Background:The root of Polygonum multiflorum Thunb. is a common traditional Chinese medicine. In recent years, the wild resources of P. multiflorum have been seriously broken, and the cultivated varieties have been degrading. The germplasm resources of P. multiflorum need protection and preservation. So far, no in vitro germplasm preservation of P. multiflorum has been reported.Objective:To explore a method for the in vitro germplasm preservation of P. multiflorum.Materials and Methods:A large number of buds from seed explants were induced by tissue culture. The single buds were used as experimental materials to study the effects of plant growth regulator, temperature, and osmotic pressure on the preservation time, growth recovery, and genetic stability.Results:When the buds were inoculated onto Murashige and Skoog (MS) basal media containing 4% w/v sucrose, 2% w/v mannitol, and 1% w/v sorbitol, supplemented with paclobutrazol (PP333) 1.0 mg/l, abscisic acid (ABA) 5.0 mg/l, and daminozide (B9) 30.0 mg/l in an illuminated chamber under a 16 h photoperiod of 1500 lx light intensity at 15°C for 10 months, the survival rate was over 70% with good growth recovery and genetic stability.Conclusion:The results of this study can be used for medium-term in vitro germplasm preservation of P. multiflorum, and meeting actual needs of research and production.