Workshop-Plant Biotechnology - Sodininkystė ir daržininkystė

INSTITUTE OF HORTICULTURE,
LITHUANIAN RESEARCH CENTRE FOR
AGRICULTURE AND FORESTRY
WORKSHOP-SEMINAR
PLANT BIOTECHNOLOGY
ADVANCES IN AGRICULTURE
Programme and Abstracts
Kaunas district, Lithuania,
October 27–28, 2011

UDK 575:581.19:631.523
ORGANIZING COMMITTEE
Prof. habil. dr. Vidmantas Stanys (Institute of Horticulture, LRCAF) – chairman
Prof. dr. Česlovas Bobinas (Institute of Horticulture, LRCAF)
Dr. Audrius Sasnauskas (Association of Agriculture and Forestry Research
Institutes)
Dr. Danas Baniulis (Institute of Horticulture, LRCAF)
Dr. Vidmantas Bendokas (Institute of Horticulture, LRCAF)
Birutė Frercks (Institute of Horticulture, LRCAF)
VENUE
October 27th – hotel “Best Western Santaka”, J. Gruodžio st. 21 Kaunas.
October 28th – the Institute of Horticulture, LRCAF, Kauno st. 30, Babtai,
Kaunas dist.
ACKNOWLEDGMENTS
The workshop-seminar is funded by the European Social Fund and supported in
part by AB Bruker Daltonics Scandinavia and UAB Analytical Solutions.
Address of the volume in internet www.lsdi.lt
© Institute of Horticulture Lithuanian Research Centre for Agriculture and Forestry

PROGRAMME
October 27 (Thursday)
Hotel “Best Western Santaka”, Kaunas
9 30 –10 00 REGISTRATION
10 00 –10 05 OPENING OF THE WORKSHOP – SEMINAR
10 05 –15 00 PLENARY SESSION
10 05 –11 00 BIOTECHNOLOGICAL APPLICATIONS OF THE KNOWLEDGE ON
MOLECULAR HOST-VIRUS INTERACTIONS IN PLANT DISEASE
CONTROL
Prof. Jari Valkonen
Department of Agricultural Sciences, Helsinki University
11 00 –11 15 C o f f e e b r e a k
11 15 –12 10 PLANT VIRUS INTERACTIONS WITH NUCLEUS
Dr. Minna Rajamaki
Department of Agricultural Sciences, Helsinki University
12 10 –13 10 L u n c h
13 10 –14 05 PHOSPHOPROTEOMIC DISSECTION OF PLANT IMMUNE
SIGNALLING
Dr. Hirofumi Nakagami
Plant Science Centre, RIKEN Institute
14 05 –15 00 SIGNAL TRANSDUCTION IN PLANT CELLS
Dr. Irutė Meškienė
Max F. Perutz Laboratories, University of Vienna
15 00 –16 30 POSTER SESSION
October 28 (Friday)
Institute of Horticulture, LRCAF, Babtai, Kaunas district
10 00 –12 00 Discussion “ADVANCES OF BIOTECHNOLOGICAL RESEARCH ON
PLANT ADAPTABILITY AND APPLICATION IN AGRICULTURE”
12 00 –13 00 L u n c h
13 00 Post-conference excursion

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
PLENARY SESSION ABSTRACTS
Biotechnological applications of the knowledge on molecular
host-virus interactions in plant disease control
Jari P. T. Valkonen
Department of Agricultural Sciences, PO Box 27, FI-00014 University of
Helsinki, Finland, E-mail jari.valkonen@helsinki.fi
Viruses are among the most dangerous pathogens of plants. Their transmission to
new crops in seeds, seed tubers, cuttings and other planting materials results in particularly
heavy yield losses. Viruses cannot be controlled by chemical treatments during the
growing season. Transmission of viruses is also difficult to control in the field. Due to
these limitations, use of virus-free planting materials and virus-resistant cultivars remain
as the main approaches to control plant viruses.
Cryotherapy represents a biotechnological application that can be used to obtain
virus-free plants from infected materials. It makes use of the plants’ natural ability to
protect the meristematic tissue against virus infection and utilizes the methodology of
cryopreservation. In cryotherapy, shoot tips are treated briefly in liquid nitrogen using
adjusted cryopreservation protocols and healthy plants are regenerated from the surviving
pathogen-free meristematic tissue. Freedom of meristematic, undifferentiated cells from
viruses is related to the phenomenon described as recovery of plants from virus infection.
Recovery is based on the basal antiviral defence mechanism called RNA silencing. It is
able to recognise double-stranded RNA (dsRNA) specifically and target it to degradation.
The dsRNA-specific cellular RNases (Dicers) cleave dsRNA to small interfering RNAs
(siRNA) that are used to target homologous single-stranded RNA molecules, in this case
viral RNA genomes or viral gene transcripts. The large amounts of virus-derived siRNA
hence produced can be used for diagnostic purposes to detect viruses. For this purpose, the
siRNA pool from the plant is isolated and sequenced, the overlapping siRNA sequences
are assembled to large contigs, and the contigs are compared to the viral sequences available
in databases to identify the viruses in the sample.
However, viruses have evolved various means of evade or suppress antiviral defense
in their host plants. Active suppression of RNA silencing is used by all viruses. One of the
most studied viral RNA silencing suppressors is HCpro of Potyviruses, the largest group of
plant-infecting RNA viruses. HCpro interacts with the translation initiation factors eIF4E
and eIF(iso)4E for as yet unknown reasons. However, these interactions are most likely
important to virus infection because many recessive resistance genes used for breeding
of resistance to Potyviruses in crop plants are mutated forms of the eIF4E and eIF(iso)4E
genes. Further understanding of the protein complexes formed by viral and host proteins
is expected to raise new possibilities to develop virus-resistant plant cultivars.
5

Plant virus interactions with nucleus
Minna Rajamäki
Department of Agricultural Sciences, PO Box 27, FI-00014 University of
Helsinki, Finland, E-mail minna.rajamaki@helsinki.fi
Positive-strand RNA viruses form the largest group of plant-infecting viruses. Unlike
plant DNA viruses that replicate in the host cell nucleus, the positive-strand RNA viruses
replicate in membranous structures in the cytoplasm. However, some of their proteins
are targeted to the nucleus and nucleolus, the reasons of which have remained largely
unknown. Recent studies have indicated that viral protein may localize in the nucleus in
order to recruit nuclear proteins to be used in virus infection, to sequester or redistribute
some nuclear components to disrupt nuclear functions or counteract plant host defence
responses. For example, the ability of Groundnut rosette virus (GRV; genus Umbravirus)
to move over long distances through plant phloem is dependent on the nucleolar targeting
of its ORF3 protein. In the nucleus, the ORF3 protein interacts with the major nucleolar
protein, fibrillarin, recruiting some of it to the cytoplasm, which is required for the formation
of long distance movement competent viral ribonucleoprotein particles. Potyviruses,
which constitute the largest group of plant RNA viruses, also target certain viral proteins
to the nucleus. The majority of the multifunctional nuclear inclusion protein a (NIa) of
Potato virus A (PVA; genus Potyvirus) is found in the nucleus and nucleolus of virusinfected
cells. Two regions at the N-proximal VPg domain of NIa control both nuclear and
nucleolar localization. Mutations that debilitate nuclear and nucleolar localization of NIa
are detrimental for virus amplification suggesting that nuclear localization of NIa is important
for virus infection. NIa also interacts with fibrillarin, but the role of this interaction is
likely different from Umbraviruses as fibrillarin depletion in plants does not compromise
viral long distance movement but rather reduces PVA accumulation. Interestingly, the data
suggest a link between nuclear/nucleolar localization of NIa and interference with host
antiviral defences (RNA silencing). The results thus demonstrate that the nucleus may
have essential role also in plant RNA virus infection and could provide new insights of
strategies how to control plant virus infections in the future.
6

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Phosphoproteomic dissection of plant immune signaling
Hirofumi Nakagami
Plant Proteomics Research Unit, RIKEN Plant Science Center,
1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan,
E-mail hironakagami@psc.riken.jp
Proteomics is one of the best available tools for studying posttranslational modifications
(PTMs). Therefore, it is well suited for dissecting signaling pathways which often
utilize PTMs as a means of transmitting signals. Among the several PTMs described
thus far, phosphorylation is the most extensively studied and has been shown to play an
important role in plant immune signaling.
At first, we have developed a plant phosphoproteomics platform, which enables
monitoring phosphorylation events in plant cells at the cellular level. Besides developing
the phosphoproteomics platform, we have also established a method for the differential
analysis of plant samples which enables phosphoproteome dynamics to be monitored.
In order to identify novel players involved in plant immunity, we examined phosphoproteome
changes in Arabidopsis upon MAMP (microbe-associated molecular pattern)
treatment and identified 569 proteins whose phosphorylation status significantly changes
in response to flg22 and/or chitin treatment. Most of the identified proteins have not been
reported yet to take part in MAMP-triggered immunity, but included well known MAMPsignaling
regulators such as MPK3/4/6, MKK1/2/4/5, MEKK1, MKP1, and RbohD.
To verify involvement of the identified proteins in MAMP-triggered responses,
we have been isolating T-DNA insertion lines for these proteins and characterizing
flg22-induced ROS (reactive oxygen species) production in the isolated mutants. So far we
have identified 28 genes as negative regulators of MAMP-induced ROS production, and
are expecting to isolate more MAMP-signaling regulatory genes with continued screening.
These results suggest that phosphoproteomics-based screening will be powerful and
effective approach to identify novel signaling components in plants.
Furthermore, we are developing a novel method, which utilizes the phosphoproteomics
technique, to identify substrates of protein kinases involved in plant immunity.
7

Signal transduction by MAPK pathways in plant cells
Irute Meškienė, Alois Schweighofer
Institute of Biotechnology, Vilnius, Lithuania; Max F. Perutz
Laboratories, University of Vienna; Medical Universty of Vienna,
Austria, E-mail irute.meskiene@univie.ac.at
Environmental and developmental inputs are perceived at the cell membranes and
communicated to the nucleus to regulate cellular functions. Intracellular signalling in all
eukaryotes is employing a highly conserved mechanism, which is based on reversible
protein phosphorylation. Protein phosphorylation is an essential posttranslational modification
mechanism of signalling proteins.
Phosphorylation transiently activates mitogen-activated protein kinase (MAPK)
signalling pathways, which are regulated by opposing actions of protein kinases and protein
phosphatases. The core module of this signaling system consists of three interlinked
protein kinases: a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK) and a
MAPK, that transmit a signal via sequential phosphorylation events.
MAPKs activation leads to reprogramming of plant cellular activities, including
changes in gene expressions and plant hormone levels to generate appropriate plant
responses. A “Switch-off” mechanism of this signaling is essential to ensure the correct
cellular response to a given stimulus. This mechanism involves protein phosphatases that
inactivate MAPKs by dephosphorylation ensuring the transient mode of signalling. MAPK
inactivation can be achieved by evolutionary distant protein phosphatases, including serine/
threonine protein phosphatases of PP2C-type and tyrosine or dual-specificity (tyrosine
and serine/threonine) phosphatases.
In the group of serine/threonine protein phosphatases plants evolved the large family
of PP2C-type and there a cluster of proteins, which control MAPK activities (Schweighofer
et al., 2004; Umbrasaite et al., 2010). Four PP2Cs AP2C1, AP2C2, AP2C4 and AP2C3
retain the protein structure for MAPK interaction (Schweighofer et al., 2004) and function
as MAPK phosphatases (Schweighofer et al., 2007; Umbrasaite et al., 2010). The woundand
biotic stress-induced AP2C1 is a negative regulator of MAPKs and modulator of plant
innate immunity, affecting plant stress hormone production, such as wound-jasmonate
and ethylene (Schweighofer et al., 2007). The closely related AP2C3 is expressed during
stomata development and regulates MAPKs during signaling in stomata developmental
pathway. Stomata are cells controlling plant gas exchange and transpiration, essential
processes for our ecosystem.
Recent characterisations of Arabidopsis protein phosphatase mutants, identification
of substrate MAPKs and related signalling pathways highlight the important roles
8

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
of MAPK-phosphatases in the pathways regulating stress responses, plant hormone
biosynthesis, protection against herbivores, stomata cell development, and cell cycle. It
suggests that these signalling pathways and the components involved are determining
essential plant functions.
Schweighofer et al. (2004). Plant PP2C phosphatases: emerging functions in stress signaling.
Trends Plant Sci 9, 236–243.
Schweighofer et al. (2007). The PP2C-Type Phosphatase AP2C1, Which Negatively Regulates
MPK4 and MPK6, Modulates Innate Immunity, Jasmonic Acid, and Ethylene Levels in
Arabidopsis. Plant Cell 19, 2 213–2 224.
Umbrasaite et al. (2010). MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal
cells leading to stomata development in Arabidopsis. PLoS ONE 5, e15357.
9

POSTER SESSION ABSTRACTS
Identification and analysis of winter wheat genes induced at low
temperatures
Rita Armonienė, Žilvinas Liatukas, Gintaras Brazauskas
Lithuanian Research Centre for Agriculture and Forestry, Institute of
Agriculture, Akademija, LT-58344, Kėdainiai distr., Lithuania,
E-mail rita.armoniene@lzi.lt
Freezing temperature is one of the most severe abiotic stresses limiting wheat growth,
productivity, and distribution. Freezing tolerance in plants develops through acclimation
to cold by growth at low, above-freezing temperatures. Large number of genes are known
to have differential expression during the period of cold acclimation. Main task of our
study is to find genes expressed at different time points of cold acclimation and to find
out if these genes – candidates are responsible for freezing tolerance formation. The first
assignment of the study was the evaluation of freezing tolerant and susceptible wheat lines
using freezing test in natural and artificial conditions. Thirty winter wheat lines/varieties
were subjected to the freezing test in the plant growth chamber. Leaves were cut off near
the crowns after cold treatment and re-growing plants were counted, as well as chlorophyll
fluorescence parameter Fv/Fm was measured. Three most freezing tolerant wheat lines/
varieties were identified: 68, 66 and 67. The correlation coefficient between the Fv/Fm
parameter with freezing tolerance studies was 0.84, p < 0.001. The same thirty wheat
lines were grown in the field experiment during the winter 2010/2011 and the evaluation
in natural conditions was conducted in April 2011 on a 1–9 score basis. The result of the
evaluation in natural conditions showed that most freezing tolerant wheat lines/varieties
were ‘Zentos’, 29 and ‘Ada’. We will also present future work of our study for identification
and analysis of genes induced at low temperatures by employing cDNA-AFLP
profiling and TILLING methods in winter wheat. We hope that identification and analysis
of new genes will lead to better understanding of the genetic regulation of winter wheat
cold acclimation and freezing tolerance formation.
10

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Bioactive potential of lectin-enriched protein fractions from
Thymus vulgaris L. and Urtica dioica L. grass
V. Bakšenskaitė 1 , L. Bistrovaitė 2 , G. Balčiūnaitė 3 , V. Kavaliauskis 3 ,
V. Bendokas 4 , D. Baniulis 4 , V. Stanys 4 , D. Majienė 2 , J. Liobikas 2 ,
J. Bernatonienė 3 , A. Savickas 3
1
Department of Biochemistry and Biotechnologies, Faculty of Natural
Sciences, Vytautas Magnus University, Vileikos 8, Kaunas, Lithuania;
2
Institute of Neurosciences, Medical Academy, Lithuanian University of
Health Sciences, Eiveniu 4, Kaunas, Lithuania;
3
Department of Drug Technology and Social Pharmacy, Faculty of
Pharmacy, Medical Academy, Lithuanian University of Health Sciences,
Mickeviciaus 9, Kaunas, Lithuania;
4
Institute of Horticulture, Lithuanian Research Centre for Agriculture
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,
E-mail vaida.baksenskaite@fc.vdu.lt
Medical herbs and spice plants present a valuable source for manufacturing biopharmaceutical
medicines, and substantial amounts of the herbs are consumed for food
worldwide. Herb production is established as one of priority areas for non-traditional
agriculture in Lithuania. Lamiaceae family of plants include a number of valuable medical
and spice plants that are represented by wild species or being common feature of gardens
and agricultural production in Lithuania. Common thyme (Thymus vulgaris L.) is valued
for the rich content of etheric oils, vitamins and minerals, and it is popular in food industry
as a spice. Stinging nettle (Urtica dioica L.), a member of Urticaceae family, is another
plant common in Lithuania and has an established medical value.
Although content of mineral and organic substances of the medical herbs is well
characterised, presence of bioactive polypeptides is much less appreciated. Lectins
(phytohemagglutinins) are glycoproteins common in plants that demonstrate a broad
spectrum of bioactive effects. Recent investigations revealed a potential for use of lectins
as anticancer agents.
Meanwhile mainly leaves or stems are consumed in case of a number of popular
medical and spice herbs, including the nettle and thyme, seeds and roots of plants have
been established as a common source for isolation of lectins from plants, so far. Therefore
data on the presence of lectins in the leaves or stems of the two herbs is ambigous. In this
study, we prepare lectin-enriched protein fractions from extracts of the two herbs and
11

assess their blood cell agglutinating activity using tests employing rabbit erythrocytes and
macrophage J774 cells. The study revealed that lectin-like glycoprotein-enriched fractions
from Common thyme and Stinging nettle grass contained specific hemagglutination activity
up to 1.3 and 3.0 mg protein/ml, respectively. Application of the fractions containing the
hemagglutination activity induced aggregation of macrophage J774 cells, and a highest cell
death induction was observed in case of extracts obtained from herb of Common thyme.
The results suggest presence of substantial hemagglutination activity content in grass of
the two common medical herbs and bioactive potential of the lectin proteins.
12

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
mtDNA polymorphism of Scots pine
D. Danusevičius 1 , J. Buchowska 1 , V. Stanys 2 , J. B. Šiksnianiene 2 ,
D. Baniulis 2
1
Facultry of Forest and Ecology, Aleksadras Stulginskis Univeristy,
Studentu 11, Akademija, LT-53361 Kaunas reg., Lithuania;
2
Institute of Horticulture, Lithuanian Research Centre for Agriculture
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,
E-mail darius.danusevicius@asu.lt
The earlier studies on the post-glacial colonisation and DNA polymorphism in Scots
pine lack material from the eastern range of the species distribution. The hypothetical
post-glacial refugium may be in the central Europe and regions around Ural mountains
needs further examination. Aim of out study is to use the maternally inherited mtDNA
CAPS markers to investigate mtDNA polymorphism and the migration patters based
on the large-scale genetic material from the provenance trials with eastern Scots pine
populations. Samples were collected in the provenance trials to obtain complete and even
coverage of the eastern range, which will reach Japan in the east. The results revealed
two major post-glacial refugial zones: (a) in southern Europe and southern Asia with
prevalence of the Nad7-1 mitotype AA2, which was commonly referred to as universal
as it was fond in Scotland as well as in Far east, and (b) much more restricted to the in
north eastern European part of Russia, with maximum occurrence of the Nad7-1 mitotype
BA2 in the region west of central Ural mountains; this constitutes an important finding
with previously least tested genetic material from eastern Russia, (c) found only locally
in one Turkish population mitotype CA2. Central European populations possessed grated
mtDNA diversity and more than others contained both A and B alleles, what indicates a
convergence of the southern and northern refugial lines in central Europe.
13

Effect of far-red light on the growth of Chrysanthemum
plantlets in vitro
Stasė Dapkūnienė 1, 2 , Anželika Kurilčik 1, 3 , Genadij Kurilčik 4 ,
Pavelas Duchovskis 3 , Akvilė Urbonavičiūtė 3 , Silvija Žilinskaitė 1 ,
Arturas Žukauskas 4
1
Botanical Gardens of Vilnius University, Kairėnų 43, LT-10239, Vilnius,
Lithuania;
2
Plant Gene Bank, Stoties 2, LT-58343 Akademijas, Kėdainiai disrt. ,
Lithuania;
3
Institute of Horticulture, Lithuanian Research Centre for Agriculture
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;
4
Institute of Materials Science and Applied Research, Vilnius University,
Saulėtekio 9-III, LT-10222 Vilnius, Lithuania
The influence of far-red light on chrysanthemum growth was studied using a
light-emitting diode (LED)-based illuminator. Four groups of chrysanthemum plantlets
(Chrysanthemum morifolium Ramat. ‘Ellen’) were simultaneously grown under independently
set different illumination regimes. The study showed that far-red light hinders
the chrysanthemum growth processes in vitro, but increases growth of the roots length.
Meanwhile with an increase of far-red light photon flux density, the rooting rate of explants
decreases. Fresh and dry weight accumulation was depended on the rooting processes
of chrysanthemum plantlets. The study also show that phytohormones are involved in
drymatter accumulation and rhizogenesis processes. The presence of far-red light reduces
the content of all photosynthetic pigments. This shows that by adjusting of the incident
spectrum using a multiwavelength solid-state illuminator, the growth process of chrysanthemum
culture in vitro can be controlled.
14

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Assesment of genetic diversity within Lithuanian sweet cherry
genetic resources collection using AFLP markers
Birutė Frercks, Inga Stepulaitienė, Dalia Gelvonauskienė,
Jūratė Bronė Šikšnianienė, Vidmantas Stanys
Institute of Horticulture, Lithuanian Research Centre for
Agriculture and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr.,
Lithuania, E-mail b.frercks@lsdi.lt
Northern border of the secondary spreading of the sweet cherry is located in the Baltic
region. Populations of wild sweet cherry adapted to local growth conditions are found in
Western part of Lithuania. These sweet cherry populations were source for development of
the earliest cultivars in Lithuanian. The use of molecular techniques that detect variations at
DNA level is more objective and independent from environmental influences in contrast to
morphological traits, and it makes possible to develop cultivar-specific DNA fingerprints.
In this study the genetic diversity among 31 sweet cherry accessions was evaluated
using amplified fragment lenght polymorphism (AFLP) markers. Using 9 AFLP primer
combinations 485 fragments were produced, 350 of them (72 %) were polimorphic.
Polymorphism percentage, PIC and resolving power were calculated to assess the discriminatory
power of nine primer combinations used. Polymorphism percentage (PP)
ranged from 55.1 % to 100 % , in average 79.26 %. Average fragment PIC for each primer
combination ranged from 0.30 to 0.42 (mean 0.36). Resolving power varied from 11.42 to
31.74 with an average 20.75. In our study the RP was determinated as the real attribute for
AFLP to characterise the discriminatory power of primer combination. All of the 31 sweet
cherry accessions tested could be clearly differentiated with 9 AFLP primer combinations
used in this study. For cluster analysis 350 polimorphic fragments were used. The results
of the cluster analysis showed the consistence between AFLP markers analysis and the
known genetic background of cultivars and asseccions.
15

Genetic variation among Lithuanian populations of
Impatiens parviflora determined by RAPD-PCR markers
Rasa Jankauskaitė 1 , Milda Jodinskienė 1 , Judita Žukauskienė 1 ,
Steven B. Janssens 2 , Algimantas Paulauskas 1 ,
Eugenija Kupčinskienė 1
1
Vytautas Magnus University, Department of Biology, Vileikos 8,
LT-44404 Kaunas, Lithuania;
2
Laboratory of Plant Systematics, Institute of Botany and
Microbiology, K. U. Leuven, Kasteelpark Arenberg 31, P. O. Box 2437,
BE-3001 Leuven, Belgium, E-mail e.kupcinskiene@gmail.com
Human activities are resulting in the spread of many non-indigenous species to habitats
in which they are non-native. One of the most invasive alien species in the woods of
Central and Northern Europe is small balsam (Impatiens parviflora), an Asiatic species.
Its introductions appear to be increasing in forest ecosystems and are considered harmful.
This species is affecting ground-layer vegetation displacing native community components
having similar biology. Small balsam is valuable model for invasiveness investigation.
Due to small unattractive flowers intentional anthropogenic spread of this species might be
excluded. It has gradually been recognized that ecological attributes alone are insufficient
to explain why some plant species became invasive. This has led for more genetic studies
of introduced species. Various different mechanisms might be implicated in generating the
genetic variation underlying rapid adaptive evolution and the colonization of new habitats.
Till now the exact source of genetic variation causing traits important to successful
invasions remains uncharacterized. The objective of this study was to evaluate genetic
variability of Lithuanian populations of I. parviflora. Plant material was collected from
twenty one site (totally 420 individuals) different in geography, habitats and population
size. Populations of I. parviflora were characterised by the random amplified polymorphic
DNA method (RAPD) using OPD-20, OPQ-11, 269, 340, 250, 222, 474, 516, OPB-7,
OPA-20 primers. Percentage of polymorphic DNA bands for populations of small balsam
was low and ranged between 7 and 45. Mean value for population was 22 %. For the separate
populations expected heterozygosity interval was 0.021–0.140. Shannon’s information
index ranged between 0.033 and 0.215 being the lowest for Jonava-Upninkai and Preila
forest edges and the highest for park near the blacktop street in Kaunas A. Sanciai area.
Populations with more hemerobic habitats especially with bigger extent of soil erosion
and traffic intensity, showed higher genetic variation.
16

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Analyses of genetic variability in wintering Brassica napus
considering to different temperatures
Algimantas Paulauskas 1 , Milda Jodinskienė 1, 2 , Loreta Griciuvienė 1 ,
Judita Žukauskienė 1 , Irena Brazauskienė 3 , Eglė Petraitienė 3
1
Department of Biology, Faculty of Nature Sciences, Vytautas Magnus
University, Vileikos str. 8, LT-44404, Kaunas, Lithuania;
2
Laboratory of Plant physiology, Institute of Botany of Nature Research
Centre, Akademijos str. 2, LT-08412, Vilnius, Lithuania;
3
Institute of Agriculture, Lithuanian Research Centre for Agriculture
and Forestry, Instituto av. 1, LT-58344 Akademija, Kėdainiai distr.,
Lithuania, E-mail milda.jodinskiene@botanika.lt
Winter oilseed rape is grown in many countries for its valuable oil. The growing conditions
in Lithuania are different according to the winter temperatures. Low temperature is
very important for plant growth and survival, which is a characteristic for over wintering of
oilseed rape. Hard winter in 2010 was critic for the most of oilseed rape cultivars – many
farmers in Lithuania had loss of their crops.
Eleven winter oilseed rape cultivars – ‘DK Secure’, ‘Sunday’, ‘Sw Celsius’, ‘Titan H’,
‘Visby’, ‘Hornet H’, ‘Kronos H’, ‘Vision’, ‘Libea’, ‘Silvia’, ‘Valesca’ – were examined.
They were grown in the experimental fields in Lithuanian Institute of Agriculture
(Kėdainiai region). 10 to 14 samples of leaves from individual plants were collected in
spring 2010 within each population. 6 to 10 samples were collected from plants which
survived winter period. 4 to 6 samples were collected from plants which managed to
outgrowth from their roots.
The aim of the work was to search for the links between good over wintering and
genetic diversity. Random amplified polymorphic DNA (RAPD) markers offer quick
screening of different regions of the genome for genetic polymorphisms. DNA from 10
to 14 individuals per cultivar was analysed using nine different primers; the 73 detected
bands were polymorphic. Genetic diversity in Brassica napus cultivars was estimated
using AMOVA program. The results demonstrated that genetic polymorphism among 11
tested winter oilseed rape cultivars was high. The dendrogram obtained by using genetic
distances clusters showed the clear separation of all 11 populations to different clusters.
As a result, ‘DK Secure’ was separated from other cultivars. Evaluation of the results of
11 different oilseed rape cultivars over wintering showed that the last mentioned cultivar
had some different growth peculiarities comparing to others and also had the most quantity
of the survived individuals in the spring.
17

Quality, plant resistance and productivity of organically grown
carrots hybrids and cultivars
Rasa Karklelienė, Audrius Radzevičius, Edita Dambrauskienė,
Elena Survilienė, Česlovas Bobinas, Laisvūnė Duchovskienė,
Danguolė Kavaliauskaitė
Institute of Horticulture, Lithuanian Research Centre for Agriculture and
Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,
E-mail r.karkleliene@lsdi.lt
Investigation was carried out at the Institute of Horticulture in 2008–2009. Carrot
(Daucus sativus RöhL.) cultivars ‘Svalia’ F 1
, ‘Skalsa’ F 1
, ‘Bolero’ F 1
, ‘Noveno’ F 1
,
‘Garduolės’, ‘Vaiguva’, ‘Vytėnų nanto’, ‘Šatrija’, ‘Monanta’, ‘Tito’, ‘Samson’, ‘Magi’
and ‘Crona’ were investigated. Growing vegetables organically there were used natural
fertilizers. Perennial investigation shows that carrot root-crop quality depends not only
on soil type and growing conditions but also on genotype. ‘Bolero’ F 1
and ‘Noveno’ F 1
(74.7 t ha -1 and 61.7 t ha -1 ) had a highest yield. ‘Noveno’ F 1
has a rich content of carotene
(18.7 mg per 100 g -1 ). ‘Skalsa’, ‘Tito’ and ‘Monanta’ was most damaged by carrot foliage.
It was found that cultivars ‘Skalsa’ F 1
, ‘Tito’, ‘Garduolės’, ‘Vytėnų Nanto’, ‘Bolero’ and
‘Magi’ were damaged by mycoplasma disease. Radicina Alternaria, Phoma sp., Botrytis
cinerea, Sclerotinia sclerotiorum and bacteria in stored carrots caused rot.
18

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Low temperature storage of Rosaceae (Fragaria ananassa and
Pyrus communis) plants in vitro
Vanda Lukoševičiūtė 1 , Rytis Rugienius 1 , Gražina Stanienė 1 ,
Asta Blažytė 1, 2 , Dalia Gelvonauskienė 1 , Bronislovas Gelvonauskis 2 ,
Audrius Sasnauskas 1 , Vidmantas Stanys 1
1
Institute of Horticulture, Lithuanian Research Centre for Agriculture
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;
2
Plant Gene Bank, Stoties 2, LT-58343 Akademija, Kedainiai distr.,
Lithuania
The biodiversity is important not only for preservation of natural populations but
also for development of new cultivars of cultivated plants. Genetic resources of fruit and
berries are preserved mainly as field gene banks. This system requires a large land area,
labor and capital investment. Many of accessions in the collections are unique. Climatic
conditions, plant diseases and pests constitute a serious hazard to genetic resources stored
in field collections. In vitro culture techniques including storage under growth limiting
conditions and cryopreservation provide alternative storage forms for protecting important
germplasm. During the past few decades, different in vitro conservation methods have
been developed, however methods that are suitable for storage of particular cultivars or
genotype groups in the large collections often are not suitable or less suitable to other
genotype. Strawberry and pear, representing woody and grass species of Rosaceae family
are important cultivated crops and could be used as model species for development and
adjusting methodology of in vitro storage.
The present study aims to develop a procedure for in vitro low temperature storage of
strawberry and pear by investigate the effect of different culture media and incubation at
low temperature on state of plants of various genotypes. Twenty strawberry and nine pear
accessions were used for the study in the Institute of Horticulture, Lithuanian Research
Centre for Agriculture and Forestry in 2009–2011.
After medium term storage (15 months for strawberry and 6 months for pear) at 4 °C
temperature microshoots survival exceeded 80 %. However, state of the plants varied depending
on genotype. Condition of strawberry microshoots decreased considerably after
12 months of the storage. At the end of the storage (after 15 months) 40 % of strawberry
plants in vitro were in good condition (average score exceeded 2), 60 % of plants in poor
and bad condition (average score below 2). It was established that state of pear microshoots
remained the best (score 4–5) after 6 month storage in 4 °C temperature on MS medium
with 0.75 mg/l BAP and 3 % sucrose.
19

Effect of genotype and medium composition on linseed
(Linum usitatissimum L.) ovary culture
Ramunė Masienė, Natalija Burbulis, Aušra Blinstrubienė
Aleksandras Stulginskis University, Department of Crop Science and
Animal Husbandry, Agrobiotechnology laboratory, Studentų 9, LT-53361
Akademija, Kaunas district, Lithuania, E-mail ramunee7@hotmail.com
Breeding linseed (Linum usitatissimum L.) using haploid techniques allows breeders
to develop new cultivars in a shorter time period. Many research groups successfully
created new linseed genotypes through anther culture; however ovary culture has been
the subject of only a few earlier studies. In the present study, the effect of genotype and
growth regulators combination on callus induction and shoots regeneration in ovary culture
of nine commercially important linseed cultivars was investigated. Ovaries were cultured
on modified MS medium supplemented with three different combinations of plant growth
regulators. Variable callogenic responses were expressed by all of the genotypes tested
on different induction media. The results suggested that specific combination of growth
regulators for callus induction must be designed for each genotype. Shoot regeneration
from ovary derived callus is a critical phase of the whole gynogenetic process. Differences
in adventitious shoot formation frequency among genotypes were demonstrated and four
responsive genotypes have been selected. Ovary derived callus from cultivar ‘Mikael’
manifested the highest adventitious shoot formation frequency with a high number of
shoots per explant. The optimum ratio of growth regulators for shoot regeneration was
shown to depend on the genotype. Cultivars ‘Linola’, ‘Mikael’ and ‘Szaphir’ showed the
highest shoot regeneration frequency when callus had originated from induction medium
supplemented with 2 mg l -1 BAP and 2 mg l -1 NAA while combination of 1 mg l -1 BAP and
2 mg l -1 IAA promoted shoot formation in ovary-derived callus of ‘Barbara’. The highest
rate of shoots per explant has been obtained in second subculture.
20

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
A genetic linkage map for Cecidophyopsis ribis resistance in
Ribes nigrum based on microsatellite and AFLP markers
Ingrida Mažeikienė, Vidmantas Bendokas, Vidmantas Stanys,
Tadeušas Šikšnianas
Institute of Horticulture, Lithuanian Research Centre for Agriculture and
Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,
E-mail mazeikiene@hotmail.com
Genetic linkage maps provide essential information for molecular breeding. It is
known, that the P gene provides resistance of blackcurrant to Cecidophyopsis ribis that is
a vector of blackcurrant reversion virus (BRV). C. ribis is one of the most damaging pests
of blackcurrants. Therefore, it is important to select resistant blackcurrants germplasm.
In our study, a linkage map around the resistance locus controlled by predicted P gene
was constructed. The genetic paternal linkage map of R. nigrum was constructed using a F 1
progeny of 158 individuals from three families. In blackcurrant crossings, three R. nigrum
genotypes with a different sensitivity to C. ribis were used as the maternal plants, and
resistant to gall mite cultivar ‘Dainiai’ was used as the paternal. Forty three (43) AFLP
and twenty two (22) microsatellite markers obtained from analysis of the crosses with
‘Dainiai’ were mapped onto 5 linkage groups, 2 triples and 1 pair at the linkage criteria
LOD 3.0.Sixteen markers could not be linked to any linkage group at a LOD threshold
3.0.The obtained consensus map covers 715.067 cM, with an average marker spacing of
14.593 cM. AFLP fragment CTA-ACC-107 was closely linked with resistance to C. ribis
and was detected in the sixth linkage group. This marker was 37.742 cM and 17.469 cM
from morphologic trait of resistance to gall mite identified in blackcurrant evaluation trials
in 2010 and 2011, respectively.
The presented map provides crucial information for future genetic studies of Ribes
nigrum in particular for quantitative trait loci (QTL) mapping of economically important
traits. Further testing of gall mite resistant R. nigrum genotypes would establish association
of the 107 bp AFLP fragment as a part of the P resistance in R. nigrum.
21

Dynamics of ascospore dispersal and pathogenicity of L. maculans
and L. biglobosa on winter oilseed rape
Agnė Piliponytė, Irena Brazauskienė, Eglė Petraitienė,
Gintaras Brazauskas
Lithuanian Research Centre for Agriculture and Forestry, Institute of
Agriculture, Akademija, LT-58344, Kėdainiai distr., Lithuania,
E-mail agne.piliponyte@lzi.lt
Phoma stem canker, caused by L. maculans and L. biglobosa is the most important
disease of oilseed rape in Europe. These both species co-exist in Lithuania. Information
on timing of ascospore release is an essential tool in the protection of oilseed rape against
Phoma stem canker. Spore release was monitored using a Burkard 7-day volumetric spore
trap from September to November in 2010.The number of spores was counted under optical
microscope. The results show that first peak of ascospore was observed on 2 September
(105 ascospores per day) and the second peak was detected on 9 September (191 ascospores
per day). Later on ascospore dispersal decreased and low ascospore numbers were
observed during October. The abudance of ascospores in the air was dependant on the
weather factors, especially amount and frequency of rainfall. Real time–PCR will be used
to investigate species-specific ascospore spread dynamics.
It is known that two fungi cause differential host defence response to infection. In
controlled environment experiments winter oilseed rape plants were inoculated using
suspensions of L. maculans and L. biglobosa conidium. The results show that incubation
period was longer on the plants inoculated with L. maculans (11–12 days) than
using L. biglobosa (6–7 days). cDNA - AFLP technique will be employed to identify
pathogenesis related differentially expressed genes. We expect that our results will lead
to better understanding of these two phytopathogenic fungi and will give useful insights
into disease control improvement.
22

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Genetic polymorphism of wild pear population in Lithuania
Rytis Rugienius 1 , Asta Blažytė 1, 2 , Vanda Lukoševičiūtė 1 ,
Juratė Bronė Šikšnianienė 1 , Dalia Gelvonauskienė 1 ,
Bronislovas Gelvonauskis 2 , Audrius Sasnauskas 1 , Danas Baniulis 1 ,
Vidmantas Stanys 1
1
Institute of Horticulture, Lithuanian Research Centre for Agriculture
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;
2
Plant Gene Bank, Stoties 2, LT-58343 Akademija, Kedainiai distr.,
Lithuania, E-mail r.rugienius@lsdi.lt
Wild pear (Pyrus pyraster (L.) Burgsd.) is pear species growing naturally in Lithuania
and is related to cultivated pear (Pyrus communis L.). In some cases, plants identified as
P. pyraster represent various stages of hybridization between original P. pyraster and
P. communis. Therefore, a boundary between cultivated pear, naturalized plants and wild
pear is ambiguous. There is limited information on genetic variation and structure of
P. pyraster population. Pear genetic resources have not been extensively characterized
due to low morphological diversity, lack of differentiating characters among species and
widespread crossability. Traditional methods of characterization, based on agronomic and
morphological parameters, are being affected by environmental conditions and phenological
stages of the plants. Therefore, an employment of molecular techniques would provide
deeper understanding of genetic background of the wild populations and hybrids of the crop.
The aim of our study was to characterize microsatellite loci of pear originally collected
from different localities of Lithuania as naturally growing specimens, presently
grown in germplasm collection at the Institute of Horticulture Lithuanian Research Centre
for Agriculture and Forestry and evaluate possible link between microsatelite alleles and
morphological parameters of the accessions. Microsatelite analysis was performed on
84 pear clones, grouped to three groups: wild, cultivated or hybrid genotypes, according
prevailing morphological traits. Characterization of 9 polymorphic microsatellite loci of
pear accessions identified 154 polymorphic alleles. The allele number per locus ranged
from 12 to 20 alleles. The most polymorphic microsatellite loci (EMPc106, EMPc117,
NB109a and CH02c11) as well as unique alleles, characteristic to each from of the three
genotype groups were identified. High genetic polymorphism was demonstrated by genetic
relationship and heterozygosity analysis among accessions. Lower Ho than He values
was identified proclaiming comparatively often occasions of self fertilisation in naturally
growing pears. It was shown that variability of morphological traits of Pyrus accessions
weakly reflects genetic variation. To some extend, only presence of initial hairiness and
number of seeds in the fruit could be used as morphological markers of the genetic proximity
of Pyrus accessions.
23

Identification of nepoviruses in tomatoes
Donatas Šneideris, Irena Zitikaitė, Banga Grigaliūnaitė,
Marija Žižytė, Juozas Staniulis
Nature Research Centre, Institute of Botany, Žaliųjų Ežerų Str. 49,
LT-08406, Vilnius, E-mail Donatas.sneideris@gmail.com
The nepoviruses (nematode-transmitted, polyhedral viruses) are a group of about
46 viruses that infect many plant families. They cause diseases of economical importance in
a wide range of cultivated annual, perennial and woody plants, and are of serious concern
to quarantine authorities worldwide. Their wide host range combined with ability to be
transmitted by nematodes, seed or pollen makes them particularly dangerous.
Few nepoviruses (Arabis mosaic nepovirus, Tobacco ringspot nepovirus, Tomato
ringspot nepovirus) have already been detected and identified in ornamental plants in
Lithuania. Now we identified two nepoviruses – Tobacco ringspot nepovirus (TRSV) and
Tomato black ring nepovirus (TBRV) in tomatoes. Infected tomato plants were assayed by
DAS-ELISA tests. Extracts of plants that showed positive reaction in DAS-ELISA were
inoculated to more than twenty different indicator plants. Local lesions and systemic infection
were observed in plants belonging to Nicotiana, Chenopodium, Datura, Gomphrena
and Tetragonia genera. Transmission electron microscopy (EM) of extracts from leaves of
infected test plants revealed presence of icosahedral virus particles of ~ 30 nm in diameter
that are characteristic of nepoviruses. Presence of TRSV and TBRV in infected test plants
was also detected by RT-PCR using virus specific primers. Thus, detection and identification
of TRSV and TBRV in naturally infected tomatoes and test-plants was confirmed by
DAS-ELISA, EM and RT-PCR.
24

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Characterization of microsatellite marker loci of traditional and
indigenous apple tree cultivars in Lithuania.
Sidona Sikorskaitė, Dalia Gelvonauskienė, Vidmantas Stanys,
Danas Baniulis
Institute of Horticulture, Lithuanian Research Centre for Agriculture and
Forestry, Babtai LT-54333, Kaunas reg., Lithuania,
E-mail d.baniulis@lsdi.lt
Apple fruits constitute an important part of horticultural production in Lithuania
and worldwide. Tools for assessment of genetic polymorphism and genotyping are required
for breeding and research on apple genetic resources. The aim of present study
was to characterize microsatellite loci of indigenous and traditional Lithuanian apple tree
(Malus × domestica) cultivars and to select primer pairs suitable for genotyping of the
cultivars. Thirty-seven traditional cultivars and indigenous cultivars developed during
the last century, and eleven standard cultivars available at the collection of apple genetic
resources at the Institute of Horticulture, LRCAF were assessed. Apple genotyping was
performed using eleven PCR primers specific for polymorphic microsatellite loci of
Malus × domestica and recommended by ECPGR Malus workgroup. All microsatellite
loci exhibited a high level of polymorphism – 8 to 14 alleles with mean value of 10.45
for traditional and indigenous cultivars and 5 to 12 alleles 7.55 in average for reference
cultivars. The observed heterozygosity varied from 0.70 to 0.97 with an average of 0.84
for traditional and indigenous cultivars and from 0.64 to 1.00 with mean value of 0.90 for
standard cultivars. Locus CH01g12 was the most polymorphic micorsatellite locus in the
group of 37 traditional and indigenous cultivars, while CH02b10 locus exhibited the highest
polymorphism in the set of standard cultivars. The polymorphism of the microsatellite
markers allowed to specifically identify 35 of the traditional and indigenous cultivars.
25

Isolation of apple tree (Malus × domestica Borkh.) cell nuclei for
proteome analysis
Sidona Sikorskaitė 1 , Sandra Zavadskytė 2 , Vidmantas Stanys 1 ,
Danas Baniulis 1
1
Institute of Horticulture, Lithuanian Research Centre for Agriculture
and Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania;
2
Department of Biochemistry and Biotechnologies, Vytautas Magnus
University, Vileikos st. 8-201, Kaunas LT-44404, Lithuania,
E-mail d.baniulis@lsdi.lt.
Apple tree is economically important plant of the Rosaceae family. Additionally,
relatively small genome size, availablility of apple tree genome sequence and molecular
marker tools, makes apple tree attractive as a model species for genomic studies on woody
plants of the Rosaceae family. Proteome analysis has been a valuable tool in studies on
complex molecular mechanisms involved in plant development, productivity and response
to environmental stimuli. However, proteome of entire plant cell comprises a largely
complex picture presents high demands on dynamic range and sensitivity of protein and
analysis procedures. Therefore, subcellular fractions are often used in functional genomics
studies to reduce sample complexity. Signaling pathways set off to changes of gene expression
lead to cell nucleus that encloses a number of important signaling messengers. Due
to complexity of procedures required for nuclear protein preparation, studies addressing
nuclear proteome in plants have been scarce to date, and information on nuclear proteome
of plants of the Rosaceae family remains vague. Therefore the aim of this study was to
develop procedure nuclear protein preparation from fresh leave tissue of apple tree suitable
for proteomics analysis. To achieve the objective, issues of efficiency of breakage of
the woody plant cells, nuclei stabilization and isolation of pure factions of the organelle,
stability and solubilization of the isolated protein were addressed. Several previously
published procedures on plant nuclei preparation for genetic and proteome analysis were
evaluated and conditions were optimized. Disruption of apple tree leave tissue by grinding
in liquid nitrogen ensured efficient cell breakage and filtration followed by differential
centrifugation led to separation of organellar fraction. Further, a concentration of detergent
was optimized for differential lysis of organelles, and it was found that treatment with 1 %
Triton X-100 was optimal for isolation of stable nuclei. The nuclei were fractionated by
equilibrium centrifugation on combined sucrose and percoll gradient. Purity of protein
of the isolated nuclei was established using immunoblot analysis and organellar markers.
The developed procedure yielded microgram quantities of protein suitable for proteome
analysis from several grams of leave tissue.
26

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Detection and molecular identification of alien viruses affecting stone
fruit and vegetable crops in Lithuania
Juozas Staniulis 1 , Irena Zitikaitė 1 , Marija Žižytė 1 ,
Elena Jackevičienė 2 , Laima Urbanavičienė 1 , Donatas Šneideris 1
1
Institute of Botany, Nature Research Centre, Zaliuju Ezeru 49,
LT-08406, Vilnius;
2
The State Plant Service under the Ministry of Agriculture of the
Republic of Lithuania, Ozo g. 4A, LT-08200, Vilnius
Alien virus species infecting stone fruit and vegetable crops in Lithuania are considered:
Plum pox virus (PPV) affecting plums (Prunus domestica L.), Beet necrotic yellow
vein virus (BNYVV) affecting sugar beets (Beta vulgaris var. saccharifera L.) and Pepino
mosaic virus (PepMV) affecting tomatoes (Lycopersicon esculentum MilL.). For the first
time in Lithuania PPV was detected 15 years ago, BNYVV – 6 years and PepMV just
few years ago. The viruses were isolated from natural infection sources and mechanically
transmitted to susceptible test-plants. Identity of PPV in symptomatic plum tree leaves
and test-plants (Chenopodium foetidum, Pisum sativum), BNYVV in sugar beet roots and
test-plants (Chenopodium quinoa, C. amaranticolor, Tetragonia expansa) and PepMV in
tomato fruits and Nicotiana test-plant species was confirmed by immuno enzyme DAS-
ELISA test, electron microscopy, and molecular RT-PCR procedure. RT-PCR, sequencing
and RFLP analysis have confirmed that all PPV isolates appeared to be attributed to PPV-D
strain group. PPV appeared to have limited distribution in few administrative regions of
central and northern part of Lithuania. BNYVV was detected in several locations of four
administrative regions. Multiplex reverse transcription PCR analysis of the coat protein
(CP) gene sequences (RNA-2) showed that in Lithuania occur A and B types of BNYVV.
PepMV at first was detected and identified in imported commercial tomato fruits. Later
on this virus was detected in tomato fruits grown in commercial tomato greenhouses in
Lithuania. Isolates from fruits of tomato were identified as PepMV on the bases of symptom
expression on the fruits and test-plants, morphology of virions, results of DAS-ELISA
tests and polymerase chain reaction.
27

Analysis of genetic structure of Botrytis spp. isolates from
horticultural plant hosts
Alma Valiuškaitė, Elena Survilienė, Danas Baniulis,
Laisvūnė Duchovskienė, Neringa Rasiukevičiūtė
Institute of Horticulture, Lithuanian Research Centre for Agriculture and
Forestry, Kauno 30, LT-54333 Babtai, Kaunas distr., Lithuania,
E-mail a.valiuskaite@lsdi.lt
Analysis of the genetic diversity of plant pathogen population is the most important
area. The polymorphic diversity could provide significant information relating to the
pathosystem. Genetic identification is increasingly used in plant pathology for the indentification
of individual at specific and intra-specific levels of plant pathogens. Botrytis spp.
wide spread fungi causing diseases in many horticultural plants. This study performed the
analysis the variation of genetic structure of Botrytis spp. isolates originating from different
horticultural plant hosts from six locations of Lithuania. Isolates collected from fruits of
Malus × domestica, Fragaria × ananassa, and vegetables Allium cepa, Brassicae oleracea
var. capitata, Lycopersicon esculentum were analysed using molecular markers. Thirty
isolates with distinct morphological-cultural properties specific to B. cinerea, B. squamosa
and B. aclada were prepared. The isolates differed in their optimum growth, conidial formation
and size of conidia on Potato dextrose agar. Assessment of genetic polymorphism
using AFLP method revealed high genetic diversity among the selected Botrytis spp. isolates.
An assessment of genetic polimorphism of the thirty selected Botrytis spp. isolates
using AFLP method with three pairs of primers with one selective nucleotide resulted in
identification of 1968 polymorphic alleles. Dendrograms, analysis of genetic structure
of Botrytis spp. revealed the existence of genetic differentiation in specimens between
isolates from the different host plants. There were found seventeen groups which genetic
distance between isolates was less than 0.5. Analysis of the genetic diversity confirmed
the Botrytis spp. polyphagia characteristic feature: the closely related isolates were distributed
in different horticulture plant hosts. Genetic distance between isolates from different
geographic regions was 0.9 it showed that Botrytis spp. isolates are characterized by high
polymorphism. Great variation of genetic isolates is not a consequence of geographic
isolation, because closely related isolates were found in different areas.
28

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
RAPD analysis of genetic differences among Lithuanian
populations of Impatiens glandulifera
Lina Zybartaitė 1 , Milda Jodinskienė 1 , Judita Žukauskienė 1 ,
Steven B. Janssens 2 , Algimantas Paulauskas 1 ,
Eugenija Kupčinskienė 1
1
Vytautas Magnus University, Department of Biology, Vileikos 8,
LT-44404 Kaunas, Lithuania;
2
Laboratory of Plant Systematics, Institute of Botany and Microbiology,
K. U. Leuven, Kasteelpark Arenberg 31, P. O. Box 2437, BE-3001
Leuven, Belgium, E-mail e.kupcinskiene@gmail.com
Nowadays many reports are providing evidence about rapid spread and adaptation
of invasive species. Such changes may result in genetic differentiation between local
populations. First introduced to Europe (England) from Nepal in 1838/1939, Impatiens
glandulifera quickly became a garden favorite. In Nepal I. glandulifera seeds are harvested
as a food source, their oil is used for cooking. Albeit a very attractive and usefull one
the Himalayan balsam or policeman‘s helmet, I. glandulifera, had become a prodigious
weed. Impatiens glandulifera is highly invasive in almost whole Europe and appears in
various habitats. In Lithuania it is distributed through all the country. No accounts exists
for evidence of local differentiation concerning I. glandulifera in the Baltic region. The
objective of this study was to evaluate genetic variability of Lithuanian populations of
I. glandulifera. Twenty populations (totally 400 individuals) were collected from various
districts of the country. I. glandulifera was characterised by the randomly amplified
polymorphic DNA method (RAPD) using eight 10-mer oligonucleotids. Selected RAPD
primers generated between 18 and 30 bands each. Totally 188 bands were recorded
and all of them were polymorphic. Percentage of polymorphic DNA bands for separate
populations of I. glandulifera was not very high and ranged between 40 and 56. For the
separate populations number of effective alleles ranged between 1.188 and 1.271, expected
heterozygosity interval was 0.115–0.165. Shannon’s information index ranged between
0.183 and 0.255 being the lowest for Kruonis (semi-natural site near the groove) and the
highest for hemerobic Vilnius-Snipiskes site. Cluster analyses indicated that all populations
might be split into 4 groups among which there is no clear geographical pattern. Our
genetical analyses indicate multiple introduction of this invasive alien species in Lithuania.
Presumably several different ways of invasion of I. glandulifera took place: natural run
and predisposing it intentional and unintentional dispersal by human.
29

Molecular analysis of wild cranberry (Vaccinium oxycoccus L.)
genetic diversity using RAPD and SSR
Judita Žukauskienė 1 , Algimantas Paulauskas 1 , Laima Česonienė 2 ,
Remigijus Daubaras 2
1
Faculty of Natural Sciences of Vytautas Magnus University, Vileikos 8,
Kaunas, LT-44404, Lithuania;
2
Kaunas Botanical Garden of Vytautas Magnus University,
Ž. E. Žilbero 6, Kaunas., LT-46324, Lithuania,
E-mail j.zukauskiene@gmf.vdu.lt
Situation of Lithuanian peat bogs, force to give a thought about survival risk of wild
cranberry Vaccinium oxycoccus population’s in Lithuania. Genetic variation represents
the starting point for further evolution and is an important prerequisite for the prediction
of evolutionary responses. This is of practical significance in the light of human-caused
habitat fragmentation, alteration, or destruction. Genetic structure among and within
populations also depends on the life history of a species.
The present work was conducted to evaluate RAPD (random amplified polymorphic
DNA) and SSR (Simple Sequence Repeat) marker systems for their ability to detect genetic
diversity within and among V. oxycoccus populations and to compare the efficiency
of these two marker types.
Using nine RAPD primers we investigated genetic variation among and within 151
individuals (7 populations of different size Lithuanian and 3 populations of other countries)
of V. oxycoccus. With these profiles we evaluated 254 fragments. According to RAPD
results were selected different individuals 56 of V. oxycoccus and tested them with 10 SSR
primer pairs. These SSR primers generated 62 fragments.
According to RAPD data genetic variation among populations varied from 7 to 25 %
(RAPD) and from 16 to 14 % (SSR) molecular variance (AMOVA)).
The higher level of polymorphism detected by RAPD markers has contributed to the
lower genetic similarity estimates based on RAPD markers as compared to SSR markers.
The dendrograms generated with hierarchicalUPGMA (Unweighted Pair Group
Method with Arithmetic mean) cluster analysis revealed mixed and homogenous V. oxycoccus
lineages. These lineages confirm the prediction that Lithuanian populations were
derived from one population before glaciations.
30

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
The technique of “artificial seeds” creation for propagation and
storage strawberry plant material
Ivan Kulikov, Valeriy Vysotsky, Lilia Alekseenko
All-Russian Horticultural Institute for Breeding, Agrotechnology and
Nursery of Russian Academy of Agrarian Science, Moscow, 115598,
Zagorievskaya str. , 4, E-mail vstisp@vstisp.org
The investigation for revealing the possibility of obtaining “artificial seeds” of
strawberry (Fragaria × ananassa Duch.) was carried out. In experiments were involved
varieties ‘Roxana’, ‘Pokahontas’, ‘Divnaya’. Microshoots and buds have been encapsulated
into alginate coat.
The “artificial seeds” of strawberry which were able to germination on nutrient media,
had the ability to store at low temperature during one month, normally grew and developed
in the soil. The influence of sucrose concentration on germination of encapsulated shoot
apices of strawberry has been studied. The percentage of “artificial seeds” germination
for ‘Roxana’ variety contained in the coat 3 % of sucrose was 65, and increased up to 69
when the sucrose content was 6 %.
The ability to germinate depended on nutrient media composition for explants
preparing before encapsulation and variety’s peculiarities. Encapsulated shoot apices of
strawberry variety ‘Pokahontas’ cultivated on nutrient media with sucrose concentration
6 % and relatively low cytokinin content stored at the temperature +2…+4 °С during
30 days germinated in the soil on 56.8 %. The percentage of germinated “artificial seeds”
of the same variety from the nutrient media with paclobutrazol was only 43.2. Explants
of variety ‘Divnaya’ from the similar nutrient media gave 76.8 of germinated “artificial
seeds” in the soil.
The prolongation of the storage term of capsules could be ensured by means of the
substances inhibiting explants growth (abscisic acid, paclobutrazol etc.), explants preparing
before encapsulation on the nutrient media with sucrose concentration 6 % and low
6-benzylaminopurine content (0.2 mg/l). Promising technique could be double encapsulation
for increasing of alginate coat durability and reducing of water lost.
31

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
PARTICIPANTS
Name Institution Country E-mail
Lilia
All-Russian Horticultural Russia allex7897@mail.ru
Alekseenko Institute for Breeding,
Agrotechnology and
Nursery
Rita
Institute of Agriculture, Lithuania rita.armoniene@lzi.lt
Armonienė LRCAF
Vaida
Bakšenskaitė
Vytautas Magnus
University
Lithuania vaida.baksenskaite@fc.vdu.lt
Gabrielė
Balčiūnaitė
Danas
Baniulis
Vidmantas
Bendokas
Erna
Biveinytė
Česlovas
Bobinas
Gintaras
Brazauskas
Odeta Buzaitė
Zenonas
Dabkevičius
Edita
Dambrauskienė
Darius
Danusevičius
Stasė
Dapkūnienė
Laisvūnė
Duchovskienė
Medical Academy,
Lithuanian University of
Health Sciences
Institute of Horticulture,
LRCAF
Institute of Horticulture,
LRCAF
Institute of Biotechnology,
Vilnius University
Institute of Horticulture,
LRCAF
Institute of Agriculture,
LRCAF
Vytautas Magnus
University
Institute of Agriculture,
LRCAF
Institute of Horticulture,
LRCAF
Facultry of Forest and
Ecology, Aleksadras
Stulginskis Univeristy
Plant Gene Bank,
Botanical Gardens of
Vilnius University
Institute of Horticulture,
LRCAF
Lithuania gabriele.balc@gmail.com
Lithuania d.baniulis@lsdi.lt
Lithuania v.bendokas@lsdi.lt
Lithuania ernute32@yahoo.com
Lithuania c.bobinas@lsdi.lt
Lithuania gintaras@lzi.lt
Lithuania o.buzaite@gmf.vdu.lt
Lithuania lammc@lammc.lt
Lithuania e.dambrauskiene@lsdi.lt
Lithuania darius.danusevicius@asu.lt
Lithuania stase.dapkuniene@gf.vu.lt
Lithuania laisve.d@lsdi.lt

Name Institution Country E-mail
Birutė Frercks Institute of Horticulture, Lithuania b.frercks@lsdi.lt
LRCAF
Dalia Institute of Horticulture, Lithuania d.gelvonauskiene@lsdi.lt
Gelvonauskienė LRCAF
Bronislovas Plant Gene Bank Lithuania b.gelvonauskis@agb.lt
Gelvonauskis
Perttu Haimi University of Helsinki Finland perttu.haimi@gmail.com
Rasa
Vytautas Magnus Lithuania ra.jankauskaite@gmail.com
Jankauskaitė University
Milda Nature research centre, Lithuania milda.jodinskiene@botanika.lt
Jodinskienė Vytautas Magnus
University
Monika Kairytė Vytautas Magnus Lithuania mnk.kairyte@gmail.com
University
Linas Kalėda Institute of
Lithuania linas@nomadsbio.lt
Biotechnology, Vilnius
University
Rasa KarklelienėInstitute of
Lithuania r.karkleliene@lsdi.lt
Horticulture, LRCAF
Danguolė
Kavaliauskaitė
Institute of Horticulture, Lithuania d.kavaliauskaite@lsdi.lt
LRCAF
Vilius
Kavaliauskis
Vaiva
Kazanavičiūtė
Ivan
Kulikov
Eugenija
Kupčinskienė
Julius
Liobikas
Vanda
Lukoševičiūtė
Medical Academy,
Lithuanian University of
Health Sciences
UAB Nomads
Lithuania v.kavaliauskis@gmail.com
Lithuania vaiva@ibt.lt
All-Russian Horticultural Russia vstisp@vstisp.org
Institute for Breeding,
Agrotechnology and
Nursery
Vytautas Magnus Lithuania e.kupcinskiene@gmail.com
University
Institute of
Lithuania ljulius@vector.kmu.lt
Neurosciences, Medical
Academy, Lithuanian
University of Health
Sciences
Institute of Horticulture, Lithuania v.lukoseviciute@lsdi.lt
LRCAF

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
Name Institution Country E-mail
Daiva Majienė Institute of
Lithuania daivamaj@vector.kmu.lt
Neurosciences, Medical
Academy, Lithuanian
University of Health
Sciences
Ramunė Department of Crop Lithuania ausra.blinstrubiene@asu.lt
Masienė Science and Animal
Husbandry, Aleksandras
Stulginskis University
Ingrida
Mažeikienė
Institute of Horticulture, Lithuania mazeikiene@hotmail.com
LRCAF
Irutė
Meškienė
Hirofumi
Nakagami
Šarūnas
Paškevičius
Agnė
Piliponytė
Audrius
Radzevičius
Minna
Rajamäki
Neringa
Rasiukevičiūtė
Aušra
Ražanskienė
Max F. Perutz
Laboratories, University
of Vienna
Plant Proteomics
Research Unit, RIKEN
Plant Science Center
Institute of
Biotechnology,
Vilnius University
Institute of Agriculture,
LRCAF
Institute of Horticulture,
LRCAF
Department of
Agricultural Sciences,
University of Helsinki
Institute of Horticulture,
LRCAF
Institute of
Biotechnology,
Vilnius University
Austria
Japan
irute.meskiene@univie.ac.at
hironakagami@psc.riken.jp
Lithuania paskevicius.sarunas@gmail.com
Lithuania agne.piliponyte@lzi.lt
Lithuania a.radzevicius@lsdi.lt
Finland
minna.rajamaki@helsinki.fi
Lithuania neringa_ra@yahoo.com
Lithuania ausra@nomadsbio.lt
Rytis Rugienius Institute of Horticulture, Lithuania r.rugienius@lsdi.lt
LRCAF
Sidona Institute of Horticulture, Lithuania ssidona@yahoo.com
Sikorskaitė LRCAF
Tadeušas Institute of Horticulture, Lithuania v.bendokas@lsdi.lt
Šikšnianas LRCAF
Jūratė Bronė
Šikšnianienė
Institute of Horticulture, Lithuania j.siksnianiene@lsdi.lt
LRCAF

Name Institution Country E-mail
Donatas Institute of Botany, Lithuania donatas.sneideris@gmail.com
Šneideris Nature Research Centre
Audrius Institute of Horticulture, Lithuania a.sasnauskas@lsdi.lt
Sasnauskas LRCAF
Juozas Institute of Botany, Lithuania juozas.staniulis@botanika.lt
Staniulis Nature Research Centre
Vidmantas Institute of Horticulture, Lithuania v.stanys@lsdi.lt
Stanys LRCAF
Urtė Starkevič Institute of
Lithuania bonckute@gmail.com
Biotechnology,
Vilnius University
Inga
Institute of Horticulture, Lithuania i.stepulaitiene@lsdi.lt
Stepulaitienė LRCAF
Elena Survilienė Institute of Horticulture, Lithuania e.surviliene@lsdi.lt
LRCAF
Laima
Urbanavičienė
Institute of Botany of
Nature Research Centre
Lithuania laima.urbanaviciene@botanika.
lt
Alma Institute of Horticulture, Lithuania a.valiuskaite@lsdi.lt
Valiuškaitė LRCAF
Jari P. T .
Valkonen
Department of
Agricultural Sciences,
University of Helsinki
Finland jari.valkonen@helsinki.fi
Valeriy
Vysotskiy
Sandra
Zavadskytė
Marija Žižytė
Judita
Žukauskienė
Lina Zybartaitė
All-Russian Horticultural Russia vstisp@vstisp.org
Institute for Breeding,
Agrotechnology and
Nursery
Vytautas Magnus Lithuania sandra.zavadskyte@fc.vdu.lt
University
Institute of Botany, Lithuania marija.zizyte@botanika.lt
Nature Research Centre
Vytautas Magnus Lithuania j.zukauskiene@gmf.vdu.lt
Univesity
Vytautas Magnus Lithuania lina.zybartaite@gmail.com
University

Workshop-Seminar “Plant Biotechnology Advances in Agriculture”
2011 10 27–28
INDEX
Alekseenko L. /31/
Armonienė R. /10/
Bakšenskaitė V. /11/
Balčiūnaitė G. /11/
Baniulis D. /11/13/23/25/26/28/
Bendokas V. /11/21/
Bernatonienė J. /11/
Bistrovaitė L. /11/
Blažytė A. /19/23/
Blinstrubienė A. /20/
Bobinas Č. /18/
Brazauskas G. /10/22/
Brazauskienė I. /17/22/
Buchowska J. /13/
Burbulis N. /20/
Česonienė L. /30/
Dambrauskienė E. /18/
Danusevičius D. /13/
Dapkūnienė S. /14/
Daubaras R. /30/
Duchovskienė L. /18/28/
Duchovskis P. /14/
Frercks B. /15/
Gelvonauskienė D. /15/19/23/25/
Gelvonauskis B. /19/23/
Griciuvienė L. /17/
Grigaliūnaitė B. /24/
Jackevičienė E. /27/
Jankauskaitė R. /16/
Janssens S. B. /16/29/
Jodinskienė M. /16/17/29/
Karklelienė R. /18/
Kavaliauskaitė D. /18/
Kavaliauskis V. /11/
Kulikov I. /31/
Kupčinskienė E. /16/29/
Kurilčik A. /14/
Kurilčik G. /14/
Liatukas Ž. /10/
Liobikas J. /11/
Lukoševičiūtė V. /19/23/
Majienė D. /11/
Masinė R. /20/
Mažeikienė I. /21/
Meškienė I. /8/
Nakagami H. /7/
Paulauskas A. /16/17/29/30/
Petraitienė E. /17/22/
Piliponytė A. /22/
Radzevičius A. /18/
Rajamäki M. /6/
Rasiukevičiūtė N. /28/
Rugienius R. /19/23/
Sasnauskas A. /19/23/
Savickas A. /11/
Schweighofer A. /8/
Sikorskaitė S. /25/26/
Staniulis J. /24/27/
Stanys V. /11/13/15/19/21/23/25/26/
Stepulaitienė I. /15/
Stanienė G. /19/
Survilienė E. /18/28/
Šikšnianienė J. B. /13/15/23/
Šikšnianas T. /21/
Šneideris D. /24/27/
Urbanavičienė L. /27/
Urbonavičiūtė A. /14/
Valiuškaitė A. /28/
Valkonen Jari P. T. /5/
Vysotsky V. /31/
Zavadskytė S. /26/
Zitikaitė I. /24/27/
Zybartaitė L. /29/
Žilinskaitė S. /14/
Žižytė M. /24/27/
Žukauskas A. /14/
Žukauskienė J. /16/17/29/30/

The abstracts have been printed as received, and no proofreading or corrections
have been made. The contents of the abstracts are entirely the responsibility
of the authors.
Abstraktų kalba neredaguota.
Kompiuteriu maketavo Larisa KAZLAUSKIENĖ
SL 1070. 2011 10 20 2,5 sp. 1. Tiražas 100 egz.
Išleido Lietuvos agrarinių ir miškų mokslų centro filialas
Sodininkystės ir daržininkystės institutas,
Kauno g. 30, LT-54333 Babtai, Kauno r.
Spausdino UAB „Mudu2“ Studentu g. 12-64,
LT-50243 Kaunas
Užsakymo Nr. 9634