Improvements in Protoplast Isolation Protocol of Dendrobium antennatum Lindl

This study aimed to determine the effect of a combination of enzyme concentrations, incubation time, and osmoticum concentrations on protoplast isolation mesophyll of leaves of Dendrobium antennatum Lindl. This was an experimental study using a 3×3×3 factorial design and three replications.The treatment variable was a combination of 3 levels of enzyme concentration (0.5% cellulase + 0.2% pectinase, 1% cellulase + 0.2% pectinase, and 1.5% cellulase + 0.2% pectinase), 3 levels of long incubation time (6 hours, 9 hours, and 12 hours), and 3 levels of osmoticum concentration (0.3 M sucrose, 0.4 M sucrose, and 0.5 M sucrose). The data on protoplast density and the number of viable protoplasts were analyzed using the Kruskal-Wallis test followed by the Whitney test. The results showed that the combination of the three treatment variables influences protoplast density and the number of viable protoplasts. The highest protoplast density was achieved in the combined treatment (1.5% cellulase + 0.2% pectinase), 6 hours of incubation time, and 0.3 M sucrose. The highest number of viable protoplasts was achieved in the combined treatment (0.5% cellulase + 0.2% pectinase), 6 hours of incubation time, and 0.5 M sucrose.