The use of the two T-DNA binary system to derive marker-free transgenic soybeans

A binary vector, pPTN133, was assembled that harbored two separate T-DNAs. T-DNA one contained a bar cassette, while T-DNA two carried a GUS cassette. The plasmid was mobilized into the Agrobacterium tumefaciens strain EHA101 . Mature soybean cotyledonary node explants were inoculated and regenerated on medium amended with glufosinate. Transgenic soybeans were grown to matu rity in the greenhouse. Fifteen primary transformants (T-0) representing 10 independent events were characterized. Seven of the 10 independent To even ts co-expressed GUS. Progeny analysis was conducted by sowing the T-1 seeds and monitoring the expression of the GUS gene after 21 d. Individual T-1 p lants were subsequently scored for herbicide tolerance by leaf painting a u nifoliate leaf with a 100 mg 1(-1) solution of glufosinate and scoring the leaf 5 d post application. Herbicide-sensitive and GUS-positive individuals were observed in four of the 10 independent events. Southern blot analysis confirmed the absence of the bar gene in the GUS positive/herbicide-sensit ive individuals. These results demonstrate that simultaneous integration of two T-DNAs followed by their independent segregation in progeny is a viabl e means to obtain soybeans that lack a selectable marker.