Cytological Studies Of Male Sterile Mutant Ms1 In Alfalfa (Medicago Sativa L.)

Alfalfa (Medicago sativa L.,2n=4x=32) is one of the most important legumes for its high nutritional quality and forage yield in the world. Male sterility characteristics have important theoretical significance and application value in studying genetic mechanism of male sterility and obtaining heterosis. In our previous study, male sterile mutant ms1 was derived form a large-scale screen of sterile mutants in a national alfalfa breeding programme. The objectives of this study were to carry out cytological observation and to reveal the mechanism of male sterility of this new spontaneous mutant in alfalfa. The main results are summarized as follows:1. From 2009-2011, pollen production and pollen viability of ms1 as well as wild type plant were tested using blood cell counting method and TTC method, the three years’continuous observations showed that the pollen production of the mutant was significantly less than that of wild type plant; The pollen viability of ms1 was 4.8% in average, which was significantly lower that of 69.4% in WT plant. The transverse-sections of mature anther showed that there was no normal appearing pollen grain in mutant plant while a large number of pollen grains were observed in WT plant. The phenotypic results above indicated that pollen abortion maybe happen at the late stage of anther development. The stability of male sterility of this new mutant was also could be confirmed.2. The microsporogenesis and microgametogenesis have been examined in ms1 and wild type plant under light and scanning electron microscopy. The results showed that six developmental stages were divided for the normal anther development in alfalfa:1) sporogenous mass stage,2) meiocyte stage,3) dyad stage,4) tetrad stage,5) microspore stage and 6) pollen stage. Before dyad stage, there were no differences in the epidermis, endothecium, middle layer and tapetum between ms1 and wild type plant. Up to dyad stage, the middle layer has been disappeared both in wild type plant and male sterile mutant. While normal male meiosis occurred to form tetrads, the tapetum was still no obvious difference in wild type and male sterile anthers. After tetrad stage, the tapetal cells appeared delayed vacuolization and degeneration in male sterility anthers compared to tapetal cells of wild type. The microspore in male sterile anther failed to form a normal pollen wall at the uninucleate microspore stage, which indicated that the tapetal cell plays an important role in pollen exine formation.The observations of pollen grains under fluorescence microscopy showed that shape of pollens from ms1 were shrunken and irregular with too much callose deposition on the cell wall, while normal pollens were regular and tightly connected each other. The pollen exine formation and structure in the wild type and ms1 were investigated furthermore by scanning electron microscopy. The results showed that regular shape and plump pollen grains were observed in wild type plant and sporopollenin deposition on the pollen wall was also observed. Contrast to that of wild type plant, pollen grains of ms1 mutant had a shrunken shape and the pollen wall had an abnormal smooth surface. These results indicated that pollen exine formation was defective in msl. So the abnormal vacuolization and degeneration of tapetum caused the absence of the pollen exine. Thus, we may conclude that the tapetum abnormal development and the absence of the pollen exine were responsible for male sterility in this study.3. Meiosis in pollen mother cells (PMCs) of the male sterile plant was similar to that of wild type plants. However, the pairing and segregation of chromosomes in the PMC of the male sterility plants was different from wild type plants. Unparallel chromosome and massing chromosome were found in metaphase of the pollen mother cells at the meiosis stage in ms1. These unparallel chromosomes underwent segregation to form the unequal tetrad in this study. We also observed that the phenomenon of laggard chromosomes and chromosome bridges during the stage of anaphase. The laggard chromosomes may result in the pentanuclear microspore and the chromosome bridges may lead to obtain the triad microspore. An interesting phenomenon was observed during the stage of tetrad, the pole of the tetrad appeared to degenerate. During PMC meiosis, many irregular chromosomes segregation was examined and these abnormalities maybe result in the pollen abortion in ms1.All in all, our results indicated that msl belong to sporogenous male sterility with normal stamen development but abnormal development at late microsporogenesis. To obtain heterosis in alfalfa, utilization of male sterile line is one of the ways to avoid inbreeding depression. For that purpose, male sterile mutants will provide a potential starting point for the cytological and genetic investigation of anther and pollen development in the future.