Establishment And Development Of The Agrobacterium-mediated Genetic Transformation System Of White Clover (Trifolium Repens)

Many legume plants may produce the beneficial isoflavones , which are vital to the formation of nitrogen-fixing root-nodules and the disease resistance response of plants. White clover(trifolium repens), a high quality legume forage, however, has little isoflavones. To improve the isoflavone contents of such forage, an available strategy is to introduce gene(s) encoding the key enzyme(s) in the biosynthesis pathway of insoflavones. As part of the project to improve isoflavone contents of legume forage by introducing isoflavone synthase (IPS) gene, in this work, we have established the Agrobacterium-mediated transformation system of white clover.Agobacterium tumefaciens strain A311 carrying the plant tranfer vector pB1121, which contains the neomycin phosphotransferasell gene(nptll) and P -glucuronidase reporter gene (GUS) both under the control of the CaMV 35S promoter, was used in the establishment of the genetic tranformation of white clover. The factors affecting the transformation efficiency and regerneration efficiency of white clover have been investigated and optimized.To transform white clover, the cotyledon with petiole from 4~5days old seedlings were precultured for 2 days, and then co-cultivated with the A. tumefaciens strain A311 for 4-5 days. The cotyledon were co-cultivated, and then incubated on the regeneration medium (MB+1 mg. L-16-BA+0. 1mg. L-1NAA+5mg.L-1'AgNO3 containing 40mg. L1 kanamycin and400mg. L 'carbennicillin for 6 weeks, the transformed buds appeared on the explants obivously. the regeneration percentage of transformed buds was about 22. 6%. The transformed buds grew up into subculture rooting medium(1/2MB+0.1 mg. L1NAA) containing 30 mg. L1kanamycin and 400 mg.L1 carbennicillin. the percentage of transformation is about 6. 5%. So the established transformation system of white clover can be implied to transfer the IPS gene.