Plant regeneration system derived from hairy roots of Datura arborea L. was established. The optimum medium for inducing adventitious buds by ‘one-step method’ was MS+6-BA 2.0 mg L-1+NAA 0.2 mg L-1. Using ‘two-step method’, callus was induced on MS medium supplement with 6-BA 4.0 mg L-1+KT 0.5 mg L-1+2,4-D 0.5 mg L-1 at first, then adventitious buds were differentiated from the induced callus on MS medium supplemented with 6-BA 4.0 mg L-1+NAA 0.2 mg L-1. The optimum rooting medium for adventitious buds was 1/2MS + IBA 0.1 mg L-1. By using PCR technique, the gene segments rolB and rolC were obtained from the leaves of regeneration plant. The results of HPLC showed that the contents of tropane alkaloids (TA) in regeneration plants was much more than those in the wild plants.