Isolation and callus formation of Gracilariopsis bailiniae(Gracilariales, Rhodophyta) protoplasts

This paper reports the first successful isolation of protoplasts from G racilariopsis bailiniae and their callus formation. The base solution type, concentration of isolating enzymes, concentration of sorbitol, incubation time, temperature and pH of the enzyme solution were tested to optimize the protoplast yield. The optimized isolation conditions were: 40% base solution 3(deionized water containing 25 mmol/L MESTris and 25 mmol/L CaCl 2 ·2 H 2 O) and 60% crude Marinomonas sp. YS-70 agarase solution, containing 2% w/v cellulase, 1% w/v macerozyme R-10 and 0.4 mol/L sorbitol, with incubation for 4 h at 28°C and pH 6.5. The highest yield of viable protoplasts, which was obtained in these conditions, was(1.75±0.25)×10 6 cells/g fresh weight. Cell wall regeneration of most protoplasts from G. bailiniae was complete within 60 h and the first division of cells happened after ≥3 days. Two division types were observed in the first division of protoplasts from G. bailiniae— asymmetric division and symmetric division. After the first division, the cells underwent a series of divisions to form callus cell masses.