坛紫菜单个体细胞克隆的丝状体途径

Morphogeny of conchocelis thalli from single somatic cell clone cultivation of Porphyra haitanensis

用海螺酶酶解挑选出的具有优良性状的坛紫菜(Porphyra haitanensis)叶状体制备游离的营养细胞,在显微镜下选取单个细胞放入96孔板中进行隔离培养。一部分细胞通过2种发育途径形成了丝状体:一种途径是单细胞生长一段时间产生突起后发育形成丝状体;另一种途径是单细胞先形成愈伤组织,愈伤组织解体放散出类似"孢子"的细胞,"孢子"再发育形成丝状体。将获得的丝状体扩大培养作为纯系并下海养殖。同普通品系相比,选育的品系在产量和质量上表现出一定的优势。在纯系培育方面,由单倍的叶状体细胞发育形成的二倍丝状体,遗传物质一步纯合,后代叶状体个体遗传性状高度一致,可以保证优良经济性状不丢失。这种紫菜纯系培育方法大幅度缩短了纯系培育周期。

The release of free-living vegetative cells were successfully achieved by enzymolysis of Porphyra haitanensis fronds.The fronds with good configuration, such as color and length,were selected from field net in Xiangshan,Zhejiang Province. The blades were dried in darkness at room temperature and stored at - 20 ℃ .Before experiment, the fronds were taken out and immerged into seawater for two or four days to make cell retrieve activity. After enzymolysis, single somatic cells were picked out into 96 well microplates under invert microscope, with the help of capillary tube. Then single cell was cultivated at 20 ℃ , 12L-12D and light density 1500 - 2 000 lx. The medium was refreshed after 3 to 5 d.The development of somatic cells was observed continuously and described as follows:in the procedure of cultivation,majority of cells died only except a small portion of the somatic cell which can develop into conchocelis thallus, blade and calluse-like cell group.Conchocelis thalli can be formed through two routes:in the first case, single somatic cell produced pustutes from some parts of the cell,then these pustutes elongated and produced forks, finally grew into conchocelis thalli. In this case,some cells only produced pustutes from the parts where pigments concentrated, and the others produced pustutes too,but cytoplasm color change couldn' t be observed under microscope. In the second case, somatic cell formed callus-like cell group primarily. After cultivated for some time,the cal-lus-like cell guoup disintegrated and released spore-like cells. These spores developed into conchocelis thalli, while a few spore-like cells germinated into small blades. But some cell.groups didn't break,just formed conchocelis thalli from the live cells in the cell-group. In order to distinguish the development of somatic cell from sexual cell (carpogonium and carpospore) , single marginal cells of female blade were cultivated as the method as somatic cell cultivation. The results showed that it was effective to differentiate somatic cell and carpospore by the size and germinating fashion. It was important for the establishment of pure line diploid conchocelis.After three months or more, these conchocelis thalli produced from single cell,as pure lines, were cultivated largely through suspension cultivation method to multiplied, and further cultured the same as the tra-ditional mariculture. Comparing with un-selected line, the pure lines showed some superior traits, including the output of blades and other economic characters.On the point of pure line establishment,from haploid blade cell to diploid conchocelis thallus, the origin genetic material was reduplicated directly.So this cultivation method gives a guarantee to descendant blades to possess high similarity in genetic background. This technique has two conceivable advantages which are preventing the loss of desired economic characteristics and shortening the cultivating time compared with traditional pure line establishemt method.