AN ANTIBIOTIC PROTOCOL FOR PREPARING AXENIC CELL-CULTURES OF PORPHYRA-LINEARIS

A procedure for producing axenic cell suspension cultures of Porphyra is described. Bacteria from a gametophytic frond of P. linearis were evaluated for their sensitivity to different antibiotics. The most effective were ampicillin, carbenicillin, erythromycin, kanamycin, neomycin and ticarcillin. Cells derived from P. linearis clonal cell line CMAC-25, which were contaminated with bacteria, were treated in D-11 medium containing a mixture of these antibiotics at a concentration of 2 mg mL-1 of each. Next, an abalone enzyme mixture was applied to remove the antibiotic-treated cell walls that normally harbour bacteria. After regulating the osmolality of the protoplasts derived from the cells, single protoplasts were isolated and incubated in an antibiotic solution for 24 h before transferring to D-11 medium for propagation. Axenicity was confirmed in sterility testing media. A success rate of 85-100% was achieved.