Screening For Alfalfa Mutants In Male Sterility And Their Cytological Observations

Alfalfa (Medicago sativa L.) is an important leguminous forage, which is monoclinic and polyandry as well as a typical cross-pollinated plant. Male sterility characteristics have important theoretical significance and application value in studying genetic mechanism of male sterility and hybrid heterosis etc. Screening for alfalfa mutants in male sterility could provide the basis material for carrying out more research in this field. In this paper, based on the wide molecular and phenotypic genetic diversity in alfalfa germplasm we studied, we analysed the genetic variance of pollen quantity and pollen vigor in 20 alfalfa varieties. The objectives of this study were to establish a screening method of alfalfa mutant in male sterile and carry out cytological observations with the process of reproductive development furthermore. The main results are summarized as follows:1. A new phenotypic screening mutant system was established, named MS-alfalfa method. Using pollen quantity classification and pollen viability TTC staining method, we analysed genetic variation of pollen quantity and pollen viability in 20 alfalfa varieties, and the results showed that:1) There were significant difference (p<0.001) of alfalfa pollen quantity and pollen viability within varieties and among varieties, and both of the variations within varieties were larger than those variations among varieties, which indicated that the individual method should be used to screen the male sterile mutants in alfalfa; 2) No significant difference was observed between different reproductive branches in alfalfa. Whereas the Number of florets between 4 different grades of pollen quantity in alfalfa plants showed that there was 58.54% floret with pollen quantity of grade 4, the floret number of grade 3 accounted 32.32%, and florets with pollen quantity of grade 2 and grade 1 were 6.97% and 2.22%, respectively. The probability of mutant obtained is about 2% to screen by using pollen quantity classification method; 3) Using pollen viability (TTC staining) to determine the rate of pollen abortion of alfalfa found that pollen abortion rate range within individual plants was 0.53% to 73.14%, but the abortion rate range within varieties was only 15.72% to 31.31%, which indicated that pollen of alfalfa floret showed part abortion or incomplete abortion. Our results showed that the suitable method to screen alfalfa male sterile mutants was individual pollen quantity method. 2. Totally we discovered 16 male sterile mutants, which including 14 non-pollen male sterile alfalfa mutants (MS 1-14) and 2 multi-pistil non-pollen male sterile alfalfa mutants (M-fonl and M-fon2). The mutants above were selected from Graze401+z (5), Golden queen (2), Qingyang (1), Big west (2), Longzhong (1), Gannong No.2 (2), Giant (2) and Algonguin (1) seperately. Compared the fertility between male sterile alfalfa mutants MS1-14, M-fon1 and M-fon2 with other normal alfalfa plants, we found that the stigma receptivity and the pistil fertility of these MS1-14 mutant plants were similar compared with those of normal alfalfa plants. The stigma receptivity and ovule number of M-fon1 and M-fon2 were lower than those of normal alfalfa plants. The pistils of M-fonl also showed a high degree of degradation. These results indicated that M-fonl was not only a male sterile but also a female sterile mutant plant.Research on the seed set of M-fon1, M-fon2, and MS1-14 alfalfa mutants found that:M-fonl, M-fon2 and MS 1-14 showed self-incompatibility. Under artificial hybridization and natural crossing, the seed set of Ms1-14 were higher than those of M-fon1 and M-fon2. These results indicated that female sterility of M-fonl and M-fon2 resulted in the lower seed set but male sterile mutants MS 1-14 had reproductive advantage.3. During the different reproductive development stages, the cytological observations was compared and we found there were four types of mutant in male sterile of alfalfa plants:1) no pollen mutant types showed that the anther shriveled to crack in the late stages of development and no pollen grains in anthers; 2) anther dehiscence mutant types were those had normal anthers full of pollen grains but no dehiscence observed during tripping process; 3) anther develop mutant types showed small anthers and short flower shrubs in the late development stages as well as little pollen grains in the anthers; 4) ultrastructure observation of pollen develop abortion types showed that the pollens had irregular shapes. The surface of pollen began to shrink from germinal furrow, then different planes, and at last result in we can not see the contraction surface.To select male sterile mutant plants was useful to explore the genetic mechanisms and the regulation of genes related to male fertility in alfalfa. Our results above indicated that there exist wide genetic variations in male fertility in different alfalfa varieties, and there was larger variation within varieties than those among varieties. The MS-alfalfa method was a suitable screening system for alfalfa male sterility mutant selection. The male sterile mutant plants (MS-14, M-fon1 and M-fon2) we had obtained and therir cytological observations will provide the database and materials for the researches on male sterility genetic analysis and hybrid seeds production furthermore, and provide basic materials for male sterile, female sterile and their relationship, so as to lay the foundation for speeding up the process of alfalfa breeding.