Secondary Metabolite Enhancement of Pokeweed (Phytolacca americana L.) Calli Using Drought and Salinity Stress Under In Vitro Condition

The pokeweed (Phytolacca americana L.) plant is native to North America and contains bioactive compounds with medicinal potential, particularly phenolics and saponins. This study enhanced the production of secondary metabolites in pokeweed callus cultures using sodium chloride (NaCl) and polyethylene glycol (PEG) as elicitors under aseptic conditions. Pokeweed seeds were cultured on Murashige and Skoog (MS) medium for 8 weeks. Fully expanded leaves from the second to third position from the shoot were excised and induced to form calli on MS medium supplemented with 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) for 5 weeks. Fully developed calli were elicited with PEG6000 at concentrations of 0, 1.25, 2.5, and 5% (w/v) in combination with NaCl at concentrations of 0, 100, 200, and 300 mM for 15 days. Callus growth was recorded, followed by drying and extraction using methanol (MeOH) for biochemical analysis. Calli elicited with 2.5% PEG and 300 mM NaCl exhibited the highest total phenolic content (TPC) (21.063 µg GAE/mg DW) and total flavonoid content (TFC) (1.927 µg QUE/mg DW). The highest antioxidant activities determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS), and ferric ion reducing antioxidant potential (FRAP) assays were 0.998, 1.574, and 0.998 µg TE/mg DW, respectively. The elicitation of pokeweed calli with 300 mM NaCl yielded the highest amount of Esculentoside A (EsA) (15.753 µg/mg DW). All the elicitor treatments significantly enhanced metabolite accumulation compared to the control group (p < 0.05). The findings indicated that elicitation with PEG and NaCl effectively enhanced the production of secondary metabolites in P. americana callus cultures. This study offers a promising alternative approach for utilizing P. americana in pharmaceutical and medicinal applications.