Cell-Wall Regeneration by Protoplasts Isolated from Higher Plants

Abstract

When first observed, the plant cell was effectively defined as a space between cell walls. In dead tissues, such spaces are empty, but the cell walls that delimit these spaces exist only because they were secreted by living protoplasts that once filled the spaces. The protoplast is the plant cell, as we now know it. Among higher plants particularly, protoplasts rarely exist naturally in the absence of their cell walls, and experimentalists have long sought to isolate them. Such isolated protoplasts were first prepared by cutting plasmolyzed tissues (von Klercker, 1892, Plowe, 1931). When the cut passed between the protoplast and the wall, the undamaged protoplast could be released. Only low yields of protoplasts could be obtained by this “mechanical” method, and it was not until the 1960s that higher yields were obtained as a result of the application to plants of the concept of protoplast isolation by enzymatic wall degradation (Cocking, 1960), a technique used previously for isolating bacterial and fungal protoplasts. Even higher yields were obtained once enyzmes of fungal origin were used (Gregory and Cocking, 1965; Takebe et al., 1968), and protoplasts are now isolated by such means from many species for numerous experimental purposes. In principle, the technique is simple. The tissue is placed either within, or on the surface of, a hypertonic solution (commonly of mannitol) containing a suitable mixture of cell-wall hydrolases.