Experiments to maximize the isolation and purification of viable protoplasts from shoot cultures of asparagus (Asparagus oficitzalis L.) were conducted. Important factors for high yield of viable protoplasts included: the use of in vitro etiolated shoots as source material; 0.6 M glucose as an osmoticum in a modified KM medium; a combination of pectinase, cellulase, and hemicellulase, each at 1% (wlv) for enzymatic digestion of cell walls; and physical factors such as the volume of enzyme

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... on and speed of gyratory shaking. Protoplasts were purified by suspending digested etiolated shoot tissue in 0.6 M sucrose, overlaid with KMG medium and centrifugation at 650 g. The asparagus genotype had a marked influence on protoplast yield, with some genotypes yielding up to 18.4 X 10h protoplastslg fresh etiolated shoot tissue with 90% viability.