The two-line pollination control system, which usually depends on the utilization of thermosensitive or photoperiod genic male-sterile lines, has been widely used in various crops. However, this system is susceptible to instability issues caused by uncontrollable weather fluctuations. A stable and handy two-line pollination control system is highly desirable in many crop species for heterosis exploitation. Oxophytodienoic acid reductase 3 (OPR3) was proven to be involved in jasmonate biosynthesis. In the present study, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat) was utilized to mutate two OPR3 homologs in Brassica napus. After two OPR3 homologs were simultaneously mutated, mutants exhibited complete male sterility, and fertility could be easily restored by exogenous MeJA treatment. Hybrids produced from crosses between the opr3 sterile lines and normal varieties exhibited heterosis. This new two-line system based on OPR3 mutation provides higher stability and convenience than traditional systems. By using exogenous MeJA treatment to restore fertility, the system enables more precise control of male fertility transition, which has great potential to significantly contribute to the maneuverable production of hybrid seeds in rapeseed as well as other Brassica species crops.

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