Isolation Conditions for High Yields of Protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae)

In an investigation of the main factors determining protoplastyield in Laminaria saccharina and L. digitata, protoplasts wereisolated from epidermal, cortical and medullary cells of vegetativethallus by incubation with commercial cellulases, crude andpurified mannuronate lyases and purified guluronate lyases.Treatment of the tissue with the calcium chelator EGTA beforeenzymatic digestion greatly increased the protoplast yield.Preplasmolysis was also necessary to obtain large numbers ofhealthy protoplasts and this was most effective when carriedout during chelation with EGTA. Purification of the mannuronatelyases by ion exchange chromatography reduced the toxicity ofthe crude enzyme preparation. The activities of the wall degradingenzymes were differentially influenced by pH and the optimumfor alginate-lyase activity (8.0) was higher than that for cellulaseactivity (<6.0). Protoplast yield decreased linearly withincreasing pH in the enzyme medium over the range tested (6.0–8.0),and this suggests that cellulases are more critical to walldigestion than alginate-lyases. Ionic osmotica gave improvedyields compared with sugar alcohols or sugars. Increasing thecalcium concentration of the enzyme medium brought about anexponential decrease in protoplast yield and wall digestionwas almost completely inhibited at concentrations exceeding8.0 mol m^–3. However, low levels of calcium (<2.0 molm^–3) were beneficial to protoplast viability. Yields of10⁷ to 10⁸ protoplasts g^–1 fr. wt. were consistently obtainedand 20% to 30% of these regenerated new cell walls within 1–2d of culture. Key words: Laminaria protoplasts, cell wall, alginate-lyases