Development of Protoplasts from Grateloupia sparsa and G. filicina (Halymeniaceae, Rhodophyta)

For isolation of protoplasts from Grateloupia sparsa and G. filicina, commercial enzymes of cellulase, macerozyme, agarase, abalone acetone powder and papain were used in seven combinations. A maximum yield of protoplasts of 7 × 10 8 g −1 (fw) was obtained with a mixture of 4% cellulase, 2% macerozyme, 50 U/mL or 1000 U/mL agarase and 4% papain. Very few protoplasts (10 3 g −1 fw) were isolated with mixtures of cellulase and macerozyme. After isolation, protoplasts were incubated in 0.7 M mannitol-PES medium for 10 days then transferred to PES medium to obtain the highest number of regenerated protoplasts. Protoplasts excreted amorphous matrix on their surface as soon as they were isolated, and deposited cell wall after 4 days of culture