H₂O₂-GA₃-Na₂WO₄ Synergistically Promotes Germination of Immature Winter Wheat Grains for Speed Breeding

Seed germination is a critical initial stage of the plant life cycle, regulated by signaling pathways such as phytohormones and reactive oxygen species (ROS). However, the low germination rate of immature grains is a key bottleneck limiting wheat speed breeding. This study used immature grains of the winter wheat cultivar Kenong 199 (KN199) collected 18 days post anthesis to establish an efficient germination protocol. By screening individual and combined treatments of hydrogen peroxide (H₂O₂, 1%), gibberellin (GA₃, 20 μM), and varying concentrations of abscisic acid (ABA) synthesis inhibitor sodium tungstate (Na₂WO₄), alongside transcriptome analysis, we identified the optimal reagent combination and gained preliminary insight into its molecular basis. The triple reagent combination of 0.5 mM Na₂WO₄ + 20 μM GA₃ + 1% H₂O₂ exhibited the highest germination rate of 80%, approximately sevenfold higher than single reagent treatments, with germination rate peaking after 4 days. Transcriptome profiling revealed that this combination modulated the expression of key genes related to dormancy release and germination, including upregulation of GA biosynthesis gene GA3ox2 and ABA catabolism gene TaCYP707A2, and downregulation of ABA biosynthesis and signaling genes (ABI5, TaNCED1, etc.). Additionally, genes associated with energy metabolism and transport pathways were enhanced. This optimized reagent combination significantly improves immature grain germination, shortens the breeding cycle, and provides a practical tool for achieving "five generations per year" speed breeding in winter wheat. Our findings contribute to seed biology by offering a chemical strategy to overcome dormancy in immature cereal grains.