Mg2+ Ions Stimulate both Helical Protoplast-Callose-Fiber Formation and Protoplast Division in a Mangrove Tree, Sonneratia caseolaris: Analysis of Sub-fibril Structures of PCF by Atomic Force Microscopy

Abstract

The effects of four salts, NaCl, KCl, MgCl2 and CaCl2, on protoplast division and protoplast-callose-fiber (PCF) formation were examined using suspension cultured cells of a mangrove tree, Sonneratia caseolaris. Basal medium was hormone-free Murashige & Skoog’s medium containing 0.8 M sorbitol and 3% sucrose. Addition of 50-100 mM of Mg2+ ions highly stimulated both cell division and PCF formation, while addition of Ca2+ ions was inhibitory. Addition of Na+ ions at 10-25 mM and K+ ions at 50-100 mM stimulated protoplast division but not PCF formation. Helical PCF rapidly elongated from a specific site of cell division plate after 3 days of culture. The β-1,3-glucan (callose) component of the protoplast-fibers was stained with Aniline Blue fluorescent dye and was re-certified enzymatically using laminarinase. After selecting the PCF using a micromanipulator, we analyzed the fibril- and sub-fibril- structures using laser confocal scanning microscopy (LCSM), and atomic force microscopy (AFM). We discuss the uniqueness of the PCF of S. caseolaris as compared to PCFs of several plants.