Optimization Of Subculture And Differentiation And Regeneration For Alfalfa Anther Callus Culture

The experimental materials is alfalfa anther, induced callus, and study optimization of culture conditions for subculture and differentiation and regeneration. The experiments also studied preliminary ploidy identificationthe of alfalfa chromosomes by flow cytometry method. It provide a scientific basis to improve the construction of alfalfa DH. The results are as follows:①. The alternation of Ms and N6 was more effective medium than using single macroelement in subculture, and it is the better increment and colour of callus.②The addition amount of auxin is 2mg/l in the 1-5th generation subculture . It is generally 0～0.5 mg/l in the increment culture stage. It is 1.5-2.5 mg/l subculture in last. It is the optimum combination of auxin to add to AC(0.8g/l) for preventing browning in 6-10th generation subculture.③LH is not only increase biomass of callus but also prevent browning to add to 0.8-1g/l. .④Cytokinin will reduce concentration in time when callus growed loose green dot in the differentiation culture, and it should be less than 2.0 mg/l, Otherwise callus easy to browning or malformed seedling.⑤The results show that the orthogonal of macroelement and hormone, The difference of increased volume was generally significant(F﹥3.096,P﹤0.05)to 2,4-D and macroelement in callus growth. Weakly and strong light has little effect. The difference of 6-BA,LH and AC change significantly to different light. Requirement of callus was different obviously to components of medium under different light conditions.⑥The optimal medium was 1/2MS+0.01-0.05 mg/lNAA+1g/l LH+20g/l sucrose+6.5g/l agar for inducing root.It is the best regenerated plant for transplanting whose basal part have strong roots and no callus..⑦Ploidy analysis of callus and regenerated plant chromosomes by flow cytometry shows that there has little correlation between ploidy of callus and times of subculture.