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

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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