Objective To establish a tissue culture system of Pueraria thomsonii and to cultivate a new breed autotetraploid.Methods Using the MS medium as the basic medium,the explants shoot apexes and stem segments were cultured to differentiate the regenerations.The autotetraloid with genetic material doubled was achieved by treating shoot tips with colchicine of different concentrations for different long times.Results The proper media MS+6-BA 1.0 mg/L + 2,4-D 1.0 mg/L+NAA 0.2 mg/L is for pro-(tocorn) inducing,MS+6-BA 1.0 mg/L+2,4-D 0.2 mg/L for diffrentiation and propagation,and 1/2(MS+) IBA 0.2 mg/L for rooting.The best effect of the autotetraploid induction can be achieved by treating the 0.3-0.5 cm shoot tips cut from the 1 cm plantlets with 0.4%-0.5% colchicine for 48 h,and it is unlikely to succeed by treating shoot tips with colchicine of very low or high concentration or for very long or short time.Conclusion The autotetraploid gives a chance to improve the content of medical materials in the root of P.thomsonii.