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.