Lysozyme-GFP dual gene in the transformation in callus of alfalfa.

Hypocotyls of 2 varieties of alfalfa(Gan-nong No.1 and Gan-nong No.3) were used as receptors respectively for transformation of Lyz-GFP genes by Agrobacterium tumefaciens mediated since December 2007.The callus containing dual genes of Lyz-GFP genes were harvested in this research.The results of fluorescence observation and PCR amplification showed that the Lys-GFP genes could express their function in alfalfa callus successfully.The effects of transformation of Lys-GFP dual genes in Gan-nong No.1 and Gan-nong No.3 were also studied in this paper,and better transformation conditions were established.A large number of resistant callus had been received.The results indicated that 75 mg·L-1 Kanamycin could restrain growth of alfalfa callus,300 mg·L-1 Cefotaxine could inhibit growth of Agrobacterium tumefaciens LBA4404.The optimum conditions for gene transformation in alfalfa were that hypocotyls were conducted to be precultured for 3-4 days,Agrobacterium tumefaciens whose concentration was OD600 0.4-0.5 was needed to inoculate alfalfa explants for 10 minutes.And MS medium supplemented with 2.0 mg·L-1 2,4-D,0.5 mg·L-1 KT,0.6% agar and 2.5% sucrose was needed to induce production of callus which resist Kanamycin from hypocotyls of alfalfa after these explants being incubated with Agrobacterium tumefaciens for 3 days.The induction rates of callus which resist Kanamycin of Gan-nong No.1 and Gan-nong No.3 were 35% and 32%,respectively.