Improvement of Wheat and Barley Cultivation Through Seed Priming with UV, Ozone, and Nutripriming (Fe, Zn, and B)

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This work evaluated the efficacy of various seed priming techniques, which are easy to implement, rapid, economical, and environmentally friendly. These approaches could enhance grain yield in a practical and scalable manner.

Abstract

In the context of exponential global population growth, climate change, and increasingly stringent regulations limiting the use of agrochemical inputs, it is essential to explore sustainable alternatives that can enhance crop productivity. This study contributes to the search for innovative solutions to address these challenges by evaluating more efficient and environmentally friendly agricultural practices. Among such alternatives, seed priming has emerged as a promising and cost-effective technique to improve crop performance. In this work, the responses of wheat and barley seeds to treatments involving low-dose ultraviolet (UV) radiation, ozone, and nutripriming—a technique based on soaking seeds in nutrient solutions containing boron (B), iron (Fe), and/or zinc (Zn), were evaluated. All treatments were initially assessed under in vitro conditions using a Petri dish assay, followed by a tray cell experiment to evaluate their impact on various seedling biometric parameters. For nutripriming, an additional experiment was conducted under deficit irrigation to examine its effectiveness under water stress. A field trial was subsequently performed to evaluate the transferability of the results to real-world conditions. Seed priming with UV and ozone significantly enhanced root development in the in vitro assay for both crops, but these effects were not consistently observed in the tray experiment. In the field trial, the UV treatment increased thousand grain weight (TGW) in wheat, although no improvements in final yield were detected. Nutripriming treatments produced positive effects in both the Petri dish and tray experiments. Individual nutrient treatments mitigated early water stress in wheat and enhanced root development in barley. Combined nutrient treatments generally showed no significant effects, with the exception of the Zn+B combination, which improved shoot development in barley. Although no statistically significant differences were observed in the field trial, positive trends were identified, supporting the need for further research under diverse field conditions.

1. Introduction

Modern agriculture is facing multiple interrelated challenges that compromise its capacity to meet future food demands. Among the most pressing is the continuous growth of the human population. According to current projections, the global population may reach 9.7 billion by 2050 [1]. This demographic pressure translates into an increasing demand for food, animal feed, and raw materials, placing additional strain on agricultural systems that must produce more with limited land, water, and mineral-nutrition resources [2]. At the same time, climate change has emerged as a major threat to crop productivity. The rising frequency and intensity of extreme weather events—such as droughts, heatwaves, floods, and unseasonal frosts—have caused substantial crop losses worldwide. These phenomena not only reduce crop yields, but also threaten the stability of agricultural production at a global scale [3]. In parallel, agricultural practices are being reshaped by increasingly stringent regulatory frameworks. In regions such as the European Union, policies are progressively restricting the use of chemical inputs, particularly synthetic fertilizers and pesticides. While these measures aim to reduce environmental pollution and promote more sustainable production systems, they also limit the availability of conventional tools for crop management, forcing farmers to adopt alternative strategies to sustain productivity within tighter legal constraints [4].

Cereals remain the most important crops for food, feed, and global food security. Among them, wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) play a central role in global agriculture. Wheat is the most widely cultivated cereal worldwide, with approximately 220 million hectares under cultivation, and ranks third in total grain production, yielding nearly 800 million metric tons annually [5]. Wheat flour is used in a wide variety of products—including bread, pasta, cookies, and other derivatives [6]—serving as a dietary staple in many regions where it accounts for 18–21% of human caloric intake [7]. This dependence implies that fluctuations in wheat availability and pricing directly affect global food security [8].

Barley, in turn, is the fourth most important cereal in terms of harvested area, with about 46 million hectares, contributing roughly 146 million metric tons to global production [5]. It plays a key role in animal feed due to its favorable nutritional profile [8], and in the brewing industry, where it is the essential raw material for malt production, being irreplaceable in the manufacture of beer and other fermented beverages [9]. Furthermore, barley is highly adaptable to stress conditions; among C3 cereals, it exhibits the highest water use efficiency [10], making it particularly suitable for cultivation in drought-prone areas. Given their agronomic and economic importance, improving the performance and resilience of wheat and barley represents a strategic priority for ensuring future food security.

One promising strategy to address current agricultural challenges is the biotechnological enhancement of seed material. Among these approaches, seed priming has emerged as a particularly promising technique. It involves the controlled activation of metabolic processes prior to germination, with the aim of improving early plant development [11]. This approach enhances seed performance and promotes vigorous seedling establishment, making it a viable and sustainable treatment option. In this study, three seed priming techniques were evaluated for their potential integration into agricultural systems. The first was priming with ultraviolet (UV) radiation, which belongs to the category of physical agent-based priming [11]. This technique has been investigated across all three UV spectrum regions (UV-A, UV-B, and UV-C) [12,13,14] and is based on exposing seeds to a specific radiation dose. The underlying hypothesis is that UV treatments stimulate the production of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂) and superoxide (O₂⁻), which act as stress signals and activate defense mechanisms, occasionally resulting in beneficial physiological responses [15,16]. Laboratory trials have demonstrated such effects: for instance, UV-C treatment increased the antioxidant capacity of seeds compared to the control, along with higher levels of total phenolic compounds, without inhibiting germination [17]. UV treatments have also been reported to influence physiological parameters, including increased concentrations of chlorophyll, carotenoids, and anthocyanins [18,19].

The second method evaluated was ozone exposure, another physical seed priming technique [11]. Ozone is a strong oxidizing agent that induces oxidative stress in seeds by promoting the accumulation of ROS, which subsequently trigger a cascade of physiological responses [20]. These changes can ultimately enhance plant growth and development, as demonstrated in several studies [21,22,23].

Finally, micronutrient seed priming (nutripriming) consists of soaking seeds in a nutrient solution for a defined period, followed by drying at room temperature until their original moisture content is restored, enabling subsequent sowing [24]. This treatment induces pre-germinative metabolic changes, enhancing seedling vigor and early stress resilience. Plant defense mechanisms are influenced by certain micronutrients, as the production of ROS is closely related to them. Moreover, many micronutrients are components of plant defense enzymes and can accelerate specific signals related to germination [25]. On the other hand, nutrients penetrate the seed, where they are stored and subsequently mobilized by the seedling during the early stages of development, a process particularly beneficial in soils deficient in the specific minerals applied [26]. Ultimately, it contributes to better plant establishment and crop yield [25]. Nutripriming can be performed using various micronutrients, such as magnesium (Mg) [27], manganese (Mn) [28], molybdenum (Mo) [11], chlorine (Cl) [11], copper (Cu) [29], zinc (Zn) [30], boron (B) [31], or iron (Fe) [32]. In this work, we selected B, Zn, and Fe for nutripriming treatments, based on prior research showing their effectiveness in promoting seedling vigor and enhancing early plant development [31,32,33]. Zn is involved in chlorophyll synthesis, auxin formation, and is also present in various antioxidant enzymes [34]. B is associated with the production of certain enzymes linked to starch synthesis, which may explain the positive effects observed on germination [35]. Fe is essential for chlorophyll synthesis and the formation of antioxidant enzymes [36].

It is important to highlight that in cereal crops, such as wheat and barley, where seeding rates often exceed 150 kg/ha [6], it becomes essential to develop seed treatment technologies that are not only effective but also scalable. Treating such large volumes requires practical methods that can be efficiently implemented under real farming conditions. Therefore, any proposed priming strategy must demonstrate both agronomic efficacy and economic feasibility to ensure its adoption by growers, particularly by providing a consistent economical return on investment. The strategies evaluated in this study align with these requirements as they are intended to be simple, cost-effective, and suitable for large-scale seed treatment applications.

This study aimed to evaluate the plant growth-promoting (PGP) effects of three seed priming techniques: UV radiation, ozone exposure, and nutripriming (with B, Fe, and Zn), on two major cereal crops, wheat and barley. The underlying hypothesis is that applying these treatments at the seed stage, early plant development can be enhanced, resulting in more robust seedlings with greater resilience to abiotic stress, an improved capacity to maintain growth under reduced input conditions, and a positive impact on crop establishment and final yield. Specifically, the effect of the priming treatments was evaluated in vitro (on germination, and radicle and shoot length), in growth chamber (on day of emergence, shoot height, and shoot and root dry weight), and in the field (on tiller density, plant height, aerial biomass, grain yield, thousand grain weight, and grain bulk density).

2. Materials and Methods

2.1. Plant Material

The plant material used included bread-making wheat (T. aestivum L. cv. Rimbaud) and two-row barley (H. vulgare L. var. distichum cv. Lavanda). Wheat was developed by RAGT Semences (Rodez, France) and used as second-generation certified seed (R-2). Barley was developed by Florimond Desprez (Cappelle-en-Pévèle, France) and also used as R-2. Wheat seeds had previously been treated with a commercial mixture of tebuconazole and prothioconazole (Raxil Plus, Bayer CropScience, Leverkusen, Germany), whereas barley seeds were treated with fludioxonil (Syngenta, Basilea, Switzerland) and tebuconazole (Tansil, UPl, Barcelona, Spain). Wheat had a thousand grain weight (TGW) of 39.8 g with 11% moisture content, and barley had a TGW of 47 g with 10.5% moisture content.

2.2. Physical Treatments

UV and ozone treatments were applied using a commercial device (DIVI, INGEMOV, Palencia, Spain). For UV exposure, the device was fitted with a UV-C lamp (TUV PL-L 95W/4P HO 1CT/25, Philips, Amsterdam, The Netherlands) emitting radiation at 423 W·m⁻². The lamp was positioned 60 cm above the seeds. UV-C was selected based on previous reports indicating its effectiveness for seed priming [14]. The applied UV dose was calculated according to López-Rubira et al. [37].

D = (I × t)/1000,

(1)

where D is the applied radiation dose (kJ·m⁻²), I is the radiant flux (W·m⁻²), and t is exposure time (s). Ten exposure times, corresponding to specific doses (Table S1), were tested, based on prior studies.

Ozone treatments were carried out using the same device, equipped with an ozone generator. During treatment, the operator used a protective mask with an ozone-specific filter. The applied ozone dose was calculated following Avdeeva et al. [22].

D = C × t,

(2)

where D is the ozone dose (g·s·m⁻³), C is the ozone concentration in the chamber (g·m⁻³), and t is the exposure time (s). Ozone concentration was maintained constant at 0.0206 g·m⁻³, as measured by an ozone sensor (JXBS-GAS, Weihai JXCT Electronic Technology, Weifang, China). Seven exposure times, each corresponding to a specific dose (Table S2), were tested, selected based on previous reports.

2.3. Nutripriming Treatments

For nutripriming, 400 g of seeds were immersed in 1 L glass bottles containing the corresponding nutrient solution for 16 h. Seeds were then air-dried on paper filters at room temperature (20–25 °C) for 3 days. Iron treatments used iron sulfate heptahydrate (FeSO₄·7H₂O, Olmix Group, Bréhan, France), zinc treatments used zinc sulfate heptahydrate (ZnSO₄·7H₂O, Ekmekçioğulları, Mersin, Turkey) for the Zn treatments, and boron treatments used borax (Na₂B₄O₇·10H₂O, Eti Maden, Çankaya, Ankara, Turkey). The control (hydropriming) consisted of soaking the seeds in distilled water. Three concentrations were tested for each nutrient: 5000, 500, and 50 mg·L⁻¹ for Fe and Zn; 1000, 100, and 10 mg·L⁻¹ for B.

To verify nutrient uptake, seeds were analyzed for total micronutrient content using inductively coupled plasma mass spectrometry (ICP-MS, 7800 ICP-MS with Octopole Reaction System, Agilent Technologies, Santa Clara, CA, USA) at the Laboratory of Instrumental Techniques, University of Valladolid. Samples were ground with an electric grain mill (Fidibus, Komo, Kochen, Germany) and subjected to acid digestion prior to ICP-MS. Nutripriming treatments, both individual and combined, effectively enhanced seed micronutrient content in a dose-dependent manner (

Table 1. Micronutrient concentrations in wheat and barley seeds (μg·g⁻¹ dry weight) after nutripriming with individual or combined micronutrients.

	Wheat			Barley
Treatment	B (μg/g)	Fe (μg/g)	Zn (μg/g)	B (μg/g)	Fe (μg/g)	Zn (μg/g)
Control	1.73	32.9	19.3	0.68	30.7	18.2
B 1000 mg/L	35.60	31.5	20.6	40.40	31.7	18.7
B 100 mg/L	6.70	37.0	22.1	4.86	26.6	16.9
B 10 mg/L	2.16	33.5	19.7	1.50	28.6	17.8
Fe 5000 mg/L	0.90	437.0	19.2	0.65	452.0	17.6
Fe 500 mg/L	0.76	89.3	19.4	0.67	105.0	18.6
Fe 50 mg/L	0.94	37.6	21.5	0.59	36.0	20.2
Zn 5000 mg/L	1.39	32.7	>500	0.67	32.4	>500
Zn 500 mg/L	1.09	29.2	94.7	0.75	27.8	95.8
Zn 50 mg/L	1.19	30.4	25.6	0.83	29.3	28.1
Fe 500 mg/L+B 100 mg/L	4.69	89.2	22.0	4.02	95.6	18.7
Zn 500 mg/L+B 100 mg/L	4.10	28.0	97.7	4.05	28.5	114
Zn 500 mg/L+Fe 500 mg/L	0.68	82.8	94.6	0.74	93.8	91.0
Zn 500 mg/L+Fe 500 mg/L+B 100 mg/L	4.11	93.4	106.0	3.75	84.4	85.2

).

2.4. In Vitro Experiments

Two independent in vitro germination trials were conducted: one to evaluate UV and ozone treatments, individually and in combination, and another focusing on nutripriming. After treatments, five seeds were placed in Petri dishes (90 mm Ø) between two 60 g m⁻² filter papers, with 3 mL of distilled water added per dish. All materials were sterilized in an autoclave at 120 °C for 20 min. UV and ozone treatments were replicated four times, and nutripriming treatments, three times. The dishes were incubated in a growth chamber (IGCS 900 HR-LED, Ibercex, Madrid, Spain) at 25 °C, 70% relative humidity, and a 16 h light/8 h dark photoperiod (80–100 µmo m² s⁻¹). Dishes were randomly distributed and repositioned every 2 days. Germination percentage, radicle, and shoot lengths were measured at 8 days after sowing (DAS). Seeds were considered germinated when the radicle exceeded 2 mm.

2.5. Seed Tray Experiments

Two separate experiments were conducted: one for UV/ozone treatments and another for nutripriming. Both were carried out under identical conditions for wheat and barley. Seeds were sown in seedling trays with individual cells (27 × 27 × 60 mm) filled with perlite at a depth of 3 cm. Trays were placed in the same growth chamber as above. For UV/ozone experiments, the best treatments identified in vitro were tested: 3 min UV, 5 min ozone, their combination, and an untreated control. Shoot length was measured at 7 and 14 DAS; germination percentage, root length, and root dry weight were recorded at 14 DAS. In this case, 24 seeds per treatment per crop were sown in a completely randomized design.

For nutripriming, the most effective concentrations from in vitro experiments were tested: 500 mg·L⁻¹ Fe, 500 mg·L⁻¹ Zn, and 100 mg·L⁻¹ B, with hydropriming as control. Two trials were conducted: (i) nutrients applied individually (Fe: pH 3.9, electrical conductivity, EC, 375 μS/cm; Zn: pH 5.3, EC 358 μS/cm; B: pH 9, EC 56.5 μS/cm) and (ii) nutrients applied in combination (Zn+Fe: pH 4.2, EC 365 μS/cm; Zn+B: pH 7.1, EC 204 μS/cm; Fe+B: pH 5.9, EC 201 μS/cm; Zn+Fe+B: pH 5.5, EC 258 μS/cm). In the individual trial, 18 seeds per treatment per crop were sown in a completely randomized design; in the combined trial, 15 seeds were used per treatment due to the higher number of combinations. Both trials were repeated under two irrigation regimes: optimal (watering to field capacity every 2 days) and deficit (watering every 5 days). Emergence day, shoot height at 7 and 14 DAS, and shoot and root dry weight at 14 DAS were recorded for both crops.

2.6. Field Experiments

Two field trials were conducted in Soto de Cerrato, Palencia, Spain (41°56′09.8″ N, 4°26′03.8″ W), in a continental Mediterranean climate characterized by cool, wet winters and hot, dry summers, typical of rainfed cereal systems in Castilla y León [38,39,40]. Both trials were conducted under rainfed conditions following standard local practices. Figure S1 shows monthly precipitation and temperatures (average, minimum, and maximum); Table S3 provides soil characteristics. Seasonal rainfall totaled 363 mm.

One trial evaluated UV+ozone treatments, and the other assessed nutripriming. The field had been fallow during the preceding season. Soil preparation involved deep loosening with a chisel plough followed by shallow tillage with a spring-tine harrow. Sowing was performed on 22 November 2023 with a single-row precision seeder (Model 1001B, Earthway Products Inc., Bristol, IN, USA), at 16 cm row spacing and a target density of 400 plants·m⁻². Experimental plots measured 3 × 2 m and were arranged in a completely randomized design with three replicates per treatment.

Intermediate measurements (tiller number, plant height, fresh and dry aerial biomass) were taken on 17 April 2024 (147 DAS). Final harvest occurred on 23 July 2024 (244 DAS). Grain yield was determined after threshing with a laboratory thresher (Model HEGE 16, H. U. Hege Maschinen GmbH, Waldenburg, Germany). Kernel number was assessed with an electromechanical counter (Numigral, NUM 3, Tripette et Renaud, Asnières-sur-Seine, France), and bulk density with an electronic meter (Pfeuffer Hecto, Kitzingen, Germany). Treatments were the same as in the seed tray experiments.

2.7. Statistical Analysis

Statistical analyses were performed to evaluate treatment effects on germination and early seedling growth in the in vitro experiments. For germination percentage, a chi-square test with Yates’ correction was applied. Analysis of variance (ANOVA) and multiple comparison procedures were used to assess treatment effects on biometric parameters, after verifying variance homogeneity (Bartlett’s test) and normality (Shapiro–Wilk test). When assumptions were not met, robust methods were employed, including Welch’s ANOVA and a 10% trimmed mean transformation. For seed tray experiments, seedling emergence was analyzed using nonparametric survival analysis (Kaplan–Meier estimator) with the “Survival” package version 3.8.3 [41] in R software. Survival curves were generated with the Survfit function, and differences between treatments were tested with Survdiff. For the field experiment, treatment effects on the different parameters measured were analyzed using the ANOVA followed by LSD tests when statistically significant results were found in the ANOVA, and assumptions of normality and homoscedasticity were met [42]. All analyses were conducted in R version 4.5.1 (R Foundation for Statistical Computing, Vienna, Austria).

3. Results

3.1. Physical Treatments

3.1.1. In Vitro Test

In wheat, no significant differences in germination percentage were detected among treatments according to the chi-square test (χ² = 22.39, p = 0.28), with values consistently above 90%. In contrast, significant differences were observed in barley (χ² = 60.21, p < 0.001). All treatments achieved germination percentages above 90%, except for the 1-min UV treatment (55%) and the 15-min ozone treatment (70%).

The ANOVA revealed a significant effect of treatment on both root and shoot length in wheat and barley (

Table 2. One-way ANOVA summary (degrees of freedom, F-values, and p-values) for the effect of physical treatments on radicle and shoot length in wheat and barley seedlings at 7 DAS.

		Wheat		Barley
Analyzed Parameters	Degrees of Freedom	F-Value	p-Value	F-Value	p-Value
Shoot length 7 DAS	19	2.12	0.008	2.22	0.006
Root length 7 DAS	19	77.67	<0.001	11.59	<0.001

).

Significant increases in wheat seedling shoot length were observed exclusively under the 5-min ozone treatment, which showed a clear improvement compared to the control (Figure 1a). No significant effects were detected for the remaining treatments, including all UV exposures. Regarding root length, ozone treatments generally produced significant increases compared to both the control and UV treatments (Figure 1b), with the exception of the 1-min ozone exposure, which did not differ significantly from the control. Conversely, none of the UV treatments enhanced root length relative to the control.

In barley seedlings, shoot length was significantly affected by the priming treatments, although none of them resulted in improvements compared to controls (Figure 1a). The only significant difference observed was between the 3-min UV and the 20-min ozone treatments. In contrast, root length exhibited a broadly positive response to the treatments. All ozone and UV applications induced significant increases compared to the control, except for the 1-min UV and 20-min ozone treatments, which did not differ significantly (Figure 1b).

3.1.2. Seed Tray Test

The tray cell experiment included the overall best-performing treatments from the Petri dish assay (3 min UV and 5 min ozone), as well as a combined treatment (3 min UV + 5 min O₃). The ANOVA revealed a significant effect on wheat shoot length at 14 DAS (

Table 3. Effect of UV, ozone, and combined UV+ozone treatments on early seedling growth parameters in wheat and barley. Values represent means (n = 24) ± standard error of shoot length at 7 and 14 days after sowing (DAS), root length, and root dry weight at 14 DAS. Data include standard error of the mean (SEM), F-value, and p-value from the ANOVA. Different letters within the same column indicate significant differences at p < 0.05 according to the LSD test.

Treatment	Shoot Length 7 DAS (mm)		Shoot Length 14 DAS (mm)		Root Length 14 DAS (mm)		Root Dry Weight 14 DAS (mg)
	Wheat	Barley	Wheat	Barley	Wheat	Barley	Wheat	Barley
Control	56.90 ± 2	62.36 ± 5	98.03 ± 3 ab	115.29 ± 5	140.78 ± 5	124.19 ±5	8.8 ± 0.9	10.5 ± 0.8
UV	61.45 ± 1	69.90 ± 3	100.70 ± 4 ab	134.17 ± 4	131.45 ± 8	123.54 ± 4	10.1 ± 0.2	12.1 ± 0.8
Oz	52.60 ± 3	60.20 ± 5	92.46 ± 3 b	116.36 ± 6	125.28 ± 5	119.13 ± 6	8.9 ± 1.1	11.7 ± 1.0
UV+Oz	58.69 ± 3	56.17 ± 5	104.96 ± 4 a	118.00 ± 6	124.70 ± 7	119.58 ± 6	8.15 ± 4.4	11.9 ± 2.7
SEM±	1.31	2.27	1.68	2.79	3.12	2.6	0.4	0.6
F-value	2.002	1.6	2.832	2.59	2.097	0.25	1.05	0.27
p-value	0.128	0.194	0.049	0.057	0.115	0.860	0.417	0.846

). Although no significant differences were detected by the LSD test between any of the treatments and the control, the combined treatment showed a positive trend toward increasing this growth parameter (Table 3).

3.1.3. Field Experiment

In wheat, the ANOVA did not reveal statistically significant effects of the treatment on any measured agronomic parameter (

Table 4. Effect of UV, ozone, and combined treatments on tiller density, plant height, aerial biomass, and yield components in wheat. Values represent treatment means (n = 3) ± standard error. Standard error of the mean (SEM), eta squared (η²), and ANOVA p-values are provided for each variable.

Treatment	Number of Tillers (1 m2)	Plant Height (cm)	Fresh Aerial Biomass per m2 (g)	Dry Aerial Biomass per m2 (g)	Yield (Kg/ha)	TGW (g)	Bulk Density (Kg/hl)
Control	578.7 ± 94	50.7 ± 3.8	2.2 ± 0.1	0.48 ± 0.02	1930.5 ± 294	41.86 ± 3.0	71.23 ± 3.9
UV	581.3 ± 88	46.7 ± 4.2	2.4 ± 0.3	0.57 ± 0.07	1510.6 ± 180	49.46 ± 3.1	74.36 ± 1.0
Oz	509.3 ± 30	46.0 ± 3.5	2.3 ± 0.3	0.52 ± 0.09	1795.0 ± 200	45.24 ± 0.2	70.28 ± 4.3
UV+Oz	557.3 ± 150	44.7 ± 2.1	2.0 ± 0.2	0.41 ± 0.03	1773.4 ± 238	47.79 ± 1.2	62.90 ± 5.5
SEM±	26.1	1.08	0.11	0.03	294.5	1.28	2.14
Eta squared (η2)	0.11	0.385	0.177	0.346	0.176	0.449	0.357
F-value	0.33	1.67	0.57	1.41	0.57	2.18	1.48
p-value	0.80	0.25	0.65	0.31	0.65	0.10	0.29

). When considering eta squared, the thousand grain weight (TGW) was the parameter whose variability was best explained by the treatment. In this case, the treatments that used UV (either alone or in combination with ozone) tended to produce the highest values (Table 4).

In barley, the ANOVA revealed a significant effect of the treatment only on dry aerial biomass (

Table 5. Effect of UV, ozone, and combined treatments on tiller density, plant height, aerial biomass, and yield components in barley. Values represent treatment means (n = 3) ± standard error. Standard error of the mean (SEM), eta squared (η²), and ANOVA p-values are provided for each variable. Different letters within a column indicate significant differences according to LSD test (p < 0.05).

Treatment	Number of Tillers (1 m2)	Plant Height (cm)	Fresh Aerial Biomass per m2 (g)	Dry Aerial Biomass per m2 (g)	Yield (Kg/ha)	TGW (g)	Test Weight (Kg/hl)
Control	573.3 ± 78	50.7 ± 3.5	2.3 ± 0.1	0.45 ± 0.01 ab	1483.9 ± 71	53.75 ± 0.6	62.09 ± 2.9
UV	528.0 ± 48	55.0 ± 3.5	1.8 ± 0.2	0.31 ± 0.04 a	1437.9 ± 85	53.08 ± 1.5	61.49 ± 3.7
Oz	738.7 ± 76	54.3 ± 5.2	2.7 ± 0.3	0.52 ± 0.08 b	1449.3 ± 211	56.85 ± 2.9	65.21 ± 2.3
UV+Oz	616.0 ± 28	53. 7 ± 2.6	2.0 ± 0.2	0.34 ± 0.04 a	1466.4 ± 196	50.91 ± 2.7	64.39 ± 3.11
SEM±	35.4	1.70	0.14	0.03	65.9	1.12	1.39
Eta squared (η2)	0.447	0.086	0.505	0.594	0.006	0.329	0.113
F-value	2.15	0.25	2.72	3.91	0.02	1.31	0.34
p-value	0.17	0.86	0.11	0.05	0.99	0.34	0.80

). For this parameter, the ozone treatment produced the highest values, although they did not differ significantly from the control. Besides dry aerial biomass, fresh aerial biomass and tiller density also showed high eta-squared values, indicating that the treatment may account for a large proportion of the variability in these parameters (Table 5). In these cases, the ozone treatment tended to increase their values.

3.2. Nutripriming

3.2.1. In Vitro Test

The chi-square test indicated no significant differences among treatments in germination percentage for either wheat (χ² = 15.285, p = 0.085) or barley (χ² = 12.94, p = 0.165), both of which showed values above 90%. Regarding biometric measurements at 7 days, the ANOVA (

Table 6. One-way ANOVA summary (degrees of freedom, F-values, and p-values) for the effect of nutripriming treatments on root dry weight, shoot dry weight, and shoot length in wheat and barley seedlings at 7 DAS.

		Wheat		Barley
Analyzed Parameters	Degrees of Freedom	F-Value	p-Value	F-Value	p-Value
Root dry weight	9	6.30	<0.001	4.63	0.022
Shoot dry weight	9	3.49	0.010	6.78	<0.001
Shoot length	9	11.76	<0.001	9.96	<0.001

) revealed a significant effect of treatment on all evaluated parameters. In wheat, a general improvement was observed across all measured variables (radicle weight, shoot weight, and shoot length), with intermediate-dose treatments being the most effective for all three nutrients (Figure 2).

However, although no nutrient treatment significantly outperformed the control overall, the post hoc test revealed specific pairwise differences within each nutrient group. The concentration factor showed significant effects within nutrients, with intermediate concentrations of Fe and Zn resulting in significantly higher radicle weight and shoot length compared with other concentrations. In barley, the ANOVA also revealed significant treatment effects of the treatment on the response variables. However, multiple comparison tests did not show significant differences for root dry weight. For shoot dry weight and shoot length, treatment with Fe at 500 mg·L⁻¹ significantly improved both parameters compared with the control (Figure 2).

3.2.2. Seed Tray Test

No significant differences in seedling emergence time were observed under any experimental condition, in either wheat or barley, regardless of the nutrient priming treatment or irrigation level. Seed emergence occurred uniformly across treatments, indicating that neither the type of micronutrient applied nor the water regime had a significant effect on emergence timing (

Table 7. Summary of survival analysis for seedling emergence in wheat and barley under individual and combined nutrient priming treatments and contrasting water availability.

	Crop	Water Conditions	Degrees of Freedom	χ2-Value	p-Value
Individual nutrients	wheat	Normal	3	0.26	0.97
		Deficit	3	3.92	0.22
	barley	Normal	3	1.13	0.77
		Deficit	3	3.3	0.35
Combined nutrients	wheat	Normal	4	1.67	0.79
		Deficit	4	1.69	0.79
	barley	Normal	4	2.96	0.56
		Deficit	4	3.87	0.42

).

A two-way ANOVA was used to assess the effects of water availability and nutripriming treatment when micronutrients were applied individually (

Table 8. Effect of individual nutripriming treatments (Control, C, B, Fe, and Zn) and irrigation regime (N: normal and D: deficit) on early seedling growth parameters in wheat. Values represent means (n = 18) ± standard error of shoot length at 7 and 14 days after sowing (DAS), shoot dry weight at 14 DAS, and root dry weight at 14 DAS. Data include F-value and p-value from the ANOVA. Different letters within the same column indicate significant differences at p < 0.05 according to the LSD test.

Treatment	Water	Shoot Length 7 DAS	Shoot Length 14 DAS	Shoot Dry Weight 14 DAS	Root Dry Weight 14 DAS
C	N	4.20 ± 0.24 b	7.60 ± 0.5	7.86 ± 0.6	7.19 ± 0.8
	D	2.37 ± 0.6 a	7.11 ± 0.6	7.68 ± 0.8	7.56 ± 1.3
B	N	3.27 ± 0.4 ab	6.63 ± 0.6	7.2 ± 0.8	5.98 ± 0.9
	D	4.02 ± 0.3 b	7.0 ± 0.3	7.87 ± 0.5	7.61 ± 0.7
Fe	N	3.5 ± 0.4 b	7.38 ± 0.4	7.43 ± 0.6	8.39 ± 0.6
	D	3.65 ± 0.4 b	7.28 ± 0.6	6.91 ± 0.8	8.40 ± 1.0
Zn	N	3.78 ± 0.3 b	6.9 ± 0.6	7.35 ± 0.7	9.93 ± 0.6
	D	3.61 ± 0.4 b	8.04 ± 0.4	7.69 ± 0.7	8.18 ± 0.8
Water (W)	F-value	1.03	0.38	0.02	0.01
	p-value	0.31	0.54	0.87	0.91
Treatment (T)	F-value	0.44	0.61	0.24	2.85
	p-value	0.72	0.61	0.87	0.04
W × T	F-value	4.00	0.88	0.29	1.37
	p-value	0.009	0.45	0.84	0.26

and

Table 9. Effect of individual nutripriming treatments (Control, C, B, Fe, and Zn) and irrigation regime (N: normal and D: deficit) on early seedling growth parameters in barley. Values represent means (n = 15) ± standard error of shoot length at 7 and 14 days after sowing (DAS), shoot dry weight at 14 DAS, and root dry weight at 14 DAS. Data include F-value and p-value from the ANOVA. Different letters within the same column indicate significant differences at p < 0.05 according to the LSD test.

Treatment	Water	Shoot Length 7 DAS	Shoot Length 14 DAS	Shoot Dry Weight 14 DAS	Root Dry Weight 14 DAS
C	N	4.02 ± 0.3	8.69 ± 0.3	7.35 ± 0.7	7.79 ± 0.7 a
	D	4.54 ± 0.3	10.02 ± 0.7	8.12 ± 0.6	8.99 ± 0.7ab
B	N	5.29 ± 0.2	8.65 ± 0.3	7.43 ± 0.7	11.25 ± 0.7 cd
	D	4.43 ± 0.4	9.67 ± 0.4	8.26 ± 0.6	9.72 ± 0.6 abc
Fe	N	5.24 ± 0.3	10.53 ± 0.4	9.45 ± 0.6	12.04 ± 0.5 d
	D	4.88 ± 0.4	10.01 ± 0.7	8.24 ± 1.1	10.48 ± 1.0 bcd
Zn	N	4.85 ± 0.3	9.01 ± 0.5	8.46 ± 0.7	7.69 ± 0.7 a
	D	4.72 ± 0.4	9.58 ± 0.4	12.12 ± 3.7	9.49 ± 0.8 abc
Water (W)	F-value	0.79	2.88	0.55	0.01
	p-value	0.37	0.09	0.46	0.96
Treatment (T)	F-value	2.02	2.68	1.28	7.29
	p-value	0.11	0.049	0.28	<0.001
W × T	F-value	1.49	0.89	1.05	2.91
	p-value	0.22	0.45	0.37	0.037

, for wheat and barley, respectively).

For shoot length at 7 DAS, no significant main effects were detected in either wheat or barley, although a significant water × treatment (W × T) interaction was found in wheat. At 14 DAS, no significant effects were detected for wheat. In barley, shoot dry weight at 14 DAS was not significantly affected by any factor. By contrast, root dry weight at 14 DAS was significantly affected by treatment in both wheat and barley. The W × T interaction was also significant in barley, but not in wheat. Water regime alone did not significantly affect any measured parameter in either crop.

In wheat, due to the significant W × T interaction for shoot length at 7 DAS, all treatment combinations were compared (Table 8). Under optimal water conditions, none of the nutrient treatments significantly improved shoot length compared with the control. Under water stress, however, the control group exhibited a marked reduction in shoot length, whereas seedlings treated with B, Fe, or Zn maintained shoot lengths comparable to the well-watered control. Although the treatment factor significantly affected root dry weight, post hoc analyses did not reveal significant differences between individual treatments and the control (Figure 3a). In barley, treatment significantly affected root dry weight (Table 9), with B and Fe treatments significantly improving this parameter compared with the control (Figure 3b). Since the W × T interaction was also significant, all treatment × water regime combinations were analyzed (Table 9). Under optimal water conditions, B and Fe significantly increased root dry weight compared with the control. Under water deficit conditions, however, no treatment outperformed the control, and no significant differences were detected among treatments.

When micronutrients were applied in combination, water availability significantly affected root dry weight in wheat (

Table 10. Effect of combined nutripriming treatments (Control, Fe+B, Zn+B, Zn+Fe, and Zn+Fe+B) on early seedling growth parameters in wheat. Values represent means (n = 15) ± standard error of shoot length at 7 and 14 days after sowing (DAS), shoot dry weight at 14 DAS, and root dry weight at 14 DAS. Data include F-value and p-value from the two-way ANOVA.

Treatment	Water	Shoot Length 7 DAS	Shoot Length 14 DAS	Shoot Dry Weight 14 DAS	Root Dry Weight 14 DAS
C	N	3.37 ± 0.5	10.02 ± 0.6	14.02 ± 3.1	8.94 ± 1.9
	D	2.02 ± 0.3	9.14 ± 0.5	7.34 ± 0.7	17.42 ± 2.7
Fe+B	N	2.61 ± 0.4	9.15 ± 0.4	9.55 ± 0.8	10.55 ± 1.6
	D	2.64 ± 0.3	8.25 ± 0.6	8.72 ± 0.6	17.06 ± 0.9
Zn+B	N	2.34 ± 0.4	8.65 ± 0.5	12.30 ± 3.9	10.42 ± 1.5
	D	1.94 ±0.3	7.9 ± 0.6	7.0 ± 0.6	17.75 ± 0.9
Zn+Fe	N	2.37 ± 0.4	8.56 ± 0.7	8.35 ± 0.9	10.18 ±1.2
	D	2.65 ± 0.3	8.55 ± 0.5	11.97 ± 2	15.57 ± 0.8
Zn+Fe+B	N	2.54 ± 0.4	8.65 ± 0.8	11.23 ± 0.9	12.74 ± 1.4
	D	2.25 ± 0.2	8.84 ± 0.4	9.7 ± 0.5	16.67 ± 0.9
Water (W)	F-value	2.02	1.74	2.19	48.59
	p-value	0.16	0.19	0.14	<0.001
Treatment (T)	F-value	0.66	1.53	0.15	0.52
	p-value	0.62	0.19	0.96	0.72
W × T	F-value	1.3	0.44	1.57	0.76
	p-value	0.27	0.78	0.19	0.55

), leading to a reduction (normal irrigation, 16.90 ± 0.63; deficit watering, 10.57 ± 0.64). No significant effects were observed for other factors or parameters (Table 10).

In barley, water deficit caused significant decreases in all measured parameters (

Table 11. Effect of combined nutripriming treatments (Control, Fe+B, Zn+B, Zn+Fe, and Zn+Fe+B) on early seedling growth parameters in barley. Values represent means (n = 15) ± standard error of shoot length at 7 and 14 days after sowing (DAS), shoot dry weight at 14 DAS, and root dry weight at 14 DAS. Data include F-value and p-value from the two-way ANOVA.

Treatment	Water	Shoot Length 7 DAS	Shoot Length 14 DAS	Shoot Dry Weight 14 DAS	Root Dry Weight 14 DAS
C	N	3.88 ± 0.4	12.14 ± 0.6	12.62 ± 0.9	19.1 ± 1.4
	D	2.72 ± 0.4	9.76 ± 0.6	8.59 ± 0.8	17.25 ± 1.3
Fe+B	N	3.0 ± 0.4	11.34 ± 0.4	10.93 ± 0.5	18.52 ± 1.2
	D	2.39 ± 0.5	9.53 ± 0.7	7.92 ± 0.8	10.99 ± 1.3
Zn+B	N	2.81 ± 0.4	10.01 ± 0.5	13.66 ± 0.7	20.69 ± 1.0
	D	2.81 ± 0.5	9.96 ± 0.7	10.6 ± 0.6	14.78 ± 0.8
Zn+Fe	N	3.15 ± 0.4	10.91 ± 0.5	11.46 ± 0.8	17.67 ± 1.1
	D	2.52 ± 0.5	9.39 ± 0.8	9.27 ± 0.8	14.28 ± 1.1
Zn+Fe+B	N	4.23 ± 0.3	12.11 ± 0.3	10.41 ± 0.6	22.81 ± 0.8
	D	3.25 ± 0.3	10.54 ± 0.3	8.71 ± 0.4	13.48 ± 0.7
Water (W)	F-value	6.49	14.95	38.01	18.90
	p-value	0.01	<0.001	<0.001	<0.001
Treatment (T)	F-value	2.21	1.74	4.42	1.13
	p-value	0.07	0.15	<0.001	0.34
W × T	F-value	0.55	1.03	1.05	1.14
	p-value	0.69	0.39	0.39	0.34

): shoot length at 7 and 14 DAS (7 DAS: normal watering, N: 3.41 ± 0.19, deficit watering, D: 2.74 ± 0.2; 14 DAS: N: 11.3 ± 0.3, D: 9.8 ± 0.3), shoot dry weight at 14 DAS (N: 11.8 ± 0.3, D: 9.0 ± 0.3), and root dry weight at 14 DAS (N: 19.8 ± 0.9, D: 14.2 ± 0.9). The treatment factor had a significant effect only on shoot dry weight, with the multiple comparison test indicating that the Zn+B treatment was significantly superior to the other treatments and the control (Figure 4). No significant W × T interaction was found for any of the evaluated parameters (Table 11).

3.2.3. Field Experiment

Under field conditions, the ANOVA did not yield p-values below 0.05 in wheat for any of the agronomic parameters studied (

Table 12. Effect of nutripriming treatments on tiller density, plant height, aerial biomass (fresh and dry), and yield components (grain yield, thousand grain weight, TGW, and seed bulk density, SBD) in wheat. Values represent treatment means (n = 3) ± standard error. Standard error of the mean (SEM), eta squared (η²), and ANOVA F-value and p-values are provided for each variable.

Treatment	Number of Tillers (1 m2)	Plant Height (cm)	Fresh Aerial Biomass (Kg)	Dry Aerial Biomass (Kg)	Yield (Kg/ha)	TGW (g)	SBD (Kg/hl)
Control	578.7 ± 67	50.7 ± 2	2.226 ± 0.1	0.480 ± 0.1	1930.5 ± 294	41.86 ± 3.0	71.23 ± 3.9
Hydropriming	570.7 ± 143	49.0 ± 1	2.243 ± 0.3	0.497 ± 0.1	1728.7 ± 170	43.69 ± 0.8	73.72 ± 2.8
B	589.3 ± 88	51.0 ± 4	2.478 ± 0.6	0.533 ± 0.1	1734.4 ± 211	48.51 ± 2.0	80.17 ± 0.4
Fe	650.7 ± 8	50.0 ± 2	3.057 ± 0.2	0.720 ± 0.1	1832.3 ± 224	41.88 ± 1.0	73.62 ± 3.7
Zn	490.7 ± 32	50.0 ± 1	2.374 ± 0.2	0.557 ± 0.1	1670.5 ± 195	42.80 ± 1.0	65.49 ± 5.5
Fe+B	605.3 ± 98	48.7 ± 2	2.298 ± 0.3	0.507 ± 0.1	1820.5 ± 100	48.94 ± 1.3	76.63 ± 0.3
Zn+B	464.0 ± 66	44.7 ± 3	2.098 ± 0.4	0.480 ± 0.1	1617.0 ± 83	44.80 ± 2.5	72.11 ± 1.2
Zn+Fe	560.0 ± 85	49.0 ± 2	2.651 ± 0.2	0.613 ± 0.1	1547.6 ± 119	43.64 ± 1.4	76.03 ± 2.3
Zn+Fe+B	514.7 ± 29	47.3 ± 3	1.807 ± 0.2	0.393 ± 0.1	1417.4 ± 96	43.47 ± 1.6	65.64 ± 7.0
SEM±	63.30	2.32	0.32	0.07	239.1	1.77	3.78
Eta squared (η2)	0.277	0.239	0.350	0.417	0.161	0.492	0.423
F-value	0.86	0.71	1.21	1.61	0.43	2.18	1.65
p-value	0.56	0.68	0.35	0.10	0.89	0.08	0.18

).

The eta-squared values indicated that for TGW, SBD, and dry aerial biomass, a notable proportion of their variability was explained by the treatment (Table 12). For dry aerial biomass, the Fe treatment tended to produce higher values, whereas for TGW and SBD, the treatments containing B (B and Fe+B) tended to increase their values, although without significant differences (Table 12). In barley, the ANOVA did not reveal any significant treatment effects on the studied parameters, and no eta-squared values were particularly remarkable (

Table 13. Effect of nutripriming treatments on tiller density, plant height, aerial biomass (fresh and dry), and yield components (grain yield, thousand grain weight, TGW, and seed bulk density, SBD) in barley. Values represent treatment means (n = 3) ± standard error. Standard error of the mean (SEM), eta squared (η²), and ANOVA F-value and p-values are provided for each variable.

Treatment	Number of Tillers (1 m2)	Plant Height (cm)	Fresh Aerial Biomass (Kg)	Dry Aerial Biomass (Kg)	Yield (Kg/ha)	TGW (g)	SBD (Kg/hl)
Control	573.3 ± 98	50.7 ± 3.5	2.337 ± 0.1	0.450 ± 0.01	1483.97 ± 71	53.75 ± 0.6	62.1 ± 2.9
Hydropriming	714.7 ± 35	60.3 ± 1.8	3.260 ± 0.3	0.523 ± 0.04	1833.77 ± 96	52.41 ± 0.3	62.2 ± 3.2
B	680.0 ± 74	56.0 ± 2.5	2.337 ± 0.2	0.407 ± 0.05	1785.00 ± 99	53.27 ± 1.8	63.0 ± 4.4
Fe	696.0 ± 75	52.0 ± 4.2	2.440 ± 0.3	0.430 ± 0.05	1750.80 ± 257	49.79 ± 4.5	67.1 ± 7.0
Zn	701.3 ± 64	55.3 ± 7.7	2.567 ± 0.6	0.480 ± 0.12	1360.17 ± 290	53.85 ± 1.3	63.9 ± 4.4
Fe+B	698.7 ± 16	59.0 ± 2.3	3.098 ± 0.5	0.480 ± 0.10	1612.10 ± 127	54.10 ± 0.8	60.5 ± 4.0
Zn+B	829.3 ± 43	59.7 ± 0.9	3.437 ± 0.6	0.567 ± 0.10	1460.80 ± 24	53.22 ± 2.0	60.2 ± 6.4
Zn+Fe	616.0 ± 74	57.7 ± 5.4	2.218 ± 0.4	0.347 ± 0.05	2057.60 ± 363	49.94 ± 2.3	67.1 ± 5.8
Zn+Fe+B	837.3 ± 19	56.7 ± 6.0	3.142 ± 0.6	0.503 ± 0.07	1321.77 ± 129	57.45 ± 0.9	73.2 ± 5.7
SEM±	27.99	4.36	0.45	0.07	211.3	2.03	5.04
Eta squared (η2)	0.322	0.206	0.326	0.259	0.374	0.363	0.231
F-value	1.07	0.59	1.09	0.79	1.35	1.28	0.68
p-value	0.43	0.77	0.41	0.62	0.28	0.31	0.70

).

4. Discussion

The results obtained in this study showed no significant effect of UV and ozone priming treatments on the germination percentage of barley and wheat seeds. The use of certified seed lots with germination rates exceeding 95% inherently limits the potential for further improvement of this parameter. In contrast, previous studies have reported enhancements in germination percentage following UV or ozone treatments, particularly when initial germination rates were below 80% [23,43,44,45,46]. Future investigations could focus on evaluating the effects of these priming techniques on seed lots with lower initial germination to assess their potential under suboptimal conditions. Importantly, none of the applied treatments resulted in a reduction in germination percentage in either crop, indicating the absence of phytotoxic effects at the applied doses.

In the in vitro trial, noticeable morphometric changes were observed. In wheat, several ozone doses resulted in increased root length compared to the control. In barley, nearly all doses of both UV and ozone significantly enhanced root length. These findings are consistent with previous studies in cereals [43,44] and other crops such as cotton [47], in which UV-C exposure induced similar responses. However, this increase in root length was not observed in the seedling tray experiments, where no treatment significantly outperformed the control in either crop. This discrepancy may be attributed to differences in the timing of measurement—root length was assessed on day 7 in the in vitro assay, whereas it was evaluated on day 14 in the seedling tray experiment.

The priming effect may be more pronounced during early developmental stages and thus may have dissipated by the later measurement. Both trials were conducted under controlled conditions at 25 °C. Given that the base developmental temperature for wheat and barley is close to 0 °C, future studies could evaluate whether physical priming enhances seedling performance under lower temperature field conditions during early development.

Although no statistically significant differences were detected in wheat by day 14, the combined UV-ozone treatment showed a positive trend, suggesting potential as a growth-promoting strategy. If validated in future studies, the observed effect could be attributed to a synergistic interaction between UV and ozone. In barley, the ANOVA yielded a p-value near the conventional significance threshold of 0.05, suggesting a possible treatment effect on growth parameters. Similar responses have been reported in UV-primed maize [14], rice [13], or wheat [43,44], as well as in ozone-treated maize [21]. Notably, the sequential application of ozone followed by UV radiation may offer specific benefits. UV radiation could degrade residual ozone [48], thereby reducing its accumulation on the seed surface and minimizing potential harm. Since ozone degradation generally requires 20 to 50 min [49], a sequential treatment design may ensure that ozone’s effect is confined to the programmed exposure time.

In the field trial, no significant effects of the physical priming treatments were observed on any agronomic parameter in either crop. This lack of observable impact may be due to the absence of stress conditions during the growing season. One of our initial hypotheses was that the treatments might enhance plant resilience under water deficit conditions by promoting root development, thereby improving access to deeper soil moisture. However, the field trial experienced typical weather, with no extreme events. Temperature conditions remained within normal ranges, and rainfall was slightly above average [50], particularly in June—a critical period for grain filling [51]. Water availability during this phase influences key yield parameters such as TGW and seed bulk density [52]. Thus, drought stress, which might have amplified the effects of priming, did not occur during the trial.

Nevertheless, some treatment trends were observed. In wheat, TGW exhibited a positive trend with UV treatment. As TGW is an important quality parameter, particularly for milling performance [53], this may suggest a potential physiological benefit, possibly via improved photoassimilate translocation [54]. While TGW has not been directly assessed in other UV priming studies, several have reported physiological responses potentially linked to this outcome [15]. In barley, a slight increase in aerial biomass was observed following ozone treatment, although it was not statistically significant. Aerial biomass is particularly relevant in cereals due to its association with photosynthetic capacity and yield potential [27,55]. These trends, although not significant, are in line with previous research on Arachis hypogaea, where UV-C treatment increased biomass accumulation [56]. Additional multi-year field studies under variable climatic conditions will be necessary to confirm these trends.

In the nutripriming trials, laboratory analyses confirmed increased nutrient content in seeds following treatment, consistent with findings in other studies reporting increased Fe in rice [57] and increased Zn in maize [28], wheat, and barley [29], even when seeds were rinsed after soaking [58]. In the in vitro assay, no germination inhibition was observed in either crop, suggesting that the nutrient concentrations applied were not phytotoxic. However, high concentrations of borax have been reported to inhibit germination in other studies [31]. In wheat, although no treatment significantly improved biometric parameters over the control, intermediate nutrient doses showed a generally positive trend, justifying their selection for further testing. In other in vitro studies on other crops, nutripriming had yielded positive outcomes using B [24], Zn [22,59], or Fe [28,31]. In barley, the intermediate Fe treatment significantly increased shoot length and weight, while the highest Fe dose (5000 mg L⁻¹) had a phytotoxic effect, reducing leaf size. This is consistent with prior reports of high Fe doses inducing Fenton reactions and generating reactive oxygen species (ROS), leading to cellular oxidative damage [60].

In the seedling tray experiment, survival analysis indicated no effect of treatments on time to emergence. While this confirms the absence of phytotoxicity, it may also reflect a lack of positive effects under optimal conditions. Certified seeds and optimal temperatures may have minimized observable differences compared to the untreated control. However, some biometric parameters showed significant improvements. In wheat, treatments enhanced drought tolerance, a promising result that aligns with previous findings where B application reduced drought impact [61]. In barley, treatment effects were more evident in root development, with B and Fe showing stimulatory effects, consistent with previous findings in wheat [31,62], maize [28,63], and rice [57]. Enhanced early root growth can play a crucial role in improving cereal crop performance and yield [64].

Most combined treatments did not produce significant effects, except for a notable improvement in aerial biomass in barley with the Zn+B combination, a nutrient pairing not previously explored. However, Zn has been successfully combined with Mn, P, Fe, and Mg [26,27,28,32,58], suggesting its broad compatibility due to its solubility across a wide pH range [65]. In contrast, combining Fe and B may be less effective or even detrimental, as borax can increase solution pH, leading to Fe precipitation [66], as observed in the present study (Fe solution: pH 3.9; Fe+B solution: pH 5.9). Using boric acid instead may stabilize pH and mitigate this issue [67]. Additionally, combined treatments may increase total salt concentrations, particularly in triple-nutrient applications, which could lead to osmotic stress and adverse effects on seeds. However, this fact was not so evident after checking out the EC values obtained for each solution.

Under field conditions, nutrient priming treatments were evaluated in the absence of climatic stressors. As with physical priming, this limited our ability to evaluate their role in stress mitigation. The field’s inherent variability, along with optimal growing conditions, likely masked any subtle treatment effects. Furthermore, the use of fungicide-treated seeds, while representative of regional agricultural practices, may have influenced the priming response. All these aspects should be further investigated in multiple situations to better evaluate the actual potential of these techniques in the real world.

Despite this, Fe+B presented a certain trend to improve TGW in wheat. Although not significant, this tendency aligns with previous studies in wheat, where B treatments increased TGW [31], and in peanut, where Fe priming also improved TGW [68]. A similar trend was observed in dry aerial biomass, where Fe treatment led to slight improvements. While these did not translate into higher grain yields, they may reflect a physiological trade-off, where increased biomass does not equate to improved yield due to intra-plant competition or resource allocation imbalances [69]. This is plausible given Fe’s known role in biomass production [70]. This aspect is also consistent with previous studies in maize, where Fe treatments improved aerial biomass [28].

As with physical treatments, the positive effects observed under controlled conditions were not fully replicated in the field. The transient nature of early physiological responses may explain this, as laboratory assessments were conducted at 14 DAS, while field evaluations occurred later. Furthermore, early field temperatures averaged below 10 °C, in contrast to the constant 25 °C in the growth chamber, which may have suppressed the expression of early vigor. Nevertheless, in scenarios involving water stress or temperature extremes—conditions not encountered in the present study—these early responses may translate into meaningful agronomic benefits.

An important aspect to consider for the future success of the priming technique is its potential for scaling up to a commercial level. For UV and ozone treatments, scalability in cereals appears feasible at the final stage of seed selection and processing, particularly for seeds intended for sowing, where the incorporation of a UV/ozone treatment system could be readily implemented. However, the prototype used in our experiments would need to be adapted for integration into seed selection lines in order to achieve sufficient processing capacity without delaying current processing times. Given the short duration required for priming, its implementation does not appear to represent a major obstacle. In contrast, the application of the nutripriming technique may be more challenging due to the soaking and subsequent drying periods, which are time-consuming. Nevertheless, it would be worthwhile to explore whether the soaking period could be reduced without compromising effectiveness, or whether the incorporation of a forced hot air drying system could shorten the drying phase, thereby facilitating its application in seed processing facilities for sowing.

5. Conclusions

This study demonstrated that several seed priming treatments can promote plant growth under laboratory conditions, particularly during early developmental stages in wheat and barley. Notably, certain micronutrient treatments, when applied individually, were able to mitigate the negative effects of water deficit, especially in wheat. However, these positive effects were not fully translated to the field, where favorable weather conditions and limited replication may have obscured treatment benefits. Whether these treatments could exert more pronounced effects under stress conditions warrants further investigation. Nonetheless, both physical and nutritional seed priming techniques remain promising due to their simplicity, low cost, and potential to enhance early vigor under challenging field conditions. Future research should focus on multi-year field trials under a broader range of environmental scenarios to fully evaluate their agronomic potential.