Laser Microperforation of Medicago Sativa Root Hair Cells

Microperforation of the plant cell wall of root hairs from Medicago sativa can be performed without mechanical contact by using a UV laser microbeam under plasmolytical conditions. Through a gentle and progressive plasmolysis the cells remain viable and the plasma membrane stays intact. The cellular polarity of the root hairs is basically maintained, and it is possible to gain direct access to distinct membrane areas. The plasma membrane is free of visible traces of cell wall fragments, as shown by fluorescence microscopy with Tinopal. The opening in the cell wall (from 1 to 5 μm) leads to a probably turgor-driven movement of protoplasm. The protoplasm can be completely extruded forming a spherical, turgid protoplast which contains the nucleus and shows cytoplasmic streaming. In many cases, only a partial extrusion of the cell content leads to subprotoplasts of various sizes which are still connected to the root hair. Protoplast viability was tested by staining with fluorescein diacetate. The movement of protoplasm depends on the calcium concentration of the extracellular medium. Addition of 0.5 to 1 mM calcium chloride stabilizes the process and clearly increases the yield of successfully opened root hairs. Laser microsurgery avoids the side effects of enzymatic digestion of the plant cell wall. Furthermore, the cell-to-cell interactions through plasmodesmata can be maintained. This preparative laser microtechnique should provide primary material for the study of membrane properties, for example, by using the patch clamp technique (Neher, 1992; Sakmann, 1992).