Effective Priming Techniques to Enhance Ghaf (Prosopis cineraria L. Druce) Seed Germination for Mass Planting

Abstract

Seed priming is a cost-effective and efficient pre-sowing technique that promotes seed germination and plant growth under abiotic stress. This study was conducted to investigate the effect of different priming treatments on breaking the dormancy of ghaf (Prosopis cineraria L. Druce) seeds. Furthermore, the effects of storage conditions and seed coating on seed germination were examined. Treatments with hot water, KNO₃, KCl, polyethylene glycol (PEG), H₂SO₄, gibberellin (GA₃), cytokinin benzyl adenine (BA), and H₂SO₄ + BA were investigated. Among all the tested treatments, seeds responded the best to the H₂SO₄ 50% 2 min + BA (200 ppm) priming treatment in terms of germination uniformity and effectiveness. This treatment initiated germination 5 days after sowing and increased the germination percentage from 11.32% in the untreated seed to 83% and 100% in the primed ones after 5 and 20 days, respectively. The germination percentage and primary root length were shown to be more responsive to this combined treatment, followed by the H₂SO₄ 50% 2 min+ BA 100 ppm. This treatment achieved the best result of 100% germination when the seeds were covered in a seed ball. Additionally, the treated seeds could be stored at room temperature or 4 °C for more than 10 days with no negative effect on the germination. Based on these findings, this treatment could be recommended to farmers and plant producers to boost the mass production of ghaf trees.

1. Introduction

Ghaf (Prosopis cineraria L. Druce) is an evergreen flowering tree of the Fabaceae family. It is native to the arid regions of Western Asia, the Middle East, and the Indian Subcontinent, including India, Pakistan, Afghanistan, Iran, the United Arab Emirates (UAE), Oman, Saudi Arabia, and Yemen [1]. The tree is a xerophytic plant that thrives in dry environments, producing more blooms and fruits in drought conditions [2]. It can also survive extreme summer temperatures (40–48 °C) and low temperatures of less than 10 °C [3].

Traditionally, different communities have valued native ghaf trees for the versatility of all their parts. Indeed, ghaf has been dubbed “King of the Desert” or “the Wonder Tree” [4]. Apart from its cultural significance for the Bedouin traditional lifestyle, it is designated as the national tree of the UAE [1]. The ghaf tree, together with the date palm and sidr, makes substantial contributions to the agricultural economy and rural development, including soil stabilization, soil nutrition (biological nitrogen fixation), shade, and food for a wide range of wild animals and domesticated species [4,5].

In the wild, the ghaf tree reproduces poorly and is difficult to propagate vegetatively [6,7]. Seeds are compressed, oval, hard, and dark brown in color, with mucilaginous endosperm enveloping the embryo; cotyledons are rounded, flat, and epigeous when germination occurs [3]. The seeds germinate in 10–20 days under suitable conditions [3], and seed dormancy after sowing results in slow seed germination and inconsistent seedling emergence, which causes varying plant stands [8]. Moreover, tender seedlings are exposed to soil-borne pathogens for a prolonged period due to the uneven seed germination and seedling emergence. Dormant seeds are the main obstacle to growing tree seedlings; hence, it is crucial to overcome them before germination can begin [9].

Seed priming is a pre-sowing technique that partially hydrates seeds without causing germination by exposing them to a specific solution [10,11]. Earlier studies have revealed that seed priming improves germination and early development of seedlings due to the changes in enzyme activation, metabolic activities, protein synthesis, the biochemical process of cell repair, and an improvement in the antioxidant defense system [9,12]. Various priming methods have been developed to boost the speed and synchronicity of seed germination, such as hydro-/osmo-, nutrient-, chemical-, bio-, and physical priming [9].

Hydro-priming is a simple and inexpensive approach in which seeds are soaked in water for a certain amount of time and then dried to a particular moisture level before sowing [13]. This technique has been used effectively to improve the germination capacity and uniformity of seedling emergence in many crops [12,14]. In osmo-priming, osmotica such as polyethylene glycol (PEG), sugars (e.g., mannitol and sorbitol), and salts (e.g., NaCl, NaNO₃, MgCl₂, and KNO₃) are added to the solution to suppress water uptake [15]. The physiological responses of seed germination to osmotic stress have been described [9,16,17]. It was also shown that seeds priming with sulfuric acid improved seed germination and disrupted the dormancy of Sabina vulgaris and Adansonia digitata seeds by enhancing water absorption by the embryo and softening the seed coat tissue [18,19].

Among pre-sowing treatment approaches, hormonal priming using plant hormones has been highly effective [20,21]. It has been reported that seed priming treatment with plant growth hormones, such as auxins, gibberellins, cytokinins, salicylic acid, and abscisic acid significantly improved the germination performance of many crop species under both normal and stressed conditions [21,22]. For instance, in dicot plants, the stimulatory effects of cytokinins on seed germination have been widely investigated.

To boost germination, seeds have been coated with a wide range of materials [23]. Seed coating is conducted to enhance protection, germination, and water absorption [23]. For instance, in Iceland, diatomaceous earth was used as a seed coat for Leymus arenarius to extend storage and serve as a mechanical barrier during sowing [24].

Seeds must also be stored in a way that maintains their viability for long periods. Recommendations on storage conditions have been proposed by the International Board for Plant Genetic Resources’ Advisory Committee on Seed Storage. Cold storage preserves plant tissue and improves storability and shelf life [25,26]. Seeds with a moisture content of 3–7% at base collection should be stored in sealed containers. Although subzero temperatures are acceptable, temperatures of −18 °C or below are desirable. It is recommended that seeds with ≤7% moisture content be stored at less than 15 °C in sealed bags, especially in tropical areas [27].

Although numerous studies investigating the influence of seed priming treatments on the germination performance of seeds from various plant species have been conducted, the literature contains limited information on ghaf seed germination in the literature. Therefore, this study was initiated to establish the most effective seed priming treatment for enhancing the germination of ghaf seeds and to determine the optimal conditions for storing of the primed seeds. In addition, improving the growth performance of seedlings in sandy soil using seedball with different compositions was also investigated.

2. Materials and Methods

2.1. Place and Experiment Conditions

The research was conducted during September 2021–May 2022. All priming treatments and seed germination tests in Petri dishes were conducted in the lab at room temperature 22 ± 1 °C and humidity 50–60%. The soil pot experiment was done in a polycarbonate greenhouse at the experimental farm, United Arab Emirates University, Al Ain, UAE (co-ordinate latitude and longitude of 24.2191° N and 55.7146° E), with natural light, and controlled evaporative cooling system with average daily temperature 19.2–28.4 °C, and relative humidity 40–60%.

2.2. Biological Material and Substances Used for Seed Priming

Ghaf seeds (Prosopis cineraria L. Druce) were collected from the Environmental Protected Areas Authority in Sharjah, UAE) 25.28260° N, 55.6951 °E(, and stored in plastic boxes at 4 °C before being tested for germination. Chemicals used for seed priming treatments (H₂SO₄, gibberellin [GA₃], and BA) were purchased from Sigma-Aldrich Canada Co., Ltd. (Oakville, ON, Canada), other chemical reagents (PEG, KCl, and KNO₃) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.3. Seeds Priming Experimental Set-Up

Osmo-, hydro-, acid-, and hormone-priming treatments were investigated. Before use, seeds weighing 10 g were surface sterilized with 100 mL of sodium hypochlorite (0.1%) for 2 min, rinsed with distilled water three times and then treated. Immediately after each priming treatment, seeds were rinsed with distilled water three times and dried on blotting paper at room temperature (22 ± 1 °C) overnight in darkness before sowing. Untreated control ghaf seeds were only surface sterilized, rinsed with distilled water and dried before sowing. All solutions were prepared with distilled water.

2.3.1. Hydro-Priming (With Hot Water)

Ghaf seeds were immersed in hot water (100 °C) for 1 or 5 min. After treatment, the seeds in a mesh were immediately placed in cold tap water for 5 min to stop the heating reaction and then washed with distilled water three times, and dried as described above before sowing.

2.3.2. Osmo-Priming

Osmo-priming treatments were performed using KNO₃ (0.5% and 2%), KCl (1% and 2%), and PEG 6000 (10% and 20%). The seeds were fully immersed in the solutions for 24 h in light, and the flasks were sealed with a hole on top and aeration was provided through a narrow tube (6 mm inner diameter) connected to a small aquarium pump for supplying air into each solution.

2.3.3. Acid-Priming with Sulfuric Acid

For acid-priming, ghaf seeds were immersed in 25% H₂SO₄ (for 10, 20, or 30 min) and 50% concentration (for 2, 5, or 10 min). Then, the seeds were washed with distilled water three times and dried at room temperature (22 ± 1 °C) overnight in darkness before sowing.

2.3.4. Hormone Priming

Stock solutions (1 mg/mL) of GA₃ and BA hormones were prepared by mixing 100 mg of the hormone powder and 3–5 mL of solvent (Ethanol for GA₃ and KOH for BA). Once dissolved, the volume was completed to 100 mL by adding distilled water. The stock solutions were used to prepare different concentrations of each hormone. Ghaf seeds were immersed in GA₃ solution at concentration of 750, 100, or 1500 ppm, or with cytokinin BA at 50, 100, or 200 ppm for 24 h in light, then washed with distilled water three times and dried at room temperature overnight in darkness before sowing.

2.3.5. Acid-Hormone Priming

For treatment with the combination of sulfuric acid and hormone, ghaf seeds were treated with 50% H₂SO₄ for 2 min, then washed with distilled water three times followed by immersing in BA (100 or 200 ppm) for 16 h (overnight in light). Seeds were then washed with distilled water three times and dried at room temperature overnight in darkness before sowing.

2.4. Seed Response to Priming Treatments

2.4.1. Color, Size, and Physical Changes

Immediately after each priming treatment, seeds were sorted according to physical changes, i.e., seed weight, size (swollen seed), and color. Specifically, the seed dimensions (length, width), and weight of 50 seeds were recorded. Seed color values (L, a, b) were also measured using a colorimeter (HunterLabInc., Reston, VA, USA). Then, the number of seeds that responded to the treatments (swollen) was recorded and the response percentage was calculated using the following equation [28]:

Responsive seed (%) = number of seeds that responded to the priming treatment/
total number of seeds × 100.

2.4.2. Seeds Germination Indicators and Seedling Growth

Germination in Petri Dishes and the Primary Radicle Length

After each treatment, 30 seeds were placed on Whatman No.1 filter paper (sterilized at 105 °C for 1 h) in 8 cm diameter Petri dishes. Five replicates of Petri dishes were prepared for sowing and kept at room temperature (22 ± 1 °C). During the germination experiment, the seeds were supplied with 2 mL distilled water on a daily basis as needed. Seed germination was observed daily, and data on the number of germinated seeds was recorded every 5 days for up to 20 days. Seeds were considered germinated when the radicles appeared and were roughly >5 mm in length. After 10 days of sowing, the primary root radicle length was measured for all treatments. The germination percentage was calculated in accordance with the method reported by Patil et al. [28] as follows:

Seed germination (%) = The number of germinated seeds at a specific time
interval of trial/number of initial sowed seeds × 100.

Seedlings Emergence in Sandy Soil

Seedling emergence was also investigated in sandy soil. Seeds treated with BA and/or H₂SO₄ were placed in pots of 7 cm in height and 10 cm in top circumference, with 450 g of sand in each pot. Ten pots (replications) were used for each treatment, and 10 seeds were planted in each pot. The pots were watered with 120 mL of water on the day of planting to wet the sand, and then every other day with 60 mL of water. Seedling’s emergence (when the cotyledon leaf emerged above the soil surface) was observed every day, and data were collected every 5 days. The emergence percentage was calculated as follows:

Seedling emergence (%) = The number of emerging seedlings at a specific time
interval of trial/number of initial seeds planted × 100.

Seed Ball Preparation and Seedlings Emergence in Sandy Soil Substrate

To protect and support the treated ghaf seed during planting in the desert, seed balls were designed to cover the seeds. The seed ball was prepared from various components in different quantities. The final combination that produced a firmly integrated ball contained the following: 100 g of sand, 34 g of bentonite clay, 1.3 g of biochar, 65 g of compost, 10 g of mycorrhiza granules, and 120 mL of water. All the seed ball ingredients were obtained from Emirates biofertilizer factory, Al Ain, UAE, except the biochar was obtained from Shalimar Biotech Industries LLC, Dubai, UAE. The mixture pH was 7.22 and EC was 0.65 dSm/m. The mixture was hand-blended until a coherent ball was produced. Seed balls with diameters of 1.5–1.7 cm, and weights of 8.6–9.5 g were prepared; two treated seeds were placed in each seed ball. Seed balls were planted in pots with sandy soil in the greenhouse and watered every other day. Seedling’s emergence of the seed was observed every day, and data were collected every 5 days. Seedling length and leaf number were recorded every 5 days for a period of 20 days.

2.5. Effect of Primed Seed Storage (Conditions and Duration) on Germination Parameters

Seeds from the combined priming treatment (H₂SO₄ + BA) were divided into 27 piles of 150–200 seeds (3 bags × 3 replicates × 3 storage conditions) plus three piles for control no storage (immediately planted after priming treatment). The plastic bags (groups of 18 bags) were vacuum-sealed and stored at one of three different storage conditions: room temperature (22 ± 1 °C), 4 °C, or −20 °C, for a period of 3, 7 and 10 days. After each storage period, three bags were withdrawn from each storage condition, and seed sowing was performed in Petri dishes (as described above). Germination was monitored daily, and data on the number of germinated seeds were recorded every 3 days for up to 15 days. The germination percentage was calculated as described above.

2.6. Statistical Analysis

The results of the experiment in Petri dishes and soil regarding the effects of priming treatment were statistically analyzed by one-way analysis of variance (ANOVA). The data from the Petri dish experiments are presented as mean values across replicates, with five repeats (30 seeds in each Petri dish), and with 10 replicates for soil experiments (10 seeds in each pot, or two seed balls in each pot) for each treatment. The analysis was performed using SAS (SAS Institute Inc., Cary, NC, USA). The mean values were compared using Fisher’s least significant differences (LSD) test at a 5% level of significance (p ≤ 0.05).

3. Results

3.1. Seed Response to Priming Treatment

Ghaf seed imbibition (swelling) was observed after each treatment, as evidenced by changes in seed weight, size, and color.

Table 1. Responsive seeds percentage after different priming treatments.

Treatments	Responsive Seed (%)
Control	20.11 ± 1.11 g
Hot water (1 min)	48.22 ± 2.13 d
Hot water (5 min)	33.72 ± 2.71 f
KCl (1%) 24 h	14.88 ± 1.07 h
KCl (2%) 24 h	20.99 ± 1.33 g
KNO3 (0.5%) 24 h	10.60 ± 0.84 h
KNO3 (2%) 24 h	19.45 ± 1.73 g
PEG6000 (10%) 24 h	20 ± 1.28 g
PEG6000 (20%) 24 h	20 ± 0.98 g
H2SO4 (25%), 10 min	33 ± 0.91 f
H2SO4 (25%), 20 min	40 ± 1.01 e
H2SO4 (25%), 30 min	64 ± 1.32 c
H2SO4 (50%), 2 min	73 ± 0.68 b
GA3 (750 ppm)	30 ± 1.10 f
GA3 (1000 ppm)	40 ± 1.15 e
GA3 (1500 ppm)	80 ± 1.28 a
Cytokinin BA (50 ppm)	27 ± 1.04 f
Cytokinin BA (100 ppm)	35 ± 0.88 f
Cytokinin BA (200 ppm)	32 ± 1.21 f
H2SO4 (50%) for 2 min + BA (100 ppm)	70.2 ± 1.77 b
H2SO4 (50%) for 2 min + BA (200 ppm)	82 ± 5.13 a

The values are the mean ± standard error. Means followed by different letters are significantly different (LSD test; p ≤ 0.05). Polyethylene glycol (PEG), gibberellin (GA3), cytokinin Benzyl adenine (BA).

shows that ghaf seeds responded differently to different priming treatments. The seed-response percentage varied according to the concentration/duration of treatment and ranged from 10% to 82%. The highest response was observed upon treatment with 50% H₂SO₄ for 2 min + BA. Seeds responding to the priming treatment had significantly (p ≤ 0.05) greater weight, length, and width than those that did not respond to the treatment or the original seeds (Figure 1). It was also noted that the responsive seeds were lighter in color (higher L value) than the original seeds. Additionally, the seeds that did not show any sign of response were smaller in size and weight and darker in color (Figure 1).

3.2. Seed Germination in Petri Dishes and Primary Root Length

The effects of different priming treatments, durations, and concentrations on germination percentage and primary root length of ghaf seeds in Petri dishes are shown in

Table 2. The effect of different priming treatments on ghaf seeds germination in Petri dishes.

Treatments	Day 0	Germination (%), 5 DAS	Germination (%), 20 DAS	Primary Root Length (cm)_10 DAS
Control	10	11 ± 0.73 g	23.33 ± 1.11 gh	0.55 ± 0.01 e
Hot water (1 min)	10	20 ± 1.2 f	50 ± 4.46 e	0.65 ± 0.09d e
Hot water (5 min)	10	21 ± 0.8 f	35 ± 1.23 fg	0.50 ± 0.06 e
KCl (1%) 24 h	5	12 ± 0.73 g	16.67 ± 0.98 h	1.15 ± 0.11 bc
KCl (2%) 24 h	5	15 ± 0.52 g	21.67 ± 1.21 h	1.15 ± 0.13 bc
KNO3 (0.5%) 24 h	5	3 ± 0.25 h	11.67 ± 0.79 h	0.7 ± 0.03 d
KNO3 (2%) 24 h	5	7 ± 0.53 gh	21.67 ± 1.03 h	0.9 ± 0.07 cd
PEG6000 (10%) 24 h	5	7 ± 0.34 gh	20 ± 1.07 h	1.1 ± 0.20 c
PEG6000 (20%) 24 h	5	10 ± 0.33 g	20 ± 1.07 h	1.15 ± 0.13 bc
H2SO4 (25%), 10 min	5	47 ± 2.7 d	93.33 ± 1.97 b	0.85 ± 0.07 d
H2SO4 (25%), 20 min	5	47 ± 2.5 d	96.67 ± 2.25 b	1.05 ± 0.08 c
H2SO4 (25%), 30 min	5	64 ± 1.9 c	80 ± 2.34 c	1.15 ± 0.13 bc
H2SO4 (50%), 2 min	5	73 ± 4.1 b	100 ± 3.06 a	1.2 ± 0.11 b
GA3 (750 ppm)	5	3 ± 0.07 h	30 ± 1.10 g	0.55 ± 0.05 e
GA3 (1000 ppm)	5	8 ± 1.0 gh	40 ± 1.15 f	0.70 ± 0.05 d
GA3 (1500 ppm)	5	13 ± 1.11 g	80 ± 1.28 c	0.75 ± 0.03 d
Cytokinin BA (50 ppm)	5	27 ± 2.1 e	60 ± 5.81 e	0.50 ± 0.02 e
Cytokinin BA (100 ppm)	5	35 ± 1.8 e	70 ± 3.11 d	0.50 ± 0.04 e
Cytokinin BA (200 ppm)	5	32 ± 2.4 e	100 ± 4.37 a	1.15 ± 0.12 bc
H2SO4 (50%) for 2 min + BA (100 ppm)	5	43 ± 4.6 d	76.7 ± 2.21 cd	1.2 ± 0.13 b
H2SO4 (50%) for 2 min + BA (200 ppm)	5	83 ± 5.4 a	100 ± 4.65 a	1.45 ± 0.10 a

The values are the mean ± standard error (n = 5). Means followed by different letters are significantly different (LSD test; p ≤ 0.05). DAS, days after sowing, polyethylene glycol (PEG), gibberellin (GA3), cytokinin Benzyl adenine (BA).

and Figure 2 and Figure 3. After 5 and 20 days after sowing (DAS), there were significant differences (p ≤ 0.05) in the germination parameters of ghaf seeds between the different treatments, at all times and concentrations. For all treatments, the germination percentage increased as the concentration increased, or the duration of the priming treatment increased.

The seeds subjected to hydro-priming with hot water for 1 min exhibited a significantly (p ≤ 0.05) higher germination percentage (50%) than those subjected to the 5 min treatment (35%) and the control group (23.33%) (Table 2). Meanwhile, the germination continued to increase at 5, 10, and 15 DAS, reaching its maximum at 20 DAS (Figure 2A).

All osmo-priming treatments (KNO₃, KCl, and PEG) negatively affected the germination percentage, with decreases in the range of 11–20% when compared with the control (23.33%) and other treatments, after 5 and 20 DAS (Table 2). Among all of the treatments, treatment with KNO₃ (0.5%) was associated with the lowest germination percentage (3%). Increasing the concentration improved the germination percentage across all of the osmo-priming treatments (Table 2, Figure 2B,C). Meanwhile, KCl and PEG treatments significantly (p ≤ 0.05) improved primary root length compared with that in the control group.

Seeds treated with 50% H₂SO₄ for 5 and 10 min were severely damaged, and no germination was detected; hence, only the 2 min treatment was used for further investigation. In accordance with the present findings (Table 2 and Figure 2), acid priming treatment appeared to be the best option for enhancing the germination percentage of ghaf seeds with this rate ranging from 47% to 73% at 5 DAS, and 80% to 100% at 20 days. The concentration of 25% significantly (p ≤ 0.05) improved the germination percentage from 23.33% in the control group to 96.67% at 20 DAS. Meanwhile, the 10 min treatment with 50% H₂SO₄ damaged the cotyledons. Treatment for 2 min duration gave the highest germination percentages at 5 (73%) and 20 DAS (100%) (Table 2).

Table 2 and Figure 2E,F show the impacts of BA and GA₃ on the germination percentage, and primary root length of ghaf seeds. The results show that in the seeds primed with BA (at 50, 100, and 200 ppm), there were significant (p ≤ 0.05) increases in the germination percentage at 5 days (27%, 32%, and 35%, respectively) and 20 days after sowing (60%, 70%, and 100%, respectively) compared with the findings in the control group and groups with other treatments. Meanwhile, treatment with GA₃ (750, 1000, and 1500 ppm) had a significantly (p ≤ 0.05) lower effect than the control. For both hormones, increasing concentration significantly (p ≤ 0.05) improved the germination percentage (Figure 3).

Although BA showed an optimal effect on the germination percentage (100% after 20 days) in the Petri dish experiment, the response of the ghaf seeds to this treatment ranged from 27% to 32% after 5 days; this was lower than the response for sulfuric acid treatment which gave faster germination ranged from 33% to 73%. This indicates that the acid treatment is more advantageous than the hormone treatment (Table 1). The combination of acid and hormone treatments were also tested. The results showed that combined treatment of acid and hormone (50% H₂SO₄ 2 min + 200 ppm BA) achieved significantly (p ≤ 0.05) better results for ghaf germination with germination percentages of 83% and 100% after 5 and 20 days after sowing, respectively. Among all the tested treatments, the germination percentage, and primary root length were shown to be more responsive to the combined treatment of 50%H₂SO₄ 2 min + 200 ppm BA, followed by the 50% H₂SO₄ 2 min + 100 ppm BA (Table 2, Figure 2G). Based on these results, the most successful treatments were further examined for their effectiveness in soil.

3.3. Seedling Emergencein Sandy Soil

Figure 4 shows the seedling emergence of hormone-primed ghaf seeds in soil pots experiment. All cytokinin-primed seeds had significantly (p ≤ 0.05) higher emergence percentages than the control (Figure 4A). At 20 days after planting, seeds primed with 200 ppm BA alone had the highest emergence percentage (80), followed by those subjected to 100 ppm, and 50 ppm treatments. Seeds primed with 50% H₂SO₄ for 2 min in combination with 200 ppm BA had a significantly (p ≤ 0.05) higher emergence percentage (80%) than those primed with 50% H₂SO₄ + 100 ppm BA and the unprimed ones (Figure 4B). These results are consistent with the results of the Petri dish experiment above.

3.4. Seed Ball Experiment Results

Figure 5 shows the germination percentage of ghaf seeds in seed balls). In all treatments seedling emergence occurred after 10 days, whereas in the control group, it began after 15 days. The emergence increased over time and peaked 20 days after planting. Ghaf seed balls with seeds primed using 50% H₂SO₄in conjunction with 200 ppm BA had a significantly (p ≤ 0.05) the higher emergence percentage (84%) than the seeds primed with sulfuric acid only and the control (Figure 5B). The length of seedlings and the number of true leaves were also recorded for the ghaf seedlings growing in a pot with sandy soil (Figure 5C,D). The combined treatment produced longer seedlings and more true leaves than the treatment with H₂SO₄ alone and the control treatment because emergence was initiated earlier in the former group (Figure 5C).

3.5. Storage of Primed Seeds

Ghaf seeds primed with the combined treatment (H₂SO₄ + BA) were stored at room temperature (22 ± 1 °C), 4 °C, or −20 °C for 10 days. At 3 and 6 DAS, control seeds (no storage) had a significantly higher germination percentage than the stored seeds (Figure 6). Storage at room temperature and at 4 °C had no adverse effect on seed germination. There were no significant differences (p ≥ 0.05) in the germination percentage between seeds stored at room temperature and those stored at 4 °C, regardless of the storage period (3, 7 or 10 days) (Figure 6). At 3, 6, and 9 DAS, the seeds stored for 3 days at room temperature had germination percentages of 20%, 66%, and 80%, respectively. Meanwhile, seeds stored for 7 and 10 days under similar conditions had ~50% germination at 3 DAS. A similar trend was observed for seeds stored at 4 °C as the seed germination continued to increase with time up to 15 DAS. All the seeds stored at −20 °C were damaged, and only one of them germinated. In this group, at 12 DAS, no germination was recorded (Figure 6).

4. Discussion

The hydro-priming of ghaf seeds with hot water was shown to typically enhance the germination characteristics under laboratory conditions, which is consistent with the previous studies on other plant species [9,29]. Similar results were described for the hydro-priming of broad bean (Vicia faba L.) seeds, with significant improvements compared with unprimed seeds [14]. Immersing seeds in water has various impacts on their germination and subsequent seedling growth, ranging from beneficial effects to adverse effects or having no effects at all, depending on the type of seed used, priming conditions, duration of immersion, and moisture content of the seeds [12]. In this study, hydro-priming treatment enhanced the germination percentage of ghaf seeds with that in the control group, this effect was lower than that for the other treatments.

Although several reports have shown that seed priming using osmo-priming is an efficient approach for enhancing seed germination, seedling emergence, and stress tolerance of a variety of agricultural plants [9,17], in the present study, the germination percentage was adversely affected by all osmo-priming treatments, to varying degrees. These results are consistent with those reported by Tahaei et al. [30], who found that priming fennel seeds with 0.4% KNO₃ in Petri dishes did not significantly increase their germination. In addition, when Conopodium majus (Gouan) Loret seeds were exposed to various concentrations of KNO₃, no significant improvement in the embryo development rate was noted [31].

The immersion of ghaf seeds in concentrated sulfuric acid for different durations was clearly shown to be effective at disrupting their dormancy. A similar finding was reported by Manga and Sen [8], who found that treatment with H₂SO₄ (95%) for 10 or 15 min significantly improved the germination of ghaf seeds, as the slow and poor germination could be due to physical dormancy. It is thus essential that seeds are subjected to some type of chemical or physical pretreatment prior to sowing for rapid and uniform germination. The findings on acid priming treatment in the present study could be attributed to the breaking of physical dormancy caused by the dissolution of the waxy structure of the epidermis and dissolving the impermeable seed coat. If the seed coat is removed, whether chemically or physically, water is absorbed, oxygen will diffuse to the embryo, and germination takes place, indicating that the seed coat is the source of hardness [18]. In several tree species, increased water absorption has been shown to overcome seed dormancy [32].

Regarding hormone priming, it has been reported that plant hormones such as cytokinin GA₃ play significant roles in promoting germination [24,33]. In addition, GA₃ has been reported to promote seed germination and stem elongation and to mediate a variety of plant developmental processes [34]. According to Demir et al. [33], the use of GA₃ dramatically accelerated the germination of eggplant (Solanum melongena) seeds. Similarly, seed priming with GA₃ improved the percentage and rate of seed germination in several crop species, including maize, lentils, and wheat [34,35]. It also boosted growth and yield. However, there is still a need for further investigation of the mechanisms by which GA₃ priming acts on seed germination.

BA priming treatment showed a high but slow effect on the ghaf seed germination. The germination peaked (100%) at 200 ppm but only after 20 DAS. Similar findings have been reported by Mangena [36], who found that priming soybean seeds with BA improved the amount of root biomass, and rates of blooming, and fruiting in drought-stressed plants. In addition, priming old groundnut (Arachis hypogaea L.) seeds with BA (150 ppm) improved germination and seedling indexes by boosting antioxidant enzyme activities and reducing oxidative damage [37]. However, the exact mechanisms by which priming with cytokinin improves germination have not been characterized. The exogenous application of this plant growth regulator was reported to reduce abscisic acid ABA-induced stomatal closure, because cytokinin plays a key role in stomatal movement [36].

Despite the fact that the maximum seed germination (100%) was obtained in the three treatments: acid alone, BA alone, and the combined treatment of acid + BA 20 DAS, the combined treatment showed the fastest effect as the germination percentage reached 83% at 5 DAS. This could be attributed to the combined effect of acid and cytokinin. Thus, this treatment was further examined for its effectiveness in soil.

Considering the outcomes of the current study, the treatment with 200 ppm BA alone or in combination with 50% H₂SO₄ can be recommended as the optimal treatment for enhancing the germination of ghaf seeds in soil. Owing to the structural differences in the embryos of different seed types and the tissues that surround the embryos, germination and priming mechanisms may differ significantly among different seeds at the cytological and biochemical levels [17]. In addition, because of genetic variation, ambiguous blooming and seed development patterns, and other natural phenomena, seed populations usually conceal significant variations in structure and physiological conditions, which affect how long it takes a seed to germinate [17]. Understanding such species-specific physiological characteristics might provide useful insights to improve mass production, at least within the seed industry. Despite this, the techniques used in our research are simple for farmers to use and produce results quickly, enabling one to test the most recommended priming chemicals and immersion times before beginning any large-scale planting.

To improve seed protection, germination, and water absorption, seed ball was developed as an effective supporting strategy during planting in sandy soil (imitating the desert soil). Seed cover has been used for seed protection purposes in different species [23,38]. Leymus arenarius L. seeds were coated with diatomaceous earth to enhance storage and provide a mechanical barrier to protect seeds during germination [24]. In this study, the seed balls were made from various components at different proportions. These components included clay, compost, sand, and biochar to provide essential nutrients for the seedlings as well as improve the seed ball’s physical structure. Adding vesicular arbuscular mycorrhiza (VAM) granules was also essential for enhancing the root establishment of the ghaf seedling and increasing the uptake of nutrients from the seed balls. Primed ghaf seeds showed a high germination percentage even with a seed ball when planted in pots with sandy soil in the greenhouse. Therefore, this seed ball has the potential to be used for the mass planting of ghaf in the desert to support the seedlings during the growth season.

Regarding the storage of primed seeds, ghaf seeds behaved differently when stored at various temperatures (room temperature, 4 °C, and −20 °C). The treated seeds did not withstand storage at −20 °C; specifically, the seed peel had disintegrated, and a malodorous liquid was noted. For these seeds, no germination was observed throughout the experiment, indicating that the seeds had lost their viability. The treated seeds had a higher weight (Figure 1), indicating that their moisture level was still relatively high, and storing them at −20 °C would damage the embryo, leading to germination failure. Similar results from previous studies indicated that the storage of seeds at temperatures less than −18 °C causes significant damage, especially in tropical areas [39,40]. In ghaf seeds stored at room temperature or 4 °C, there were no adverse effects on seed viability regardless of the storage period (3, 7, or 10 days). This confirms the earlier reports describing that those seeds with a moisture level of 7% can be stored at ≤15 °C in sealed bags [27].

5. Conclusions

Ghaf is a significant part of the hot desert vegetation system as it can combat desertification and improve soil fertility in arid environments. The different priming treatments and immersion times applied in this study were associated with different responses and the potential for enhancing the germination behavior of ghaf seed. Among different treatments, seeds responded the best to the priming treatment with 50% H₂SO₄ + BA (200 ppm), in terms of effectiveness and germination uniformity. The treatment boosted germination 5 days after sowing and increased the germination percentage from 11.32% in untreated seeds to 83% in primed ones. Being covered with seed balls, these seeds showed a high germination percentage when planted in sandy soil. Furthermore, primed ghaf seeds could be stored at room temperature or 4 °C for more than 10 days with no adverse effects on seed germination. This will benefit the transporting of these seeds for long distance or even exporting abroad. Therefore, this treatment may be suggested as the most effective option for enhancing the germination of ghaf seeds and hence for assisting in desert reforestation.