Optimization of parameters for isolation of protoplasts from the antarctic sea ice alga Chlamydomonas sp ICE-L

The present study was initiated to provide a systematic protocol for producing protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L suitable for physiological studies. The results showed that the mixtures of 3.0% Cellulase R-10 and 2.0% Macerozyme R-10 were most effective for isolating protoplasts from this alga. Optimum pH and temperature for hydrolytic enzyme reaction were pH 6.0 and 15 degrees C, respectively. Mannitol and sorbitol were found to be the excellent osmotic stabilizers. Growth conditions of the algae prior to enzyme treatment also influenced the yield of protoplasts greatly. At the optimized condition, protoplast production was 47.8%, and the viability of isolated protoplasts was more than 97.6% as confirmed by Evan's blue staining.