Cytology and Molecular Analysis on Sterile Mechanism of SaNa-1A CMS Derived from Somatic Hybrid Between Brassica napus and Sinapis alba

Cytoplasm male sterile (CMS), as an ideal system regulating pollination, has greatly contributed to the improvement in rapeseed output. Somatic hybridization is effective in creating new CMS lines from potential mitochondrial genome recombination of parents. A sterile line (SaNa-1A) was selected from the backcross progenies of Brassica napus-Sinapis alba somatic hybrids. By comparing different anther developmental stages between sterile and maintainer line (SaNa-1B) through microscope observation, we found tetrads in sterile lines were covered with thick callose, and tapetum cells were severely vacuolated during the tetrad stage. In the early uninucleate stage, microspores in sterile line were vacuolated with few inclusions; tapetum was condensed and separated with the intermediate cells. In the late uninucleate stage, microspores were adhered together and embraced with the tapetum cells, which were located in the center of pollen sac. Physiological and biochemical analysis of developing anther and mitochondria in sterile and maintainer lines revealed that SOD and POD responses were induced with the accumulation of ROS in aborted anther of SaNa-1A. Meanwhile, we compared the F₁F₀-ATPase activity, ATP content in mitochondria, and COX activity in anther between sterile and maintainer lines and found that the activity of F₁F₀-ATPase was significantly downregulated with the anther development of sterile line, which was lower than in maintainer lines. By using the TdT-mediated dUTP Nick-End Labeling method to identify the PCD during anther development, we identified that tapetum cells in normal anthers undergo PCD since the tetrad stages and maximized it at the uninucleate stage. However, few hybridization signals were identified in the tetrads of sterile lines. Meanwhile, PCD was not obvious in the tapetum cells of sterile lines and was identified in tapetum cells since the uninucleate stage.

Keywords: Brassica napus, Cytoplasm male sterile (CMS), Physiological and biochemical analysis, Mechanism