First report of genetic transformation and CRISPR/Cas12a-mediated gene editing of European beech (Fagus sylvatica L.)

Fagus sylvatica L. (European beech) is a dominant hardwood forest tree species across Central Europe, supporting diverse ecosystem services and forming the basis of a significant market for high-value timber. However, climate change increasingly threatens beech vitality and productivity, making molecular insights into its stress resilience and functional validation of underlying genes urgently needed. Here, we report an improved protocol for protoplast isolation from seedling leaves and demonstrate, for the first time, transient genetic transformation and CRISPR/Cas-mediated genome editing in F. sylvatica. PEG-mediated transformation was sequentially optimized, achieving efficiencies of 59 {+/-} 6.19%. Transformation efficiency was strongly influenced by season, which also affected protoplast yield. Both, a basic molecular toolkit for functional genomics and future biotechnological applications were established by testing a set of promoters and reporters. For proof-of-concept genome editing, we achieved 4.75 to 32.69% editing efficiencies in the PHYTOENE DESATURASE gene (FsPDS) using temperature-tolerant LbCas12a (ttLbCas12a). The establishment of a reliable protoplast transformation and editing system provides a crucial foundation for future genetic improvement and functional studies in this non-model tree species.