Agrobacterium-Mediated insect resistance gene (cryIAb) Transfer studies in cabbage CV. pride of India

Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAb) transfer in cabbage using Agrobacterium-mediated gene transfer technique. Agrobacterium tumefaciens strain containing npt-II and cryIAb genes in binary vector pbinbt1 was used for genetic transformation studies. high frequency plant regeneration protocol was standardized using two types of explants viz. cotyledon and hypocotyl. these explants were used from seven days old aseptically grown seedlings. hypocotyl explants showed better shoot regeneration response (92.50%) as compared to cotyledon (80.0%) on MS medium supplemented with 0.1 mg/l IBA was found best (100%) for root regeneration. Regenerated plantlets with well developed root and shoot system were acclimatized in the mixture of sand: soil: FYM (1: 1: 1). ninety per cent of the plants survived during acclimatization. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of tissues (segments of cotyledon and hypocotyl) and to select transformed cells and transgenic shoots during transformation experiment. Kanamycin sensitivity (10–50 mg/l) was checked by fresh weight of the explants which was measured at the interval of 7 days till 35 days. From the relative growth of the explants, it was found that the concentration as low as 10mg/l is toxic to the explants. After cocultivation, explants were transferred to selective shoot regeneration medium for selection. Effect of pre-incubation and cocultivation times was studied on the transformation frequency in cotyledon and hypocotyl explants of cabbage. Pre-incubation of 72 hours and cocultivation of 48 hours was found optimum as it gave maximum transgenic shoot regeneration on selective medium. twelve putative Bt transformants were randomly selected and subjected for molecular analysis. PcR with cry IAb gene specific primers amplified 1.0 kb fragments from genomic DNA of kanamycin resistant shoots, thereby indicating the presence of transgenes in the regenerated plantlets. out of 12 putative transgenic plantlets, only 4 plantlets showed the amplification of the gene cryIAb in the genome of transgenic plantlets.