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P - Posters legume grains whose proteins have a low level of methionine. Therefore, the At D-CGS was seed-specific expressed in soybean seeds. The level of soluble methionine increased up to seven-fold, while the methionine that was incorporated into the protein seeds increased up to 18-fold. The phenotype and germination rate did not alter in the transgenic seeds compared to the wild-type seeds. These results have shown new ways of elevating methionine content in seeds, thus enhancing their nutritional qualities. P05-024: AN IMPROVED PROTOCOL FOR TRANSFOR- MATION OF ANTIRRHINUM MAJUS Manchado Rojo, M.* - Portero Martinez, M.A. - Weiss, J. - Egea- Cortines, M. Universidad Politécnica De Cartagena *Corresponding author e-mail: maria.manchado@upct.es Genetic transformation is a cornerstone to obtain information of gene functions. We have developed an improved protocol for transformation and regeneration of Antirrhinum majus, obtaining a highly reproducible method that has yielded up to a high efficiency, close to 10% (the final results will be discussed in the congress). Several aspects affect transformation efficiency. We tested two lines, 165E and Vilmorin Nain and two different explants, leaf discs and hypocotyls from seedling of two and four weeks. As a proof of concept we transformed A. majus with a pHellsgate12 construct expressing RNAi of the homeotic gene Deficiens. Two week old hypocotyls explants from the line Vilmorin Nain had the highest transformed rate. Putative transformants were tested by PCR using NPTII primers and by their phenotypes. The resulting plants showed classic phenotypes corresponding to hypomorphic alleles of Def, which included sepaloid petals and sterile stamens and fell somewhere in strength between deficiens chlorantha and deficiens nicotianoides. P05-025: MAKING A TRANSGENIC RICE WITHOUT SE- LECTION Jung, H.* - Bang, S.W. – Kim, Y.S. – Kim, J.K. School of Biotechnology and Environmental Engineering, Myongji University *Corresponding author e-mail: harin0723@gmail.com Over the past several years, consumer and environmental groups have expressed concern about the use of antibiotic-and herbicideresistance genes against ecological and food safety perspective. Although no scientific basis has been established for these concerns, generating marker-free plants would certainly contribute to the public acceptace of transgenic crops. Here we present a technology that allows us to make transgenic plants without using selectable markers. This technology relies on an efficient Agrobacterium-mediated transformation method and PCR-based selection of transformants. After co-cultivated, transformed cells were allowed to regenerate on MS medium without any antibiotics or herbicides. In about 2-3 weeks after regeneration, a leaf disc of regenerated plants from one callus and was punched and pooled in one tube. We performed PCR with the leaf discs for the presence of transgene in the pools. We then select the positive pools and punched a leaf disc of individual rice plants in the positive pools. Second PCR from individual plants of the PCRpositive pools led us to identify transgenic plants. This series of process was referred to as the Clean T-DNA technology. Such transgenic rice plants are marker-free and further analyzed after grown in a paddy field. Thus, our Clean T-DNA technology may provide marker-free transgenic rice plants that can be used directly for commercial purpose P05-026: USSING OF TRANSGENOSIS FOR INDUCTION OF VIRUS RESISTANCE IN PE Hanacek, P.¹* - Rohrer, M.¹ - Reinöhl, V.¹ - Prochazka, S.¹ - Safarova, D.² - Navratil, M.² - Horacek, J.³ - Svabova, L.³ - Smykal, P.³ - Griga, M.³ ¹Mendel University in Brno ²Palacky University Olomouc ³Agritec, Research, Breeding & Services,Ltd. *Corresponding author e-mail: hanacek@mendelu.cz Viral diseases are one of the serious problems that can cause great economical loss during pea cultivation. In the Czech Republic Pea enation mosaic virus (PEMV) and Pea seed-borne mosaic virus (PSbMV) were the most frequently detected viruses in samples collected from inspected pea fields. Integrated plant protection is a modern trend against pest and diseases, and includes also creation of resistant varieties. Breeding for resistance could be used, but the process is lengthy and sometimes the resistance sources are not available. This goal can be achieved utilizing biotechnological tools, especially transgenosis. Here we present results of testing of this alternative approach – the use of genetic modification to achieve the induction of resistance to viral diseases by inverted repeats post-transcriptional gene silencing (IR-PTGS). For both PEMV and PSbMV we cloned sense and antisense CP cDNA between the 35S promoter and CAMV terminator that result in hairpin (hp)RNA. This dsRNA is processed by a dsRNase resulting in 21-23 nt siRNAs (small interfering RNA). Incorporation of the siRNAs into a nuclease complex that degrades ssRNA in a sequence specific manner induces viral resistance. Preparation and testing of plasmid vectors as well as results of testing of transgenic pea plants will be presented. This research was supported by a project of the Czech Ministry of Agriculture QI91A229. P05-027: PHYTOREMEDIATION OF STABLE CESIUM AND LEAD FROM SOLUTIONS BY CHENOPODIUM ALBUM Moogouei, R.¹*- Borghei, M.² - Arjmandi, R.¹ - Vosoughi, M.² ¹Department of Environmental Management, Faculty of Environment and Energy, Science and Research Branch, Islamic Azad University, Tehran, Iran ²Sharif University of Technology *Corresponding author e-mail: r_moogoui@iau-tnb.ac.ir Chenopodium album plants were tested for their potential to remediate stable cesium and lead from solutions in 15 d. Hydroponically grown Plants were exposed to CsCl and Pb(C 2 H 3 O 2 ) 2 solutions at three different concentrations (0.6, 2 and 5 mg l -1 ). When plants were incubated in CsCl solutions 68.08 ± 2.12%, 39.66 ± 3.48% and 56.37 ± 1.90% cesium was found to be remediated after 15d, respectively. Moreover more than 99% lead was removed from the Pb(C 2 H 3 O 2 ) 2 solution in all three concentrations after 15d. When both CsCl and Pb(C 2 H 3 O 2 ) 2 were supplemented together to the solution, 14.53 ± 1.62%, 47.25 ± 0.96% and 48.01 ± 1.43% cesium and 71.22 ± 0.25%, 94.31 ± 0.24% and 98.40 ± 0.05% lead were removed after 15d. The present study suggests that hydroponically grown Chenopodium album could be used as a potential candidate plant for phytoremediation Cesium and lead from solutions, however plants were found to be more efficient for the remediation of lead than cesium. P05-028: THE DEVELOPMENT OF TRANSGENIC FLAX WITH ENHANCED HEAVY METALS BINDING CAPABI- LITY Vrbová, M.¹* - Horacek, J.¹ - Vetrovcova, M.¹ - Macek, T.² - Griga, M.¹ ¹AGRITEC Ltd., Plant Biotechnology Department, Šumperk, Czech Republic ²Joint Laboratory of IOCB and ICT Prague, Institute of Organic Chemistry and Biochemistry CAS, Prague, and Department of Biochemi *Corresponding author e-mail: vrbova@agritec.cz Flax is an industrial crop utilized mainly for technical purposes and for this reason is a good candidate for phytoextraction of P
FESPB 2010 - XVII Congress of the Federation of European Societies of Plant Biology heavy metals (HM) from polluted soils. Because the HM accumulation by commercial flax varieties is not high enough, genetically modified flax with inserted heavy metal binding gene may improve heavy metal binding and detoxification capacity. Agrobacterium tumefaciens strain EHA 105 containing binary vector pBI-αMT with α-domain of metal-binding mammalian metallothionein α-MT (with potential for heavy metal detoxification) was used in the experiments. With the aim to obtain the genotype with high transformation ability, we screened 16 flax/linseed genotypes for sensitivity/recalcitrance to transformation. The genotype AGT-0917 was found as the most responsive one, and thus used for further experiments. The transgene integration in GUS-positive transformants was confirmed by PCR. The difference between stable (non-segregating) transgenic and non-transformed regenerants of T2 generation was observed in cadmium (Cd) effect on growth parameters in vitro as well as in Cd-accumulation by explants grown in vitro. The Cd-content in stems and roots from GM and non-GM flax plants grown in the field was determined. The results indicate enhanced heavy metals binding capability of transgenic flax with inserted α-MT gene. Acknowledgement: Authors thank for support of projects 1M06030, MSM 2678424601, MSM 6046137305 and Z 40550506 of the Czech Ministry of Education. P05-029: PROPAGATION AND CALLUS INDUCTION IN DIANTHUS ANTICARIUS SUBSP. SAORINII Agulló Antón, M.* - Bañón-Arnao, M. – Acosta, M. Universidad de Murcia. Department of Plant Biology (Plant Physiology) *Corresponding author e-mail: maagullo@um.es Dianthus anticarius subsp. Saorinii is a vulnerable species, whose few individuals are restricted to the Sierra de Almenara (SW Murcia, Spain). In this study, different biotechnology techniques are applied in an attempt to propagate and conservate this subspecie. Saorinii seeds were germinated in vitro on solid MS medium after being decontaminated. Root and stem node explants obtained from the in vitro seedlings were cultured on MS medium supplemented with different concentrations of 2,4-D and kinetin (KN). Callus induction, root and stem regeneration were assessed. The best response for callus induction was observed from stem node segments on MS medium containing 0.5 mgL -1 or more 2,4-D. No calli were observed in the absence of 2,4-D in either kind of explant and, in the case of root explants, no calli were observed at low concentrations (0.2 mgL -1 ), either. For stem node explants, balanced or high auxin/cytokinin (2,4-D/KN) ratios in the culture media seemed to be beneficial for callus formation. Root explants generally resulted in a lower callus formation than stem explants, and required higher auxin/cytokinin ratios. Regarding plant propagation, only the node segments produced shoots or complete plants. Root and shoot growth was specially evident from stem node explants when no 2,4-D was applied and the KN concentration was below 1 mgL -1 . In the case of root explants, high root elongation was observed when no 2,4- D was applied. The results described provide useful information for developing tools for the large-scale multiplication and conservation of germplasm of Dianthus anticarius subsp. Saorinii. ACKNOWLEDGEMENTS: Project CARMurcia/PEPLAN (S4 and S13) and CARMurcia-Dirección General de Patrimonio Natural y Biodiversidad (F.J. Sánchez Saorín). P05-030: PROTEINACEOUS ELICITOR OF INDUCED RESISTANCE FROM LEPTOSPHAERIA MACULANS Valentova, O- .Dinh Kim, P. – Sasek, V.² - Burketova, L.² ¹Institute of Chemical Technology Prague ²Institute of Experimental Botany AS CR *Corresponding author e-mail: olga.valentova@vscht.cz The efficiency of plant defence mechanisms against pathogens depends on the ability of the plant to recognise its effector molecules followed by signal transduction leading to the expression of pathogenesis related (PR) proteins. These proteins represent a group of de novo synthesized proteins, first described in plant tissues infected with pathogens but later on also in plants treated with various elicitors and chemical compounds. They are suggested to be in a tight correlation with disease resistance and/or tolerance, as well as with the level of systemic acquired resistence (SAR). Elicitors are molecules secreted by pathogen during microbial entry or derived from their cell walls capable to activate plant defence mechanisms. Cultivation media of Leptosphaeria maculans, fungus causing “blackleg” of oilseed rape (Basic napus) induce the expression of systemic acquired resistence marker genes (PR1 and WRKY 70) in cotyledons of B. napus plants. Analysis of extracellular proteins of cotyledons treated with cultivation media showed induction of at least two acidic chitinase isoenzymes. In biological assay we show that cultivation media induces resistance towards L. maculans. For further characterization the compounds with the elicitation activity were partially purified by dialysis, ultrafiltration, ion exchange and size exclusion chromatography. Digestion of the active fractions with trypsin, beta-glucosidase and alphaamylase indicated the proteinaceous character of the elicitor supported by its heat instability. Acknowledgement This study was supported by grants of Ministry of Agriculture no. QH 81201 and Czech Science Foundation no. 522/08/1581. P05-031: THE ROLE OF TS AND BAPT GENE EXPRES- SION IN THE TAXOL BIOSYNTHETIC PATHWAY IN ELICITED CELL CULTURES OF TAXUS BACCATA Moyano, E.¹* - Onrubia, M.¹ - Bonfill, M.² - Palazón, J.² - Cusidó, R.M.² ¹Departament de Ciències Experimentals i de la Salut (UPF) ²Facultad de Farmacia, Universidad de Barcelona *Corresponding author e-mail: elisabeth.moyano@upf.edu Biotechnological production of valuable secondary metabolites in plant cell or organ cultures is an alternative to their extraction from whole plant material. However, the use of plant cell factories has had only limited commercial success. The biotechnological production of the anticancer compound taxol and related taxanes has become a commercial reality for various companies, but the low productivity of Taxus cell cultures requires a wide use of elicitors. Here, we describe the action of methyl jasmonate (MeJ 100 mM) and vanadyl sulphate (VS 50 mM), as well as the joint action of both elicitors on a two-stage Taxus baccata culture. The elicitors were added when cells had adapted to the production medium, 8 days after being transferred from the growth medium and the transcript levels of the genes encoding taxadiene synthase (TXS) and baccatin III 13-O-(3-amino-3-phenylpropanoyl) transferase (BAPT) were determined by qPCR. The results were related to taxol and related taxane production in the selected T. baccata cell line. Elicitation with MeJ, VS or both caused quite marked changes in the total taxane production, varying according to the elicitor. Cell cultures treated with MeJ achieved the highest levels of total taxanes almost throughout the experiment. Regarding individual taxane production, MeJ significantly increased both taxol and baccatin III accumulation, but VS only activated taxol production to the same extent. Regarding gene expression, MeJ clearly increased the transcription level of the txs and bapt genes but the presence of VS in the medium only increased the accumulation of the bapt gene mRNA. These results suggest that the elicitors have different and probably interfering mechanisms of action in taxane biosynthesis. P05-032: A PLATFORM FOR HIGH LEVEL ISOPRENOID PRODUCTION IN NICOTIANA TABACUM van Deenen. N.¹* - Post, J.¹ - Schulze Gronover, C.² - Prüfer, D.¹ ¹Department for Plant Biochemistry and Biotechnology, Westphalian Wilhelm´s University Muenster, Germany
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FESPB 2010 - XVII Congress of the F
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PLENAR SESSION LECTUR
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PARALL SESSION LECTUR
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POST 04 POSTERS • Environmental S
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Author Index Aarts, M.G. S18-002 Ab
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Cattivelli, L. P01-031 Cawly, J. P1
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Gallego, B. P17-037 Gallego, P.P. P
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Kersten, B. P10-017 Keskin, O. P05-
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Miguel, C. S02-001, P01-122 Milhinh
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Ramiro, M. P09-018 Ramon, M P13-002
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Tanimoto, E. P01-045 Tarakanov, I.
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