Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409, USA.
BMC Plant Biol. 2024 Oct 11;24(1):946. doi: 10.1186/s12870-024-05674-5.
Heterosis is a common phenomenon in plants and has been extensively applied in crop breeding. However, the superior traits in the hybrids can only be maintained in the first generation but segregate in the following generations. Maintaining heterosis in generations has been challenging but highly desirable in crop breeding. Recent study showed that maternally produced diploid seeds could be achieved in rice by knocking out three meiosis related genes, namely REC8, PAIR1, OSD1 to create MiMe in combination with egg cell specific expression of BBM transcription factor, a technology called clonal seeds. Interestingly, there has been very limited reports indicating the feasibility of this approach in other crops.
In this study, we aimed to test whether clonal seeds could be created in cotton. We identified the homologs of the three meiosis related genes in cotton and used the multiplex CRISPR/Cas9 gene editing system to simultaneously knock out these three genes in both A and D sub-genomes. More than 50 transgenic cotton plants were generated, and fragment analysis indicated that multiple gene knockouts occurred in the transgenic plants. However, all the transgenic plants were sterile apparently due to the lack of pollen. Pollination of the flowers of the transgenic plants using the wild type pollens could not generate seeds, an indication of defects in the formation of female sexual cells in the transgenic plants. In addition, we generated transgenic cotton plants expressing the cotton BBM gene driven by the Arabidopsis egg cell specific promoter pDD45. Two transgenic plants were obtained, and both showed severely reduced fertility.
Overall, our results indicate that knockout of the clonal seeds related genes in cotton causes sterility and how to manipulate genes to create clonal seeds in cotton requires further research.
杂种优势是植物中的一种常见现象，已广泛应用于作物育种。然而，杂种中的优良性状仅能在第一代中保持，而在随后的几代中则会分离。在作物育种中，保持杂种优势是具有挑战性但又非常理想的。最近的研究表明，通过敲除三个减数分裂相关基因 REC8、PAIR1 和 OSD1，并与卵母细胞特异性表达的 BBM 转录因子结合，可在水稻中产生母源二倍体种子，这一技术称为克隆种子。有趣的是，在其他作物中，关于这种方法可行性的报道非常有限。
在这项研究中，我们旨在测试克隆种子是否可在棉花中产生。我们鉴定了棉花中这三个减数分裂相关基因的同源物，并使用多重 CRISPR/Cas9 基因编辑系统同时敲除这三个基因在 A 和 D 亚基因组中的表达。生成了 50 多个转基因棉花植株，片段分析表明，多个基因在转基因植株中发生了敲除。然而，所有的转基因植株都明显不育，显然是由于花粉缺乏所致。用野生型花粉对转基因植株的花进行授粉不能产生种子，表明转基因植株中雌性生殖细胞的形成存在缺陷。此外，我们还生成了表达由拟南芥卵母细胞特异性启动子 pDD45 驱动的棉花 BBM 基因的转基因棉花植株。获得了两个转基因植株，它们的育性都严重降低。
总的来说，我们的结果表明，棉花中克隆种子相关基因的敲除导致不育，以及如何操纵基因来产生棉花克隆种子还需要进一步研究。
