Gardenia jasminoides Ellis is a commercially important medicinal and ornamental plant; however, its functional genomics remain poorly understood because of the lack of efficient cell-based research tools. To address this limitation, we established an optimized method for isolating viable protoplasts from petal and mesophyll tissues of G. jasminoides and developed a polyethylene glycol (PEG)-mediated transient expression system. For petal protoplast isolation, the optimal enzyme combination consisted of 3.0% cellulase R-10 and 1.0% macerozyme R-10 supplemented with 0.5 M D-mannitol, yielding 5.26 × 106 protoplasts per gram fresh weight (FW) with 80.63% viability. For mesophyll protoplast isolation, 1.5% cellulase R-10 and 0.5% macerozyme R-10 supplemented with 0.5 M D-mannitol produced 8.75 × 106 protoplasts g−1 FW with 84.55% viability. PEG-mediated transient transformation was optimized at 20% PEG4000 for petal protoplasts and 40% PEG4000 for mesophyll protoplasts, resulting in efficient GFP expression. This system was successfully applied to subcellular localization analyses of floral regulatory proteins (GjAP3, GjPI, and GjSEP) and defense-related proteins (GjNPR1 and GjTGA2), as well as to the validation of protein–protein interactions between GjSEP and GjPI and between GjNPR1 and GjTGA2 using bimolecular fluorescence complementation and yeast two-hybrid assays. Collectively, these results establish a reliable and species-specific protoplast-based platform for rapid functional characterization of genes in G. jasminoides, providing an effective tool for future studies on gene regulation, metabolic engineering, and molecular breeding in this horticultural plant species.
Chen et al. (Thu,) studied this question.