Sodium selenite (Na₂SeO₃) (MW of 172.94) was purchased from Hi Media, India. Fresh leaves of C. auriculata were collected from Tiruvannamalai district, Tamil Nadu, India (latitude 12.3841 and longitude 79.1178). The amaranthus variety used in the study is Arka Suguna, which is resourced from IIHR, Bangalore, India.

Cassia auriculata leaf powder was tested at three concentrations (0.5, 2.5, and 5 g) suspended in 100 mL of double-distilled water (ddH₂O) for optimal extract preparation. The suspensions were maintained in a water bath at 60°C with continuous stirring (200 rpm) for 30 min. The resulting mixtures underwent sequential filtration: primary filtration through sterile muslin cloth to remove particulate matter, followed by vacuum filtration using Whatman No. 42 filter paper to eliminate residual macro particles. The clarified, particle-free extracts were aseptically transferred to sterile reagent bottles and stored at 4°C for immediate use in SeNP synthesis. Sodium selenite (Na₂SeO₃; Molecular Weight: 172.94 g/mol; HiMedia Laboratories) solutions were prepared in five concentrations (5, 8, 10, 12, and 15 mM) using ddH₂O as solvent. For SeNP synthesis, each concentration was combined with the standardized leaf extract (5 g/100 mL) in a 1:4 (v/v) ratio. The 1:4 ratio was chosen due to its demonstrated efficacy in prior optimization studies of NP production (Khurana et al., 2019; El-Saadony et al., 2021). The reaction mixtures were incubated at 75°C (±1°C) for 25 min in a hot plate magnetic stirrer (800 rpm) to ensure complete reduction of Se⁴⁺ to Se⁰. Temperature and stirring speed were rigorously controlled to maintain consistent reaction kinetics across all trials.

To achieve optimal nanoparticle stability, the biosynthesized SeNP solution underwent controlled incubation in an orbital shaker (120 rpm) under sequential temperature conditions: initial stabilization at 72°C (±0.5°C) for 3 h, followed by gradual cooling to 37°C (±0.5°C) for extended incubation periods (6, 12, 24, 48, 60, and 72 h). At each time interval, aliquots were aseptically collected and analyzed by UV-Vis spectroscopy to monitor plasmon resonance peak stability at 230 nm. This dual-phase thermal protocol ensured progressive nanoparticle maturation while preventing aggregation.

The synthesized biogenic selenium nanoparticle’s physicochemical properties were characterized using advanced analytical techniques. UV-Vis spectral analysis was carried out on a Genesys 180 spectrophotometer (Thermo Fisher Scientific, United States), with absorbance measurements recorded across 200–800 nm wavelengths at 1 nm intervals. Nanoparticle morphology and elemental composition were evaluated through transmission electron microscopy (Quanta 200 FEG, FEI, Netherlands) coupled with energy-dispersive X-ray spectroscopy (Oxford Instruments X-MaxN EDX system), performed at 200 kV accelerating voltage at Tamil Nadu Agricultural University’s facility. Crystalline structure was determined by X-ray diffraction analysis (Bruker D8 Advance, United States) employing a 2θ range of 10°–80° with 0.02° step resolution. Surface functional group identification was achieved using FT-IR spectroscopy (PerkinElmer Spectrum Two) scanning the 4,000–400 cm⁻¹ spectral range at Central University of Tamil Nadu’s analytical laboratory. For quantitative analysis, TEM micrographs were processed using ImageJ software (v1.45, NIH) with threshold-based particle detection. Size distribution histograms and statistical parameters (mean ± SD) were generated using OriginPro 2022 (OriginLab Corporation) with Gaussian curve fitting. All measurements were performed in triplicates.

The biocompatibility of biosynthesized SeNPs was evaluated against two beneficial soil microorganisms: Bacillus subtilis (MTCC 121) and Pseudomonas aeruginosa (MTCC 7903). Sterile nutrient agar (20 mL; HiMedia, M001) was supplemented with 5 mL of SeNP solution (100 ppm) in Petri plates (90 mm diameter). Bacterial cultures (100 µL of 10⁸ CFU/mL suspension) were spread-plated and incubated for 48 h at 37°C (±0.2°C). Microbial growth was assessed by measuring colony diameter (mm) and optical density (600 nm) compared to untreated controls.

Seeds of Amaranthus var. Arka Suguna underwent surface sterilization using 0.4% (v/v) sodium hypochlorite solution for 5 min, followed by three thorough rinses with sterile distilled water. After air-drying under aseptic conditions in a laminar flow hood, the sterilized seeds were transferred to Petri plates containing 2% plain agar media (HiMedia, GRM026), which had been pre-treated with either 100 ppm SeNPs solution or control treatments. The plates were maintained in a growth chamber set at 25°C with a 16-h photoperiod/8-h dark cycle. Daily observations were conducted over 7 days to quantify germination percentage and measure radicle elongation (in mm).

Artificial drought conditions were established using polyethylene glycol 6000 (PEG-6000; HiMedia, PCT1306). Four osmotic stress levels were tested: 16.7 mM (−0.05 MPa), 25 mM (−0.10 MPa), 37.5 mM (−0.20 MPa), and 56.3 mM (−0.40 MPa) PEG solutions. Each concentration was applied at 5 mL per potray cell (4 cm diameter × 5 cm depth) containing the growth medium. The optimal PEG concentration was determined based on germination percentage and Amaranthus var seedling vigor index. A standardized growth medium was prepared by homogenizing farmyard manure (FYM), river sand, and vermicompost in a 1:1:1 (v/v/v) ratio. Physical properties of the substrate were characterized (pH 6.8 ± 0.2, EC 1.2 ± 0.3 dS/m).

Surface-sterilized Amaranthus var. Arka Suguna seeds were nano-primed via incubation in 25 or 100 ppm biosynthesized SeNP solutions (1:2 seed: solution ratio) for 60 min at 25 rpm (25°C ± 1°C) using a benchtop tube rotator before sowing. Soil application was performed on day 3 post-germination (D3) via root zone drenching with 100 ppm SeNP solution (5 mL/portray cell). In comparison, foliar spraying (25 ppm, 1 mL/portray cell) was conducted at the first true leaf stage (D6) using an atomizer (50–100 μm droplets) during peak stomatal activity to optimize uptake. All treatments were employed in controlled environmental conditions (28°C ± 2°C, 70% RH).

Amaranthus var. Arka Suguna microgreens were harvested at physiological maturity (9 days post-sowing) for growth and yield evaluation. Germination percentage was calculated as (number of germinated seeds/total seeds sown) × 100, with radicle emergence ≥2 mm as the germination criterion. Plant height was measured from the growth medium surface to the apical meristem using digital calipers (Mitutoyo, ±0.01 mm precision). For yield analysis, fresh weight was recorded immediately post-harvest (Shimadzu balance, ATX224R, Japan), while dry weight measurements followed 48-h dehydration in a forced-air oven at 60°C ± 1°C (P-Lab, Precession lab industries, India) until constant mass was achieved. All measurements were conducted in triplicate across three independent experimental runs.

Selenium concentration in Amaranthus microgreens was determined using a modified (Duff and Chessin, 1965) acid digestion method. 0.5 g of tissue was digested in a 10 mL acid mixture (7.5:2.5 v/v nitric: perchloric acids) at 85°C–100°C until white fumes appeared. After cooling, 3 mL 30% hydrogen peroxide (H2O2) was added and reheated (5–10 min) to complete oxidation. The digestate was reacted with 10 mL 3% hydrazine sulfate (reducing agent) and 3 mL 2.5% gum arabic (stabilizer) in a boiling water bath (10 min), producing an orange-red Se-hydrazine complex. The final volume was adjusted to 25 mL using deionized water, and absorbance was recorded at 420 nm relative to sodium selenite standards (0–5 ppm). Spike recovery tests (90%–105%) and reagent blanks validated the protocol, with results expressed as mg Se/g dry weight (triplicate measurements).

Three independent biological replicates were performed for all experimental treatments. Data analysis was conducted using R Statistical Software (version 4.2.3) with specialized packages for agricultural research (dplyr v1.1.0, agricolae v1.3-6, and multicomp v1.4-23). Treatment comparisons were assessed through one-way analysis of variance (ANOVA) followed by post hoc testing using Tukey’s honestly significant difference (HSD) method at α = 0.05. Significant differences between treatment groups were indicated using compact letter display notation following standard agricultural research conventions. For additional verification, Fisher’s least significant difference (LSD) test was performed at the 5% probability level. All graphical representations were generated using GraphPad Prism software (version 8.4.3) to visualize treatment effects and statistical relationships.

The biosynthesis of selenium nanoparticles (SeNPs) was optimized using C. auriculata leaf extract with various concentrations of sodium selenite (5 mM, 8 mM, 10 mM, 12 mM, 15 mM) and incubation hours (6, 12, 24, 48, 60, 72 h). Among tested concentrations, 5% leaf extract combined with 12 mM sodium selenite yielded the most effective SeNP synthesis, exhibiting peak absorbance at 230 nm with high intensity (Figure 1A). The reduction process commenced immediately upon mixing. Subsequent stabilization studies revealed that a 72-h incubation period at 120 rpm produced SeNPs with the highest absorbance peak (Figure 1B). These standardized conditions 5% leaf extract, 12 mM sodium selenite, and 72-h incubation were thus established as optimal for efficient and stable SeNP biosynthesis.

To establish an effective in vitro drought induction system, polyethylene glycol (PEG) was tested at varying concentrations (16.7, 25, 37.5, 56.3 mM). Results demonstrated that 37.5 mM and 56.3 mM PEG most effectively simulated drought stress (Figure 2), as evidenced by pronounced physiological responses. Thus, 37.5 mM PEG concentration was selected for subsequent experiments to evaluate drought-induced changes in plant biochemical and physiological parameters.

The biocompatibility assessment of biosynthesized SeNPs (100 ppm) demonstrated a stimulatory effect on beneficial soil microorganisms, including P. aeruginosa and B. subtilis. Unlike exhibiting inhibitory effects, the SeNPs enhanced microbial growth compared to untreated controls (Figures 3A,B). These findings suggest that the biosynthesized SeNPs maintain compatibility with essential soil microbiota while potentially promoting their proliferation. A toxicity assessment of biosynthesized SeNPs was conducted on Amaranthus var. Arka Suguna microgreens using four concentrations (100, 250, 500 and 1,000 ppm). While the highest concentration (1,000 ppm) exhibited phytotoxic effects, impairing plant development, lower concentrations (100–500 ppm) showed no adverse effects and instead promoted normal growth (Figure 3C). These results establish a concentration-dependent response to SeNPs, with 1,000 ppm representing the toxicity threshold for this cultivar.

UV-Vis spectroscopic analysis of biosynthesized SeNPs (prepared with 12 mM sodium selenite and 5% C. auriculata leaf extract) revealed characteristic absorbance peaks at 230 nm for all tested samples, including 100 ppm SeNPs, pure selenium powder, and crude leaf extract (Figure 4A). Dynamic light scattering analysis indicated the synthesized nanoparticles had an average size distribution of 337.3 nm with a polydispersity index (PdI) of 0.321 (Figure 4B), demonstrating moderate size variation among the nanoparticles. The particle size measurements, polydispersity index (PDI), and zeta potential were carried out in Milli-Q water as the dispersion medium. Nevertheless, it is established that such physicochemical features are highly affected by the matrix surrounding it, e.g., ionic strength, pH, and the occurrence of macromolecules like root exudates and soil colloids (El-Saadony et al., 2021; Iqbal et al., 2025). Thus, under real application conditions like seed coating, soil correction, or foliar spraying, nanoparticle behavior can differ significantly from that seen in water. Environmental interactions may influence nanoparticle stability, uptake efficiency, and biological efficacy. Future studies should consider evaluating nanoparticles’ size distribution and surface charge directly in the relevant matrices to better correlate their physicochemical properties with plant responses and agronomic outcomes.

FT-IR spectroscopy confirmed the involvement of functional groups from C. auriculata leaf extract in the reduction and stabilization of biosynthesized SeNPs. Comparative analysis of the leaf extract (Figure 4C), sodium selenate (Figure 4D), and biosynthesized SeNPs (Figure 4E) identified key vibrational frequencies, including a broad OH stretch at 3291.09 cm⁻¹ (indicative of aromatic rings and ether-methoxy groups) and C–H asymmetric bending at 1066.03 cm⁻¹ and 92.99 cm⁻¹. The observed redshift in the OH band of SeNPs suggested hydroxyl group interactions with nanoparticle surfaces, a critical factor in their stabilization. Spectral changes, including the retention or disappearance of–OH/COO–peaks, further supported the role of these functional groups in capping and stabilizing the SeNPs. These findings align with reported mechanisms of plant-mediated nanoparticle synthesis and stabilization. The X-ray diffraction (XRD) result reveals that the biosynthesized SeNPs are amorphous in nature (Figures 4F, G). The result matches with the previous reported values (Chen et al., 2009).

TEM analysis revealed spherical selenium nanoparticles (SeNPs) with distinct size distributions: chemically synthesized SeNPs ranged from 100 to 200 nm (Figure 5A), while biosynthesized SeNPs using C. auriculata leaf extract exhibited smaller sizes (80.6–135 nm; Figures 5B,C). EDAX confirmed Se presence in all samples, with characteristic absorption peaks at 0.1–0.5 keV and 10–10.5 keV (Figures 5D–F). The 5 mM Na₂SeO₃-derived SeNPs showed higher peak intensity in the lower energy range than 12 mM Na₂SeO₃ or ascorbic acid-synthesized particles. Size distribution analysis further demonstrated that ascorbic acid-produced SeNPs had the largest mean area (953.7 nm²) and particle size (163.71 nm). At the same time, biosynthesized SeNPs (5 mM and 12 mM Na₂SeO₃) displayed smaller, more uniform dimensions (Figures 5G–L). Copper signals in EDAX spectra originated from the TEM grid substrate.

Se is generally considered a non-essential micronutrient for the plant growth (Zhan et al., 2021). Recent studies highlighted the potential of SeNp to improve germination percentage, growth and stress tolerance in various crops (Abouelhamd et al., 2023; Ghanbari et al., 2023). The study investigated the effects of biogenic synthesized SeNp on the growth attributes of amaranth microgreens (Amaranthus var Arka Suguna) under artificially induced drought conditions with PEG @ 37.5 mM). The combined application method involves seed treatment (ST) and soil application of SeNp at 100 ppm conc. (SA) and foliar application at 25 ppm conc. (FA) had a significant impact on all growth parameters. The combined application (ST+SA+FA) enhanced seed germination to 96.67% under artificial drought stress while also increasing plant height (5.63 cm), fresh weight (332.24 mg) and dry weight (24.14 mg) of harvested amaranth microgreens (Table 1; Figure 6). The seed treatment combined with the soil application (ST+SA) followed the ST+SA+FA treatment in terms of germination percentage, and plant height, showing similar results. However, the ST+SA treatment did not significantly differ in fresh weight (283.87 mg) and dry weight (23.93 mg) compared to the combined application. The lowest germination percentage of 72.50 percent, plant height of 4.43 cm, fresh weight 205.48, and dry weight 14.91 mg was observed in control (Figure 6). The findings of this study align with previous research indicated that biosynthesized SeNPS significantly increases the germination percentage of Amaranth microgreens. Similar growth promotion by SeNp has been reported in carrot plants (Abouelhamd et al., 2023), green beans (Ismail et al., 2023), and potatoes (Perfileva et al., 2023) increased plant height and physiological tolerance (Nagdalian et al., 2023). reported that applying SeNp at 20 mg/L concentration enhances the growth in barley. These results support the role of SeNp in enhancing plant growth under stress conditions (Figures 6A–D).

Application of SeNp significantly increased both fresh and dry weight of amaranth microgreen, which aligns with the findings of de los Santos-Vázquez et al. (2016), who reported that foliar application of Se at 5 mg/L increased the biomass production and vitamin C content in lettuce by overwhelmed stress. Similarly, dry matter production and essential oil content were improved by applying SeNp under drought conditions in Basil (Asghari et al., 2023). The current study findings correlate with these findings and show that the combined application of biosynthesized SeNp (ST+SA+FA) can enhance drought tolerance and biomass production in amaranth microgreens under drought conditions and highlight the potential of SeNp as a sustainable tool for enhancing crop resilience and productivity in stress environments.

The biochemical parameters of amaranth microgreens (Amaranthus var. Arka Suguna) viz., chlorophyll content, carotenoids, total phenols, flavonoids, antioxidants, carbohydrates, vitamin c, soluble proteins, total soluble sugars (TSS), enzymatic activity and Se accumulation, was influenced by biogenic synthesized SeNp. The results obtained in the study revealed that SeNp significantly enhances these biochemical parameters, promoting plant growth and stress tolerance under drought conditions. The photosynthetic efficiency of the crop plants was determined by the accumulation of chlorophyll content in the plant systems. In our study, chlorophyll content in the amaranth microgreen was significantly influenced by the combined application of SeNp (ST+SA+FA), recording the highest chlorophyll A and B (1.03 and 0.24 mg/100 g) content, along with higher carotenoid level (79.7 mg/g) followed by ST+SA (Table 1; Figures 7A–C). All other methods of application of SeNp show statistically similar results at the 5 percent significance level. These results were supported by the previous studies conducted by Abbas (2012), who reported that Se influences Chlorophyll and carotenoid levels by controlling the redox status of leaves. Also, Vijayarengan (2013) and Marisamy et al. (2015) identified that Se application has protective effects on chloroplast enzymes and improves photosynthetic pigments. Additionally, applying selenium at 10 and 5 µM enhanced the photosynthetic pigments in cucumber leaves exposed to salt stress (Hawrylak-Nowak, 2009). The result suggested that applying biosynthesized SeNPs increases photosynthetic pigments of amaranth microgreens.

Combined application of SeNp (ST+SA+FA) recorded the highest phenolic content (6.23 mg/g), while ST+SA+FA also showed increased flavonoid levels (11.4 mg/g). The total antioxidant availability was highest in ST+SA (23.6 mg/g), followed by the combination of ST+SA+FA of SeNp (22.3 mg/g) under drought conditions (Table 2; Figures 8A–C). These results are closely related to the findings of Chu et al. (2009), who observed that Se application reduces ROS and malondialdehyde content while increasing phenolic and flavonoid levels in wheat under cold stress. Similarly, antioxidant activity and robust antioxidant potential of SeNps under both saline and non-saline conditions were reported by Shahraki et al. (2022) and Guleria et al. (2020), implying their role in mitigating oxidative stress and enhancing plant growth and development. Earlier studies demonstrated that selenium nanoparticles (SeNPs) synthesized via green methods possess significant antioxidant activity (Kondaparrthi et al., 2019; Mellinas et al., 2019; Boroumand et al., 2019; Dumore and Mukhopadhyay, 2020). These findings align with the observed enhancement in total phenols, flavonoids, and antioxidant levels in Amaranthus microgreens following treatment with biosynthesized SeNPs (Table 2; Figures 8A–C).

Carbohydrate content was significantly higher in amaranth microgreens treated with ST+SA+FA of SeNp, recording a maximum of 951 mg/100 g, followed by ST+SA of SeNp-treated microgreens (782 mg/100 g) under drought conditions (Table 2; Figure 7G). Consistent with the reports of Alam et al. (2022) and Zhou et al. (2022), selenium nanoparticles were shown to boost growth performance in stressed plants through enhanced carbohydrate metabolism. Likewise, vitamin C content is also higher in amaranth microgreen treated with ST+SA+FA of SeNp (36.7 g/100 g) under drought conditions (Table 2; Figure 8D), in line with the findings of de los Santos-Vázquez et al. (2016), reported that application of SeNp improves antioxidant and vitamin C content in tomatoes and melons. These results suggest that SeNp enhances nutrient accumulation and drought tolerance in amaranth microgreens.

SeNPs and selenite significantly enhance total selenium accumulation and soluble protein content in crops such as soybean sprouts and potato tubers. However, the effects vary by species, in soybeans (Sarwar et al., 2020) leads to an increase in the accumulation of soluble proteins, whereas in soybean sprouts, soluble proteins get reduced (Rao et al., 2022). In this study, biosynthesized SeNPs elevated total soluble proteins in Amaranthus microgreens, with the highest levels in ST+SA+FA (377.2 mg/100 g) and the lowest in the control (65 mg/100 g) (Table 2; Figure 7F). Glucose content was highest in ST+FA (1.13% w/w), comparable to ST (1.10% w/w), while ST+SA+FA and ST+SA showed slightly lower values (1.03% w/w) (Table 2; Figure 7E). Total soluble solids (TSS) peaked in ST+SA+FA and ST+SA (1.33% Brix), followed by ST and ST+FA (1.20% Brix), with the control exhibiting the lowest (0.76% Brix) (Table 2; Figure 7D). These findings align with reports that SeNPs improve sugar accumulation by enhancing stress resilience and metabolic activity, as seen in tomatoes treated with 10 mg/L SeNPs (Hernández-Hernández et al., 2019). The present study confirms that biosynthesized SeNPs boost total soluble sugars in Amaranthus microgreens (Table 2; Figures 7D,E).

Selenium is an essential component of antioxidant enzymes, critically involved in scavenging reactive oxygen and nitrogen species to mitigate oxidative cellular damage. In the current investigation, catalase (CAT) activity reached its maximum in the ST+SA+FA treatment (13.50 U/mg/min), with ST+SA (12.77 U/mg/min) showing comparable results, whereas the control group displayed the lowest activity (10.65 U/mg/min) (Table 2; Figure 9A). A parallel trend was observed for superoxide dismutase (SOD), where ST+SA+FA exhibited the highest activity (0.065 U/mg/min), significantly surpassing the control (0.032 U/mg/min), with intermediate treatments showing no statistically distinct effects (Table 2; Figure 9B). These results corroborate earlier studies highlighting selenium’s capacity to upregulate antioxidant enzymes, enhancing plant resilience under stress. For instance, Jiang et al. (2017) documented elevated SOD activity in selenium-supplemented maize, and Hussein et al. (2019) reported analogous antioxidant enzyme modulation. Further evidence from Wu et al. (2016) and Marslin et al. (2017) underscores selenium’s ability to augment proline accumulation, peroxidase, and glutathione peroxidase (GPX) activity, collectively attenuating oxidative stress and lipid peroxidation. The present findings validate that biosynthesized selenium nanoparticles (SeNPs) markedly enhance CAT and SOD activity in Amaranthus microgreens (Table 2; Figure 9), solidifying selenium’s pivotal role in fortifying antioxidant defense mechanisms in plants.

Selenium is primarily absorbed by plant roots through sulfur transporters and incorporated into organic compounds, with its accumulation in shoots influenced by soil selenium availability and organic matter content (Terry et al., 2000; Arscott and Goldman, 2012). Similar dose-dependent selenium accumulation patterns have been reported in wheat, alfalfa, sunflower (Lintschinger et al., 2000; Lyons et al., 2005), Brassica species (Banuelos et al., 1997), kale (Lefsrud et al., 2006), and onion (Barak and Goldmon, 1997). The selenium content in Amaranthus microgreens was highest in the ST+SA+FA treatment (10.69 mg/g), followed by ST+SA (8.16 mg/g), while the control exhibited minimal accumulation (0.60 mg/g) (Table 2; Figure 10). These findings align with previous studies demonstrating that soil-applied SeNPs significantly enhance selenium uptake in plants, as observed in bok choy, where root concentrations exceeded shoot levels (Ramos et al., 2010).

The current study confirms that biosynthesized SeNPs effectively promote selenium biofortification in Amaranthus microgreens (Table 2; Figure 10), highlighting their potential for enhancing nutritional quality in leafy vegetables.