Effect of Hormonal Priming and Osmopriming on Germination of Winter Savory (Satureja montana L.) Natural Population under Drought Stress

Abstract

Winter savory (Satureja montana L.) is an important medicinal, aromatic, and honey plant. In Croatia, it is widely distributed along the Adriatic coast, where it is frequently exposed to droughts. First, the winter savory natural population with the highest germination across different drought treatments after hydropriming was selected. Nine hundred seeds from each of the three natural populations (P1, P2, and P3) were hydroprimed (dH₂O) for 48 h. The seeds were then germinated in drought treatments with different concentrations of polyethylene glycol (PEG 6000) (−0, −0.2, −0.4, −0.8, −1.2, −1.6, −2, −2.5, −3.0 MPa). Since P1 showed the best results in germination parameters, it was used for the second phase of the experiment, where the effect of hormonal priming (100 and 400 ppm GA3, 48 h in the dark) and osmopriming (0.2% and 0.6% w/v KNO₃, 72 h in the dark) on seed germination and seedling morphological parameters of the selected winter savory population under drought stress conditions (−0.8 and −2.5 MPa) was evaluated. Although winter savory grows in dry areas, this study showed that extremely dry conditions (−3.0 and −2.5 MPa) negatively affected seed germination, but this effect can be mitigated by priming treatments, especially with the hormonal priming (GA3 400 ppm).

1. Introduction

Climate change will certainly be felt all over the world, and the increase in temperature and change in precipitation are expected to exacerbate water problems [1]. Drought stress, which occurs due to temperature dynamics, light intensity, and low rainfall, is considered one of the most destructive abiotic stresses worldwide and has a significant impact on crop production [2,3]. It strongly affects the morphological, physiological, biochemical, and molecular characteristics of plants [1,2,4]. Early developmental stages of perennial species are most affected by water deficits [5], and water is the most important factor stimulating germination, as water uptake is the first stage of germination [6].

Improving germination, plant development, and yield under drought stress is increasingly sought in plant breeding [7] and can be partially improved by seed priming treatments [4,8]. Seed priming is a widely used method to improve seed germination and further plant growth and development [9]. Seed priming is an alternative, low-cost, and viable technique that can improve drought stress tolerance through improved and advanced seed germination [3]. It is a technique that reduces the time of water and essential nutrients absorption and enables rapid and uniform germination and reduces the sensitivity of seeds to external environmental factors [10]. The selection and success of priming techniques depend on the plant species, morphology, and physiology of the seed [11]. There are several common types of priming treatments: hydropriming, osmopriming, nutrient priming, thermopriming, biopriming, chemical priming, and hormonal priming [10,11]. Hydropriming, as the simplest, is also low-cost and the most environmentally friendly seed priming technique, which implies the soaking of seeds in water [3,9]. Osmopriming is the most common priming method in which seeds are also hydrated in a low-osmotic, aerated solution having more advantages than hydropriming (earlier germination and seedling emergence and response to stress conditions) [3]. Hormonal priming, such as priming with gibberellic acid, usually increases the emergence, growth, and extensiveness of root systems [12].

Winter savory (Satureja montana L.) is an evergreen, perennial, semishrubby plant species in the Lamiaceae family that inhabits dry, sunny, rocky regions, and karst [13,14,15]. It is native to the Mediterranean region, where it grows as a wild plant, but is cultivated throughout Europe [14,15]. In Croatia, it is widely distributed along the Adriatic coast, where it is often subjected to drought events. It is an important medicinal, aromatic, and honey plant, traditionally used as a spice and natural preservative [13,16]. The leaves of winter savory have a high content of secondary metabolites such as steroids, flavonoids, tannins, and essential oils, hence their importance in the pharmaceutical, food, and perfume industries [17]. Winter savory has been found to have antioxidant, antibacterial, antifungal, antiviral, antiparasitic, diuretic, anti-HIV-1, antidiarrheal, antiproliferative, and immunostimulatory effects, as well as antiproliferative activity on cancer cells [18,19,20]. The essential oil recently gained importance, especially in Europe, as a natural food preservative [21,22] and has a herbicidal effect [23]. Of all species in the genus Satureja, S. montana has the greatest economic importance and is cultivated and used worldwide [14].

Given the importance of winter savory, the management plan should be based on exploration and selection of the best native populations and establishment of cultivation of native winter savory. Therefore, our experiment was conducted in two phases; first, the aim was to select the winter savory population with the highest germination across different drought treatments after hydropriming. The second aim was to evaluate the effect of hormonal priming and osmopriming on seed germination and morphological parameters of the selected winter savory population under drought stress conditions.

2. Materials and Methods

2.1. Plant Material

The research material included seeds collected from three natural populations of winter savory (Satureja montana L.). Seeds were collected in September and October 2018 at three different locations in Croatia (Figure 1): Stolac (44.93 N, 14.99 E; Population 1 (P1); MAP02980), Rončević dolac (44.96 N, 14.96 E; Population 2 (P2); MAP02981), and Trbušnjak (44.98 N, 14.92 E; Population 3 (P3); MAP02982). All three locations are in karst areas with a sub-Mediterranean climate and frequent water deficit periods. The collected seed is held as a part of the Collection of Medicinal and Aromatic Plants of the Department of Seed Science and Technology at the University of Zagreb Faculty of Agriculture.

Figure 1. Locations of the collected seeds of three natural populations of winter savory [(a)—Stolac (P1); (b)—Rončević Dolac (P2); (c)—Trbušnjak (P3)].

2.2. The First Phase of the Experiment

Hydropriming and Seed Germination Test

In the first phase of the experiment, the 900 seeds of each winter savory population were subjected to hydropriming. The seeds were placed in distilled water (dH₂O) for 48 h. Then, the seeds were taken out and washed under running water and under distilled water. Finally, the seeds were dried to the initial moisture content and germinated over nine (9) different drought treatments (Table 1), which were produced using different concentrations of polyethylene glycol (PEG 6000; Sigma-Aldrich Co, St. Louis, MO, USA) (Table 1).

The seeds were germinated on germination paper (Munktell 21/N, 580 × 580 mm, 80 g/qm) with a thin layer (3 mm) of cotton wool in 10 cm diameter Petri dishes (Steriplan^®, DU-RAN^®, DWK Life Sciences GmbH, Mainz, Germany) in a germination chamber at a constant temperature (22 °C ± 1 °C) with a photoperiod of 16 h of light and 8 h of darkness. The experimental design was two-factor factorial arranged in a randomized complete design with four repetitions (4 Petri dishes) with 25 seeds each. The germination test lasted 21 days [24], and the number of germinated seeds was counted every 48 h. The seed was considered germinated when the radicle was ≥2 mm, indicating the seed’s ability to produce a normal seedling. Abnormal seedlings such as damaged seedlings, deformed seedlings, and decayed seedlings were not included in the germination count because they rarely survive to produce plants and were considered as non-germinated seeds [24].

Table 1. Osmotic pressure in treatment solutions used in the experiment for the seed germination of three winter savory natural populations. Osmotic pressure was produced using polyethylene glycol (PEG 6000) [25].

Treatments	Osmotic Pressure in Treatment Solutions
T1 (control)	dH2O
T2	−0.2 MPa
T3	−0.4 MPa
T4	−0.8 MPa
T5	−1.2 MPa
T6	−1.6 MPa
T7	−2.0 MPa
T8	−2.5 MPa
T9	−3.0 MPa

Table 1. Osmotic pressure in treatment solutions used in the experiment for the seed germination of three winter savory natural populations. Osmotic pressure was produced using polyethylene glycol (PEG 6000) [25].

Treatments	Osmotic Pressure in Treatment Solutions
T1 (control)	dH2O
T2	−0.2 MPa
T3	−0.4 MPa
T4	−0.8 MPa
T5	−1.2 MPa
T6	−1.6 MPa
T7	−2.0 MPa
T8	−2.5 MPa
T9	−3.0 MPa

2.3. The Second Phase of the Experiment

Hormonal Priming and Osmopriming

In the first phase of the experiment, Population 1 showed the best results in measured germination parameters and was selected for the second phase of the experiment, in which the effect of hormonal priming and osmopriming on seed germination and morphological parameters under drought stress conditions were evaluated. Hormonal priming was performed by soaking the seeds in gibberellic acid (GA3; Sigma-Aldrich Co, St. Louis, MO, USA) at concentrations of 100 ppm and 400 ppm for 48 h in the dark, whereas osmopriming was performed by soaking the seeds with potassium nitrate (KNO₃; Sigma-Aldrich Co, St. Louis, MO, USA) at concentrations of 0.2% w/v and 0.6% w/v for 72 h also in the dark. Then, the seeds were taken out, washed under running water, then under distilled water and dried to the initial moisture content. A total of 1200 seeds were used, with 300 seeds included in each treatment (Table 2).

Table 2. Combination of seed priming treatments and the osmotic pressure in drought treatment solutions in the second phase of the experiment of the seed germination of winter savory Population 1 (P1).

Seed Priming Treatments	Drought Treatments (Osmotic Pressure)
PT1—100 ppm GA3	TR1 TR2 TR3	dH2O −0.8 MPa −2.5 MPa
PT2—400 ppm GA3	TR1 TR2 TR3	dH2O −0.8 MPa −2.5 MPa
PT3—0.2 w/v% KNO3	TR1 TR2 TR3	dH2O −0.8 MPa −2.5 MPa
PT4—0.6 w/v% KNO3	TR1 TR2 TR3	dH2O −0.8 MPa −2.5 MPa

The germinability of seeds was tested at different osmotic pressure treatments produced using respective concentrations of polyethylene glycol (PEG 6000) [25]. In Treatment 1 (TR1), germination was performed on distilled water (control), in Treatment 2 (TR2) on a PEG solution of −0.8 MPa, and in Treatment 3 (TR3) on a PEG solution of −2.5 MPa. Table 2 shows all combinations of priming treatments and osmotic pressure in drought treatments used in the second phase of the experiment. The seeds were germinated under the same conditions and germination count was done as in the first phase of the experiment.

2.4. Measurements

At the end of the second phase of the experiment (21st day), morphological parameters of seedlings (shoot length (SL; cm), root length (RL; cm), and total (seedling) length (TL; cm)) were measured. The scanner Epson Perfection V700 (Seiko Epson Corporation, Nagano, Japan) was used to scan the seedlings, and WinRhizo Pro software (Regent Instruments Inc., Quebec, QC, Canada) was used to analyze the above-mentioned traits.

2.5. Data Analysis

The germination parameters were calculated at the end of the first and second phases of the experiment.

Germinability (G, %) represents the number of germinated seeds in percentage [26] and was calculated according to the formula

$$G= \frac{n_k}{ n} \times 100$$

where n_k indicates the number of germinated seeds, and n is the total number of seeds in the experiment.

Mean germination time (MGT, day) is the germination time in days and was calculated according to the formula:

$$\mathit{MGT}=\frac{{{\displaystyle \sum }}_{i=1}^k{n}_i{t}_i}{{{\displaystyle \sum }}_{i=1}^k{n}_i}$$

where t_i is the time from the beginning of the experiment to the observation time (ith), n_i is the number of germinated seeds in the ith time, and k is the last day of germination [26].

The coefficient of variation of germination time (CV_t; %) or homogeneity was calculated as follows:

$${\mathit{CV}}_t=\frac{s_t}{\overline{t}}·100$$

where s_t is the standard deviation of germination time, and $\overline{t}$ is the mean germination time [26].

The mean germination rate (MR) is calculated as the reciprocal of the mean germination time:

$$\mathit{MR}=\frac{ 1}{T}$$

where T is the mean germination time and CV is the coefficient of velocity [26].

The uncertainty of the germination process (U) is given by the expression:

$$U=-{\displaystyle \sum }_{i=1}^k{f}_i{\mathit{log}}_2{f}_i$$

being,

$$f_i=\frac{n_i}{{{\displaystyle \sum }}_{i=1}^k{n}_i}$$

where n_i is the number of germinated seeds during the ith time, and k is the last day of observation [26].

The synchrony of the germination process (Z) is the quotient between the sum of the partial combinations of the number of germinated seeds in each t_i, two by two, and the two-by-two combination of the total number of germinated seeds at the end of the experiment, assuming that all seeds that germinated did so simultaneously. It is calculated by the expression:

$$Z=\frac{{{\displaystyle \sum }}_{i=1}^k{C}_{n_{i}2}}{C_{\sum n_{i}2}}$$

being,

$$C_{n_i,2}=\frac{n_{i }{ (n}_{i }-1)}{2}$$

where C_ni,₂ is the combination of seeds germinated in the ith time, two by two, and n_i is the number of seeds germinated in the ith time [26].

Germination index (GI) was calculated using the formula [27]:

$$\mathit{GI}={\displaystyle \sum }_{i=1}^k\frac{n_i}{t_i}$$

Germination on the fifth day is expressed as a percentage (G5; %).

Number of normal seedlings in relation to the total number of seedlings germinated, expressed as a percentage (N, %).

2.6. Statistical Analysis

Using SAS software PROC GLM [28], a two-way analysis of variance (ANOVA) was conducted to determine if there were significant differences in measured germination and seedling morphological parameters among the population and treatments. Tukey’s test (p ≤ 0.05) was used to compare the mean differences between the values of the quantitative variables of the treatments. The original variables of G, CV_t, G5, and N, expressed as percentages, were transformed before the analysis.

3. Results and Discussion

3.1. The First Phase of the Experiment

The germination of hydroprimed seeds of three winter savory natural populations (P1, P2, and P3) were studied at different drought treatments (T1–T9) simulated by different osmotic pressure produced using different concentrations of polyethylene glycol (PEG 6000). A significant number of seeds germinated in each treatment, except in T9 (−3 MPa), in which only four seeds from P1 and three seeds from P3 germinated. These results showed that winter savory can successfully germinate under intense drought conditions, as high as −2.5 MPa. Based on the validity of the statistical analysis, −3 MPa treatment was excluded from further analysis. Table S1 shows interaction effect of populations and drought treatment for the germination parameters of three winter savory natural populations (P1, P2 and P3)

Analysis of variance (ANOVA) (Table 3 and Table 4) revealed a significant influence of population and drought treatment on germinability (G), mean germination time (MGT), mean germination rate (MR), and germination index (GI). The statistical significance of the population was determined by the coefficient of variation of germination time (CV_t), while the statistical significance of the treatment was determined by the difference in the uncertainty of the germination process (U), the number of normal seedlings (N), and the synchrony, as well as the germination process (Z).

Table 3. Differences between three winter savory natural populations (P1, P2, and P3) in the first phase of the experiment for the germination parameters under drought stress.

Source of Variability	G	MGT (Day)	CVt	MR	U	Z	GI	G5	N
P1	46.75 A	6.57 B	60.57 A	0.16 A	2.21 A	0.17 A	2.55 A	8.63 A	68.67 A
P2	35.88 B	9.14 A	42.60 B	0.12 B	2.13 A	0.16 A	1.25 C	5.38 A	75.91 A
P3	42.00 AB	8.19 A	45.60 B	0.13 B	2.17 A	0.17 A	1.62 B	8.00 A	70.51 A
p (F)	***	***	***	***	ns	ns	***	ns	ns

G (Germinability), MGT (mean germination time), CV_t (coefficient of variation of the germination time), MR (mean germination rate), U (uncertainty of the germination process), Z (synchrony of the germination process), GI (germination index), G5 (germination in the fifth day after the start of the experiment), N (number of normal seedlings). P1 (Population 1), P2 (Population 2), P3 (Population 3). Values followed by the same letter in each column are not significantly different based on the Tukey test at 0.05 probability. ns—not statistically significant p > 0.05, * 0.05 > p > 0.01, ** 0.01 > p > 0.001, *** p < 0.001.

Table 4. Differences between treatments for the germination parameters in the first phase of the experiment of the seed germination of three winter savory natural populations under drought stress.

Source of Variability	G	MGT (Day)	CVt	MR	U	Z	GI	G5	N
T1	51.33 A	8.29 A	50.64 A	0.12 BC	2.50 A	0.14 A	1.96 ABC	9.33 A	85.46 A
T2	41.33 A	5.76 B	55.51 A	0.18 A	1.90 BC	0.24 A	2.44 A	5.00 A	38.39 B
T3	45.67 A	8.28 A	55.48 A	0.12 BC	2.41 AB	0.13 A	1.81 ABC	6.00 A	78.56 A
T4	44.00 A	8.85 A	50.92 A	0.12 BC	2.40 AB	0.14 A	1.72ABC	8.33 A	82.00 A
T5	41.00 A	8.65 A	51.03 A	0.12 BC	2.17 ABC	0.16 A	1.61 BC	6.00 A	80.66 A
T6	39.33 A	8.92 A	49.04 A	0.12 C	2.20 ABC	0.16 A	1.42 C	6.67 A	70.88 A
T7	45.00 A	6.94 AB	42.02 A	0.16 AB	1.97 BC	0.23 A	2.29 AB	12.67 A	70.70 A
T8	24.67 B	8.06 A	42.07 A	0.14 ABC	1.83 C	0.15 A	1.21 C	4.67 A	66.92 A
p (F)	***	***	ns	***	***	*	***	ns	***

G (germinability), MGT (mean germination time), CV_t (coefficient of variation of the germination time), MR (mean germination rate), U (uncertainty of the germination process), Z (synchrony of the germination process), GI (germination index), G5 (germination in the fifth day after the start of the experiment), N (number of normal seedlings). T1 (dH₂O, control), T2 (−0.2 MPa), T3 (−0.4 MPa), T4 (−0.8 MPa), T5 (−1.2 MPa), T6 (−1.6 MPa), T7 (−2 MPa), T8 (−2.5 MPa). Values followed by the same letter in each column are not significantly different based on the Tukey test at 0.05 probability. ns—not statistically significant p > 0.05, * 0.05 > p > 0.01, ** 0.01 > p > 0.001, *** p < 0.001.

A significant difference was found between populations in germinability (G) (Table 3). The highest percentage of average germination (G) was found for P1 (46.75%), followed by P3 (42.00%) and P2 (35.88%). In mean germination time (MGT), which reflects germination speed, P1 (6.57 days) was significantly different from P2 (9.14 days) and P3 (8.19 days), while there was no significant difference between P2 and P3. Germination index (GI) was highest in P1 (2.55), followed by P3 (1.62) and P2 (1.24). From these results, it is evident that P1 showed the best results for germination parameters, and it was selected for the second phase of the experiment of the research.

The lowest percentage of germination was found in T8 (−2.5 MPa) (G; 24.67%) and the highest in control (T1) (51.33%) (Table 4). Therefore, Treatment T8 was repeated in the second phase of the experiment as the highest level of drought at which seeds germinated to see the effect of GA3 and KNO₃ priming treatments on winter savory P1 seed germination. T2 (−0.2 MPa) and T7 (−2 MPa) showed the best results for mean germination time (MGT), mean germination rate (MR), and germination index (GI) but also had the lowest percentage of normal seedlings (N) in all three populations. In the second phase of the experiment of winter savory P1 seed germination, T4 (−0.8 MPa) was repeated as moderate drought.

In similar studies, hydropriming had a positive effect on the germination parameters of natural populations of Dalmatian pyrethrum (Tanacetum cinerariifolium/Trevir./Sch. Bip) [29] and of pyrethrum seeds under drought and salinity conditions [30] and also results in a higher percentage of cotton (Gossypium herbaceum L.) seed germination under drought and temperature stress [31].

3.2. The Second Phase of the Experiment

In the second phase of the experiment, the effect of osmopriming and hormonal priming on winter savory natural population (P1) seed germination and seedling morphological parameters under drought stress conditions were examined.

Analysis of variance (ANOVA) (Table 5) revealed a significant influence of priming treatments on all parameters studied. A significant influence of drought treatments (Table 6) was found for all studied parameters, except for the coefficient of variation of the germination time (CV_t) and the number of normal seedlings (N).

Table 5. Differences between priming treatments for the germination parameters of natural winter savory Population 1 in the second phase of the experiment under drought stress.

Source of Variability	G	MGT	CVt	MR	U	Z	GI	G5	N
PT1	75.33 A	3.59 B	31.95 B	0.29 A	1.18 B	0.48 A	6.14 A	71.67 A	93.52 A
PT2	78.67 A	4.12 B	30.82 B	0.24 AB	1.02 B	0.59 A	5.24 B	74.67 A	93.28 A
PT3	48.33 B	5.23 A	24.85 B	0.21 B	1.25 AB	0.47 A	3.18 C	36.67 B	93.03 A
PT4	40.33 B	4.15 B	55.66 A	0.26 A	1.71 A	0.30 B	2.77 C	31.67 B	69.22 B
p (F)	***	***	***	***	**	***	***	***	***

G (germinability), MGT (mean germination time), CV_t (coefficient of variation of the germination time), MR (mean germination rate), U (uncertainty of the germination process), Z (synchrony of the germination process), GI (germination index), G5 (germination in the fifth day after the start of the experiment), N (number of normal seedlings). PT1 (100 ppm GA3), PT2 (400 ppm GA3), PT3 (0.2 w/v% KNO₃), PT4 (0.6 w/v% KNO₃). Values followed by the same letter in each column are not significantly different based on the Tukey test at 0.05 probability. ns—not statistically significant p > 0.05, * 0.05 > p > 0.01, ** 0.01 > p > 0.001, *** p < 0.001.

Table 6. Differences between treatments for the germination parameters of natural winter savory Population 1 in the second phase of the experiment under drought stress.

Source of Variability	G	MGT	CVt	MR	U	Z	GI	G5	N
TR1	67.75 A	3.94 B	38.26 A	0.26 A	1.26 AB	0.48 AB	5.13 A	63.00 A	88.84 A
TR2	61.25 A	3.70 B	33.50 A	0.28 A	1.11 B	0.53 A	4.76 A	58.50 A	87.40 A
TR3	53.00 B	5.18 A	35.69 A	0.21 B	1.49 A	0.38 B	3.11 B	39.50 B	85.55 A
p (F)	***	***	ns	***	*	*	***	***	ns

G (germinability), MGT (mean germination time), CV_t (coefficient of variation of the germination time), MR (mean germination rate), U (uncertainty of the germination process), Z (synchrony of the germination process), GI (germination index), G5 (germination in the fifth day after the start of the experiment), N (number of normal seedlings). TR1 (dH₂O), TR2 (−0.8 MPa PEG), TR3 (−2.5 MPa PEG). Values followed by the same letter in each column are not significantly different based on the Tukey test at 0.05 probability. ns—not statistically significant p > 0.05, * 0.05 > p > 0.01, ** 0.01 > p > 0.001, *** p < 0.001.

The results of this study showed that drought stress has significant inhibitory effects on the germination of winter savory. However, hormonal primed seeds (PT1 and PT2) showed improved germination parameters compared to osmoprimed seeds (PT3 and PT4) (Table 5). Additionally, the effect of osmopriming is not favorable under more intense drought conditions (Table 5 and Table S2).

Suppression of germination by higher KNO₃ concentrations was also observed in Salvia cyanescens Boiss. & Bal. [32] and Sorbus pohuashanensis (Hance) Hedl. [33]. On the other hand, GAs have been found to play an important role in many essential plant growth and development processes, including seed germination, stem elongation, leaf spread, flower and fruit development, and transition to fruit set [34]. They are commonly used to overcome seed dormancy and can significantly improve seed germination in many species, mainly by activating embryo vegetative growth, mobilizing reserves, and weakening a growth-inhibiting endosperm layer [35,36]. In a similar study, hormonal priming (GA3) and osmopriming (KNO₃) significantly increased germination in two Papaver species (P. rhoeas L. and P. dubium L.), with maximum germination parameters obtained with hormonal priming in both species [37]. Sukifto et al. [38] found that a priming treatment with GA3 for 12 h significantly improved the germination performance of Malaysian indica rice (Oryza sativa L.), compared to unprimed seeds. Primed seeds showed better germination percentage, increased germination index, and reduced mean germination time. In a study conducted by Delač et al. [29], hydropriming of Dalmatian pyrethrum seeds showed better results than osmopriming with potassium nitrate (KNO₃).

There was a statistically significant difference in the mean germination time (MGT) of priming treatments. Hormonal priming (PT1; 100 ppm GA3) gave the shortest mean germination time, which increased significantly at T3 (−2.5 MPa). Hormonal priming (PT2; 400 ppm GA3) showed average results for all osmotic pressures. Statistically significant, the longest mean germination time (7.34 days) was determined by a combination of PT3 (0.2 w/v% KNO₃) and TR3 (−2.5 MPa), while the shortest germination time was recorded for PT1 (100 ppm GA3) × TR1 (control) (3.10 days) and PT1 (100 ppm GA3) × TR2 (−0.8 MPa) (3.19 days) (Table S2).

Germination index (GI) was also higher in hormonal priming than in osmopriming (Table 5) and is approximately the same with germination in TR1 (control) and TR2 (−0.8 MPa), while it decreases significantly with increasing osmotic pressure up to −2.5 MPa (TR3; Table 6). A similar study on wild rye (Secale montanum Guss.) was carried out by Ansari et al. [39], where hormonal priming (GA3) improved germination parameters under drought stress conditions.

The number of abnormal seedlings increased with the increased osmotic pressure in the osmoprimed seeds, whereas in hormonal priming (PT1), the number of normal seedlings significantly increased in TR3 and PT2 and was approximately the same as in TR1 and TR2. Kaya et al. [40] found that osmopriming (KNO₃) of sunflower (Helianthus annuus L.) seeds did not increase the number of normal seedlings under drought stress conditions. In a study by Rouhi and Sepehri [41], hormonal priming with GA3 significantly mitigated the negative effects of drought stress in peanut (Arachis hypogaea L.) seeds.

Table 7 shows the interaction effect of priming treatment and drought treatment on natural winter savory P1 morphological parameters in the second phase of the experiment. Hormonal priming (PT2) increased all measured morphological parameters (RL, SL and TL). In a study by Heydariyan et al. [12], hormonal priming (125 ppm, 250 ppm, and 500 ppm GA3) of caper (Capparis spinosa L.) seeds was found to increase germination percentage, germination index, and caper root length at different drought stress levels. Additionally, in the study of Tsegay and Andargie [42], hormonal priming (GA3) was found to significantly improve germination rate, reduce average germination time, and increase shoot and root length in maize (Zea mays L.), pea (Pisum sativum L.), and axe (Lathyrus sativus L.) under conditions of osmotic stress or salt stress. Hormonal priming (PT2, 400 ppm GA3) had the best effect on total seedling length (TL) in TR2 (−0.8 MPa). Regarding the effect of treatment, it was observed that shoot length (SL) was significantly reduced at the highest osmotic stress (−2.5 MPa, TR3) in osmopriming (PT4).

Table 7. Interaction effect of priming treatment and drought treatment of natural winter savory Population 1 morphological parameters in the second phase of the experiment.

Source of Variability	RL	SL	TL
PT1*TR1	2.45 ABC	1.00 BCD	3.45 BC
PT1*TR2	2.61 AB	1.15 AB	3.73 AB
PT1*TR3	2.13 BCD	0.80 CDE	2.93 CD
PT2*TR1	2.97 A	1.09 ABC	4.06 AB
PT2*TR2	2.98 A	1.34 A	4.33 A
PT2*TR3	2.48 ABC	0.99 BCD	3.47 BC
PT3*TR1	2.12 BCD	1.33 A	3.45 BC
PT3*TR2	2.68 AB	1.07 ABC	3.75 AB
PT3*TR3	2.20 BCD	0.74 DE	2.94 CD
PT4*TR1	1.95 CD	0.89 BCDE	2.83 CD
PT4*TR2	1.60 D	0.81 BCDE	2.41 D
PT4*TR3	0.93 E	0.63 E	1.55 E

RL (root length, cm), SL (shoot length, cm), TL (total (seedling) length, cm). PT1 (100 ppm GA3), PT2 (400 ppm GA3), PT3 (0.2 w/v% KNO₃), PT4 (0.6 w/v% KNO₃). TR1 (dH2O), TR2 (−0.8 PEG), TR3 (−2.5 PEG). Values followed by the same letter in each column are not significantly different based on the Tukey test at 0.05 probability.

A highly significant difference was observed in osmopriming (PT4, 0.6 w/v% KNO₃). Root length (RL) and total length decreased with increasing drought stress and were significantly reduced with osmopriming (PT4) in TR2 and TR3. Kaya et al. [40] found that hydropriming and osmopriming with KNO₃ showed better root growth of sunflower under drought and salt stress conditions.

4. Conclusions

Based on the results of this study, it can be concluded that there is diversity among winter savory populations to drought stress. Extremely dry conditions have a negative effect on germination and seedling morphology. However, seed priming can improve germination parameters, especially under moderate drought conditions. Hormonal priming with GA3 showed the best results, followed by seed priming with dH₂O (hydropriming). Priming technology, especially hormonal priming, could improve germination and seedling development of winter savory under drought conditions.