Preparation of protoplasts from Chlorella vulgaris K-73122 and cell wall regeneration of protoplasts from C. vulgaris K-73122 and C-27

Protoplasts from Chlorella vulgaris K-73122 were obtained by enzymatic digestion with a mixture of Acromopeptidase, Cellulase ONOZUKA R-10, Chitosanase KI, Gluczyme, and Uskizyme. The formation of naked protoplasts was confirmed by fluorescence microscopy using fluorescent brightner 28, which stains cell walls. About 88% of C. vulgaris K-73122 cells were converted into osmotically-labile cells. Furthermore, a method for regeneration of intact cells from the protoplasts was developed. Utilization of 0.5 M sucrose as an osmoticum, Fe-EDTA as an iron source, and bacto-agar as a supporting was shown to help regeneration of the cell walls of two strains, C. vulgaris K-73122 and C-27.