| Tissue culture technique of two alfalfa culture(Medicago varia.cv.Xinmu NO.1,Medicago sativa L.cv.XinJiang Daye) was researched from leaf-derived explants and established consummate regeneration tissue culture system of alfalfa leaf. On the foundation of regeneration system for alfalfa tissue culture, the salt-tolerance gene AtNHX1 transferred to alfalfa by Agrobacterium-mediated transformation in this paper. The research results are as follows:(1) Regeneration tissue culture system establishment.â‘ The medium of induction culture: MS+2,4-D2mg/L+NAA1mg/L +KT1 mg/L, the callus ratio of Medicago varia.cv.Xinmu NO.1 on the medium was 100%, and the callus ratio of Medicago sativa L.cv.XinJiang Daye on the medium was 95.5%;â‘¡The medium of multiplication culture: MS+2,4-D2mg/L+NAA1mg/L+KT1mg/L+CH0mg/L;â‘¢The medium of differentiation culture: MS+KT4mg/L;â‘£The medium of rooting culture: 1/2MS. With 3% sugar and 0.7% agar in all above of mediums. Regenerative plant came into being and the heredity conversion operation of alfalfa regeneration system had been built up.(2) Agrobacterium-mediated AtNHX1 gene transformation system of alfalfa was established. In the Agrobacterium-mediated gene transformation, frequency of GUS+ positive explants, frequency of resistant callus that were used to optimize the alfalfa had been established. In the Agrobacterium- mediated gene transformation, frequency of GUS+ positive explants, frequency of resistant callus were used to optimize the parameters affecting transformation. In the optimization of every factor of the system, we had confirmed Kan as 50mg/L. Results showed the efficiency of transformation were varied greatly by bacterial concentration and infection time. The highest transformation efficiency was obtained when the bacterial cell density was OD600 = 0.5 with 20 minutes of infection. It was also higher to transfer efficiently under pre-culture 4 days and co-culture for 3days. |
PDF Full Text Request