Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1–sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

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.