Systematic Evaluation of Mesophyll Protoplast Isolation and PEG-Mediated Transient Expression in Rosa chinensis 'Old...

Stable transformation in rose is slow and genotype-dependent, motivating rapid cellular assays for candidate-gene testing. We systematically evaluated mesophyll protoplast isolation and PEG-mediated transient expression in Rosa chinensis 'Old Blush'. Sequential experiments compared enzyme preparations, digestion time, D-mannitol concentration, cultivar, and donor cultivation regime. Under the tested conditions, Yakult preparations containing 3.0% Cellulase R-10 and 2.0% Macerozyme R-10, 16 h digestion, and 0.4 mol/L D-mannitol yielded 4.641 ± 0.102 × 10⁶ protoplasts/g fresh weight with 94.26 ± 0.15% FDA viability. Recovery varied significantly among donor regimes and between the two cultivars tested. In a 27-combination factorial experiment with three plants as blocks, 25% PEG 4000 working solution (12.5% nominal final concentration), 8 min transfection, and 20 h dark incubation gave the highest observed mGFP6-positive fraction (7.14 ± 0.22%). This efficiency supports imaging of individually transfected cells under the tested conditions, but broader population-level or high-throughput applications require further validation. Protoplast division, plant regeneration, stable transformation, cross-date reproducibility, and transfer to other genotypes were not assessed.