A rapid, efficient method for the mass production of pollen protoplasts from Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast.

An optimized protocol was established to isolate large numbers of mature living pollen protoplasts of Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast. Intact pollen grains of P. bungeana or pollen with short tubes were incubated with gentle agitation in a solution of 2% cellulase R-10, 1.5% macerozyme R-10, 15% sucrose, 0.01% H3BO3, and 0.01% CaCl2. Intact pollen protoplasts with diameters of 40 μm were liberated, with an isolation rate of up to 70% after 6 h of enzymatic incubation. The optimal pH and temperature for the reaction were 5.8 and 24 °C, respectively, and the optimal enzymatic digestion conditions were 6 h of incubation in the above solution. The method for isolating pollen protoplasts from P. wilsonii was similar to that for P. bungeana, except that the incubation medium contained 12% rather than 15% sucrose and the optimal enzyme concentrations were 3% cellulase and 2% macerozyme. The isolated pollen protoplasts were demonstrated to be living by microscopy in a fluorochromatic reaction with fluorescein diacetate (FDA).