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CHEN Haihong, CHEN Weizhou, SHI Jingyi, CHEN Zepan, ZHANG Yi. Isolation and callus formation of Gracilariopsis bailiniae (Gracilariales, Rhodophyta) protoplasts[J]. HaiyangYuHuZhao, 2018, 36(6): 2268-2277

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

CHEN Haihong, CHEN Weizhou, SHI Jingyi, CHEN Zepan, ZHANG Yi
Marine Biology Institution, Key Laboratory of Marine Biotechnology of Guangdong Province, Shantou University, Shantou 515063, China
This paper reports the first successful isolation of protoplasts from Gracilariopsis 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 CaCl2·2H2O) 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℃ and pH 6.5. The highest yield of viable protoplasts, which was obtained in these conditions, was (1.75±0.25)×106 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.
Key words:    Gracilariopsis bailiniae|enzyme|marine bacterium|protoplast isolation|cell division|callus   
Received: 2017-07-14   Revised:
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