Glutathione metabolism maintains redox homeostasis to enhance somatic embryogenesis in hybrid larch

Somatic embryogenesis (SE) is a pivotal technique for large-scale propagation and genetic improvement of plant germplasm. While redox homeostasis is essential for SE in conifers, the molecular mechanisms that maintain this balance in oxygenation state remain poorly understood. When embryogenic suspensor masses (ESMs) of hybrid larch (Larix kaempferi × L. olgensis) with distinct browning phenotypes were examined, excessive hydrogen peroxide (H₂O₂) accumulation in browned tissues was identified as a primary driver of SE failure. By applying the H2O2 scavenger N,N′-dimethylthiourea (DMTU) alongside exogenous H2O2, a negative dose-dependent relationship between H₂O₂ levels and SE efficiency was established for the first time. Transcriptomic analysis and quantitative reverse transcriptase-PCR revealed that DMTU activates glutathione (GSH) metabolism in a spatiotemporal manner: promoting early GSH synthesis and sustained reactive oxygen species (ROS) scavenging via the glutathione S-transferase (GST) pathway, while simultaneously triggering a BABY BOOM (BBM)–LEAFY COTYLEDON 1 (LEC1)–WUSCHEL-RELATED HOMEOBOX 9 (WOX9) transcriptional cascade. Stage-specific assays further demonstrated that adding GSH during the maintenance stage alleviates oxidative damage, whereas continuous GSH supply during the induction stage further enhances SE efficiency by maintaining H2O2 homeostasis. Conversely, buthionine sulfoximine (BSO) treatment exacerbated browning and suppressed SE. Notably, the combined application of DMTU and GSH increased SE efficiency by 28% relative to DMTU alone and by 173% relative to GSH alone. Together, these findings demonstrate that targeted redox regulation can effectively enhance SE in conifers, providing a robust, redox-balance-based strategy for optimizing conifer SE systems.