Studies on Cell Wall Regeneration and Cell Division in Soybean Protoplasts Using Fluorescence and Scanning Electron Microscopy.

Abstract

Protoplasts were enzymatically isolated from soybean suspension cells and were cultured in a liquid medium. The reappearance of cell wall at the surface of naked protoplasts was studied by fluorescence and scanning electron microscopy. Using fluorescence microscopy (Calcofluor White staining method), the cultured protoplasts regenerated new cell walls only a few minutes after isolation and protoplasts were completely covered with new wall material after 9-7 12 h. The first division of the protoplasts was found 15 h after culture and often observed after 1-2 d culture. These regenerated cell walls could be digested with cellulase but not pectinase. By scanning electron microscopy, the wall materials deposited on the surface of protoplast were observed. After 9 h culture, short fibrils attached to projections on the plasma membrane. After that, long fibrils were formed and spread over the surface. After 1 d culture, the fibrous materials which had already deposited on the surface transformed to complex wall structure. The dividing protoplast was also observed after 12 h culture.