GENETIC-TRANSFORMATION OF ALFALFA SOMATIC EMBRYOS AND THEIR CLONAL PROPAGATION THROUGH REPETITIVE SOMATIC EMBRYOGENESIS

Genetically transformed alfalfa (Medicago sativa L., cv. Zajecarska 83 ) plantlets were obtained by inoculating somatic embryos with Agrobact erium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somat ic embryos, 5-7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidi fied culture medium contained mineral salts, vitamins, 40 g 1(-1) sucr ose, 1 g 1(-1) yeast extract and 0.05 mg 1(-1) BA. Five clones, transf ormed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, o n media containing 100 mg 1(-1) of kanamycin. The transformation of ka namycin-resistant clones was confirmed by assaying the activity of neo mycin phosphotransferase II and/or beta-glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/re generation system based on somatic embryogenesis may be suitable for e stablishing transgenic alfalfa Lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.