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Agrobacterium mediated cry 1Aa gene transfer in punica granatum L. cv. Kandhari Kabuli

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(Agrobacterium mediated cry 1Aa gene transfer in Punica granatumL. cv. Kandhari Kabuli)

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2013
Kanwar, Kamlesh
DYSPU
Ph.D

The present investigation aims at “Agrobacterium mediated cry 1Aa gene transfer in Punica granatum L. cv. Kandhari Kabuli”. A valuable plant regeneration and genetic transformation protocol was developed for Punica granatum L. cv. Kandhari Kabuli using both mature and juvenile explants. Regeneration from mature explants (leaf and nodal segment) procured from 8 years old selected tree and juvenile (cotyledon and hypocotyl) explants excised from 14 to 15 days old in vitro germinated seedlings ofPunica granatum L. cv. Kandhari Kabuli was carried out through indirect and direct method. Calli were initiated from mature leaf, cotyledon and hypocotyl explants. The best media for callus induction from mature (leaf) and juvenile explants were MS medium supplemented with 20.0 µM NAA + 10.0 µM BA and MS medium supplemented with 12.5 µM NAA+15.0 µM BA, respectively. The highest percentage of callus was obtained from cotyledon (84.56%) explants followed by hypocotyl (77.71%) and leaf (76.72%) explants. The calli thus obtained from mature (leaf) and juvenile (cotyledon and hypocotyl) explants showed highest differentiation on MS medium supplemented with 8.0 µM BA + 2.5 µM kinetin + 2.5 µM NAA and MS medium supplemented with 11.0 µM BA + 2.5 µM NAA. The highest percentage of direct adventitious shoot bud induction from mature explants such as leaf (42.95%) and nodal segment (57.80%) was observed on MS medium supplemented with 10.0 µM BA + 2.5 µM NAA and MS medium supplemented with 9.0 µM BA. Solid MS medium supplemented with 9.0 µM BA + 8.0 µM kinetin + 2.5 µM NAA resulted in highest per cent shoot regeneration from both cotyledon (69.60%) and hypocotyl (58.80%) explants. Cotyledon explants was found to be most responsive explants for regeneration through direct as wells as indirect method. The adventitiousshoots obtained were rooted on ½ strength MS medium containing 500 mg l -1 activated charcoal. Sixty five percent of plantlets were successfully established in earthen pots containing soil and sand (1:1). Different explants such as leaf, nodal segment, cotyledon and hypocotyl were transformed using Agrobacterium tumefaciens strain EHA105 harbouring pBinAR-1Aa plasmid carrying cry1Aa (insect resistance gene) and neomycin phoshotransferase-II (nptII) marker through both indirect and direct standardized regeneration protocols. Factors affecting transformation frequency such as age of seedlings, pre-conditioning duration, wounding of explants, immersion duration, co-cultivation duration, presence of acetosyringone and osmoprotectants (betaine HCl and proline) and pH of infection and co-cultivation medium, explants type were studied. Both age of seedlings and pre-conditioning of explants affected putative transformation frequency. Co-cultivation of all the explants (wounded mildly with needle) for 2 days after immersion in Agrobacteriumsuspension for 5 minutes was found to be optimal for transformation. Inclusion of acetosyringone (100 µM) and osmoprotectants (12 µM betaineHCl and 10 µM proline) in the infection and co-cultivation medium (adjusted at 5.8 pH) led to an increase in putative transformation frequency. By applying optimized transformation conditions, the highest putative transformation frequency of 15.12% was obtained with cotyledon explants among all the explants through indirect organogenesis. On the other hand nodal segment showed highest putative transformation frequency of 9.08% as compared to all the other three explants through direct organogenesis. Efficient selection was obtained and escapes were prevented when kanamycin was used at 50 mg l -1 concentration. The putative transgenic shoots were rooted on selective rooting medium and hardened in plastic cups containing sterilized sand. PCR analysis showed integration of cry1Aa and npt-II gene in 55% of the putative transgenic plantlets. Subsequent RT-PCR analysis showed expression of cry1Aa gene in 100% of the PCR confirmed transformed plantlets. Signature of Major

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