Crown Gall Culture and Camptothecin Production in Camptotheca acuminata

Crown gall cultures of of Camptotheca acuminata were established by infected in vitro leaf explants with Agrobacterium tumefaciens strain A 208. The greatest transformation frequency - 55.2% was obtained when explants co-cultured with the medium containing 1 x 108~9 bacterial cells mL-1 and 200 M acetosyringone for 2 days. At least one crown gall was induced from a explant, in some case 15 galls more per explant were occurred. Integration of T-DNA into host genome was proven using southern blotting as probed by 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 30 day in culture, while up to 7~8 times in liquid medium after 20 days in culture. The contents of camptothecin (CPT) of 10 gall strains were analyzed by HPLC. The greatest CPT % in dry weight of cells was 0.0385%, while the poorest was near 0%, indicating the variation of CPT production in transformed galls was very great, and extensive selection of gall lines is necessary.

利用根癌农杆菌（Agrobacterium tumefaciens）A208菌株侵染喜树（Camptotheca acuminata）的离体叶片外植体，成功建立其冠瘿瘤培养体系。当外植体与含有1×10^8~10^9个细菌细胞·mL⁻¹、200 μM乙酰丁香酮的培养基共培养2天时，可获得最高转化效率，达55.2%。每个外植体至少可诱导出1个冠瘿瘤，部分外植体每株可产生15个以上的瘤体。以胭脂碱合酶基因（nopaline synthase gene, nops）为探针进行Southern印迹（Southern blotting）分析，证实T-DNA已整合至宿主基因组中。转化所得的冠瘿瘤在无激素MS培养基上可快速生长：培养30天后，固体培养基上的瘤体鲜重可达初始的4.3~6.4倍；液体培养基中培养20天后，瘤体鲜重可达初始的7~8倍。采用高效液相色谱法（High Performance Liquid Chromatography, HPLC）对10株冠瘿瘤菌株的喜树碱（camptothecin, CPT）含量进行检测，结果显示细胞干重中CPT含量最高为0.0385%，最低接近0%，表明转化冠瘿瘤的CPT合成能力差异显著，因此需对瘤株开展广泛筛选。