Workshop-Plant Biotechnology - SodininkystÄ ir daržininkystÄ
Workshop-Plant Biotechnology - SodininkystÄ ir daržininkystÄ
Workshop-Plant Biotechnology - SodininkystÄ ir daržininkystÄ
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INSTITUTE OF HORTICULTURE,<br />
LITHUANIAN RESEARCH CENTRE FOR<br />
AGRICULTURE AND FORESTRY<br />
WORKSHOP-SEMINAR<br />
PLANT BIOTECHNOLOGY<br />
ADVANCES IN AGRICULTURE<br />
Programme and Abstracts<br />
Kaunas district, Lithuania,<br />
October 27–28, 2011
UDK 575:581.19:631.523<br />
ORGANIZING COMMITTEE<br />
Prof. habil. dr. Vidmantas Stanys (Institute of Horticulture, LRCAF) – cha<strong>ir</strong>man<br />
Prof. dr. Česlovas Bobinas (Institute of Horticulture, LRCAF)<br />
Dr. Audrius Sasnauskas (Association of Agriculture and Forestry Research<br />
Institutes)<br />
Dr. Danas Baniulis (Institute of Horticulture, LRCAF)<br />
Dr. Vidmantas Bendokas (Institute of Horticulture, LRCAF)<br />
B<strong>ir</strong>utė Frercks (Institute of Horticulture, LRCAF)<br />
VENUE<br />
October 27th – hotel “Best Western Santaka”, J. Gruodžio st. 21 Kaunas.<br />
October 28th – the Institute of Horticulture, LRCAF, Kauno st. 30, Babtai,<br />
Kaunas dist.<br />
ACKNOWLEDGMENTS<br />
The workshop-seminar is funded by the European Social Fund and supported in<br />
part by AB Bruker Daltonics Scandinavia and UAB Analytical Solutions.<br />
Address of the volume in internet www.lsdi.lt<br />
© Institute of Horticulture Lithuanian Research Centre for Agriculture and Forestry
PROGRAMME<br />
October 27 (Thursday)<br />
Hotel “Best Western Santaka”, Kaunas<br />
9 30 –10 00 REGISTRATION<br />
10 00 –10 05 OPENING OF THE WORKSHOP – SEMINAR<br />
10 05 –15 00 PLENARY SESSION<br />
10 05 –11 00 BIOTECHNOLOGICAL APPLICATIONS OF THE KNOWLEDGE ON<br />
MOLECULAR HOST-VIRUS INTERACTIONS IN PLANT DISEASE<br />
CONTROL<br />
Prof. Jari Valkonen<br />
Department of Agricultural Sciences, Helsinki University<br />
11 00 –11 15 C o f f e e b r e a k<br />
11 15 –12 10 PLANT VIRUS INTERACTIONS WITH NUCLEUS<br />
Dr. Minna Rajamaki<br />
Department of Agricultural Sciences, Helsinki University<br />
12 10 –13 10 L u n c h<br />
13 10 –14 05 PHOSPHOPROTEOMIC DISSECTION OF PLANT IMMUNE<br />
SIGNALLING<br />
Dr. H<strong>ir</strong>ofumi Nakagami<br />
<strong>Plant</strong> Science Centre, RIKEN Institute<br />
14 05 –15 00 SIGNAL TRANSDUCTION IN PLANT CELLS<br />
Dr. Irutė Meškienė<br />
Max F. Perutz Laboratories, University of Vienna<br />
15 00 –16 30 POSTER SESSION<br />
October 28 (Friday)<br />
Institute of Horticulture, LRCAF, Babtai, Kaunas district<br />
10 00 –12 00 Discussion “ADVANCES OF BIOTECHNOLOGICAL RESEARCH ON<br />
PLANT ADAPTABILITY AND APPLICATION IN AGRICULTURE”<br />
12 00 –13 00 L u n c h<br />
13 00 Post-conference excursion
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
PLENARY SESSION ABSTRACTS<br />
Biotechnological applications of the knowledge on molecular<br />
host-v<strong>ir</strong>us interactions in plant disease control<br />
Jari P. T. Valkonen<br />
Department of Agricultural Sciences, PO Box 27, FI-00014 University of<br />
Helsinki, Finland, E-mail jari.valkonen@helsinki.fi<br />
V<strong>ir</strong>uses are among the most dangerous pathogens of plants. The<strong>ir</strong> transmission to<br />
new crops in seeds, seed tubers, cuttings and other planting materials results in particularly<br />
heavy yield losses. V<strong>ir</strong>uses cannot be controlled by chemical treatments during the<br />
growing season. Transmission of v<strong>ir</strong>uses is also difficult to control in the field. Due to<br />
these limitations, use of v<strong>ir</strong>us-free planting materials and v<strong>ir</strong>us-resistant cultivars remain<br />
as the main approaches to control plant v<strong>ir</strong>uses.<br />
Cryotherapy represents a biotechnological application that can be used to obtain<br />
v<strong>ir</strong>us-free plants from infected materials. It makes use of the plants’ natural ability to<br />
protect the meristematic tissue against v<strong>ir</strong>us infection and utilizes the methodology of<br />
cryopreservation. In cryotherapy, shoot tips are treated briefly in liquid nitrogen using<br />
adjusted cryopreservation protocols and healthy plants are regenerated from the surviving<br />
pathogen-free meristematic tissue. Freedom of meristematic, undifferentiated cells from<br />
v<strong>ir</strong>uses is related to the phenomenon described as recovery of plants from v<strong>ir</strong>us infection.<br />
Recovery is based on the basal antiv<strong>ir</strong>al defence mechanism called RNA silencing. It is<br />
able to recognise double-stranded RNA (dsRNA) specifically and target it to degradation.<br />
The dsRNA-specific cellular RNases (Dicers) cleave dsRNA to small interfering RNAs<br />
(siRNA) that are used to target homologous single-stranded RNA molecules, in this case<br />
v<strong>ir</strong>al RNA genomes or v<strong>ir</strong>al gene transcripts. The large amounts of v<strong>ir</strong>us-derived siRNA<br />
hence produced can be used for diagnostic purposes to detect v<strong>ir</strong>uses. For this purpose, the<br />
siRNA pool from the plant is isolated and sequenced, the overlapping siRNA sequences<br />
are assembled to large contigs, and the contigs are compared to the v<strong>ir</strong>al sequences available<br />
in databases to identify the v<strong>ir</strong>uses in the sample.<br />
However, v<strong>ir</strong>uses have evolved various means of evade or suppress antiv<strong>ir</strong>al defense<br />
in the<strong>ir</strong> host plants. Active suppression of RNA silencing is used by all v<strong>ir</strong>uses. One of the<br />
most studied v<strong>ir</strong>al RNA silencing suppressors is HCpro of Potyv<strong>ir</strong>uses, the largest group of<br />
plant-infecting RNA v<strong>ir</strong>uses. HCpro interacts with the translation initiation factors eIF4E<br />
and eIF(iso)4E for as yet unknown reasons. However, these interactions are most likely<br />
important to v<strong>ir</strong>us infection because many recessive resistance genes used for breeding<br />
of resistance to Potyv<strong>ir</strong>uses in crop plants are mutated forms of the eIF4E and eIF(iso)4E<br />
genes. Further understanding of the protein complexes formed by v<strong>ir</strong>al and host proteins<br />
is expected to raise new possibilities to develop v<strong>ir</strong>us-resistant plant cultivars.<br />
5
<strong>Plant</strong> v<strong>ir</strong>us interactions with nucleus<br />
Minna Rajamäki<br />
Department of Agricultural Sciences, PO Box 27, FI-00014 University of<br />
Helsinki, Finland, E-mail minna.rajamaki@helsinki.fi<br />
Positive-strand RNA v<strong>ir</strong>uses form the largest group of plant-infecting v<strong>ir</strong>uses. Unlike<br />
plant DNA v<strong>ir</strong>uses that replicate in the host cell nucleus, the positive-strand RNA v<strong>ir</strong>uses<br />
replicate in membranous structures in the cytoplasm. However, some of the<strong>ir</strong> proteins<br />
are targeted to the nucleus and nucleolus, the reasons of which have remained largely<br />
unknown. Recent studies have indicated that v<strong>ir</strong>al protein may localize in the nucleus in<br />
order to recruit nuclear proteins to be used in v<strong>ir</strong>us infection, to sequester or redistribute<br />
some nuclear components to disrupt nuclear functions or counteract plant host defence<br />
responses. For example, the ability of Groundnut rosette v<strong>ir</strong>us (GRV; genus Umbrav<strong>ir</strong>us)<br />
to move over long distances through plant phloem is dependent on the nucleolar targeting<br />
of its ORF3 protein. In the nucleus, the ORF3 protein interacts with the major nucleolar<br />
protein, fibrillarin, recruiting some of it to the cytoplasm, which is requ<strong>ir</strong>ed for the formation<br />
of long distance movement competent v<strong>ir</strong>al ribonucleoprotein particles. Potyv<strong>ir</strong>uses,<br />
which constitute the largest group of plant RNA v<strong>ir</strong>uses, also target certain v<strong>ir</strong>al proteins<br />
to the nucleus. The majority of the multifunctional nuclear inclusion protein a (NIa) of<br />
Potato v<strong>ir</strong>us A (PVA; genus Potyv<strong>ir</strong>us) is found in the nucleus and nucleolus of v<strong>ir</strong>usinfected<br />
cells. Two regions at the N-proximal VPg domain of NIa control both nuclear and<br />
nucleolar localization. Mutations that debilitate nuclear and nucleolar localization of NIa<br />
are detrimental for v<strong>ir</strong>us amplification suggesting that nuclear localization of NIa is important<br />
for v<strong>ir</strong>us infection. NIa also interacts with fibrillarin, but the role of this interaction is<br />
likely different from Umbrav<strong>ir</strong>uses as fibrillarin depletion in plants does not compromise<br />
v<strong>ir</strong>al long distance movement but rather reduces PVA accumulation. Interestingly, the data<br />
suggest a link between nuclear/nucleolar localization of NIa and interference with host<br />
antiv<strong>ir</strong>al defences (RNA silencing). The results thus demonstrate that the nucleus may<br />
have essential role also in plant RNA v<strong>ir</strong>us infection and could provide new insights of<br />
strategies how to control plant v<strong>ir</strong>us infections in the future.<br />
6
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Phosphoproteomic dissection of plant immune signaling<br />
H<strong>ir</strong>ofumi Nakagami<br />
<strong>Plant</strong> Proteomics Research Unit, RIKEN <strong>Plant</strong> Science Center,<br />
1-7-22 Sueh<strong>ir</strong>o-cho, Tsurumi, Yokohama 230-0045, Japan,<br />
E-mail h<strong>ir</strong>onakagami@psc.riken.jp<br />
Proteomics is one of the best available tools for studying posttranslational modifications<br />
(PTMs). Therefore, it is well suited for dissecting signaling pathways which often<br />
utilize PTMs as a means of transmitting signals. Among the several PTMs described<br />
thus far, phosphorylation is the most extensively studied and has been shown to play an<br />
important role in plant immune signaling.<br />
At f<strong>ir</strong>st, we have developed a plant phosphoproteomics platform, which enables<br />
monitoring phosphorylation events in plant cells at the cellular level. Besides developing<br />
the phosphoproteomics platform, we have also established a method for the differential<br />
analysis of plant samples which enables phosphoproteome dynamics to be monitored.<br />
In order to identify novel players involved in plant immunity, we examined phosphoproteome<br />
changes in Arabidopsis upon MAMP (microbe-associated molecular pattern)<br />
treatment and identified 569 proteins whose phosphorylation status significantly changes<br />
in response to flg22 and/or chitin treatment. Most of the identified proteins have not been<br />
reported yet to take part in MAMP-triggered immunity, but included well known MAMPsignaling<br />
regulators such as MPK3/4/6, MKK1/2/4/5, MEKK1, MKP1, and RbohD.<br />
To verify involvement of the identified proteins in MAMP-triggered responses,<br />
we have been isolating T-DNA insertion lines for these proteins and characterizing<br />
flg22-induced ROS (reactive oxygen species) production in the isolated mutants. So far we<br />
have identified 28 genes as negative regulators of MAMP-induced ROS production, and<br />
are expecting to isolate more MAMP-signaling regulatory genes with continued screening.<br />
These results suggest that phosphoproteomics-based screening will be powerful and<br />
effective approach to identify novel signaling components in plants.<br />
Furthermore, we are developing a novel method, which utilizes the phosphoproteomics<br />
technique, to identify substrates of protein kinases involved in plant immunity.<br />
7
Signal transduction by MAPK pathways in plant cells<br />
Irute Meškienė, Alois Schweighofer<br />
Institute of <strong>Biotechnology</strong>, Vilnius, Lithuania; Max F. Perutz<br />
Laboratories, University of Vienna; Medical Universty of Vienna,<br />
Austria, E-mail <strong>ir</strong>ute.meskiene@univie.ac.at<br />
Env<strong>ir</strong>onmental and developmental inputs are perceived at the cell membranes and<br />
communicated to the nucleus to regulate cellular functions. Intracellular signalling in all<br />
eukaryotes is employing a highly conserved mechanism, which is based on reversible<br />
protein phosphorylation. Protein phosphorylation is an essential posttranslational modification<br />
mechanism of signalling proteins.<br />
Phosphorylation transiently activates mitogen-activated protein kinase (MAPK)<br />
signalling pathways, which are regulated by opposing actions of protein kinases and protein<br />
phosphatases. The core module of this signaling system consists of three interlinked<br />
protein kinases: a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a<br />
MAPK, that transmit a signal via sequential phosphorylation events.<br />
MAPKs activation leads to reprogramming of plant cellular activities, including<br />
changes in gene expressions and plant hormone levels to generate appropriate plant<br />
responses. A “Switch-off” mechanism of this signaling is essential to ensure the correct<br />
cellular response to a given stimulus. This mechanism involves protein phosphatases that<br />
inactivate MAPKs by dephosphorylation ensuring the transient mode of signalling. MAPK<br />
inactivation can be achieved by evolutionary distant protein phosphatases, including serine/<br />
threonine protein phosphatases of PP2C-type and tyrosine or dual-specificity (tyrosine<br />
and serine/threonine) phosphatases.<br />
In the group of serine/threonine protein phosphatases plants evolved the large family<br />
of PP2C-type and there a cluster of proteins, which control MAPK activities (Schweighofer<br />
et al., 2004; Umbrasaite et al., 2010). Four PP2Cs AP2C1, AP2C2, AP2C4 and AP2C3<br />
retain the protein structure for MAPK interaction (Schweighofer et al., 2004) and function<br />
as MAPK phosphatases (Schweighofer et al., 2007; Umbrasaite et al., 2010). The woundand<br />
biotic stress-induced AP2C1 is a negative regulator of MAPKs and modulator of plant<br />
innate immunity, affecting plant stress hormone production, such as wound-jasmonate<br />
and ethylene (Schweighofer et al., 2007). The closely related AP2C3 is expressed during<br />
stomata development and regulates MAPKs during signaling in stomata developmental<br />
pathway. Stomata are cells controlling plant gas exchange and transp<strong>ir</strong>ation, essential<br />
processes for our ecosystem.<br />
Recent characterisations of Arabidopsis protein phosphatase mutants, identification<br />
of substrate MAPKs and related signalling pathways highlight the important roles<br />
8
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
of MAPK-phosphatases in the pathways regulating stress responses, plant hormone<br />
biosynthesis, protection against herbivores, stomata cell development, and cell cycle. It<br />
suggests that these signalling pathways and the components involved are determining<br />
essential plant functions.<br />
Schweighofer et al. (2004). <strong>Plant</strong> PP2C phosphatases: emerging functions in stress signaling.<br />
Trends <strong>Plant</strong> Sci 9, 236–243.<br />
Schweighofer et al. (2007). The PP2C-Type Phosphatase AP2C1, Which Negatively Regulates<br />
MPK4 and MPK6, Modulates Innate Immunity, Jasmonic Acid, and Ethylene Levels in<br />
Arabidopsis. <strong>Plant</strong> Cell 19, 2 213–2 224.<br />
Umbrasaite et al. (2010). MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal<br />
cells leading to stomata development in Arabidopsis. PLoS ONE 5, e15357.<br />
9
POSTER SESSION ABSTRACTS<br />
Identification and analysis of winter wheat genes induced at low<br />
temperatures<br />
Rita Armonienė, Žilvinas Liatukas, Gintaras Brazauskas<br />
Lithuanian Research Centre for Agriculture and Forestry, Institute of<br />
Agriculture, Akademija, LT-58344, Kėdainiai distr., Lithuania,<br />
E-mail rita.armoniene@lzi.lt<br />
Freezing temperature is one of the most severe abiotic stresses limiting wheat growth,<br />
productivity, and distribution. Freezing tolerance in plants develops through acclimation<br />
to cold by growth at low, above-freezing temperatures. Large number of genes are known<br />
to have differential expression during the period of cold acclimation. Main task of our<br />
study is to find genes expressed at different time points of cold acclimation and to find<br />
out if these genes – candidates are responsible for freezing tolerance formation. The f<strong>ir</strong>st<br />
assignment of the study was the evaluation of freezing tolerant and susceptible wheat lines<br />
using freezing test in natural and artificial conditions. Th<strong>ir</strong>ty winter wheat lines/varieties<br />
were subjected to the freezing test in the plant growth chamber. Leaves were cut off near<br />
the crowns after cold treatment and re-growing plants were counted, as well as chlorophyll<br />
fluorescence parameter Fv/Fm was measured. Three most freezing tolerant wheat lines/<br />
varieties were identified: 68, 66 and 67. The correlation coefficient between the Fv/Fm<br />
parameter with freezing tolerance studies was 0.84, p < 0.001. The same th<strong>ir</strong>ty wheat<br />
lines were grown in the field experiment during the winter 2010/2011 and the evaluation<br />
in natural conditions was conducted in April 2011 on a 1–9 score basis. The result of the<br />
evaluation in natural conditions showed that most freezing tolerant wheat lines/varieties<br />
were ‘Zentos’, 29 and ‘Ada’. We will also present future work of our study for identification<br />
and analysis of genes induced at low temperatures by employing cDNA-AFLP<br />
profiling and TILLING methods in winter wheat. We hope that identification and analysis<br />
of new genes will lead to better understanding of the genetic regulation of winter wheat<br />
cold acclimation and freezing tolerance formation.<br />
10
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Bioactive potential of lectin-enriched protein fractions from<br />
Thymus vulgaris L. and Urtica dioica L. grass<br />
V. Bakšenskaitė 1 , L. Bistrovaitė 2 , G. Balčiūnaitė 3 , V. Kavaliauskis 3 ,<br />
V. Bendokas 4 , D. Baniulis 4 , V. Stanys 4 , D. Majienė 2 , J. Liobikas 2 ,<br />
J. Bernatonienė 3 , A. Savickas 3<br />
1<br />
Department of Biochemistry and Biotechnologies, Faculty of Natural<br />
Sciences, Vytautas Magnus University, Vileikos 8, Kaunas, Lithuania;<br />
2<br />
Institute of Neurosciences, Medical Academy, Lithuanian University of<br />
Health Sciences, Eiveniu 4, Kaunas, Lithuania;<br />
3<br />
Department of Drug Technology and Social Pharmacy, Faculty of<br />
Pharmacy, Medical Academy, Lithuanian University of Health Sciences,<br />
Mickeviciaus 9, Kaunas, Lithuania;<br />
4<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,<br />
E-mail vaida.baksenskaite@fc.vdu.lt<br />
Medical herbs and spice plants present a valuable source for manufacturing biopharmaceutical<br />
medicines, and substantial amounts of the herbs are consumed for food<br />
worldwide. Herb production is established as one of priority areas for non-traditional<br />
agriculture in Lithuania. Lamiaceae family of plants include a number of valuable medical<br />
and spice plants that are represented by wild species or being common feature of gardens<br />
and agricultural production in Lithuania. Common thyme (Thymus vulgaris L.) is valued<br />
for the rich content of etheric oils, vitamins and minerals, and it is popular in food industry<br />
as a spice. Stinging nettle (Urtica dioica L.), a member of Urticaceae family, is another<br />
plant common in Lithuania and has an established medical value.<br />
Although content of mineral and organic substances of the medical herbs is well<br />
characterised, presence of bioactive polypeptides is much less appreciated. Lectins<br />
(phytohemagglutinins) are glycoproteins common in plants that demonstrate a broad<br />
spectrum of bioactive effects. Recent investigations revealed a potential for use of lectins<br />
as anticancer agents.<br />
Meanwhile mainly leaves or stems are consumed in case of a number of popular<br />
medical and spice herbs, including the nettle and thyme, seeds and roots of plants have<br />
been established as a common source for isolation of lectins from plants, so far. Therefore<br />
data on the presence of lectins in the leaves or stems of the two herbs is ambigous. In this<br />
study, we prepare lectin-enriched protein fractions from extracts of the two herbs and<br />
11
assess the<strong>ir</strong> blood cell agglutinating activity using tests employing rabbit erythrocytes and<br />
macrophage J774 cells. The study revealed that lectin-like glycoprotein-enriched fractions<br />
from Common thyme and Stinging nettle grass contained specific hemagglutination activity<br />
up to 1.3 and 3.0 mg protein/ml, respectively. Application of the fractions containing the<br />
hemagglutination activity induced aggregation of macrophage J774 cells, and a highest cell<br />
death induction was observed in case of extracts obtained from herb of Common thyme.<br />
The results suggest presence of substantial hemagglutination activity content in grass of<br />
the two common medical herbs and bioactive potential of the lectin proteins.<br />
12
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
mtDNA polymorphism of Scots pine<br />
D. Danusevičius 1 , J. Buchowska 1 , V. Stanys 2 , J. B. Šiksnianiene 2 ,<br />
D. Baniulis 2<br />
1<br />
Facultry of Forest and Ecology, Aleksadras Stulginskis Univeristy,<br />
Studentu 11, Akademija, LT-53361 Kaunas reg., Lithuania;<br />
2<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,<br />
E-mail darius.danusevicius@asu.lt<br />
The earlier studies on the post-glacial colonisation and DNA polymorphism in Scots<br />
pine lack material from the eastern range of the species distribution. The hypothetical<br />
post-glacial refugium may be in the central Europe and regions around Ural mountains<br />
needs further examination. Aim of out study is to use the maternally inherited mtDNA<br />
CAPS markers to investigate mtDNA polymorphism and the migration patters based<br />
on the large-scale genetic material from the provenance trials with eastern Scots pine<br />
populations. Samples were collected in the provenance trials to obtain complete and even<br />
coverage of the eastern range, which will reach Japan in the east. The results revealed<br />
two major post-glacial refugial zones: (a) in southern Europe and southern Asia with<br />
prevalence of the Nad7-1 mitotype AA2, which was commonly referred to as universal<br />
as it was fond in Scotland as well as in Far east, and (b) much more restricted to the in<br />
north eastern European part of Russia, with maximum occurrence of the Nad7-1 mitotype<br />
BA2 in the region west of central Ural mountains; this constitutes an important finding<br />
with previously least tested genetic material from eastern Russia, (c) found only locally<br />
in one Turkish population mitotype CA2. Central European populations possessed grated<br />
mtDNA diversity and more than others contained both A and B alleles, what indicates a<br />
convergence of the southern and northern refugial lines in central Europe.<br />
13
Effect of far-red light on the growth of Chrysanthemum<br />
plantlets in vitro<br />
Stasė Dapkūnienė 1, 2 , Anželika Kurilčik 1, 3 , Genadij Kurilčik 4 ,<br />
Pavelas Duchovskis 3 , Akvilė Urbonavičiūtė 3 , Silvija Žilinskaitė 1 ,<br />
Arturas Žukauskas 4<br />
1<br />
Botanical Gardens of Vilnius University, Ka<strong>ir</strong>ėnų 43, LT-10239, Vilnius,<br />
Lithuania;<br />
2<br />
<strong>Plant</strong> Gene Bank, Stoties 2, LT-58343 Akademijas, Kėdainiai disrt. ,<br />
Lithuania;<br />
3<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;<br />
4<br />
Institute of Materials Science and Applied Research, Vilnius University,<br />
Saulėtekio 9-III, LT-10222 Vilnius, Lithuania<br />
The influence of far-red light on chrysanthemum growth was studied using a<br />
light-emitting diode (LED)-based illuminator. Four groups of chrysanthemum plantlets<br />
(Chrysanthemum morifolium Ramat. ‘Ellen’) were simultaneously grown under independently<br />
set different illumination regimes. The study showed that far-red light hinders<br />
the chrysanthemum growth processes in vitro, but increases growth of the roots length.<br />
Meanwhile with an increase of far-red light photon flux density, the rooting rate of explants<br />
decreases. Fresh and dry weight accumulation was depended on the rooting processes<br />
of chrysanthemum plantlets. The study also show that phytohormones are involved in<br />
drymatter accumulation and rhizogenesis processes. The presence of far-red light reduces<br />
the content of all photosynthetic pigments. This shows that by adjusting of the incident<br />
spectrum using a multiwavelength solid-state illuminator, the growth process of chrysanthemum<br />
culture in vitro can be controlled.<br />
14
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Assesment of genetic diversity within Lithuanian sweet cherry<br />
genetic resources collection using AFLP markers<br />
B<strong>ir</strong>utė Frercks, Inga Stepulaitienė, Dalia Gelvonauskienė,<br />
Jūratė Bronė Šikšnianienė, Vidmantas Stanys<br />
Institute of Horticulture, Lithuanian Research Centre for<br />
Agriculture and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr.,<br />
Lithuania, E-mail b.frercks@lsdi.lt<br />
Northern border of the secondary spreading of the sweet cherry is located in the Baltic<br />
region. Populations of wild sweet cherry adapted to local growth conditions are found in<br />
Western part of Lithuania. These sweet cherry populations were source for development of<br />
the earliest cultivars in Lithuanian. The use of molecular techniques that detect variations at<br />
DNA level is more objective and independent from env<strong>ir</strong>onmental influences in contrast to<br />
morphological traits, and it makes possible to develop cultivar-specific DNA fingerprints.<br />
In this study the genetic diversity among 31 sweet cherry accessions was evaluated<br />
using amplified fragment lenght polymorphism (AFLP) markers. Using 9 AFLP primer<br />
combinations 485 fragments were produced, 350 of them (72 %) were polimorphic.<br />
Polymorphism percentage, PIC and resolving power were calculated to assess the discriminatory<br />
power of nine primer combinations used. Polymorphism percentage (PP)<br />
ranged from 55.1 % to 100 % , in average 79.26 %. Average fragment PIC for each primer<br />
combination ranged from 0.30 to 0.42 (mean 0.36). Resolving power varied from 11.42 to<br />
31.74 with an average 20.75. In our study the RP was determinated as the real attribute for<br />
AFLP to characterise the discriminatory power of primer combination. All of the 31 sweet<br />
cherry accessions tested could be clearly differentiated with 9 AFLP primer combinations<br />
used in this study. For cluster analysis 350 polimorphic fragments were used. The results<br />
of the cluster analysis showed the consistence between AFLP markers analysis and the<br />
known genetic background of cultivars and asseccions.<br />
15
Genetic variation among Lithuanian populations of<br />
Impatiens parviflora determined by RAPD-PCR markers<br />
Rasa Jankauskaitė 1 , Milda Jodinskienė 1 , Judita Žukauskienė 1 ,<br />
Steven B. Janssens 2 , Algimantas Paulauskas 1 ,<br />
Eugenija Kupčinskienė 1<br />
1<br />
Vytautas Magnus University, Department of Biology, Vileikos 8,<br />
LT-44404 Kaunas, Lithuania;<br />
2<br />
Laboratory of <strong>Plant</strong> Systematics, Institute of Botany and<br />
Microbiology, K. U. Leuven, Kasteelpark Arenberg 31, P. O. Box 2437,<br />
BE-3001 Leuven, Belgium, E-mail e.kupcinskiene@gmail.com<br />
Human activities are resulting in the spread of many non-indigenous species to habitats<br />
in which they are non-native. One of the most invasive alien species in the woods of<br />
Central and Northern Europe is small balsam (Impatiens parviflora), an Asiatic species.<br />
Its introductions appear to be increasing in forest ecosystems and are considered harmful.<br />
This species is affecting ground-layer vegetation displacing native community components<br />
having similar biology. Small balsam is valuable model for invasiveness investigation.<br />
Due to small unattractive flowers intentional anthropogenic spread of this species might be<br />
excluded. It has gradually been recognized that ecological attributes alone are insufficient<br />
to explain why some plant species became invasive. This has led for more genetic studies<br />
of introduced species. Various different mechanisms might be implicated in generating the<br />
genetic variation underlying rapid adaptive evolution and the colonization of new habitats.<br />
Till now the exact source of genetic variation causing traits important to successful<br />
invasions remains uncharacterized. The objective of this study was to evaluate genetic<br />
variability of Lithuanian populations of I. parviflora. <strong>Plant</strong> material was collected from<br />
twenty one site (totally 420 individuals) different in geography, habitats and population<br />
size. Populations of I. parviflora were characterised by the random amplified polymorphic<br />
DNA method (RAPD) using OPD-20, OPQ-11, 269, 340, 250, 222, 474, 516, OPB-7,<br />
OPA-20 primers. Percentage of polymorphic DNA bands for populations of small balsam<br />
was low and ranged between 7 and 45. Mean value for population was 22 %. For the separate<br />
populations expected heterozygosity interval was 0.021–0.140. Shannon’s information<br />
index ranged between 0.033 and 0.215 being the lowest for Jonava-Upninkai and Preila<br />
forest edges and the highest for park near the blacktop street in Kaunas A. Sanciai area.<br />
Populations with more hemerobic habitats especially with bigger extent of soil erosion<br />
and traffic intensity, showed higher genetic variation.<br />
16
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Analyses of genetic variability in wintering Brassica napus<br />
considering to different temperatures<br />
Algimantas Paulauskas 1 , Milda Jodinskienė 1, 2 , Loreta Griciuvienė 1 ,<br />
Judita Žukauskienė 1 , Irena Brazauskienė 3 , Eglė Petraitienė 3<br />
1<br />
Department of Biology, Faculty of Nature Sciences, Vytautas Magnus<br />
University, Vileikos str. 8, LT-44404, Kaunas, Lithuania;<br />
2<br />
Laboratory of <strong>Plant</strong> physiology, Institute of Botany of Nature Research<br />
Centre, Akademijos str. 2, LT-08412, Vilnius, Lithuania;<br />
3<br />
Institute of Agriculture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Instituto av. 1, LT-58344 Akademija, Kėdainiai distr.,<br />
Lithuania, E-mail milda.jodinskiene@botanika.lt<br />
Winter oilseed rape is grown in many countries for its valuable oil. The growing conditions<br />
in Lithuania are different according to the winter temperatures. Low temperature is<br />
very important for plant growth and survival, which is a characteristic for over wintering of<br />
oilseed rape. Hard winter in 2010 was critic for the most of oilseed rape cultivars – many<br />
farmers in Lithuania had loss of the<strong>ir</strong> crops.<br />
Eleven winter oilseed rape cultivars – ‘DK Secure’, ‘Sunday’, ‘Sw Celsius’, ‘Titan H’,<br />
‘Visby’, ‘Hornet H’, ‘Kronos H’, ‘Vision’, ‘Libea’, ‘Silvia’, ‘Valesca’ – were examined.<br />
They were grown in the experimental fields in Lithuanian Institute of Agriculture<br />
(Kėdainiai region). 10 to 14 samples of leaves from individual plants were collected in<br />
spring 2010 within each population. 6 to 10 samples were collected from plants which<br />
survived winter period. 4 to 6 samples were collected from plants which managed to<br />
outgrowth from the<strong>ir</strong> roots.<br />
The aim of the work was to search for the links between good over wintering and<br />
genetic diversity. Random amplified polymorphic DNA (RAPD) markers offer quick<br />
screening of different regions of the genome for genetic polymorphisms. DNA from 10<br />
to 14 individuals per cultivar was analysed using nine different primers; the 73 detected<br />
bands were polymorphic. Genetic diversity in Brassica napus cultivars was estimated<br />
using AMOVA program. The results demonstrated that genetic polymorphism among 11<br />
tested winter oilseed rape cultivars was high. The dendrogram obtained by using genetic<br />
distances clusters showed the clear separation of all 11 populations to different clusters.<br />
As a result, ‘DK Secure’ was separated from other cultivars. Evaluation of the results of<br />
11 different oilseed rape cultivars over wintering showed that the last mentioned cultivar<br />
had some different growth peculiarities comparing to others and also had the most quantity<br />
of the survived individuals in the spring.<br />
17
Quality, plant resistance and productivity of organically grown<br />
carrots hybrids and cultivars<br />
Rasa Karklelienė, Audrius Radzevičius, Edita Dambrauskienė,<br />
Elena Survilienė, Česlovas Bobinas, Laisvūnė Duchovskienė,<br />
Danguolė Kavaliauskaitė<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture and<br />
Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,<br />
E-mail r.karkleliene@lsdi.lt<br />
Investigation was carried out at the Institute of Horticulture in 2008–2009. Carrot<br />
(Daucus sativus RöhL.) cultivars ‘Svalia’ F 1<br />
, ‘Skalsa’ F 1<br />
, ‘Bolero’ F 1<br />
, ‘Noveno’ F 1<br />
,<br />
‘Garduolės’, ‘Vaiguva’, ‘Vytėnų nanto’, ‘Šatrija’, ‘Monanta’, ‘Tito’, ‘Samson’, ‘Magi’<br />
and ‘Crona’ were investigated. Growing vegetables organically there were used natural<br />
fertilizers. Perennial investigation shows that carrot root-crop quality depends not only<br />
on soil type and growing conditions but also on genotype. ‘Bolero’ F 1<br />
and ‘Noveno’ F 1<br />
(74.7 t ha -1 and 61.7 t ha -1 ) had a highest yield. ‘Noveno’ F 1<br />
has a rich content of carotene<br />
(18.7 mg per 100 g -1 ). ‘Skalsa’, ‘Tito’ and ‘Monanta’ was most damaged by carrot foliage.<br />
It was found that cultivars ‘Skalsa’ F 1<br />
, ‘Tito’, ‘Garduolės’, ‘Vytėnų Nanto’, ‘Bolero’ and<br />
‘Magi’ were damaged by mycoplasma disease. Radicina Alternaria, Phoma sp., Botrytis<br />
cinerea, Sclerotinia sclerotiorum and bacteria in stored carrots caused rot.<br />
18
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Low temperature storage of Rosaceae (Fragaria ananassa and<br />
Pyrus communis) plants in vitro<br />
Vanda Lukoševičiūtė 1 , Rytis Rugienius 1 , Gražina Stanienė 1 ,<br />
Asta Blažytė 1, 2 , Dalia Gelvonauskienė 1 , Bronislovas Gelvonauskis 2 ,<br />
Audrius Sasnauskas 1 , Vidmantas Stanys 1<br />
1<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;<br />
2<br />
<strong>Plant</strong> Gene Bank, Stoties 2, LT-58343 Akademija, Kedainiai distr.,<br />
Lithuania<br />
The biodiversity is important not only for preservation of natural populations but<br />
also for development of new cultivars of cultivated plants. Genetic resources of fruit and<br />
berries are preserved mainly as field gene banks. This system requ<strong>ir</strong>es a large land area,<br />
labor and capital investment. Many of accessions in the collections are unique. Climatic<br />
conditions, plant diseases and pests constitute a serious hazard to genetic resources stored<br />
in field collections. In vitro culture techniques including storage under growth limiting<br />
conditions and cryopreservation provide alternative storage forms for protecting important<br />
germplasm. During the past few decades, different in vitro conservation methods have<br />
been developed, however methods that are suitable for storage of particular cultivars or<br />
genotype groups in the large collections often are not suitable or less suitable to other<br />
genotype. Strawberry and pear, representing woody and grass species of Rosaceae family<br />
are important cultivated crops and could be used as model species for development and<br />
adjusting methodology of in vitro storage.<br />
The present study aims to develop a procedure for in vitro low temperature storage of<br />
strawberry and pear by investigate the effect of different culture media and incubation at<br />
low temperature on state of plants of various genotypes. Twenty strawberry and nine pear<br />
accessions were used for the study in the Institute of Horticulture, Lithuanian Research<br />
Centre for Agriculture and Forestry in 2009–2011.<br />
After medium term storage (15 months for strawberry and 6 months for pear) at 4 °C<br />
temperature microshoots survival exceeded 80 %. However, state of the plants varied depending<br />
on genotype. Condition of strawberry microshoots decreased considerably after<br />
12 months of the storage. At the end of the storage (after 15 months) 40 % of strawberry<br />
plants in vitro were in good condition (average score exceeded 2), 60 % of plants in poor<br />
and bad condition (average score below 2). It was established that state of pear microshoots<br />
remained the best (score 4–5) after 6 month storage in 4 °C temperature on MS medium<br />
with 0.75 mg/l BAP and 3 % sucrose.<br />
19
Effect of genotype and medium composition on linseed<br />
(Linum usitatissimum L.) ovary culture<br />
Ramunė Masienė, Natalija Burbulis, Aušra Blinstrubienė<br />
Aleksandras Stulginskis University, Department of Crop Science and<br />
Animal Husbandry, Agrobiotechnology laboratory, Studentų 9, LT-53361<br />
Akademija, Kaunas district, Lithuania, E-mail ramunee7@hotmail.com<br />
Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders<br />
to develop new cultivars in a shorter time period. Many research groups successfully<br />
created new linseed genotypes through anther culture; however ovary culture has been<br />
the subject of only a few earlier studies. In the present study, the effect of genotype and<br />
growth regulators combination on callus induction and shoots regeneration in ovary culture<br />
of nine commercially important linseed cultivars was investigated. Ovaries were cultured<br />
on modified MS medium supplemented with three different combinations of plant growth<br />
regulators. Variable callogenic responses were expressed by all of the genotypes tested<br />
on different induction media. The results suggested that specific combination of growth<br />
regulators for callus induction must be designed for each genotype. Shoot regeneration<br />
from ovary derived callus is a critical phase of the whole gynogenetic process. Differences<br />
in adventitious shoot formation frequency among genotypes were demonstrated and four<br />
responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’<br />
manifested the highest adventitious shoot formation frequency with a high number of<br />
shoots per explant. The optimum ratio of growth regulators for shoot regeneration was<br />
shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaph<strong>ir</strong>’ showed the<br />
highest shoot regeneration frequency when callus had originated from induction medium<br />
supplemented with 2 mg l -1 BAP and 2 mg l -1 NAA while combination of 1 mg l -1 BAP and<br />
2 mg l -1 IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest<br />
rate of shoots per explant has been obtained in second subculture.<br />
20
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
A genetic linkage map for Cecidophyopsis ribis resistance in<br />
Ribes nigrum based on microsatellite and AFLP markers<br />
Ingrida Mažeikienė, Vidmantas Bendokas, Vidmantas Stanys,<br />
Tadeušas Šikšnianas<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture and<br />
Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,<br />
E-mail mazeikiene@hotmail.com<br />
Genetic linkage maps provide essential information for molecular breeding. It is<br />
known, that the P gene provides resistance of blackcurrant to Cecidophyopsis ribis that is<br />
a vector of blackcurrant reversion v<strong>ir</strong>us (BRV). C. ribis is one of the most damaging pests<br />
of blackcurrants. Therefore, it is important to select resistant blackcurrants germplasm.<br />
In our study, a linkage map around the resistance locus controlled by predicted P gene<br />
was constructed. The genetic paternal linkage map of R. nigrum was constructed using a F 1<br />
progeny of 158 individuals from three families. In blackcurrant crossings, three R. nigrum<br />
genotypes with a different sensitivity to C. ribis were used as the maternal plants, and<br />
resistant to gall mite cultivar ‘Dainiai’ was used as the paternal. Forty three (43) AFLP<br />
and twenty two (22) microsatellite markers obtained from analysis of the crosses with<br />
‘Dainiai’ were mapped onto 5 linkage groups, 2 triples and 1 pa<strong>ir</strong> at the linkage criteria<br />
LOD 3.0.Sixteen markers could not be linked to any linkage group at a LOD threshold<br />
3.0.The obtained consensus map covers 715.067 cM, with an average marker spacing of<br />
14.593 cM. AFLP fragment CTA-ACC-107 was closely linked with resistance to C. ribis<br />
and was detected in the sixth linkage group. This marker was 37.742 cM and 17.469 cM<br />
from morphologic trait of resistance to gall mite identified in blackcurrant evaluation trials<br />
in 2010 and 2011, respectively.<br />
The presented map provides crucial information for future genetic studies of Ribes<br />
nigrum in particular for quantitative trait loci (QTL) mapping of economically important<br />
traits. Further testing of gall mite resistant R. nigrum genotypes would establish association<br />
of the 107 bp AFLP fragment as a part of the P resistance in R. nigrum.<br />
21
Dynamics of ascospore dispersal and pathogenicity of L. maculans<br />
and L. biglobosa on winter oilseed rape<br />
Agnė Piliponytė, Irena Brazauskienė, Eglė Petraitienė,<br />
Gintaras Brazauskas<br />
Lithuanian Research Centre for Agriculture and Forestry, Institute of<br />
Agriculture, Akademija, LT-58344, Kėdainiai distr., Lithuania,<br />
E-mail agne.piliponyte@lzi.lt<br />
Phoma stem canker, caused by L. maculans and L. biglobosa is the most important<br />
disease of oilseed rape in Europe. These both species co-exist in Lithuania. Information<br />
on timing of ascospore release is an essential tool in the protection of oilseed rape against<br />
Phoma stem canker. Spore release was monitored using a Burkard 7-day volumetric spore<br />
trap from September to November in 2010.The number of spores was counted under optical<br />
microscope. The results show that f<strong>ir</strong>st peak of ascospore was observed on 2 September<br />
(105 ascospores per day) and the second peak was detected on 9 September (191 ascospores<br />
per day). Later on ascospore dispersal decreased and low ascospore numbers were<br />
observed during October. The abudance of ascospores in the a<strong>ir</strong> was dependant on the<br />
weather factors, especially amount and frequency of rainfall. Real time–PCR will be used<br />
to investigate species-specific ascospore spread dynamics.<br />
It is known that two fungi cause differential host defence response to infection. In<br />
controlled env<strong>ir</strong>onment experiments winter oilseed rape plants were inoculated using<br />
suspensions of L. maculans and L. biglobosa conidium. The results show that incubation<br />
period was longer on the plants inoculated with L. maculans (11–12 days) than<br />
using L. biglobosa (6–7 days). cDNA - AFLP technique will be employed to identify<br />
pathogenesis related differentially expressed genes. We expect that our results will lead<br />
to better understanding of these two phytopathogenic fungi and will give useful insights<br />
into disease control improvement.<br />
22
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Genetic polymorphism of wild pear population in Lithuania<br />
Rytis Rugienius 1 , Asta Blažytė 1, 2 , Vanda Lukoševičiūtė 1 ,<br />
Juratė Bronė Šikšnianienė 1 , Dalia Gelvonauskienė 1 ,<br />
Bronislovas Gelvonauskis 2 , Audrius Sasnauskas 1 , Danas Baniulis 1 ,<br />
Vidmantas Stanys 1<br />
1<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;<br />
2<br />
<strong>Plant</strong> Gene Bank, Stoties 2, LT-58343 Akademija, Kedainiai distr.,<br />
Lithuania, E-mail r.rugienius@lsdi.lt<br />
Wild pear (Pyrus pyraster (L.) Burgsd.) is pear species growing naturally in Lithuania<br />
and is related to cultivated pear (Pyrus communis L.). In some cases, plants identified as<br />
P. pyraster represent various stages of hybridization between original P. pyraster and<br />
P. communis. Therefore, a boundary between cultivated pear, naturalized plants and wild<br />
pear is ambiguous. There is limited information on genetic variation and structure of<br />
P. pyraster population. Pear genetic resources have not been extensively characterized<br />
due to low morphological diversity, lack of differentiating characters among species and<br />
widespread crossability. Traditional methods of characterization, based on agronomic and<br />
morphological parameters, are being affected by env<strong>ir</strong>onmental conditions and phenological<br />
stages of the plants. Therefore, an employment of molecular techniques would provide<br />
deeper understanding of genetic background of the wild populations and hybrids of the crop.<br />
The aim of our study was to characterize microsatellite loci of pear originally collected<br />
from different localities of Lithuania as naturally growing specimens, presently<br />
grown in germplasm collection at the Institute of Horticulture Lithuanian Research Centre<br />
for Agriculture and Forestry and evaluate possible link between microsatelite alleles and<br />
morphological parameters of the accessions. Microsatelite analysis was performed on<br />
84 pear clones, grouped to three groups: wild, cultivated or hybrid genotypes, according<br />
prevailing morphological traits. Characterization of 9 polymorphic microsatellite loci of<br />
pear accessions identified 154 polymorphic alleles. The allele number per locus ranged<br />
from 12 to 20 alleles. The most polymorphic microsatellite loci (EMPc106, EMPc117,<br />
NB109a and CH02c11) as well as unique alleles, characteristic to each from of the three<br />
genotype groups were identified. High genetic polymorphism was demonstrated by genetic<br />
relationship and heterozygosity analysis among accessions. Lower Ho than He values<br />
was identified proclaiming comparatively often occasions of self fertilisation in naturally<br />
growing pears. It was shown that variability of morphological traits of Pyrus accessions<br />
weakly reflects genetic variation. To some extend, only presence of initial ha<strong>ir</strong>iness and<br />
number of seeds in the fruit could be used as morphological markers of the genetic proximity<br />
of Pyrus accessions.<br />
23
Identification of nepov<strong>ir</strong>uses in tomatoes<br />
Donatas Šneideris, Irena Zitikaitė, Banga Grigaliūnaitė,<br />
Marija Žižytė, Juozas Staniulis<br />
Nature Research Centre, Institute of Botany, Žaliųjų Ežerų Str. 49,<br />
LT-08406, Vilnius, E-mail Donatas.sneideris@gmail.com<br />
The nepov<strong>ir</strong>uses (nematode-transmitted, polyhedral v<strong>ir</strong>uses) are a group of about<br />
46 v<strong>ir</strong>uses that infect many plant families. They cause diseases of economical importance in<br />
a wide range of cultivated annual, perennial and woody plants, and are of serious concern<br />
to quarantine authorities worldwide. The<strong>ir</strong> wide host range combined with ability to be<br />
transmitted by nematodes, seed or pollen makes them particularly dangerous.<br />
Few nepov<strong>ir</strong>uses (Arabis mosaic nepov<strong>ir</strong>us, Tobacco ringspot nepov<strong>ir</strong>us, Tomato<br />
ringspot nepov<strong>ir</strong>us) have already been detected and identified in ornamental plants in<br />
Lithuania. Now we identified two nepov<strong>ir</strong>uses – Tobacco ringspot nepov<strong>ir</strong>us (TRSV) and<br />
Tomato black ring nepov<strong>ir</strong>us (TBRV) in tomatoes. Infected tomato plants were assayed by<br />
DAS-ELISA tests. Extracts of plants that showed positive reaction in DAS-ELISA were<br />
inoculated to more than twenty different indicator plants. Local lesions and systemic infection<br />
were observed in plants belonging to Nicotiana, Chenopodium, Datura, Gomphrena<br />
and Tetragonia genera. Transmission electron microscopy (EM) of extracts from leaves of<br />
infected test plants revealed presence of icosahedral v<strong>ir</strong>us particles of ~ 30 nm in diameter<br />
that are characteristic of nepov<strong>ir</strong>uses. Presence of TRSV and TBRV in infected test plants<br />
was also detected by RT-PCR using v<strong>ir</strong>us specific primers. Thus, detection and identification<br />
of TRSV and TBRV in naturally infected tomatoes and test-plants was conf<strong>ir</strong>med by<br />
DAS-ELISA, EM and RT-PCR.<br />
24
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Characterization of microsatellite marker loci of traditional and<br />
indigenous apple tree cultivars in Lithuania.<br />
Sidona Sikorskaitė, Dalia Gelvonauskienė, Vidmantas Stanys,<br />
Danas Baniulis<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture and<br />
Forestry, Babtai LT-54333, Kaunas reg., Lithuania,<br />
E-mail d.baniulis@lsdi.lt<br />
Apple fruits constitute an important part of horticultural production in Lithuania<br />
and worldwide. Tools for assessment of genetic polymorphism and genotyping are requ<strong>ir</strong>ed<br />
for breeding and research on apple genetic resources. The aim of present study<br />
was to characterize microsatellite loci of indigenous and traditional Lithuanian apple tree<br />
(Malus × domestica) cultivars and to select primer pa<strong>ir</strong>s suitable for genotyping of the<br />
cultivars. Th<strong>ir</strong>ty-seven traditional cultivars and indigenous cultivars developed during<br />
the last century, and eleven standard cultivars available at the collection of apple genetic<br />
resources at the Institute of Horticulture, LRCAF were assessed. Apple genotyping was<br />
performed using eleven PCR primers specific for polymorphic microsatellite loci of<br />
Malus × domestica and recommended by ECPGR Malus workgroup. All microsatellite<br />
loci exhibited a high level of polymorphism – 8 to 14 alleles with mean value of 10.45<br />
for traditional and indigenous cultivars and 5 to 12 alleles 7.55 in average for reference<br />
cultivars. The observed heterozygosity varied from 0.70 to 0.97 with an average of 0.84<br />
for traditional and indigenous cultivars and from 0.64 to 1.00 with mean value of 0.90 for<br />
standard cultivars. Locus CH01g12 was the most polymorphic micorsatellite locus in the<br />
group of 37 traditional and indigenous cultivars, while CH02b10 locus exhibited the highest<br />
polymorphism in the set of standard cultivars. The polymorphism of the microsatellite<br />
markers allowed to specifically identify 35 of the traditional and indigenous cultivars.<br />
25
Isolation of apple tree (Malus × domestica Borkh.) cell nuclei for<br />
proteome analysis<br />
Sidona Sikorskaitė 1 , Sandra Zavadskytė 2 , Vidmantas Stanys 1 ,<br />
Danas Baniulis 1<br />
1<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture<br />
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;<br />
2<br />
Department of Biochemistry and Biotechnologies, Vytautas Magnus<br />
University, Vileikos st. 8-201, Kaunas LT-44404, Lithuania,<br />
E-mail d.baniulis@lsdi.lt.<br />
Apple tree is economically important plant of the Rosaceae family. Additionally,<br />
relatively small genome size, availablility of apple tree genome sequence and molecular<br />
marker tools, makes apple tree attractive as a model species for genomic studies on woody<br />
plants of the Rosaceae family. Proteome analysis has been a valuable tool in studies on<br />
complex molecular mechanisms involved in plant development, productivity and response<br />
to env<strong>ir</strong>onmental stimuli. However, proteome of ent<strong>ir</strong>e plant cell comprises a largely<br />
complex picture presents high demands on dynamic range and sensitivity of protein and<br />
analysis procedures. Therefore, subcellular fractions are often used in functional genomics<br />
studies to reduce sample complexity. Signaling pathways set off to changes of gene expression<br />
lead to cell nucleus that encloses a number of important signaling messengers. Due<br />
to complexity of procedures requ<strong>ir</strong>ed for nuclear protein preparation, studies addressing<br />
nuclear proteome in plants have been scarce to date, and information on nuclear proteome<br />
of plants of the Rosaceae family remains vague. Therefore the aim of this study was to<br />
develop procedure nuclear protein preparation from fresh leave tissue of apple tree suitable<br />
for proteomics analysis. To achieve the objective, issues of efficiency of breakage of<br />
the woody plant cells, nuclei stabilization and isolation of pure factions of the organelle,<br />
stability and solubilization of the isolated protein were addressed. Several previously<br />
published procedures on plant nuclei preparation for genetic and proteome analysis were<br />
evaluated and conditions were optimized. Disruption of apple tree leave tissue by grinding<br />
in liquid nitrogen ensured efficient cell breakage and filtration followed by differential<br />
centrifugation led to separation of organellar fraction. Further, a concentration of detergent<br />
was optimized for differential lysis of organelles, and it was found that treatment with 1 %<br />
Triton X-100 was optimal for isolation of stable nuclei. The nuclei were fractionated by<br />
equilibrium centrifugation on combined sucrose and percoll gradient. Purity of protein<br />
of the isolated nuclei was established using immunoblot analysis and organellar markers.<br />
The developed procedure yielded microgram quantities of protein suitable for proteome<br />
analysis from several grams of leave tissue.<br />
26
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Detection and molecular identification of alien v<strong>ir</strong>uses affecting stone<br />
fruit and vegetable crops in Lithuania<br />
Juozas Staniulis 1 , Irena Zitikaitė 1 , Marija Žižytė 1 ,<br />
Elena Jackevičienė 2 , Laima Urbanavičienė 1 , Donatas Šneideris 1<br />
1<br />
Institute of Botany, Nature Research Centre, Zaliuju Ezeru 49,<br />
LT-08406, Vilnius;<br />
2<br />
The State <strong>Plant</strong> Service under the Ministry of Agriculture of the<br />
Republic of Lithuania, Ozo g. 4A, LT-08200, Vilnius<br />
Alien v<strong>ir</strong>us species infecting stone fruit and vegetable crops in Lithuania are considered:<br />
Plum pox v<strong>ir</strong>us (PPV) affecting plums (Prunus domestica L.), Beet necrotic yellow<br />
vein v<strong>ir</strong>us (BNYVV) affecting sugar beets (Beta vulgaris var. saccharifera L.) and Pepino<br />
mosaic v<strong>ir</strong>us (PepMV) affecting tomatoes (Lycopersicon esculentum MilL.). For the f<strong>ir</strong>st<br />
time in Lithuania PPV was detected 15 years ago, BNYVV – 6 years and PepMV just<br />
few years ago. The v<strong>ir</strong>uses were isolated from natural infection sources and mechanically<br />
transmitted to susceptible test-plants. Identity of PPV in symptomatic plum tree leaves<br />
and test-plants (Chenopodium foetidum, Pisum sativum), BNYVV in sugar beet roots and<br />
test-plants (Chenopodium quinoa, C. amaranticolor, Tetragonia expansa) and PepMV in<br />
tomato fruits and Nicotiana test-plant species was conf<strong>ir</strong>med by immuno enzyme DAS-<br />
ELISA test, electron microscopy, and molecular RT-PCR procedure. RT-PCR, sequencing<br />
and RFLP analysis have conf<strong>ir</strong>med that all PPV isolates appeared to be attributed to PPV-D<br />
strain group. PPV appeared to have limited distribution in few administrative regions of<br />
central and northern part of Lithuania. BNYVV was detected in several locations of four<br />
administrative regions. Multiplex reverse transcription PCR analysis of the coat protein<br />
(CP) gene sequences (RNA-2) showed that in Lithuania occur A and B types of BNYVV.<br />
PepMV at f<strong>ir</strong>st was detected and identified in imported commercial tomato fruits. Later<br />
on this v<strong>ir</strong>us was detected in tomato fruits grown in commercial tomato greenhouses in<br />
Lithuania. Isolates from fruits of tomato were identified as PepMV on the bases of symptom<br />
expression on the fruits and test-plants, morphology of v<strong>ir</strong>ions, results of DAS-ELISA<br />
tests and polymerase chain reaction.<br />
27
Analysis of genetic structure of Botrytis spp. isolates from<br />
horticultural plant hosts<br />
Alma Valiuškaitė, Elena Survilienė, Danas Baniulis,<br />
Laisvūnė Duchovskienė, Neringa Rasiukevičiūtė<br />
Institute of Horticulture, Lithuanian Research Centre for Agriculture and<br />
Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,<br />
E-mail a.valiuskaite@lsdi.lt<br />
Analysis of the genetic diversity of plant pathogen population is the most important<br />
area. The polymorphic diversity could provide significant information relating to the<br />
pathosystem. Genetic identification is increasingly used in plant pathology for the indentification<br />
of individual at specific and intra-specific levels of plant pathogens. Botrytis spp.<br />
wide spread fungi causing diseases in many horticultural plants. This study performed the<br />
analysis the variation of genetic structure of Botrytis spp. isolates originating from different<br />
horticultural plant hosts from six locations of Lithuania. Isolates collected from fruits of<br />
Malus × domestica, Fragaria × ananassa, and vegetables Allium cepa, Brassicae oleracea<br />
var. capitata, Lycopersicon esculentum were analysed using molecular markers. Th<strong>ir</strong>ty<br />
isolates with distinct morphological-cultural properties specific to B. cinerea, B. squamosa<br />
and B. aclada were prepared. The isolates differed in the<strong>ir</strong> optimum growth, conidial formation<br />
and size of conidia on Potato dextrose agar. Assessment of genetic polymorphism<br />
using AFLP method revealed high genetic diversity among the selected Botrytis spp. isolates.<br />
An assessment of genetic polimorphism of the th<strong>ir</strong>ty selected Botrytis spp. isolates<br />
using AFLP method with three pa<strong>ir</strong>s of primers with one selective nucleotide resulted in<br />
identification of 1968 polymorphic alleles. Dendrograms, analysis of genetic structure<br />
of Botrytis spp. revealed the existence of genetic differentiation in specimens between<br />
isolates from the different host plants. There were found seventeen groups which genetic<br />
distance between isolates was less than 0.5. Analysis of the genetic diversity conf<strong>ir</strong>med<br />
the Botrytis spp. polyphagia characteristic feature: the closely related isolates were distributed<br />
in different horticulture plant hosts. Genetic distance between isolates from different<br />
geographic regions was 0.9 it showed that Botrytis spp. isolates are characterized by high<br />
polymorphism. Great variation of genetic isolates is not a consequence of geographic<br />
isolation, because closely related isolates were found in different areas.<br />
28
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
RAPD analysis of genetic differences among Lithuanian<br />
populations of Impatiens glandulifera<br />
Lina Zybartaitė 1 , Milda Jodinskienė 1 , Judita Žukauskienė 1 ,<br />
Steven B. Janssens 2 , Algimantas Paulauskas 1 ,<br />
Eugenija Kupčinskienė 1<br />
1<br />
Vytautas Magnus University, Department of Biology, Vileikos 8,<br />
LT-44404 Kaunas, Lithuania;<br />
2<br />
Laboratory of <strong>Plant</strong> Systematics, Institute of Botany and Microbiology,<br />
K. U. Leuven, Kasteelpark Arenberg 31, P. O. Box 2437, BE-3001<br />
Leuven, Belgium, E-mail e.kupcinskiene@gmail.com<br />
Nowadays many reports are providing evidence about rapid spread and adaptation<br />
of invasive species. Such changes may result in genetic differentiation between local<br />
populations. F<strong>ir</strong>st introduced to Europe (England) from Nepal in 1838/1939, Impatiens<br />
glandulifera quickly became a garden favorite. In Nepal I. glandulifera seeds are harvested<br />
as a food source, the<strong>ir</strong> oil is used for cooking. Albeit a very attractive and usefull one<br />
the Himalayan balsam or policeman‘s helmet, I. glandulifera, had become a prodigious<br />
weed. Impatiens glandulifera is highly invasive in almost whole Europe and appears in<br />
various habitats. In Lithuania it is distributed through all the country. No accounts exists<br />
for evidence of local differentiation concerning I. glandulifera in the Baltic region. The<br />
objective of this study was to evaluate genetic variability of Lithuanian populations of<br />
I. glandulifera. Twenty populations (totally 400 individuals) were collected from various<br />
districts of the country. I. glandulifera was characterised by the randomly amplified<br />
polymorphic DNA method (RAPD) using eight 10-mer oligonucleotids. Selected RAPD<br />
primers generated between 18 and 30 bands each. Totally 188 bands were recorded<br />
and all of them were polymorphic. Percentage of polymorphic DNA bands for separate<br />
populations of I. glandulifera was not very high and ranged between 40 and 56. For the<br />
separate populations number of effective alleles ranged between 1.188 and 1.271, expected<br />
heterozygosity interval was 0.115–0.165. Shannon’s information index ranged between<br />
0.183 and 0.255 being the lowest for Kruonis (semi-natural site near the groove) and the<br />
highest for hemerobic Vilnius-Snipiskes site. Cluster analyses indicated that all populations<br />
might be split into 4 groups among which there is no clear geographical pattern. Our<br />
genetical analyses indicate multiple introduction of this invasive alien species in Lithuania.<br />
Presumably several different ways of invasion of I. glandulifera took place: natural run<br />
and predisposing it intentional and unintentional dispersal by human.<br />
29
Molecular analysis of wild cranberry (Vaccinium oxycoccus L.)<br />
genetic diversity using RAPD and SSR<br />
Judita Žukauskienė 1 , Algimantas Paulauskas 1 , Laima Česonienė 2 ,<br />
Remigijus Daubaras 2<br />
1<br />
Faculty of Natural Sciences of Vytautas Magnus University, Vileikos 8,<br />
Kaunas, LT-44404, Lithuania;<br />
2<br />
Kaunas Botanical Garden of Vytautas Magnus University,<br />
Ž. E. Žilbero 6, Kaunas., LT-46324, Lithuania,<br />
E-mail j.zukauskiene@gmf.vdu.lt<br />
Situation of Lithuanian peat bogs, force to give a thought about survival risk of wild<br />
cranberry Vaccinium oxycoccus population’s in Lithuania. Genetic variation represents<br />
the starting point for further evolution and is an important prerequisite for the prediction<br />
of evolutionary responses. This is of practical significance in the light of human-caused<br />
habitat fragmentation, alteration, or destruction. Genetic structure among and within<br />
populations also depends on the life history of a species.<br />
The present work was conducted to evaluate RAPD (random amplified polymorphic<br />
DNA) and SSR (Simple Sequence Repeat) marker systems for the<strong>ir</strong> ability to detect genetic<br />
diversity within and among V. oxycoccus populations and to compare the efficiency<br />
of these two marker types.<br />
Using nine RAPD primers we investigated genetic variation among and within 151<br />
individuals (7 populations of different size Lithuanian and 3 populations of other countries)<br />
of V. oxycoccus. With these profiles we evaluated 254 fragments. According to RAPD<br />
results were selected different individuals 56 of V. oxycoccus and tested them with 10 SSR<br />
primer pa<strong>ir</strong>s. These SSR primers generated 62 fragments.<br />
According to RAPD data genetic variation among populations varied from 7 to 25 %<br />
(RAPD) and from 16 to 14 % (SSR) molecular variance (AMOVA)).<br />
The higher level of polymorphism detected by RAPD markers has contributed to the<br />
lower genetic similarity estimates based on RAPD markers as compared to SSR markers.<br />
The dendrograms generated with hierarchicalUPGMA (Unweighted Pa<strong>ir</strong> Group<br />
Method with Arithmetic mean) cluster analysis revealed mixed and homogenous V. oxycoccus<br />
lineages. These lineages conf<strong>ir</strong>m the prediction that Lithuanian populations were<br />
derived from one population before glaciations.<br />
30
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
The technique of “artificial seeds” creation for propagation and<br />
storage strawberry plant material<br />
Ivan Kulikov, Valeriy Vysotsky, Lilia Alekseenko<br />
All-Russian Horticultural Institute for Breeding, Agrotechnology and<br />
Nursery of Russian Academy of Agrarian Science, Moscow, 115598,<br />
Zagorievskaya str. , 4, E-mail vstisp@vstisp.org<br />
The investigation for revealing the possibility of obtaining “artificial seeds” of<br />
strawberry (Fragaria × ananassa Duch.) was carried out. In experiments were involved<br />
varieties ‘Roxana’, ‘Pokahontas’, ‘Divnaya’. Microshoots and buds have been encapsulated<br />
into alginate coat.<br />
The “artificial seeds” of strawberry which were able to germination on nutrient media,<br />
had the ability to store at low temperature during one month, normally grew and developed<br />
in the soil. The influence of sucrose concentration on germination of encapsulated shoot<br />
apices of strawberry has been studied. The percentage of “artificial seeds” germination<br />
for ‘Roxana’ variety contained in the coat 3 % of sucrose was 65, and increased up to 69<br />
when the sucrose content was 6 %.<br />
The ability to germinate depended on nutrient media composition for explants<br />
preparing before encapsulation and variety’s peculiarities. Encapsulated shoot apices of<br />
strawberry variety ‘Pokahontas’ cultivated on nutrient media with sucrose concentration<br />
6 % and relatively low cytokinin content stored at the temperature +2…+4 °С during<br />
30 days germinated in the soil on 56.8 %. The percentage of germinated “artificial seeds”<br />
of the same variety from the nutrient media with paclobutrazol was only 43.2. Explants<br />
of variety ‘Divnaya’ from the similar nutrient media gave 76.8 of germinated “artificial<br />
seeds” in the soil.<br />
The prolongation of the storage term of capsules could be ensured by means of the<br />
substances inhibiting explants growth (abscisic acid, paclobutrazol etc.), explants preparing<br />
before encapsulation on the nutrient media with sucrose concentration 6 % and low<br />
6-benzylaminopurine content (0.2 mg/l). Promising technique could be double encapsulation<br />
for increasing of alginate coat durability and reducing of water lost.<br />
31
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
PARTICIPANTS<br />
Name Institution Country E-mail<br />
Lilia<br />
All-Russian Horticultural Russia allex7897@mail.ru<br />
Alekseenko Institute for Breeding,<br />
Agrotechnology and<br />
Nursery<br />
Rita<br />
Institute of Agriculture, Lithuania rita.armoniene@lzi.lt<br />
Armonienė LRCAF<br />
Vaida<br />
Bakšenskaitė<br />
Vytautas Magnus<br />
University<br />
Lithuania vaida.baksenskaite@fc.vdu.lt<br />
Gabrielė<br />
Balčiūnaitė<br />
Danas<br />
Baniulis<br />
Vidmantas<br />
Bendokas<br />
Erna<br />
Biveinytė<br />
Česlovas<br />
Bobinas<br />
Gintaras<br />
Brazauskas<br />
Odeta Buzaitė<br />
Zenonas<br />
Dabkevičius<br />
Edita<br />
Dambrauskienė<br />
Darius<br />
Danusevičius<br />
Stasė<br />
Dapkūnienė<br />
Laisvūnė<br />
Duchovskienė<br />
Medical Academy,<br />
Lithuanian University of<br />
Health Sciences<br />
Institute of Horticulture,<br />
LRCAF<br />
Institute of Horticulture,<br />
LRCAF<br />
Institute of <strong>Biotechnology</strong>,<br />
Vilnius University<br />
Institute of Horticulture,<br />
LRCAF<br />
Institute of Agriculture,<br />
LRCAF<br />
Vytautas Magnus<br />
University<br />
Institute of Agriculture,<br />
LRCAF<br />
Institute of Horticulture,<br />
LRCAF<br />
Facultry of Forest and<br />
Ecology, Aleksadras<br />
Stulginskis Univeristy<br />
<strong>Plant</strong> Gene Bank,<br />
Botanical Gardens of<br />
Vilnius University<br />
Institute of Horticulture,<br />
LRCAF<br />
Lithuania gabriele.balc@gmail.com<br />
Lithuania d.baniulis@lsdi.lt<br />
Lithuania v.bendokas@lsdi.lt<br />
Lithuania ernute32@yahoo.com<br />
Lithuania c.bobinas@lsdi.lt<br />
Lithuania gintaras@lzi.lt<br />
Lithuania o.buzaite@gmf.vdu.lt<br />
Lithuania lammc@lammc.lt<br />
Lithuania e.dambrauskiene@lsdi.lt<br />
Lithuania darius.danusevicius@asu.lt<br />
Lithuania stase.dapkuniene@gf.vu.lt<br />
Lithuania laisve.d@lsdi.lt
Name Institution Country E-mail<br />
B<strong>ir</strong>utė Frercks Institute of Horticulture, Lithuania b.frercks@lsdi.lt<br />
LRCAF<br />
Dalia Institute of Horticulture, Lithuania d.gelvonauskiene@lsdi.lt<br />
Gelvonauskienė LRCAF<br />
Bronislovas <strong>Plant</strong> Gene Bank Lithuania b.gelvonauskis@agb.lt<br />
Gelvonauskis<br />
Perttu Haimi University of Helsinki Finland perttu.haimi@gmail.com<br />
Rasa<br />
Vytautas Magnus Lithuania ra.jankauskaite@gmail.com<br />
Jankauskaitė University<br />
Milda Nature research centre, Lithuania milda.jodinskiene@botanika.lt<br />
Jodinskienė Vytautas Magnus<br />
University<br />
Monika Ka<strong>ir</strong>ytė Vytautas Magnus Lithuania mnk.ka<strong>ir</strong>yte@gmail.com<br />
University<br />
Linas Kalėda Institute of<br />
Lithuania linas@nomadsbio.lt<br />
<strong>Biotechnology</strong>, Vilnius<br />
University<br />
Rasa KarklelienėInstitute of<br />
Lithuania r.karkleliene@lsdi.lt<br />
Horticulture, LRCAF<br />
Danguolė<br />
Kavaliauskaitė<br />
Institute of Horticulture, Lithuania d.kavaliauskaite@lsdi.lt<br />
LRCAF<br />
Vilius<br />
Kavaliauskis<br />
Vaiva<br />
Kazanavičiūtė<br />
Ivan<br />
Kulikov<br />
Eugenija<br />
Kupčinskienė<br />
Julius<br />
Liobikas<br />
Vanda<br />
Lukoševičiūtė<br />
Medical Academy,<br />
Lithuanian University of<br />
Health Sciences<br />
UAB Nomads<br />
Lithuania v.kavaliauskis@gmail.com<br />
Lithuania vaiva@ibt.lt<br />
All-Russian Horticultural Russia vstisp@vstisp.org<br />
Institute for Breeding,<br />
Agrotechnology and<br />
Nursery<br />
Vytautas Magnus Lithuania e.kupcinskiene@gmail.com<br />
University<br />
Institute of<br />
Lithuania ljulius@vector.kmu.lt<br />
Neurosciences, Medical<br />
Academy, Lithuanian<br />
University of Health<br />
Sciences<br />
Institute of Horticulture, Lithuania v.lukoseviciute@lsdi.lt<br />
LRCAF
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
Name Institution Country E-mail<br />
Daiva Majienė Institute of<br />
Lithuania daivamaj@vector.kmu.lt<br />
Neurosciences, Medical<br />
Academy, Lithuanian<br />
University of Health<br />
Sciences<br />
Ramunė Department of Crop Lithuania ausra.blinstrubiene@asu.lt<br />
Masienė Science and Animal<br />
Husbandry, Aleksandras<br />
Stulginskis University<br />
Ingrida<br />
Mažeikienė<br />
Institute of Horticulture, Lithuania mazeikiene@hotmail.com<br />
LRCAF<br />
Irutė<br />
Meškienė<br />
H<strong>ir</strong>ofumi<br />
Nakagami<br />
Šarūnas<br />
Paškevičius<br />
Agnė<br />
Piliponytė<br />
Audrius<br />
Radzevičius<br />
Minna<br />
Rajamäki<br />
Neringa<br />
Rasiukevičiūtė<br />
Aušra<br />
Ražanskienė<br />
Max F. Perutz<br />
Laboratories, University<br />
of Vienna<br />
<strong>Plant</strong> Proteomics<br />
Research Unit, RIKEN<br />
<strong>Plant</strong> Science Center<br />
Institute of<br />
<strong>Biotechnology</strong>,<br />
Vilnius University<br />
Institute of Agriculture,<br />
LRCAF<br />
Institute of Horticulture,<br />
LRCAF<br />
Department of<br />
Agricultural Sciences,<br />
University of Helsinki<br />
Institute of Horticulture,<br />
LRCAF<br />
Institute of<br />
<strong>Biotechnology</strong>,<br />
Vilnius University<br />
Austria<br />
Japan<br />
<strong>ir</strong>ute.meskiene@univie.ac.at<br />
h<strong>ir</strong>onakagami@psc.riken.jp<br />
Lithuania paskevicius.sarunas@gmail.com<br />
Lithuania agne.piliponyte@lzi.lt<br />
Lithuania a.radzevicius@lsdi.lt<br />
Finland<br />
minna.rajamaki@helsinki.fi<br />
Lithuania neringa_ra@yahoo.com<br />
Lithuania ausra@nomadsbio.lt<br />
Rytis Rugienius Institute of Horticulture, Lithuania r.rugienius@lsdi.lt<br />
LRCAF<br />
Sidona Institute of Horticulture, Lithuania ssidona@yahoo.com<br />
Sikorskaitė LRCAF<br />
Tadeušas Institute of Horticulture, Lithuania v.bendokas@lsdi.lt<br />
Šikšnianas LRCAF<br />
Jūratė Bronė<br />
Šikšnianienė<br />
Institute of Horticulture, Lithuania j.siksnianiene@lsdi.lt<br />
LRCAF
Name Institution Country E-mail<br />
Donatas Institute of Botany, Lithuania donatas.sneideris@gmail.com<br />
Šneideris Nature Research Centre<br />
Audrius Institute of Horticulture, Lithuania a.sasnauskas@lsdi.lt<br />
Sasnauskas LRCAF<br />
Juozas Institute of Botany, Lithuania juozas.staniulis@botanika.lt<br />
Staniulis Nature Research Centre<br />
Vidmantas Institute of Horticulture, Lithuania v.stanys@lsdi.lt<br />
Stanys LRCAF<br />
Urtė Starkevič Institute of<br />
Lithuania bonckute@gmail.com<br />
<strong>Biotechnology</strong>,<br />
Vilnius University<br />
Inga<br />
Institute of Horticulture, Lithuania i.stepulaitiene@lsdi.lt<br />
Stepulaitienė LRCAF<br />
Elena Survilienė Institute of Horticulture, Lithuania e.surviliene@lsdi.lt<br />
LRCAF<br />
Laima<br />
Urbanavičienė<br />
Institute of Botany of<br />
Nature Research Centre<br />
Lithuania laima.urbanaviciene@botanika.<br />
lt<br />
Alma Institute of Horticulture, Lithuania a.valiuskaite@lsdi.lt<br />
Valiuškaitė LRCAF<br />
Jari P. T .<br />
Valkonen<br />
Department of<br />
Agricultural Sciences,<br />
University of Helsinki<br />
Finland jari.valkonen@helsinki.fi<br />
Valeriy<br />
Vysotskiy<br />
Sandra<br />
Zavadskytė<br />
Marija Žižytė<br />
Judita<br />
Žukauskienė<br />
Lina Zybartaitė<br />
All-Russian Horticultural Russia vstisp@vstisp.org<br />
Institute for Breeding,<br />
Agrotechnology and<br />
Nursery<br />
Vytautas Magnus Lithuania sandra.zavadskyte@fc.vdu.lt<br />
University<br />
Institute of Botany, Lithuania marija.zizyte@botanika.lt<br />
Nature Research Centre<br />
Vytautas Magnus Lithuania j.zukauskiene@gmf.vdu.lt<br />
Univesity<br />
Vytautas Magnus Lithuania lina.zybartaite@gmail.com<br />
University
<strong>Workshop</strong>-Seminar “<strong>Plant</strong> <strong>Biotechnology</strong> Advances in Agriculture”<br />
2011 10 27–28<br />
INDEX<br />
Alekseenko L. /31/<br />
Armonienė R. /10/<br />
Bakšenskaitė V. /11/<br />
Balčiūnaitė G. /11/<br />
Baniulis D. /11/13/23/25/26/28/<br />
Bendokas V. /11/21/<br />
Bernatonienė J. /11/<br />
Bistrovaitė L. /11/<br />
Blažytė A. /19/23/<br />
Blinstrubienė A. /20/<br />
Bobinas Č. /18/<br />
Brazauskas G. /10/22/<br />
Brazauskienė I. /17/22/<br />
Buchowska J. /13/<br />
Burbulis N. /20/<br />
Česonienė L. /30/<br />
Dambrauskienė E. /18/<br />
Danusevičius D. /13/<br />
Dapkūnienė S. /14/<br />
Daubaras R. /30/<br />
Duchovskienė L. /18/28/<br />
Duchovskis P. /14/<br />
Frercks B. /15/<br />
Gelvonauskienė D. /15/19/23/25/<br />
Gelvonauskis B. /19/23/<br />
Griciuvienė L. /17/<br />
Grigaliūnaitė B. /24/<br />
Jackevičienė E. /27/<br />
Jankauskaitė R. /16/<br />
Janssens S. B. /16/29/<br />
Jodinskienė M. /16/17/29/<br />
Karklelienė R. /18/<br />
Kavaliauskaitė D. /18/<br />
Kavaliauskis V. /11/<br />
Kulikov I. /31/<br />
Kupčinskienė E. /16/29/<br />
Kurilčik A. /14/<br />
Kurilčik G. /14/<br />
Liatukas Ž. /10/<br />
Liobikas J. /11/<br />
Lukoševičiūtė V. /19/23/<br />
Majienė D. /11/<br />
Masinė R. /20/<br />
Mažeikienė I. /21/<br />
Meškienė I. /8/<br />
Nakagami H. /7/<br />
Paulauskas A. /16/17/29/30/<br />
Petraitienė E. /17/22/<br />
Piliponytė A. /22/<br />
Radzevičius A. /18/<br />
Rajamäki M. /6/<br />
Rasiukevičiūtė N. /28/<br />
Rugienius R. /19/23/<br />
Sasnauskas A. /19/23/<br />
Savickas A. /11/<br />
Schweighofer A. /8/<br />
Sikorskaitė S. /25/26/<br />
Staniulis J. /24/27/<br />
Stanys V. /11/13/15/19/21/23/25/26/<br />
Stepulaitienė I. /15/<br />
Stanienė G. /19/<br />
Survilienė E. /18/28/<br />
Šikšnianienė J. B. /13/15/23/<br />
Šikšnianas T. /21/<br />
Šneideris D. /24/27/<br />
Urbanavičienė L. /27/<br />
Urbonavičiūtė A. /14/<br />
Valiuškaitė A. /28/<br />
Valkonen Jari P. T. /5/<br />
Vysotsky V. /31/<br />
Zavadskytė S. /26/<br />
Zitikaitė I. /24/27/<br />
Zybartaitė L. /29/<br />
Žilinskaitė S. /14/<br />
Žižytė M. /24/27/<br />
Žukauskas A. /14/<br />
Žukauskienė J. /16/17/29/30/
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