agronomy
Article
Effect of Hormonal Priming and Osmopriming on
Germination of Winter Savory (Satureja montana L.) Natural
Population under Drought Stress
Monika Vidak 1, * , Boris Lazarević 2 , Monika Nekić 3 , Zlatko Šatović 1,4
Citation: Vidak, M.; Lazarević, B.;
Nekić, M.; Šatović, Z.;
Carović-Stanko, K. Effect of
Hormonal Priming and
Osmopriming on Germination of
Winter Savory (Satureja montana L.)
Natural Population under Drought
Stress. Agronomy 2022, 12, 1288.
https://doi.org/10.3390/
agronomy12061288
Academic Editors: Alessandra
Carrubba and Mauro Sarno
Received: 22 April 2022
Accepted: 25 May 2022
Published: 27 May 2022
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4.0/).
Agronomy 2022, 12, 1288. https://doi.org/10.3390/agronomy12061288
and Klaudija Carović-Stanko 1,4
1 Centre of Excellence for Biodiversity and Molecular Plant Breeding (CoE CroP-BioDiv), Svetošimunska
cesta 25, HR-10000 Zagreb, Croatia; zsatovic@agr.hr (Z.Š.); kcarovic@agr.hr (K.C.-S.)
2 University of Zagreb Faculty of Agriculture, Department of Plant Nutrition, Svetošimunska cesta 25,
HR-10000 Zagreb, Croatia; blazarevic@agr.hr
3 University of Zagreb Faculty of Agriculture, Svetošimunska cesta 25, HR-10000 Zagreb, Croatia;
monikanekic7@gmail.vcom
4 University of Zagreb Faculty of Agriculture, Department of Seed Science and Technology, Svetošimunska
cesta 25, HR-10000 Zagreb, Croatia
* Correspondence: mvidak@agr.hr
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 (dH2 O) 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).
Keywords: germination parameters; gibberellic acid; polyethylene glycol; potassium nitrate
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 in-
creasingly 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
https://www.mdpi.com/journal/agronomy
Agronomy 2022, 12, 1288 2 of 11

nutrients absorption and enables rapid and uniform germination and reduces the sensitiv-
ity 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 [13–15].
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 immunos-
timulatory effects, as well as antiproliferative activity on cancer cells [18–20]. 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.
Agronomy 2022,12,
Agronomy2022, 12,1288
x FOR PEER REVIEW 33 of
of1112

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

2.2.The
2.2. TheFirst
FirstPhase
Phaseofofthe
theExperiment
Experiment
Hydropriming
Hydropriming and Seed GerminationTest
and Seed Germination Test
InInthe
thefirst
firstphase
phaseofofthetheexperiment,
experiment,the the900
900seeds
seedsofofeach
eachwinter
wintersavory
savorypopulation
population
were subjected to hydropriming. The seeds were placed in distilled
were subjected to hydropriming. The seeds were placed in distilled water (dH water (dH O)
2 2O) forfor
48 h.
48
Then, the seeds
h. Then, werewere
the seeds takentaken
out and
outwashed underunder
and washed running water and
running water under
and distilled water.
under distilled
Finally,
water. the seedsthe
Finally, were
seedsdried
wereto dried
the initial
to themoisture content and
initial moisture germinated
content over nineover
and germinated (9)
different
nine (9) drought
differenttreatments (Table 1), which
drought treatments (Tablewere produced
1), which were using different
produced using concentrations
different con-
ofcentrations
polyethylene glycol (PEG 6000;
of polyethylene glycolSigma-Aldrich Co, St. Louis,Co,
(PEG 6000; Sigma-Aldrich MO,St. USA)
Louis,(Table
MO, USA)1). (Ta-
The
ble 1). 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 ®,
The seeds were germinated on germination paper (Munktell 21/N, 580 × 580 mm, 80
DU-RAN ® , DWK Life Sciences GmbH, Mainz, Germany) in a germination chamber at
g/qm) with a thin layer (3 mm) of cotton wool in 10 cm diameter Petri dishes (Steriplan®,
aDU-RAN
constant®temperature (22 ◦ C ±
, DWK Life Sciences 1 ◦ C) Mainz,
GmbH, with a photoperiod
Germany) in of 16 h of lightchamber
a germination and 8 h at ofa
darkness. The experimental design was two-factor factorial arranged in
constant temperature (22 °C ± 1 °C) with a photoperiod of 16 h of light and 8 h of darkness. a randomized
complete design with
The experimental four was
design repetitions (4 Petri
two-factor dishes)
factorial with 25in
arranged seeds each. The germination
a randomized complete de-
test lasted 21 days [24], and the number of germinated seeds was
sign with four repetitions (4 Petri dishes) with 25 seeds each. The germination counted every 48testh.lasted
The
seed was considered germinated when the radicle was ≥ 2 mm, indicating
21 days [24], and the number of germinated seeds was counted every 48 h. The seed was the seed’s ability
toconsidered
produce a germinated
normal seedling.
whenAbnormal
the radicleseedlings
was ≥2 mm, suchindicating
as damaged theseedlings, deformed
seed’s ability to pro-
seedlings, and decayed seedlings were not included in the germination
duce a normal seedling. Abnormal seedlings such as damaged seedlings, deformed seed- count because they
rarely
lings,survive to produce
and decayed plantswere
seedlings and not
wereincluded
considered as non-germinated
in the germination count seeds [24]. they
because
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
Tablewinter savorypressure
1. Osmotic natural populations.
in treatmentOsmotic
solutionspressure
used inwas produced using
the experiment polyethylene
for the glycol
seed germination
(PEG 6000) [25].
of three winter savory natural populations. Osmotic pressure was produced using polyethylene
glycol (PEG 6000) [25].
Treatments Osmotic Pressure in Treatment Solutions
Treatments
T1 (control) Osmotic Pressure
dH2 O in Treatment Solutions
T2 T1 (control) −0.2 MPa dH2O
T3 T2 −0.4 MPa
−0.2 MPa
T4 −0.8 MPa
T3 −0.4 MPa
T5 −1.2 MPa
T6 T4 −0.8 MPa
−1.6 MPa
T7 T5 −1.2 MPa
−2.0 MPa
T8 T6 −2.5 MPa
−1.6 MPa
T9 −3.0 MPa
T7 −2.0 MPa
T8 −2.5 MPa
2.3. The Second Phase ofT9the Experiment −3.0 MPa
Hormonal Priming and Osmopriming
2.3. The Second Phase of the Experiment
In the first phase of the experiment, Population 1 showed the best results in measured
Hormonal Priming
germination andand
parameters Osmopriming
was selected for the second phase of the experiment, in which
the effect of hormonal priming and osmopriming
In the first phase of the experiment, on seed
Population germination
1 showed the bestand morphological
results in measured
parameters under drought stress conditions were evaluated. Hormonal
germination parameters and was selected for the second phase of the experiment, priming wasin
Agronomy 2022, 12, 1288 4 of 11

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 solu-
tions in the second phase of the experiment of the seed germination of winter savory Population 1 (P1).

Seed Priming Treatments Drought Treatments (Osmotic Pressure)

TR1 dH2 O
PT1—100 ppm GA3 TR2 −0.8 MPa
TR3 −2.5 MPa
TR1 dH2 O
PT2—400 ppm GA3 TR2 −0.8 MPa
TR3 −2.5 MPa
TR1 dH2 O
PT3—0.2 w/v% KNO3 TR2 −0.8 MPa
TR3 −2.5 MPa
TR1 dH2 O
PT4—0.6 w/v% KNO3 TR2 −0.8 MPa
TR3 −2.5 MPa

The germinability of seeds was tested at different osmotic pressure treatments pro-
duced using respective concentrations of polyethylene glycol (PEG 6000) [25]. In Treat-
ment 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
nk
G= × 100
n
where nk 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:
∑ k 1 ni ti
MGT = i=
∑ik=1 ni
Agronomy 2022, 12, 1288 5 of 11

where ti is the time from the beginning of the experiment to the observation time (ith), ni 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 (CVt ; %) or homogeneity was calcu-
lated as follows:
st
CV t = ·100
t
where st is the standard deviation of germination time, and t is the mean germination time [26].
The mean germination rate (MR) is calculated as the reciprocal of the mean
germination time:
1
MR =
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:

k
U = − ∑ f i log2 f i
i =1

being,
ni
fi = k
∑ i =1 ni
where ni 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 ti , 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:
∑k Cn 2
Z = i =1 i
C∑ ni 2
being,
n i ( n i −1)
Cni ,2 =
2
where Cni ,2 is the combination of seeds germinated in the ith time, two by two, and ni is the
number of seeds germinated in the ith time [26].
Germination index (GI) was calculated using the formula [27]:

k
ni
GI = ∑
t
i =1 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, CVt , G5, and N, expressed as
percentages, were transformed before the analysis.
Agronomy 2022, 12, 1288 6 of 11

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) (Tables 3 and 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 (CVt ),
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), CVt (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), CVt (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 (num-
ber of normal seedlings). T1 (dH2 O, 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 signifi-
cantly 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.
Agronomy 2022, 12, 1288 7 of 11

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 prim-
ing 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 prim-
ing 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 (CVt ) 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), CVt (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.

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 (Tables 5 and S2).
Agronomy 2022, 12, 1288 8 of 11

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), CVt (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 (dH2 O), 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.

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 com-
bination of PT3 (0.2 w/v% KNO3 ) and TR3 (−2.5 MPa), while the shortest germina-
tion 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 osmoprim-
ing (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 ex-
periment. Hormonal priming (PT2) increased all measured morphological parameters
Agronomy 2022, 12, 1288 9 of 11

(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 fol-
lowed 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 ger-
mination parameters, especially under moderate drought conditions. Hormonal priming
with GA3 showed the best results, followed by seed priming with dH2 O (hydropriming).
Priming technology, especially hormonal priming, could improve germination and seedling
development of winter savory under drought conditions.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/agronomy12061288/s1, Table S1: The interaction effect of popula-
tions and drought treatment for the germination parameters of three winter savory natural population
(P1, P2, and P3) in the first phase of the experiment.; Table S2: The interaction effect of pretreatment
and drought treatment for the germination parameters of natural winter savory Population 1 in the
second phase of the experiment.
Author Contributions: Conceptualization, K.C.-S. and Z.Š.; formal analysis, Z.Š. and B.L.; inves-
tigation, M.N. and K.C.-S.; resources, M.N. and B.L.; writing—original draft preparation, M.V.;
writing—review and editing, B.L. and K.C.-S. All authors have read and agreed to the published
version of the manuscript.
Agronomy 2022, 12, 1288 10 of 11

Funding: This research received no external funding.

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: This work is part of the research program on conservation of medicinal and
aromatic plants carried out by the Working Group on Medicinal and Aromatic Plants financed by
the National Program for the Conservation and Sustainable Use of Plant Genetic Resources for Food
and Agriculture of the Republic of Croatia. The publication was supported by the Open Access
Publication Fund of the University of Zagreb Faculty of Agriculture.
Conflicts of Interest: The authors declare no conflict of interest.

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