In recent years, plant protoplasts have become increasingly important in plant biology research (see reviews 5,23,25). For example, protoplasts have been used in the studies of intracellular localization of metabolic processes (2,3,18), cell wall biosynthesis (21), organelle isolation and characterization (2,3,13,18,19), and protoplast fusion in genetic research (4). Because of the lack of methodology in the large scale isolation of viable root protoplasts, no studies have yet been conducted on ion uptake in isolated protoplasts from root cells. Recent reports by Mettler and Leonard (15,16) Delaware, 19898. 2Prehminay results had been reported at the "Plant Membrane Transport Workshop," Toronto, Canada, July, 1979. showed that this treatment inhibited cell wall digestion by cellulase during the isolation procedure. As shown diagrammatically in Figure 1, the segments were then incubated in a cell wail digestion enzyme solution (3 g tissue/20 ml enzyme solution) consisting of 2% Cellulysin (Calbiochem), 1% hemicellulase, and 0.5% pectinase in a solution of 0.2 mM CaC12 and 0.6 M mannitol at pH 5.5 for 3.5 to 4.5 h with constant shaking (50 cycles/min) at 30 C. After the cuticle and some undigested materials were removed with a pair of forceps or a spatula, the remaining crude protoplast mixture was layered onto 20 ml of a solution containing 5% Ficoll (type 400), 25 mm Mes (pH 5.5), 0.2 mm CaCl2, and 0.5 mm DTT3 in 0.7 M mannitol. The mixture was then centrifuged at 400g for 10 min to pellet the starch grains and other large debris. The isolation procedure was continued by transferring the upper layer, which still contained most of the intact protoplasts, to another centrifuge tube where they were diluted with 15 to 20 ml of 0.7 M mannitol containing 25 mm Mes (pH 6.0), 0.2 mm CaCl2, and 0.5 mm DTT (protoplast suspension solution). Protoplasts were immediately spun down at 2,000g for 10 min and then washed once with an additional 20 ml of suspension solution.Final protoplast pellets were resuspended in 7 ml of the protoplast suspension solution to which 10%o Ficoll had been added, and 7 ml each of 8, 5, and 0%o Ficoll-containing protoplast suspension solutions were then layered on top in sequence. TheFicoll gradients were then centrifuged at 300g for 20 min. Based on light microscopic analysis, intact viable protoplasts were found at the interface of the 0 and 5% Ficoll gradients. These were removed with a pipet, washed once with 20 ml protoplast suspension solution, followed by a 10-min, 2,000g-pelleting centrifugation. Morphologically, intact protoplasts could also be found in other interfaces ofthe Ficoll gradient. However, vital staining tests showed that most of these protoplasts were not viable. With the exception of incubation, all procedures were performed at room temperature. All solutions used were sterilized by passing them through a 0.
