Establishment of protoplast isolation, purification and transient transformation system from Rhododendron petals

The genus Rhododendron, comprising approximately 1200 species, is the largest within its family and possesses significant ornamental value. However, functional genomics studies in this genus are hampered by an inefficient Agrobacterium-mediated stable transformation system. To facilitate rapid gene function verification, we developed a protoplast-based transient expression system using petal tissues. We optimized the enzymatic hydrolysis conditions (2.0% cellulase, 1.00% macerozyme, 0.6 mol/L mannitol, 8-h digestion) and established a two-step purification protocol involving centrifugal precipitation followed by flotation. For polyethylene glycol (PEG)-mediated transformation, the optimal parameters were 40% PEG4000, 0.10 mol/L CaCl₂, 35 °C, 25 μg of plasmid, and a 20-min incubation. The system's feasibility for subcellular localization was confirmed by expressing HSFC1a-eGFP and RCI2B-eGFP fusion proteins in R. pulchrum protoplasts. Furthermore, qRT-PCR analysis showed that the transcript levels of HSFC1a and RCI2B peaked at 12 h post-transformation, demonstrating time-dependent expression dynamics.