Analysis of the Formation of Protoplasts and Regeneration of Cells in Phycomyces blakesleeanus

It is possible ot prepare protoplasts of the zygomycete fungus, Phycomyces blakesleeanus, by digesting the cell wall of spore germlings with commercially available chitinase and chitosanase. However, the cells without any cell walls immediately form large aggregates, and thus, it is difficult to isolate the individually separated protoplasts. Inherent problem with the formation of aggregates in preparing protoplasts could be solved by the use of bovine serum albumin (BSA). As a result, we were able to prepare a large number of single protoplsts quickly and easily. We took time-lapse photomicrographs of the formation of protoplasts, and found that there were certain regions of the cell wall of spore germlings that were sensitive to chitinase and chitosanase, although the cell wall of the original spores is known to be insensitive to these enzymes. There are two kinds of cell walls on a spore germling; one with a bound wheat germ agglutinin (WGA), and the other a bound concanavalin A (ConA). Furthermore, only cells with walls which had bound WGA were able to regenerate, while those with walls with bound ConA were not able to regenerate.