Production of protoplasts from the brown alga, Sargassum muticum (Yendo) Fensholt (Phaeophyta)

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Abstract
A variety of enzymes were tested for their ability to produce protoplasts from Sargassum muticum (Yendo) Fensholt both singly and in various combinations. The most successful combination of enzymes was 2% Cellulase Onozuka RS, 10% Limpet Acetone Powder Type I (Sigma) in 0.6 M sorbitol (w/v sterile sea water). Protoplasts were produced in large quantities from the meristematic regions and adjacent stipes using these enzymes in combination with gentle grinding of the tissue. The viability of protoplasts released was confirmed by exclusion of Evans Blue stain and undiminished chlorophyll fluorescence of the protoplasts. Complete absence of cell wall material was demonstrated using Calcofluor White. The applicability of these methods to other species of Sargassum was shown with S. polyphyllum J. Agardh and S. echinocarpum J. Agardh.

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