Study on rebuilding of sea forest by means of tissue culture of seaweed for cleaning up of heavy oil polluted seashore

为了在短时间内重建海洋森林，将植物组织培养技术应用于海藻的培养。对海洋食草动物的培养条件进行了优化研究，并对其摄食抑制剂的筛选进行了研究。(1)进行了海藻的组织培养。在光照(46μ摩尔~(-1)~(-2))的条件下，在PESI培养基中培养了赤子爱胜蚓愈伤组织，培养20d后，细胞质量增加了15倍。建立了藻类愈伤组织生长监测的图像分析系统，能够清晰地检测培养一周后细胞质量的差异。为了改善光照条件，采用了发光二极管的全彩照射系统。在红色、蓝色和绿色三种颜色中，蓝光被发现是有效的。当蓝光发光二极管(5.3μ摩尔-1)以每天10小时的光照周期照射时，获得1.5倍的生长。(2)液体…中植株的再生更多的m经常被介质引起的流动应力所抑制。为了消除栽培过程中的剪切力等流动应力，提出了添加粘性添加剂的栽培基质。选用了羧甲基纤维素作为粘性添加剂。以胡萝卜愈伤组织作为胚性愈伤组织的模型进行培养时，再生频率可达3倍左右。此外，延长了试管苗的生产时间，并保持了1年左右的高再生能力。(3)为了清理被重油污染的海岸，选育能够表达石油降解基因的重组藻类是非常有吸引力的。对藻类愈伤组织进行了基因转移。以GFP或葡萄糖苷酶基因为报告基因构建了植物细胞表达载体，尝试了两种物理方法：基因轰击法和激光穿透法。然而，没有获得转化子，也没有检测到瞬时表达。作为下一步的方法，对附着在海藻上的细菌进行了分离，以将它们用作基因表达的宿主菌株。分离出了各种菌株。其中一株Cytophaga sp.对海洋藻类的附着活性较弱。(4)由于藻类幼苗有时以腹足类等海洋食草动物为食。取食抑制剂受到了人们的广泛关注。对龟甲中的糖苷酶进行了抑菌实验。从茶叶中提取的多酚类物质如表没食子儿茶素没食子酸酯(EGCG)对酶活性有强烈的抑制作用。在用纤维素片进行摄食行为测试时，EGCG对腹足动物Cantharidus Japan onicus的摄食表现出极高的抑制作用。较少

In order to rebuilt a sea forest without much time, techniques of plant tissue culture was applied for the culture of seaweed. The optimization of culture condition was investigated and the screening of feeding inhibitors for marine herbivores was also studied.(1) Tissue culture of marine alga was carried out. Callus of Eisenia bicyclis was cultivated in PESI medium under the light (46 μmols^<-1>m^<-2>) and cell mass increased 15 times in amount after 20-d culture. The image analysis system for monitoring of alga callus growth was established and difference of cell mass after one week culture was able to detect explicitly. In order to improve condition of light irradiation, full color irradiation system of light emitting diode was used. Among three colors such as red, blue, and green, blue light was found to be efficient. When blue LED (5.3 μmols^<-1>m^<-2>) was irradiated at photo period of 10 h per day, 1.5 times higher growth was obtained.(2) Regeneration of plantlet in liquid mediu … More m has been frequently suppressed by flow stress caused by medium. In order to remove the flow stress such as shear stress during cultivation, viscous additive supplemented medium was proposed. As a viscous additive, carboxymethyl cellulose was selected. When carrot callus as a model of embryogenic plant callus was used and cultivated in the medium, regeneration frequency reached about three times higher level. In addition, prolonged production of plantlets was achieved and high regeneration ability kept for about 1 year.(3) In order to clean up heavy oil polluted seashore, breeding of recombinant alga which can express oil degrading gene is much attractive. Gene transfer to alga callus was carried out. Plasmid vector with GFP or glucronidase gene as a reporter gene was constructed According to the method reported in plant cells, two physical methods such as particle bombardment method, and laser penetration method were attempted. However, no transformants was obtained and no transient expression was detected. As a next approach, isolation of bacteria attached on marine algae was carried out to use them as a host strain for gene expression. Various strains were isolated. One strain of Cytophaga sp.showed weak activity on the attachment to marine algae.(4) Since algae plantlets are sometimes fed by marine herbivores such as gastropods. the feeding inhibitors has received much attention. lnhibitory effect on glycosidase from turban shell was tested. Polyphenols extracted from tea leaf such as epigallocatechin gallate (EGCG) strongly inhibited the enzyme activity. When test of feeding behavior carried out using cellulose sheet, EGCG showed extremely high inhibitory effect on feeding of Cantharidus japonicus, one of gastropods. Less