Methyl jasmonate and salicylic acid as powerful elicitors for enhancing the production of secoiridoid glycoside from cell suspension cultures of Enicostema axillare (Poir. ex Lam.) A. Raynal

Enicostema axillare (Poir. ex Lam.) A. Raynal is an immense medicinal plant that has been extensively used to treat malaria, diabetes, stomachache, and fever in traditional medicine. This plant contains high amounts of secoiridoid glycosides (gentiopicroside and swertiamarin), which have numerous health benefits. However, no leaf callus or cell suspension culture has been established for E. axillare for the production of beneficial metabolites. As a result, the goal of this work was to standardize an efficient and robust method for callus induction, callus and cell biomass, as well as swertiamarin and gentiopicroside accumulation. The highest callus induction frequency (85.40 ± 0.51%) and formation of leaf into callus with a mean callus biomass (1.283 ± 0.02 g/explant fresh weight (FW) and 0.176 ± 0.00 g/explant dry weight (DW)) and metabolite contents (swertiamarin 0.53 ± 0.01 mg/g and gentiopicroside 0.64 ± 0.05 mg/g) were obtained on Murashige and Skoog (MS) medium augmented with 2,4-D (1.0 mg/L) + Kin (0.5 mg/L) after 12 weeks of culture. The induced leaf callus was studied for cell suspension growth kinetics (biomass) and metabolite production. After 60th days of culture, the maximum of the leaf cell suspension (5.25 ± 0.05 g FW (50 mL medium) and 0.39 ± 0.00 g DW (50 mL medium) and secoiridoid glycosides (gentiopicroside, 1.44 ± 0.05 mg/g, and swertiamarin, 0.88 ± 0.01 mg/g DW) were obtained, which corresponded to 4.19 and 36 times greater than the lag phase (after 10th days). Cultured leaf cell suspensions were treated with methyl jasmonate (MJ) and salicylic acid (SA) at 50, 100 and 200 μM after 60th day of cell growth and evaluated for swertiamarin and gentiopicroside contents after 24 h of treatment. The greatest swertiamarin (2.43 ± 0.05 mg/g DW, 2.76-fold) and gentiopicroside (2.41 ± 0.09 mg/g DW, 1.67-fold) levels were observed in the treatments with 200 and 100 μM SA elicitor, respectively. These findings can lead to the use of this plant as an alternative source to improve the commercial production of secoiridoid glycosides in large-scale cultures.