ISOLATION OF PROTOPLASTS FROM KAPPAPHYCUS-ALVAREZII VAR TAMBALANG (RHODOPHYTA) AND SECRETION OF IOTA-CARRAGEENAN FRAGMENTS BY CULTURED-CELLS

A method for generating protoplasts from the carrageenan-producing red alga Kappaphycus alvarezii was developed. Digestions with cellulase a nd kappa-carrageenase produced only a few cortical cell protoplasts, w hile digestions with cellulase and iota-carrageenase only produced epi dermal cell protoplasts. When both carrageenases were used in the dige stion media with cellulase, protoplasts were released from all cell ty pes and yields ranged from 1.0 to 1.2 x 10(7) cells g-1 with sizes fro m 5 to 200 mum diameter. Protoplasts were subsequently cultured to stu dy cell wall regeneration. Calcofluor-positive material (probably cell ulose) was detected within 6 h after removal of protoplasts from the w all digestion media, whereas, iota-carrageenan fragments were detected in all regenerating protoplast cultures 24 h after removal from the d igestion media. Protoplasts continued to produce Calcofluor-positive m aterial and secrete carrageenan fragments into culture media for sever al days. However, cells cultured in media augmented with K+ ions stopp ed secreting carrageenan fragments after 24 h. Cells cultured for 48 h in seawater labelled weakly with an iota-carrageenan hybridization pr obe, but not at all with a corresponding kappa-probe. Cells cultured f or 48 h, blotted to nylon membranes and probed with anti-carrageenan m onoclonal antibodies, showed the presence of gelling carrageenan subun its in the cell walls.