1989/01/01 by DECLAN M. BUTLER, Kjetill Østgaard, KJETILL ØSTGAARD +8 · 4 citations
Earth and Planetary Sciences · #Marine and coastal plant biology #Marine and coastal ecosystems #Ocean Acidification Effects and Responses
paper · doi:10.1093/jxb/40.11.1237
In an investigation of the main factors determining protoplast yield in Laminaria saccharina and L. digitata, protoplasts were isolated from epidermal, cortical and medullary cells of vegetative thallus by incubation with commercial cellulases, crude and purified mannuronate lyases and purified guluronate lyases. Treatment of the tissue with the calcium chelator EGTA before enzymatic digestion greatly increased the protoplast yield. Preplasmolysis was also necessary to obtain large numbers of healthy protoplasts and this was most effective when carried out during chelation with EGTA. Purification of the mannuronate lyases by ion exchange chromatography reduced the toxicity of the crude enzyme preparation. The activities of the wall degrading enzymes were differentially influenced by pH and the optimum for alginate-lyase activity (≈8.0) was higher than that for cellulase activity (<6.0). Protoplast yield decreased linearly with increasing pH in the enzyme medium over the range tested (6.0–8.0), and this suggests that cellulases are more critical to wall digestion than alginate-lyases. Ionic osmotica gave improved yields compared with sugar alcohols or sugars. Increasing the calcium concentration of the enzyme medium brought about an exponential decrease in protoplast yield and wall digestion was almost completely inhibited at concentrations exceeding 8.0 mol m−3. However, low levels of calcium (<2.0 mol m−3) were beneficial to protoplast viability. Yields of 107 to 108 protoplasts g−1 fr. wt. were consistently obtained and 20% to 30% of these regenerated new cell walls within 1–2 d of culture.