However, the mean viability of isolated protoplasts decreased to 54% and 52% after 12 h
digestion of leaves and 20 h of callus, respectively. Donor tissues were pre-plasmolyzed in CPW with either 0.7 M mannitol or sorbitol for 30, 60, or 90 min. significant differences
in protoplast yield and viability were observed among osmotica and pre-plasmolysis durations. Sixty-min preplasmolysis of in vitro leaves and 90 min pre-plasmolysis of callus in 0.7 M sorbitol was the most efficient pretreatment for further achievement of protoplasts (Table 3).