Zhou Y H, Wang S J
Shanghai Fisheries University.
Chin J Biotechnol. 1989;5(4):223-30.
Protoplasts, as important media in genetic engineering, had been isolated from some genera of seaweeds in the beginning of this decade. Using a mixed enzyme solution composed of 3% Sea snail enzyme, 3% Cellulase Onozuka R-10, 1% Macerozyme and 0.5% Pectinase, viable protoplasts were isolated from Caloglossa leprieurii (Mont) J. Ag. (Ceramiales, Rhodophyta). The viability of protoplast was determined by its exclusion of Evans Blue stain. The absence of cell walls were observed in the protoplasts, and were determined by Calcofluor White, reduced osmolarity, electron microscopic examination and self-fusion. About half of the protoplasts survived and divided at a high rate when they were cultivated in a thin liquid layer of modified medium No.5 (MES as basic medium, supplemented with MS medium, vitamins and 1ppm IAA, 1.5 ppm KT). Regenerated plantlet appeared after 45 days. Tetrasporangia appeared in the blade of some regenerated plants after 4 months. Due to its properties of Being large, polynucleal and regenerative, protoplasts of C. leprieurii offer a good prospect in the field of cell fusion and genetic operation of seaweeds.
原生质体作为基因工程中的重要媒介，在本世纪初已从一些海藻属中分离出来。使用由3% 海螺酶、3% 纤维素酶Onozuka R-10、1% 离析酶和0.5% 果胶酶组成的混合酶溶液，从绳藻（Ceramiales, Rhodophyta）中分离出了有活力的原生质体。原生质体的活力通过其对伊文思蓝染色的排斥来确定。通过荧光增白剂、渗透压降低、电子显微镜检查和自我融合观察到原生质体中没有细胞壁。当在改良的5号培养基（以MES为基础培养基，添加MS培养基、维生素和1ppm IAA、1.5 ppm KT）的薄液层中培养时，约一半的原生质体存活并以高速度分裂。45天后出现再生植株。4个月后，一些再生植株的叶片中出现了四分孢子囊。由于绳藻原生质体具有体积大、多核和可再生的特性，在海藻细胞融合和基因操作领域具有良好的应用前景。
