HIGH-FREQUENCY EMBRYOGENESIS FROM COTYLEDONS OF BIRDS-FOOT TREFOIL (LOTUS-CORNICULATUS) AND ITS EFFECTIVE UTILIZATION IN AGROBACTERIUM-TUMEFACIENS-MEDIATED TRANSFORMATION

We have developed a rapid and simple procedure to regenerate bird's-fo ot trefoil (Lotus corniculatus L.) plants from hypocotyl and cotyledon explants of 10 to 14-days-old seedlings. Both explant types regenerat ed well on the same medium with only minimal interfacing callus format ion but with different modes of morphogenesis. While hypocotyls respon ded primarily by forming shoot apices, cotyledons mostly underwent emb ryogenesis. Remaining on the original explantation medium, both struct ures developed into rootless shoots. The highest shoot regeneration fr equency was obtained on BS-based medium containing 0.5 mg/L 6-benzylam inopurine with approximately 80 % of all explants forming on average 1 9 shoots in four weeks. Only two media were used in the entire regener ation process, the second being the rooting medium of half-strength B5 supplemented with 0.01 mg/L alpha-naphthaleneacetic acid. Rooting fre quency and survival rate after potting were about 100%. This regenerat ion protocol has been successfully applied to Agrobacterium tumefacien s-mediated transformation. Cotyledon segments were used as acceptor ti ssue. The explants were co-cultivated with A. tumefaciens strain LB440 4 carrying the plasmid vector pBI121. This vector contains the neomyci n phosphotransferase II gene (NPTII) and beta-glucuronidase reporter g ene (GUS), both under the control of the CaMV 35S promoter. Kanamycin- resistant plants regenerated within 45 days after transfer to selectiv e media. On a selection medium containing 100 mg/L kanamycin, shoots w ere Formed by 19.0 % of the explants. The histological GUS assay showe d that 7.0 % of the resistant shoots also expressed the GUS gene in a variety of tissues. The stable integration of this gene was confirmed by polymerase chain reaction (PCR) analysis. Using the embryogenic cot yledon regeneration system transgenic bird's-foot trefoil plants were obtained within 2-3 months.