Table 1 Solution recipes for protoplast isolation and transformation

Enzyme solution (resuspension solution)

10 mM MES, 1.5% (wt/vol) cellulase R10, 0.75% (wt/vol) macerozyme R10, and 20 mM KCl. 10 mM CaCl2, 0.1% BSA, 1–5 mM β-mercaptoe- thanol (optional) and mannitol (0.6 M), pH 5.7

Room temp. (freshly prepared)

Leaf strips lysis

W5 solution

2 mM MES, 154 mM NaCl, 125 mM CaCl2 and 5 mM KCl, pH 5.7

4 °C

Release and wash protoplasts

MMg solution

4 mM MES, 0.4 M mannitol and 15 mM MgCl2, pH 5.7

4 °C

Resuspend protoplast pellet

PEG-Ca²⁺ solution (resuspension solution)

20% (wt/vol) PEG4000, 100 mM CaCl₂ and mannitol (0.3 M)

Room temp. (freshly prepared)

Transform plasmids (10ug is used in this study) into protoplasts