Developmental regulator-mediated transcriptional reprogramming reveals novel regulators of somatic embryogenesis in sorghum

Recalcitrance to genetic transformation remains a fundamental bottleneck in crop improvement, driven by genotype-specific barriers that include entrenched defense transcriptional states, limited embryogenic competence, and inadequate hormonal reprogramming potential. Here, we present a multi-layered transcriptomic analysis of somatic embryogenesis (SE) and regeneration in two sorghum genotypes (forage Tx2910 and grain Tx2783), two explant types (immature embryos and leaf bases), and three developmental regulator (DR) treatments (GRF-GIF, WUS, and BBM+WUS). This design lets us separate genotype, explant, and DR-specific regulatory programs. Using in-depth transcriptomics analysis (bulk RNAseq and single nucleus transcriptomics), we identified the molecular logic by which DRs override genotype-specific barriers and redirect somatic cells toward embryogenic and regeneration-competent states. Compared to Tx2910, the transformation-recalcitrant sorghum line Tx2783 shows a stronger defense response to both Agrobacterium infection plus culture stress and depends more heavily on DR-driven transcriptional reprogramming to exit this defense-locked state. WUS acts as an early, broad suppressor of defense and stress signaling while activating fate-specification programs, whereas GRF-GIF drives a delayed but pronounced transcriptional shift toward cell proliferation and morphogenesis. Integrative gene network analyses further identified a prioritized set of sorghum-specific transcription factors with potential to increase regeneration efficiency. Among the top candidates, we identified that SbEREB167 and SbEREB174 (AP2/EREBP family) efficiently induce SE and regeneration in sorghum. Collectively, this study establishes the transcriptional architecture underlying DR-mediated transformation across sorghum types and identifies novel regulators that hold potential to improve plant transformation across recalcitrant cereal species.