Separation and Purification of Protoplast Types fromCommelina communisL. Leaf Epidermis

Guard cell and epidermal/subsidiary cell protoplasts obtained by enzymic digestion of peeled Commelina communis leaf epidermis were separated and purified by discontinuous density gradient ccntrifugation with media based on Percoll (Pharmacia Fine Chemicals AB, Uppsala, Sweden). The cell types were recovered over 99.9% pure at yields exceeding 50% efficiency, and mesophyll contamination could be virtually eliminated when desired. Osmotic characteristics of the protoplast types were evaluated and compared to in vivo values, and the viability of the protoplasts, assessed using a range of criteria, was found to be high. Purified Commelina guard cell protoplasts were able to evolve O2 when illuminated, and this was substantially reduced in the presence of the inhibitor DCMU, indicating that they possess photosystem II activity. Specific advantages of this method of protoplast purification, and the potential uses of separate suspensions of guard cells and epidermal/subsidiary cells in experiments on stomatal physiology are discussed.