# Osmopriming Increases Seed Germination of Amaranthus cruentus (L.)

Arnaud Busquère, Dominique Lefebvre, Patrice Galaup, Lucas Tricoulet, Charline Musset, Eric Lacroux, Othmane Merah

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Arnaud Busquère, Dominique Lefebvre, Patrice Galaup, Lucas Tricoulet, Charline Musset, et al. "Osmopriming Increases Seed Germination of Amaranthus cruentus (L.). Journal of New Seeds, 2025, 4 (3), pp.37. 10.3390/seeds4030037. hal-05250376

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Article

# Osmopriming Increases Seed Germination of *Amaranthus cruentus* (L.)

Arnaud Busquère<fnref n="1" />, Dominique Lefebvre<fnref n="2" />, Patrice Galaup<fnref n="3" />,†, Lucas Tricoulet<fnref n="3" />,‡, Charline Musset<fnref n="3" />,§,
Eric Lacroux<fnref n="1" /> and Othmane Merah <fnref n="1" />,<fnref n="2" />,*

## Abstract

Amaranth is considered a gluten-free, protein-rich pseudocereal. However, seed dormancy affects the germination rate and production. The aim of this study was to investigate the effects of osmopriming, hydropriming, priming with Algal and chia-seed extract biostimulants, scarification, and different combinations of them on seed germination. The results showed that hydropriming, osmopriming, (at least by 13%), and speed (two days earlier) of germination compared to the control. The same trend was observed, to a lesser extent, for priming with a biostimulant. The production of osmopriming has recently started in France. However, seed priming offers a promising solution to overcome the lack of germination. The aim of this study was to test several priming methods and their impact on amaranth seed germination. An imbibition curve was first established and showed that the first two germination phases were reached at 12 h after imbibition. Duration had no effect on germination compared to the control. In contrast, increasing the rate measurement of polyphenol oxidase (PPO) activity revealed a peak during the first few hours. The largest peak was observed for this. These results suggest growing amaranth by increasing the germination rate.

**Keywords:** soaking; imbibition; scarification; osmopriming; amaranth; PEG; polyphenol oxidase activity; seed

## 1. Introduction

The genus *Amaranthus* (L.) includes up to eight hundred species of amaranth, herbaceous annual dicotyledonous plants considered to be pseudocereals, as well as quinoa, buckwheat, or millet. Today, amaranth (*Amaranthus* spp.) is an essential part of the diets of people in Latin America and Africa [1]. Consumed as a vegetable mainly for its leaves, it is also found in the diets of certain pre-Columbian civilizations. Amaranthus species are a very protein-rich food, higher than most common cereals, with almost 18% protein content [2], close to quinoa. This gluten-free pseudocereal represents a potentially especially important nutritional source for meeting the world's food challenges. Its use as a vegetable or snack is developing in new territories, with nutritional values close to those of current

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Academic Editor: Aria Dolatabadian

Received: 1 June 2025

Revised: 20 July 2025

Accepted: 6 August 2025

Published: 12 August 2025

**Citation:** Busquère, A.; Lefebvre, D.; Galaup, P.; Tricoulet, L.; Musset, C.; Lacroux, E.; Merah, O. Osmopriming Increases Seed Germination of *Amaranthus cruentus* (L.). *Seeds* **2025**, *4*, 37. https://doi.org/10.3390/seeds4030037

**Copyright:** © 2025 by the authors.
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<footnote n="1">¹ Laboratoire de Chimie Agro-Industrielle (LCA), Université de Toulouse, INRAE, F-31030 Toulouse, France; busquerea@gmail.com (A.B.); eric.lacroux@ensiacet.fr (E.L.)</footnote>

<footnote n="2">² Département Génie Biologique, IUT, Université de Toulouse, F-32000 Auch, France; dominique.lefebvre@iut-tlse3.fr</footnote>

<footnote n="3">³ Ovalie Innovation, 2 Rue Marguerite Duras, F-32000 Auch, France</footnote>

<footnote>* Correspondence: othmane.merah@ensiacet.fr</footnote>

<footnote>† Current address: Natais Company, F-32130 Bézéril, France.</footnote>

<footnote>‡ Current address: LEGTA Beaulieu Lavacant, F-32550 Pavie, France.</footnote>

<footnote>§ Current address: Regar Association, F-32000 Auch, France.</footnote>
flours. This plant contains the highest squalene content per seed in the plant world [3–5], making it a crop of great interest. Squalene, an isoprenoid, has several benefits for human health. It is an intermediate for the biosynthesis of all steroid hormones and cholesterol in animals and plants [6]. The squalene is used in various fields of application such as the growth of tumors in the colon, skin, lungs, and breast. It is also used as an emollient in vaccines [7,8]. In the cosmetics industry, this molecule is used in moisturizing creams as an agent that rapidly penetrates the skin without leaving oily traces or sensations on the skin [1].

Amaranth is able to provide mature seeds with tolerance to the summer conditions of temperature and humidity. The physiological particularity of C4 photosynthesis enables it to develop a high biomass over a very short period [9]. There are three main species of seeded Amaranthus cultivated worldwide. These are *A. hyponcondriacus*, *A. caudatus* and *A. cruentus*, the latter being native to North and Central America [10]. It is a dioecious plant, with broad leaves and a very short cycle, enabling rapid, moderate growth (two meters long at maturity). With a low thousand-grain weight (TGW) of around one gram, the seeds are very small (0.5 to 1 mm) and white, golden, or pink in color [11]. This very small diameter presents an obstacle for amaranth cultivation, as sowing techniques are not sufficiently developed to make it a main crop. Recent interest in amaranth is linked to the nutritional composition of its grains, mainly due to their high protein and starch content. The absence of gluten is another factor in its appeal [11]. However, these species suffer from emergence difficulties depending on environmental and intrinsic parameters [12–14]. It is therefore important to have uniform and sufficient germination to preserve emergence and ensure the production of these species. Indeed, germination tests also revealed contrasting germinative capacities. Depending on the agronomic parameters of the planting zone and the depth of sowing, the success rate at emergence is lower than the estimated potential of the amaranth seed. Moreover, germination can be erratic, as the seeds do not all sprout in the same manner or at the same moment. This may lead to differences in growth and dry matter production [15–19].

Several solutions can be proposed to overcome this limitation. Chemical, physical, or hormonal treatments, or miscellaneous methods have been used [17,18,20–22]. Scarification has been among the first used methods to break dormancy or tegument's inhibition [18,23–25]. Chemical priming is another possibility to increase germination [17,26,27]. Other studies have used osmopriming, hydropriming, halopriming, hormonal priming [17,19], or nanopriming [28].

In the Amaranthus species, seed priming has been assessed to improve seed germination. These techniques have been used in order to improve crop yield. Unfortunately, there are few studies that have examined different solutions for improving amaranth germination using hydropriming and osmopriming with polyethylene glycol (PEG) 6000. This work has highlighted the impact of priming on the germination of *A. cruentus* and *A. hypochondriacus* [29]. Moreover, this report has focused on hydro- and osmopriming without establishing an imbibition curve. There is no study of the effect of scarification on the germination of amaranth seeds or its combination with priming techniques. Furthermore, the effect of the biostimulant has not been studied. In order to determine the optimum conditions for priming *A. cruentus*, the establishment of an imbibition curve should be addressed. There are no studies that have reported other methods to enhance germination in amaranth seeds. Therefore, the aim of this study was to investigate the comparative effect of different priming techniques, scarification, or both, on the germination rate. Moreover, the activity of the polyphenol oxidase PPO, as a key enzyme involved in oxygen and phenol regulation, was also examined.
## 2. Materials and Methods

Seeds of Panam and Biocoop genotypes of *Amaranthus cruentus* were purchased from Biocoop (Auch, France) and from Panam Cie (Villemur-sur-Tarn, France). This second genotype of *A. cruentus* has been used in the whole experiment. The pre-germination treatments were studied on seeds that had been disinfected with 1% calcium hypochlorite for 15 min (Thermofisher formerly Acros Organic, 2021, Seneffe, Belgium) and then rinsed with distilled water.

### 2.1. Kinetics of Imbibition Seed of *Amaranthus Cruentus*

In order to determine the optimal imbibition time for the hydropriming of those two genotypes of *A. cruentus*, imbibition kinetics were achieved. We measured the weight gained, following the water entry into the seed over the course of 24 h. To obtain all the values needed, two samples of seeds (with the same dry weight) were used. The weight measurements of the first sample were performed during the first 11 h. The weight measurements of the second sample were performed for 11 h, after 13 h of imbibition. The trial was performed with 100 seeds of each sample, spread into 5 repetitions of 20 seeds, for the two genotypes. Seeds are placed on top of 3 layers of Wattman paper (Dutcher, 2013, Issy-les-Moulineaux, France), soaked with 5 mL of water, into a 55 mm wide Petri dish. The seeds were dry weighed on a precision scale, every hour, to obtain an evolution curve of the average seed weight. In order to determine the curve of the kinetics of imbibition as a function of the measurement of the percentage of water accumulated in the seed over 24 h, the following formula was applied:

$$ \text{Seed water uptake percentage} = \frac{(\text{Weight}(t) - \text{Dry Weight})}{\text{Dry Weight}} \times 100 $$

In order to determine what could explain the difference between those two genotypes, we measured the moisture content of the seeds at the same time. We measure the initial weight of 5 repetitions of 20 seeds of each genotype. After calcination at 100 °C for 48 h, the mean mass was measured again. The moisture content is expressed as a percentage by means of the following formula:

$$ \text{moisture content}(\%) = \frac{(\text{Fresh Weight} - \text{Dry weight})}{\text{Dry Weight}} \times 100 $$

All data were expressed as mean standard deviation. The analysis of variance and average germination rates were carried out using Fisher's and Student's tests, respectively.

### 2.2. Germination Test

The factor experiments were conducted on the impact of hydropriming, osmopriming, and mechanical scarification on germination rate and speed. The experiment was carried out with five repetitions. For each modality of each factor, all seeds were treated for 15 min with a 10% sodium hypochlorite antifungal treatment. Seeds were then washed three times for three minutes with distilled water. For each treatment, 10 seeds were deposited in a Petri dish and repeated five times, for a total of 50 seeds. Manual shelling is not easy because of the size of the seed (0.6–0.8 mm) [23]. Therefore, mechanical scarification was carried out using a G76 mechanical shaker (New Brunswick Scientific Co. Inc., 2012, Edison, NJ, USA) at 200 rpm and at 25 °C. Seeds in a beaker, with the lower fraction of sand sieved at 500 µm, were stirred for 15 min. After separating seeds and sand by a new 500 µm sieve, seeds were either packed or subjected to three hours of hydropriming in water or half-diluted natural chia biostimulant (as recommended by the provider). Unprimed and not scarified seeds were used as controls.
The imbibition for osmopriming and hydropriming was achieved using a G76 mechanical shaker (New Brunswick Scientific Co. Inc., 2012, Edison, NJ, USA) at 200 rpm, and each period of lamination was followed by dehydration in the oven for 24 h under the protection of light, before germination. To avoid this germination and to use the seeds later, we dried them in an oven. Hydropriming was studied over one or two soaking periods, with different soaking times, coupled with the use of a chia-based biostimulant. The hydropriming controls were carried out over three hours with water in the negative control and a 5% and 1% algal root biostimulant for the positive control. The priming tests with 50% chia extract were conducted on a single or double coating for 1 h or 3 h. Osmopriming was studied by soaking seeds in a PEG 6000 solution, following the method described by Moosavi et al. [29]. Osmotic priming was tested for three and six hours with an osmotic potential of -10 bar according to the Kaufmann equation [30]. The seeds were rinsed in three 10 min baths in distilled water before drying for 24 h at 30 °C in an oven, sheltered from light. The soaking time of six hours for osmopriming is based on the scientific literature [29]. The choice of three hours for hydropriming is based on the major imbibition period of the seed (seed imbibition curve, see Section 3).

Petri dishes were incubated closed, at room ambient conditions (temperature of 22 °C). Natural conditions at Auch in May 2024 were 15 h light/9 h dark. Germination tests included measurements every 24 h for four days and then after one week. To determine the germination rate, the following formula was applied:

$$Germination (\%) = \frac{\text{Number of germinated seeds} \times 100}{\text{Total seeds}}$$

The T50 germination rate was measured with the time (in hours) required for 50% of seeds to germinate. All data were expressed as mean ± standard deviation. The analysis of variance and average germination rates were carried out using Fisher's and Student's tests, respectively.

## 2.3. Determination of Polyphenol Oxidase Activity

The plant material used is the seeds of the disinfected amaranth to which the different priming modalities have been applied. A control with only one disinfection was retained. Hydropriming was tested for three hours, and osmopriming with six hours of PEG 6000 (Sigma-Aldrich, formerly Merck-Schuchardt, 2014, Schnelldorf, Germany), with an osmotic potential of -10 bar according to the Kaufmann equation. With control, the plant material used for enzymatic extractions will be extracted from 0.5 mg of seed per sample.

### 2.3.1. PPO Extraction

The seeds were germinated in Petri dishes for 0 h, 12 h, and 24 h. Seeds of each phase of germination were ground separately in a mortar and homogenized according to the procedure of Mishra and Patra [31], in 2 mL of 0.1 mol.L⁻¹ phosphate buffer at pH 6.8. The samples were swirled and then kept in ice until centrifuged at 8000× g for 10 min. Based on the Moosavi et al. [29] method, the sample was used to measure enzymatic activity. The extracted supernatant was kept in the freezer (−20 °C) until analysis.

### 2.3.2. Assay of PolyPhenol Oxidase (PPO) Activity

The procedure is based on the method established by Mayer et al. [32] and Avallone et al. [33] and modified by Romuald [34]. Seed extract (100 µL) was added to a mixture containing 1.25 mL of phosphate buffer (Na₂HPO₄/NaH₂PO₄ at 0.2 mol.L⁻¹, pH 6.8), and 150 µL of catechol at 50 mmol.L⁻¹. The blank is composed of only 1.5 mL of phosphate buffer, and the enzyme control for each modality is composed of 1.4 mL of phosphate buffer and 100 µL of the sample. The activity of PPO was determined by measuring the kinetics
of the enzyme at 25 °C for 240 s at 420 nm in a 3 mL PMMA vessel using a Jenway 7315 spectrophotometer (Bibby Scientific, Saint Ouen, France) capable of performing kinetics. Polyphenol oxidase activity is expressed in units per milliliter (U.mL⁻¹) of extract. One enzymatic unit (U) is defined as the amount of enzyme required to catalyze the oxidation of 1 µmol of catechol per minute at 25 °C and pH 6.8.

**3. Results and Discussion**

The objectives of this study were to evaluate different priming techniques on Amaranthus seed germination. In order to determine the duration of imbibition, the curve of water absorption was established.

Imbibition curves are displayed in Figure 1. For both amaranth seeds, the water absorption increased significantly during the first three hours. For example, the water content in the Panam seed rose three times during the first three hours.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Kinetics of seed imbibition of *Amaranthus cruentus* L. Different letters indicate significant mean differences based on (test t of Student) at 0.05 probability level.</figcaption></figure>

In order to determine the optimum priming times for *Amaranthus cruentus* seeds, the imbibition kinetics curve was run on two genotypes. The time required for optimum water absorbance over the first few hours was determined. Water uptake after three hours of treatment was greater than 35% of the initial seed weight. After 6 h of soaking, the seeds absorbed more than 40% and 45% for Panam and Biocoop, respectively. Finally, these seeds absorbed 50% of their initial weight in water in just 9 h.

This is the first report on the kinetics of water absorption by amaranth seeds. Nevertheless, there are numerous studies on other species.

The average mass of the Biocoop seed is more than 20% higher than Panam (Table 1). We observed moisture content by dehumidification at 100 °C, and the Biocoop seed contained 2% less water than Panam (Table 1). Moreover, the thickness of the hull was 0.21 mm and 0.22 mm for Panam and Biocoop (Figure 2), respectively. All these reasons may explain the observed difference in imbibition speed between the two genotypes.
<table><caption>**Table 1.** Comparing initial mass and moisture content of two genotypes of *Amaranthus cruentus*.</caption><thead><tr><th>Genotype</th><th>Initial Mass (mg)</th><th>Initial Seed Moisture (%)</th></tr></thead><tbody><tr><td>Panam</td><td>0.782 ± 0.018 <sup>a</sup></td><td>16.13 ± 0.012 <sup>a</sup></td></tr><tr><td>Biocoop</td><td>1.082 ± 0.016 <sup>b</sup></td><td>14.15 ± 0.009 <sup>b</sup></td></tr></tbody></table>

Different letters within the same column indicate significant mean differences based on test t of Student at a 0.05 probability level.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Longitudinal section of seeds from two amaranth genotypes. (a) Panam and (b) Biocoop.</figcaption></figure>

The soaking phase of *Amaranthus cruentus* can be estimated over the first twelve hours. Continuing to hydrate the amaranth seeds until their latent phase has the potential to improve germination [29]. Without going beyond this phase, hydration would enable earlier DNA replication, embryonic growth, facilitated RNA production, and better ATP availability. On the other hand, once the irreversible phase III, known as the growth phase, has been reached, with cell elongation and radicle protrusion, priming is impossible.

Studies have already investigated the imbibition kinetics of *Vigna unguiculata* seeds to determine the end of imbibition phase I, just before the start of the latent phase [16]. This period represents the first germination phase stricto sensu, i.e., a still reversible phase in which germination initiation takes place. Most water enters through the seed coat during these first few hours [35]. During phase II of germination, rehydration of the seed does not cause any damage (does not affect the ability to germinate). It is then possible, when this stage is reached, to carry out a (hydro)priming treatment as a pre-germination treatment to stimulate germination. The third phase, characterized by metabolic activation, means that rehydration would be lethal for the seed. In chia seeds, the imbibition phase has a duration of 6 h [36–38].

Germination speed and germination rate were measured with different pre-germination treatments, and the results are displayed in Table 2. The duration of the hydropriming and osmopriming treatments was chosen according to the imbibition curve in order to optimize the hydric and osmotic treatment process on the seed (Figure 1). After six to twelve hours, the imbibition of the *A cruentus* seed tended towards a maximum of 150 to 160% of its initial mass. The germination speed decreased by 24 h, induced by priming with water or a root biostimulant (95% germination), consistent with other reports [15,16]. Double hydropriming for 1 h with a natural biostimulant based on chia mucilage resulted in 24 h less germination than the control. The germination rate was not affected by the hydropriming treatment (Table 2). The duration of priming is the critical factor in hydropriming. Seeds must not be primed for too long so as not to initiate metabolic processes in phase III of germination. This fact complicates the creation of a universally applicable protocol. For example, the ideal response time for chia is 6 h [36–38], whereas it is around 100 h for
*Allium cepa* [39]. Hydropriming was proposed as a valuable, safe technique to increase germination in several species [20]. On the contrary, other studies have concluded that hydropriming is not effective for certain species [40–42]. Moreover, in our study, we tested two hydration times based on the imbibition curve (Figure 1). There was no difference between the imbibition times in terms of germination speed, although the shorter times of imbibition resulted in a higher germination rate (Table 2). The same trend was observed in *Hibiscus sabdariffa* [43]. Therefore, it seems important to clearly define the parameters that facilitate hydration, such as the treatment temperature, its duration, the quantity of water, the environment, as well as the plant species, and in some cases, even the variety used [42–46].

<table><caption>**Table 2.** Effects of priming and scarification on germination speed and germination rate of *A. cruentus* after seven days.</caption><thead><tr><th>Modality</th><th>Germination Speed T50 (hours)</th><th>Germination Rate at T50 (%)</th><th>Germination Rate (%)</th></tr></thead><tbody><tr><td>Control seeds</td><td>72</td><td>71 ± 5.85 <sup>b</sup></td><td>84 ± 5.85 <sup>b</sup></td></tr><tr><td>Scarification</td><td>72</td><td>71 ± 9.65 <sup>a</sup></td><td>84 ± 3.7 <sup>a</sup></td></tr><tr><td>Scarification and hydropriming</td><td>48</td><td>73 ± 9.25 <sup>a</sup></td><td>86 ± 9.65 <sup>a</sup></td></tr><tr><td>Scarification with algal biostimulant</td><td>72</td><td>68 ± 13.25 <sup>a</sup></td><td>81 ± 9.7 <sup>a</sup></td></tr><tr><td>Control with chia extract</td><td>48</td><td>57 ± 8.7 <sup>a</sup></td><td>80 ± 6.3 <sup>a</sup></td></tr><tr><td>Control with algal biostimulant</td><td>48</td><td>56 ± 11.1 <sup>a</sup></td><td>95 ± 5.45 <sup>b</sup></td></tr><tr><td>Simple hydropriming 1 h</td><td>72</td><td>62 ± 12.85 <sup>a</sup></td><td>75 ± 11.4 <sup>a,c</sup></td></tr><tr><td>Simple hydropriming 3 h</td><td>72</td><td>52 ± 7.45 <sup>a</sup></td><td>61 ± 3.7 <sup>c</sup></td></tr><tr><td>Dual hydropriming 1 h</td><td>48</td><td>63 ± 11.4 <sup>a</sup></td><td>79 ± 11.1 <sup>a</sup></td></tr><tr><td>Dual hydropriming 3 h</td><td>72</td><td>58 ± 5.05 <sup>a</sup></td><td>68 ± 12.45 <sup>c</sup></td></tr><tr><td>Osmopriming 3 h</td><td>24</td><td>63 ± 6.41 <sup>b</sup></td><td>97 ± 3.75 <sup>b</sup></td></tr><tr><td>Osmopriming 6 h</td><td>24</td><td>74 ± 8.74 <sup>a</sup></td><td>100 ± 2.45 <sup>b</sup></td></tr></tbody></table>

Different letters within the same column indicate significant mean differences based on test t of Student at a 0.05 probability level.

The osmopriming treatments had a significant effect on the germination speed and germination rate of *Amaranthus cruentus*. Indeed, the germination of osmoprimed seeds was three times faster than the control (Table 2). The germination rate was nearly 20% higher than control seeds (Table 2). The impact of priming has already been demonstrated in numerous studies for different species [15,28,47–49]. The germination rate is improved after soaking in water or an osmotic solution (Table 2). Emergence is faster and more uniform than in the absence of priming [15], and there is an increase in vigor, a reduction in germination speed, and longer root length [29]. Osmopriming has been found as an effective method in numerous plant species [27,29,39,47–50].

Scarification has no positive impact on the germination of *Amaranthus cruentus*, except in combination with priming (Table 2). This result can be explained by the fact that priming is more effective on germination. Moreover, this result mirrored that the lack of germination is not due to mechanical inhibition but rather to a chemical one. This finding confirms reports previously published on other species [16,20,26]. Chemical scarification resulted in a better germination rate [51] but is less effective than mechanical scarification on species such as Fabaceae [52], with a more practical and chemically safer treatment on seeds [23,53].

Seed priming is a pre-germination breeding technique that will lead to the early emergence of seedlings by controlling metabolic processes in the first phases of germination [54]. It improves uniform germination by reducing the duration of imbibition [55], enhancing the activation of pre-germinative enzymes, and boosting the production of metabolites [56].
Polyphenol oxidase activity peaks in the first few hours after water imbibition at sowing [31]. The measurement of polyphenol oxidase activity was determined from an average of the initial velocities on seeds with or without priming for nine modalities (Figure 3). Priming led to very high activity on the imbibition part, as the control without pre-germinative treatment upstream of the VIC showed zero activity. The enzymatic activity of PPO was significantly higher for the hydropriming and osmopriming treatments at the time of germination (Figure 3). PPO activity peaks at around 12 h with the hydropriming treatment, earlier than the control, during the first of the three phases of seed germination [16,35]. With the osmopriming treatment, PPO activity is already at a maximum as soon as VIC is initiated, which correlates with the results of the study and the early stage induced by soaking in the osmotic PEG 6000 solution. With a germination speed of 24 h and a germination rate of almost 100%, an osmopriming treatment on Amaranthus cruentus offers a solution to the germination difficulties of *Amaranthus cruentus*.

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Polyphenol oxidase activity of amaranth seeds primed with different methods. Different letters indicate significant mean differences based on test t of Student at a 0.05 probability level; 1 U is defined as the amount of enzyme that catalyzes the transformation of 1 µmol of catechol in 1 min under the assay conditions.</figcaption></figure>

The presence of polyphenols in the seed coat has been demonstrated for many species [23]. The study on the priming of *A. cruentus* and *A. hypochondriacus* showed an increase of around 50% in peroxidase and 40% in polyphenol oxidase [29]. It now appears that this activity is not constant over time. Priming forces early and much higher activity of polyphenol oxidase during the first few hours. This activity is closely correlated with the entry of more water into the plant [35] during the first few hours of the imbibition phase [57]. The role of water and PEG 6000 in priming would have a greater impact on the
phenolic compounds present in the seed coat [58-60]. The hypothesis of a physical phenomenon of leaching by priming is not accurate. Priming is thought to trigger polyphenol oxidase activity. The high level of PPO activity would lead to partial chemical degradation of the inhibiting substances in the integuments, which would block germination [23,61,62]. The increased water entry during the first hours of priming imbibition has a triggering effect on the first phase of germination and leads to the early degradation of polyphenols, the antioxidants present in the tegument upstream of germination. Moreover, primed seeds showed an increase in antioxidant activities, higher solubilization of stored proteins, and a decrease in the peroxidation of lipids [54,63]. An increased activity of PPO was reported in safflower treated with zinc oxide nanoparticles [64]. The treatment of potatoes with low-temperature plasma jet technology resulted in a clear increase in PPO activity during germination [65]. Likewise, spermine treatment improved drought-induced osmotic stress by raising polyphenol oxidase activities in several crop species [66-69]. This is associated with improved signaling, which contributes to plant adaptation to water stress [67,69].

One of the main types of dormancy involves rigid teguments that limit the diffusion of water and oxygen. It is induced by germination inhibitors (like phenols) that suppress seed germination [70]. The presence of phenols in the seed coat strongly limits the diffusion of oxygen to the embryo [71,72]. Polyphenol oxidase catalyzes the hydroxylation of monophenols into o-diphenols. The latter are then converted to quinones with molecular oxygen as the electron acceptor [64,73]. Their activities are enhanced when plants are exposed to abiotic stress. It is well known that PEG simulates water stress. However, the rapid hydration caused by priming could probably induce an early release of phenols present in the seed coats [69,71,72,74], leading to an increased PPO [65-68].

**4. Conclusions**

The present study aimed to examine the effects of different ways of priming in order to improve amaranth seed germination. The observed results showed that osmopriming presented a higher germination rate than the control and other investigated methods. This is probably due to the stimulation of polyphenol oxidase activity observed in this study. These findings highlight the potential interest of osmopriming to improve the germination rate of amaranth seeds. This study has been carried out under controlled laboratory conditions. Therefore, the results should be ascertained by experiments under field conditions to establish the benefits of this method for this species. Moreover, the benefits of osmopriming should be studied on seedling emergence and vigor as well as on advanced growth stages and seed production. Moreover, it could be interesting to complete this study by investigating the behavior of primed seeds under different environmental conditions.

**Author Contributions:** Conceptualization, O.M., P.G., L.T., C.M. and A.B.; methodology, A.B., D.L., O.M., E.L. and P.G.; software, A.B., D.L., E.L. and O.M.; validation, O.M., D.L. and P.G.; formal analysis, A.B., D.L., C.M. and O.M.; investigation, A.B., O.M., D.L., C.M. and L.T.; resources, O.M., P.G., L.T. and D.L.; data curation, A.B. and E.L.; writing—original draft preparation, A.B., L.T., C.M. and E.L.; writing—review and editing, O.M., D.L. and P.G.; visualization, O.M. and D.L.; supervision, O.M.; project administration, O.M.; All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author due to potential industrial applications.
**Conflicts of Interest:** Authors Patrice Galaup, Lucas Tricoulet, and Charline Musset were employed by the company Ovalie Innovation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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