Enhancement of Male Sterility Stability in Indica Rice by Dual Thermo-Sensitive Genic Male Sterile Genes

Low-temperature-induced fertility restoration in thermo-sensitive genic male sterile (TGMS) lines severely impairs hybrid seed purity, which is a major bottleneck for two-line hybrid rice production. Most commercial TGMS lines rely on the single tms5 locus, leading to high climatic vulnerability. In this study, we developed a dual-locus strategy by target genome editing of TMS5 and MS1 in indica rice GH89. Adenine base editing at the MS1 locus exhibited a high editing efficiency of 93.5%. Transgene-free homozygous single mutants (GH89-tms5 and GH89-MS1) and double mutant (GH89-tms5 + MS1) were generated for phenotypic analysis. The double mutant GH89-tms5 + MS1 remained completely sterile for 5 and 10 days under controlled low temperature (23.5 °C), with only minimal fertility restoration after 15 days. In the field, it maintained complete sterility for 84 consecutive days and was fully insensitive to short-term low temperature fluctuations, outperforming single mutants and commercial control Y58S. Moreover, the double mutant retained most key yield-related agronomic traits of the wild type with only minor variations. This dual mutation forms a "double-lock" fertility regulatory system, significantly increasing the low-temperature duration threshold for fertility restoration. The GH89-tms5 + MS1 line exhibits promising potential for future rice breeding applications.