Transformation and regeneration of creeping bentgrass (Agrostis palustris Huds) protoplasts

An efficient plant regeneration system from protoplasts of creeping bentgrass (Agrostis palustris Huds.) cultures is essential for gene transfer to these grasses through direct DNA uptake into protoplasts. A simple and efficient plant regeneration system for protoplasts isolated from embryogenic suspension cultures of seven creeping bentgrass cultivars was established. Suspension cultures were derived from embryogenic callus cultures established from surface-sterilized mature seeds. Four creeping bentgrass cultivars were tested to determine their effect as feeders on plating efficiencies, callus development, and regeneration of protoplasts. Embryogenic suspension cultures from all four cultivars worked as feeders with plating efficiencies ranging from 0.05 to 0.32%. Protoplast derived calli formed within 3 wk after isolation and were regenerated with, or without, the addition of cytokinin to the regeneration medium, Some cultivars required the use of a particular feeder to regenerate plants, Plants were regenerated from all the cultivars tested. The bar gene, which confers resistance to the herbicide bialaphos [2-amino-4-(hydroxymethylphosphinyl)butanoic acid], was transformed into protoplasts by means of either PEG or electroporation. Bialaphos-resistant colonies were obtained from Eve creeping bentgrass cultivars. Resistant colonies of the cultivar Cobra were regenerated into plants, One hundred fifty-three Cobra transformants analyzed were resistant to five times the field rate of commercial formulation of the herbicide, Molecular characterization of the transformants revealed the stable integration of the bar gene into the genome and expression of transcripts corresponding to the bar gene.