SRIVASTAVA, D.K.AGGARWAL, GAURAV2016-06-222016-06-222012http://krishikosh.egranth.ac.in/handle/1/67721ABSTRACT Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in Himalayan poplar. Agrobacterium tumefaciens strain containing npt-II and cryIAa genes in binary vector pBin-1Aa was used for genetic transformation studies. Plant regeneration studies were carried out using two different types of explant viz. leaf and petiole. Leaf and petiole explants were procured from glass house grown plants of Populus ciliata. Petiole explants showed better shoot regeneration as compared to leaf explants. In leaf explants, the high frequency shoot regeneration (80.00%) was obtained on medium supplemented with 0.024mg/l TDZ + 79.7 mg/l adenine. Whereas, in petiole explants the high frequency shoot regeneration (85.70%) was obtained on MS medium supplemented with 0.044mg/l TDZ + 79.7 mg/l adenine. MS medium supplemented with 0.10 mg/l IBA was found best for root regeneration from in vitro developed shoots. The 198 regenerated plantlets were acclimatized on a mixture of sand and soil. Effect of different concentrations of cefotaxime was studied on the regeneration potential in leaf and petiole explants of Himalayan poplar. The maximum per cent (75%) and (78%) shoot regeneration were obtained on MS regeneration medium with 300mg/l cefotaxime in leaf and petiole explants, respectively. Effect of different concentrations of cefotaxime and kanamycin (50 mg/l) were studied on the growth of agrobacterial cells and regeneration potential of leaf and petiole tissues after cocultivation. In leaf and petiole explants the growth of agrobacterial cells were controlled at concentration of 400 mg/l cefotaxime and maximum per cent shoot regeneration 33.00% and 48.00% was obtained on MS medium supplemented with 400 mg/l cefotaxime, respectively. Preculturing of leaf explants for 48 hrs and co-cultivation with agrobacterial cells for 48 hrs worked out to be the best treatment as it gave the highest transformation frequency (42.86%) in leaf explants. Whereas in petiole explants 72 hrs. preculturing and 72 hrs. cocultivation was worked out to be the best treatment as it gave the highest transformation frequency (50.50%). In petiole explants effect of different concentrations of acetosyringone were studied to enhance the transformation frequency in Himalayan poplar. The maximum percent shoot regeneration (76.08%) was obtained from those explants cultured on shoot regeneration medium containing 100 μM acetosyringone at standardized preculturing and cocultivation time interval i.e. 72 hrs. The presence/integration of transgene (cry IAa) into the genome of Himalayan poplar was confirmed by PCR using gene specific primers and Southern blot analysis using radioactive labelled DNA probe. The Southern blot analysis has also been used to confirm copy number of transgene into the genome of Himalayan poplar. For PCR analysis, 48 putative transgenic shoots of Himalayan poplar were randomly selected and out of 48 putative transgenic shoots/plantlets, 16 shoots were found to be +ve for the integration of transgene i.e. cry IAa into the genome of Himalayan poplar. For Southern blot analysis 10 PCR +ve shoots were randomly selected out of 16 PCR +ve shoots. Out of the 10 PCR +ve shoots, 4 shoots were confirmed +ve for integration of transgene cry IAa into the genome of Himalayan poplar with 1 to 4 copies of gene insertion. The confirmation of expression of the transgene cry IAa into the genome of Himalayan poplar at transcriptional level was confirmed by Reverse Transcriptase PCR, Multiplex RTPCR and Real Time PCR. A protocol for high frequency plant regeneration and insect resistance gene transfer in Himalayan poplar have been standardized.en---poplar ,Genetic transformationThesis