Formation and regeneration of protoplast from a unicellular green alga Haematococcus pluvialis

An effective method of preparing osmotically-labile protoplasts from Haematococcus pluvialis was established. Microalgal cells grown semi-synchronously under weak blue light was most effective in obtaining viable protoplasts with Proteinase K (0.06%) treatment in the presence of 0.2 M sorbitol/mannitol. The frequency of the osmotically-sensitive cells reached maximum in as short as 15 min protease treatment at 35°C. Protoplast formation was also verified from structural changes of the algal cells under microscopic observation. Protoplast regeneration was successfully carried out by stepwise reduction of sorbitol/mannitol concentrations from 0.2 M to 0.05 M followed by incubation on a plate under various osmotic condition. This stepwise reduction of the osmosis enabled the protoplasts to regenerate the algal cell wall with significantly improved yield.