Optimized and Reliable Protoplast Isolation for Transient Gene Expression Studies in the Gymnosperm Tree Species Pinus densiflora

Efficient protoplast isolation and gene transfection remain significant challenges in gymnosperms, particularly in Pinus species, where stable transformation is highly limited. Conventional pine protoplast preparation methods have resulted in extremely low transfection efficiencies, hindering functional genomic studies. This study presents an optimized method for isolating high-yield, viable protoplasts from Pinus densiflora (Korean red pine), providing a robust system for transient gene expression assays. Splitting one-month-old cotyledons produced the highest mesophyll protoplast yield (5.0 × 106 cells/g FW), which further increased to 1.2 × 107 cells/g FW after optimizing the enzyme mixture (4.5% cellulase, 0.7% pectinase, 3% hemicellulase), maintaining viability above 86%. Developing xylem and whole-stem protoplasts were also successfully isolated by mitigating resin leakage and debris contamination, with a 17% sucrose gradient yielding 7.4 × 104 cells/g FW at 81.9% viability. Overcoming prior inefficiencies, this protocol significantly enhances gene transfection efficiency, achieving 94.1% GFP transformation with 82.9% viability. Furthermore, transient activation assays confirmed strong activation of pine-derived reporters by native effectors, underscoring the assay’s suitability for studying gymnosperm-specific gene regulation. Given the limited stable transformation strategies available for Pinus species, this optimized protoplast transient gene expression system provides a practical and reliable platform for transient gene expression analysis, offering valuable opportunities for studying gene function and regulation in gymnosperms.