Study on algal growth factor in agar and production of bioactive substance by tissue culture

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1. Identification of algal growth factor in agar. Gracilaria agar has an unknown algal growth factor having low molecular weight. Using reversed-phase (Shimadzu Shim-pack CLC-ODS) and silica gel (TOSO TSK gel Amido-80) HPLC, X-ray fluorescence analysis and atomic absorption spectrometry, sugars, organic acids, amino acids, nucleic acid-related compounds, inorganic elements were analyzed. Gracilaria agar contained tartaric, malonic and citric acids which are not detected in Gelidium agar. When those three acids were added to culture medium, growth rate of green alga Ulva pertusa increased to 149% of that of control. Those three organic acids, there for, were identified as the growth factor in Gracilaria agar. 2. Physicochemical properties of the agar for algal tissue culture. The 0.7% Gracilaria agar showed the best growth of algal tissue. The gel strength, pH, viscosity, SO_4 contents, syneresis after 200 h of the 0.7% agar were 24, 6.6, 7.9 cp, 0.79%, 2 g, respectively. The most suitable agar for algal tissue culture is considered to have moderate gel strength and small syneresis. 3. Production of antitumor substance by tissue culture. Axenic tissue from brown alga Heterochordaria abietina, green algae Codium pugniformis, and C. fragile were cultured in PESI medium at 15 C on a 14:10 L:D cycle under 1200 lx. Yield of antitumor glycoprotein was 4.7%, 2.1% and 2.2% in H. abietina, C. pugniformis and C. fragile, respectively. Inhibitory effects of the glycoprotein on growth of WI-38 VA-13 were examined. IC_<50> was 9.1, 300.8 and 274.9 in H. abietina, C. pugniformis and C. fragile, respectively. These values are almost the same as that of naturally grown algae in ocean. By the axenic tissue culture, therefore, antitumor glycoprotein was produced successfully.