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喜樹腫瘤細胞培養與喜樹鹼生產
Crown Gall Culture and Camptothecin Production of Camptotheca acuminata
摘要
本研究建立喜樹葉片轉殖野生型農桿腫瘤菌A 208(Agrobacterium tumefaciens A 208)之方法，當農桿菌A208的菌液濃度為1×10(上標 8~9) cells mL^(-1)，菌液添加200μM acetosyringone，與葉片共同培養2天，葉片的轉殖率可達最高的55.2%。每個葉片產生的腫瘤細胞最少1個，最多可達15個以上。以南方墨點分析法(Southern blot)證明農桿腫瘤菌的nops基因確實插入腫瘤細胞。腫瘤細胞可在不含植物生長調節劑的MS培養基中生長快速，在固體培養基培養30天，細胞鮮重可增加4.3~6.4倍；以液體培養基培養，細胞生長更快，培養20天細胞鮮重可增加7~8倍。利用HPLC分析10個不同轉殖系的喜樹鹼含量，最高可達0.0385%（細胞乾重），最低則趨近於0，顯示不同腫瘤的喜樹鹼含量差異很大，因此廣泛篩選轉殖系是很重要的。
並列摘要
Crown gall cultures of Camptotheca acuminata were established by infecting in vitro leaf explants with Agrobacterium tumefaciens strain A 208. The highest transformation frequency of 55.2% was obtained when explants were co-cultured in medium containing 1×10(superscript 8~9) bacterial cells mL^(-1) and 200μM acetosyringone for 2d. At least 1 crown gall was induced from an explant, and in some cases, 16 galls per explant also occurred. Integration of T-DNA into the host genome was proven using Southern blotting as probed by the nops gene. Transformed galls grew rapidly in hormone-free MS medium. Their fresh weight increased up to 4.3~6.4 times on solid medium after 30d of culture, while it increased up to 7~8 times in liquid medium after 20d of culture. The contents of camptothecin (CPT) of 10 gall strains were analyzed by high performance liquid chromatography. The highest percent CPT of the dry weight of cells was 0.0385%, while the lowest was nearly 0%, indicating that variation in CPT production in transformed galls was great, and extensive selection of gall lines is necessary to produce maximum camptothecin levels.