SHARMA, S.K.DHIMAN, KARUNA2017-02-172017-02-172009http://krishikosh.egranth.ac.in/handle/1/5810001378ABSTRACT An efficient protocol for in vitro plant regeneration and genetic transformation has been developed in broccoli (Brassica oleracea L. var. italica cv. Solan green head) tissues. Hypocotyl and cotyledon were used as explants for in vitro plant regeneration studies in broccoli. The explants were excised from sterile germinated seedlings and placed on shoot induction medium. The hypocotyl and cotyledon explants showed high frequency of shoot regeneration (62.50% and 60.00 %) on MS medium supplemented with 2.5 mg/l BAP and 2.0 mg/l NAA and MS medium supplemented with 3.5 mg/l Kn and 0.1 mg/l NAA respectively. MS medium supplemented with 0.2 mg/l NAA was found to be best for root regeneration (90.00%). The broccoli plantlets were able to regenerate plantlets within 6-7 weeks. Regenerated plantlets were successfully acclimatized. The high frequency regeneration system served as an excellent tool for the establishment of an efficient transformation method for broccoli. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter 􀈕- 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. After co-cultivation only the transformed cells were able to grow on selective shoot regeneration medium (50mg/lt Kanamycin and 500mg/lt cefotaxime) whereas non-transformed cells/explants died on the selective medium. Transformation experiment could be scored as early as 3-4 weeks after selection. The effect of pre-culturing and co-cultivation was studied. Explants pre-cultured for 72 hours prior to co-cultivation for 48 hours resulted in improved transformation frequency. Putative transformed calli and shoot from hypocotyl explants obtained, which were able to grow on the selective medium containing 50mg/lt Kanamycin. The presence of npt- II and uid A (gus) was confirmed by Polymerase chain reaction using designed primers. Out of 4 putative transformed calli and 1 shoot, 2 calli and a shoot have shown the amplification of npt-II and uid A (gus) genes. Expression of uid A (gus) gene was analyzed in these transformed calli by GUS assay and two transformed calli which showed presence of npt-II and uid A (gus) genes were also GUS positive. A protocol for plant regeneration and genetic transformation in broccoli tissues has been standardized.en---Thesis