After incubation, the reaction mixture was filtered through a glass filter (15 AG 2) and the filtrate was refiltered through a glass filter (15 AG 3). The filtrate was centrifuged at 700 g for 5 min. Sedimented protoplasts were suspended in 10 ml of hypertonic liquid MM containing 0.6 M NaCl and centrifuged twice at 700 g for 5 min and finally the pellets were resuspended in 1 ml of hypertonic liquid MM.