The two-line hybrid rice breeding technology relies on thermo-sensitive genic male sterile (TGMS) lines carrying the tms5 locus. This locus originates from a natural mutation and exhibits varying sterility onset temperatures across different genetic backgrounds. Additionally, genetic drift may occur in subsequent generations. Thus, elevated sterility onset temperatures and genetic drift are key bottlenecks restricting the development of two-line hybrid rice technology.

Using the japonica rice variety Zhonghua 11 (ZH11) as material, we established a method for creating P/TGMS lines. Rice seeds were treated with ethylmethanesulfonate (EMS), and sterile individuals were screened from the M₂ population under high-temperature conditions. After ratooning, these fertility-restored plants were obtained under low-temperature conditions.

Through years of experimentation, approximately 10% of the sterile plants were able to self-fertilize under low temperatures. Seeds from these fertility-restored lines were collected and further validated for sterility under high temperatures and fertility under low temperatures. These validated P/TGMS lines were subjected to genetic population construction combined with BSA-seq (Bulked Segregant Analysis sequencing) for gene mapping, resulting in the identification of 18 new P/TGMS lines in the ZH11 background. Among these, ostms15, ostms16, ostms18, and ostms19 have been published. Notably, the fertility transition temperature of ostms15 is significantly lower than that of the widely used tms5 locus in the same genetic background. Currently, more than 20 research institutions have utilized this sterile line for hybrid rice breeding.

The P/TGMS line creation technology proposed in this study can generate a series of novel P/TGMS lines, potentially overcoming the bottleneck of P/TGMS line scarcity in two-line hybrid rice breeding and supporting the sustainable development of hybrid rice breeding.