EVALUATION OF A COTYLEDONARY NODE REGENERATION SYSTEM FOR AGROBACTERIUM-MEDIATED TRANSFORMATION OF PEA (PISUM-SATIVUM L)

A rapid regeneration system was used for studies of Agrobacterium-mediated transformation in Pisum sativum L. Cotyledonary node explants were inoculated with Agrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS), beta-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R1 progeny. NPTII activity was observed in the R2 generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.