Cultivation Of Alfalfa Haploid And Analysis Of The Hybrid Fertility And Heterosis Of Main Characters

The haploid plants were obtained by tissue culture of autotetraploid alfalfa anther(pollen).Many valuable genes carried by the wild germplasm can be introduced into the autotetraploid alfalfa varieties by crossing the haploid plants with the diploid wild germplasm.It has important research value and practical significance for widening the genetic basis of alfalfa and accelerating the breeding of new breakthrough varieties.In this study,anther tissue of Medicago Sativa L.'Xinjiang Daye' were cultured under optimized conditions to obtain the haploid plants and the ploidy of these plants were confirmed by Flow cytometry.Followed by applying different concentrations of colchicine to the haploid plants,the double haploid plants were obtained and used for interspecific distant hybridization at the diploid level.The genetic distance between parents and the authenticity of hybrids were further investigated by SRAP markers.In addition,the difference of heterosis effect among different ploidy combinations was determined by the plant performance of hybrid,such as height and biomass,in the first growth year,providing the basis for selecting new germplasm and hybrid parents in future research.The main results are as follows:(1)The optimized regeneration system of alfalfa anther tissue culture improved the differentiation rate of callus to 87.5%and the acquisition rate of haploid alfalfa plants(diploid)to 23.7%.The optimal rooting medium for haploid plants(diploid)was 1/2MS+0.1mg/L NAA+2%sucrose+0.7%agar.When the seedlings were transplanted with sterile nutrient soil,the highest survival rate could be achieved.Additionally,the double haploid alfalfa plants were obtained through tissue culture with the leaves of the above haploid plants as explants under the condition of 0.2%colchicine+1.5%DMSO in the liquid suspension oscillation culture for 24h.The acquisition rate of the double haploid in regenerated plants was 2.86%?8.6%.(2)Fourteen reciprocal cross combinations were made at the diploid level.Through analyzing the performance data in different cross combinations,there was a significant difference in the pod setting rate,the seed number per pod and the relative seed setting rate between different hybridization periods.Most of the hybrid combinations had better seed bearing at the initial and final flowering stages.(3)The genetic distances of the 12 parental materials were analyzed by 14 pairs of SRAP primers,and the authenticity of 20 hybrid combinations was identified.The purity of hybrids was 75-100%.The true hybrids mainly showed the complementary band pattern of parents,while the 12.5%?66.67%true hybrids showed the missing band pattern of parents and the new band type,which was also a manifestation of the heterosis.(4)Among the combinations which showed strong growth performance,2/3 of the combinations were from the parents with a large genetic distance,for example,the interspecific cross between haploid alfalfa plants and dioploid M.ruthenica plants showed positive heterosis in yield traits.(5)The difference of ploidy and the genetic distance between parents had significant influence on heterosis formation,and there was a certain equivalence on the yield performance of the diploid level cross combinations as well as the tetraploid level cross combinations.Besides,the heterosis of diploid cross combinations was stronger than tetraploid cross combinations.