Elicitors directed in vitro growth and production of stevioside and other secondary metabolites in Stevia rebaudiana (Bertoni) Bertoni.

Stevia rebaudiana (stevia) is a plant in the Asteraceae that contains several biologically active compounds including the antidiabetic diterpene glycosides (e.g. stevioside, rebaudioside and dulcoside) that can serve as zero-calorie sugar alternatives. In this study, an elicitation strategy was applied using 5% polyethylene glycol (PEG), sodium chloride (NaCl; 50 and 100 mM) and gibberellic acid (2.0 and 4.0 mg/L GA3) to investigate their effect on shoot morphogenesis, and the production of phenolics, flavonoids, total soluble sugars, proline and stevioside, as well as antioxidant activity, in shoot cultures of S. rebaudiana. Herewith, the media supplemented with 2 mg/L and 4 mg/L GA3 exhibited the highest shooting response (87% and 80%). The augmentation of lower concentrations of GA3 (2 mg/L) in combination with 6-benzylaminopurine (BAP) resulted in the maximum mean shoot length (11.1 cm). The addition of 100 mM NaCl salts to the media led to the highest observed total phenolics content (TPC; 4.11 mg/g-DW compared to the control 0.52 mg/g-DW), total flavonoids content (TFC; 1.26 mg/g-DW) and polyphenolics concentration (5.39 mg/g-DW) in shoots cultured. However, the maximum antioxidant activity (81.8%) was observed in shoots raised in media treated with 50 mM NaCl. The application of 2 mg/L of GA3 resulted in the highest accumulation of proline (0.99 µg/mL) as compared to controls (0.37 µg/mL). Maximum stevioside content (71 µL/mL) was observed in cultures supplemented with 100 mM NaCl and 5% PEG, followed by the 4 mg/L GA3 treatment (70 µL/mL) as compared to control (60 µL/mL). Positive correlation was observed between GA3 and stevioside content. Notably, these two compounds are derived from a shared biochemical pathway. These results suggest that elicitation is an effective option to enhance the accumulation of steviosides and other metabolites and provides the groundwork for future industrial scale production using bioreactors.

Stimulation of secondary metabolites by copper and gold nanoparticles in submerge adventitious root cultures of Stevia rebaudiana (Bert.).

Nanotechnology is one of the advance technologies that almost found implications in every field of science. The importance is due to the unique properties of nanoparticles. In this study, bimetallic alloys of copper (Cu) and gold (Au) were tested in submerge root cultures of Stevia rebaudiana for production of biomass and secondary metabolites. A known amount of inoculum roots were submerged in liquid Murashige and Skoog medium containing combination of naphthalene acetic acid (NAA; 0.5 mg l-1) and different ratios of nanoparticles (NPs). NAA augmented medium was used as control. The addition of nanoparticles (30 µg l-1) stimulated biomass accumulation (1.447 g/flask) on 27th day of log phases. The maximum total phenolics content (TPC; 16.17 mg/g-DW) and total flavonoids content (TFC; 4.20 mg/g-DW) were displayed using AuCu-NPs (1:3) and NAA. The same combinations enhanced total phenolic production (TPP; 116 mg/L) and total flavonoid production (TFP; 29.5 mg/L) in submerged cultures. A strong correlation was observed between phenolics, flavonoids and dry biomass. Moreover, maximum 1, 1-diphenyl-2-picrylhydrazyl (DPPH) activity of 79% was displayed by addition of AuCu (1:3) nanoparticles. In conclusion, nanoparticles application has shown a positive effect in enhancing biomass and secondary metabolites production in adventitious root cultures of Stevia rebaudiana.