Genetic Transformation Of Dysosma Versipelis With Agrobacterium Rhizogenes And Podophyllotoxin Production In Hairy Roots

Dysosma versipellis (Hance) M.Cheng is a local med herb that belongs to the Berberidaceae. Several natural chemical have been isolated from Dysosma Versioellis (Hance)M. Cheng. It is a source of highly valued podophyllotoxin which is currently being investigated for carinogenicity, cytotoxcity and antitumor activity.Based on these,we adopted techniques of plant physiology , molecular biology, pharmaceutical analysis interactively, established a system to transform hairy roots of Dysosma versipellis and gained the podophyllotoxin by the hairy roots culture. We also studied the content of podophyllotoxin, which would establish bedrock to obtain secondary metabolites of medicinal plants by hairy roots culture for industrialization.The main results were as follows: An efficient transformation system for the medicinal plant Dysosma versipelis was originally established with Agrobacterium rhizogenes 15834. Hairy roots could be obtained directly from the cut edges of stem explants 30 days after inoculation with the bacteria. The high frequency of transformation was obtained by three-day's pre-culture on MS medium supplementeds with NAA 1.0 mg/1 and KT 0.2 mg/1 ,three-day's co-culture on growth regulator free MS medium and addition AS(hydroxyacetosyringone) 100 umol/l, the inducing rate of hairy root was up to 30.9% . Hairy roots clones grew rapidly on solid growth regulator free 1/2 MS medium. The PCR, PCR-Southern analysis indicated that Ri plasmid left-hand T-DNA fragment(564bp) integrated into the genome of transformed roots.HPLC analysis showed that the contents of podophyllotoxin in hairy roots (0.25%) were higer than in vitro culture roots (0.17%) and close to wild roots (0.29%);the contents of podophyllotoxin in hairy roots under illumination was higer than hairy roots under gloominess(0.11%);that the contents of podophyllotoxin of callus from hairy roots(0.064%) was higer than callus from non-transformed roots and lower than callus from stems and leaves(0.17%).