Study On Factors Of Alfalfa Transformation With Atriplex Dimorphostegia NHX Gene Mediated By Agrobacterium Tumefaciens

Tissue culture technique of two alfalfa varieties (Medicago varia.cv.Xinmu NO.1 and Medicago sativa L.cv.XinJiang Daye) was researched from cotyledon-derived explants and established regeneration tissue culture system of alfalfa cotyledon. On the foundation of regeneration system for alfalfa tissue culture, the salt-tolerance gene Atriplex dimorphostegia NHX was transferred into alfalfa by Agrobacterium- mediated transformation. The results are as follows:(1) Regeneration tissue culture system was established.â‘ The medium of induction culture: MS+2,4-D 2mg·L^-1 +KT0.5 mg·L^-1, the callus ratio of Medicago varia.cv.Xinmu NO.1 and Medicago sativa L.cv.XinJiang Daye were 98.3% and 97.5% respectively;â‘¡The medium of differentiation culture: MS+6-BA 0.5 mg·L^-1 +NAA 0.1 mg·L^-1, the green shoots ratio and the regenerated plantlet ratio of Medicago varia.cv.Xinmu NO.1 were 38.4% and 19.3% respectively; the green shoots ratio and the regenerated plantlet ratio of Medicago sativa L.cv.XinJiang Daye on the medium were 43.3% and 22.5% respectively;â‘¢The medium of rooting culture: 1/2MS. The rooting ratio and transplanting survival ratio of Medicago varia.cv.Xinmu NO.1 were 45.7% and 78.3% respectively. The rooting ratio and transplanting survival ratio of Medicago sativa L.cv.XinJiang Daye were 62.6% and 74.2% respectively.(2) Agrobacterium-mediated Atriplex dimorphostegia NHX gene transformation system of alfalfa was established.Kanamycin restrained the callus regeneration when it exsisted in callus inducing period, so we delayded 15 days to select resistant callus and this method improved the transformation frequency. In the Agrobacterium- mediated gene transformation, the rate of resistant callus and Kan-resistant somatic embryo were used to optimize the parameters affecting transformation in the paper. The cotyledon was used as tested materials.The highest transformation efficiency was obtained when the bacterial cell density was OD600 = 0.4⁰.5 with10¹5minutes of infection. It was also higher to transfer efficiently under 3 days pre-culture , 4days co-culture and AS concentration with20³0 mg·L^-1. Carb optimum concentration was 200 mg·L^-1, Kan as 50 mg·L^-1 was for resistant callus and 10 mg·L^-1 for resistant somatic embryo. By PCR, the results showed that Atriplex dimorphostegia NHX gene had been integrated into genomic DNA of alfalfa.