Integrated optimization of somatic embryogenesis and genetic transformation reveals novel regulators of regeneration competence in peanut

Peanut ( Arachis hypogaea L. ) is an important global oil and cash crop. Its functional genomics and molecular breeding are severely hindered by its complex allotetraploid background and recalcitrant genetic transformation. Here, we established a multi-factor integrated optimization platform that combines nutritional regulation, transformation timing, and transcriptomic profiling to overcome the transformation bottleneck in peanut. Among 12 screened cultivars, the Valencia-type NDH108 showed superior embryogeneic capacity with a somatic embryogenesis (SE) induction rate over 90%. Fine-tuning the carbon-to-nitrogen (C/N) ratio (1.4 C:1 N, NO₃⁻/NH₄⁺ ≈4.4:1) greatly alleviated genotype differences in SE competence, elevating SE rates in low-responsive cultivars from below 30% to over 85%. we also defined the ideal somatic embryo developmental window (approximately one month post-induction) for Agrobacterium infection. Further transcriptomic profiling of the SE induction process uncovered coordinated reprogramming of carbon and nitrogen metabolism and a widespread gene expression switch triggered by light-to-dark culture transitional. We further characterized the embryo-specific ATS3B gene family as a candidate factor potentially associated with somatic embryo morphogenesis. ATS3B expression was correlated with auxin efflux carrier genes PIN1 and PIN3 . Over-expression ATS3B facilitated the formation of well-organized somatic embryos, wheres gene silencing resulted in chaotic, callus-like proliferaton. Collectively, this work provides an integrated technical framework combining germplasm screening, nutritional adjustment, environmental signal regulation and transformation windiow screening to overcome transformation recalcitrance in peanut and allied crops.