Stage-specific DNA methylation and transcriptional responses accompany 5-azacytidine-enhanced somatic embryogenesis in Chinese fir (Cunninghamia lanceolata)

Somatic embryogenesis (SE) is a key technique for clonal propagation and genetic improvement of forest trees and is strictly regulated by epigenetic modifications. Here, we evaluated the responses of three Chinese fir ( Cunninghamia lanceolata ) genotypes to 5‑azacytidine (5‑AzaC), a DNA methylation inhibitor, and selected genotype 4098‑1, which exhibited the highest tolerance and proliferation capacity, for further integrated morphological, physiological, and transcriptomic analyses. The results showed that appropriate concentrations of 5-AzaC significantly promoted callus proliferation and improved SE efficiency. Additionally, 5-AzaC treatment significantly accelerated somatic embryo development. Global DNA methylation declined and subsequently rose during SE, and 5-AzaC significantly reduced methylation levels between days 15 and 30. Antioxidant assays showed that 1.5 μM 5-AzaC enhanced superoxide dismutase (SOD) and peroxidase (POD) activities at the mid-SE stage (days 15–30) and reduced hydrogen peroxide accumulation at the late stage of SE. Transcriptomic analysis revealed pronounced time‑dependent reprogramming, with day 7 as a key transition point featuring the most differentially expressed genes, including the downregulation of genes involved in metabolism, defense, and DNA repair. Weighted gene co‑expression network analysis (WGCNA) further revealed significant correlations between antioxidant‑responsive modules and DNA methylation modules, indicating coordinated regulation. Key enriched pathways included phenylpropanoid biosynthesis, flavonoid biosynthesis, glutathione metabolism, plant MAPK signaling, and plant hormone signal transduction. Collectively, these findings indicate that 5-AzaC-enhanced SE is associated with coordinated changes in DNA methylation dynamics, redox homeostasis, and developmental signaling networks. Thereby providing insights into the epigenetic regulatory mechanisms underlying conifer SE and informing strategies for its efficient induction.