For transformation, 100 ll protoplast solution was gently and
thoroughly mixed with 10 ll plasmid DNA solution (containing
10–20 lg plasmid DNA/dsRNA) and 110 ll PEG solution (40% w/v
PEG-4000, 0.6 M mannitol, 100 mM CaCl2). The mixture was
incubated for 15–25 min at room temperature, and then the cells
were washed once in 440 ll W5 solution. The protoplasts were
incubated in 1 mL WI solution (4 mM MES, pH 5.7, 0.6 M mannitol,
20 mM KCl) in the dark for 12–48 h. The protoplasts were
harvested and stored at 80 C until use.