Effect of culture conditions on Agrobacterium-mediated transformation in datura.

ABSTRACT

A two step selection procedure is described for high frequency transformation and regeneration of transgenic plants by coculture of leaf discs of Datura innoxia with Agrobacterium tumefaciens carrying binary vectors. Leaf discs were cocultured with disarmed A. tumefaciens vectors pGS Glucl, pGSTRN943, pGV2260 and pBI121, and subcultured on regeneration media containing kanamycin. Kanamycinresistant, putatively "transformed" callus and vegetative buds were isolated, and subcultured on media containing reduced amounts of growth regulators and kanamycin to induce shooting. Rooted shoots produced normal fertile plants. Transformation frequency was related to duration of preculture, co-culture, and the bacterial strain used. With pGS Glue 1, a 3 day co-culture resulted in 70% of leaf discs being transformed. Transformation was confirmed by histochemical test for GUS activity, by the ability of leaf discs to initiate callus and from NPTII test, and Southern blot analysis. Progeny of the transgenic plants showed Mendelian segregation for kanamycin resistance.