Quercus suber L. (Q. suber) is an evergreen tree species known for producing high-quality cork. Traditional seed propagation of Q. suber has low viability and is time-consuming. Therefore, we used young stem segments of 2~3-year-old seedlings as explants, and optimized protocols for tissue culture and rapid micropropagation of Q. suber. The best disinfection method was 0.10% HgCl₂ (v/v) for 5 min. 0.50 g·L⁻¹ Poly Vinyl Pyrrolidone (PVP) is the best anti-browning agent with a significant reduction in browning by nearly 1.76-fold (58.89% → 33.33%). Woody Plant Medium supplemented with micronutrients and vitamins from Murashige and Skoog Medium (WPMS) was found to be the most suitable for shoot formation. The optimal hormone ratio for development of shoots from axillary buds was 0.60 mg·L⁻¹ 6-benzyladenine (6-BA). Among the cytokinins tested, 0.50 mg·L⁻¹ 6-BA was the most suitable for development of shoots from axillary buds. In additon, the highest percentage of rooting explants (66.67%) and rooting number (3.03) was obtained on WPM basal medium supplemented 0.20 mg·L⁻¹ IBA and 0.20 mg·L⁻¹ NAA. In summary, we have established a set of protocols for tissue culture and rapid micropropagation of Q. suber. These findings lay the foundation for rapid micropropagation and genetic improvement.

Anthocyanins are among the flavonoids that serve as the principal pigments affecting the color of plants. During leaf growth, the leaf color of ‘Zhonghuahongye’ gradually changes from copper-brown to yellow-green. At present, the mechanism of color change at different stages has not yet been discovered. To find this, we compared the color phenotype, metabolome, and transcriptome of the three leaf stages. The results showed that the anthocyanin content of leaves decreased by 62.5% and the chlorophyll content increased by 204.35%, 69.23%, 155.56% and 60%, respectively. Differential metabolites and genes were enriched in the pathway related to the synthesis of ‘Zhonghuahongye’ flavonoids and anthocyanins and to the biosynthesis of secondary metabolites. Furthermore, 273 flavonoid metabolites were detected, with a total of eight classes. DFR, FLS and ANS downstream of anthocyanin synthesis may be the key structural genes in reducing anthocyanin synthesis and accumulation in the green leaf of ‘Zhonghuahongye’. The results of multi-omics analysis showed that the formation of color was primarily affected by anthocyanin regulation and its related synthesis-affected genes. This study preliminarily analyzed the green regression gene and metabolic changes in ‘Zhonghuahongye’ red leaves and constitutes a reference for the molecular breeding of ‘Zhonghuahongye’ red leaves.

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.