In this study, adventitious roots of Panax quinquefolium L. have been successfully established. The highest induction rate of roots was obtained in MS medium containing 3 mg L−1 IBA after 4 weeks of culture. The culture conditions of adventitious root were optimized and evaluated with response surface methodology. The best culture conditions for root growth seemed to be 0.75 salt strength MS medium, 4.70 g L−1 inoculum size and 40 days of culture. The active component contents of adventitious roots were compared with those of native roots. The total saponins content was found to be 16.28 mg g−1 in native root and 4.64 mg g−1 in adventitious root. The polysaccharide content of the adventitious root was 1.5 times higher than that in the native P. quinquefolium (30.54 vs. 20.28 mg g−1).

•Treating roots with fungal elicitors efficiently enhanced content of ginsenosides.•Strong correlation of ginsenosides, signal molecules and key genes was observed.•Profiles of ginsenosides in P. ginseng roots treated with A. niger were obtained.In this work, we selected three fungi strains (Aspergillus niger, Aspergillus flavus and Aspergillus oryzae) as elicitors prepared from mycelium or fermentation broth to improve ginsenosides production in adventitious roots culture. The results showed that ginsenosides production (29.90 ± 4.67 mg g−1) was significantly enhanced upon elicitation with 200 mg L−1 A. niger elicitor prepared from mycelium, which was 3.52-fold of untreated group. HPLC-ESI–MSn analysis was performed, showing that ginsenoside Rb3 was present after treatment with the A. niger. Furthermore, we found that A. niger significantly enhanced accumulation of Nitric oxide (NO), salicylic acid (SA) and jasmonic acid (JA) involved in plant defense response, and significantly up-regulated the expression of the geranyl diphosphate synthase (GPS), farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), dammarenediol synthase (DS), Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) and three UDP-glycosyltransferases (UGT) genes (UGTAE2, UGT94Q2 and UGTpg100).