Authors：Shu-Hwa Chang, Fen-Hui Chen, Jeen-Yin Tsay, Jung Chen, Chih-Yun Huang, Wen-Ling Lu, Cheng-Kuen Ho

Year：2014

Key words：Nothapodytes nimmoniana, Agrobacterium rhizogenes, hairy roots, camptothecin

     Nothapodytes nimmoniana hairy roots were induced by infecting stem and leaf segments
with 3 wild-type Agrobacterium rhizogenes strains, AR281, AR1600, and ATCC15834.
Results showed great variations in the transformation efficiencies among different A.
rhizogenes strains and N. nimmoniana clones in vitro. Strain ATCC15834 had the highest
transformation ability. Among all 10 clones, 7 clones were infected, and hairy roots were
successfully induced. These hairy roots grew vigorously and were highly branched on
phytohormone-free solid medium. Three fast-growing hairy root lines, for which integration
of the rol B gene into the root genome was verified using PCR and Southern blotting, were
used for the growth and alkaloid production tests. After being cultured for 40 d, the growth
index of hairy roots was 2.6~3 and camptothecin (CPT) contents were 0.0537~0.1555%
dry weight (DW). When cultured in liquid medium, hairy roots grew up to 1.8-times faster
than they did in solid medium. After 30 d in liquid culture, the CPT concentration was
9.8 mg/L. Approximately 93.9% (9.2 mg/L) of the total CPT produced was excreted into the
culture medium. With continual harvesting of CPT from the liquid medium, this system will
benefit the development of large-scale bioreactor culture in the future.