礁膜原生质体的分离及培养  

Isolation and cultivation of protoplasts of Monostroma nitidum (Chlorophyta, Monostromaceae)

作　　者：谢恩义[1] 马家海[1] 

机构地区：[1]上海水产大学生命科学与技术学院,上海200090

出　　处：《水产学报》2004年第1期62-67,共6页Journal of Fisheries of China

基　　金：浙江省科委科技兴海专项项目(200204)

摘　　要：为了开发礁膜人工育苗的新技术,以4％果胶酶和2％纤维素酶的甘露醇溶液,分离酶解经无菌处理的礁膜叶状体,在40mmol·L^(-1)CaCl_2,0.7mol·L^(-1)甘露醇,pH5.5,温度28℃,摇床速度50r·min^(-1)旋转振荡3h的条件下,1.0g湿重礁膜可获原生质体约2×10~6个。原生质体分离后依次用比重为1.030、1.026、1.022的消毒海水(添加N、P、Fe^(2+)、IAA、KB、Vit C)培养,2d后已形成明显的再生细胞壁,3～4d后细胞开始分裂,8d后形成由4～8个细胞组成的细胞团,以后原生质有不同的发育形态,较常见的是形成由一共同胶质膜包被的类似亲本的叶状体组织块,另一种是原生质体形成孢子囊,成熟后,可产生许多游动孢子,再由游动孢子发育成正常形态的膜状配子体,第三种发育形态为偏离亲本正常形态的管状体。In order to develop a new cultivation technology of Monostroma nitidum Wittr, this study was to isolate and cultivate the protoplasts of this green alga. Protoplasts of the alga were isolated by enzymatic degradation from the thallus. The optimization of isolation conditions was carried out in this experiment. The result showed that the optimized parameters were 4% pectinase, 2% cellulase, 0.7 mol·L^(-1) Mannitol, 40mmol·L^(-1) CaCl_2, pH5.5, 28℃ and 50r·min^(-1) shaker speed for 3 hours, with a yield of 2×10~6 spherical, green viable protoplasts per gram of fresh thallus. Purified protoplasts were cultured in autoclaved seawater medium supplemented with N, P, Fe^(2+), IAA, KB, and Vit C, and the specific gravity of the medium gradually decreased from 1.030 to 1.026 and 1.022 in turn in early culture. Protoplasts usually began to form new cell wall within two days after isolation and began to divide from day 3 to day 4 in medium. 4 to 8 cell clusters were formed after 8 days. Even when regeneration under uniform conditions, protoplasts followed three different developmental patterns. Usually, protoplasts underwent repeated cell divisions and developed directly to a monostromatic thallus cell clusters where the cell number was different and all cells wrap by a common gelatine. Some protoplasts formed a cell wall and then developed into sporangia. After sporangia matured, they liberated zoospores that settled and grew into thallus similar to the parent thallus in successive cultures. The third developmental patterns was to form tubular thallus which is different from the parent thallus.

关 键 词：礁膜 原生质体 分离 培养 绿藻 人工育苗 无性繁殖 

分 类 号：S968.412[农业科学—水产养殖]