Studies on Agrobacteriummediated insect resistance gene transfer in Cabbage (Brassica oleracea L. var. capitata) and molecular analysis of regenerated plantlets
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Date
2013
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YSPU
Abstract
Genetic transformation studies were carried out to standardize a protocol for insect resistance gene (cryIAa) transfer in cabbage (Brassica oleracea L. var. capitata cv. Pride of India). 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. cotyledon, hypocotyl, leaf and petiole Cotyledon and hypocotyl explants were used from seven to nine days old aseptically grown seedlings whereas, leaf and petiole explants were procured from glass house 20-25 days old grown seedlings of cabbage. Hypocotyl explants showed better shoot regeneration as compared to other explants. High efficiency shoot regeneration was obtained in cotyledon (91.11 %), hypocotyl (94.44 %), leaf (91.11 %) and petiole (88.88%) explants on MS medium supplemented with 0.33mg/l TDZ + 79.7 mg/l Adenine, 0.22mg/l TDZ + 0.088mg/l IAA, 0.22mg/l TDZ + 0.02mg/l NAA and 0.33mg/l TDZ + 0.02mg/l NAA, respectively.MS medium supplemented with 0.10mg/l NAA was found best for root regeneration from in vitro
developed shoots. The regenerated plantlets were acclimatized successfully on cocopeat. Kanamycin sensitivity experiment was carried out to study the effect of antibiotic on relative growth of leaf and petiole tissues and to select transgenic shoots during transformation experiment. Kanamycin sensitivity (10-60mg/l) was checked by fresh weight of the 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 cotyledon and hypocotyl explants of cabbage and found no much effect of cefotaxime on regeneration potential. Effect of different concentrations of cefotaxime and kanamycin (50mg/l) were studied on the growth of agrobacterial cells and regeneration potential of cotyledon and hypocotyl tissues after cocultivation. In both the explants the growth of agrobacterial cells were controlled at concentration of 400mg/l cefotaxime and maximum per cent shoot
regeneration in cotyledon (35.55 %) and hypocotyl (48.15 %) was obtained on MS medium supplemented with 400mg/l cefotaxime, respectively. Effect of preculturing and co-cultivation was studied on the transformation frequency. Preculturing of cotyledon and hypocotyl explants for 72 hours and co-cultivation with agrobacterial cells for 48 hours worked out to be the best treatment as it gave the highest transformation frequency (4.66 %) and (14.50 %) in respective explants. Effect of different concentrations of acetosyringone was studied in cotyledon and hypocotyl explants to enhance the transformation frequency. The maximum percent shoot regeneration (18.66 %) and (32.00 %) was obtained from cotyledon and hypocotyl explants cultured on shoot regeneration medium containing 100μM acetosyringone at standardized preculturing and cocultivation time interval i.e. 72 hours and 48 hours. The presence/integration of transgene (cryIAa) into the genome of cabbage 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 cabbage. For PCR analysis, 40 putative transgenic shoots were randomly selected and out of 40 putative transgenic shoots/plantlets, 20 shoots were found to be +ve for the presence/integration of transgene i.e. cryIAa into the genome of
cabbage. For Southern blot analysis 10 RT-PCR +ve shoots were selected. Out of the 10 PCR +ve shoots, 5 shoots were confirmed +ve for integration of transgene cryIAa into the genome of cabbage with 1 to 3 copies of gene insertion. The confirmation of expression of the transgene cryIAa into the genome of cabbage 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 cabbage (Brassica oleracea L. var. capitata cv. Pride of India) has been standardized.
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Keywords
regeneration, cabbages, genes, planting, genetic processes, antibiotics, transgenics, concentrates, auxins, bacteria