Efficient clonal seeds sorting for apomictic hybrid rice using a pollen-specific gene switch system

Significant progress in apomictic hybrid rice development faces challenges like achieving high induction rates and seed-setting efficiencies, and distinguishing clonal from zygotic embryos. To address the challenge of selecting clonal seeds, we developed a dual-fluorescence labelling gene switch system using the recombinase Cre/LoxP + FRT. Initially, this system was tested in callus tissue under a constitutive promoter; then, we replaced the promoter with a pollen-specific one to develop the pollen-specific gene switch (PSGS) system. The effectiveness of PSGS in rice pollen was subsequently validated. After confirming its functionality, we co-transformed the PSGS vectors with apomixis vectors in hybrid rice Yongyou 2640 (YE) and Yongyou 4949 (YS) using Agrobacterium-mediated transformation. Finally, we identified 18 MiMe mutants carrying the PSGS; the progeny of 16 lines were all red fluorescence seeds (zygotic embryo). Surprisingly, line L47-4 and L151-1 yielded 418 (n = 418) and 218 (n = 1279) non-fluorescent seeds in the T₁ generation, respectively. The ploidy detection of non-fluorescent seeds showed that 57 (n = 68) and 64 (n = 72) were diploid in Line L47-4 and L151-1, individually. This phenomenon was reproducible in the T₂ generation; 97 (n = 121) and 164 (n = 187) non-fluorescent seeds were diploid from line L47-4 and L151-1, respectively. This study demonstrates the ability of PSGS to distinguish between clonal seeds and zygotic seeds, with a sorting accuracy rate ranging from 80.2% to 88.9%, which is essential for improving clonal seed purity and advancing apomixis in rice cultivation.