Book of Abstracts - Geyseco

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P05 Biotechnology P05-001: GENETIC DIVERSITY STUDIES OF COARSE AND FINE RICE USING RAPD MARKERS Iqbal, M.* - Iqbal, A. - Saeed, A. - Nisar, A. CABB, University of Agriculture, Faisalabad, Pakistan *Corresponding author e-mail: ahsanuaf2003@yahoo.com The availability of genetically diverse gene pool is vitally important in the varietal development program. Molecular Markers are being extensively utilized to explore the genetic diversity among native and exotic germplasm. This study was designed to reveal the genetic diversity and patterns of relationships among the 20 accessions/genotypes representative of Basmati and Non-Basmati Rice from the existing rice gene pool using RAPD (Random Amplified Polymorphic DNA) Markers. Employing RAPD, Thirty (30) decamer oligonucleotide primers were used to estimate the genetic diversity. Out of these thirty primers, seventeen (17) primers give the polymorphic results and produced a total of 116 fragments, out of which 101 were polymorphic (87.06%) while 15 fragments were monomorphic (12.93%). Similarity coefficients had ranged from 0.47 to 0.90. The average genetic similarity was calculated 0.68 (68%). In this study, the Coarse rice genotypes showed more polymorphism (85.84%) than the Fine rice genotypes (61.76%). Genotypes were clustered into 8 distinct groups: A, B, C, D, E, F, G and H but two genotypes i.e. Shadab and Kangni-27 showed divergence from all the genotypes of the groups. So, these diverse genotypes should be included in the breeding programme. P05-002: VARIATIONS IN BARE-1 INSERTION PAT- TERNS IN BARLEY CALLUS CULTURES Gozukirmizi, N.* - Evrensel, C. – Yilmaz, S. – Temel, A. Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics *Corresponding author e-mail: nermin@istanbul.edu.tr Stability of aging barley calli was investigated with BARE-1 transposon specific Inter-Retrotransposon Amplified Polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS (Murashige and Skoog) medium supplemented with 3 mg/L 2,4-D and maintained on same medium for 30, 45 and 60 days. Ten IRAP primers were used in 25 different combinations and as a result, the variation level of DNA isolated from 30-, 45- and 60-days old calli, have been found to change 14-25%, depending on the mature embryo material and the age of callus. While the similarity level between 30- days and 45-days old calli is 84%, it is 79% between 30-days and 60-days old calli and it is 76% between 45-days and 60-days old calli. As a result, culture conditions cause genetic variations and there are also evident BARE-1 transposon alterations. The findings are expected to contribute to genetic engineering studies to get better results and also to understand, how transposons contribute to features like tissue culture – especially callus tissue – formation and the ability of regeneration thereof. To our knowledge, this is the first report of employment of IRAP technique in barley in terms of callus development. Keywords Hordeum vulgare L.- Transposon - Tissue culture – BARE-1 – Inter Retrotransposon Amplified Polymorphism P - Posters P05-003: METABOLIC ENGINEERING OF POTATO (SO- LANUM TUBEROSUM) FOR ENHANCED ASCORBIC ACID (VIT C) ACCUMULATION Upadhyaya, C.P.* - Hemavathi – Nookaraju, A. – Mayank, A.G. – Park, S.W. Department of Molecular Biotechnology, College of Life & Environmental Sciences, Konkuk University, Seoul, Korea *Corresponding author e-mail: cpupadhyay@gmail.com Potato (Solanum tuberosum L.) is an important vegetable crop which ranks fourth among the staple foods of mankind after wheat, rice and maize. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. The potato (Solanum tuberosum L. cv. Taedong Valley) was engineered for enhanced ascorbic acid (Vit C) ovr-accumulation which also leads to increase in tolerance for various abiotic stresses. A gene encoding D-galacturonic acid reductase (GalUR) isolated from strawberry and l-gulono-γ-lactone oxidase (GLOase gene), from rat cells under the control of CaMV 35S promoter was introduced in potato plants resulted in 1.6-3.0 folds increase in AsA in transgenic potato. Integration of the GalUR and GLOase gene in the plant genome was confirmed by PCR, RT-PCR and Southern blotting. Ascorbic acid (AsA) levels in transgenic tubers were determined by highperformance liquid chromatography (HPLC). The increases in levels of AsA were positively correlated with increased GalUR and GLOase activity in both kinds of transgenics. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen (MV), NaCl or mannitol as compared to control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of GalUR or GLOase gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. P05-004: ANIONIC SOYBEAN PEROXIDASE AND ITS APPLICATION IN ENZYME IMMUNOASSAY Sakharov, I.* M.V.Lomonosov Moscow State University *Corresponding author e-mail: sakharovivan@gmail Cationic horseradish peroxidase (HRP) is usually used in сhemiluminescent enzyme immunoassay (CLEIA), one of highly sensitive analytical methods. However, HRP has drawbacks connecting with quick decay of CL signal and, in some cases, insufficiently high sensitivity. We demonstrated that anionic plant peroxidase isolated from soybeans (SbP) is more effectively biocatalysts than HRP-C and is able to oxidize luminol in the absence of enhancers. In addition, unlike HRP-C, SbP produces a long-term CL signal. Although SbP is a more potent biocatalyst in luminol oxidation than HRP-C itself, in the presence of enhancers HRP-C produces a higher CL intensity than SbP. On the other hand, HRP-C enhancers do not practically increase SbPinduced CL. At screening of some phenothiazines we showed that 3-(10’- phenothiazinyl)propane-1-sulfonate (SPTZ) is a first potent enhancer of CL induced by anionic SbP. Also, the simultaneous introduction of SPTZ and 4-morpholinopyridine (MORP) in the substrate mixture resulted in an additional increase of CL as well as a decrease of the lower detection limit (LDL) of SbP. The SbPcatalyzed chemiluminescent signals in presence of SPTZ and SPTZ/MORP are long-term. At comparison of the three fully optimized systems, SbP–SPTZ– MORP versus HRP-C–SPTZ– MORP versus HRP-C– PIP, it demonstrated that the SbP system possessed significantly higher sensitivity and lower LDL value. The SbP-SPTZ-MORP system was successfully employed in CL-EIA for determination of human thyroglobulin, one of mar- P
FESPB 2010 - XVII Congress of the Federation of European Societies of Plant Biology kers of thyroid gland cancer. These results open up very promising perspectives for using the SbP-SPTZ-MORP system in ultra-sensitive immunoassays. P05-005: CALCIUM REGULATES TUBERIZATION IN POTATO THROUGH ENHANCED EXPRESSION OF CALMODULIN, CALCIUM DEPENDANT PROTEIN KI- NASE AND LIPOXYGENASE Won, P.* - Nookaraju, A. – Upadhyaya, C.P. - Chun, S.C. - Kim, D.H. Department of Molecular Biotechnology, Konkuk University, Seoul, Korea *Corresponding author e-mail: sewpark@konkuk.ac.kr Calcium plays an important role in plant physiology and various plant cell signaling pathways, and alters many biochemical processes by activating specific enzymes. Cytosolic Ca 2+ regulates the activity of different Ca 2+ dependant protein kinases, which specifically phosphorylate various key metabolic enzymes. In the present study, the expression of two Ca 2+ regulator proteins, calmodulin (CaM1) and Calcium Dependant Protein Kinase (StCDPK; EC 2.7.1.37), as well as the lipoxygenase (LOX; EC 1.13.11.12) were studied upon Ca 2+ application to the single node segments of potato cv. Desiree inoculated for tuberization. Calcium at higher levels (6 mM) significantly improved the tuber number, tuber growth and tuber yield under in vitro conditions. The mRNA transcript expression levels of the CaM1, StCDPK and LOX were found to be significantly higher in stolons showing positive correlation with supplemental Ca 2+ and tuberization response. Similar trends were observed with LOX enzyme activity, which has increased by 18% and 25% with the addition of Ca 2+ at 3 and 6 mM, respectively to the tuber induction medium, when compared to control. The present study reports that the increase in tuberization, tuber growth and tuber yield with the supplementation of Ca 2+ could be attributed to the increased expression of the Ca 2+ dependant proteins and enhanced lipoxygenase activity that played an important role in tuberization in potato. P05-006: MOLECULAR INVESTIGATION OF TERPENE SYNTHASE GENES FROM SALVIA FRUTICOSA AND SALVIA OFFICINALIS Bacu, A.¹* - Makris A.¹ – Owen, C.² ¹Department of Biotechnology, Faculty of Natural Sciences, University of Tirana, Albania ²Maich, CIHEAM, Greece *Corresponding author e-mail: a_bacu@yahoo.com The aim of this study was to investigate the extent of variation in monoterpene synthase genes in the sage plants Salvia fruticosa and Salvia officinalis. Gene fragments from four different but closely related monoterpene synthase genes from Salvia fruticosa and three from Salvia officinalis were cloned. This was achieved by using a PCR amplification and cloning strategy based on similarities between previously reported monoterpene synthase genes from different plant species. The isolated gene fragments were sequenced and compared with each-other and with previously isolated genes. The results of sequence analysis showed that the isolated clones represented novel sequences derived from monoterpene synthase genes, which had not been previously described. Subsequently, efforts were made to isolate the full-length gene sequence of the most interesting Salvia fruticosa clone. In order to produce monoterpe synthase-specific polyclonal antibodies fusion proteins were produced from two of the clones. To achieve this, subfragments of the two genes corresponding to open reading frames were PCR amplified and then cloned into the vector pBAD-TOPO. The His-tag produced fusion proteins have been used for the production of polyclonal antibodies by immunizing rabbits. It is anticipated that these antibodies will be of value to demonstrate the expression patterns of these genes. P05-007: UPTAKE AND ACCUMULATION OF CD IN TWO WILLOW CLONES EXPOSED TO ELEVATED CONCENTRATIONS OF CD, NI AND PB-EDTA Borisev, M.¹ – Pajevic, S.² - Nikolic, N.² - Krstic, B.² - Pilipovic, A.³ - Orlovic, S.³ ¹Department for Biology and Ecology, Faculty of Sciences, University of Novi Sad ²Department for biology and ecology, Faculty of Sciences, Serbia ³Institute for Lowland Forestry and Environment, Serbia *Corresponding author e-mail: milan.borisev@dbe.uns.ac.rs Two willow clones (Salix alba – clone 68/53/1 and Salix nigra – clone 0408) were exposed to elevated concentrations of Cd, Cd+Ni, Cd+Pb-EDTA and Cd+Ni+Pb-EDTA, each in two concentrations (10 –4 M and 10 -5 M) in water culture solutions. Cd content accumulated in the root, shoot and leaves was measured and analysed in order to determine phytoextraction potential of investigated genotypes. Results display supstantial amounts of Cd accumulated in aboveground tissues, proving considerable Cd phytoextraction possibilities. Inhibition of uptake and translocation of Cd was detected in the presence of Ni in the hydroponic solution indicating to antagonism between these two metals. Similar inhibiton effect in the presence of Pb-EDTA was less expresed because of the heavy metal helation by EDTA. Competition of Cd, Ni and Pb to same metal transporters and carriers is the probable cause of the detected antagonism. P05-008: A NEW STRATEGY TO ENHANCE THE PRO- DUCTION OF PHYTOSTEROLS IN DAUCUS CAROTA CELL CULTURES Sabater, A.* - Sabater-Jara, AB. – Almagro, L. - Belchí-Navarro, S. – Pedreño, M.A. Dpto Biología Vegetal. Facultad de Biología. Universidad de Murcia. Spain. *Corresponding author e-mail: asj00482@um.es We have developed a new innovative procedure to produce phytosterols from Daucus carota cell cultures (1). The method is based on elicitation using cyclodextrins and methyljasmonate (MJ), separately or in combination. MJ is a key compound of the signal transduction pathway involved in the production of secondary metabolites (2) and cyclodextrins are cyclic oligosaccharides which induce a cascade of cellular events that gives rise to the accumulation of some metabolites (3), in this case, phytosterols. This new strategy to enhance phytosterol production combines the addition of these inducing factors at their optimal concentrations, optimal UV irradiation dose and time of elicitation at an optimal culture growth stage of D. carota cells. The results open up a new strategy for producing phytochemicals with nutraceutical and medical applications. References 1.S abater-Jara, A.B. Almagro, L. Bru, R and Pedreño, MA. 2008. P200803107 2.Gundlach H, Müller MJ, Kutchan TM and Zenk MH. 1992. PNAS 89: 2389–2393 3.Bru, R; Sellés, S; Casado, J; Belchí-Navarro, S; and Pedreño, MA. 2006. J. Agric.Food Chem.54: 65- 71 Acknowledgements Sabater-JaraA, .B. and Almagro L. held a grant from the MI- CINN. This work has been partially supported by MICINN (BIO2008-02941), by Consejería de Educación, Ciencia e Investigación de la Región de Murcia (BIO BVA 07 01 0003) and, by Fundación Séneca (08799/PI/08). P05-009: EARLY SIGNALLING EVENTS IN RESPONSE TO METHYLJASMONATE AND CYCLODEXTRINS IN TOBACCO CELLS Pedreño, M.¹* - Almagro, L. – Pugin, A.² ¹Dpto de Biología Vegetal. Facultad Biología. Universidad de Murcia. Spain
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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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