Development of a xylem protoplast expression system for gene function analysis and genome editing in Fraxinus mandshurica Rupr

Protoplast-based engineering is a powerful tool in plant genetic studies and genome editing, yet its application in Fraxinus mandshurica (a species crucial for environmental, agroforestry, and industrial purposes) has been limited. This study aimed to develop a method for isolating and transfecting stem differentiating xylem (SDX) protoplasts from F. mandshurica, thereby providing a platform for genetic manipulation and functional genomics. The optimized protocol yielded a maximum protoplast count of 7.35 × 10⁶/g with 81.8 % viability, and an improved transfection efficiency of 64.71 %. Transient protein subcellular localization was achieved; and virus induced gene silencing (VIGS) assays demonstrated gene silencing efficiencies ranging from 64.5 % to 70.3 %; protein-protein interactions between glycogen synthase kinase 3 (GSK3) and BRI1-EMS-SUPPRESSOR1 (BES1) were confirmed using co-immunoprecipitation and bimolecular fluorescence complementation experiments; additionally, CRISPR/Cas9-based gene editing achieved an editing efficiency of 8.6 % in F. mandshurica protoplasts, while adenine base editing (ABE) utilizing Cas9 nickase (nCas9) introduced precise -A to -G conversions in the target gene FmBES1 with efficiencies ranging from 1.05 % to 3.4 %. The study demonstrates the successful application of protoplast-based technologies in F. mandshurica, especially CRISPR/Cas9 and base editing, which offers a promising tool for accelerating genetic improvements. The integration of protoplast technology with traditional breeding methods could shorten breeding cycles and enhance desirable traits such as improved wood quality and bioenergy production in F. mandshurica.