High-frequency of twin-embryo seeds with clonal and sexually produced embryos drives apomixis in hybrid rice

Abstract Heterosis is a common phenomenon in biology, and fixation of heterosis can be achieved via artificially induced apomixis. Sundaresan's team used mitosis instead of meiosis (MiMe) rice mutants that ectopically expressed BBM1 in egg cells to induce the production of clonal seeds. In this study, we ectopically expressed BBM1 in MiMe mutants, and 62.50% of the seeds produced by Yongyou 4949 hybrid rice were clonal. More importantly, a high frequency of twin seedlings (33.56%) was detected. Moreover, a low frequency (1.41%) of twin seedlings was obtained only when BBM1 was ectopically expressed, and no twin seedlings were found when the genome of the MiMe mutant was edited. These findings suggest that the high frequency of twin seedlings resulted from the ectopic expression of BBM1 combined with MiMe. The embryo sacs of the twin seedlings were removed and subjected to cytological observations. One sperm combined with the polar nucleus to develop into the endosperm, and the other sperm combined with the egg cell to form a zygotic embryo. Moreover, a synergid cell near the egg cell developed into an embryo through apogamy, thus resulting in the formation of two embryos. The results showed that AtDD45-driven BBM1 could induce synergid cells to develop into embryos autonomously through normal fertilization of egg cells, resulting in the formation of twin seedlings. This study provides a theoretical reference and new ideas for research on apomixis in hybrid rice and is important for promoting the fixation of heterosis of hybrid rice.