Objective This study aims to reveal the dynamic changes in the main cell wall components of mesophyll cells of Japanese chestnut during leaf development, to clarify their relationship with protoplast isolation efficiency, and to establish a protoplast preparation system for Japanese chestnut, thereby laying a solid foundation for subsequent somatic cell hybridization, exogenous gene transformation, and related studies.

Method Young leaves of Japanese chestnut seedlings at different growth stages (3 days, 6 days, and 9 days after leaf unfolding) were used as materials. The main cell wall components and their contents were determined, and the protoplast isolation efficiency was compared among these stages. Pearson correlation analysis and principal component analysis (PCA) were employed to comprehensively evaluate the relationship between cell wall components and protoplast isolation efficiency, and to screen the optimal leaf growth stage for enzymatic digestion. On this basis, the effects of different enzyme combinations, enzymatic digestion time, mannitol concentration, and sucrose concentration on protoplast isolation were investigated. The number of protoplasts was counted by hemocytometry, and protoplast viability was assessed by fluorescein diacetate (FDA) staining.

Result  (1) With leaf growth and development, the contents of all cell wall components (cellulose, hemicellulose, lignin, protopectin, and water-soluble pectin) increased significantly (p < 0.05). The contents of all cell wall components were the lowest in leaves at 3 d after leaf unfolding. The protoplast number, viability, and net yield of leaves at 3 d and 6 d after leaf unfolding were significantly higher than those at 9 d (p < 0.05). (2) Correlation analysis showed that cellulose content, lignin content, protopectin content, and water-soluble pectin content were significantly negatively correlated with protoplast number and viability (p < 0.05), whereas hemicellulose showed weak correlations with all protoplast indicators. (3) Further analysis of the main cell wall component contents at different leaf growth stages revealed that leaves at 3 d after leaf unfolding had the lowest comprehensive score (F_n = 4.344), indicating the lowest cell wall component contents and the best protoplast isolation effect; thus, leaves at 3 d after leaf unfolding were the optimal growth stage for enzymatic digestion of Japanese chestnut. (4) Based on these findings, the optimal preparation conditions were screened as follows: leaves at 3 d after leaf unfolding were digested in cell-protoplast washing solution containing 2.5% cellulase R-10, 1.0% macerozyme R-10, 1.0% hemicellulase, and 0.4 mol/L mannitol, at 25 ℃ in darkness for 4 h. Under these conditions, the maximum protoplast number reached 1.98 × 10⁶ protoplasts/g, with viability reaching 82.69%. After enzymatic digestion, high-quality protoplast pellets were obtained by sucrose density gradient centrifugation at 20%–25%.

Conclusion In this study, Japanese chestnut mesophyll cells were used to systematically analyze the dynamic changes in the main cell wall components during leaf development. The stage at 3 d after leaf unfolding was identified as the optimal growth stage for enzymatic digestion. An efficient protoplast isolation and purification system suitable for Japanese chestnut mesophyll cells was established. These results provide a theoretical basis for optimizing enzymatic digestion and purification conditions in other woody plants based on the dynamic characteristics of cell wall components during development.