Isolation of Viable Protoplasts from Gracilaria salicomia (Rhodophyta)

Abstract

The study aimed to present the technique for the isolation of protoplasts from Gracilaria salicornia using commercial enzymes agarase, cellulase and macerozyme R-10. Surface sterilization of the specimens prior to the treatments was carried out with 4% Betadine® to prevent bacterial contamination. The pH of the isolation medium and temperature for the enzyme reaction was 6.5 and 20°C, respectively. Mannitol (l.OM) v/as used as the osmotic stabilizer. When the tissue sample (0.1 g, fresh wt.) of G. salicornia was digested with an enzyme mixture consisting of 100 units of agarase, 30 units Cellulase, 1% Macerozyme, and 1.0 M mannitol in 20 niM MES buffer (pH 6.5) and sterile seawater as base fbr 90 min at 20°C and 60 rpm, 1.85 x 109 viable protoplasts were successfully liberated whose viability was tested, using 0.1 % Evans Blue staining.