Plasmolysis of Glycine max mesophyll cells: the use of octylguanidine and its implications in protoplast isolation

Abstract

Soybean ( Glycine max Merr cv. ‘Ransom’) mesophyll cells were difficult to plasmolyze when placed in mannitol. Plasmolysis behavior depended largely on plant age and cell type. Difficulty in plasmolyzing mesophyll cells increased with plant age. Cells of the the second palisade layer were more difficult to plasmolyze than those of the first layer and spongy parenchyma. No plasmolysis occured in cells from podding stage plant material (16 weeks old). Brief treatment of cells with 0.3 mM octylguanidine (OG) in 0.4 M mannitol decreased plasmamembrane adherence to the cell wall and enhanced plasmolysis in all cell types except starch filled palisade cells of the second layer of 16-week-old material. Light starvation of tissue and OG treatment or 0.75 M CaCl 2 as plasmolyticum improved plasmolysis in cells of the second palisade layer of leaves of 10–16-week-old plants. These observations indicate that plasmolysis of soybean mesophyll cells is difficult due to either strong adherence of the plasma membrane to the cell wall, and/or mechanical resistance to protoplast contraction as a consequence of starch accumulation in chloroplasts. Our observations on the plasmolytic behavior of mesophyll cells and the high protoplast yield at microscale isolation in situ suggest that plasmolysis using OG will result in higher protoplast yield and quality.