Objective Pentacyclic triterpenoids in birch bark exhibit significant pharmacological activity; however, their natural availability is limited, thereby restricting their further development and utilization. The accumulation of birch triterpenoids is jointly regulated by plant cell engineering technologies and exogenous hormone induction. This study aimed to investigate the regulatory effects of exogenous abscisic acid (ABA) and ethephon on triterpenoid accumulation and the expression of key genes involved in triterpenoid biosynthesis in birch tissue-cultured plantlets.

Methods Clustered birch plantlets were used as experimental materials. Different concentrations of ABA and ethephon were applied on the 7th day of culture. High-performance liquid chromatography (HPLC), colorimetric assays, and quantitative real-time PCR (RT-qPCR) were employed to determine triterpenoid contents, antioxidant enzyme activities, and the expression levels of related genes.

Results (1) Treatment with 10 μmol/L ABA significantly promoted the accumulation of triterpenoids and their components in birch plantlets. The contents of total triterpenoids, betulin, and oleanolic acid reached 82.43, 24.61, and 33.13 mg/g, respectively, representing increases of 46.29%, 28.73%, and 22.78% compared with the control. Under ethephon treatment at 5 mmol/L, the highest contents of total triterpenoids, betulin, and oleanolic acid were 78.53, 26.54, and 35.84 mg/g, respectively, corresponding to increases of 39.07%, 54.04%, and 27.02% over the control.(2) Both hormone treatments upregulated the expression of triterpenoid biosynthesis-related genes (HMGR, BPY, and DS) and stress-responsive genes (CYP450 and CP) to varying degrees, with HMGR showing the most pronounced response. In addition, the activities of antioxidant enzymes, including CAT, SOD, APX, and PAL, were significantly enhanced. (3) Key genes in the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were markedly induced by ABA and ethephon treatments. Compared with the control, JAZ1/2 and NPR1 were upregulated, whereas JAZ3/4 and NPR3 were downregulated. Furthermore, important transcription factors in the JA signaling pathway, including MYC2, bHLH44/42, and TGA2/5/7, were significantly upregulated.

Conclusion Treatment with 10 μmol/L ABA or 5 mmol/L ethephon effectively promoted triterpenoid accumulation in birch plantlets. These treatments also activated the antioxidant defense system and induced the expression of genes involved in JA and SA signaling pathways as well as triterpenoid biosynthesis. This study provides a theoretical basis and technical reference for enhancing triterpenoid production in birch through plant cell engineering approaches.