Fluctuation in secondary metabolite production and antioxidant defense enzymes in in vitro callus cultures of goat’s rue (Galega officinalis) under different abiotic stress treatments

Exogenous abiotic stress treatments are an effective way of accelerating stress tolerance by modulation of accumulation of various secondary metabolites in pharmaceutical, medicinal, and economically important plant species. This research aimed to evaluate the effects of nutrient, chemical, temperature, and ultraviolet (UV) radiation abiotic stress on fluctuations in selected secondary metabolites and the antioxidant defense system of Galega officinalis L. (goat’s rue) callus tissue cultures. Stress treatments were achieved by exposing callus cultures of goat’s rue to five different strengths of Murashige and Skoog (MS) medium (2MS, MS, MS/2, MS/4, and MS/8), four different temperatures (4 °C, 22 °C, 35 °C, and 45 °C), two chemicals [mercury chloride (HgCl₂) and copper sulfate (CuSO₄)], and three types of UV radiation (UV-A, UV-B, and UV-C) for three different durations (15, 30, and 60 min). The amounts of selected phenolic secondary metabolites (apigenin, p-coumaric acid, genistein, kaempferol, luteolin, rutin hydrate, trans-ferulic acid, salicylic acid, chlorogenic acid, naringenin, and isorhamnetin) in callus cultures of goat’s rue was determined by liquid chromatography-electrospray ionization-multistage/mass spectrometry (LC–ESI–MS/MS). The dominant molecule was p-coumaric acid in all stress treatments. The highest amounts of luteolin, genistein, p-coumaric, naringenin, apigenin, trans-ferulic acid, salicylic acid, and rutin were determined in the MS/8, MS/8, MS/2, MS/2, MS/4, UV-A (15 min), UV-A (15 min), and 4 °C treatments, respectively. Interestingly, the percentage increase (379.01%) in the total selected phenolic molecules was highest in the MS/8 treatment. The superoxide dismutase and catalase antioxidant enzyme activity were highest in the CuSO₄ and MS/4 treatments, respectively. Moreover, the MS/4 treatment resulted in the highest proline content. The total phenolic content was highest after the temperature (4 °C) and UV radiation (UV-A 30 min) treatment. The UV-A (15 min) treatment had the strongest antioxidant activity, with the highest total flavonoid content and the lowest concentration inhibiting 50% of DPPH (IC₅₀) value. These outcomes indicated that different abiotic stress factors caused extraordinary variations in antioxidant levels and secondary metabolite accumulation as a way of regulating the plant’s defense system. The newly developed protocol in the current study can be used to provide the desired amount of special secondary metabolite production and establish a new valuable herbal medicine to cure many chronic diseases, as well as in the pharmaceutical area and other related areas.