SRIVASTAVA, D.K.SHARMA, SHIKHA2016-11-152016-11-152013http://krishikosh.egranth.ac.in/handle/1/85685ABSTRACT An efficient protocol for in vitro plant regeneration and genetic transformation has been developed in pea (Pisum sativum L. var. Lincon) tissues. Hypocotyl, root, leaf and cotyledon were used as explants for in vitro plant regeneration studies in pea. The explants were excised from 10-12 days old in vitro grown seedlings and placed on shoot induction medium. High frequency shoot regeneration from hypocotyl (81.45%), root (83.53%) and cotyledonary node (72.76%) explants was obtained on MS + 4.5 mg/l BAP + 1.6 mg/l NAA, MS + 2.0 mg/l TDZ and MS + 4.5mg/l BAP + 1.8 mg/l IBA, respectively. MS medium supplemented with 0.20 mg/l IBA was found to be best for root regeneration (63.33%) from in vitro developed shoots. The pea plantlets were able to regenerate 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 pea. For genetic transformation, disarmed Agrobacterium tumefaciens LBA 4404 strain containing a reporter β- glucuronidase [uid A (gus)] gene in binary vector (pBI 121) system along with kanamycin resistance gene (nptII) for selection in both bacteria and plant was used for co- cultivation experiment to transfer uid A (gus) and npt-II genes in pea. After co-cultivation only the transformed cells were able to grow on selective shoot regeneration medium containing 50mg/l Kanamycin and 500mg/l cefotaxime, whereas non-transformed cells/explants turned brown to black and died on the selective medium. Pre incubation of 48 hrs and cocultivation of 48 hrs was found optimum as it gave maximum transgenic shoot regeneration on selective medium. The transformation frequency was low in hypocotyl explants (2.66%) as compared to root explants (3.55%) on selective shoot regeneration medium (50mg/l Kanamycin and 500mg/l cefotaxime). The putative transformants were randomly selected for the amplification of npt-II and gus genes with specific designed primer by polymerase chain reaction. Out of 10 putative transformed calli and 3 shoot, 6 calli and a shoot have shown the amplification of uid A (gus) genes. These positive samples were then further analysed by PCR using designed primers for npt-II genes. Out of 6, only 3 of the transformed calli showed the presence of npt-II genes in pea genome. The expression of gus gene was analyzed by using biochemical and histochemical techniques of GUS assay. The gus gene was expressed in the PCR positive transformed calli of pea. A protocol for genetic transformation in pea with npt-II and gus genes has been standardized.enpea (Pisum sativum L. var. Lincon),genetic transformationThesis