Transcriptional differences between repeatedly recultured and freshly induced embryogenic calli reveal candidate developmental regulators

Peanut (Arachis hypogaea L.) is an economically important oilseed and food legume, yet genetic transformation remains challenging because of genotype dependence and the lengthy tissue-culture and regeneration process. A better understanding of the molecular mechanisms underlying callus development and proliferation could help identify candidate morphogenic genes and developmental regulators (DRs) for peanut improvement. Here, we used RNA sequencing to compare embryogenic calli from the cultivar Georgia Green that had been maintained for three months (new callus; NC) or repeatedly subcultured for four years (old callus; OC). The two callus types displayed distinct morphologies: NC was pale cream, expanded, and cauliflower-like, whereas OC was more yellow, compact, and nodular. We identified 1,141 genes significantly upregulated in NC and 2,086 in OC (adjusted p < 0.05; log2FC; fold change > 2). Transcription factors were prominent among 13 categories of differentially expressed genes, including members of the AP2/ERF, WOX, GRAS, LBD, and NAC families, several of which are known DRs in other crops. qRT-PCR analysis of seven selected genes strongly corroborated the RNA-seq results (r = 0.944; R2 = 0.891). Highly upregulated candidates included C2H2/BED-type zinc-finger proteins, calreticulin/calnexin, a glycine-rich protein, a late embryogenesis abundant protein, glycoside hydrolases, and SWEET sugar transporters. These findings provide a resource for investigating callus development in peanut and prioritize candidate genes for functional validation and future improvement of transformation and regeneration systems.