Transgenic plants of Lolium multiflorum, Lolium perenne, Festuca arundinacea and Agrostis stolonifera by silicon carbide fibre-mediated transformation of cell suspension cultures

Seven Lolium multiflorum, one Lolium perenne, 12 Festuca arundinacea and six Agrostis stolonifera plants were regenerated following transformation with a hygromycin resistance gene and hygromycin selection, from cell suspension colonies treated with silicon-carbide whiskers. Transformation was confirmed by PCR and Southern blotting; the latter also showed that six of the L. multiflorum plants were independent transformants (insufficient molecular evidence was obtained for the seventh), nine of the 12 F. arundinacea plants were independent transformants, but that all the A. stolonifera plants were derived from a single transformation event. Most plants tested contained fewer than five integrated transgene copies. Transgene expression was confirmed by reverse transcriptase-PCR (RT-PCR). Of the one A. stolonifera and three L. multiflorum transformants regenerated after co-transformation with both the hygromycin resistance gene and the β- glucuronidase (gus A) gene, none were found to express GUS activity. L. multiflorum regenerants from older (14-16 week old) cell suspensions showed loss of female fertility, but analysis of the progeny from three plants showed that the transgenes were being inherited as a single dominant allele with a high frequency of transmission of hygromycin resistance.