微拟球藻原生质体制备与再生

Preparation and regeneration of protoplasts from Nannochloropsis oculata

综合考虑酶混合液处理时间和初始细胞密度两个因素,选取终浓度为4%半纤维素酶和2%崩溃酶的酶混合液对一株海洋经济微藻——微拟球藻(Nannochloropsis oculata)进行原生质体的制备与再生,Calcofluor White染料染色可在荧光显微镜下观察到完整的原生质体细胞。实验结果表明:同一初始密度藻细胞酶处理1～3 h制备率较高;酶处理相同时间较高初始密度(2×107～3×107cells/mL)的藻细胞制备率较高,并且原生质体在再生培养基上可再生,生长趋势与野生型细胞一致。考虑到酶处理时间过长或者密度过大会对原生质体的再生产生影响,本实验选择最适酶处理时间为1h,初始细胞密度为2×107cells/mL。

The influence of enzyme digestion time and initial algal density on algal cells were considered.Protoplasts of Nannochloropsis oculata,a marine microalga,were prepareded by digestion with the mixture of 4% Hemicellulase and 2% Driselase.The protoplasts were examined after the treatment of Calcofluor White strain.The results shown that the highest preparation efficiency occurred at the initial cells density of 2×107~3×107 cells/mL and a digestion of 1～ 3h digestion.The resulting protoplasts could regenerate on regeneration medium.The growth of protoplasts was consistent with wild algal cells.Considered the influence of regeneration,we selected the optimal treatment time of 1h and initial algal density of 2×107 cells/mL.