The recessive genic male sterility (RGMS) method has several benefits in hybrid seed production; yet, it is seldom employed in industrial hybrid seed production owing to the difficulties of producing an ample number of pure male sterile seeds. In this study, we present an efficient methodology for developing a two-line strategy to produce hybrid seed through targeted mutation of BnaMS1 and BnaMS2 in conjunction with the RUBY reporter in Brassica napus. In this method, male sterile lines were successfully created directly from different elite rapeseed breeding lines through CRISPR/Cas9-mediated mutagenesis and enhanced Agrobacterium-mediated transformation. To establish an efficient transgenic maintainer, three SPT vectors carrying a functional BnaMS1 gene linked to different reporters (DsRed, BnaA07.PAP2, and RUBY) were tested and compared in rapeseed. The results indicated that the PMR-based reporter possesses advantages such as phenotypic stability and ease of identification at early stages, making it an ideal tool for rapid and efficient screening. Subsequently, ideal transgenic maintainer lines with a single hemizygous copy of the SPT cassette were successfully developed in the context of Bnams1Bnams2 double mutants. The progeny from crossing the maintainer line with its male-sterile counterpart exhibited a 1:1 segregation pattern of nontransgenic male-sterile and male-fertile maintainer plants, distinguishable by seedling color. This biotechnological approach to male sterility offers promising prospects for improving the propagation of recessive genic male-sterile plants and the development of hybrid seeds in rapeseed. Furthermore, it is simple to adapt this technique to more Brassica crops.

中文翻译：

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BnaMS1/BnaMS2 的靶向突变与 RUBY 报告基因相结合，为甘蓝型油菜杂交种子生产提供了高效的两系系统


隐性雄性不育（RGMS）方法在杂交种子生产中具有多种优点；然而，由于难以生产大量纯雄性不育种子，因此很少用于工业杂交种子生产。在这项研究中，我们提出了一种有效的方法，用于开发两系策略，通过 BnaMS1 和 BnaMS2 的定向突变以及甘蓝型油菜中的 RUBY 报告基因来生产杂交种子。在该方法中，通过 CRISPR/Cas9 介导的诱变和增强的农杆菌介导的转化，直接从不同的优良油菜育种系成功创建雄性不育系。为了建立有效的转基因维持子，在油菜籽中测试并比较了三个携带与不同报告基因（DsRed、BnaA07.PAP2 和 RUBY）连接的功能性 BnaMS1 基因的 SPT 载体。结果表明，基于PMR的报告基因具有表型稳定、易于早期识别等优点，是快速高效筛选的理想工具。随后，在 Bnams1Bnams2 双突变体的背景下成功开发了具有 SPT 盒单个半合子副本的理想转基因维持系。保持系与其雄性不育对应物杂交的后代表现出非转基因雄性不育和雄性可育保持系植物的 1:1 分离模式，可通过幼苗颜色进行区分。这种雄性不育的生物技术方法为改善隐性雄性不育植物的繁殖和油菜杂交种子的开发提供了广阔的前景。此外，这种技术很容易适用于更多的芸苔属作物。