High-frequency embryogenesis from cotyledons of bird's-foot trefoil ( Lotus corniculatus) and its effective utilization in Agrobacterium tumefaciens-mediated transformation

Abstract

Summary We have developed a rapid and simple procedure to regenerate bird's-foot trefoil (Lotus corniculatus L.) plants from hypocotyl and cotyledon expiants of 10 to 14-days-old seedlings. Both expiant types regenerated well on the same medium with only minimal interfacing callus formation but with different modes of morphogenesis. While hypocotyls responded primarily by forming shoot apices, cotyledons mostly underwent embryogenes is. Remaining on the original explantation medium, both structures developed into rootless shoots. The highest shoot regeneration frequency was obtained on B5-based medium containing 0.5 mg/L 6-benzylaminopurine with approximately 80% of all expiants forming on average 19 shoots in four weeks. Only two media were used in the entire regeneration process, the second being the rooting medium of half-strength B5 supplemented with 0.01 mg/L α-naphthaleneacetic acid. Rooting frequency and survival rate after potting were about 100%. This regeneration protocol has been successfully applied to Agrobacterium tumefaciens-mediated transformation. Cotyledon segments were used as acceptor tissue. The expiants were co-cultivated with A. tumefaciens strain LB4404 carrying the plasmid vector pBI121. This vector contains the neomycin phosphotransferase II gene (NPTII) and β-glucuronidase reporter gene (GUS), both under the control of the CaMV 35S promoter. Kanamycin-resistant plants regenerated within 45 days after transfer to selective media. On a selection medium containing 100 mg/L kanamycin, shoots were formed by 19.0% of the expiants. The histological GUS assay showed 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 cotyledon regeneration system transgenic birds-foot trefoil plants were obtained within 2–3 months.