An efficient woody plant protoplast platform enables transgene-free multiplex genome editing and rapid trait validation in pear

Multiplex editing is crucial for analyzing complex multiple-gene traits in woody plants, yet its application remains limited by low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, this study establishes an efficient protoplast isolation protocol for Pyrus, employing 1.0% cellulase R10 and 0.4% macerozyme R10 with an 8.5 h digestion, and demonstrates its broad applicability across seven economically important woody plants. Coupling a 40% PEG-4000-mediated transfection regimen with DNA-free CRISPR/Cas9 ribonucleoprotein (RNP) delivery enabled multiplex genome editing in isolated protoplasts. Using this platform, simultaneous disruption of PbrARC3, PbrPARC6, and PbrFtsZ2-1a, key components of the chloroplast division apparatus, consistently reproduced macro-chloroplast abnormalities, confirming effective multigene perturbation within a single cellular context. Notably, chloroplast division failure activated chloroplast-to-nucleus retrograde signaling, evidenced by induction of nuclear stress-response genes PbrRBOHD and PbrZAT12, a concomitant surge in reactive oxygen species, and progression to severe cellular deformation. These results establish a rapid, cross-genus protoplast-RNP workflow that enables DNA-free multiplex editing and accelerates genotype-to-phenotype analyses in woody perennials. The approach provides a practical foundation for functional genomics and supports advances in non-transgenic precision breeding of tree crops.