross beta.
INTRODUCTION
Radiation of natural origin at the earth’s surface consists
of two components namely cosmic rays and radiation
from the radioactive nuclides in the earth’s crust. The
latter component is the terrestrial radiation, which mainly
originates from the so-called primordial radioactive
nuclides that were made in the early stage of the formation
of the solar system. Uranium, thorium and potassium
are, however, the main elements contributing to natural
terrestrial radioactivity (UNSCEAR, 2000).
Naturally-occurring radioactive material (NORM) is the
*Corresponding author. E-mail: eesmeray@selcuk.edu.tr. Tel:
+90-332-2232080, +90-542-5629808.
#This study has been presented as poster at the VIth
International Conference of Modern Management of Mine
Producing, Geology and Environmental Protection (SGEM),
Albena-Varna, Bulgaria, 12-16 June 2006.
term used to describe materials that contain radionuclides
that exist in the natural environment. Long-lived
radioactive elements of interest include uranium, thorium
and potassium and any of their radioactive decay
products, such as radium and radon. These elements
have always been present in the earth’s crust and within
the tissues of all living species. Studies of terrestrial
natural radiation are of great importance for various
reasons. They serve as useful tracers for atmospheric
variation studies. It is usually realized that natural environmental
radiation mainly depends on geological and
geographical conditions. Also, an understanding of
natural radioactivity was used to determine the age of the
earth using the radioactive decay of 238 U to 206 Pb and
235 U to 207 Pb. Each isotope has, however, a unique decay
rate so that the ratio of the number of daughter atoms to
parent isotopes in rocks and minerals depends on the
time which has elapsed since the system became closed
(Jamal, 2002). Many of the mining and processing

4314 Afr. J. Biotechnol.
Figure 1. A map of the study area and location of the sampling points.
(physical, chemical and termal): uranium, thorium, gold,
silver, copper, nickel, iron, aluminium, molybdenum, tin,
titanium, vanadium, etc. industries such as aforementioned
cause natural radiation.
Extraction of minerals
This was undertaken by conventional underground or
open pit mining techniques or acid leaching. Mineral processing
can involve dry techniques including electrostatic
or magnetic separation or wet techniques such as acid or
alkaline leaching and chemical flotation or electrical or
furnace smelting. All of these processes can affect the
concentrations of radionuclides in both waste and product
streams.
Mineral sands production
Mineral sands production (including ilmenite, leucoxene,
rutile, zircon, monazite and xenotime) leads to several
waste materials from primary processing of ore. Some of
these waste materials are returned to the mined out pit
for disposal.
In copper production
Wastes containing NORM arise in tailings from the
flotation stage and furnace slags from the smelting stage.
In tin and tantalum production, dry and wet separation
stages produce tailings slurry that is further treated and
disposed off in a tailings dam close to the mine site.
Tantalum products also contain low levels of NORM. Iron
and steel production wastes include blast furnace slags
and dusts and fumes from the sinter plant and off gas
cleaning in the blast furnace operation.
In phosphate ore processing
NORM is found in tailings from fertiliser production that
are normally used as backfill at the mine site and also in
fertilizer product at levels which do not cause any
significant increase in the uranium and thorium levels in
soil treated with fertilizer. In phosphoric acid production,
the majority of the NORM is left in the phosphogypsum,
which can be stockpiled on site or disposed of as landfill.
Phosphogypsum may also be used in fertilizer, soil
conditioner, building material (plasterboard, cement
aggregate) and in road construction (ARPANSA, 2004).
A lot of studies can be found on natural radioactivity in
earth soil and water samples. In this study, natural radioactivities
in water samples were investigated from mining
origin.
MATERIAL AND METHODS
Site description
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).
Radionuclide WHO (WHO,2006) Institute of Turkish Standards, (ITS 266, 2005)
Gross alpha 0.01 Bq/l (2.7 pCi/l) 0.0037 Bq/l (0.1 pCi/l)
Gross beta 0.1 Bq/l (27 pCi/l) 0.037 Bq/l (1 PCi/l)
in the Turkey’s central Anatolia region. Konya has a lot of mine
resources. These are barite, bentonite, mercury, porcelain clay,
clay, limestone, chrome, meerschaum, marble, talcum, asbestos,
lignite, etc. (MTA, 2010). Naturally, these mining resources and
their processing activity lead to natural radiation on nearest water
supplies and soil.
Sampling
In this study, gross alpha and gross beta activities were measured
for the samples which have been taken from eight different drinking
water resource (1 surface water, 6 well water, 1 tap water) in Konya
city for two different seasons (winter and summer). Sampling points
are shown in Figure 1. The values of measurements of the samples
which were taken in the Centrum of Konya show that there is no
natural radiation problem in water resources. Although, some
measurements result to higher gross alpha parameter according to
TS 266 (Turkish Standards of drinking water), but are still lower
than WHO standards, so this causes no problem. Furthermore,
gross beta activity values are fairly low according to TS 266 also,
WHO standards, so beta radioactivity causes no danger (TS 266,
2005). Drinking water standards of gross alpha and gross beta are
shown in Table 1.
Determination of natural radioactivity
Primarily, the aim of this study was to determine the level of natural
radioactivity in different drinking water supplies in Konya city. Eight
sampling sites for drinking water supplies have been selected in
Konya city. Figure 1 gives a detailed description of all sampling
sites. Sample bottles were rinsend with pure water, then filled
completely to minimize headspace. The sample bottles were
cleaned using a modified procedure before collection. (Laxen and
Harrison, 1981). Whole drinking water samples were collected by 1
L capacity of polypropylene bottles and acidified pH 2 with pure
HNO3 until analysis. Measurements of radioactivity level in all water
samples were performed by the method of EPA using the gross
alpha and gross beta counting system Epa method 900 (EPA,
1980). An aliquot of a preserved drinking water sample is evaporated
to a small volume and transferred quantitatively to a tared
2-inch stainless steel counting planchet. The sample residue is
dried to constant weight, re-weighed to determine dry residue
weight, then, counted for alpha and/or beta radioactivity. Counting
efficiencies for both alpha and beta particle activities were selected
according to the amount of sample solids from counting efficiency
vs sample solids standard curves (EPA, 1980). Gross α and βactivities
were determined by means of a Tennelec LB 1000 low
background proportional counter subsequently (Tennelec, 1989).
The counter was calibrated with 241 Am and 90 Sr– 90 Y standards by
using potentials of 1100 and 1600 V corresponding to the α and βplateaus,
respectively. The total yields obtained for the α and βcountings
were 16 and 34%, respectively.
Calculations
Equation 1: The following equation was used to calculate the alpha
radioactivity:
Esmeray and Aydin 4315
Where, A is the net alpha count rate (gross alpha count rate minus
the background count rate) at the alpha voltage plateau; C is the
alpha efficiency factor read from the graph of efficiency versus mg
of water solids per cm 2 of planchet area, (cpm/dpm); V is the
volume of sample aliquot, (ml) and 2.22 is the conversion factor
from dpm/pCi (EPA, 1980).
Also, equations that were used to calculate beta radioactivity are
given as:
Equation 2: Is used if there are no significant alpha counts when
the sample is counted at the alpha voltage plateau, the beta activity
can be determined from the following equation:
Where, B is the net beta count rate (gross count rate minus the
background count rate at the beta voltage plateau); D is the beta
efficiency factor read from the graph of efficiency versus mg of
water solids per cm 2 of planchet area, (cpm/dpm); V is the volume
of sample aliquot, (ml) and 2.22 is the conversion factor from
dpm/pCi (EPA, 1980).
Equation 3: Is used when counting beta radioactivity in the
presence of alpha radioactivity by gas-flow proportional counting
systems (at the beta plateau), alpha particles are also counted.
Since alpha particles are more readily absorbed by increasing
sample thickness than beta particles, the alpha/beta count ratios
vary with increasing sample thickness.
Therefore, it is necessary to prepare a calibration curve by
counting standards containing americium-241 with increasing
thickness of solids on the alpha plateau and then, on the beta
plateau, plotting the ratios of the two counts versus density
thickness.
The alpha amplification factor (E) from that curve is used to
correct the amplified alpha count on the beta plateau. When
significant alpha activity is indicated by the sample count at the
alpha voltage plateau, the beta activity of the sample can be
determined by counting the sample at the beta voltage plateau and
calculating the activity from the following equation:
Where, B (as defined in equation 2); D = (as defined equation 2); A
= (as defined in equation 2); E is the alpha amplification factor read
from the graph of the ratio of alpha counted at the beta
voltage/alpha counted at the alpha voltage versus sample density
(1)
(2)
(3)

4316 Afr. J. Biotechnol.
Table 2. Winter season, activity concentrations of gross alpha and gross beta in water
supplies samples in Konya city. (Esmeray and Aydın,2006)
Sample location α Radioactivity (Bq/l) β Radioactivity (Bq/l)
Apa Dam 0.072 ± 0.0023 0.20 ± 0.004
Anit 0.079 ± 0.0026 0.08 ± 0.004
Kongaz II 0.007 ± 0.0021 0.06 ± 0.004
Kas III 0.0062 ± 0.0026 0.05 ± 0.004
Forestry directorate 0.052 ± 0.0025 0.10 ± 0.004
City center (KOSKI) 0.007 ± 0.0020 0.01 ± 0.003
Marangozlar 0.014 ± 0.0022 0.09 ± 0.004
Meram II 0.0077 ± 0.0011 0.18 ± 0.004
Table 3. Spring Season, Activity concentrations of gross alpha and gross beta in
water supplies samples in Konya City. (Esmeray and Aydın,2006)
Sample location α Radioactivity (Bq/l) β Radioactivity (Bq/l)
Apa Dam 0.018 ± 0.0019 0.11 ± 0.004
Anit 0.035 ± 0.0023 0.07 ± 0.003
Kongaz II 0.006 ± 0.0019 0.09 ± 0.004
Kas III 0.0012 ± 0.0018 0.04 ± 0.003
Forestry directorate 0.008 ± 0.0023 0.13 ± 0.004
City center (KOSKI) 0.006 ± 0.0017 0.04 ± 0.003
Marangozlar 0.065 ± 0.0028 0.18 ± 0.004
Meram II 0.007 ± 0.0021 0.18 ± 0.004
thickness; V is the volume of sample aliquot, (ml) and 2.22 is the
conversion factor from dpm/pCi (EPA, 1980).
Finally, all results were converted between units (pCi/l) to (Bq/l)
and used for experimental results. (Esmeray, 2005)
RESULTS AND DISCUSSION
Activity concentrations of gross alpha and gross beta in
drinking water in Konya are shown in Tables 2 and 3. As
shown in Table 2, Apa Dam, Anit and Forestry Directorate’s
gross alpha activity concentrations are over than
the standards of ITS and nevertheless, all results of
gross beta activity concentrations are permissible limits
for both WHO and ITS regulations for drinking water.
Only Marangozlar’s gross alpha activity concentration is
more than ITS standards, as shown in Table 3, but all
other gross alpha activity concentrations are permissible
limits for both WHO and ITS regulations for drinking
water standards. In addition, all gross beta and gross
alpha activity concentrations are generally good and
acceptable for WHO and ITS’s recommended levels of
drinking water (Table 1).
All experimental results show that, the gross alpha
activity ranges between (0.0062 (Meram II) and 0.79
(Anıt) Bq.l -1 -winter season) (0.0012 (Kas III) and 0.065
(Koski) Bq.l -1 -spring season). Besides, gross beta activity
lies between (0.001(Koski) and 0.2 (Apa Dam) Bq.l -1 -
winter season) (0.004 (Koski, Kas III) and 0.18 (Meram II,
Marangozlar) Bq.l -1 -spring season). Activity concentrations
of gross alpha and gross-beta in water supplies
in Konya are shown in Table 3 (winter and spring
season).
As shown in Tables 2 and 3, the lowest average were
0.0062 ± 0.0026 Bq/l (gross alpha) and 0.01 ± 0.003 Bq/l
(gross beta) in winter and the highest seasonal average
were 0.079 ± 0.0026 Bq/l (gross alpha) and 0.20 ± 0.004
Bq/l (gross beta) in winter. Also, the lowest average were
0.0012 ± 0.0018 Bq/l (gross alpha) and 0.04 ± 0.003 Bq/l
(gross beta) in spring and the highest seasonal average
were 0.065 ± 0.0028 Bq/l (gross alpha) and 0.18 ± 0.004
Bq/l (gross beta) in spring. Evaporation conditions, temperature
changes, solubility of radioactive materials can
be causes of seasonal changes in gross alpha and gross
beta activities in Konya. Besides, the concentration of
radioactivity increases in spring and summer due to high
evaporation rates and the increase of solubility of salts
due to the higher temperature of the water (Hosseini,
2007). Additionally, high levels of natural radioactivity in
drinking water are accompanied with potential health
risks for the population by increasing the radiation dose.
Hence, water must be purified before using it. There are
several methods known to remove radioactivity from water

such as aeration to remove granular activated
carbon (GAC), ion exchange methods (IX), reverse
osmosis (RO) to remove “Gross alpha and gross beta,
uranium etc.” and various adsorption methods to remove
other natural radioactivity sources (Esmeray and Aydin,
2008).
Conclusions
The levels of gross alpha and gross beta activity in Konya
region located in central anatolia of Turkey have been
studied. Experimental results observed show that, gross
alpha and gross beta activity concentrations are lower
than standards of WHO and ITS (WHO, 2006; ITS,
2001). This kind of the study must be continued for other
parts of Konya and Turkey. The results and data obtained
in this work are a baseline which can be used to evaluate
possible future changes.
ACKNOWLEDGEMENT
The authors thank the Selcuk University Research Fund
(BAP) for its financial support of the work undertaken
here (2004/70), that this paper has been derived from a
part of MSc. thesis of Ertugrul ESMERAY.
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