Initially, protoplasts were isolated to detect various parameters using flow cytometric analysis. The most efficient ratio of cells to enzyme solution, for digestion of cell walls, needed to be established. To detect whether the time of incubation with the enzyme solution influenced the state or viability of the protoplasts, they were observed periodically under the light microscope during digestion at different concentrations of enzyme solution. After 2 h digestion with light swirling every 20 min, the protoplasts were still intact (Figure 1), and viable as detected with Trypan blue staining (results not shown). Increasing the digestion period led to a decrease in cell membrane integrity. The size of the protoplasts varied between 60 mm and 90 mm. Figure 1 shows the difference between cells before digestion with an enzyme solution and protoplasts after digestion. Size determination of protoplasts was important since the flow tip of the flow cytometer is limited to 100 mm and if the protoplasts exceeded this size, could lead to blockages in the flow tip of the flow cytometer, with ineffective readings and a time consuming clean-up process. / Dr. Marianne J. Cronje