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.