Optimization of Somatic Embryogenesis and Transcriptomic Analysis of the Early Stage of Callus Redifferentiation in...

Quercus suber L. (Q. suber) is an ecologically and industrially valuable species, yet faces challenges in propagation in China. This study optimized somatic embryogenesis (SE) protocols using two-year-old Q. suber leaves, focusing on petioles and leaf veins as the most responsive explants, with May as the optimal sampling time. The MSSH medium (a combination of Murashige and Skoog Medium (MS) major elements and Schenk and Hildebrandt Medium (SH) minor elements and vitamins) under darkness maximized transdifferentiation. Additionally, the highest callus induction was achieved with 0.50 mg/L 6-benzyladenine (6-BA) and 1.00 mg/L 1-Naphthaleneacetic acid (NAA). Liquid culture with 1.00 g inoculum and 0.50 mg/L 6-BA + 0.20 mg/L NAA achieved the best proliferation. Redifferentiation peaked at 0.15 mg/L NAA + 0.20 mg/L 6-BA. Transcriptome profiling identified 4534 differentially expressed genes (DEGs) between embryogenic callus (E1) and global embryos (E2), with key pathways linked to cell wall remodeling, stress responses, and photosynthesis. Key regulators identified during the early stage of callus redifferentiation include cytokinin oxidase 3 (CKX3), gibberellin-responsive protein (GH3.6), and pectin lyase 5 (PL5), among others. This study provides insights into efficient SE of Q. suber and the genes underlying early callus redifferentiation, laying the groundwork for future research.