Agrobacterium-mediated transformation and regeneration of cotton plants

Cotton (Gossypium hirstitum L.) was transformed by the EHA 10 1 strain of Agrobacterium tumefaciens harboring a binary vector pGA482GG plasmid carrying the marker genes for neomycin phosphotransferase II (nptII) determining resistance to kanamycin and beta-glucuronidase (GUS). The cotyledons, hypocotyls, shoot meristem tissue. and its segments taken from in vitro growing seedlings were used as explants. Explants were cultured in a Murashige and Skoog (MS) medium containing various hormone combinations to induce shoot regeneration. The highest frequency of shoot formation was obtained from the shoot meristem. After selection in the MS medium containing kanamycin (50 mg/l), these tissues were tested by histochemical GUS assav. Shoots regenerated from excised shoot incristems or their halves were cultured for 4-6 weeks to obtain rooted plants. which then produced fully-developed plants and seeds in pots. Genomic integration of the kanamycin-resistance gene was detected by the PCR analysis. Seed germination percentage was 95% after the F1 seeds of transgenic cotton plants were cultured on half-strength MS medium supplemented with 50 mg/l kanamycin. Thus a protocol for effective Agrobacterium-mediated genetic transformation of cotton was optimized.