Abstract
Elongated and spiral β-1,3-glucan (callose) fibers were obtained by new factors from protoplasts cultured in liquid medium from suspension cultured cells of two salt-tolerant mangrove species; Avicennia alba and Bruguiera sexangula. Differences in salt factor for protoplast-fiber formation were compared with those of the callose fibers developed from protoplasts of non-mangrove tree plants, Larix leptolepis and Betula platyphylla, which high concentrations of divalent cations, Mg2+ (50 mM) or Ca2+ (100 mM), were stimulatory. In the halophilic A. alba protoplasts, whose cell division was stimulated by up to 400 mM NaCl, addition of Mg2+, Ca2+, K+ ions inhibited protoplast-fiber formation. In B. sexangula, protoplast-fibers were rapidly and efficiently formed only by another new factor, electric cell fusion treatment of protoplasts. Spiral fibers developed from mangrove protoplasts were detected under an inverted microscope, and their specific blue-green color for callose after staining with Aniline Blue dye was detected under a fluorescence microscope. Enzymatic certification of callose was further performed with laminarinase, specific for callose, in comparison with cellulase CBH1, specific for cellulose. Differences in sub-structures, fibrils and sub-fibrils of two mangrove protoplast-fibers were analyzed using laser confocal scanning microscopy, atomic force microscopy and image J analysis. Tube-like fine structure was observed using transmission electron microscopy in single protoplast-fiber of B. sexangula selected with a micromanipulator.
Highlights
Sasamoto et al (2003) reported novel protoplast-fiber formation using protoplast cultures of Betula platyphylla leaves and embryogenic cells of a conifer, Larix leptolepis, in liquid medium containing high concentrations of CaCl2 (100 mM), and MgCl2 (50 mM), respectively
Inhibition of fiber formation of A. alba by divalent cations is a phenomenon different from that previously reported in protoplast-callose-fibers developed from protoplasts of non-mangrove tree plants, Larix and Betula which required a high concentration of divalent cations, Mg2+ (50 mM) and Ca2+ (100 mM), respectively (Sasamoto et al, 2003; Fukumoto et al, 2005)
High concentrations of divalent cations Mg2+ and Ca2+, which are factors of protoplast-callose-fiber formation in non-mangrove tree species, Larix and Betula, were not effective for protoplast-fiber formation in the halophilic mangrove plant, A. alba and salt-tolerant mangrove plant, B. sexangula. In the former A. alba protoplast, addition of a low concentration of NaCl or without additional salts were effective for formation of protoplast-callose-fiber. In the latter B. sexangula protoplasts, though cell division was stimulated by divalent cations, protoplast-fibers were not formed with the salts
Summary
Sasamoto et al (2003) reported novel protoplast-fiber formation using protoplast cultures of Betula platyphylla leaves and embryogenic cells of a conifer, Larix leptolepis, in liquid medium containing high concentrations of CaCl2 (100 mM), and MgCl2 (50 mM), respectively. A spiral elongated fiber developed from the surface of a spherically enlarged protoplast. These findings suggested the importance of divalent cations in protoplast-fiber formation. The spiral protoplast-fibers could be observed under an inverted microscope after two weeks to more than one month of protoplast culture. They were stained with Aniline Blue dye which showed a β-1,3-glucan (callose) component. The protoplast-fibers were studied further using laser confocal scanning microscopy (LCSM) with Alexafluor 488 phalloidin staining, and using single cell TEM to visualize the site of protoplat-fiber elongation from a single Larix protoplast selected with a http://jps.ccsenet.org
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