Plant regeneration, genetic transformation and expression of gus gene in broccoli

Plant regeneration and genetic transformation studies were conducted to standardize a protocol for high frequency plant regeneration and Agrobacterium-mediated gene transfer in broccoli (Brassica oleracea L. var. italica). The hypocotyl (62.50%) and cotyledon (60.00%) explants showed high frequency of shoot regeneration on Murashige and Skoog (MS) medium supplemented with 2.5 mg/l 6-Benzylaminopurine (BAP) and 0.2 mg/l Naphthalene acetic acid (NAA) and MS medium supplemented with 3.5 mg/l Kinetin and 0.1 mg/l NAA respectively. Highest percentage of root regeneration (90.00%) in in vitro developed shoots was obtained on MS half strength basal medium supplemented with 0.2 mg/l NAA. Regenerated plantlets were successfully acclimatized. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter b-glucuronidase [uid A (gus)] gene in binary vector (pBI 121) system along with kanamycin resistance gene (npt-II) for selection in both bacteria and plant was used for co-cultivation experiment to transfer uid A (gus) and npt-II genes in broccoli. Putative transformed calli and shoot were obtained from hypocotyl explants on the selective medium containing 50mg/l kanamycin. The presence of npt-II and uid A (gus) genes were confirmed by polymerase chain reaction and expression of uid A (gus) gene was analyzed by expression of a chimeric bacterial gene that encodes b-glucuronidase.