Studies on developments of cell and tissue culture

(1)在密松未成熟胚胚胚性细胞（ECs）传代培养中，添加1250mg/l l -谷氨酰胺和1000mg/l聚乙烯吡咯烷酮（PVP）的mDCR培养基培养条件较好，ECs增殖率高，质量好。在体胚形成过程中，添加30μM的脱落酸（ABA）、10%的麦芽糖和分子量为8000的聚乙二醇（PEG）均对体胚发育有促进作用。(2)通过****形态观察和内源氨基酸的定量分析，阐明了leptolepis落叶松、云杉和日本柳杉培养细胞的特征，从而证实了时间氨基酸对ECs继代培养的影响，添加一定浓度的氨基酸促进了ECs发育，胚区清晰大，胚柄长。由此提示体细胞胚形成的可能性。在像柳杉（Cryptomeria japonica）这样较多的植物中，通过内源氨基酸含量的监测来分析和评价的方案对于早期选择具有高分化能力的细胞，最终形成体胚是有效的。(3)原生质体的分离条件以及原生质体增殖的培养基和植物激素的最佳组合通常因植物种类而异。微孔板（24孔）测定法。为白杨原生质体培养建立的培养基，适用于研究白杨和日本白杨等针叶树的培养条件。通过少量原生质体在96孔微孔板中培养，可以很容易地确定植物激素的最佳种类、浓度和细胞密度。(4)我们确定了GjNdly除5'-非翻译启动子区域外的整个核苷酸序列。虽然在裸子植物的基因产物中没有检测到富含脯氨酸的区域，但我们在从日本隐毛菊推断的氨基酸序列的氨基末端区域发现了这样一个区域。少

(1)In subculture of embryogenic cells (ECs) from immature embryo of Pinus densiflora, more effevtve culture conditions were mDCR medimun which added L-glutamine of 1250mg/l and polyvinyl pyrrolidone(PVP) of 1000mg/l, resulting in higher proliferation of ECs with good quality. Furthermore, in formation of somatic embryo from ECs, additions of abscisic acid (ABA) of 30μM, maltose of 10% and polyethylene glycols (PEG) of molecular weight 8000 were effective in the development.(2)In Larix leptolepis, Picea jezoensis and Crypbomeria japonica, characteristics of the culture cells were clarified b**** the morphological observation and a quantitative analysis of endogenous amino acid, using micro-HPLC Consequently, effect of time amino acid was confirmed in the subculture of ECs, and an addition of a certain concentration of amino acid promoted development of ECs with clear and large embryo region and long suspensors. Thus result suggests possibility of somatic embryo formation. In recalcitran … More t species, like Cryptomeria japonica, it is considered that protocols of the analysis and evaluation by monitoring endogenous amino acid contents are effective for selecting the cells with high differential ability in the early stage, which result finally in somatic embryo(3)A suitable condition for isolation of protoplast and an optimum combination of medium and plant hormone for proliferation of protoplast generally vary according to plant species. Micro-plate (24 well) assay method that had been. established for protoplast culture of Populus alba, is adaptable to investigate culture conditions of coniferous tree such as L.leptolepis and C.japonica. By culturing small amount of protoplasts in a well of 96 well micro-plate, an optimal kind and concentration of plant hormone(s), and/or cell density was easily determined.(4)We determined the entire nucleotide sequence of GjNdly with the exception of the 5'-untranslationed promoter region. Although no proline-rich region has previously been detected in the gene product from gymnosperms, we found such a region in the amino-terminal region of the deduced amino acid sequence from Cryptomeria japonica. Less