Table 1 Solutions for protoplast isolation and transfection

Enzyme solution

20 mM MES KOH, 1.5% (W/V) Cellulase R-10, 0.5% (W/V) Macerozyme R-10, 10 mM CaCl₂, 20 mM KCl, 0.1% BSA (W/V) and 0.5 M Mannitol pH = 5.7

Freshly prepared

Digestion of cell wall material

WS2

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

4 °C

Washing protoplasts

MMG

0.4 M Mannitol, 15 mM MgCl₂, 4 mM MES pH 5.7

Freshly prepared

Resuspend the protoplast for counting and transfection

PEG-Ca²⁺ solution

40% (W/V) PEG 4000, 0.2 M Mannitol and 0.1 M CaCl₂

Freshly prepared

Plasmid transformation

W1

0.5 M Mannitol, 20 mM KCL, 4 mM MES pH = 5.7

Freshly prepared

Resuspend protoplast after transfection