Protoplast isolation from shoots of asparagus cultures

Abstract

Experiments to maximize the isolation and purification of viable protoplasts from shoot cultures of asparagus (Asparagus officinalis 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% (w/v) for enzymatic digestion of cell walls; and physical factors such as the volume of enzyme solution 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 10⁶ protoplasts/g fresh etiolated shoot tissue with 90% viability.