Objective To increase the ultimate yield of periplocin in Periploca sepium adventitious root cultures by a two-stage culture based on nitrogen source.Methods Firstly,the effects of nitrogen source(NH4+-NO3-) at different ratios and different total initial nitrogen amounts on the accumulation of biomass and secondary metabolites in adventitious root cultures of P.sepium were investigated,and growth and production media for the two-stage culture based on the above results were established.Results The highest biomass and periplocin content were obtained in the culture medium of 15 mmol/L total nitrogen amount with NH4+-NO3-(1:2) and 30 mmol/L total nitrogen amount with nitrate as the sole nitrogen source.By adopting a fed-batch cultivation strategy,the dry weight adventitious root,periplocin content and yield were increased by 136%,108%,and 389%,respectively when compared with those of the control,reaching up to 8.13 g/L,157.15 μg/g,and 1277.63 μg/L,respectively.Furthermore,it was found that in the process of two-stage culture,the adventitious roots grew thicker significantly after they were transferred into production medium directly.Conclusion The ultimate yield of periplocin in P.sepium adventitious root cultures could be significantly increased by a two-stage culture based on nitrogen source. Objective To increase the ultimate yield of periplocin in Periploca sepium adventitious root cultures by a two-stage culture based on nitrogen source. Methods Firstly, the effects of nitrogen source (NH4 + -NO3-) at different ratios and different total initial nitrogen amounts on the accumulation of biomass and secondary metabolites in adventitious root cultures of P. sepium were investigated, and growth and production media for the two-stage culture based on the above results were established. Results of the highest biomass and periplocin content were obtained in the culture medium of 15 mmol / L total nitrogen amount with NH4 + -NO3- (1: 2) and 30 mmol / L total nitrogen amount with nitrate as the sole nitrogen source. By adopting a fed-batch cultivation strategy, the dry weight adventitious root, periplocin content and yield were increased by 136%, 108%, and 389%, respectively when compared with those of the control, reaching up to 8.13 g / L, 157.15 μg / g, and 1277.63 μg / L, respectively. that in the process of two-stage culture, the adventitious roots grew thicker significantly after they were transferred into production medium directly. Confc The Ultimate yield of periplocin in P. sepium adventitious root cultures could be significantly increased by a two-stage culture based on nitrogen source.