Abstract: Little information is available about the research on plant pollen protoplasts，especially the isolation of apple pollen protoplasts. In this study，the protoplasts from mature pollen of Malus × domestica Borkh.‘Fuji’by the method of‘germination-enzymatic treatment’were isolated. To screen the most effective condition of isolation，some key factors were analyzed. The results indicated that：The isolation percentage was up to 6.83% when the pollens germinating for 45 min and then treated with mixed enzyme solution including 1% Cellulase Onozuka R-10，1% Macerozyme R-10 and 18% Mannitol for 6 h. The cell walls of isolated protoplasts were degraded entirely according to the examination of 0.1% calcoflower white staining. 0.01% fluorescein diacetate（FDA）staining indicated the protoplasts were viable. Therefore，the proroplasts can be used for further research.

Key words: apple, pollen, protoplast, isolation