Priming ‘Santa Isabel’ Pea (Pisum sativum L.) Seeds with NaCl and H₂O₂ as a Strategy to Promote Germination

Abstract

Peas possess significant nutritional properties due to their high protein levels, carbohydrates, fiber, and vitamins. Increased climate variability can lead to water stress in crops like peas. Therefore, priming plants through seed priming is a technique that has proven effective as a pre-conditioning method for plants to cope with more severe future stresses. Different doses and soaking times of ‘Santa Isabel’ pea seeds in NaCl and H₂O₂ were evaluated to enhance and promote germination. Two experiments were conducted under controlled conditions (average temperature 15.8 °C) through a completely randomized design with a 4 × 3 factorial arrangement, comprising 12 treatments in each trial. In the first trial, NaCl doses (0, 50, 100, or 150 mM) and the soaking time of the seeds in NaCl (12, 24, or 36 h) were examined. In the second trial, H₂O₂ doses (0, 20, 40, or 60 mM) were tested with the same imbibition times. The 50 mM NaCl dose at 24 h demonstrated the best values for germination rate index, mean germination time, germination rate (GR), and germination potential (GP). Seed imbibition for 24 h in NaCl, as well as in H₂O₂, is the ideal time to achieve the best GR and GP. The dry mass of leaf and stipule recorded the highest values with a 60 mM dose of H₂O₂ and 24 h of imbibition. An application of 150 mM NaCl resulted in the highest values of germinated seed dry mass, while causing lower dry mass in roots, stems, leaves, and stipules; however, it maintained similar total dry mass values.

1. Introduction

Peas are legumes with various uses, from human consumption to green fodder. Their importance lies in their nutritional health benefits, due to their high content of protein, carbohydrates, fiber, and vitamins A, B, and C [1]. The main producing countries include Canada, Russia, China, the United States, India, and Australia, with global production ranging between 10 and 11 million tons (t) across a planted area of 6.2 million hectares (ha) [2]. By 2022, Colombia reached the production of 63,217.5 tons over an area of 27,166.12 ha, with Nariño, Cundinamarca, and Boyacá being the most prominent producing departments [3].

One of the biggest problems in the germination of pea seeds during field sowing is the low uniformity of germination, which results in uneven plants and complicates crop management. Therefore, enhancing the speed and uniformity of seed germination is crucial to optimizing the crop cycle [4]. Rapid and uniform germination facilitates synchronous seedling emergence, making it easier to plan and execute agronomic tasks such as pest and disease management, fertilization, and irrigation [5]. Additionally, reducing the time between sowing and emergence decreases exposure to adverse conditions, such as water deficit or pathogen pressure, ultimately leading to more successful crop establishment and higher yields [6].

Priming is a strategy to mitigate the effects of water stress. It is used to improve germination rates and seed uniformity, and it helps prepare the plant to face subsequent stressful conditions [7]. In this context, priming is defined as a pre-germination technique that induces rapid and uniform seed emergence and can activate the stress response system through a stimulus that confers seed tolerance to specific future stress conditions [8]. Among the substances used in seed priming are sodium chloride (NaCl) and hydrogen peroxide (H₂O₂), which can enhance plant tolerance to stress conditions, such as osmotic or water stress. Furthermore, they have advantages over other substances due to their availability and ease of implementation [9].

According to Kiran et al. [10], plants produced from seed priming with NaCl increased their osmotic adjustment capacity due to a higher endogenous Na⁺ and Cl⁻ content in the roots, as well as an increase in osmoprotective and antioxidant substances that reduce oxidative DNA damage caused by reactive oxygen species (ROS). In this regard, Pandolfi et al. [11] found that pea plants grew more at a concentration of 80 mM NaCl when previously exposed to 10 mM NaCl, compared to those exposed to 25 mM NaCl. Likewise, Senturk and Sivritepe [12] reported that priming pea seeds with 150 mM NaCl allowed the plants to develop salinity tolerance during germination and the seedling stage.

Priming with H₂O₂ induces an inductive pulse that helps protect plants under abiotic stress by restoring redox homeostasis and mitigating oxidative stress, thereby preventing damage to membranes, proteins, and lipids through modulation of stress signaling pathways [13]. In this regard, Moussa and Mohamed [14] found that pea seedlings treated with a seed priming of 70 mM H₂O₂ exhibited higher total chlorophyll content, carotenoids, photosynthetic activity, growth, and synthesis, along with accelerated production of proline and antioxidant enzymes, as well as reduced oxidative damage under water stress.

Therefore, this research aimed to evaluate various doses and exposure times of ‘Santa Isabel’ pea seeds to NaCl and H₂O₂ to enhance and promote germination.

2. Materials and Methods

2.1. Location

The experiment took place in a plastic-covered mesh house at the Universidad Pedagógica y Tecnológica de Colombia (UPTC) Tunja campus, located in Boyacá, at coordinates 5°33.10″ N, 73°21.30″ W, and an altitude of 2692 m. The average temperature during the experiments was 15.8 °C, relative humidity was 60%, and sunlight was provided for 4 to 6 h a day, as shown in Figure 1.

2.2. Plant Material

The Santa Isabel variety was used, one of the most cultivated in Colombia, acquired from the seed company LERL (Distribuciones Semillas LERL Ltda, Bogotá, Colombia), with a purity of 98%. The seed was stored at room temperature and inside an airtight bag before the start of the experiment. This variety achieves an average yield of 1013.6 kg of dry peas per hectare in the Ipiales and Pasto areas [15]. It adapts to altitudes between 2000 and 3000 m, in environments with temperatures ranging from 13 to 18 °C, with an average maximum of 21 °C and a minimum of 9 °C; also, it requires approximately 250 to 380 mm of water, well distributed throughout the growing cycle, with increased water demand between the growth and flowering stages [16].

2.3. Experimental Design and Field Establishment

For the development of the research, two experiments were conducted under a completely randomized design with a 4 × 3 factorial arrangement, comprising 12 treatments in each case. In the first trial, the first factor was the dose of NaCl (Carl Roth GmbH + Co KG, Karlsruhe, Germany) (0, 50, 100, or 150 mM), and the second factor was the imbibition time of the seeds in NaCl (12, 24, or 36 h). In the second trial, the first factor was the dose of H₂O₂ (Merck KGaA, Darmstadt, Germany) (0, 20, 40, or 60 mM), and the second factor was the imbibition time of the seeds in H₂O₂ (12, 24, or 36 h). Each trial treatment had five replicates, totaling 60 experimental units (EU) per trial. Each EU consisted of 48 seeds, which were sown in 72-cavity germination trays of type A-BA72 (A and P de, Tenjo, Colombia) at a depth of 5 mm. The substrate used was Seed Pro 8020 XL (Projar Group, Valencia, Spain), a mixture of blond peat (80%) and black peat (20%). The substrate was watered daily to maintain humidity and thus promote germination. The first trial was sown on August 16 and completed on September 6, while the second trial was sown on September 16 and completed on 6 October 2023.

2.4. Response Variables

Germination is the emergence of the radicle; however, given that in field conditions, the visible variable to measure is when the epicotyl emerges through the substrate, this was considered a germinated seed. Seeds germinated four days after sowing (DAS); the number of germinated seeds and the time to germination in days were measured daily. From these data, the germination rate (GR), germination potential (GP), mean germination time (MGT), and germination rate index (GRI) were calculated according to the equations in

Table 1. Equations used to calculate germination parameters .

Variable	Equation	Units
Germination rate	$GR=\left({\displaystyle \frac{N_S}{N}}\right)\ast 100$	%
Germination potential	$GP=\left({\displaystyle \frac{Nmax}{N}}\right)\ast 100$	%
Mean germination time	$MGT={\displaystyle \frac{{\sum }_{i=1}^k{n}_i{t}_i}{{\sum }_{i=1}^k{n}_i}}$	Days to germination
Germination rate index	$GRI={\displaystyle \sum _{i=1}^k}\left({\displaystyle \frac{n_i}{t_i}}\right)$	Seeds germinated by day

. Once the plants were ready for transplantation at 21 and 16 DAS, destructive sampling was performed for the first and second trials, where the dry mass of the roots, stems, leaflets, stipules, germinated seeds, and total mass was measured. The dry mass was determined using a Boeco Bas 31 plus balance (Boeckel & Co. and GmbH & Co. KG, Hamburg, Germany) with a precision of 0.0001 g, after subjecting the different plant organs to 70 °C for 48 h in a Memmert UNB500 drying oven (Memmert GmbH, and Co. KG, Schwabach, Germany). The stipule leaf area (SLA) and leaflet area (LA) were determined by photographic analysis, with images processed using ImageJ software 1.54h, following the methodology of [17], in which the total leaf area (TLA) was also calculated as the sum of SLA and LA and expressed in cm².

2.5. Statistical Analysis

The data were initially assessed for normality using the Kolmogorov–Smirnov test to ensure adherence to the assumptions required for subsequent statistical analyses. After confirming normality (p < 0.05), an analysis of variance (ANOVA) was performed, revealing significant differences among treatments. Post hoc comparisons were conducted using Tukey’s test (p < 0.05) to categorize treatment groups. Likewise, a Pearson correlation test was performed between the parameters evaluated for each trial and for variables exhibiting significant interaction effects. Response surface models were developed using the RSREG procedure in SAS and adapted to Equation (1). All statistical analyses were conducted using SAS OnDemand for Academics 9.4M8 (SAS Institute Inc., Cary, NC, USA). Figures were generated with SigmaPlot version 14.0.0.124 (Systat Software, Inc., San Jose, CA, USA). Additionally, data visualization and figure customization were performed using the open-source programming language Python 3.11 within the Visual Studio Code 1.98.2 environment, employing the NumPy 2.2.0 and Matplotlib 3.10.3 libraries.

Y = a + b × I + c × D + d × I² + e × I×D + f × D²,

(1)

where Y is the response variable, a, b, c, d, e, f are the estimated parameters of the model, I is the imbibition time, and D is the doses of NaCl or H₂O₂.

3. Results and Discussion

3.1. Effect of Imbibition Time and NaCl Doses on Germination Parameters in Pea Seeds

The GR showed highly significant differences between imbibition times and NaCl doses, with only the interaction exhibiting significant differences (

Table 2. Average germination parameters in pea seeds subjected to different imbibition times and NaCl doses.

Imbibition Time (Hours)	Doses of NaCl (mM)	GR (%)	GP (%)	MGT (Days)	GRI (Number of Seeds Per Day)
12	0	82.92 b	34.17 ab	6.21 a	5.19 d
	50	98.33 a	34.58 ab	5.30 cd	10.12 ab
	100	94.17 ab	35.00 ab	5.62 abcd	8.16 abcd
	150	93.75 ab	33.75 ab	6.04 ab	6.56 cd
24	0	92.08 ab	37.92 ab	5.44 bcd	7.85 abcd
	50	98.75 a	42.92 a	5.17 d	10.61 a
	100	96.25 a	32.50 ab	5.73 abcd	8.80 abc
	150	89.58 ab	24.58 b	6.01 abc	7.27 bcd
36	0	97.50 a	41.67 a	5.04 d	9.73 abc
	50	97.08 a	42.92 a	5.07 d	9.72 abc
	100	98.75 a	44.58 a	5.07 d	9.15 abc
	150	97.50 a	37.50 ab	5.91 abc	7.57 abcd
Factor	Level
Imbibición time (hours)	12	92.29 b	34.38 b	5.79 a	7.51 b
	24	94.17 ab	34.48 b	5.59 a	8.63 ab
	36	97.71 a	41.67 a	5.27 b	9.04 a
Doses of NaCl (mM)	0	90.83 b	37.92 ab	5.56 b	7.59 bc
	50	98.06 a	40.14 a	5.18 c	10.15 a
	100	96.39 a	37.36 ab	5.47 bc	8.70 ab
	150	93.61 ab	31.94 b	5.98 a	7.13 c
ANOVA		Significance
I		**	**	**	**
D		**	*	**	**
I × D		*	ns	**	*

I: imbibition time; D: doses of NaCl; GR: germination rate; GP: germination potential; MGT: mean germination time; GRI: germination rate index. ns: no significant, * and ** indicate a significant effect (p < 0.05 and p < 0.01, respectively). Different lowercase letters indicate significant differences according to the grouping of interactions and individual factor levels, according to Tukey’s test (p < 0.05).

, Figure 2a). The 36 h imbibition produced 5.8% more GR than the 12 h imbibition. Similarly, the 50 mM NaCl dose yielded 7.9% more GR than the control. The evaluated factors indicated that 24 h imbibition at 50 mM NaCl resulted in 6.2% more PG than 12 h imbibition at the control.

A 36 h long imbibition likely allows for uniform hydration of embryonic tissues, increasing gibberellin levels, which facilitates the activity of key hydrolytic enzymes in starch degradation [18]. In contrast, 12 h imbibition may lead to incomplete hydration, which limits the mobilization of reserves and decreases germination. Priming with NaCl (50 mM) and decreased abscisic acid (ABA) levels and increased gibberellins (GA), which activated hydrolytic enzymes like α-amylases that break down starch into soluble sugars, speeding up root emergence. At the same time, the expression of stress-related genes involved in osmoprotection mechanism, such as proline and aquaporins, as well as antioxidant enzymes like SOD and catalase, was upregulated to reduce oxidative damage and make better use of reserves. These processes explain the lower mean germination time (MGT) and the higher germination rate index (GRI), especially with 24 h imbibition, when hormonal synchronization was at its best [19]. In comparison, the control does not promote this stimulus, and seed germination relies solely on endogenous signals.

The GP showed highly significant differences in imbibition time and significant differences in NaCl dose. The interaction between factors did not show significant differences. Imbibition for 36 h yielded 21.2% more GP than that for 12 h, while the 50 mM dose resulted in 25.6% more GP than the 150 mM dose. In terms of the interaction between factors, the combination of 100 mM NaCl and 36 h of imbibition produced 81.3% more GP than 150 mM NaCl for 24 h. Imbibition for 36 h allows for optimal hormonal synchronization; moreover, it is likely that with this imbibition time, ABA is progressively degraded, while gibberellins increase, stimulating cell expansion and uniform rupture of the seed coat [20], which raises the GP values. Regarding the NaCl dose, the application of 50 mM induces mild stress, activating enzymes and enhancing hormonal signaling, which acts as a tolerance priming mechanism that activates stress genes, such as osmoprotectors and aquaporins, to prepare the seedling for future challenges [21]. In contrast, the control does not provide a stimulus, likely due to residual latency or slower metabolic activation, which reduces uniformity in germination.

The MGT showed highly significant results regarding imbibition times, NaCl doses, and the interaction of the evaluated factors (Figure 2b). Imbibing pea seeds for 12 h resulted in a 9.8% higher MGT than imbibing for 36 h. Similarly, the application of 150 mM NaCl increased the MGT by 15.4% compared to the 50 mM dose, which aligns with [12]. They found that, under salinity conditions with a priming of 150 mM NaCl in pea cv. ‘Bolero’, the MGT averaged 12.25 days, higher than the 5.55 days obtained in the present study. They also noted a reduction in total emergence. The combination of 12 h of imbibition with zero mM of NaCl produced the highest MGT, 23.2% greater than the MGT of the treatment with 36 h of imbibition and zero mM of NaCl. Imbibition for 36 h likely allows for the complete hydration of the embryo, accelerating the mobilization of reserves, as optimal activation of enzymes such as amylases occurs. This leads to the degradation of starch and proteins, releasing energy and sugars toward the radicle, which facilitates the rapid breaking of the seed coat and the onset of germination [18]. Furthermore, 36 h imbibition enhances cell expansion through ABA degradation and increased levels of gibberellin [20].

The GRI of pea seeds showed highly significant differences based on imbibition times and priming with different NaCl doses. Imbibition for 36 h resulted in a GRI that was 20.4% higher than that of imbibition for 12 h. In terms of the imbibition dose, 50 mM led to a GRI that was 42.3% higher than the GRI observed with 150 mM NaCl. The interaction between the imbibition factor and NaCl dose also exhibited significant differences, with the combination of 24 h of imbibition at 50 mM yielding the highest GRI, which is 104% faster than the GRI from imbibition for 12 h at a dose of 0 mM.

Similarly, the GRI reported by [12] was 11.09 for treatments with priming, whereas it was 13.2 for treatments without priming. This aligns with the findings of the present study, which showed GRI values of 7.13 and 7.59 for treatments with and without priming, respectively, indicating that 150 mM NaCl delays the germination rate in peas due to the energy expenditure involved in NaCl preparation, during which genes encoding osmoprotectants (proline synthesis genes (P5CS)) and ion transporters (NHX1 for Na⁺/H⁺ exchange) are upregulated to mitigate ionic toxicity [22].

3.2. Effect of Imbibition Time and NaCl Doses on the Dry Mass Distribution of Different Organs in Pea Seedlings

Root dry mass showed significant differences based on imbibition times and highly significant differences regarding NaCl doses (Figure 2c and Figure 3a). The control treatment dose (0 mM) exhibited the highest values, while increasing NaCl doses led to a significant decrease in root dry mass, with the application of 150 mM NaCl resulting in a 21.9% reduction. Similarly, imbibition in NaCl for 24 h produced the most significant root dry mass (Figure 3b). The interaction between imbibition and dose displayed significant differences, with 24 h imbibition and 0 mM yielding the highest values. In comparison, imbibing 150 mM NaCl for 36 h resulted in the lowest root dry mass, which was 40.3% lower.

Similarly, Naz et al. [23] reported that imbibition in the highest doses of NaCl (80 mM) reduced root length in pea seedlings compared to the control. They also noted that nutrient absorption and root growth rates decrease under salinity conditions. Souza et al. [24] found that imbibition of Myracrodruon urundeuva Allemäo seeds in 50 mM NaCl reduced fresh root mass by 57%. This reduction in root dry mass is attributed to NaCl-induced salt stress, which diminishes the plant’s ability to absorb water, leading to decreased growth and the accumulation of toxic ions within the plant, which disrupts the osmotic balance [25]. In this regard, Atta et al. [26] mention that osmotic stress and water deficit increase the production of abscisic acid (ABA), which alleviates the effects of salinity on photosynthesis and growth due to the accumulation of K⁺, Ca²⁺, and proline in root vacuoles, counteracting the absorption of Na⁺ and Cl^-. Similarly, Smolko et al. [27] state that exposure to salinity reduces the size of the root meristem, inhibits the formation of lateral roots, and limits auxin transport due to decreased levels of certain proteins.

Stem dry mass showed highly significant differences between imbibition times and significant differences between doses, while the interaction between the two factors did not reveal any significant differences. Imbibition for 24 h resulted in the highest values of stem dry mass, whereas imbibition for 36 h decreased stem dry mass gain by 20.2%. The 150 mM NaCl dose lowered stem dry mass accumulation by 2.6% compared to the control treatment. In this regard, salt stress reduces water uptake, which impacts cell elongation [26]. Additionally, the accumulation of ABA due to water stress may influence the biosynthesis and transport of auxins, hormones responsible for stem elongation [27].

Both leaflets and stipules exhibited highly significant differences in dry mass based on imbibition time, NaCl dose, and their interaction (Figure 2d,e). Imbibition for 36 h resulted in a 21.9% and 16.7% increase in leaf dry mass accumulation, respectively, compared to imbibition for 12 and 24 h. Likewise, the dry mass of stipules from seedlings obtained from seeds soaked for 36 h was 28.1% and 33.6% higher than those soaked for 24 and 12 h, respectively, indicating that a longer soaking time positively influences stipule dry mass accumulation. Regarding NaCl dose, the application of 150 mM reduced the dry mass of leaves and stipules by 18.8% and 32.7%, respectively, compared to the average of the other doses applied. For the interaction between factors, the combination of 36 h of imbibition and 0 mM of NaCl produced the most significant dry mass of leaves and stipules. In comparison, the lowest results were observed with imbibition for 24 h at 150 mM of NaCl, which was 46% and 63.9% lower, indicating that salinity has a more significant impact on the growth of stipules than on that of leaves.

This is attributed to the fact that ionic toxicity generated by salinity alters cellular homeostasis, inducing the accumulation of ABA, which causes stomatal closure and leads to decreased photosynthesis, ultimately resulting in reduced cell expansion. Similarly, salt-induced water deficit limits cell division in organs such as leaves and stipules [26]. Furthermore, due to oxidative stress, auxins and cytokinins exhibit diminished effects on leaf development [27]. Therefore, it is logical that the hormonal response prioritizes plant survival over plant growth, which would explain the more significant mass losses in stipules, the most sensitive tissues.

The dry mass of the germinated seeds showed highly significant differences between the two evaluated factors individually, while it displayed significant differences for the interaction (Figure 2f). The 36 h soaking period resulted in the highest dry mass of germinated seeds, whereas the 24 h soaking yielded 19.7% less. Regarding the NaCl dosage, the 150 mM dose produced the highest dry mass of germinated seeds, 27.1% greater than the average of the other treatments. Concerning the interaction, soaking pea seeds in 150 mM NaCl for 36 h resulted in 86.6% more dry mass of germinated seeds compared to soaking for 24 h in 0 mM NaCl.

The above suggests that the imbibition of pea seeds in NaCl alters the distribution of seed mass accumulation in seedling organs, as the 150 mM dose limits or delays the translocation of photoassimilates from the seed to the other seedling organs. This results in lower dry mass of roots, stems, leaves, and stipules. In this regard, Atta et al. [26] state that salt stress delays water absorption and favors the accumulation of ABA, which inhibits the activity of hydrolytic enzymes such as α-amylase on starch in the cotyledons. This affects the mobilization of reserves from the seed to the other plant organs, decreasing the initial growth and development of the seedling and generating greater residual dry mass of the seed at the time of measurement.

The total dry mass of pea seedlings showed significant differences in the imbibition time factor; however, no significant differences were observed in the NaCl doses or their interaction. The 36 h imbibition treatment yielded the highest total dry mass values, with increases of 8.9% and 11.4% compared to the 12 and 24 h periods, respectively. The different NaCl doses applied to pea seeds generated an average total seedling dry mass of 9.66 g. Although this difference was not statistically significant, the 150 mM dose resulted in a 0.3% reduction compared to the control treatment. Despite the saline stress, the total dry mass remained unchanged, suggesting a compensation strategy in peas where resource redistribution occurs. The 150 mM dose reduced the dry matter of roots, leaves, and stipules, but did not affect that of the stems, and maintained a higher dry matter content in germinated seeds compared to the control treatment. This suggests a delay in the transport of reserves, likely caused by the effects of salt stress as described by Atta et al. [26].

3.3. Effect of Imbibition Time and NaCl Doses on the Leaf Area of Pea Seedlings

The LA and SLA showed significant differences between the evaluated factors, imbibition time and NaCl dose, while the interaction did not present significant differences for either of the two variables (

Table 3. Average leaf areas of pea seedlings from seeds subjected to different imbibition times and NaCl doses.

Imbibition Time (Hours)	Doses of NaCl (mM)	SLA (cm2)	LA (cm2)	TLA (cm2)
12	0	114.11 cd	257.77 cd	371.88 de
	50	83.15 d	233.97 d	317.12 de
	100	123.12 cd	263.42 cd	386.54 de
	150	90.91 d	221.85 d	312.75 de
24	0	183.19 abc	288.23 cd	471.41 bcde
	50	146.80 bcd	290.03 cd	436.84 cde
	100	184.95 abc	310.33 cd	495.28 bcd
	150	87.08 d	199.38 d	286.47 e
36	0	246.08 a	470.16 a	716.24 a
	50	214.57 ab	447.60 ab	662.16 ab
	100	208.26 ab	392.93 abc	601.18 abc
	150	154.95 bcd	315.27 bcd	470.22 bcde
Factor	Level
Imbibición time (hours)	12	102.82 c	244.25 b	347.07 c
	24	150.51 b	271.99 b	422.50 b
	36	205.97 a	406.49 a	612.45 a
Doses of NaCl (mM)	0	181.13 a	338.72 a	519.85 a
	50	148.17 a	323.87 a	472.04 a
	100	172.11 a	322.22 a	494.34 a
	150	110.98 b	245.50 b	356.48 b
ANOVA		Significance
I		**	**	**
D		**	**	**
I × D		ns	ns	ns

I: imbibition time; D: NaCl doses; SLA: stipules; LA: leaflet area; TLA: total leaf area. ns: no significant, ** indicate a significant effect (p < 0.01, respectively). Different lowercase letters indicate significant differences according to the grouping of interactions and individual factor levels, according to Tukey’s test (p < 0.05).

). A 36 h imbibition increased the SLA and LA by 100.3% and 66.4%, respectively, compared to a 12 h imbibition. In this sense, prolonged imbibition (36 h) likely allows for complete hydration of embryonic tissues, which increases the activity of amylase and protease enzymes that rapidly degrade starch [18], providing more substrates for the initial growth of stipules and leaflets.

On the other hand, the application of control treatment resulted in a 72.2% and 37.9% increase in SLA and LA, respectively, compared to a 150 mM NaCl dose. This decrease can be attributed to the fact that a high concentration of NaCl (150 mM) limits water uptake by the plant, which restricts the cell turgor necessary for the expansion of both leaves and stipules [28]. Additionally, the salt stress caused by the NaCl dose can raise ABA levels, which may induce stomatal closure, thereby reducing the photosynthetic rate and cell growth [26].

When analyzing the TLA, highly significant differences were observed for the two evaluated factors, but not for the interaction, suggesting a parallel or additive effect between the factors. Soaking for 36 h yielded the highest TLA values, 76.5% higher than the TAR generated by seedlings whose seeds were soaked for 12 h. Conversely, the 150 mM NaCl dose decreased TLA by 31.4% compared to the control, which can be attributed to the possibility that seed priming may have induced osmotic stress, thereby limiting water absorption and affecting cell expansion, and ultimately influencing the total leaf area [28].

3.4. Correlation Analysis Between the Parameters Evaluated for the NaCl Applications

The analysis of the correlation coefficients shows that TLA is strongly related to LA (0.97) and SLA (0.94) (Supplementary Table S2), indicating that the total leaf area mainly derives from these two components. The stipule dry mass has high correlations with leaflets dry mass (0.83), SLA (0.76), and TLA (0.78), suggesting that this organ significantly contributes to vegetative development. In contrast, germinated seed dry mass correlates negatively with most morphological variables, especially with root dry mass (−0.68) and stem dry mass (−0.55), indicating a potential trade-off in resource allocation, as previously discussed. MGT shows high negative correlations with variables such as GRI (−0.72) and GR (−0.66), demonstrating that a longer time to reach germinative mass negatively impacts seedling quality and vigor.

3.5. Effect of Imbibition Time and H₂O₂ Doses on Germination Parameters in Pea Seeds

The GR, GP, MGT, and GRI exhibited highly significant differences based on imbibition times, but not in relation to H₂O₂ doses or the interaction of factors (

Table 4. Average germination parameters in pea seeds subjected to different imbibition times and doses of H₂O₂.

Imbibition Time (Hours)	Doses of H2O2 (mM)	GR (%)	GP (%)	MGT (Days)	GRI (Number of Seeds Per Day)
12	0	95.42 ab	37.92 a	5.68 ab	8.31 cd
	20	97.50 ab	37.08 a	5.81 a	8.30 cd
	40	92.92 b	41.25 a	5.70 a	8.06 d
	60	95.00 ab	40.42 a	5.75 a	8.14 d
24	0	99.17 a	51.67 a	5.06 c	9.64 ab
	20	96.25 ab	51.25 a	5.05 c	9.42 ab
	40	99.58 a	47.08 a	5.02 c	9.80 a
	60	97.92 ab	48.75 a	5.18 bc	9.30 abc
36	0	98.75 a	42.08 a	5.66 ab	8.58 bcd
	20	98.33 ab	47.08 a	5.57 ab	8.68 bcd
	40	97.08 ab	41.25 a	5.45 abc	8.84 abcd
	60	97.08 ab	40.00 a	5.44 abc	8.84 abcd
Factor	Level
Imbibición time (hours)	12	95.21 b	39.17 b	5.74 a	8.20 c
	24	98.23 a	49.69 a	5.08 c	9.54 a
	36	97.81 a	42.60 b	5.53 b	8.74 b
Doses of H2O2 (mM)	0	97.78 a	43.89 a	5.47 a	8.84 a
	20	97.36 a	45.14 a	5.48 a	8.80 a
	40	96.53 a	43.19 a	5.39 a	8.90 a
	60	96.67 a	43.06 a	5.46 a	8.76 a
ANOVA		Significance
I		**	**	**	**
D		ns	ns	ns	ns
I × D		ns	ns	ns	ns

I: imbibition time; D: doses of H₂O₂; GR: germination rate; GP: germination potential; MGT: mean germination time; GRI: germination speed index. ns: no significant, ** indicate a significant effect (p < 0.01, respectively). Different lowercase letters indicate significant differences according to the grouping of interactions and individual factor levels, according to Tukey’s test (p < 0.05).

). A 24 h imbibition period resulted in 3.1% and 26.8% higher GR and GP, respectively, compared to a 12 h imbibition period. Similarly, the 24 h imbibition increased GRI by 16.3% compared to the 12 h imbibition. However, the 12 h imbibition produced the highest MGT, which was 12.9% greater than that of the 24 h imbibition. The average GR across all treatments was 97.1%, while the GP was 43.8%, the MGT was 5.4 days to germination, and the GRI was 8.8 germinated seeds per day. Soaking for 24 h provides sufficient hydration, creating an ideal balance for germination by reducing ABA and increasing gibberellins concentration [20], which lowers MGT [29]. Additionally, Hernández [30] notes that during germination, significant changes occur in the seeds’ redox status, featuring a reduction in ABA that depends on H₂O₂. He also indicates that ROS contributes to lowering the ABA/GA ratio, which promotes germination.

The absence of significant differences in the germination parameters of pea seedlings subjected to different doses of H₂O₂ can be attributed to the fact that, in the absence of prior stress, the application of H₂O₂ did not need to regulate any metabolic processes internally. It is known that H₂O₂ plays an important role in signal transduction to increase tolerance to abiotic stress, thereby enhancing the survival capacity of plants under adverse conditions [31]. However, it has been demonstrated that under drought stress conditions, priming with low levels of H₂O₂ stimulates a positive response of genes to stress, resulting in increased vigor indices and germination percentages [32]. In this regard, it is known that H₂O₂ priming triggers ROS signaling genes, including RBOHD (NADPH oxidase) and MAPK signaling pathways (PsMAPK2), which enhance stress memory and promote germination by modulating the ABA/GA ratio [33]. Likewise, Hemalatha et al. [31] mention that the germination percentage of rice seeds increased on average by 12% in treatments with H₂O₂ priming in both sensitive and tolerant varieties to salinity. Furthermore, soaking pea seeds in H₂O₂ enhances germination, seedling growth, and seedling vigor indices because H₂O₂ acts as a signaling molecule at the onset of germination, which involves specific changes at the hormonal and proteomic levels [33].

3.6. Effect of Imbibition Time and H₂O₂ Doses on the Dry Mass Distribution of Different Organs in Pea Seedlings

The root dry mass of pea seedlings showed highly significant differences for both imbibition times and hydrogen peroxide (H₂O₂) doses; however, the interaction between the factors did not show significant differences. The root dry mass obtained after 12 h of imbibition was 17.3% lower compared to that obtained after 24 and 36 h (Figure 4a). Conversely, the 60 mM H₂O₂ dose resulted in the highest root dry mass, which was 4.2% and 26.3% higher than the control treatment and the 20 mM H₂O₂ dose, respectively (Figure 4b). Likely, 12 h of imbibition does not provide sufficient hydration of the seed tissues, thus limiting the activation of hydrolytic enzymes and all the processes associated with germination [18]. Similarly, Rodrigues et al. [34] state that imbibition time affects the expression of genes related to the formation of adventitious roots.

At low concentrations, H₂O₂ serves as a cellular signaler and can activate antioxidant enzymes such as superoxide dismutase, catalase, and peroxidases, which enhance stress tolerance and promote growth. Furthermore, H₂O₂ is known to regulate the expression of genes related to meristematic cell proliferation in the root [35], which may explain the increased root dry mass obtained with the 60 mM dose.

Stem dry mass exhibited highly significant differences across imbibition times, while H₂O₂ doses and their interaction did not reveal significant differences for stem dry mass in pea seedlings. The 36 h imbibition resulted in 22.6% and 60.8% more stem dry mass compared to the 12 h imbibition, with the latter showing an average value of 0.88 g per seedling by the end of the evaluation. This can be attributed to the complete hydration of the embryo induced by the 36 h imbibition. Conversely, the evaluated H₂O₂ concentrations were likely insufficient to induce a priming effect. Additionally, pea seeds contain enzymatic systems that neutralize H₂O₂, preventing oxidative stress [13] and leading to the absence of significant differences in stem dry mass.

Highly significant differences were observed in imbibition times, as well as significant differences in H₂O₂ doses and the interaction between factors for the leaflets’ dry mass (Figure 5a). Imbibitions for 24 and 36 h resulted in 24.7% and 30.4% more leaf dry mass, respectively, compared to imbibition for 12 h. This is attributed to the fact that longer imbibition times lead to more efficient metabolic and hormonal activation, which are essential for faster leaf growth and development [36].

The 0 mM H₂O₂ dose produced 22.7% more leaf dry mass than the 60 mM H₂O₂ dose, which had the lowest values. Regarding the interaction, 36 h imbibition and 0 mM showed the highest leaf dry mass values, 72.4% higher than the 12 h imbibition and 60 mM H₂O₂ dose, which exhibited the lowest results. According to Barba-Espín et al. [33], the dose of 60 mM of H₂O₂ can be considered high, potentially inducing oxidative stress that causes damage to lipids, proteins, and DNA, leading to inhibition of cell division and elongation. This, in turn, affects leaf growth, as the process will require additional energy expenditure to activate defense mechanisms.

Stipule dry mass showed highly significant differences in imbibition times and significant differences in the interaction; however, the H₂O₂ dose factor did not display statistical differences. Imbibition for 24 h was 21.2% and 47.4% greater than for 36 and 12 h, respectively. The combination of a 24 h imbibition time and a 60 mM H₂O₂ dose produced the highest stipule dry mass, 94.1% higher than the biomass reported for the treatment with 12 h of imbibition and 20 mM H₂O₂.

The dry mass of germinated seeds exhibited highly significant differences concerning the imbibition time factor and the interaction, but it did not show differences regarding the H₂O₂ dose factor. A 12 h imbibition period resulted in 20.4% and 89.9% more germinated dry matter than the 24- and 36 h imbibition periods, respectively. The combination of factors that maximized the accumulation of germinated seed dry matter in pea seedlings was a 12 h imbibition period with a dose of 60 mM H₂O₂, which was 124.4% higher than the combination of 36 h and 60 mM, the latter having the lowest values. This indicates that adequate imbibition promotes the translocation of photoassimilates to other emerging parts of the plant. In contrast, a mere 12 h imbibition period does not ensure complete seed hydration [36], leading to an initial delay in seedling development.

The total dry mass of pea seedlings showed highly significant differences between imbibition times and their interaction, while significant differences were observed only for the H₂O₂ dose (Figure 5b). Imbibition for 24 h resulted in the highest total dry mass values, measuring 2.9% and 17.5% greater than those for imbibition times of 12 and 36 h, respectively. Concerning the dose factor, the control treatment had, on average, 5.7% more total seedling dry mass compared to the treatments that received H₂O₂ application. According to Makhaye et al. [36], the imbibition process can be divided into three phases: in the first, rapid water absorption occurs; in the second, metabolic activation takes place; and in the third, embryo growth begins. Therefore, it is likely that 24 h imbibition allows seeds to adequately complete these phases, especially Phase II, which is crucial for activating hydrolytic enzymes. The fact that H₂O₂ application improved seed vigor by increasing the mass of different organs without enhancing germination parameters may be due to H₂O₂ acting as a signaling molecule that regulates redox balance, but not necessarily accelerating initial germination [33]. In this context, the 60 mM dose of H₂O₂ did not improve GR or GP; however, its effect was to induce stress tolerance by activating antioxidant enzymes, which supported post-germination growth. It is known that high concentrations of H₂O₂, in addition to the oxidative damage they produce, alter hormone balance, as H₂O₂ stimulates the catalysis of ABA, inhibiting the germination process and generating an imbalance in the ABA/GA ratio [37].

3.7. Effect of Imbibition Time and H₂O₂ Doses on the Leaf Area of Pea Seedlings

The SLA showed highly significant differences between imbibition times and was significant among the different H₂O₂ doses, while the interaction demonstrated highly significant differences (

Table 5. Average leaf areas of pea seedlings from seeds subjected to different imbibition times and H₂O₂ doses.

Imbibition Time (Hours)	Doses of H2O2 (mM)	SLA (cm2)	LA (cm2)	TLA (cm2)
12	0	247.15 bce	542.38 ab	789.53 abcd
	50	215.29 cde	383.00 b	598.29 cd
	100	230.63 bcde	433.59 b	664.22 bcd
	150	200.01 e	329.52 b	529.53 e
24	0	336.69 ab	545.54 ab	882.23 abc
	50	283.85 abcde	432.84 b	716.69 bcd
	100	306.01 abcde	528.34 ab	834.35 abc
	150	319.75 abc	552.30 ab	872.05 abc
36	0	310.93 abcd	510.02 ab	820.94 abcd
	50	291.74 abcde	724.12 a	1015.86 a
	100	205.40 de	544.28 ab	749.68 abcd
	150	357.51 a	551.22 ab	908.73 ab
Factor	Level
Imbibition time (hours)	12	223.27 b	422.12 b	645.39 b
	24	311.57 a	514.75 a	826.33 a
	36	291.39 a	582.41 a	873.80 a
Doses of H2O2 (mM)	0	298.25 a	532.65 a	830.90 a
	20	263.63 ab	513.32 a	776.95 a
	40	247.35 b	502.07 a	749.42 a
	60	292.42 ab	477.68 a	770.10 a
ANOVA		Significance
I		**	**	**
D		*	ns	ns
I × D		**	**	**

I: imbibition time; D: H₂O₂ doses; SLA: stipules; LA: leaflet area; TLA: total leaf area. ns: no significant, * and ** indicate a significant effect (p < 0.05 and p < 0.01, respectively). Different lowercase letters indicate significant differences according to the grouping of interactions and individual factor levels, according to Tukey’s test (p < 0.05).

and Figure 5c). Imbibition for 24 h was 39.4% and 6.9% higher than imbibition for 12 and 36 h, respectively. Regarding H₂O₂ doses, seedlings from the control treatment displayed the highest SLA values, surpassing all tested H₂O₂ doses, specifically 20.5% higher than the SLA recorded with the 40 mM H₂O₂ dose. Conversely, the combination of 36 h of imbibition and 60 mM H₂O₂ resulted in the highest SLA, which was 78.7% higher than the application of 60 mM H₂O₂ with 12 h of imbibition.

Significant differences were observed for both LA and TLA regarding the imbibition factor and the interaction, while LA and TLA did not exhibit significant differences for the various H₂O₂ doses evaluated. The 36 h imbibition period resulted in LA and TLA increases of 37.9% and 35.4%, respectively, compared to the 12 h imbibition treatment. Conversely, the control indicated 11.5% and 10.8% higher LA and TLA, respectively, than the applications of 60 and 40 mM H₂O₂. Concerning the interaction, the combination of a 36 h imbibition and a 20 mM H₂O₂ dose achieved the highest LA and TLA, at 119.7% and 91.8% greater than the 12 h imbibition with a 60 mM dose, illustrating the significant impact that LA has on TLA.

3.8. Analysis of the Response Surface Models Obtained for Pea Seed Germination Parameters and Seedling Biomass

Table 6. Estimated parameters of the response surface equation for pea seedlings from seeds subjected to different imbibition times and NaCl doses.

Estimated	GR	MGT	RDM	LDM	SDM	DMGS
a	84.583333	6.292854	1.474960	1.982127	0.497687	6.332820
b	0.114583	−0.020423	0.092714	−0.060040	−0.010087	−0.259080
c	0.216667	−0.015844	0.009269	0.008608	0.003287	−0.011612
d	0.005787	−0.000392	−0.001329	0.001854	0.000573	0.004968
e	−0.002222	0.000234	−0.000288	−0.000204	−0.000095650	0.000415
f	−0.001000	0.000088895	−0.000042440	−0.000038553	−0.000015473	0.000065800
r2	0.3036	0.5580	0.4111	0.5164	0.6027	0.5706

GR: germination rate; MGT: mean germination time; RDM: root dry mass; LDM: leaflets dry mass; SDM: stipule dry mass; DMGS: dry mass of germinated seed; r²: determination coefficient.

presents the estimated coefficients of the response surface model obtained for the variables of seed germination and seedling growth of pea for NaCl doses. The GR shows a high base value (a = 84.58%) but a low coefficient of determination (r² = 0.3036), suggesting that other factors independent of those evaluated influence the GR. The MGT decreases with increasing NaCl doses and imbibition time (negative coefficients for b and c), with a moderate coefficient of determination (r²) (0.5580), indicating that these factors partially explain the observed variability. The dry mass of roots and leaves showed contrasting responses, since root dry mass increases with imbibition time (positive b), while leaflet dry biomass decreases (negative b). Stipule dry mass and germinated seed dry mass exhibited the highest r² values (0.6027 and 0.5706, respectively), indicating that the model best explains these variables. Salt stress and imbibition time differentially affect biomass distribution, with a notable impact on germination and initial seedling growth.

Table 7. Estimated parameters of the response surface equation for pea seedlings from seeds subjected to different imbibition times and H₂O₂ doses.

Estimated	LDM	TDM	SLA
a	0.445370	8.197823	78.391033
b	0.115916	0.298814	19.797850
c	0.001729	−0.023079	−4.055098
d	−0.001893	−0.006829	−0.376688
e	−0.000316	−0.000886	0.037388
f	0.000006875	0.000700	0.049815
r2	0.3469	0.4763	0.4126

LDM: leaflets dry mass; TDM: total dry mass; SLA: stipule leaf area; r²: determination coefficient.

presents the estimated coefficients for leaf and total dry mass, as well as for stipule leaf area (SLA) in response to the H₂O₂ assay. The SLA intercept (a) value is the highest at 78.39, indicating that stipules are sensitive to oxidative stress. Conversely, the negative coefficients for c (H₂O₂ dose) on total dry mass and stipule leaf area suggest that high concentrations reduce these parameters. However, the low R² values (0.3469 and 0.4763, respectively) indicate that other factors influence the variability, such as the relatively low average temperature (15.8 °C) and the high relative humidity (60%) in the greenhouse, which can affect enzymatic activity and water absorption. Likewise, the substrate may impact water retention and aeration, influencing the initial hydration of the embryo [38]. Additionally, the relatively few hours of sunlight can affect gibberellin synthesis, which is crucial for mobilizing reserves, and the innate genetic variability of the seeds may have contributed to the stress response [39]. The estimated value for the product of imbibition time and dose (e) has a slight but significant effect on SLA, suggesting that imbibition time modulates the response to H₂O₂. Overall, H₂O₂ impacted leaf biomass more than total biomass.

3.9. Correlation Analysis Between the Parameters Evaluated for the H₂O₂ Applications

Based on the analysis of the correlation coefficients between variables (Supplementary Table S2), it was observed that the variable TLA shows strong positive correlations with (0.94) and SLA (0.70), suggesting a close relationship between foliar and total inputs. Likewise, germinated seed mass has notable negative correlations with root dry mass (−0.42), stem dry mass (−0.64), and TLA (−0.50), indicating a possible antagonism in resource allocation. On the other hand, GRI presents a positive correlation with GR (0.64) and GP (0.62), indicating that higher seedling vigor is associated with better weight and germination. In contrast, MGT is negatively correlated with several variables, including GRI (−0.94), suggesting that a longer time to reach mass is linked to reduced vigor. These results highlight key relationships among growth, biomass allocation, and germination performance.

4. Conclusions

Under controlled conditions (15.8 °C and 60% relative humidity), using 50 mM NaCl for 24 h effectively improved pea germination by increasing the germination rate by 7.9% and shortening the mean germination time. For practical purposes, applying 50 mM NaCl is recommended as an effective and scalable treatment. While priming with H₂O₂ promoted seedling growth, it did not significantly enhance germination parameters, likely due to variety-specific responses or environmental factors such as the substrate used or the limited sunlight hours (4 to 6 h) during the experiment. Future research should aim to optimize H₂O₂ usage across different pea varieties and explore its mechanisms on seed physiology. Additionally, further studies will test the treatments identified here under actual production growing conditions.