Isolation of Protoplasts fromKappaphycus alvareziivar.tambalang(Rhodophyta) and Secretion of i-Carrageenan Fragments by Cultured Cells

Abstract

A method for generating protoplasts from the carrageenan-producing red alga Kappaphycus alvarezii was developed. Digestions with cellulase and _k-carrageenase produced only a few cortical cell protoplasts, while digestions with cellulase and i-carrageenase only produced epidermal cell protoplasts. When both carrageenases were used in the digestion media with cellulase, protoplasts were released from all cell types and yields ranged from 1·0 to 1·2×10⁷ cells g⁻¹ with sizes from 5 to 200 μm diameter. Protoplasts were subsequently cultured to study cell wall regeneration. Calcofluor-positive material (probably cellulose) was detected within 6 h after removal of protoplasts from the wall digestion media, whereas, i-carrageenan fragments were detected in all regenerating protoplast cultures 24 h after removal from the digestion media. Protoplasts continued to produce Calcofluorpositive material and secrete carrageenan fragments into culture media for several days. However, cells cultured in media augmented with K⁺ ions stopped secreting carrageenan fragments after 24 h. Cells cultured for 48 h in seawater labelled weakly with an i-carrageenan hybridization probe, but not at all with a corresponding _k-probe. Cells cultured for 48 h, blotted to nylon membranes and probed with anti-carrageenan monoclonal antibodies, showed the presence of gelling carrageenan subunits in the cell walls.