Construction of a GFP-BAR plasmid and its use for switchgrass transformation

A dual marker plasmid comprising the reporter gene sgfp (green fluorescent protein) and the selectable bar gene (Basta tolerance) was constructed by r eplacing the uidA (beta -glucuronidase, GUS) gene in a uidA-bar construct w ith sgfp. A particle inflow gun was used to propel tungsten particles coate d with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and p ollen of transgenic plants. Nearly 100 plants tolerant to Pasta were obtain ed from the experiments, and Southern blot hybridization confirmed the pres ence of both the bar and sgfp genes. Plants regenerated from in vitro cultu res of transgenic plants grew on medium with 10 mg l(-1) bialaphos. When th e pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T-1 plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene.