Study On High Efficient Regeneration System Of Alfalfa Mutant Induced By N～+ Ion Beams

The experiment after implanted seeds of two alfalfa (Medicago varia.cv.Xinmu NO.1,Medicago sativa L.cv.XinJiang Daye) varieties by N+ at the energy of 35keV between 0×1016 N+/cm2 to 12×1016 N+/cm2. Tissue culture technique of two alfalfa varieties was researched from hypocotyl explants and established a high efficient N+ mutant regeneration tissue culture system of alfalfa. It was also higher efficient to transferring gene to alfalfa and offerring credible data of experiment. The research results are as follows:(1) Establishment of regeneration tissue culture system in different hormone combined medium.①The medium of induction culture: MS+2,4-D2mg/L+NAA4mg/L+6-BA1mg/L(Medicago varia.cv.Xinmu NO.1), the callus ratio is 94%. MS+2,4-D2mg/L+NAA8mg/L+6-BA1mg/L (Medicago sativa L.cv.XinJiang Daye), the callus ratio is 92.3%;②The medium of multiplication culture: MS+2,4-D2mg/L+NAA4mg/L+6-BA1mg/L+ABA1mg/L+KT2mg/L+LH400mg/L(Medicago varia.cv. Xinmu NO.1), the raise rate of callus is 1.86mm. MS+2,4-D2mg/L+NAA8mg/L+6-BA1mg/L+ABA 1mg/L+KT2mg/L+LH200mg/L (Medicago sativa L.cv.XinJiang Daye), the raise rate of callus is 2.05mm . Adding 4% sugar and 0.7 agar;③The medium of differentiation culture: MS+NAA0.5mg/L+ 6-BA0.1mg/L+KT1mg/L,the differentiation ratio of Medicago varia.cv.Xinmu NO.1 is 16.67% and Medicago sativa L.cv.XinJiang Daye is 34.78%. Adding 3% sugar and 0.8 agar;④The medium of rooting culture: 1/2MS, adding 2% sugar and 0.7 agar.(2) Established a high efficient N+ mutant regeneration tissue culture system of alfalfa①Under tissue culture condition,the median lethal dosage in Medicago varia.cv.Xinmu NO.1 and Medicago sativa L.cv.XinJiang Daye are 12×1016 N+/cm2;②Under the median lethal dosage, the somatoblast embryo intensity of Medicago varia.cv.Xinmu NO.1 is 44.55% and Medicago sativa L.cv.XinJiang Daye is 72.14% compared to CK. The seedlings form strengthen of Medicago varia.cv.Xinmu NO.1 is 40.00% and Medicago sativa L.cv.XinJiang Daye is 38.98% compared to CK.③We have selected 14 genotypes with high regeneration capability (4 genotypes come from Medicago varia.cv.Xinmu NO.1, 10 genotypes come from Medicago sativa L.cv.XinJiang Daye).The seedings were successfully transferred to soil and grew well.