Seed Nanopriming Improves Jalapeño Pepper Seedling Quality for Transplantation

Abstract

Nanopriming with metal nanoparticles (NPs) is a promising strategy for improving seedling quality in horticultural crops. This study evaluated the effects of hydropriming, ZnO, SiO₂, ZnO + SiO₂, a ZnMo nanofertilizer, and two commercial biostimulants (Osmoplant and Codasil) on the early development of Capsicum annuum L. seedlings. Morphological, physiological, and biochemical traits, including biomass, stem architecture, number of leaves, chlorophylls, carotenoids, SPAD index, and nitrate reductase (NR) activity, were measured under controlled conditions. The ZnO and ZnO + SiO₂ treatments promoted stronger root growth, higher pigment content, and higher NR activity. SiO₂ alone and ZnMo showed intermediate improvements, while Osmoplant and Codasil had more limited effects. Multivariate analyses provided complementary information: heat maps revealed correlations between traits, PCA differentiated treatment responses, and radar charts integrated performance profiles. Overall, the results provide promising evidence that seed nanopriming, particularly with ZnO and ZnO + SiO₂, improves seedling vigor and transplant potential in jalapeño peppers.

1. Introduction

During the early stages of plant development, low physiological vigor can significantly compromise photosynthetic efficiency and nitrogen assimilation, processes that are essential for the growth and productivity of horticultural crops such as Capsicum annuum L. [1]. More than 50% of the nitrogen in leaves is used for proteins involved in photosynthesis, including Rubisco, whose activity is vital for CO₂ fixation and RuBP regeneration [2]. Nitrate reductase (NR) is also considered an early marker of nitrogen nutritional status, as it catalyzes the reduction of NO₃⁻ to NO₂⁻, the first step in amino acid and protein synthesis [3]. When nitrogen availability or metabolism is restricted, photosynthetic efficiency decreases, limiting plant growth even under favorable irrigation and light conditions [4].

From a global perspective, chili peppers (C. annuum L.) are one of the most important vegetables from an economic standpoint, grown worldwide for their high commercial value and contribution to food security [5]. Within this species, the jalapeño pepper is particularly relevant because it is among the most widely consumed hot peppers worldwide, with a strong presence in both fresh and processed product markets [6]. However, its productivity is often limited by abiotic stresses such as drought and nutrient limitations, which hinder seedling establishment and crop vigor [7]. Therefore, improving seedling quality is a strategic approach to ensuring field performance and sustainable yields [8].

Traditional seed improvement techniques, such as hydropriming, have demonstrated benefits in germination and vigor, but often provide limited improvements under suboptimal or stressful conditions. This limitation has motivated the exploration of nanopriming, which involves treating seeds with metallic or metalloid NPs, such as ZnO, SiO₂, and ZnMo. Due to their high bioavailability and surface reactivity, NPs can improve water absorption, activate antioxidant pathways, regulate gene expression, and modulate energy metabolism from the early stages of development [9]. ZnO NPs have been reported to improve Rubisco activity, chlorophyll content, and photosynthetic efficiency [1], while SiO₂ NPs contribute to better water retention, nitrogen assimilation, and drought resistance [10]. Furthermore, ZnMo has been associated with increased NR activity and improved metabolic integration between photosynthesis and nitrogen nutrition [11].

However, most studies have focused on cereals such as rice and wheat [12] and legumes such as chickpeas and soybeans [13]. In contrast, reports on horticultural crops remain scarce. Jalapeño peppers, with their distinctive metabolic demands and root–stem dynamics, may respond differently to nanopriming, highlighting both the novelty and relevance of this study.

Based on this background, we hypothesized that nanopriming with ZnO, SiO₂, and ZnMo would improve photosynthetic efficiency, pigment accumulation, and NR activity in jalapeño pepper seedlings, resulting in greater vigor compared to hydropriming, commercial biostimulants, or untreated controls. The objective of this study was to evaluate the effects of these treatments on key morphological, physiological, and biochemical traits to determine their potential to produce vigorous jalapeño seedlings suitable for transplanting.

2. Materials and Methods

2.1. Experimental Site and Plant Material

The experiment was conducted between January and April 2025 at the Center for Research in Food and Development A.C. (CIAD) (Delicias, Chihuahua, Mexico). The commercial hybrid variety Imperial F1 of jalapeño pepper seeds (C. annuum L.) was used, characterized by its high vigor and uniformity, designed for intensive production systems with high fruit demand. The surface of the seeds was sterilized with a 4% sodium hypochlorite (NaClO) solution under constant agitation for five min, a concentration commonly applied to chili seeds to ensure the elimination of pathogens without reducing germination capacity [14].

2.2. Characterization of Zinc Oxide, Silicon Dioxide NPs

The zinc oxide (ZnO) and silicon dioxide (SiO₂) NPs used in this study were purchased from Investigación y Desarrollo de Nanomateriales S.A. de C.V., based in San Luis Potosí, Mexico. According to the supplier, the ZnO NPs had a wurtzite crystal structure with a purity of 99.7% and an average particle size of 50 nm, while the SiO₂ NPs had a purity of 99.9%, a light-colored fine powder appearance, and an average size of 80 nm. Both types of NPs were structurally and morphologically characterized by transmission electron microscopy (TEM) provided by the supplier as part of their quality control process, confirming their homogeneity and stability (Figure 1).

In addition, the commercial nanofertilizer BROADACRE^® ZnMo (Agrichem Fluagri, Guadalajara, Jalisco, Mexico) was used. This formulation contains 62% Zn, 5% Mo, and a seaweed extract-based chelating agent designed to prevent precipitation in suspension. It was included as a comparative treatment due to its dual micronutrient composition, which is commonly associated with nitrogen assimilation.

2.3. Nanopriming Treatments

Tri-distilled water was used as the solvent in all treatments, and chitosan (Ch) (Quitofyt^® Poly-D-glucosamine) was added as a stabilizing agent to improve nanoparticle dispersion and colloidal stability. Each formulation was prepared in a final volume of 1 L. Homogenization was achieved in two steps: magnetic stirring for 20 min followed by sonication at 40 kHz for 30 min, which ensured stable suspensions [15].

The exact concentrations and chemical compositions of all primer treatments are presented in

Table 1. Treatment, chemical composition of priming’s and doses applied on C. annuum L. seeds.

Treatment	Chemical Composition	Doses
Control	Not applicable	Not applicable
Hydropriming	Tridistilled water	Not applicable
NPs ZnO + Ch	50 nm, 99.9% and Poli-D-glucosamine	(100 and 100 mg L−1)
NPs SiO2 + Ch	80 nm, 99.9% and Poli-D-glucosamine	(10 and 100 mg L−1)
NPs ZnO + SiO2 + Ch	50 nm, 99.9%, <80 nm, 99.9% and Poli-D-glucosamine	(100, 10 and 100 mg L−1)
NPs Zn + Mo	Liquid solution composed of 62% Zn, 5% Mo, and 5% of an algae extract-based chelating agent.	(124 and 10 mg L−1)
Osmoplant®	Liquid solution composed of 6% free amino acids, 2.4% nitrogen and 3.3% potassium.	(2000 mL L−1)
Codasil®	Liquid solution with a high concentration of soluble silicon composed of 20% silicon, 4% free amino acids and 11.20% potassium.	(2000 mL L−1)

.

2.4. Experimental Design

A completely randomized design with eight treatments, three replicates, and 100 seeds per replicate was used, including a control treatment with untreated seeds. The sample size of 100 seeds per replicate is commonly used in research on seed pre-germination and germination to ensure statistical robustness and reduce variability [16].

Seed preparation was carried out by imbibition, immersing 100 seeds in 30 mL of the corresponding treatment solution described in Section 2.3, for 24 h in the dark at a temperature of 25 ± 1 °C. After the pre-germination period, the seeds were removed, rinsed three times with distilled water, and gently dried at room temperature. These steps were performed to remove excess pre-germination solution and restore seed moisture to levels compatible with germination, avoiding osmotic stress during subsequent evaluation.

2.5. Crop Management

After drying at room temperature for 24 h, the seeds were sown in polystyrene trays with 338 cavities each, using a substrate mixture in a 1:1:1 (v/v/v) ratio of vermiculite, perlite, and sphagnum peat moss. The seeds were sown at a depth of 3 mm and kept under controlled germination conditions for 7 days in total darkness at 25–30 °C and 60–80% relative humidity. After this period, the seedlings were transferred to a greenhouse, where the temperature (12–33 °C) and relative humidity (30–45%) varied naturally during the day but were controlled to ensure uniform exposure in all treatments.

During the leaf development phase (days 1–30), automated foliar fertilization was applied with a volume of 500 mL of nutrient solution per tray, directly onto the foliage.

The formulation of this solution was designed using Haifa Group’s NutriNet^® software v. 2025 [17] and consisted of commercial Haifa brand fertilizers: 6 mM total nitrogen (from Haifa Poly-Feed 17-10-27+ME), 1.6 mM phosphorus, 0.3 mM additional potassium, 4 mM calcium (Haifa Cal GG), and 1.4 mM magnesium (Haifa Mag). Chelated micronutrients were also added: 5 µM iron (Haifa Micro Fe 13%), 2 µM manganese (Haifa Micro Mn 13%), 0.25 µM copper (Haifa Micro Cu 15%), 0.3 µM molybdenum, and 0.5 µM boron. This solution was applied at three-day intervals during the first 30 days of vegetative development.

2.6. Plant Sampling

All treatments showed uniform seedling emergence, and no abnormalities were observed during the germination phase, ensuring uniform initial growth conditions. Thirty-one days after sowing (DAS), 10 jalapeño pepper seedlings were randomly selected per treatment for subsequent physiological and biochemical analysis. The samples were carefully washed three times with distilled water, followed by an additional wash with a 1% solution of nonionic detergent to remove surface residues and ensure tissue cleanliness prior to analysis. This procedure has been described as a safe method that does not affect internal physiology [18].

2.7. Plant Analysis

2.7.1. Measurements of Morphological Parameters

Morphological variables were evaluated at 31 DAS. Ten seedlings per treatment were measured to determine root length, stem length, number of leaves, and stem diameter using a 153 mm digital caliper (model HER-411; Steren^®, Mexico City, Mexico) [19]. Root length was determined from the base of the hypocotyl to the apex of the radicle, and stem length was measured from the junction of the radicle and hypocotyl to the base of the cotyledons.

The fresh weight of roots, shoots, and whole seedlings was quantified using a high-precision analytical balance (model HR-120-C; A&D Weighing^®, San Jose, CA, USA). From these measurements, the total fresh biomass per seedling was calculated and expressed in mg.

2.7.2. Concentration of Photosynthetic Pigments

Pigment content was determined in triplicate using 10 fresh leaf disks (7 mm in diameter) per treatment, obtained from photosynthetic tissue without physical damage or central veins. The disks were incubated in 10 mL of 99% methanol (v/v) for 24 h in the dark at 25 ± 1 °C. Absorbance was recorded at 666, 653, and 470 nm using a UV-VIS spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA), following the methodology of Wellburn [20]. Pigment concentrations were calculated using the following equations:

$$chl a=[15.65 (A666)-7.34 (A653\left)\right]$$

(1)

$$chl a=[15.65 (A666)-7.34 (A653\left)\right]$$

(2)

$$Carotenoids=\left(\right[\left(1000 \left(A470\right))-2.86 (chl a)-129.2 (chl b)\right])/(\left(221\right))$$

(3)

$$Total chlorophyll=chl a+chl b$$

(4)

The results were expressed in mg g⁻¹ fresh weight (FW).

2.7.3. Measurements of Chlorophyll Index

The relative chlorophyll content was measured at 25 DAS using a portable SPAD-502 chlorophyll meter (Konica Minolta Sensing, Inc., Osaka, Japan) [21]. For each treatment, three fully expanded leaves per plant were selected, avoiding midribs and damaged tissue. Three random readings were taken from each leaf under high-light conditions to ensure standardization of measurements. Results were expressed in SPAD units.

2.7.4. “In Vivo” Nitrate Reductase Activity Assay

The “in vivo” activity of nitrate reductase (NR, EC 1.7.1.1) was quantified following the methodology by Sánchez [22]. Undamaged leaf segments 0.5 g without central veins were incubated in 10 mL of potassium phosphate buffer (100 mM, pH 7.5) with 1% (v/v) propanol as a permeabilizing agent. To differentiate between endogenous and induced NR activity, two incubation media were used: one supplemented with 50 mM KNO₃ (induced activity) and another without nitrate (endogenous activity).

The samples were infiltrated at a pressure of 0.8 bar and incubated for 1 h at 30 °C in the dark to promote nitrite accumulation. The reactions were stopped by heating the samples in a water bath at 100 °C for 20 min. Subsequently, 1 mL of the reaction medium was sequentially mixed with 2 mL of 1% (w/v) sulfanilamide in 1 M HCl and 2 mL of 0.02% (w/v) N-naphthylenediamine dihydrochloride (NED) in 0.2 M HCl. The colored azo complex was quantified spectrophotometrically at 540 nm using a standard nitrite calibration curve.

2.8. Pearson Correlation Heatmap

The relationships between physiological and biochemical variables were evaluated using a Pearson correlation matrix based on treatment means. The results were visualized as a heat map generated in Python (v 3.11), chosen for its flexible, high-quality graphical visualization tools that complemented the univariate analyses performed in SAS. Correlation values close to +1 represent strong positive associations, values close to −1 represent strong negative associations, and values close to 0 indicate a weak or non-existent association [23].

2.9. Principal Component Analysis (PCA)

Principal component analysis (PCA) was applied to reduce dimensionality and identify the main sources of variation among the physiological and biochemical traits in the dataset. This approach allowed us to detect patterns related to treatment by transforming the original variables into a new set of orthogonal components. The results were visualized in a two-dimensional scatter plot, in which the samples were projected onto the first two principal components. This visualization not only summarized the variability but also highlighted the grouping of treatments, facilitating the interpretation of similarities and differences in their multivariate responses [24].

2.10. Radar Chart: Multivariate Comparison by Priming Treatment

To simultaneously compare multiple physiological and biochemical traits between treatments, radial graphs (also known as spider or star graphs) were used. This visualization integrates multivariate data into a two-dimensional space, where each radial axis corresponds to a variable and the polygon formed illustrates the overall performance profile of each treatment. Radial graphs thus complemented PCA by allowing direct visual comparison of treatments across multiple traits in a single figure [25].

2.11. Statistical Analysis

The data were analyzed using analysis of variance (ANOVA), after confirming the assumptions of normality and homogeneity of variances. The means of the treatments were compared using Fisher’s LSD test with p ≤ 0.05. Fisher’s LSD test was chosen because it offers high sensitivity for detecting differences between treatments in factorial experiments with a moderate number of comparisons, which was appropriate for our experimental design [26].

For multivariate analyses, including principal component analysis (PCA), Pearson correlation heat maps, and radar charts, Python was used to take advantage of its advanced visualization libraries, while SAS (version 9.0) was used for univariate analyses due to its robustness and wide acceptance in agronomic and physiological research. The combined use of SAS and Python ensured both rigorous statistical testing and high-quality data visualization.

3. Results and Discussion

3.1. Morphological Parameters

3.1.1. Stem Length

Stem elongation is a critical morphological parameter, as it determines the initial architecture of the seedling, its light interception capacity, and its stability during transplanting. Excessive elongation can result in weak and etiolated plants, while moderate development promotes mechanical resistance and acclimatization in field conditions [27].

In this study, significant differences (p ≤ 0.05) were observed between treatments. The control showed the longest stem length (22.4 ± 1.2 mm), closely followed by hydropriming (21.8 ± 1.0 mm), with a reduction of 2.7%. The treatments with ZnO + SiO₂ NPs (19.1 ± 0.9 mm), ZnMo (18.5 ± 1.1 mm), and Osmoplant (19.3 ± 0.8 mm) showed reductions of 14.7%, 17.4%, and 13.8%, respectively. ZnO NPs (17.1 ± 0.7 mm), SiO₂ NPs (16.8 ± 0.8 mm), and Codasil (17.0 ± 0.6 mm) recorded the shortest stems, with decreases of 23.7%, 25.0%, and 24.1% (Figure 2a).

Hydropriming maintained stem elongation similar to that of the control, while nanopriming treatments promoted more compact seedlings, a characteristic associated with greater stability during handling, reduced lodging, and improved transplant survival. These traits are agronomically relevant, as seedlings with shorter stems generally show lower mortality after transplanting and better early establishment in the field [28].

Consistently, Gallegos-Cedillo et al. [27] reported that morphological parameters, such as height, are part of the predictive indices of plant quality (e.g., Dickson’s quality index), highlighting the importance of balanced architecture for field performance. Similarly, Tatari et al. [28] demonstrated that compact seedlings with a balanced stem-to-root ratio improve tolerance under drought conditions. In our study, nanopriming treatments resulted in significantly shorter stem length compared to hydropriming and the control, resulting in a more compact morphological profile.

Although drought tolerance and transplant performance were not directly measured, the observed architectural adjustment is consistent with traits related to improved seedling quality, survival, and adaptation in horticultural crops.

3.1.2. Stem Diameter

Stem diameter is a fundamental morphological parameter, as it reflects the seedling’s support capacity, water and nutrient conduction potential, and biomass allocation to support tissues. It is also closely related to survival after transplanting and field performance [29].

Our study revealed statistically significant variations (p ≤ 0.05) between treatments. Hydropriming produced the largest stem diameter (3.1 ± 0.1 mm). Compared to this value, the control and ZnO NPs reached 2.9 ± 0.1 mm (−6.5%), SiO₂ NPs 2.7 ± 0.1 mm (−12.9%), ZnO + SiO₂ NPs 2.8 ± 0.1 mm (−9.7%), ZnMo 2.9 ± 0.1 mm (−6.5%), while Osmoplant (2.6 ± 0.1 mm; −16.1%) and Codasil (2.5 ± 0.1 mm; −19.4%) recorded the lowest values (Figure 2b).

These results indicate that hydropriming favors radial stem expansion, while nanopriming treatments maintain adequate diameters within a more compact seedling profile. It is important to note that the moderate reductions observed in nanoprimed seedlings did not compromise structural integrity, as the stems remained within functional thresholds. In fact, maintaining structural robustness along with compactness may confer adaptive advantages under stress conditions, where excessive elongation or thickening does not necessarily improve survival.

The literature confirms that stem diameter, along with height and biomass, are strong predictors of seedling quality and field performance. For example, studies in woody and horticultural crops have shown that seedlings with moderate but stable diameters can perform well under abiotic stress and transplant conditions [27,29]. In this regard, our results suggest that nanopriming generates seedlings with a functionally robust but compact morphology, which could improve establishment and resilience in variable field environments.

3.1.3. Number of Leaves

The number of leaves is a decisive morphological trait, as it defines the available photosynthetic surface area, regulating light and CO₂ capture and biomass accumulation [30]. A higher number of functional leaves has been associated with better establishment and vigor after transplanting.

In our experiment, the analysis showed significant differences (p ≤ 0.05) between treatments. Hydropriming produced the highest value (9.1 ± 0.4 leaves). In comparison, the control, ZnO NPs, and SiO₂ NPs showed values of 8.3 ± 0.3, 8.5 ± 0.3, and 8.0 ± 0.3 leaves, respectively, with reductions of 8.8%, 6.6%, and 12.1%. ZnO + SiO₂ and ZnMo NPs averaged 7.9 ± 0.3 leaves (−13.2%), while Osmoplant (7.5 ± 0.3) and Codasil (7.0 ± 0.3) recorded the lowest values, with reductions of 17.6% and 23.1% compared to hydropriming (Figure 2c).

These results indicate that hydropriming favors leaf production, while nanopriming maintains enough leaves to ensure photosynthetic functionality within a compact architecture. This balance is important because it combines structural robustness with adequate leaf area, favoring seedling vigor and yield potential in transplanted crops.

Consistent with this, Chen et al. [30] reported that leaf number and quality are key determinants of transplant survival in strawberries, and Leskovar and Othman [31] demonstrated the role of leaf development in root dynamics and yield in artichokes. Previous studies further emphasize that the combination of an adequate number of leaves and strong stems predicts superior adaptation and productivity in the field [32].

3.2. Biomass

3.2.1. Total Fresh Weight

Total fresh biomass is a comprehensive indicator of seedling vitality and physiological quality, as it reflects photosynthetic efficiency, water storage capacity, and balanced biomass distribution between aerial and root organs. This parameter has been widely used as a predictor of field yield, as it correlates with establishment capacity and subsequent agricultural yield [27].

Our findings demonstrated significant differences (p ≤ 0.05) between treatments. The highest value was recorded for ZnO NPs (3450 ± 95 mg), with a 10.3% increase compared to the control (3126 ± 88 mg). This was followed by ZnO + SiO₂ NPs (3310 ± 84 mg; +5.9%) and ZnMo (3258 ± 80 mg; +4.2%). The Hydropriming treatment (3189 ± 86 mg) showed a similar value to the control (+2.0%). In contrast, the lowest values corresponded to Osmoplant (2982 ± 81 mg; −4.6%) and Codasil (2894 ± 79 mg; −7.4%) (Figure 3a).

These results demonstrate that treatments with NPs, especially ZnO, promoted greater accumulation of total fresh biomass, suggesting a positive effect on photosynthetic efficiency and water absorption. The biostimulants Osmoplant and Codasil, on the other hand, showed less favorable effects on total biomass accumulation.

Previous studies have indicated that fresh and dry biomass are solid variables for predicting establishment potential in the field. Karwat et al. [32] highlighted that biomass accumulation is directly related to enzyme activity associated with nitrogen metabolism, while de Oliveira Ferreira [33] reported that enzymes such as nitrate reductase and glutamine synthetase are reliable indicators of the nutritional status and vigor of forest seedlings. Furthermore, it has been documented that an increase in total biomass allows for better acclimatization under conditions of salt or nutritional stress, reinforcing its usefulness as a parameter of physiological quality [34,35,36].Our study provides evidence that nanopriming, particularly with ZnO and ZnO + SiO₂, improves total fresh biomass accumulation in C. annuum L. seedlings, suggesting greater physiological and agronomic potential for successful transplanting and field productivity.

3.2.2. Aerial Fresh Weight

Fresh aboveground biomass is a key indicator of photosynthetic potential and reserve accumulation in the upper organs of the seedling. Higher values reflect greater leaf and stem development, which increases light capture and photoassimilate production, essential for initial growth and survival after transplanting [37].

In this study, analysis of variance indicated significant effects of the treatments (p ≤ 0.05). The control recorded the highest fresh above-ground biomass (1980 ± 70 mg), followed by hydropriming (1896 ± 66 mg; −4.2% compared to the control). ZnO, SiO₂, and ZnO + SiO₂ NPs reached intermediate values (1850 ± 64, 1822 ± 61, and 1810 ± 63 mg), with reductions of 6.6%, 8.0%, and 8.6%, respectively. ZnMo showed the greatest decrease (1745 ± 59 mg; −11.8%), while Osmoplant (1688 ± 55 mg; −14.8%) and Codasil (1604 ± 53 mg; −19.0%) recorded the lowest values (Figure 3b).

These results indicate that, although the control and hydropriming favored greater above-ground biomass, nanopriming promoted a more balanced distribution between shoot and root tissues. This balance between roots and shoots is relevant from an agronomic point of view, as seedlings with moderate above-ground growth but stronger root systems tend to show higher survival under transplant stress and in water-limited environments [10,29].

The literature reinforces this approach. Nile et al. [37] highlighted that nanopriming can modulate biomass distribution between organs, improving physiological efficiency and stress resistance. Similarly, Mahakham et al. [12] demonstrated that nanopriming with silver nanoparticles in aged rice stimulated balanced accumulation of shoot and root biomass, improving vigor. These findings are consistent with our results, suggesting that nanoparticle-induced biomass redistribution can improve the quality and resilience of C. annuum L. seedlings.

3.2.3. Fresh Root Weight

Fresh root biomass is an essential parameter because it reflects soil exploration capacity, water and nutrient absorption, and reserve storage, which are determining factors for seedling establishment in field conditions [38]. A robust root system ensures higher survival after transplanting and confers tolerance to abiotic stresses such as salinity, drought, or nutritional deficiencies.

In this study, significant differences (p ≤ 0.05) were observed between treatments (Figure 3c). The highest value was recorded for ZnO NPs (1450 ± 48 mg; +28.2% compared to the control, 1131 ± 42 mg). ZnO + SiO₂ NPs (1372 ± 46 mg; +21.3%) and ZnMo (1325 ± 44 mg; +17.2%) also showed significant increases, while Hydropriming reached 1248 ± 43 mg (+10.3%). Osmoplant (1195 ± 41 mg; +5.7%) and Codasil (1074 ± 40 mg; −5.0%) recorded the lowest values.

In addition to the numerical results, Figure 4 provides visual confirmation of root development under different priming treatments. Nanopriming, particularly with ZnO and ZnO + SiO₂, favored seedlings with longer and more robust roots, while Codasil produced shorter and less vigorous root systems.

These results indicate that nanoparticle treatments, especially ZnO, promoted vigorous root development, which is directly related to improved transplant survival and greater long-term yield potential. The promotion of root growth through nanopriming has been associated with increased photosynthetic efficiency and stress tolerance due to improved nutrient uptake and water conductance [37]. Wang et al. [38] demonstrated that root biomass accumulation in potatoes subjected to salt stress correlated with photosynthetic pigmentation and antioxidant enzyme activity, while Boonupara et al. [39] reported that root development predicts the physiological potential of plants under field conditions.

Together, quantitative and visual evidence confirms that nanopriming, particularly with ZnO, favors resource allocation to roots, generating seedlings with greater survival capacity and agronomic adaptability, which are advantageous traits for sustainable vegetable production.

3.3. Photosynthetic Pigments

Chlorophyll accumulation is a key indicator of photosynthetic efficiency and the ability of seedlings to maintain initial growth. Chlorophyll a is the main pigment in photosystems, while chlorophyll b acts as an auxiliary pigment, broadening the absorption spectrum; the sum of both (total chlorophyll) reflects the overall potential for carbon fixation and productivity [20].

In this study, significant differences (p ≤ 0.05) were observed between treatments. The control had chlorophyll a values of 1.55 ± 0.04 mg g⁻¹ FW, while ZnO NPs reached 2.15 ± 0.05 mg g⁻¹ FW, representing an increase of 38.7% (Figure 5a). Chlorophyll b was also higher in ZnO NPs (1.12 ± 0.03 mg g⁻¹ FW) compared to the control (0.75 ± 0.02 mg g⁻¹ FW), with an increase of 49.3% (Figure 5b). Consequently, total chlorophyll in ZnO NPs reached 3.27 ± 0.07 mg g⁻¹ FW compared to 2.30 ± 0.06 mg g⁻¹ FW in the control (+42.2%). The treatments with ZnO + SiO₂ nanoparticles (3.05 ± 0.06 mg g⁻¹ FW; +32.6%) and ZnMo (3.10 ± 0.06 mg g⁻¹ FW; +34.8%) also showed significant increases, while Codasil and SiO₂ nanoparticles did not differ statistically from the control (Figure 5c).

These results indicate that nanopriming, particularly with ZnO NPs, improves chlorophyll biosynthesis and contributes to greater photosynthetic functionality. Beyond pigment accumulation, this improvement is relevant because higher chlorophyll levels are associated with greater stress tolerance, increased seedling vigor, and improved establishment potential in the field.

The literature reinforces this interpretation. Wellburn [20] laid the foundation for spectrophotometric quantification of chlorophyll as a physiological marker. Shah et al. [30] reported that chlorophyll content responds to salinity and nutrition conditions, allowing the prediction of wheat vigor. Carreño-Siqueira et al. [35] confirmed its usefulness in selecting bean genotypes under fertilization regimes, while Lai et al. [36] demonstrated that total chlorophyll correlates with growth potential in tree species.

Taken together, these findings confirm that NP treatments, particularly ZnO and ZnMo, not only improve photosynthetic status but also contribute to producing more vigorous and resistant seedlings that are better able to adapt to field conditions.

Carotenoids play a fundamental role in the photoprotection of chloroplasts, as they dissipate excess light energy and protect against reactive oxygen species. They also stabilize antenna complexes and improve electron transport, complementing the function of chlorophylls in the photosynthetic mechanism [36].

This study revealed statistically significant variations (p ≤ 0.05) between treatments. The carotenoid content in the control was 0.21 ± 0.01 mg g⁻¹ FW, while the highest values were observed in ZnMo (0.31 ± 0.01 mg g⁻¹ FW) and Osmoplant (0.30 ± 0.01 mg g⁻¹ FW), with increases of 47.6% and 42.8%, respectively. ZnO NPs also induced a significant increase (0.27 ± 0.01 mg g⁻¹ FW; +28.6%), while SiO₂ NPs and Codasil did not differ from the control (Figure 5d).

The superior response to ZnMo may be related to its dual contribution of Zn and Mo, elements involved in enzyme systems such as nitrate reductase and antioxidant metabolism, which indirectly influence pigment biosynthesis. Similarly, Osmoplant, enriched with amino acids and potassium, may have enhanced metabolic activity and stress protection pathways, leading to greater carotenoid accumulation. In contrast, the treatments that most increased chlorophyll levels (ZnO and ZnO + SiO₂) did not always produce the highest carotenoid levels, suggesting that different preparation agents prioritize different physiological pathways.

The literature supports this dual interpretation. Karwat et al. [32] reported that accessory pigments, such as carotenoids, are sensitive indicators of stress modulation, while de Oliveira Ferreira [33] highlighted their value in combination with chlorophylls as predictors of metabolic efficiency.

These findings confirm that nanopriming with ZnMo and the Osmoplant biostimulant not only increased carotenoids but also complemented the improvements in chlorophyll content observed with other treatments. This balance between chlorophyll and carotenoids may represent a synergistic strategy, improving both photosynthetic capacity and photooxidative protection, ultimately contributing to greater vigor and resistance of C. annuum L. seedlings.

3.4. Chlorophyll Index

The SPAD index is widely recognized as a rapid and non-destructive method for estimating relative chlorophyll content and is often used as an indirect indicator of nitrogen status and photosynthetic efficiency [33]. In our study, SPAD values ranged from 37.2 ± 1.0 to 39.8 ± 1.2, with the highest averages in hydropriming and ZnO NPs and the lowest in SiO₂ NPs and Codasil. However, no statistically significant differences (p > 0.05) were detected between treatments (Figure 6).

The stability of SPAD readings contrasts with the significant increases in chlorophyll a, b, and total chlorophyll obtained by spectrophotometric analysis, suggesting that SPAD may lack sensitivity to detect subtle biochemical changes under the conditions tested. This finding is consistent with previous reports indicating that SPAD accuracy may vary depending on species, genotype, and stress level [35,36].

Although not significant, these results are relevant because they highlight the methodological limitation of relying solely on SPAD for physiological assessments in C. annuum L. seedlings. SPAD remains a useful tool in the field, but our findings support the recommendation to supplement it with direct pigment quantification or calibration models for greater accuracy.

3.5. Nitrate Reductase Activity “In Vivo”

3.5.1. Endogenous Nitrate Reductase

Nitrate reductase (NR) activity is a key indicator of nitrogen assimilation in plants, as it catalyzes the reduction of nitrate (NO₃⁻) to nitrite (NO₂⁻), the first step in incorporating this essential nutrient into organic compounds. Higher enzyme activity is generally associated with greater nitrogen use efficiency, increased growth, and higher productivity.

In our study, pairwise comparisons (LSD test) confirmed significant differences between treatments (p ≤ 0.05). The highest endogenous NR values were recorded in SiO₂ NPs (2.8 ± 0.1 µmol NO₂⁻ g⁻¹ h⁻¹ FW) and ZnO + SiO₂ NPs (2.5 ± 0.1 µmol NO₂⁻ g⁻¹ h⁻¹ FW), representing a 133% and 108% increase compared to the control (1.2 ± 0.1 µmol NO₂⁻ g⁻¹ h⁻¹ FW). Hydro-primed and ZnO NPs showed intermediate increases (1.9 and 1.7 µmol NO₂⁻ g⁻¹ h⁻¹ FW), while Osmoplant and ZnMo recorded the lowest values (1.1–1.3 µmol NO₂⁻ g⁻¹ h⁻¹ FW) (Figure 7).

The strong stimulation of NR activity by SiO₂ can be explained by its known role in improving root surface development and nitrate absorption efficiency, as well as in activating antioxidant and signaling pathways that upregulate enzymes involved in nitrogen metabolism [17]. When combined with ZnO (ZnO + SiO₂), this effect is reinforced, suggesting synergistic regulation of NR activity under nanopriming conditions.

In contrast, Osmoplant and ZnMo produced lower values, which may be related to their formulation. Osmoplant, composed mainly of amino acids, may provide nitrogen in reduced forms that bypass NR activity, resulting in poor stimulation of this enzyme. Similarly, ZnMo, although it provides micronutrients, may not directly activate NR under the conditions tested, resulting in reduced enzymatic responses compared to SiO₂ or ZnO-based treatments.

Overall, these results highlight that not all priming agents equally stimulate nitrogen assimilation. While SiO₂ and ZnO-based nanopriming improved NR activity and nitrogen metabolism, Osmoplant and ZnMo showed limited effects, underscoring the importance of treatment selection to optimize nitrogen use efficiency in C. annuum L. seedlings.

3.5.2. NO₃⁻ Induced Nitrate Reductase

In the case of NR activity measured with NO₃⁻ supplements, the highest levels were recorded in SiO₂ NPs (4.8 ± 0.2 µmol NO₂⁻ g⁻¹ h⁻¹ FW), ZnMo (5.0 ± 0.2 µmol NO₂⁻ g⁻¹ h⁻¹ FW), and ZnO + SiO₂ (4.6 ± 0.2 µmol NO₂⁻ g⁻¹ h⁻¹ FW) NPs, representing an increase of more than 300% compared to the control (1.2 ± 0.1 µmol NO₂⁻ g⁻¹ h⁻¹ FW). Osmoplant and Codasil showed moderate values, while hydropriming induced significantly higher activity than the control, but lower than the NP treatments (Figure 8).

These results suggest that SiO₂ and ZnMo were particularly effective because they act through complementary mechanisms. SiO₂ improves root architecture, water retention, and ion absorption, which increases nitrate availability in plant tissues and stimulates NR activity. ZnMo, on the other hand, provides Zn and Mo, both of which are essential cofactors for enzymatic activity. Zn stabilizes protein structure, while Mo is a critical component of the molybdenum cofactor (Moco) of the NR enzyme, directly improving catalytic efficiency. The combined ZnO + SiO₂ treatment integrates these benefits, resulting in a synergistic effect.

This mechanistic perspective is supported by previous research. Karwat et al. [32] highlighted NR as a reliable marker of nitrogen metabolism in forage grasses, and de Oliveira Ferreira [33] demonstrated that NR and GS activity indicates nitrogen status in woody species. Taken together, these findings reinforce the idea that priming agents that enhance nitrate availability (SiO₂) or provide enzymatic cofactors (ZnMo) can strongly stimulate nitrogen assimilation pathways.

Our study provides evidence that nanopriming with SiO₂, ZnMo, and ZnO + SiO₂ not only improves morphological development but also modulates critical biochemical processes. By targeting different physiological mechanisms, these treatments consolidate their role as effective strategies for improving nitrogen use efficiency in C. annuum L. seedlings.

3.6. Correlation Heatmap

Pearson’s correlation analysis allows us to identify patterns of association between morphological, physiological, and biochemical parameters, providing information on the integrated metabolic responses of seedlings [23].

In our study, the correlation matrix revealed highly significant associations between photosynthetic pigments. Very strong positive correlations (r > 0.95, p ≤ 0.001) between chlorophyll a, chlorophyll b, and total chlorophyll confirmed the joint regulation of their biosynthesis in contrasting treatments. Carotenoids also showed moderate positive correlations with chlorophyll a (r = 0.60, p ≤ 0.05) and chlorophyll b (r = 0.54, p ≤ 0.05), consistent with their role in stabilizing antenna complexes and their contribution to photoprotection [25] (Figure 9).

Interestingly, NO₃⁻-dependent nitrate reductase (NR NO₃⁻) activity showed strong negative correlations with chlorophyll a (r = −0.77, p ≤ 0.001) and total chlorophyll (r = −0.78, p ≤ 0.001). This suggests a possible metabolic trade-off: seedlings that allocate more resources to pigment biosynthesis may invest less in nitrogen assimilation pathways, or vice versa. This competition between nitrogen assimilation and pigment accumulation has been observed in crops where nitrogen limitation or regulation alters the balance between photosynthetic capacity and nutrient metabolism [30,31]. In contrast, endogenous NR (END NR) showed positive associations with root and stem biomass, indicating that greater vegetative vigor is accompanied by greater enzymatic capacity to reduce nitrates, reinforcing the physiological basis for seedling growth.

Among morphological traits, stem diameter was positively correlated with the number of leaves (r = 0.65, p ≤ 0.01), reinforcing its importance as a quality index. In contrast, stem length showed no strong associations, confirming that compact seedlings may not compromise other growth parameters. The SPAD index showed no significant correlations with chlorophyll content, supporting its limited sensitivity under these conditions, consistent with reports questioning its reliability in different species and environments [39].

Taken together, these results highlight two key ideas: (i) strong co-regulation between photosynthetic pigments ensures efficient light capture and photoprotection, and (ii) the observed trade-off between nitrate reductase activity and chlorophyll accumulation reflects the close integration—and, at times, competition—between nitrogen metabolism and photosynthetic regulation in C. annuum L. seedlings.

3.7. Multivariate Analysis

Principal component analysis (PCA) was used to explore the multivariate structure of the dataset and identify clustering patterns among treatments [40]. In our study, principal component 1 (PC1) explained 34.64% of the variance and principal component 2 (PC2) explained 23.88%, which together represent 58.52% of the total variation (Figure 10).

PC1 was mainly associated with total biomass (fresh, aerial, root) and photosynthetic pigments, while PC2 reflected nitrate reductase activity and shoot growth. This indicates that the treatments influenced both biomass accumulation and nitrogen assimilation pathways.

The treatments with ZnO NPs, ZnO + SiO₂ nanoparticles, and ZnMo were clearly separated in the upper right quadrant, associated with higher pigment content, biomass production, and NR activity. In contrast, the control, hydropriming, and SiO₂ NPs were closer to each other in the central and lower left quadrants, indicating less pronounced responses. The proximity of SiO₂ to the control suggests that its physiological effects, although present, were more moderate under the test conditions. Osmoplant and Codasil occupied intermediate positions, reflecting partial improvements without the consistency observed in nanoparticle-based treatments.

The ellipses in Figure 10 represent 95% confidence intervals generated with kernel density estimation (KDE). These ellipses illustrate not only the separation of treatments but also the degree of variability and overlap within groups. For example, the overlap between hydropriming and the control suggests a similarity in their effect on seedling traits, while the clear clustering of ZnO-based treatments reflects stronger and more consistent improvements.

In summary, PCA revealed that ZnO and ZnMo treatments were the most influential in shaping seedling physiology, while SiO₂ showed weaker effects under these conditions. Beyond separating the treatments, PCA highlighted the integrated contribution of biomass, pigments, and NR activity in defining seedling vigor, supporting the role of nanoparticle priming as a promising strategy in jalapeño production.

3.8. Radar Chart Analysis

Radial graph analysis integrates multiple physiological and biochemical traits into a single visualization, allowing for quick comparison of treatment performance [41]. In this study, the graph revealed clear contrasts between preparation strategies (Figure 11).

The ZnO, ZnO + SiO₂, and ZnMo treatments showed the broadest profiles, mainly due to greater biomass accumulation (shoots and roots) and higher chlorophyll content, which are directly related to photosynthetic efficiency and seedling vigor. In contrast, Codasil and SiO₂ NPs formed smaller profiles, indicating weaker responses. Interestingly, some treatments, such as SiO₂ and Codasil, showed relatively higher nitrate reductase activity but lower biomass, suggesting a metabolic trade-off in which nitrogen assimilation was stimulated without proportional structural growth.

The radial graph complements the PCA by providing an intuitive visualization of magnitude and distribution. While PCA explains the clustering and structure of variance, the radial plot highlights how individual traits contribute to treatment profiles. From an agronomic standpoint, these integrative profiles can help identify treatments that not only maximize vigor but also balance growth and nitrogen metabolism, traits related to transplant survival and yield potential.

3.9. General Discussion

The results of this study demonstrate that nanopriming with ZnO, SiO₂, and ZnMo nanoparticles, as well as the use of commercial bioproducts such as Codasil and Osmoplant, induce various physiological and biochemical responses in jalapeño pepper (Capsicum annuum L.) seedlings. The improvements observed in seedling quality are attributed to modulations in photosynthetic pigment accumulation, nitrogen metabolism (through nitrate reductase activity), and biomass allocation patterns. These findings reinforce the potential of seed nanopriming as a non-invasive, low-dose strategy to improve initial seedling vigor, an important trait for successful transplanting and establishment under variable environmental conditions.

Interestingly, ZnO and ZnMo treatments not only promoted robust morphological traits but also stimulated chlorophyll biosynthesis and NR activity, suggesting a dual role in structural development and metabolic enhancement. In contrast, SiO₂ NPs showed a distinct profile, favoring biochemical responses (e.g., NR activity, carotenoids) over biomass accumulation, consistent with their known role in stress modulation rather than direct growth stimulation. These differences highlight the specific effects of nanomaterial compounds, which may depend on their physicochemical properties, dosage, and interaction with seed metabolism during imbibition.

The relatively stable SPAD values observed in all treatments, despite variations in chemically determined chlorophyll content, suggest a physiological balance in the regulation of chlorophyll density. This disconnect between the SPAD index and pigment concentration may reflect genotype-specific factors or environmental uniformity under greenhouse conditions and is consistent with previous evidence questioning the accuracy of SPAD as the sole indicator of pigment content in certain scenarios.

Although this study did not directly quantify the internalization or translocation of NPs within seedling tissues, previous work has shown that NPs can penetrate seed coats and exert systemic effects [42,43]. These findings support the hypothesis that the observed biochemical modulations may not be limited to surface contact but may involve deeper physiological interactions.

Overall, the integrative analysis combining the radar chart and PCA confirms that treatments with ZnO, ZnO + SiO₂, and ZnMo produce the most comprehensive physiological response profiles, supporting their agronomic potential. However, further studies are needed to explore the long-term fate of NPs in plant systems, their biosafety, and their interaction with environmental stress factors under open-field conditions.

4. Conclusions

This study confirms that nanopriming with ZnO and ZnO + SiO₂ significantly enhances the physiological quality of Capsicum annuum L. seedlings, producing compact and functional profiles that facilitate transplanting and early establishment. For nurseries and horticultural producers, this technology represents a promising alternative to conventional priming methods by promoting stronger root development, balanced biomass distribution, and greater metabolic activation.

However, the experiments were conducted under greenhouse conditions and focused only on early developmental stages. Therefore, further validation under field conditions, long-term yield evaluation, and careful consideration of environmental safety and economic feasibility are required before recommending large-scale adoption.