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082			_a630.28 _bHOM/AG
100			_aHomey Cheriyan
245			_aAgrobacterium -mediated genetic transformation in Black Pepper (Piper rigrum L.)
260			_aVellanikkara _bDepartment of Plant Breeding and Genetics, College of Horticulture _c2000
502			_bPhD
520	3		_aInvestigations on genetic transformation in black pepper (Piper nigrum L.), variety Panniyur-l , using Agrobacterium tumefaciens strain AGL-1; 1303 harbouring antibiotic resistant selectable marker genes (npt II and hpt IV) and GUS and GFP reporter genes were carried out in Plant Tissue Culture Laboratory, College of Forestry, Kerala Agricultural University, Vellanikkara, Thrissur, during the period from November, 1997 to August, 2000. For the collection of explants for transformation work and related studies, axenic seedlings of black pepper were raised. Of the different media used for this, double sterilised moist sand was found to be the best. Callus induction on cotyledonary leaf explants and callus growth was found to be maximum in half MS medium supplemented with 1.0 mg }"1 each of IAA and BA compared to other combinations ofIAA, BA and kinetin tried. Of the different growth regulator combinations tried in half MS medium for regeneration, only when callus cultured in half MS supplemented with 1.5 mg r' IAA + 1.0 mg rl BA were transferred to half MS supplemented with 1.0 mg rl each ofIAA and BA produced regeneration to the extent of three per cent. In order to produce embryogenic calli for somatic embryogenesis, zygotic embryos were cultured in liquid basal SH medium under different cultural conditions. Somatic embryos were suspected to be produced when embryo with endosperm were cultured in liquid basal SH medium on rotary shaker. However, embryogenic calli were not generated. Black pepper tissues, both leaf and callus, were found to be susceptible to kanamycin and hygromycin when freshly prepared antibiotic stocks were used and tissues subcultured at two week interval. Callus induction on leaf was completely inhibited at 50 mg rl of kanamycin and callus growth at 100 mg r'. Hygromycin at 10 mg rI completely suppressed callus induction on leaf and callus growth at 30 mg r'. Cefotaxime at a strength of 500 mg r' effectively killed the Agrobacterium tumefaciens strain AGL-l; 1303 in pure bacterial suspension cultures with an O.D.S6Omn = 1.0. Proline was found to Increase the rate of multiplication of A. tumefaciens strain AGL-l; 1303. Leaf transformation was carried out by varying different factors affecting transformation. None of the leaf explants showed callus induction in screening media. Cefotaxime at strengths of 500 and 1000 mg rI could not eliminate Agrobacterium effectively from the leaf tissue. Prolonged survival of the Agrobacterium in the tissues could be the reason for non-multiplication of transformed cells. Callus transformation was carried out varying different factors affecting transformation. Excessive exudation of phenols was noticed in all the treatments compared to the controls. Heavy overgrowth of Agrobacterium was seen on withdrawal of cefotaxime from the screening media. Cefotaxime at 500, 1000 and 1500 mg r' could not eliminate the Agrobacterium effectively from callus tissues. Ineffective elimination of Agrobacterium, the super virulent character of the strain and the hypersensitive response of the callus to bacterial infection and wounding could be the reasons for failure of transformed cells to multiply.
700			_aVijayakumar N K (Guide)
856			_uhttp://krishikosh.egranth.ac.in/handle/1/5810015102
942			_2ddc _cTH