Cytological Investigation On The Female And Male Sterility Of FM195A(Brassica Napus L.) And Genetic Improvement Of Theseed Setting

Genic male sterility(GMS) is regarded as an efficient pollination control system for hybrid production in rapeseed(Brassica napus L.). The male-sterile plants(FM195A) from a GMS two-type line FM195 AB displays stable and complete male-sterility as well as an extremly low seed setting. Previous genetic analysis has shown that the male steility of FM195 A is controlled by one multiple-alleles locus designated as Bn Ms5. Selfing of a fertile plant(FM195B) genotyped as Bn Ms5 aBn Ms5b would give rise to fertile individuals with the genotypes of Bn Ms5 aBn Ms5a or Bn Ms5 aBn Ms5b, and sterile individuals with the genotype of Bn Ms5 bBn Ms5b, which are ideal plant materials for the study of biological functions of Bn Ms5. In this study, we employed different exprimental approaches like histological sections, feluorescenc in situ hybridization(FISH) and immunolocalization to uncover the cytological mechanism of male and female sterility in FM195 A, and discussed the potential biological function of Bn Ms5. Furthermore, we attempted to improve the female sterility of FM195 A by specifically expressing the restorer gene Bn Ms5 a in ovules. The main results are listed as follows:1. Pollen grains could germinate typically on the stigma and pollen tube can normally extend to the vicinity of ovules in both FM195 A and FM195 B plants. However, it seems that the pollen tubes in FM195 A lost the guidance of ovules as they were seriously coiled and gradually gathering around the ovules, and finally they failed to enter the micropyle. This fact suggests that some developmental defects occur in the ovules of FM195 A.2. The anther and embryo sac development were observed by confocal laser scanning microscopy and paraffin section stainning, respectively. Results showed that both the male and female sterility in FM195 A are caused by the arrested meiocytes in reproductive organs, which fail to develop into tetrads.3. Callose-stained ovule primordia were examined to monitor the cytokinesis of female meiocytes. Though the dyad and tetrad stages were clearly observed in FM195 B, no dyad and the subsequent stages could be detected in FM195 A, indicating that the first meiocyte cytokinesis is interrupted in FM195 A. Thus, the megaspores experiencea very early developmental arrest during meiosis. We also observed that the deposition of callose around PMC was much thinner in FM195 A than that of FM195 B.4. Meiotic chromosome spread of PMCs were examined to further characterize the meiosis defects in microsporocyte. Obviously abnormal chromosome behavior was observed between the transition between leptotene and zygotene in FM195 A, characterized that the meiotic chromosomes failed to condense to form short and thick bivalents and finally the whole nucleus became concentrated and degraded. Therefore, Bn Ms5 likely plays an important role in regulating the meiotic chromosome structure during early prophase I.5. Through the analyses based on fluorescence in situ hybridization, we showed that the homologous chromosome pairing was completed in the centromere regions of FM195A; however, this process failed to extend to the whole chromosome arms. Immunolocalization analyses suggested that the protein localization of three meiosis marker proteins such as Bn SWI1, Bn SYN1 and Bn MLH3 are not affected in FM195 A, further supported that Bn Ms5 may be involved in the regulation of chromosome structure in early meiosis.6. Transimission electron microscope(TEM) analysis revealed that the nucleolus of male meiocytes from FM195 A plants cannot move to one polar of the nucleus during the zygotene stage, and is not normally degraded in the following zygotene and pachytene meiotic stages.7. A construct p AGL:Bn Ms5a(harboring the ovule-specific promoter of AGL1 fused to the resoter gene Bn Ms5a) was transformed to completely male-sterile line( F1 plants from the cross between Rs1046 A and 7-5). From one transgene-positive T0 plants in which the Bn Ms5 a gene has a high expression in ovules, we observed that the female setting was significantly improved when compared to the transgene-negative plants and the recipient control, while the male-sterility phenotype was well maintained. This fact showed that the low female setting caused by arrested megaspore can be efficiently rescued by a transgene approach, which would be favorable for the application of the GMS line.