Abstract

Effects of four salts addition (10-200 mM), NaCl, KCl, MgCl₂, and CaCl₂ were examined on the growth of protoplasts and formation of helical 1.5 mm long, protoplast-callose-fibers (PCF) in Arabidopsis thaliana liquid leaf protoplast cultures. The basal medium was Murashige and Skoog’s medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine, 3% sucrose, and 0.4 M mannitol. The protoplast division was highly stimulated by the addition of 50 mM Ca²⁺ ion but was totally inhibited by 50 mM Mg²⁺ ion. Inhibition by K⁺ and Na⁺ ions was in-between. By contrast, PCF formation, whose numbers were counted under a fluorescence inverted microscope after Aniline Blue staining for β-1,3-glucan (callose), was stimulated by both K⁺ and Ca²⁺ ions but inhibited by Mg²⁺ ion. After selecting Arabidopsis PCF using a micromanipulator, the sub-fibril ultrastructure was examined by transmission electron microscopy (TEM). The PCFs of Betula platyphylla and Larix leptolepis, cultured with 200 mM Ca²⁺ and 50 mM Mg²⁺ ions, respectively, after long-term storage and rehydration, were examined by TEM. The effects of different factors were discussed on PCF formation and sub-structures in different herbaceous and tree plant species.

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