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.
