Plant Regeneration and Agrobacterium-Mediated Gene Transfer in Bell Pepper (Capsicum Annuum L.)

An investigation was carried out to standardize a protocol for plant regeneration and genetic transformation in bell pepper (Capsicum annuum L. cv. ‘California Wonder’) tissues. Cotyledon and hypocotyl were used as explants from in vitro grown seedlings. Using cotyledon as explant, high frequency of shoot regeneration was obtained on MS medium supplemented with 6.0 mg/l BAP and 0.3 mg/l IAA, whereas, in case of hypocotyl, the shoot regeneration was obtained on MS salts with B5 vitamins and 0.75 TDZ (Thidiazuron). Elongation of in vitro developed shoots was obtained on MS medium supplemented with 3.0 mg/l GA3. Root regeneration in in vitro developed shoots was obtained on MS medium supplemented with 0.5 mg/l NAA. Bell pepper plantlets were able to regenerate within two months. Genetic transformation studies were carried out using disarmed Agrobacterium tumefaciens LBA 4404 strain containing p-glucuronidase (gus) gene in binary vector pBI 121 along with kanamycin resistance gene i.e. neomycin phosphotransferase-ll (np-ll) for selection in both bacteria and plant. After cocultivation, only the transformed cells were able to grow on the selective medium. Transformed calli were selected on the selective medium containing 50 mg/l kanamycin. The presence of foreign gene (gus) was demonstrated by the expression of chimeric bacterial gene that encode β-glucuronidase. Most of the calli selected on the selective medium from cotyledon and hypocotyl explants were GUS positive.