Effects of silver-graphene oxide on seed germination and early growth of crop species

Synthesis and characterization of Ag-GO

Graphene oxide was prepared using a modification of Hummers’ method described elsewhere (Hummers & Offeman, 1958; Chung et al., 2015) and Ag-GO was synthesized by the glucose reduction method (Tang et al., 2013; Kim et al., 2018). Briefly, 50 mL of an aqueous GO solution (0.5 mg mL⁻¹) was ultrasonicated for 1 h using a bar sonicator (VC 750 ultrasonic processor; Sonic & Materials Inc., Newtown, CT, USA). The GO solution was mixed uniformly with 10 mL polyvinylpyrrolidone (PVP) solution (4 mg mL⁻¹, average molecular weight ∼29,000, Sigma–Aldrich, St. Louis, MO, USA) and 800 mg glucose (Sigma–Aldrich, St. Louis, MO, USA) and the solution was heated at 45 °C. Subsequently, silver-ammonium hydroxide solution (10 mL) was added and the solution was held at 45 °C for 7 min. The mixture was centrifuged at 12,298×g for 10 min and the supernatant was decanted; the remaining pellet was washed several times with deionized (DI) water to remove excess ions and dried at 80 °C.

The microscopic morphology of Ag-GO was observed by transmission electron microscopy (Tecnai 20; FEI, Hillsboro, ON, USA). Absorption spectra were recorded using a UV–VIS spectrophotometer (Varian Cary 50; Cary, NC, USA). Raman spectroscopy was conducted using a LabRAM ARAMIS IR2 (Horiba, Kyoto, Japan) to analyze the defects and strain in Ag-GO and GO. X-ray photoelectron spectroscopy (XPS) spectra were measured with an automated XPS Microprobe (PHI 5000 Versa Probe; ULVAC-PHI, Chigasaki, Kanagawa, Japan) to compare the surface bonding before and after deposition of Ag NPs on the GO sheet; the ratio of Ag to GO was calculated through XPS.

Seed exposure

Three plant species, alfalfa (Medicago sativa L.), radish (Raphanus sativus L.) and cucumber (Cucumis sativus L.), were used in our study, and the seeds of these plants which were stored after harvest were purchased from Danong Inc., Namyangju-si, South Korea. Before exposure to the nanomaterials, seeds were soaked in a 10% sodium hypochlorite solution for 10 min to sterilize them and rinsed with DI water three times (US Environmental Protection Agency, 2012). Ten sterilized seeds were transferred onto filter paper placed in a 100 × 15 mm Petri dish containing 5 mL of Ag-GO suspension at different concentrations (0, 0.2, 0.4, 0.8 and 1.6 mg mL⁻¹). The different concentrations were prepared by diluting the stock Ag-GO suspension of 1.6 mg mL⁻¹. Additionally, 1.6 mg mL⁻¹ of GO and Ag NPs were applied to seeds to compare their effects to those of Ag-GO. Ag NPs (Sigma–Aldrich, St. Louis, MO, USA) were stabilized using PVP and their diameter was 70.2 ± 4.9 nm (Yasur & Rani, 2013). Petri dishes were sealed with parafilm and incubated at 25 °C in the dark for 7 days. Four replicates of each treatment were analyzed, for a total of 40 seeds per test species. During incubation, the germination percentage was determined every 2 days by counting the number of germinated seeds that had roots longer than 2 mm; the shoot and root lengths were measured at the end of the incubation period using a ruler.

Analysis of H₂O₂ in seedlings

The Amplex^® red hydrogen peroxide/peroxidase assay kit (Invitrogen™, Carlsbad, CA, USA) was used for the determination of H₂O₂ on day 7 at the end of the experiment. Seedlings were washed with DI water to remove nanomaterials on the surface of the seedlings. Adhering moisture was removed from the seedlings before grinding them in liquid nitrogen using a mortar and pestle. A total of 50 mg of tissue powder were resuspended in 250 μl of cold phosphate buffer (20 mM K₂HPO₄, pH 6.5). After centrifugation at 4 °C, 50 μl of supernatant was incubated with 0.2 U mL⁻¹ horseradish peroxidase and 100 μm Amplex red reagent (10-acetyl-3,7-dihydrophenoxazine) at room temperature in the dark for 30 min. Subsequently, the fluorescence was measured with a Synergy HT multi-mode microplate reader (Biotek, Winooski, VT, USA) (excitation at 530 nm and emission at 590 nm).

Quantification of silver content in fresh seedlings

Ag content in seedlings was quantified on day 7, at the end of the experiment, following the method described by Geisler-Lee et al. (2013) with modifications. Seedlings of the three species were washed with DI water and their fresh weight (F.W.) was measured. Seedlings were fully digested using a mixture of 2 mL of HNO₃ used for trace-metal analysis (70%, Sigma–Aldrich, St. Louis, MO, USA) and 1 mL Co standard solution (1,000 mg mL⁻¹ in nitric acid, Sigma–Aldrich, St. Louis, MO, USA) heated at 120 °C for 30 min. After the solution cooled down, 2 mL of H₂O₂ used for trace analysis (H₂O₂, ∼30% w/w, Sigma–Aldrich, St. Louis, MO, USA) was added and the mixture was heated at 120 °C for 30 min. The digested samples were diluted with DI water to 20 mL and filtered using a 0.22-μm polyvinylidene fluoride (PVDF) syringe filter. Each diluted sample was analyzed using an inductively coupled plasma-optical emission spectrometer (ICP-OES, iCAP6300 Duo; Thermo Scientific, Waltham, MA, USA).

Statistical analyses

Statistical analyses were carried out using SAS^® version 9.4 (SAS Inc., Cary, NC, USA) to test the effects of Ag-GO, Ag NPs and GO on seed germination. One-way analyses of variance (ANOVA) were employed to determine the effects of Ag-GO treatment on germination percentage, the lengths of the root and shoot, H₂O₂ content, and Ag content in seedlings. Additionally, one-way ANOVA was conducted to compare the effects of treatments with Ag-GO, Ag NPs and GO at a concentration of 1.6 mg mL⁻¹. The effects of the treatments were accepted as significant at α = 0.05. Tukey’s honestly significant difference test was conducted to further investigate which means differed from other means within a group (p < 0.05) when the effect of each treatment was significant for the parameters studied. In addition, regression analyses were performed to determine the relationship between the concentration of Ag-GO treatment and Ag content in seedlings treated with Ag-GO.

Characterization of Ag-GO

The TEM images of Ag-GO showed that the Ag NPs were formed on the wrinkled surface of GO (Figs. 1A and 1B). The average diameter of Ag NPs was 57.5 ± 1.9 (average ± one standard error; n = 250) nm. The UV–VIS spectrum of GO (Fig. 1C) displayed no obvious absorption band in the wavelength range of 300–800 nm. In contrast, the UV–VIS spectrum of Ag-GO showed maximum absorbance at 430 nm, indicating that Ag NPs were loaded on the GO sheets. The Raman spectra of Ag-GO and GO (Fig. 1D) showed two peaks, the D band at 1,350 cm⁻¹ and the G band at 1,590 cm⁻¹. However, the intensity of the D and G bands in the Raman spectrum of Ag-GO was enhanced by surface-enhanced Raman scattering. The I_D/I_G ratios of GO and Ag-GO were 0.95 and 0.92, respectively. Analysis of surface bonding using XPS (Figs. 1E and 1F) clearly showed that the intensity of oxygenated carbon bonds decreased after Ag NP formation on GO. The XPS C1s spectra of GO showed three main peaks, for C–C, C–O and C=O bonds. The C–O and C=O bond peaks of Ag-GO showed a relatively lower intensity than those of GO. Additionally, the carbon-to-oxygen ratio increased from 2.0 to 4.1 after Ag NP formation on the GO sheet. The reduction of oxygenated carbon after Ag NP formation on a GO sheet has also been demonstrated by other groups (Tang et al., 2013; Kim et al., 2018). The weight ratio of Ag to GO was approximately 0.64.

Effects of Ag-GO on seed germination and seedling growth

Treatment with Ag-GO had no significant effect on germination percentage (Table 1). In addition, germination percentage was not altered by Ag NP or GO treatment. Regardless of the Ag-GO treatment concentration, 100% of the alfalfa and cucumber seeds germinated on day 4 and day 7, respectively, and 95–100% of radish seeds germinated on day 7. Ag-GO had both positive and negative effects on seedling growth (Fig. 2). The shoot length of alfalfa was not affected significantly by Ag-GO treatment; however, that of radish and cucumber showed a significant response to treatment with Ag-GO (Fig. 2A). More specifically, the shoot length of radish treated with Ag-GO at 0.2 to 1.6 mg mL⁻¹ increased up to 52% (p < 0.0001) and that of radish treated with Ag NPs at 1.6 mg mL⁻¹ increased by 36% when compared to the control. However, the shoot length of cucumber decreased by 16% upon treatment with Ag-GO at 0.8 mg mL⁻¹ compared to the control (p = 0.0003).

Ag-GO treatment also had a significant effect on the root growth of alfalfa and radish (Fig. 2B). Treatment with 1.6 mg mL⁻¹ of Ag-GO inhibited the root elongation of alfalfa by 41% compared to that of the control (p = 0.0011). However, the root length of radish treated with 0.2 mg mL⁻¹ of Ag-GO increased by 45% compared to that of the control (p = 0.0456) and its effect on root elongation significantly differed from that of 1.6 mg mL⁻¹ of GO (p = 0.0002). In contrast, treatment with Ag-GO had no significant effect on the root length of cucumber. Overall, the effects of Ag-GO, Ag NPs and GO treatments on root elongation differed significantly (p < 0.001).

H₂O₂ content in seedlings treated with Ag-GO

Ag-GO treatment significantly increased the H₂O₂ content in alfalfa and radish seedlings (Fig. 3). When alfalfa was treated with 1.6 mg mL⁻¹ of Ag-GO, its H₂O₂ content was more than twice that of the control (p < 0.0001) and similar to that in alfalfa treated with 1.6 mg mL⁻¹ of Ag NPs (p < 0.0001). In radish, the production of H₂O₂ was increased by treatment with Ag-GO compared to that in the control (p < 0.0001). In addition, the level of H₂O₂ significantly differed for Ag-GO, Ag NPs and GO treatments (p < 0.0001). In contrast, the content of H₂O₂ in cucumber seedlings was not altered by Ag-GO, Ag NPs, or GO treatments.

Ag content of seedlings treated with Ag-GO

The results of the regression analysis showed that the concentration of Ag-GO and the Ag content in the three seedlings were positively correlated. For example, the Ag content in alfalfa was positively correlated with the treatment concentration of Ag-GO (p < 0.001, R² = 0.85). Likewise, in the case of radish and cucumber, there was positive correlation between the concentration of Ag-GO and Ag content in these species (p = 0.004, R² = 0.71 in radish and p < 0.0001, R² = 0.94 in cucumber). According to the ANOVA results, there were no significant differences in the Ag content in alfalfa and radish at the concentrations of 0.2, 0.4 and 0.8 mg mL⁻¹ of Ag-GO. However, at 1.6 mg mL⁻¹ of Ag-GO, Ag accumulation in alfalfa and radish was significantly higher than that for the other treatments (p < 0.0001 and p = 0.0059, respectively). In cucumber, there was no significant difference in Ag content between 0.2 and 0.4 mg mL⁻¹ of Ag-GO treatments, but the Ag content was significantly higher at 0.8 and 1.6 mg mL⁻¹ of Ag-GO (p < 0.001) (Fig. 4). The Ag contents in alfalfa and radish treated with 1.6 mg mL⁻¹ of Ag-GO were 93.9 ± 11.6 ng mg⁻¹ F.W. and 43.6 ± 2.0 ng mg⁻¹ F.W., respectively. The Ag content in cucumber treated with 1.6 mg mL⁻¹ of Ag-GO was 12.5 ± 1.5 ng mg⁻¹ F.W., which was four times the content of Ag in cucumber treated with 0.2 mg mL⁻¹ of Ag-GO. Furthermore, the Ag content for Ag-GO and Ag NP treatments was significantly different in alfalfa and radish. The Ag content in alfalfa and radish exposed to 1.6 mg mL⁻¹ of Ag-GO was approximately two times that of seedlings treated with 1.6 mg mL⁻¹ of Ag NPs (p < 0.0049 and p = 0.0079, respectively). However, the Ag content in cucumber was similar for both treatments (Fig. 4).