Efficient multi-tissue protoplast isolation and transient expression system in Osmanthus fragrans

Plant protoplasts offer a versatile system for genomics, transcriptomics, proteomics, metabolomics, and epigenetics analyses. However, an efficient and reliable protocol for isolating Osmanthus fragrans protoplasts has not yet been established. In this study, we developed a protoplast isolation method using enzymatic digestion and evaluated key factors affecting isolation efficiency. The optimal yield (4.66 × 10⁷/g·FW) and viability (90.50%) were achieved by digesting second-stage leaves of ‘Yanhonggui’ potted seedlings for 6 h in an enzymatic solution containing 3% Cellulase R-10, 0.75% Macerozyme R-10, 0.4–0.5% Pectolyase Y-23, 0.25% Snailase, and 0.6 M mannitol. Additionally, this method was successfully adapted for isolating protoplasts from different tissues (stems, leaves, and petals) and cultivars of O. fragrans . To further optimize transformation efficiency in mesophyll protoplasts, the effects of PEG4000 concentration, plasmid amount, and incubation time were investigated. The highest transformation efficiency (64.67%) was achieved with 40 μg of plasmids per reaction and a 15 min incubation in 40% PEG4000. This protocol was then used to localize OfMYB5 and OfCCD4 proteins to the nucleus and chloroplasts, respectively, and to analyze protein-protein interactions between OfMYB21 and OfJAZ3 in a homologous system. In addition, the positive regulatory effect of OfMYB5 on OfEXPA2 was verified. In conclusion, we have established an efficient protoplast isolation and transient expression system for O. fragrans , providing a robust platform for studying the molecular biology of this species.