Priming followed by eliciting boosts chicoric acid biosynthesis in Echinacea purpurea L. hairy roots

Chicoric acid (ChA) is a valuable phenolic compound found in Echinacea purpurea , exhibiting notable pharmacological properties. In this study, the effects of preconditioning E. purpurea hairy root culture (HRC) with spermidine (Spd) and elicitation using salicylic acid (SA) and Coniothyrium palmarum cell wall (CW) were evaluated for chicoric acid production. Initial optimization identified 0.75 mM spermidine as the most effective concentration for priming E. purpurea HRC. In subsequent experiments, HRC were preconditioned with Spd (0 and 0.75 mM) and then exposed to different concentrations of SA (0, 50, 100 µM) and CW (0, 1.25, 2.5, 5% v/v) on 24th day of the HRC cycle. ChA accumulation was monitored on the the 26th and 28th days of the HRC cycle. The highest ChA content (33.93 mg g −1 DW, representing a 6.18-fold increase compared with the non-elicited control hairy roots) was produced in the HRC preconditioned with 0.75 mM Spd, exposed to 50 µM SA and 2.5% (v/v) CW, and harvested on the 26th day of the HRC cycle. Expression levels of PAL , C4H , C3H , 4CL , HCT , HTT , HQT , and CAS genes, encoding Phenylalanine ammonia lyase, Cinnamate 4-hydroxylase, Coumarate 3-hydroxylase, 4-coumarate-CoA ligase, Hydroxycinnamoyl-CoA transferase, Hydroxycinnamoyl-CoA: tartaric acid hydroxycinnamoyl transferase, Hydroxycinnamoyl-CoA: quinate hydroxycinnamoyl transferase, and Chicoric acid Synthase, respectively, significantly increased by the preconditioning and elicitation. HCT , HTT , HQT , and CAS induction was the most sustained in the high ChA-producing culture. These results demonstrated that combinatorial priming and elicitation strategies can effectively stimulate secondary metabolite biosynthesis in plant in vitro systems, ChA biosynthesis in E. purpurea HRC responding to Spd, SA, and fungal CW as a case study. • Spermidine priming (0.75 mM) plus dual elicitation (2.5% CW + 50 µM SA) boosted chicoric acid to 33.93 mg g⁻¹ DW. • HCT showed sustained upregulation and was closely associated with peak chicoric acid accumulation. • Upstream genes were activated early, while HCT remained induced during chicoric acid buildup. • Combined priming and elicitation provide a strategy to enhance phenolic production in medicinal plants.