Abstract: INTRODUCTION: Ilex cornuta ‘Fortunei’ is widely used in landscape gardening and is a common sculptured tree species. An efficient and simple protoplast preparation and separation system is of great value for breeding and gene research of woody plants. However, there are no relevant reports on the protoplast isolation and transformation system of I. cornuta ‘Fortunei’ so far, making it necessary to carry out this study.  RATIONALE: Woody plants face challenges in protoplast isolation and transformation due to characteristics such as perennial stem lignification and leaf leatheriness. Differences in cell wall composition and tissue structure among species require optimization of protoplast systems for specific species. To establish an efficient protoplast preparation and transient transformation system for I. cornuta ‘Fortunei’, orthogonal experiments and gradient experiments were used to explore factors affecting protoplast separation, and PEG-mediated method was applied for transient transformation.  RESULTS: Compared with other enzyme combinations, the orthogonal test showed that the optimal enzyme concentration combination was 2.5% (W/V) cellulase, 1.0% (W/V) macerozyme, 0.5% (W/V) pectinase, and 0.4% (W/V) snailase. After 3.5 h of enzymolysis, the protoplast yield was the highest (1.18×107∙g–1 FW) with a viability of 68.30%. The suitable mannitol concentration for osmotic regulation was 0.30 mol·L–1, and dark pretreatment of leaves for 12 h improved the effect. For transient transformation, 40% (W/V) PEG-4000 mediated the transformation of pORE_R4-IcFPS2-GFP plasmid into protoplasts, and green fluorescent signals in the nucleus overlapping with DAPI blue fluorescent signals were observed under a laser confocal microscope, indicating successful transformation.  CONCLUSION: In conclusion, this study successfully established an efficient protoplast isolation system for I. cornuta ‘Fortunei’ with optimal conditions including specific enzyme combination, enzymolysis time, osmotic regulator, and dark pretreatment. The PEG-mediated transient transformation system was also successfully constructed. These results provide a foundation for future protoplast fusion breeding and gene function research of I. cornuta.

Key words: Ilex cornuta ‘Fortunei’,  protoplast,  enzymatic hydrolysis,  transient transformation