Improvement of Stevia rebaudiana Bertoni In Vitro Propagation and Steviol Glycoside Content Using Aminoacid Silver Nanofibers

Abstract

The food industry is interested in replacing artificial sweeteners with natural sugars that possess zero calories and carbohydrates and do not cause spikes in blood sugar levels. The steviosides leaves, synthesized at Stevia rebaudiana Bertoni, are 300 times sweeter than common table sugar. Stevia propagation is limited due to the poor viability of the seeds, the long time and low germination rate, and the poor rooting ability of vegetative cuttings. Because of this, an alternative biotechnological method for its reproduction is being studied, such as multiple shoot production through direct organogenesis using nanofibers, formed from a derivative of amino acid valine as a carrier of the biologically active agent silver atoms/particles (NF-1%Ag and NF-2%Ag). The stevia explants were cultured on a medium containing NF-1%Ag and NF-2%Ag at concentrations of 1, 10, 50, and 100 mg L^-1. The NF-1%Ag and NF-2%Ag treatment caused hormetic effects on stevia plantlets. At low concentrations of from 1 to 50 mg L^-1 of nanofibers, the stimulation of plant growth was observed, with the maximum effect being observed at 50 mg L^-1 nanofibers. However, at the higher dose of 100 mg L^-1, inhibition of the values of parameters characterizing plant growth was recorded. The presence of nanofibers in the medium stimulates stevia root formatting.

Keywords: Stevia rebaudiana Bert.; antioxidant activity; carrier of Ag particles; in vitro propagation; nanofibers.

Figure 1

Structure of the compound M6.

Figure 2

UV study of the used materials. (a) Samples from the stock solution from commercially obtained AgNPs.: 0.50 mg mL⁻¹, 0.33 mg/mL, and 0.20 mg mL⁻¹ in distilled water; (b) Absorption of the material prepared as 2% AgNPs on organic compound M6 in ethanol; (c) Focus on the absorbance of AgNPs in (b).

Figure 3

SEM image of fibers with nanosized diameter and silver particles (2%).

Figure 4

IR spectra of the pure M6 (red) and M6 carrying 2% Ag NPs (blue).

Figure 5

The activity of antioxidant enzymes superoxide dismutase (SOD) (A), catalase (CAT) (B), guaiacol peroxidase (GPX) (C), and ascorbate peroxidase (APX) (D) in Stevia rebaudiana plantlets in vitro propagated on MS medium, with BAP, and on MS medium supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of aminoacid nanofibers enriched with 1% and 2% colloidal Ag (NF-1%Ag, NF-2%Ag). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter ‘a’ or “A” for the lowest data value and ascending to the next for higher data values. The statistical analysis of NF-1%Ag (uppercase) and NF-2%Ag (lowercase) was performed separately.

Figure 6

The content of metabolites with antioxidant power (total phenolic compounds (A) and flavonoids (B), WS-AOM (C) and LS-AOM (D)) and antioxidant potential (DPPH (E), FRAP (F)) in S. rebaudiana plantlets in vitro propagated on MS medium, on MS medium with BAP, and on MS medium supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of aminoacid nanofibers enriched with 1% and 2% colloidal Ag (NF-1%Ag, NF-2%Ag). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter ‘a’ or “A” for the lowest data value and ascending to the next letters for higher-data values. The statistical analysis of NF-1%Ag (uppercase) and NF-2%Ag (lowercase) was performed separately.

Figure 7

The levels of stress markers (MDA (A), -SH groups (B), H₂O₂ (C), and proline (D)) in the S. rebaudiana plantlets in vitro propagated on MS medium, on MS medium with BAP, and on MS medium supplemented with various concentrations (1, 10, 50, 100 mg L⁻¹) of aminoacid nanofibers enriched with 1% and 2% colloidal Ag (NF-1%Ag, NF-2%Ag). Values are means ± SE, n = 20; different letters indicate significant differences assessed by the Fisher LSD test (p ≤ 0.05) after performing ANOVA one-way analysis. We used the letter ‘a’ or “A” for the lowest data value and ascended to the next letters for higher data value. The statistical analysis of NF-1%Ag (uppercase) and NF-2%Ag (lowercase) was performed separately.