Effect of light and Triton X-100 on the size of protoplasts from Ulothrix gigas

An electronic particle-size analyzer (Coulter Counter with Channelyzer) was used both to monitor the purity of large numbers of protoplasts during sequential steps in their isolation from Ulothrix gigas and also to detect changes in volume (swelling or growth) after incubation of purified protoplasts under various conditions. Protoplasts almost devoid of contaminating debris, but heterogenous in size, were obtained by pelleting partly-purified protoplasts through sucrose solutions. Pure protoplasts of discrete sizes were obtained by centrifuging these resuspended protoplasts on iso-osmotic mannitol/sucrose gradients. The protoplasts were shown to enlarge (swell) and then burst when transferred to increasingly hypotonic solutions or when incubated in Triton X-100. The rate of protoplast enlargement (growth) was also monitored on the Coulter Counter and differences in size of large populations were discernible within 2 h in protoplasts incubated under low light, and growth rates of such protoplasts could be assessed for up to 36 h. The Coulter Counter provides a rapid, reproducible and sensitive method for measuring the size distribution of large numbers of protoplasts. This, combined with the techniques described here for the isolation of protoplasts from U. gigas in high yield and purity, provides a system eminently suitable for investigating a wide array of physiological and developmental processes in plant protoplasts.