Filter the enzyme solution containing protoplasts after wetting the 100 mesh stainless cell strainer (pore size: 149 mm)
with W5 solution, and centrifuge the filtrate in a 50 ml round-bottomed tube at 79 Â g for 4 min. Remove as much
supernatant as possible without disturbing the protoplasts pellet.
Re-suspend the protoplasts in 5 ml WI by gentle swirling and centrifuge centrifuged at 79 Â g for 4 min. Remove as much
supernatant as possible and re-suspend the protoplasts in 3 ml WI by gentle swirling, and inject 5 ml of 23% (w/v) sucrose
solution into the bottom of centrifuge tube, then centrifuge at 79 Â g for 4 min.