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STUDIES ON GENETIC VARIABILITY , SELECTION AND CLONING OF ELITE LINES OF PONGAMIA PINNATA – A POTENTIAL BIOFUEL FEEDSTOCK GARGIBALA SATAPATHY SRF, BIORESOURCES ENGINEERING DEPARTMENT Dr. M. Thirunavoukkarasu Principal Scientist Bioresources Engineering Department CSIR-IMMT, Bhubaneswar 751 013

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RATIONAL BEHIND THE STUDY Pongamia pinnata is an important oilseed feedstock that grows in the semi-arid regions. The seeds oil, which has been identified as a source of bio-fuel and has medicinal value. The seed content oil varies from as low as 18 to as high as 40%. P. pinnata is highly cross-pollinated crop and exists a vide range of variation. It has been reported that a single tree yields as low as 9 kg/tree to as high as 90 kg/ tree. Another problem faced by the farmers is that the seeds of P. pinnata suffer from germination and storage problems. Hence, the challenging task, as of today is to screen the naturally available P. pinnata genetic resources to select the best planting material for higher productivity.

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Collection and screening of P. pinnata germplasm across the State for the evaluation of oil yield. Studying the variability characters (phenotypic , genotypic and yield) and develop marker for early screening of elite genotypes through molecular marker technique. Standardize in vitro clonal propagation technique for mass production of elite clones. Develop clonal garden for future use. R & D SOLUTION

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Germplasm collection Yield analysis (oil) Phenotypic characters Marker for elite lines Assessment of genetic diversity at gene level In vitro Cloning Assessment of clonal fidelity using molecular marker techniques METHODOLOGY TO BE ADOPTED High Yield genotypes Clone bank

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METHODOLOGY GERMPLASM COLLECTION Collection of pods/seeds and leaf samples from different regions of the State. Study the morphological characters (shape, size and weight of the seeds and leaf characters. Computation of data.

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OIL YIELD ANALYSIS METHODOLOGY Grind the seeds of Pongamia Take 100gm of powdered seed Transfer to sonicator Add 250ml of hexane Pass it through a round-bottomed flask Attach the condenser to the sonicator adjust temp 35º-40 ºfor evaporation of solvent (Keep the cycle of evaporation for 4-5 times till get clear appearance) Keep it in the rotavapour for evaporation of solvent Collect Pongam oil Calculation of yield Dry wt of bottle =X gm Wt. of bottle with oil=Ygm Oil extracted (Y-X) gm % oil extracted =(Y-X)/100

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The fatty acid profile of pongam will be identified using a Gas Chromatography (GC). OIL YIELD ANALYSIS Contd …… METHODOLOGY GC- ANALYSIS OF PONGAM OIL

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Preserving DNA during field collection collect the leaf samples DNA extraction CTAB DNA extraction method Required reagents 2 X CTAB extraction buffer (warmed to 65°C) Chloroform-isoamyl alcohol (24:1) Isopropanol (keep in freezer) 70% ethanol 1X TE buffer polymerase chain reaction (PCR) Check DNA quality on 1% agarose gels, compare the concentration of DNA against standards of 10 ng/μl, 20 ng/μl and 100 ng/μl lambda DNA. GENETIC VARIABILITY STUDY USING MOLECULAR MARKER TECHNIQUE METHODOLOGY

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INTER SIMPLE SEQUENCE REPEAT (ISSR) ANALYSIS Analysis consists of two steps : 1, PCR amplification using semi-specific anchored primers, in which individual oligonucleotides function in both forward and reverse directions 2, product detection. Agarose gel electrophoresis and ethidium bromide staining DATA SCORING Data provided by ISSR, each locus is scored for an individual in a ‘binary’ way, as [1] or [0] (presence or absence of a product, respectively) Genetic diversity can be quantified in terms of the richness and evenness of distribution of polymorphisms within populations or other defined groups of individuals GENETIC VARIABILITY STUDY Contd…… METHODOLOGY

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IN VITRO METHODOLOGY Elite line of Pongamia Mature explants (Shoot tips & axillary buds) In vitro Seed germination Seedling explants (cot. Node, cotyledon, hypo & epicotyls) Multiple shoot induction Rooting, hardening & field establishment Clonal fidelity study

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SOME SUCCESS STORY IN THIS LINE Micropropagation from mature tissue Micropropagation from seedling tissue Successful plant regeneration from axillary bud explant from mature tree as well as from seedling explants was achieved. Germplasm were collected from Berhampur & Bhubaneswar for oil yield. Bhubaneswar germplasm showed 22 % oil and Berhampur variety had 20% oil. Further study is in progress to get high yielding variety from Orissa regions.

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SOME SUCCESS STORY IN THIS LINE DNA ISOLATION AND PCR AMPLIFICATION Good amount of DNA could be isolated from the fresh leaves sample. PCR amplification with RAPD primer

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PRACTICAL UTILITY OF THE RESEARCH OUTCOME Early evaluation of elite lines at seedling stage itself. Technology for cloning of elite varieties. Mass production of elite varieties in a shorter span of time. Possibilities obtaining somoclones . Clone bank establishment for future use

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THANK YOU