Beneficial dose-dependent effects of Ag nanoparticles on germination do not compromise growth and metabolic profiles of Capsicum annuum seedlings

Study species

Capsicum annuum L. (Solanaceae) is a perennial, flowering shrub that comprises both the wild populations (Capsicum annuum var. glabriusculum) and domesticated varieties (Capsicum annuum var. annuum). The height of wild and domesticated plants typically ranges from 50 cm to 1.5 m (Solís-Montero, Bello-Bedoy & Munguía-Rosas, 2023). The leaves of wild plants are generally smaller and differ in shape compared to those of domesticated varieties (Serrano-Mejía et al., 2022). Both wild and domesticated plants develop a taproot system with branched secondary roots. Fruit shape, size, varies widely among varieties. The flowers are typically small and white, with five petals (Serrano Mejía, 2023).

Seed germination trials

Germination trials were conducted using seeds from the domesticated chili variety Capsicum annuum var. annuum (Serrano) and its wild counterpart, Capsicum annuum var. glabriusculum. Wild chili seeds were collected from natural populations in Sonora, Mexico, while Serrano seeds were obtained from a commercial supplier. The average seed weight for the Serrano variety was 4.16 mg (207.9 mg/50 seeds), whereas the wild chili seeds averaged 2.6 mg (132.7 mg/50 seeds).

Prior to the germination assays, seeds were surface-sterilized with 70% ethanol for 2 min and rinsed three times with distilled water. Ten seeds of each variety were placed in eight cm diameter Petri dishes lined with filter paper, which provided uniform support, ensured even moisture distribution, and prevented waterlogging during germination. Each treatment was replicated five times. Seeds were treated with five mL of one of four AgNP solutions: 0 (control), 50, 100, or 250 ppm.

Each treatment was replicated four times. The Petri dishes were sealed with Parafilm to prevent moisture loss and contamination, and then placed in a controlled-environment growth chamber maintained at 24–27 ^∘C, with a 16:8 h light/dark photoperiod and 65% relative humidity. Seeds were incubated under these conditions for 14 days.

The silver nanoparticles used are part of a commercially available veterinary formulation previously characterized in detail (see Bello-Bello et al., 2018 for a detailed description on Argovit™). These nanoparticles exhibit spheroidal morphology, a size range of 1–90 nm (mean ∼35 nm), polyvinylpyrrolidone (PVP) stabilization, and a silver content of 1.2% (w/w), as confirmed by TEM, DLS, FTIR, and UV-Vis spectroscopy (Bello-Bello et al., 2018; Stephano-Hornedo et al., 2020). In this study, a concentrated stock solution was used to prepare the experimental dilutions, ensuring precise control of the final concentrations and facilitating reproducibility and comparability with previous studies.

Germination percentage

A seed was registered as germinated when radicle emergence was observed. Germination was recorded in a binomial format: 1 = germinated, 0 = not germinated. Germination was monitored daily for 14 days following treatment, corresponding to the period during which most seeds completed germination. We calculated both total germination percentage and germination rate. Total germination percentage was defined as the ratio of germinated seeds to the total number of seeds incubated.

At germination, each seed was transplanted individually into 50-cell trays filled with sterile commercial substrate (Berger BM2) for initial growth. Once seedlings developed two fully expanded leaves, they were individually transplanted into 3.5-inch pots containing the same substrate. Subsequently, when seedlings reached four leaves, they were transferred to 2.5 L pots to allow for continued growth. Wild and domesticated chili plants develop extensive root systems; thus, seedlings were transplanted twice during the experiment to ensure adequate root space and minimize restriction. All transplanting procedures were carried out carefully to avoid root damage and reduce transplant stress. Plants were irrigated with 100 mL of distilled water every other day to maintain consistent soil moisture. Additionally, seedlings received a weekly application of 25 mL of a 3 g L⁻¹ NPK (19-19-19) nutrient solution.

Growth measurements

To assess the effects of nanopriming on the growth and development of wild and domesticated chili plants, the following traits were measured at 28 and 42 days after germination: root length, shoot length, plant height, and wet and dry biomass of both root and shoot. Plants were cultivated in a controlled-environment chamber set to 24–27 ^∘C, with a 16:8 h light/dark photoperiod and 65% relative humidity.

Measurements of plant growth plant length and biomass were carried out as follow: Root length was measured from the base of the stem to the tip of the primary root. Shoot length was measured from the stem base to the apical bud of the main stem. Wet biomass was measured by carefully removing the roots from the substrate, rinsing them thoroughly with distilled water, drying them with blotting paper, and weighing the plant material using a precision balance (Mettler Toledo MSQ205) to the nearest 0.0001 g. For dry biomass, the samples were wrapped in aluminum foil and dehydrated in a VWR convection oven at 65 ^∘C for seven days to ensure complete desiccation. The dried material was then weighed using the same precision balance.

Total polyphenol content

To quantify total polyphenol content, fresh plant leaves were analyzed at 28 and 42 days after germination using the Folin–Ciocalteu method. First, properly labeled Eppendorf tubes were weighed. Leaves were then cleaned with moistened blotting paper to remove any substrate residue. Once cleaned, each sample was individually macerated in an Eppendorf tube and weighed. Samples were then incubated with 1 mL of methanol–water solution (80:20 v/v) for 24 h in the dark to prevent light-induced degradation and oxidation of phenolic compounds, thereby preserving their chemical stability during extraction.

After 24 h, the samples were centrifuged at 13,000 rpm for 5 min. In new Eppendorf tubes, 1 mL of distilled water, 20 µL of extract from each sample, and 100 µL of Folin–Ciocalteu phenol reagent (Sigma-Aldrich, St. Louis, MO, USA) were added and mixed thoroughly. Subsequently, 300 µL of 20% (w/v) sodium carbonate (Na₂CO₃) solution was added and mixed. The reaction mixtures were incubated at room temperature for 2 h. After incubation, the absorbance of each sample was measured at 765 nm using a Hach DR 2800 spectrophotometer. Gallic acid was used as the standard for constructing a calibration curve with standard concentrations of 50, 100, 150, 250, 500, 1,000, 1,500, and 2,000 mg/L, following the protocol of Ainsworth & Gillespie (2007). The regression equation derived from the standard curve was used to calculate the gallic acid equivalent (GAE) concentration in each sample. Total polyphenol content was expressed as milligrams of GAE per gram of fresh leaf tissue. Distilled water was used as the blank control.

Chlorophyll content

To quantify chlorophyll content, the leaf chlorophyll content index (CCI) was measured using a Chlorophyll Content Meter-200 Plus (OPTI-SCIENCE, Hudson, NH, USA). This device estimates chlorophyll concentration by measuring light absorbance at 600 nm and 900 nm (±1.0 nm) over a leaf area of 0.71 cm². Measurements were taken from the fourth fully expanded true leaf of each plant. For each leaf, three readings were obtained and their average was used as the final CCI value.

Cell division indicators

To evaluate the potential cytotoxic effects of nanopriming on cell division in apical zones, roots and shoots were exposed to an AgNP solution, with water used as the control. Seeds from both wild and domesticated Capsicum annuum were germinated, and once seedlings developed three to five true leaves, 20 individuals from each variety were randomly selected for the experiment. Five plants were assigned to each treatment group. The seedlings were carefully uprooted, rinsed, and placed in 150 mL beakers containing 120 mL of either the AgNP solution (5 ppm) or distilled water. Plants remained in the treatments for 72 h. Shoot and root lengths were measured every 24 h using a ruler, and digital images were analyzed with ImageJ software.

After 72 h, root samples were collected for cytological staining to visualize dividing cells. To examine cell division and calculate mitotic and phase indices. To accomplish this, root tips were prepared following a modified version of the cell division observation protocol described by Rohami et al. (2010). Briefly, lateral roots were excised from the apical zone at approximately 1 cm from the tip. The root sections were individually placed in 1.5 mL microcentrifuge tubes containing a fixative solution of ethanol and glacial acetic acid (3:1, v/v) for 24 hours at room temperature. Subsequently, hydrolysis was performed with 1 N hydrochloric acid (HCl) for 30 minutes at room temperature on a watch glass. The roots were then individually stained on microscope slides using Aceto-Orcein (Sigma-Aldrich, St. Louis, MO, USA) for 30 minutes at room temperature. After staining, the roots were rinsed twice with acetic acid to remove the excess dye. Finally, each root tip was mounted on a microscope slide, treated with a drop of acetic acid, and gently squashed using a pencil eraser to evenly spread the cells.

Stained root tip samples were examined under a light microscope at 100× magnification using immersion oil. For each sample, between 500 and 2,000 cells were counted to calculate the mitotic index and determine the distribution of cells across the different mitotic phases. The mitotic index was calculated as the ratio of dividing cells to the total number of observed cells (Howell et al., 2007). The phase index was determined by calculating the proportion of cells in each mitotic phase—prophase, metaphase, anaphase, and telophase—relative to the total number of cells observed.

Statistical analysis

To evaluate the effects of nanopriming on wild and domesticated chili plants, a logistic ANOVA and a survival analysis was used to analyze germination data. The model included the fixed effects of plant type (wild vs. domesticated), AgNP treatment concentration, and their interaction (type × treatment). Growth and biochemical traits—including wet and dry biomass, root and shoot length, total phenolic content, and chlorophyll content—were analyzed using two-way analysis of variance (two-way ANOVA). The fixed effects included plant type, AgNP concentration, and their interaction. Additionally, variation in mitotic phase frequencies was assessed using two-way ANOVA with plant type and AgNP exposure as fixed effects, along with their interaction. All statistical analyses were performed using JMP version 10 (SAS Institute Inc., Irvine, CA, USA).

Germination

Exposure to silver nanoparticles significantly affected total seed germination, with marked differences observed between wild and domesticated genotypes (Table 1; Fig. 1). In the control group, germination reached 77%, whereas treatment with 50 ppm AgNPs increased germination to 82%, 100 ppm resulted in 90%, and 250 ppm resulted in 91%. A significant plant type × treatment interaction was detected by ANOVA, indicating that the effect of AgNP concentration differed between wild and domesticated chili plants. Only the wild variety exhibited a dose-dependent response to AgNPs, with higher germination percentages observed at 100 ppm and 250 ppm compared to its control (Fig. 1). In contrast, the domesticated variety (Serrano) did not show significant differences in germination among AgNP treatments and the control. Germination rates for wild and domesticated chilies were 86% and 83.5%, respectively, and did not differ significantly (χ² = 3.33, P = 0.06). However, within the wild variety, a significant difference was found between the control group and the 250 ppm treatment (Table S1). No significant differences in germination rate were observed among treatments for the domesticate (Table S1).

Growth indicators

At 28 days after germination, significant differences between wild and domesticated varieties were observed only in shoot length, total plant length, total wet biomass (root and shoot combined), dry shoot weight, and total dry biomass (Table S2). No significant effects of AgNP treatment or treatment × variety interactions were detected at this stage. By 42 days, a significant treatment effect was detected only for total wet biomass (p = 0.04; Table S3), although this effect was independent of plant variety. A significant treatment × variety interaction was found only for total plant length (p = 0.05; Table S3; Fig. S1), indicating that the effect of AgNP concentration on total length varied between wild and domesticated plants.

Total polyphenol content

Total polyphenol levels showed no significant differences between plant varieties or AgNP treatments at 28 and 42 days after germination (Table S4). No significant plant type × treatment interactions were detected. However, in the domesticated Serrano variety, polyphenol content tended to increase at the higher AgNP concentrations (100 and 250 ppm; Fig. S2). In contrast, the wild variety exhibited irregular and inconsistent patterns of polyphenol accumulation across treatments and time points. The lack of a consistent trend may reflect underlying differences in metabolic activity at various developmental stages.

Chlorophyll content

Chlorophyll content differed significantly between wild and domesticated varieties at both 28 and 42 days after germination (Table S5). At 42 days, the effect of AgNP treatment was variety-dependent: increasing nanoparticle concentrations led to a reduction in chlorophyll content in the domesticated variety, while the wild variety remained largely unaffected (Fig. S3).

Cell division indicators: phytotoxic and genotoxic evaluation

Measurements of stem and root length during the first 72 h of AgNP exposure showed significant differences between the wild and domesticated varieties (p < 0.0001; Table S6), but no significant effects of AgNP treatments on shoot or root elongation were observed. Two-way ANOVA of leaf area at 48 and 72 h indicated significant variety × treatment interactions (Table S6); however, subsequent Tukey tests identified significant differences only between varieties, suggesting that AgNP treatments did not induce phytotoxic effects (Table S7).

Genotoxic evaluation showed no significant effects of AgNP treatment on the mitotic index or chromosomal aberrations in root cells for either variety (Table S8). Interestingly, in the wild chili, AgNP exposure increased the number of dividing cells compared to the control, while in the domesticated variety, a reduction in all mitotic phases was observed under AgNP treatment. This contrasting response may reflect underlying genetic differences between wild and domesticated genotypes.

Variability in response to AgNPs between wild and domesticated varieties

The effects of AgNPs on plants have shown considerable variability across studies, depending on species identity and nanoparticle concentration (Thongmak et al., 2022; Mays et al., 2024). The influence of natural history and domestication status has been poorly considered, despite growing evidence that domestication profoundly alters plant genotypes, phenotypes, and their functional responses (Milla et al., 2015; Serrano-Mejía et al., 2022). To our knowledge, this is the first study to explicitly compare the effects of AgNP nanopriming between a domesticated plant and its wild relative. Our results using wild and domesticated Capsicum annuum suggest that seed biological traits are key determinants of plant response to AgNP treatments. In the wild genotype, nanopriming with higher concentrations (100 ppm and 250 ppm) significantly increased germination rates, indicating a dose-dependent benefit. In contrast, the domesticated variety exhibited no significant response to any of the tested concentrations. These findings support the hypothesis that domestication-related changes in seed biology modulate the effectiveness of nanopriming.

The contrasting germination responses observed between wild and domesticated C. annuum varieties may be partially explained by physiological mechanisms influenced by nanopriming. In wild seeds, nanopriming may promote germination by enhancing physiological processes such as water imbibition and hormonal activation. The absorption of AgNPs by seeds has been shown to induce physiological responses related to the activation of phytohormones involved in growth and dormancy release (Méndez-Argüello et al., 2016), as well as the expression of genes related to cell proliferation (Qian et al., 2013). For instance, nanopriming can promote the activity of α-amylase, a critical enzyme for breaking down starch reserves during germination (Mahakham et al., 2017). In wild chilies, low germination rates have been attributed to limited gibberellic acid (GA₃) availability under natural conditions. Treatments with exogenous GA₃ at various concentrations have led to increased germination in wild seeds (Hernández-Verdugo, Oyama & Vázquez-Yanes, 2001; Cano-Vazquez et al., 2015), suggesting that hormonal limitation plays a relevant role. Therefore, silver nanoparticles may facilitate the hormonal signals needed to trigger germination, offering a possible explanation for the enhanced germination observed in wild seeds.

An important question that arises is why the domesticated variety did not respond to AgNP nanopriming. Two potential explanations may account for this lack of response. One possibility is that the domestication process has reduced the sensitivity of Capsicum annuum seeds to nanoparticles. However, this explanation seems unlikely, as previous studies have reported variation in germination and seedling growth among domesticated C. annuum varieties in response to different types of AgNPs (Yuan et al., 2018; Sánchez-Pérez et al., 2023). A more plausible explanation is that domesticated seeds have evolved a more efficient physiological response to hydropriming, reducing their reliance on environmental signals to activate gibberellic acid or cytokinins required for germination. Therefore, while AgNPs may enhance germination in wild chilies, their application may not further improve germination in domesticated varieties that already exhibit high responsiveness to hydropriming.

Evaluation of the cytotoxic and genotoxic impact of AgNPs

An important objective of this study was to determine whether exposure to AgNPs induces cytotoxic or genotoxic effects in the meristematic tissues of wild and domesticated Capsicum annuum varieties. In our study, primed and non-primed seedlings exhibited similar growth rates, reached comparable sizes, and accumulated similar levels of foliar chlorophyll and phenolic compounds 28 days after germination (Table S1). Moreover, further assays showed no negative effects of AgNPs on apical or root meristems, supporting the conclusion that AgNPs did not exert phytotoxic effects on plant growth. These findings are consistent with previous studies reporting no phytotoxicity of silver nanoparticles at low to moderate concentrations (Budhani et al., 2019). For example, in Vanilla planifolia, concentrations of 25 and 50 mg/L promoted growth without causing significant genotoxic effects (Spinoso-Castillo et al., 2017; Bello-Bello et al., 2018). In Allium cepa, concentrations up to 100 µg/mL of AgNP solution promoted growth without causing genotoxic or cytotoxic damage (Casillas-Figueroa et al., 2020). However, studies in other plant systems have associated chromosomal abnormalities to prolonged exposure to higher concentrations (Kumari, Mukherjee & Chandrasekaran, 2009). We observed no phytotoxic effects even at higher concentrations, suggesting that both wild and domesticated Capsicum annuum can tolerate elevated levels of silver nanoparticles, supporting their potential as a safe tool for enhancing germination and growth in this plant species.

One limitation of this study is the absence of PVP and ionic silver treatments to assess their potential effects on germination and growth. The experiment was not designed to isolate the effects of PVP, as previous studies have demonstrated that the PVP coating of the AgNPs used does not induce cytotoxic or genotoxic effects at the applied concentrations (Casillas-Figueroa et al., 2020; Bello-Bello et al., 2018). Moreover, other studies have reported that the observed biological effects are primarily associated with the nanoparticles themselves, rather than the coating agent or released silver ions (Cvjetko et al., 2017). Therefore, the effects observed in this study are most likely attributable to the nanoparticles.