| Get haploid alfalfa (Medicago sativa L.) can provide raw materials for homozygous tetraploid and the creation of doubled haploid population (DH). Study on anther culture of genetic variation have a great significance for the genetic stability and the integrity. Cytological identification, in the past using the squash method have high accuracy, but complete the determination of volume was very consuming time. Flow-cytometric was used for alfalfa testing, provides a precise and fast method.The main purpose of this study was anther culture homozygous tetraploid, the process involved the major aspects are as follows:1. Between different genotypes of alfalfa callus induction capacity vary considerably. Relatively low concentration of 2.4-D (0.5 mg / L) and (KT, BA) with the use of callus and bud formation, the most suitable combination of callus induction medium is NB +2.4-D 0.5mg / L + NAA 0.3mg / L +6- BA 0.5 mg / L + KT 3.0 mg / L + sucrose 6%. 30℃high temperature 48h-72h and 4℃low temperature of 24h on the best callus induction. BA and NAA when used with the callus induction of differentiation more easily, the suitable combination of differentiation medium is MS+BA 0.2mg/L+NAA 2mg/L(3mg/L); MS + sucrose 2% + agar 0.5% is the most appropriate medium for a subculture and rejuvenation of alfalfa plants. 1 / 2 MS (MS) + NAA 0.1mg / L +1% sucrose +0.5% agar is suitable for anther culture plants of the rooting medium.2. Chromosome variation in callus is very common, and software analysis obtained through callus culture time and the coefficient of variation of DNA content was positively related ; In the following generation of 0～12 weeks of this time is increasing rapidly after that with the increase in the number of subculture DNA varied cells, the increase in the percentage rate is gradual and stable.3. KT, NAA ,6-BA on alfalfa callus DNA mutagenic effect is not obvious. 2,4-D can cause DNA content variation and the processing time is the key factor.4. Genotypes related to the incidence of callus growth rate and embryonic callus ability. Analysis of the correlation coefficient r =- 0.3261 (p <0.01), shows between the growth rate of callus and differentiation of a certain correlation. Sucrose and lactose are well for the growth and differentiation of alfalfa callus. 40g/L, 20g/L, 60g/L sucrose in callus differentiation there is no significant difference. Low temperature treatment on callus help to improve the differentiation of callus, 4℃48h has the most prominent effect, and therefore the low temperature 8℃is not favorable for callus formation.5.Alfalfa callus on colchicine tolerance ability. Suspension medium low concentrations, 300mg / L colchicine for 12 weeks was the best deal. When high concentration of 1g/L,2g/L were for 48 hours is better. Through analysis of the correlation between the incubation time and the percentage of S stage cells, we get the best period of colchicines is 3 weeks concentrated for the cell division stage.6.The results of Flow-cytometric indicated that 42 plants were tetraploid plants , 5 plants were chimerics plants of diploid plus tetraploid, 2 plants were diploids plants, 1 plants were triploid plants.
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