Protoplast isolation from seedling leaves of European beech (Fagus sylvatica L.) v1

This protocol describes the isolation of leaf protoplasts from young seedling leaves of European beech (Fagus sylvatica L.). Surface-sterilized, fully unfolded but non-hardened primary leaves are cut into narrow strips, pre-washed in basal solution, enzymatically digested with cellulase and macerozyme overnight, filtered, washed, and resuspended in W5 solution or regeneration medium. The protocol typically yields highly viable protoplasts (>90% viability; average yield approx. 5.5 × 107 protoplasts g–1 fresh weight), although yield is strongly dependent on the developmental stage of the leaves and on season. Isolated protoplasts are typically 10-25 µm in diameter and include a mixed population of chloroplast-containing and chloroplast-free cells. They can be used for subsequent culture and PEG-mediated transient transformation, while sustainable regeneration requires further optimization.