[Culture of hairy roots in Pueraria phaseoloides and its puerarin production].

An efficient transformation system for genetic transformation of medicinal plant, Pueraria phaseoloides was developed, by using agropine-type Agrobacterium rhizogenes ATCC15834. Hairy roots could be obtained via callus from the cut edges of leaf explants of P. phaseoloides 20 days after inoculation with agrobacterium. 35 days after infection, the percentage of rooted leaf explants was about 85%. Hairy roots could have an autonomous growth on solid or liquid growth regulator-free MS medium but grew more rapidly and formed no callus during culture in liquid medium. The transformation of hairy roots was confirmed by PCR amplification of rolB and rolC genes of Ri plasmid from A. rhizogenes. To investigate the physiological difference between solid and liquid culture, the mitochondrial membrane potential in hairy roots cultured for 15 days in solid and liquid medium were also detected by the method of fluorescence labeling of 3,3'-dipropylthiadicarbocyanide iodide. The results showed mitochondrial membrane potential of hairy roots in liquid medium was 11.8 times higher than that on solid medium. The content of puerarin in hairy roots reached a level of 1.190 mg/g.dry.wt and was 2.5 times as much as that in the roots of P. phaseoloides seedlings and was also 1.067 times as much as that in the crude drug of several year-old Pueraria roots. Our experiments have laid a foundation for large-scale production of puerarin in P. phaseoloides hairy roots.