PROTOPLAST ISOLATION FROM SHOOTS OF ASPARAGUS CULTURES

Experiments to maximize the isolation and purification of viable proto plasts from shoot cultures of asparagus (Asparagus officinalis L.) wer e conducted. Important factors for high yield of viable protoplasts in cluded: 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 pec tinase, cellulase, and hemicellulase, each at 1% (w/v) for enzymatic d igestion of cell walls; and physical factors such as the volume of enz yme solution and speed of gyratory shaking. Protoplasts were purified by suspending digested etiolated shoot tissue in 0.6 M sucrose, overla id with KMG medium and centrifugation at 650 g. The asparagus genotype had a marked influence on protoplast yield, with some genotypes yield ing up to 18.4 x 10(6) protoplasts/g fresh etiolated shoot tissue with 90% viability.