Multi-omics analyses reveal epigenomics basis for cotton somatic embryogenesis through successive regeneration acclimation process

Plant regeneration via somatic embryogenesis is time-consuming and highly genotype-dependent. The plant somatic embryogenesis process provokes many epigenetics changes including DNA methylation and histone modification. Recently, an elite cotton Jin668, with an extremely high regeneration ability, was developed from its maternal inbred Y668 cultivar using a Successive Regeneration Acclimation (SRA) strategy. To reveal the underlying mechanism of SRA, we carried out a genome-wide single-base resolution methylation analysis for nonembryogenic calluses (NECs), ECs, somatic embryos (SEs) during the somatic embryogenesis procedure and the leaves of regenerated offspring plants. Jin668 (R4) regenerated plants were CHH hypomethylated compared with the R0 regenerated plants of SRA process. The increase in CHH methylation from NEC to EC was demonstrated to be associated with the RNA-dependent DNA methylation (RdDM) and the H3K9me2-dependent pathway. Intriguingly, the hypomethylated CHH differentially methylated regions (DMRs) of promoter activated some hormone-related and WUSCHEL-related homeobox genes during the somatic embryogenesis process. Inhibiting DNA methylation using zebularine treatment in NEC increased the number of embryos. Our multi-omics data provide new insights into the dynamics of DNA methylation during the plant tissue culture and regenerated offspring plants. This study also reveals that induced hypomethylation (SRA) may facilitate the higher plant regeneration ability and optimize maternal genetic cultivar.