Abstract

Protoplast technology offers a unique single cell system that facilitates several aspects of modern biotechnology. In this study, an efficient protocol to isolate the protoplast from callus culture of a valuable medicinal plant in Southeast Asia,Eurycoma longifolia was developed. A range of parameters which influence the isolation of E. longifolia protoplasts were investigated by using “change-one-factor-at-a-time” method. From the results obtained, callus fresh weight (FW) of 0.2 g produced the highest number of viable protoplasts, which was 1.58 ± 0.36 × 10⁴protoplasts/gFW. The highest amount of viable protoplasts (1.75 ± 0.68 × 10⁴protoplasts/gFW) was obtained when the sorbitol concentration was maintained at 0.5 M. The optimum enzyme concentration was found to be 1.5% (w/v) of cellulase and pectinase in which 2.75 ± 1.04 × 10⁴ protoplasts/gFW were isolated. Meanwhile, an incubation period of 3 h with enzyme solution resulted in the maximum yield of protoplasts (5.58 ±1.46 × 10⁴ protoplasts/gFW).

Key words: Eurycoma longifolia, callus culture, protoplast, osmoticum, viability.