FERTILE TRANSGENIC PLANTS FROM DIRECT GENE-TRANSFER TO PROTOPLASTS OFLOLIUM-PERENNE L AND LOLIUM-MULTIFLORUM LAM

We adapted and applied a method of direct gene transfer to protoplasts of the monocots Lolium perenne L. and Lolium multiflorum Lam. A total of 20 clones, which produced green transgenic plants expressing the t ransgenes GUS gene (uidA) and meomycin phosphotransferase II gene (NPT II), have been obtained in four independent transformation experiment s. A mild selection at the early stage of protoplast culture and a ste p-by-step callus selection strategy were employed to recover the trans formed clones. A transformation frequency of nearly 5 x 10(-6) (antibi otic-resistant clones/total protoplasts) was achieved, which varied wi th the plant speices, cell suspension lines, and plasmids used. The pr oportion of albino regenerants in the in the antibiotic-resistant clon es was higher than in the non-transformed clones from control experime nts. The integration of foreign DNA into the genomes of the transgenic plants was confirmed by Southern hybridization. The expression of the GUS reporter gene (vidA) was detected by histochemical staining and f luorometric assay in the gransgenic plants, and the gransgenes were tr ansmitted and expressed in the progeny of transgenic plants. These res ults demonstrate the high efficiency of the direct gene transformation in Lolium species.