Biogenic fabrication of zinc oxide nanoparticles for enhanced germination and early seedling establishment of rice under salinity stress

Salinity stress restricts rice (Oryza sativa L.) germination and early establishment through osmotic stress, ionic toxicity, and excessive reactive oxygen species production. Although ZnO nanoparticles (ZnONPs) can enhance salinity tolerance, the influence of their biological synthesis source on nanoparticle properties and efficacy remains poorly understood. ZnONPs were synthesized using leaf extracts of sorghum (S 1), seaweed (S 2), and sugar beet (S 3) and compared with commercial ZnONPs (S 4). The nanoparticles were characterized using UV–Vis spectroscopy, X-ray diffraction, Fourier-transform infrared spectroscopy, high-resolution transmission electron microscopy, field-emission scanning electron microscopy, and zeta-potential measurements. Rice seeds were nano-primed with ZnONPs at 25–100 ppm and evaluated under salinity stress for germination, growth, biomass, water uptake, hydrolytic enzyme activity, antioxidant responses, and lipid peroxidation. All biogenic ZnONPs exhibited characteristic crystalline ZnO structures, nanoscale dimensions, and predominantly spherical morphology. Seaweed-mediated ZnONPs showed superior physicochemical properties, including an average particle size of approximately 23.7 nm, high crystallinity, and enhanced colloidal stability (−24.85 mV). Nano-priming significantly mitigated salinity-induced inhibition of germination, seedling growth, and biomass accumulation. Seaweed-derived ZnONPs at 50–75 ppm produced physiological and biochemical responses comparable to those obtained with commercial ZnONPs at 100 ppm, demonstrating greater efficacy at lower concentrations. These treatments enhanced water uptake, hydrolytic enzyme activity, and the activities of superoxide dismutase, peroxidase, and catalase while reducing lipid peroxidation. Multivariate analyses further distinguished the effective ZnONP treatments based on coordinated improvements in germination, growth, antioxidant defence, and physiological traits. The biological synthesis source significantly influenced the physicochemical properties and functional efficacy of ZnONPs. Seaweed-derived ZnONPs were particularly effective at lower application rates and therefore represent a promising nano-priming strategy for improving early rice establishment under saline conditions.

Rice (Oryza sativa L.) is a major staple food crop that is sensitive to salinity, especially in coastal and irrigated lands, with implications for global food security. Salinity stress is particularly harmful during germination and early seedling establishment, which are critical stages for successful crop development and yield potential. Excessive salt accumulation causes osmotic imbalance, ionic toxicity, impaired nutrient acquisition, and excessive production of reactive oxygen species (ROS), resulting in reduced seed germination, delayed seedling growth, impaired photosynthesis and oxidative damage (Gong et al., 2020). Thus, sustainable strategies for enhancing salinity tolerance during the early stages of rice establishment are essential for maintaining crop productivity under changing environmental conditions (Faizan et al., 2021).

Nanotechnology has attracted considerable attention as a strategy to improve plant resilience to abiotic stresses (Mahawar et al., 2023). Zinc oxide nanoparticles (ZnO NPs) are particularly promising due to their dual role as a source of essential zinc and as regulators of plant physiological and biochemical processes. Zinc contributes to enzymatic activation, photosynthetic reactions and antioxidant defence, including the activity of superoxide dismutase, a key enzyme involved in ROS detoxification (Dogan et al., 2025), (Rodrigues et al., 2024). Seed Priming with ZnO nanoparticles has been reported to enhance nutrient availability, maintain oxidative balance, improve photosynthetic efficiency, and activate stress-responsive metabolism, thereby enhancing germination, seedling growth, biomass accumulation, and stress tolerance (Li et al., 2021), (Adhikary et al., 2022) Other parameters such as particle size and synthesis route, besides the application concentration, also affect the biological performance and safety of the ZnO NPs, as these can alter the surface properties, Zn 2+ release, and interactions with biological systems. For agricultural applications, a crucial question is whether ZnO NPs produced by different biological processes can elicit effective plant responses at lower concentrations without adverse effects.

Concentration- and size-dependent toxicity of conventionally synthesized ZnO NPs has been reported previously. The cytotoxicity of the ZnO NPs in HepG2 cells was concentration- and size-dependent and correlated with Zn 2+ release, oxidative stress, and inflammatory responses (Chen et al., 2019). Moreover, Maheswaran showed that both chemically and green-synthesized ZnO NPs exhibited concentration- and time-dependent cytotoxicity, with the green-synthesized particles showing comparatively higher cell viability and lower cytotoxicity (Maheswaran et al., 2024). The results suggest that the synthesis method can affect the biological activity of the ZnO NPs, and may warrant further investigation of biologically controlled synthesis methods.

While the green synthesis of ZnO NPs has received significant interest, the biological material employed in the fabrication of nanoparticles is still an underexplored factor that affects their physicochemical and functional characteristics. Phytochemicals such as phenolics, flavonoids, proteins, and other bioactive compounds derived from plants and algae can be involved in the processes of nanoparticle formation and stabilization, thus affecting their size, morphology, surface properties, dispersion stability, and biological activity (Singh et al., 2025). Biological resources can yield ZnO NPs with unique physicochemical properties, Zn 2+ release patterns and plant responses, as a result of the different biological environments they offer. However, few studies have systematically compared ZnO NPs synthesized from biologically different sources with commercially available ZnO NPs under similar plant stress conditions. This raises an important question that remains unanswered: Do differences in efficacy and optimum concentration exist between biologically synthesized and commercially available ZnO NPs under salinity-stressed conditions?

It is crucial to address this question, especially since the identification of a biological source that can yield efficient ZnO NPs at lower application concentrations may help to reduce the amount of nanoparticles that must be applied, while providing the same plant response. The beneficial effects of ZnO nanoparticle priming on stress tolerance in plants have been demonstrated in different studies, but comparative source-dependent evaluations remain limited. Numerous earlier investigations have considered a single method of nanoparticle synthesis or compared the effects of the nanoparticles with untreated controls. However, comparisons of nanoparticles synthesized from various biological sources with commercial ZnO NPs remain limited. Thus, the link between the biological source, the physicochemical properties of the nanoparticles, and their functional performance under salinity stress is still not well understood (Kathiravan et al., 2025).

The present study aimed to fill this knowledge gap by systematically comparing the ZnO NPs derived from three different biological resources, namely, sorghum leaf extract, seaweed blade extract, and sugar beet leaf extract, with commercially available ZnO NPs. The study investigated whether source-dependent differences in the physicochemical characteristics were reflected in their effectiveness and optimum concentration for successful rice establishment under salinity stress. The impact of ZnO nanoparticle seed priming on rice germination, seedling growth, biomass accumulation, photosynthetic pigments, antioxidant defence mechanisms, oxidative stress regulation and zinc acquisition under saline conditions was investigated. Moreover, hierarchical cluster analysis and principal component analysis (PCA) were used to identify treatment-specific responses and physiological traits associated with ZnO nanoparticle-mediated salinity tolerance. Most importantly, the study identified that seaweed-derived ZnO NPs were an effective biogenic formulation at 50–75 ppm, resulting in responses comparable to those observed with 100 ppm commercial ZnO NPs. In this context, the study demonstrates that the physicochemical properties and efficacy of the ZnO NPs are influenced by their biological source, and suggests the use of source-optimized biogenic ZnO NPs for effective salinity mitigation at lower application concentrations.

Chemicals and reagents

Analytical-grade zinc acetate dihydrate, sodium hydroxide, sodium chloride, ethanol, and commercial ZnO nanopowder (99.9% purity) were procured from Sigma–Aldrich India Pvt. Ltd. Deionized water was used as the solvent.

Selection and preparation of plant extracts

Three different plant species were used in the production of ZnONPs: sorghum leaves (Sorghum bicolor), sugarbeet leaves (Beta vulgaris), obtained from the campus of the Vellore Institute of Technology, Vellore, Tamil Nadu, and Seaweed blade (Sargassum wightii), collected from AGP Marines Ltd. These biological sources were selected based on their unique and diverse phytochemical compositions. Sorghum is rich in bioactive compounds, such as alkaloids, flavonoids, phenols, tannins, carbohydrates, and proteins (Cervantes-Gaxiola et al., 2024) Seaweed contains bioactive compounds that contribute to the formation and stabilization of nanoparticles (Nagarajan and Arumugam Kuppusamy, 2013) and sugar beet is rich in betacyanins and betaxanthins, which can coordinate with Zn 2+ ions during the formation of nanoparticles (Pillai et al., 2020). This approach allowed the characteristics and biological efficacy of source-dependent ZnO NPs to be compared based on differences in their phytochemical compositions and biological origins. The aerial parts were thoroughly washed, air-dried for 5 days at room temperature and ground to a fine powder. Powdered plant material (5 g) was subjected to extraction in 100 mL of deionized water at 60 °C under reflux. The filtrate obtained was stored at 4 °C for further use. (Figure 1).

Green synthesis of zinc oxide nanoparticles using different leaf extracts

ZnO nanoparticles were synthesized with minor modifications to the method described by (MuthuKathija et al., 2023). A 0.05 M zinc acetate solution was prepared in deionized water, after which the respective aqueous extract was added. The mixture was stirred at 65 °C and left to settle; subsequently, the precipitate was collected by centrifugation, washed repeatedly with deionized water and ethanol, and dried in an oven at 40 °C. The dried sample was subsequently calcined at 450 °C, pulverized, and stored for future characterization (Figure 1). In the biogenic synthesis, the plant extracts provide phytochemicals that interact with the Zn 2+ ions to promote complexation, nucleation and subsequent formation of ZnO nanoparticles. These phytochemicals also serve as capping and stabilizing agents that affect the growth and surface properties of nanoparticles. The proposed formation mechanism is schematically illustrated in Figure 2.

Characterization of ZnO NPs

Following the synthesis of ZnO NPs, several instrumental analyses were performed to characterise their properties (Balogun et al., 2020). Initial visual observations were performed to track colour changes, which indicated successful nanoparticle production. UV-Visible spectroscopy over the range of 200–800 nm (Shimadzu UV-1800) was used to analyze the optical characteristics of ZnONPs (Nayak et al., 2025). The approach outlined by (Bhuvaneshwari et al., 2025) was employed to calculate the band gap energy (Eg) of the nanoparticles, using data obtained from UV-vis diffuse reflectance spectroscopy (DRS) along with the Tauc equation. Fourier Transform Infrared (FTIR) spectroscopy (Shimadzu, Japan; Thermo Fisher Scientific) was performed over a spectral range of 4000–500 cm −1 to determine the specific functional groups and types of bonds present in ZnONPs (Sathyabama and Firdous, 2025) using the IR Affinity-1 and Thermo Nicolet iS50 equipped with ATR. The purity and hexagonal wurtzite structure of ZnO NPs were verified by X-ray diffraction (XRD) analysis using a Bruker D8 Advance instrument, with Cu-Kα source at 30 kV, over a 2θ range of 10°–80° and a scan rate of 0.20°/min. Field emission scanning electron microscopy (FESEM) with energy-dispersive X-ray spectroscopy (EDX) (FEI Quanta 250 FEG, Thermo Fisher Scientific, United States of America) was used to analyze the surface morphology and elemental composition. A thin layer of nanoparticles was applied to carbon-coated grids, and micrographs were taken at 10000x and 25000x magnifications (Bharathy et al., 2025). Transmission electron microscopy (TEM) coupled with selected area electron diffraction (SAED) (FEI Tecnai G2-20 Twin, 200 kV) was also used to further investigate the particle size, morphology and crystallinity. The stability of the colloidal dispersion and surface charge were evaluated using zeta potential analysis. Nanoparticle tracking analysis (NTA) was conducted using a ZetaView PMX 130 instrument (Particle Metrix, mono laser), with particle size measurements performed at 90° and 173°. (Kumar Kurdekar et al., 2024).

Seed germination experiment

The germination study was conducted using the paper towel method as stipulated by the International Seed Testing Association (ISTA) (Kumar et al., 2025). Rice seeds (CO-51), a salt-sensitive variety, were sourced from the Agriculture Department Office, Katpadi Block, Vellore, Tamil Nadu. The seeds underwent surface sterilization with 70% ethanol followed by a 2 min treatment with 1% (v/v) sodium hypochlorite, and were subsequently washed with deionized water (Bharati et al., 2023), (Gamit et al., 2025). A seed-to-solution ratio of 1:2 was maintained during priming. The rice seeds were soaked in the respective ZnO nanoparticle solutions and incubated on a rotary shaker at 120 rpm for 24 h. Following priming, the seeds were air-dried to reach a moisture content close to their original state before initiating germination (Bicalho and Ferreira, 2026). For the germination experiment, 25 seeds were placed between two slightly damp corrugated brown paper towels moistened with a 100 mM NaCl solution. A preliminary screening experiment was carried out, and a concentration of 100 mM NaCl was chosen. Control towels were moistened with distilled water. The towels were then rolled, wrapped in butter paper, and placed in a seed incubator set at 25 °C ± 2 °C and 75% ± 5% relative humidity. Germination was recorded daily based on radicle protrusion.

Optimization of nanoparticle concentrations for seed priming

The concentrations of ZnO NPs were determined according to the effective concentration range reported for seed priming and stress mitigation using ZnO NPs in rice (Gamit et al., 2025; Singh et al., 2022). The nanoparticles were suspended in double-distilled water and diluted to working solutions of 25, 50, 75 and 100 ppm. The solutions were then treated with probe ultrasonication at 40 kHz for 20 min before use.

Germination parameters

Speed of germination (SOG)

The speed of germination was determined in line with (Maguire, 1962)

Where, X 1 – count of seeds germinated at first assessment.

X 2 – count of seeds emerged at the second assessment.

X n - count of seeds germinated at the n th assessment.

Y 1 - number of days from sowing to the first count.

Y 2 - number of days from sowing to the second count.

Y n - number of days from sowing to n th count.

Seedling growth parameters

After 14 days, ten healthy seedlings were randomly selected from each experimental replicate for measurement. Primary root length, shoot length and total seedling length were recorded and reported in centimetres (cm). The selected seedlings were subjected to shade-drying and subsequently oven-dried at 50 °C for 2 h. The dry biomass of ten seedlings from each group was measured and reported in milligrams.

Estimation of seedling vigour index I and II

Seedling vigour indices were determined following the method described by (Abdul‐Baki and Anderson, 1973).

Vigour index I = Germination (%) × Total seedling length (cm).

Vigour index II = Germination (%) × Dry matter production (mg).

Photosynthetic pigments

The photosynthetic pigments from fresh leaves were analyzed by extracting them using ethanol and measuring their absorbance at 663 nm and 645 nm using a spectrophotometer. The levels of total chlorophyll, chlorophyll a and b were estimated according to (Arnon, 1949) and expressed as mg g −1 FW.

Lipid peroxidation (MDA content)

Membrane lipid peroxidation was measured as malondialdehyde (MDA) concentration, an indicator of oxidative stress. The fresh leaf samples were homogenized in trichloroacetic acid and thiobarbituric acid, and the absorbances were recorded at 532 nm and 600 nm. MDA content was estimated following the method of (Heath and Packer, 1968).

Antioxidant enzyme activity

The antioxidant enzymes were extracted in phosphate buffer, and their activities were measured spectrophotometrically using established protocols. Catalase activity (CAT) was determined based on the change in absorbance at 240 nm due to H 2 O 2 degradation (Lück, 1965), and expressed as U g −1 protein. Guaiacol peroxidase (POD) activity was determined by monitoring the increase in absorbance at 420 nm due to guaiacol oxidation (Maehly and Chance, 1954). SOD activity was determined by its ability to inhibit NBT reduction at 560 nm (Giannopolitis and Ries, 1977), where 1 unit corresponds to 50% inhibition of NBT reduction.

Non-enzymatic antioxidants

Total phenolic content was determined using the Folin–Ciocalteu method (Singleton and Rossi, 1965) and expressed as mg GAE g −1 FW. Total flavonoid content, was determined using the aluminium chloride colourimetric method (Chang et al., 2002), and the results were expressed as mg of quercetin equivalents (QE) g −1 FW. The total antioxidant activity was estimated by the FRAP assay (Benzi et al., 1996), and the results were presented as µmol Trolox equivalents (TE) g −1 FW.

Zinc profiling of rice seedlings

The zinc content in rice seedlings was determined by inductively coupled plasma mass spectrometry (ICP-MS; NexION 1000, PerkinElmer) following the standard analytical protocol, with results reported on a dry-weight basis. The seedlings were oven-dried, finely ground, and digested with a mixture of nitric and perchloric acids, then diluted to the required volume.

Salinity tolerance index

The Salt Tolerance Index (STI) was estimated using the equation (Ashraf et al., 2025). where,

T salt - average dry mass at optimum salinity concentration.

T control - mean dry weight of the control treatment.

Statistical analysis and multivariate analysis

The experiment was conducted in a Completely Randomized Design (CRD) with three independent replications. Data from the 11 treatments (Figure 3) (Table 1) were analyzed using analysis of variance (ANOVA) in R software (version 4.2.1; R Core Team, 2022). When significant treatment effects were detected, differences between treatment means were identified using the least significant difference (LSD) test at p < 0.05. Data are reported as the mean ± standard deviation (SD) of three independent replications. Means followed by different lowercase letters are significantly different between treatments at p < 0.05.

For multivariate analysis, hierarchical clustering and principal component analysis were performed on the germination, growth, and physiological attributes of rice seedlings. All experimental variables were normalized by Z-score transformation. Hierarchical clustering was performed using Euclidean distance and Ward’s linkage method, and the heatmap and dendrogram were generated using the pheatmap package in RStudio. The PCA was performed with the FactoMineR package in RStudio with scaled variables, and the first two principal components were selected based on the proportion of total variance explained. The variables’ contributions to PC1 and PC2 were calculated using the factoextra package, and scree plots were generated to display the percentage of variance explained by each principal component. PCA biplots were used to visualize relationships between treatments and physiological traits.

Characterization results

Plant extracts are environmentally friendly reducing and stabilizing agents for the synthesis of NPs, serving as an alternative to conventional physicochemical methods. X-ray diffraction (XRD) and UV-Visible spectroscopy were used to analyze the structural and optical properties. In addition, the surface functional groups, particle morphology and stability of the colloidal suspension were investigated using FTIR, FE-SEM and zeta potential analysis.

Visual observation and yield of nanoparticles

A clear zinc acetate solution was prepared by dissolving the salt in distilled water, followed by its addition to reddish-brown sorghum leaf extract, dark brown seaweed extract, and dark brown to black sugarbeet leaf extract, resulting in colour changes to pale yellow, light brown and beige, respectively, as shown in Figure 4 and Table 2. The gradual colour change indicates the formation of ZnONPs (Kamarajan et al., 2022; Bizuayehu et al., 2025). The extracts exhibited a characteristic brown colour, indicative of phytochemicals that contributed to nanoparticle formation. Finally, the ZnO NPs were observed as fine white solids after calcination. The yields of ZnO-NPs obtained from the different extracts varied, with sugarbeet extract (S 3) yielding the highest amount (398.4 mg) followed by sorghum (283.4 mg) and seaweed (281.6 mg). The comparatively higher yield obtained from S 3 could be related to differences in the composition and availability of phytochemicals involved in the complexation of Zn 2+ and formation of ZnO, as well as the higher extract recovery (45 mL). However, yield was not considered when selecting optimized formulation. Moreover, the plant compounds can cap the nanoparticles, thereby inhibiting agglomeration and helping regulate their size and morphology. Bioactive compounds in the three extracts contributed to the formation of ZnO nanoparticles, which aligns with similar studies (Fouda et al., 2023); (N et al., 2024).

UV–vis spectroscopy

The synthesis of ZnO NPs was verified by UV-Vis spectroscopic analysis to determine their optical absorption properties within a wavelength range of 200–700 nm. The absorption spectrum exhibited strong peaks at 357, 364, 359, and 355 nm corresponding to estimated band gap energies of 3.11 eV, 3.19 eV, 3.16 eV, and 3.48 eV, respectively, indicating the formation of ZnO NPs. The slight difference between the absorption maxima of the different samples may be explained by differences in the phytochemical composition of the extracts. which can affect nanoparticle growth, particle size, surface chemistry and structural characteristics (Bhattacharjee et al., 2021). The absorbance values declined steadily with increasing wavelength (Figure 5). The lack of additional absorbance bands in the spectra further supports the formation of phase-pure ZnO nanoparticles. The results align with those reported by (Hayat et al., 2025), who found that extracts of Peganum harmala seeds and Foeniculum officinale shoots exhibited characteristic absorption in the wavelength range of 200–400 nm. Similar studies have reported a peak absorption between 350 and 400 nm, further supporting the presence of ZnO NPs (Jan et al., 2022; Iqbal et al., 2025; Alam et al., 2025; Loganathan et al., 2025).

The direct optical band-gap energy (Eg) was estimated using the numerical derivative of the optical absorption coefficient. The direct transition model was applied to demonstrate the relationship between the photon energy and the absorption coefficient (Eswari et al., 2022) and the relationship between band-gap energy and other optical parameters is illustrated in Figure 6, which is in tandem with the studies of (Demissie et al., 2020) and (Eswari et al., 2022). The optical band gap energies (3.11–3.19 eV) of the ZnO nanoparticles produced using green methods were slightly lower than that of the commercial ZnONP (3.48 eV). Such a red shift can frequently be attributed to phytochemical capping, surface defects and distortions in crystallinity associated with green synthesis process. The higher band gap energy observed for commercial ZnO NP may be associated with the quantum-confinement effect, which is applicable to its smaller crystallite size (S 4; 13.72 nm, 3.48 eV). In the case of biogenic ZnO NPs, this relationship was not monotonic, with the crystallite size of S 3 (27.44 nm) being larger than that of S 1 (23.62 nm), whereas its band gap (3.16 eV) was higher than that of S 1 (3.11 eV). This deviation from the expected size-dependent trend could be related to the differences in surface defects, surface chemistry, and other synthesis-related factors, indicating that crystallite size is not the sole dominant factor determining the optical properties of the biogenic ZnO NPs (Flores et al., 2025). Reduced band-gap energies may indicate changes in crystallinity, defect density, and surface chemistry, and may influence the functional characteristics of ZnO nanoparticles for applications such as photocatalysis and antimicrobial activity.

XRD

The crystalline nature of ZNO NPs was determined by XRD analysis of the different samples. The diffraction patterns displayed distinct peaks within the 2θ range of 20°–80°, with characteristic peaks at approximately 31.7°, 34.4°, 36.2°, 47.5°, 56.6°, 62.8°, and 68.0°, which are attributed to specific (hkl) planes of hexagonal wurtzite ZnO, including (100) (002) (101) (102) (110) (103), and (112). The existence of sharp, well-defined diffraction peaks and the lack of observable secondary reflections provide evidence that the obtained ZnO nanoparticles are highly crystalline and phase-pure. All observed diffraction peaks are consistent with the standard JCPDS card No. 89–1397, further supporting the phase purity of the ZnO structure.

The crystallite size of ZnO NPs was estimated using the Debye–Scherrer equation, D = Kλ/βcosθ, where β represents the FWHM of the (101) diffraction peak at 36.2°. The measured crystallite sizes for ZnO NPs derived from sorghum leaf extract, seaweed, sugarbeet, and commercial ZnO nanoparticles were 23.62 nm, 20.07 nm, 27.44 nm, and 13.72 nm, respectively (Figure 7). The size differences among the samples may be associated with differences in the phytochemical composition of the representative extracts and their influence on nucleation and crystal growth (Bizuayehu et al., 2025; Iqbal et al., 2025; Sehrawat et al., 2025; Albrahim et al., 2025).

FTIR

FT-IR spectral analysis of the three leaf extracts and synthesized ZnO nanoparticles revealed the presence of functional groups such as flavonoids, phenols, and aliphatic (–CH) groups (Adamu et al., 2024) (Figure 8). The extracts exhibited a diffuse absorption band ranging from 3360 to 3575 cm −1. These bands were attributed to O–H stretching vibrations corresponding to hydroxyl groups in flavonoids and polyphenols. Moreover, absorption bands in the mid-region (1520–1580 cm −1) were attributed to aromatic C=C stretching vibrations and amide II vibrations associated with protein/amine groups, which may act as reducing and stabilizing agents.

The FTIR spectra of the ZnO nanoparticles exhibited C–N/C–O characteristics (1040–1130 and 1320–1340 cm −1) that may originate from biomolecules attached to the nanoparticle surfaces. The peaks observed in the region of (600–800 cm −1) are assigned to Zn–O stretching modes, confirming successful ZnO nanoparticle formation (Figure 9). These studies align with previous studies (Loganathan et al., 2025; Kokabi and Ebrahimi, 2020; Wijaya et al., 2024). Compared with the commercial sample, the green ZnO samples exhibit more pronounced biomolecular bands, indicating a greater organic capping associated with the plant extracts. Some peaks identified in the aqueous extract decreased after nanoparticle formation, indicating that nanoparticle synthesis altered the corresponding functional groups. Additionally, several new peaks emerged in the ZnO nanoparticles, indicating the possible adsorption of phytochemicals onto their surfaces.

HR-TEM with SAED

Morphology, particle size distribution, crystallinity and the lattice structure of the synthesized ZnO nanoparticles were analyzed using HR-TEM, particle size histogram, and SAED analysis (Figure 10). The TEM images revealed that all samples consisted of well-defined nanoparticles with slight agglomeration, which is commonly observed for ZnO nanoparticles owing to their high surface energy. The commercial ZnO nanoparticles (S 4) showed a relatively smaller size and more uniform distribution of particles compared to the biosynthesized ZnO nanoparticles (S 1 –S 3), possibly due to greater control over nucleation and growth during industrial synthesis. The green-synthesized ZnO nanoparticles exhibited mild agglomeration and slightly larger particle sizes, which could be due to the presence of phytochemicals acting as natural reducing agents and capping agents during their preparation (Nhu et al., 2022).

The particle size histogram also confirmed the nanoscale size of all samples. For the bio synthesized nanoparticles, S 2 (seaweed extract) exhibited a comparatively narrower particle size distribution than S 1 and S 3, which can be attributed to the more uniform nucleation and crystal growth. The relatively wider size distribution of S 1 and S 3 could be due to variations in the concentration of phenolic compounds, flavonoids, proteins, and polysaccharides in the extracts of the respective biological sources, which may influence crystal growth and stabilization during the green synthesis (Nayak et al., 2026).

The SAED patterns of all the samples showed a set of clear diffraction spots and concentric diffraction rings, indicating the polycrystalline nature of the nanoparticles. The diffraction rings were indexed according to the characteristic planes of the hexagonal wurtzite ZnO crystal structure, and the XRD results agreed with the standard crystallographic data. The observation of similar SAED patterns indicating the high crystallinity of green synthesized ZnO nanoparticles has been reported earlier (Mohanasundaram and Saral, 2025).

The HRTEM images showed well-defined lattice fringes, indicating that the nanoparticles were well - crystallized. The measured interplanar spacings were 0.281 nm (S 1), 0.248 nm (S 2) and 0.260 nm (S 3), which are assigned to the (101) (100) and (002) planes of hexagonal wurtzite ZnO, respectively. The measured lattice spacing values were in close agreement with the corresponding standard crystallographic values, confirming the formation of crystalline ZnO nanoparticles from sorghum leaf, seaweed blade and sugarbeet leaf extracts.

FE-SEM with EDX

As seen in the FE-SEM micrographs (Figure 11), the biosynthesized ZnO NPs exhibited a mixture of quasi-spherical and rod-shaped morphologies. These morphological characteristics, including irregular shapes and moderate aggregation, are commonly observed in green-synthesized ZnO nanoparticles. The observed morphological variations may influence the surface properties and interaction of nanoparticles with plant tissues. The particle sizes measured using ImageJ were: S 1 (Sorghum) at 29.45 nm, S 2 (Seaweed) at 27.30 nm, S 3 (Sugarbeet) at 28.57 nm, and S 4 (Commercial) at 21.10 nm (Figure 12). The smaller size of the commercial ZnONPs (S 4) may be attributed to the greater control over nucleation and crystal growth during industrial synthesis, resulting in reduced size distributions. Comparatively, green-synthesized nanoparticles are slightly larger, possibly because of biomolecular capping layers and aggregation associated with phytochemicals.

The elemental analysis of the biosynthesized ZnO NPs was determined using the EDX analysis. The characteristic peaks of zinc and oxygen confirmed the formation of ZnO. The elemental analysis showed that S 1 had 66.8 wt% zinc and 33.2 wt% oxygen, S 2 had 73.6 wt% zinc and 26.4 wt% oxygen, S 3 had 51.7 wt% zinc and 48.3 wt% oxygen, and S 4 had 69.3 wt% zinc and 30.7 wt% oxygen, respectively (Figure 13). The slight variation in the Zn/O ratios among the samples can be explained by the differences in surface chemistry, particle morphology, or residual organic compounds associated with the green synthesis approach. The emission peaks of Zn were observed at approximately 1 keV, 8.6 keV and 9.6 keV across all four samples. These findings align with previous reports (Barzinjy and Azeez, 2020; Singh et al., 2019; Tiwari et al., 2024; Faisal et al., 2021; Jayachandran et al., 2021).

Zeta potential and particle size analysis

The surface charges of ZnONPs were assessed by zeta potential (ZP) analysis. The ZP values of the NPs derived from different biological extracts (Samples 1–3) and the commercial ZnO (S 4) are shown in Figure 14. The ZnO NPs prepared using biogenic synthesis exhibited negative surface charges of −12.58 mV (S 1), −24.85 mV (S 2), and −17.52 mV (S 3). Sample 2 (ZnO NP derived from seaweed) exhibited the most negative zeta potential (−24.85 mV), suggesting comparatively greater colloidal stability due to increased electrostatic repulsion and reduced aggregation in aqueous suspension (Abdelbaky et al., 2023); (Faisal et al., 2021; Alsolmi et al., 2025). The presence of polysaccharides, sulfated metabolites, and phenolic compounds in seaweed extracts may provide anionic functional groups (-COO-, -SO3-, -O-), which contribute to surface stabilization and enhanced dispersion stability. In contrast, the commercial ZnO nanoparticle exhibited a high positive zeta potential of +40.3 mV, indicating high colloidal stability. The positive charge may be associated with surface protonation of ZnO -OH groups, residual Zn 2+ ions, and cationic stabilizers or surfactants that are commonly utilized in industrial applications.

Particle size analysis revealed a multimodal size distribution with significant peaks shown in Figure 15. The mean diameters of the ZnO NPs were S 1 – 138.2 nm, S 2 – 111.9 nm, S 3 – 160.3 nm, and commercial ZnO nanoparticles S 4 – 95.6 nm. This apparent increase in hydrodynamic particle size can be influenced by aggregation and the presence of surface-associated biomolecules. The larger particle size observed by NTA compared with FE-SEM may be explained by the natural tendency of ZnO nanoparticles to form clusters in aqueous environments. The larger size of the ZnO nanoparticles is attributed to polydispersity and the tendency of NTA to detect larger particles or aggregates, compared with FESEM and HRTEM analyses (Fouda et al., 2023).

Effects of ZnONP seed priming on germination performance, physiological parameters, zinc accumulation, and salinity tolerance indices in rice

The initial stages of plant development, such as germination and seedling establishment, are crucial for plant performance under salinity stress. In this research, ZnO nanoparticles obtained from different biological sources exhibited distinct physicochemical characteristics that significantly impacted their biological activity. Specifically, the seaweed-derived ZnO nanoparticles demonstrated superior performance due to their smaller particle size, enhanced colloidal stability, and phytochemical surface capping, which may enhance dispersion, bioavailability, and interaction with seed tissues. These results underscore the importance of nanoparticle source and surface chemistry in determining their functional efficiency within plant systems.

The present study showed that seedling growth was significantly impaired by salt stress (100 mM NaCl), resulting in reductions in the germination rate, seedling length, and seed vigour index I (SVI-I) by 33%, 46%, and 64%, respectively, compared with the control. Hydropriming provided some relief from saline stress, increasing the germination rate and seedling length by approximately 58% and the SVI-I by around 2.5 times compared with the saline stress condition. In contrast, seed priming with ZnONPs markedly improved all measured agronomic traits, with effects dependent on concentration and dose. Among the synthesized ZnO NPs (50 ppm) was found to be the most effective, resulting in 100% germination, a 96% increase in seedling length, a 291% increase in SVI-I, a 94% increase in shoot fresh weight, and an 80% boost in dry matter yield compared to saline stress. The use of commercial ZnO nanoparticles at 100 ppm resulted in the greatest improvement, achieving 100% germination, a 109% increase in seedling length, a 319% increase in SVI-I, and a 95% increase in dry matter production compared to saline stress. Both the green-synthesized and commercial ZnO nanoparticles significantly outperformed hydropriming. The green ZnO nanoparticles at 50 ppm and 75 ppm were observed to enhance SVI-I, seedling length, and shoot fresh weight by 56%, 24%, and 15%, respectively, compared with hydroprimed seeds. Meanwhile, the commercial ZnO nanoparticles at 100 ppm increased SVI-I and seedling length by 67% and 31%, respectively (Figure 16). The effect of different doses of ZnO NPs on plant growth presents a typical U-shaped pattern, with the positive effects seen at lower concentrations and potentially inhibitory effects at higher concentrations (Sorahinobar et al., 2023). The reduced activity of the green ZnO nanoparticles (100 ppm) at the elevated concentrations indicates possible dose-dependent aggregation or phytotoxicity.

Priming with nanomaterials has been reported to improve seed germination by facilitating rapid water absorption through the seed coat. This improvement may be associated with the small size of ZnO nanoparticles, which may facilitate pore formation and enhance cellular hydration, stabilize ROS levels, and promote water transport through aquaporins, thereby accelerating metabolic reactivation and germination (Figure 17) (Prerna et al., 2020). The observed improvements may also be linked to the role of zinc in enzyme activation and metabolic processes during seed germination. The reported effects of ZnO nanopriming on germination and seedling vigour and antioxidant response may be beneficial for the early establishment of seedlings (Kumar et al., 2025). In rice, ZnO nanopriming has also been reported to improve water uptake and α-amylase activity, which plays an important role in starch mobilization during germination (Sharma et al., 2021). Higher α-amylase activity can promote the hydrolysis of stored starch into soluble sugars, providing energy for embryo growth and enhancing germination performance.

The current research is similar to previous reports on Wheat (El-Shazoly et al., 2025); tomato (Kumar et al., 2025); Rice (Gamit et al., 2025; Iqbal et al., 2025; Sharma et al., 2021; Mishra et al., 2025; Ahmad et al., 2025); Amaranthus (Geremew et al., 2025); Phaseolus vulgaris (Gupta et al., 2024); pigeon pea (Kurdekar et al., 2025); Maize (Bhuvaneshwari et al., 2025; Ashraf et al., 2025).

Salinity strongly influenced chlorophyll content in rice seedlings, with total chlorophyll levels decreasing to 0.86 mg g −1 FW under 100 mM salt stress. Hydropriming provided a partial improvement, increasing the content to 1.49 mg g −1 fresh weight. Conversely, photosynthetic pigments increased significantly under salt stress when seeds were primed with ZnO nanoparticles. Green-synthesized ZnO NPs at concentrations of 50 and 75 ppm resulted in the highest pigment accumulation, achieving total chlorophyll levels of 2.57 and 2.79 mg g −1 FW, respectively, while commercial ZnO nanoparticles at 100 ppm increased total chlorophyll to 2.63 mg g −1 FW. The concurrent rise in chlorophyll a and b suggests that improved maintenance of photosynthetic pigments, which may be attributed to the role of zinc in supporting chlorophyll biosynthesis and photosystem function, as well as protecting photosynthetic tissues from oxidative stress. Similar effects have been reported in rice and other crops (Singh et al., 2022; Yasmin et al., 2020; Alabdallah and Alzahrani, 2020).

Lipid peroxidation levels rose significantly in rice seedlings subjected to salinity, with MDA concentrations increasing to 13.23 nmol g −1 FW, compared to 10.97 nmol g −1 FW in the control group. Under saline conditions, hydropriming resulted in a moderate reduction in MDA levels to 10.32 nmol g −1 FW. Seed priming with ZnO nanoparticles further reduced lipid peroxidation, with the green-synthesized ZnO NPs demonstrating the greatest reduction in oxidative damage at concentrations of 50 ppm (7.42 nmol g −1 FW) and 75 ppm (8.06 nmol g −1 FW). Commercially prepared ZnO NPs were also effective in reducing MDA levels to a minimum of 7.74 nmol g −1 FW at 100 ppm. Lower MDA levels reflect reduced membrane lipid peroxidation and may indicate better preservation of membrane integrity under salinity stress. This response was accompanied by the upregulation of antioxidant enzyme activities (SOD, POD, CAT), indicating improved antioxidant defence and oxidative stress regulation following ZnO NP priming (Singh et al., 2022; Zeghoud et al., 2022). Overall, MDA levels provide a useful biochemical indicator of membrane protection against salinity-induced oxidative damage.

The antioxidant enzyme activity of SOD, POD, and CAT was significantly decreased by salinity stress (100 mM NaCl), with reductions of 51.1%, 33.3%, and 38.5%, respectively compared to the control. Hydropriming partially alleviated these reductions, resulting in increases of 13.5%, 20.8%, and 20.8% in SOD, POD, and CAT, respectively, relative to the control. Furthermore, green-synthesized ZnO nanoparticles at concentrations of 50 and 75 ppm significantly increased SOD to 224–227 U g −1 protein, POD to 36–40 U g −1 protein, and CAT to 22–24 U g −1 protein, exceeding the enzyme activities observed under salinity stress. The application of 100 ppm commercial ZnO nanoparticles resulted in significant antioxidant activities, with SOD measuring 229 units U g −1 protein, POD at 41 U g −1 protein, and CAT at 25 U g −1 protein. These findings suggest that ZnO NP priming under salinity stress led to enhanced antioxidant defence and improved management of oxidative stress (Figure 18) (Sharma et al., 2021).

Non-enzymatic antioxidants were significantly decreased by salinity stress (100 mM NaCl). Phenols, flavonoids, and total antioxidants were reduced by approximately 64.9%, 14.8%, and 21.1%, respectively, as compared with the control. Notably, the antioxidant levels were significantly increased by nanopriming with green synthesized ZnO nanoparticles. The highest values of phenols were 8.09 mg GAE g −1 FW at 75 ppm, flavonoids were 4.86 mg QE g −1 FW at 50 ppm, and antioxidants were 1.89 μmol TE g −1 FW at 50 ppm, which exceeded both the control and salinity treatments. Likewise, commercial ZnO nanoparticles had a significant effect, with the highest values at 100 ppm: phenols at 8.03 mg GAE g −1 FW, flavonoids at 4.89 mg QE g −1 FW, and antioxidants at 1.83 μmol TE g −1 FW. The application of ZnO nanoparticles through seed nano-priming significantly increased non-enzymatic antioxidant levels under salinity stress, particularly phenolics and flavonoids, which contribute to cellular protection. The higher levels of phenolic compounds in seedlings treated with ZnO NPs suggest enhanced antioxidant capacity, which may contribute to improved protection against oxidative damage under saline conditions. These results corroborate the findings of (Hossain et al., 2022), who reported that polyphenols (notably phenolics and flavonoids) play a crucial role in mitigating oxidative damage caused by abiotic stress. ZnO nanoparticles may enhance phenolic metabolism by influencing antioxidant-related biochemical pathways, thereby contributing to redox homeostasis and plant resilience under stress. In summary, the increase in non-enzymatic antioxidant levels following ZnO nanoparticle treatment indicates enhanced antioxidant defence and improved oxidative stress regulation in rice under salinity stress. These responses were more pronounced following ZnO nanoparticle-mediated nano-priming than conventional hydropriming, highlighting the greater potential of ZnO NPs for enhancing antioxidant defence under salinity stress.

Under salinity stress (100 mM NaCl), the zinc content in rice seedlings decreased by 29.0% compared with the control, while hydropriming increased the zinc content by 59.1% compared with salinity stress alone. The green synthesized ZnO nanoparticles resulted in Zn accumulation of 59, 63, 65 and 51 at application concentrations of 25, 50, 75 and 100 ppm respectively. The maximum Zn accumulation was recorded at 75 ppm, while a decrease was noted at 100 ppm. In contrast, Zn uptake by the commercial ZnO nanoparticles was 54, 59, 66 and 69 ppm at 25, 50, 75 and 100 ppm, respectively, reflecting a dose-dependent trend in Zn uptake (Figure 19). These findings indicate that ZnO nano priming is an effective strategy to enhance zinc bioaccumulation in saline environments, although efficacy appears to be restricted at higher concentrations, likely due to a threshold limit on zinc uptake and potential toxicity. The notable increase in Zn accumulation may be associated with the smaller particle size and the high surface area of ZnO nanomaterials, which enhance absorption, mobility, and internal distribution of Zn (Sharma et al., 2021; Itroutwar et al., 2020). Moreover, the uptake and translocation of Zn are regulated by families of Zn transporters, including ZIP, NRAMP, and YSL, and their activity can be increased under nano-priming conditions (Afzal et al., 2020). Recent studies have reported similar responses to nano-enabled biofortification, in which ZnO NP priming increased the Zn content in lettuce leaves by 3.2–12.6-fold (Francisco et al., 2024). The overall findings indicate that seed priming with ZnO nanoparticles can stimulate zinc uptake and enhance germination and seedling development, underscoring its importance for nano-biofortification and the development of stress-resistant crops.

The salinity tolerance index (STI) under salt stress decreased to 79%, indicating significant stress damage. Hydropriming improved the STI by 41.8% compared with salinity stress alone. Green-synthesized ZnO nanoparticles significantly increased STI by 62.0%, 78.5%, and 86.0% at concentrations of 25, 50, and 75 ppm, respectively, although a decrease was observed at 100 ppm (53.2%). Commercially available ZnO nanoparticles showed progressive and improved salinity stress tolerance (STI) with elevated concentrations, yielded improvements in salinity stress tolerance of 69.6%, 68.4%, 82.3%, and 93.7%, respectively (Figure 19). The increase in STI is indicative of greater physiological ability to survive in saline conditions, which is consistent with the research that points out the importance of ZnO nanoparticles in enhancing antioxidant activity, ionic homeostasis, and recovery during salt stress.

Although commercial ZnONPs are effective in stimulating seedling growth, their application generally requires higher doses and is more expensive than biosynthesized ZnONPs, which leverage phytochemicals to enhance bioavailability and, hence, lower doses are typically required to achieve similar or even better results. This method aligns with green chemistry principles by reducing the use of harmful substances typically involved in nanoparticle production. In summary, salinity stress significantly affects the growth of rice seedlings. Nonetheless, treating seeds with 50 ppm and 75 ppm of green-synthesised ZnO nanoparticles, alongside commercial ZnO at 100 ppm, effectively mitigates the impact of saline conditions and boosts seedling biomass and vigour, suggesting that ZnO nano-priming is a viable, long-term strategy for developing crops tolerant to stress. The statistical validation of the combined effects of salinity stress with nanoparticles across all assessed parameters underscores the potential of nano priming to enhance seedling performance in environments with abiotic stress.

Hierarchical clustered heat map analysis of seed germination, growth and physiological responses under salinity stress

The hierarchical cluster analysis differentiated the treatment-dependent response of rice seedlings under salinity stress and ZnO nanoparticle priming (Figure 20). The untreated saline stress treatment (T 2: 100 mM NaCl) formed a distinct cluster with predominantly negative responses across germination, growth and physiological parameters, suggesting a very strong negative response to NaCl-induced osmotic and ionic stress and oxidative damage. Under prolonged saline conditions, hydropriming (T 3) led to partial recovery relative to T 2, reflecting improved germination and early seedling establishment.

The overall physiological performance of rice seedlings was substantially improved by green-synthesised ZnONps compared with commercial ZnO nanoparticles. T 6 showed the highest positive clustering pattern, followed by T 5 and T 10 with improvements in germination, seedling growth and physiological attributes. The improved response at 75 ppm suggests an effective ZnO nanoparticle concentration for promoting stress tolerance, whereas higher concentrations (T 7 and T 11: 100 ppm) resulted in comparatively reduced benefits, indicating a possible concentration-dependent limitation.

This improved performance of the ZnO nanoparticle-primed seedlings may be associated with enhanced Zn accumulation, maintenance of photosynthetic pigments and increased antioxidant defence, which collectively may contribute to reduced oxidative damage under salinity. The results align with those of (Guo et al., 2024), who showed that ZnO nanoparticles enhanced rice physiological resilience under abiotic stress by maintaining the photosynthetic performance and promoting plant growth. Likewise, El-Shazoly (El-Shazoly et al., 2025) found that ZnO seed priming positively affected antioxidant activity, osmoprotection and the maintenance of physiological functions under drought stress. The findings of both studies support the potential role of ZnO nanoparticles in strengthening plant defence responses against abiotic stress-induced cellular damage, despite the different stress conditions examined.

Green synthesized ZnO nanoparticles at 75 ppm showed the best response, which may be attributed to phytochemical capping molecules associated with the biological synthesis process that may enhance nanoparticle stability, uptake and biocompatibility. In contrast, the lower response at 100 ppm indicates that higher ZnO nanoparticle concentrations may impose concentration-dependent limitations on physiological performance and cellular homeostasis. Overall, the heat map analysis indicated that T 6 (green-synthesized ZnO NPs @ 75 ppm) was the most effective treatment to overcome the salinity-induced growth inhibition and physiological disruption, highlighting the potential of optimized green ZnO nanopriming as an efficient strategy for improving salt tolerance in rice seedlings.

Principal component analysis of rice seedlings subjected to nanopriming of ZnONps under salinity stress

Principal component analysis (PCA) was carried out to understand the relationships among germination, growth and physiological traits and to discriminate the response pattern of rice seedlings under salinity stress and nano priming with ZnONps. The total variance explained by the first two principal components was 94.2%, with PC1 accounting for 84.9% and PC2 for 9.3%, suggesting that the chosen traits captured the overall variance due to salinity and the application of ZnO nanoparticles. The scree plot verified the PC1 dominance, indicating that most of the variation depending on treatment was related to physiological recovery and stress mitigation responses (Figure 21).

PCA biplot showed a distinct separation between salt-stressed control (T 2: 100 mM NaCl) and ZnO nanoprimed seedlings. Under salinity stress, T 2 was positioned in the negative direction along PC1 and was highly associated with malondialdehyde (MDA), which was a sign of increased membrane lipid peroxidation and oxidative damage under salinity stress. On the other hand, the treatments containing ZnO nanoparticles were primarily distributed along the positive PC1 axis and were more related with the growth and physiological parameters such as chlorophyll content (Chl a, Chl b, Total Chl), antioxidant enzymes (SOD, CAT, POD), seedling vigor index (SVI), GP %, root length (RL), shoot length (SL) and biomass related parameters.

The treatment T 6 exhibited a strong positive correlation with all the physiological traits, indicating better alleviation from salinity stress-induced damage. The grouping of the T 6 around the chlorophyll, antioxidant and growth-related vectors suggests increased photosynthetic stability, better redox regulation and better seedling establishment. Likewise, a positive association was observed for T 5 and T 10, T 11, but with a lesser contribution than T 6, revealing concentration-dependent effectiveness of the ZnO nanopriming.

Results from the variable contribution analysis showed that traits like seedling vigour index (SVI), SOG, CAT, SOD, GP%, number of seedlings (NS), DMP and MDA regulation heavily contributed to PC1. These characteristics are the main physiological mechanisms accounting for the discrimination between treatments. The high contribution of antioxidant enzymes and growth attributes indicates that ZnO nanoparticle priming enhanced the antioxidant defence system and maintained cellular homeostasis under saline conditions.

The contribution analysis of PC2 revealed that the parameters salt tolerance index (STI), seedling length, shoot length, root length, antioxidants, shoot fresh weight and flavonoid content played a greater role in the secondary variation. The characteristics are closely related to osmotic adjustment, ROS protection under stress conditions.

The observed PCA pattern is consistent with the previous studies showing that ZnO nano priming enhances abiotic stress tolerance by regulating the responses to oxidative stress, increasing photosynthetic efficiency and improving antioxidant activity. Mazhar (Waqas Mazhar et al., 2022) found that seed priming with ZnO NP in rice positively affected growth, antioxidant defence, and stress tolerance, and PCA-based analysis was able to differentiate between physiological responses associated with various treatments. Similarly, Li (Li et al., 2021) showed that ZnO NP priming improved antioxidant mechanisms and metabolic regulation to influence physiological and biochemical changes in the rice under stress conditions. The results of the multivariate analysis align with the heat map and indicate the potential of green-synthesised ZnO nanoparticles as efficient nano-enabled seed priming agents for enhancing rice stress tolerance in a saline environment.

The present study demonstrated that seaweed-mediated biogenic ZnO nanoparticles are effective and sustainable nano-enabled seed priming agents for improving rice seedling establishment under saline stress conditions. Green synthesized ZnO nanoparticles, especially at 50–75 ppm, enhanced germination performance, seedling growth, photosynthetic pigment content and antioxidant defence responses, while reducing oxidative damage and lipid peroxidation, as evidenced by enhanced activities of SOD, CAT, and POD and increased levels of non-enzymatic antioxidants. The superior biological efficiency of biogenic ZnO nanoparticles at lower concentrations compared with commercial ZnO nanoparticles underscores the potential advantages in reducing nanoparticle requirements, improving cost-effectiveness, and minimizing environmental impacts. This improved performance may be attributed to the enhanced Zn acquisition observed at lower biogenic ZnONPs concentrations based on ICP-MS analysis. In addition, hierarchical clustering and PCA analysis revealed distinct treatment responses with 75 ppm of green synthesized-ZnO nanoparticles showing the strongest association with growth, physiological and stress tolerance attributes. However, this study did not address direct measurements of ROS, molecular-level analyses, or field-scale testing, and was limited to seedlings grown under controlled environments. Hence, future research should focus on elucidating the molecular mechanisms underlying the observed responses, directly quantifying ROS dynamics, and evaluating the efficacy of optimized biogenic ZnO NPs under field conditions, together with comprehensive assessment of their environmental safety.