Anther Cultures Of Alfalfa And The Ploidy Determination Of Regenerated Plants

Medicago varia Martin. CV. Caoyuan No.1 is a perennial tetraploid plants of Medicago L., comes from Medicago sativa and Medicago falcata L. breeding of hybrids, in order to utilization of heterosis better, to selection the homozygous parents, homozygous parents can show stronger heterosis, anther tissue culture is the fastest and the most effective way to cultivate the homozygous parents.This study use the male sterile alfalfa’s anther as the explant, anther culture, through the sugar, pH, the ratio of different hormone concentration, optimization of alfalfa callus induction medium and differentiation medium. And to identification of regeneration plant of ploidy, first through indirect identification (plant height, stem thick, regeneration speed, SPAD) clustering screening, The result may be haploid plants used root somatic chromosome number and SSR molecular markers do next research. The main results were as follows:(1) By 4℃ pretreatment with bud 24h, anther callus formed suitable culture medium is:1 mg/L 2,4-D+0.25 mg/L 6-BA+0.2 mg/L NAA+1 mg/L KT+5%sugar+ 0.7%agar, pH was 5.8,the callus induction rate was 80%. The suitable differentiation media was:0.5 mg/L KT+0.2 mg/L NAA+5%sugar+0.7%agar, pH was 5.8,the callus induction rate was 25%.(2) The ploidy of 75 alfalfa anther culture regeneration plant were using indirect ploidy identification method of judging the haploid plant has 8, male fertile and male sterile flowers grow seedlings, each have 4 accounted for 10.7% of the total number. Combined with chromosome number identification results of preliminary judgment 4 plants were haploid or double haploid.(3) In each level appraisal method, the plant height, the number of stomatal guard cells combine chloroplast number analysis is more accurate.