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 replacing the uidA (β-glucuronidase, GUS) gene in a uidA-bar construct with sgfp. A particle inflow gun was used to propel tungsten particles coated with this plasmid into immature inflorescence-derived embryogenic callus of switchgrass (Panicum virgatum L.). GFP was observed in leaf tissue and pollen of transgenic plants. Nearly 100 plants tolerant to Basta were obtained from the experiments, and Southern blot hybridization confirmed the presence of both the bar and sgfp genes. Plants regenerated from in vitro cultures of transgenic plants grew on medium with 10 mg l^-1 bialaphos. When the pH indicator chlorophenol red was in the medium, the transgenic plantlets changed the medium from red to yellow. Basta tolerance was observed in T₁ plants resulting from crosses between transgenic and nontransgenic control plants, indicating inheritance of the bar transgene.