Purification of Enzymatically Isolated Mesophyll Protoplasts from C3, C4, and Crassulacean Acid Metabolism Plants Using an Aqueous Dextran-Polyethylene Glycol Two-Phase System

Abstract

Enzymatic digestion of leaf segments with 2% cellulase, in combination with a pectinase in some species, yields intact protoplasts mixed with epidermal tissue, vascular tissue, broken protoplasts, and chloroplasts. Epidermal and vascular tissue are removed with sieves of various porosity. Intact protoplasts in the filtrate are separated from other components by an aqueous two-phase system which consists of dextran-polyethylene glycol, with sorbitol and sodium phosphate. Intact protoplasts partition at the interphase, while chloroplasts and broken protoplasts partition in the lower phase when the separation is facilitated by low speed centrifugation. The optimum conditions for purification of maize mesophyll protoplasts with high yields are centrifugation of the two-phase system at 300g for 6 minutes at 2 C with a mixture including 0.46 m sorbitol, 10 mm sodium phosphate, 5.5% polyethylene glycol 6000, and 10% dextran of average molecular weight of 20,000 to 40,000. The collection of protoplasts at the inter-phase was proportional to the amount of chlorophyll added over a wide range of concentrations regardless of the initial contamination of the preparation by other cellular debris. The two-phase system is applicable for protoplast purification from a wide variety of species, including C₃, C₄, and Crassulacean acid metabolism plants, regardless of protoplast size.