A multi-omics strategy identifies a SAM-dependent methyltransferase that enhances callus differentiation from mature sorghum seeds

Despite advances in sorghum transformation technology, the regeneration of plants from callus remains the major bottleneck restricting genetic improvement and molecular research. In this study, we conducted transcriptome sequencing of calli derived from four sorghum germplasms, including two with high induction but poor differentiation capacity (IS4581, IS7250) and two with high differentiation rates (IS4698, IS5295). Differential expression analysis across four pairwise comparisons (IS4581 vs. IS4698, IS4581 vs. IS5295, IS7250 vs. IS4698, IS7250 vs. IS5295) identified 1,253 shared differentially expressed genes (DEGs). GO enrichment revealed that these DEGs were mainly associated with the extracellular region, cell wall organization, and xylan biosynthetic process, while KEGG pathway analysis highlighted enrichment in phenylpropanoid biosynthesis, starch and sucrose metabolism, and galactose metabolism. Integration of transcriptomic data with 47 GWAS-identified candidate genes from a previous study pinpointed two key genes, Sobic.003G266500 and Sobic.003G266600, linked to embryogenic callus differentiation. Functional validation through Agrobacterium-mediated transformation confirmed that Sobic.003G266600 significantly enhances callus differentiation in the recalcitrant genotype IS4581. These findings provide novel insights into the molecular regulation of sorghum callus regeneration and offer valuable genetic targets for improving transformation efficiency and developing genotype-independent regeneration systems.