Srivastava, D.K.Gaur, Ayesh2017-06-032017-06-032015http://krishikosh.egranth.ac.in/handle/1/5810016891Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in cauliflower (Brassica oleracea L. var. botrytis cv. Pusa Snowball K1). 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 four different types of explants viz. hypocotyl, cotyledon, petiole and leaf. High efficiency shoot regeneration was obtained in hypocotyl (96.00%), cotyledon (76.00%), petiole (76.00%) and leaf (56.00%) explants on MS medium supplemented with 0.44mg/l TDZ + 0.08mg/l IAA, 0.30mg/l BAP + 0.10mg/l NAA, 0.22mg/l TDZ and 0.77mg/l TDZ + 79.7mg/l Adenine, respectively. Hypocotyl explants showed better shoot regeneration as compared to other explants. MS medium supplemented with 0.10mg/l IBA was found best for root regeneration from in vitro developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat and were morphologically similar and normal. Kanamycin sensitivity (10-60mg/l) was checked by observing fresh weight of the leaf and petiole explants which was measured at the interval of 7 days. From the relative growth of the explants it was found that the concentration as low as 10mg/l is toxic to the explants. Effect of different concentrations of cefotaxime was studied on the regeneration potential in leaf and petiole explants of cauliflower and found not much effect of cefotaxime on regeneration potential. Effect of different concentrations of cefotaxime and kanamycin (20mg/l) were studied on the growth of agrobacterial cells and regeneration potential of hypocotyl and petiole tissues after cocultivation. In both the explants the growth of agrobacterial cells were controlled at concentration of 300mg/l cefotaxime and maximum per cent shoot regeneration in hypocotyl (22.60%) and petiole (13.30 %) was obtained on MS medium supplemented with 300mg/l cefotaxime, respectively. Effect of preculturing and co-cultivation was studied on the transformation frequency. Preculturing of hypocotyl and petiole explants for 48 hours and co-cultivation with agrobacterial cells for 96 hours worked out to be the best treatment as it gave the highest transformation frequency (4.28 %) and (3.28 %) in respective explants. Effect of different concentrations of acetosyringone was studied in hypocotyl and petiole explants to enhance the transformation frequency. The maximum percent shoot regeneration was obtained from hypocotyl (9.23%) and petiole (10.13%) explants cultured on shoot regeneration medium containing 100μM acetosyringone and 125μM acetosyringone respectively at standardized preculturing and cocultivation time interval. The presence/integration of transgene (cryIAa) into the genome of cauliflower was confirmed by PCR using gene specific primers and Southern blot analysis using radioactive labelled DNA probe. The expression of the transgene (cryIAa) in cauliflower at transcriptional level was confirmed by reverse transcriptase-PCR and Real Time-PCR and at translational level by Bioassay. A protocol for high frequency plant regeneration and insect resistance gene transfer in cauliflower (Brassica oleracea L. var. botrytis cv. Pusa Snowball K1) has been standardized.enregeneration, cauliflowers, genes, planting, genetic processes, fungi, antibiotics, transgenics, concentrates, auxinsThesis