sustainability

Article
Effect of Salinity and Temperature on the Seed Germination
and Seedling Growth of Desert Forage Grass
Lasiurus scindicus Henr.
Jahangir A. Malik 1 , AbdulAziz A. AlQarawi 1 , Mashail N. AlZain 2, *, Basharat A. Dar 1 ,
Muhammad M. Habib 1 and Salah Nasser S. Ibrahim 1

1 Plant Production Department, College of Food and Agricultural Sciences, King Saud University,
P.O. Box 2460, Riyadh 11451, Saudi Arabia; jmalik@ksu.edu.sa (J.A.M.); alqarawi@ksu.edu.sa (A.A.A.);
bdar@ksu.edu.sa (B.A.D.); mhabib@ksu.edu.sa (M.M.H.); salahsar2008@gmail.com (S.N.S.I.)
2 Department of Biology, College of Sciences, Princess Nourah Bint Abdulrahman University, P.O. Box 84428,
Riyadh 11451, Saudi Arabia
* Correspondence: mnalzain@pnu.edu.sa

Abstract: Lasiurus scindicus Henr. is one of the most important forage grass species of the Arabian
deserts. Temperature and soil salinity are well known to influence the germination and seedling
development of various forage species. Therefore, in the current study, the effect of temperature and
salinity and their interaction on the germination parameters, seedling growth, and physiological
parameters of L. scindicus were evaluated. For this reason, L. scindicus seeds were treated with five
salinity concentrations (i.e., 0, 50, 100, 150, and 200 mM NaCl) and incubated at two temperature
levels (T1 = 25/20 ◦ C, D/N and T2 = 35/30 ◦ C, D/N). The results indicated that the salinity and
temperature significantly affected the germination indices, seedling growth parameters, chlorophyll,
and proline content. The highest germination percentage (GP; 90%) was recorded in the non-saline-
treated seeds incubated at T1. The seeds at T2 under the non-saline treatment exhibited an increased
Citation: Malik, J.A.; AlQarawi, A.A.;
germination rate (GR = 17.5%). The interactive effect of salinity and temperature on germination
AlZain, M.N.; Dar, B.A.; Habib, M.M.;
and growth parameters was significant, indicating that the germination response to salinity depends
Ibrahim, S.N.S. Effect of Salinity and
on temperature. The germination of seeds treated with 200 mM NaCl was completely inhibited at
Temperature on the Seed
Germination and Seedling Growth of
both temperatures T1 and T2. However, the ungerminated seeds at both T1 (85%) and T2 (78%)
Desert Forage Grass Lasiurus scindicus restored their germination abilities after they were transferred to distilled water. Also, the seed vigor
Henr. Sustainability 2022, 14, 8387. index (SVI) constantly showed a decline with the increasing salinity levels especially at T2, which
https://doi.org/10.3390/su14148387 was lowest when seeds were treated with 150 mM salinity. Growth parameters (i.e., aRL, aSL, RDW,
SDW, SB, and SLA) and the chlorophyll content showed a similar pattern as that of germination.
Academic Editor: Sara Magrini
However, the proline content (shoot proline and root proline) showed a progressive increase with
Received: 22 April 2022 increasing salinity and temperature. All of these characteristics indicate that L. scindicus seeds were
Accepted: 5 July 2022 not able to germinate under extreme salinity and temperature conditions but remained viable in a
Published: 8 July 2022 state of enforced dormancy. This is most likely an important adaptive strategy of this species for
Publisher’s Note: MDPI stays neutral survival in the high-saline changing habitats of the arid region of Saudi Arabia, and thus, it can be
with regard to jurisdictional claims in an excellent choice for restoring degraded rangelands and salinity-inflicted abundant farmlands for
published maps and institutional affil- forage agriculture.
iations.
Keywords: Lasiurus scindicus; salinity; temperature; seed germination; chlorophyll; proline; restoration

Copyright: © 2022 by the authors.

Licensee MDPI, Basel, Switzerland.
1. Introduction
This article is an open access article
distributed under the terms and
Seed germination is a fundamental physiological process in the successful growth and
conditions of the Creative Commons development of plants [1,2], and is significantly affected by temperature, soil salinity, etc. [3,4].
Attribution (CC BY) license (https:// The germination and seedling phases in the plant life cycle are the most vulnerable to extreme
creativecommons.org/licenses/by/ environmental stressors, especially for desert plants [5,6].
4.0/).

Sustainability 2022, 14, 8387. https://doi.org/10.3390/su14148387 https://www.mdpi.com/journal/sustainability

Sustainability 2022, 14, 8387 2 of 17

Salinity is one of the most significant abiotic stresses impacting plant growth and de-
velopment worldwide [7,8]. It is a primary cause of soil degradation and has a detrimental
impact on ecological restoration and rebuilding in desert regions [9,10]. Salinity is prevalent
in arid regions and impacts seed germination and its subsequent phases such as seedling
establishment and growth [11,12]. Soil is saline when the electrical conductivity (EC) of
its saturation extract (ECe) at the root zone exceeds 4 dS/m [13]. According to various
reports, salt stress affects about a billion hectares of land globally [8,14]. Furthermore, for
a variety of causes, which include high surface evaporation and inadequate amounts of
precipitation, weathering of native rocks, irrigation with saline water, and poor cultural
practices, salinized areas continue to increase in size at a pace of 10% annually, especially
in arid and semiarid regions [7,15]. By the year 2050, it is anticipated that more than half of
all arable land will be salinized [7,8,16].
Salinity stress disrupts enzymes and other biological functions, causing ionic leakage
from cell membranes and hence cell malfunction [8,17]. It also induces inhibition of the
chlorophyll biosynthesis pathway, thus, decreasing plant photosynthesis [18,19]. Further-
more, salinity damages plant functioning and growth by reducing water uptake capacity,
weakening root structure, causing ionic toxicity, and osmotic stress [20]. All of these factors
influence seed germination percentages and rates, as well as root and shoot length, and dry
weight [8,21]. Salt stress has also been reported to increase proline content as a protective
mechanism [22].
The influence and importance of temperature on seed quality and germination have
long been acknowledged [23,24]. It is considered one of the most prominent environmental
factors regulating growth and development by altering all individual reactions and stages
of germination in a plant [25–27]. Extreme warming, for example, is predicted to become
more common in the future [28–30]. Seed germination could be harmed as a result of such
a shift [31,32]. Also, global warming generally increases surface evaporation, encouraging
soil salinization and making it more problematic [33]. As a result, salinity and temperature,
especially in dry and semiarid regions, would have a combined effect on seed germination,
seedling growth, chlorophyll content, and proline accumulation [34,35]. For example,
all plant species have an ideal temperature for seed germination, which allows seeds to
germinate more effectively than at other temperatures [36]. Also, at optimum temperatures,
as in the previous studies, the salinity tolerance of plants was higher, and any fluctuation
in temperature hindered germination at all salinities [37,38]. Thus, it becomes critical
to investigate the combined impact of salinity and temperature on the germination and
viability of desert plants, which will increase knowledge for understanding plants’ tolerance
to salinity under fluctuating temperatures at the germination and seedling stage [39,40].
Through different mechanisms, seeds have developed the ability to delay germination
and survive under high stresses until the arrival of favorable conditions through subsequent
environmental events [36,41]. The germination recovery test is frequently used to evaluate
if salt stress damaged seeds or just prevented them from germinating [41]. Although the
majority of the literature suggests that halophytes have better germination recovery than
glycophytes, a few studies have looked at germination recovery in glycophytes [11,42].
Grasses resist salinity stress differently at the germination stage than halophytes, which
usually have a lower tolerance.
Lasiurus scindicus Henrard (Poaceae) is a perennial, multibranched, C4 desert grass.
It is a drought-resilient plant and can grow well in areas where annual precipitation is
around 200 mm [43]. It is naturally distributed in the dry regions of Africa, Asia, and Saudi
Arabia [43–45]. It is nutritiously valuable forage, mainly for ruminants, and grows well on
sandy plains, low sand dunes, hummocks [44,46], and occasionally in the coastal habitats
of the Arabian Peninsula [47]. This plant is a moderately salt-tolerant species that plays an
important role in rangeland stability [8,48,49]. All these traits make L. scindicus a competent
candidate for the restoration of degraded ranges.
The previous studies reported that increasing salinities under various temperatures
had a negative effect on the germination and growth of the L. scindicus plant [8,42]. No
Sustainability 2022, 14, 8387 3 of 17

such work is reported from the rangelands of Saudi Arabia. Therefore, the goal of this
study was to evaluate how well L. scindicus seeds tolerate various salinity levels under
fluctuating temperatures in a controlled laboratory experiment. Also, the ability of the
seeds to recover germination capacity when transferred from saline solutions to distilled
water was investigated. This would provide a crucial framework and understanding for
the restoration of salinity-inflicted deserts through the cultivation of L. scindicus plants. We
hypothesized that (1) seed germination would consistently decrease with increasing salinity,
(2) the germination at the optimum temperature (the temperature at which L. scindicus
grows naturally) will be highest at all salinity levels, and (3) the ungerminated seeds will
recover their ability to germinate once transferred to distilled water.

2. Materials and Methods

2.1. Collection of Lasiurus scindicus Seeds
Spikes with developed seeds of Lasiurus scindicus were collected from the natural
population grown in the escarpment habitat of Thumamah National Park, Riyadh, Saudi
Arabia. Thumamah National Park is a natural habitat located around 100 km north of
Riyadh, Saudi Arabia’s capital. The park is a huge and shallow depression with an average
yearly precipitation of less than 20 mm. The average yearly temperature in the area ranges
from 18 ◦ C in the winter to 37 ◦ C in the summer (Table 1) [50]. Also, the climate data of
the Riyadh region were collected between 1999 and 2019 from https://en.climate-data.
org/asia/saudi-arabia-29/ (accessed on 15 November 2021; Supplementary Figure S1).
The different plant types associated with L. scindicus in the escarpment habitat of this park
were Panicum turgidum, Haloxylon salicornicum, Cenchrus ciliaris, Rhanterium epapposum, and
Acacia ehrenbergiana. The collected spikes were air-dried at room temperature (20–25 ◦ C)
in the Range Science Lab, College of Food Science and Agriculture, King Saud University,
Riyadh, Saudi Arabia. The seeds from the air-dried spikes were separated by manual
handpicking and stored in air-tight containers at room temperature (20–25 ◦ C) with 16%
humidity for a period of four months, until further experimentation. These stored seeds
were later surface sterilized for 3 min in a 5% sodium hypochlorite (NaClO) solution. The
seeds were rinsed thrice with sterilized distilled water to remove the solution prior to 1 h of
drying at room temperature. The seed health/quality was tested by germinating 100 seeds
in moistened Petri plates with distilled water at room temperature, replicated thrice, and
found to be ≈100%. The seeds were later used for the germination experiment under two
temperatures and five levels of salinity.

Table 1. Seed collection site and its characteristics.

Species Country Site Location Seed Color Seed Weight

Escarpment habitats of
25◦ 080 13.700 N 4.02 ± 0.1494 g/
Lasiurus scindicus Saudi Arabia Thumamah National Dark Brown
46◦ 360 00.900 E 1000 seeds
Park, Riyadh

2.2. Gemination Experiment

A controlled Petri plate germination experiment [51] on the seeds of L. scindicus was
conducted using two factors (temperature and salinity). The experiment was conducted at
the Plant Production Department, College of Food and Agricultural Sciences, King Saud
University, Riyadh. Two temperatures (i.e., 25/20 ◦ C, D/N = T1 and 35/30 ◦ C, D/N = T2)
were used to imitate the habitat temperature conditions for spring and late summer for the
Riyadh region, Saudi Arabia [50], when the plant produces fully mature seeds. Also, five
salinity levels (i.e., 0 mM NaCl concentration (Control), 50, 100, 150, and 200 mM NaCl)
were applied to study the salt tolerance level of the seeds under these two temperatures. A
total of five replicates with twenty seeds for each treatment were used. All the seeds were
evenly arranged in the sterilized plastic Petri dishes (ø: 9 cm) lined with double sterilized
Whatman No. 1 filter paper. Saline solutions (5 mL/Petri) were applied and the Petri
Sustainability 2022, 14, 8387 4 of 17

dishes were sealed in zipping lock plastic bags to avoid any moisture loss by evaporation.
Two incubators (LEEC PL33 Plant Growth and Seed Germination Cabinet, LEEC Limited,
Nottingham, UK), each set at the two applied temperatures and a 12/12 hours’ light/dark
photoperiod were used. A total of 50 Petri dishes (2 temperature × 5 salinity × 5 replicates)
were randomly placed in the two incubators.
The germination readings were carried out on daily basis throughout the experimenta-
tion. The seed was considered to be germinated only when the radicle length reached 2 mm.
After twenty days of incubation, the germination counts were stopped. The germination
parameters assessed were germination percent (GP), germination rate (GR), seed vigor
index (SVI), cumulative germination speed, and recovery germination percent. The values
of GP, GR, and SVI were calculated as per the following equations:

total number o f seeds germinated

Germination(%) = × 100
total number o f seeds in petri

Xi
Germination rate = ∑ Yi
where Xi is the number of germinated seeds and Yi is the day corresponding to Xi [52].

SVI = (SL + RL) × GP

where SL = shoot length, RL = root length, and GP = germination percentage.

It is worth noting that at the 200 mM salinity treatment, seeds from both incubating
temperatures (T1 and T2) showed no germination.

2.3. Measurement of Growth Parameters

At the end of the germination period, five seedlings were randomly selected from each
Petri dish to measure the growth parameters. The shoot (aSL = average shoot length (n = 5),
SDW = shoot dry weight, and SLA = specific leaf area) and root (aRL = average root length
(n = 5), RDW = root dry weight) traits were calculated. The SLA was calculated based on
leaf weight and leaf area (scanned and calculated by using WIN 2.0 software). Also, the
overall seedling biomass (SlB) was calculated after oven drying the whole seedlings at
70 ◦ C for 72 h.

2.4. Chlorophyll Estimation

Chlorophyll a, chlorophyll b, and total chlorophyll were obtained by mashing 100 mg
of seedling tissue in 10 mL 80% acetone solution [53]. The homogenate was centrifuged at
5000 rpm for 5 min using a Benchtop Centrifuge-5810R, Eppendorf, Hamburg, Germany.
The samples were then incubated in the dark for three hours before the absorbance at
wavelengths 645 and 663 were measured using a UV–VIS spectrophotometer (SHIMADZU,
Kyoto, Japan, UV1800).

2.5. Estimation of Proline Content

Proline (µg/gFW) for the shoot and root of the L. scindicus seedlings was calculated by
homogenizing 100 mg of fresh plant shoot and root tissue in 10 mL of sulfosalicylic acid
(3%) with a mortar and pestle. The homogenate was centrifuged at 5000 rpm for 10 min
(Benchtop Centrifuge-5810R, Eppendorf, Hamburg, Germany), and 2 mL of supernatant
was extracted in a separate test tube. The extract (2 mL) was incubated for 1 h in a boiling
water bath (at 94–100 ◦ C) with glacial acetic acid and ninhydrin (2 mL each), followed by
an ice shock. To this, 4 mL of toluene was added, and the toluene-containing chromophore
was collected in a separate tube after mixing for 20 s. The absorbance was measured at
520 nm using a UV–VIS spectrophotometer (SHIMADZU, Kyoto, Japan, UV1800) [54]. A
standard curve was obtained using known concentrations of proline.
Sustainability 2022, 14, 8387 5 of 17

2.6. Data Analysis

The experiment was conducted as a factorial completely randomized design (CRD)
with five replicates for each treatment. The experiment was repeated twice to avoid
any experimental error, and before performing statistical analysis, data from both the
experiments were averaged. The data generated in the experiment were analyzed using the
statistical package Statistics version 8.1 and SAS 9.2 (Oliver Schabenberger, SAS Institute
Inc., Cary, NA, USA). The data were subjected to a two-way analysis of variance (ANOVA)
to evaluate the effect of two main factors (temperature and salinity) and their interaction
on the germination parameters, seedling growth, and recovery germination percentage.
Prior to statistical analysis, germination percentage data were arcsine transformed so that
the homogeneity of variance was ensured. However, untransformed data are presented
in figures. When ANOVA revealed significant treatment differences by the impact of
salinity, temperature, and their interaction (salinity × temperature), Duncan’s multiple
tests (p = 0.05, p = 0.01) were carried out to perform all-pairwise comparison between the
treatment means [55].

3. Results
3.1. Effect of Temperature and Salinity on Germination Indices
Temperature (A), salinity (B), and their combination (A × B) significantly (p = 0.0001)
affected the germination percentage (GP) of L. scindicus seeds (Supplementary Table S1). The
GP was the highest in non-saline-treated seeds at both the temperatures (i.e., T1 = 25/20 ◦ C
and T2 = 35/30 ◦ C, D/N) applied, followed by the GP of seeds sown at 50 mM salinity stress
(Figure 1A). At the highest levels of salinity (i.e., 100 and 150 mM), the seeds incubated at T2
showed a significant decline in the GP. The same pattern was found in germination speed,
where the non-saline-treated seeds and those with 50 mM salinity stress germinated faster
at both the temperatures T1 and T2 (Figure 2A,B). The non-saline seeds at T2 reached the
maximum germination on the 6th day, while, as in T1, the peak germination of non-saline-
treated seeds was achieved on the 8th day. For the highest salinity levels, the germination
was inhibited for the first few days. However, the germination of L. scindicus was completely
inhibited at 200 mM NaCl at both temperature levels. The temperature had no significant
effect on the GR of the L. scindicus seeds, but it showed an abrupt decline with the increasing
salinity (p < 0001, Figure 1B). The highest GR was recorded in non-saline-treated seeds
incubated at T2 (3.5 seeds/day), followed by seeds treated with 50 mM NaCl stress at
T1. Additionally, the seed vigor index (SVI) constantly showed a decline with increasing
salinity levels (Figure 1C). The results also revealed a significant effect of temperature
on the SVI, with the seeds germinated at T2 showing a consistent decrease at all NaCl
concentrations. The highest decline was observed in 150 mM-salinity-treated seeds.
Recovery germination of the L. scindicus seeds increased with the increasing NaCl
concentrations after transferring them to distilled water (Figure 3, Supplementary Table S2).
Aside from the fact that the seeds treated with the higher salinities, such as 200 mM NaCl,
did not germinate, large proportions of these seeds recovered after being transferred to
distilled water. The highest recovery percentage was recorded in the seeds with the highest
level of salinity stress (200 mM) at the optimum temperature T1 (85%), followed by the
seeds treated with 150 mM NaCl stress at both T2 (83.5%) and T1 (82.2%).
Sustainability 2022, 14, 8387 6 of 17
Sustainability 2022, 14, x FOR PEER REVIEW 6 of 17

Figure 1. Effect of temperature and salinity on the germination parameters of L. scindicus seeds. (A)
Figure 1. Effect of temperature and salinity on the germination parameters of L. scindicus seeds.
germination percentage; (B) germination rate; and (C) seed vigor index. Bars represent the mean
(A) germination
values and nailspercentage; (B) germination
on the top indicate standardrate; and
error. (C) seed
Capital andvigor
smallindex.
lettersBars represent
indexed on thethe
topmean
of
values andrepresent
the bars nails on the
the significance
top indicateof
standard error. Capital
the temperature and small
and salinity letters
effect, indexed on
respectively, at pthe top of
= 0.05,
the bars represent
Duncan’s test. the significance of the temperature and salinity effect, respectively, at p = 0.05,
Duncan’s test.
 Sustainability 2022, 14, 8387 7 of 17
Sustainability 2022, 14, x FOR PEER REVIEW 7 of 17

Figure 2. Cumulative
Figure germination
2. Cumulative speed speed
germination of L. scindicus seeds atseeds
of L. scindicus different temperatures
at different and salinity
temperatures and salinity
levels.
Sustainability 2022, 14, x FOR PEER (A) temperature 1 and (B) temperature 2. Each bar is a representation of the mean (n = 5)
REVIEW 8 of 17
levels. (A) temperature 1 and (B) temperature 2. Each bar is a representation of the mean (n = 5)
values and the nails on the top indicate standard error.
values and the nails on the top indicate standard error.
Recovery germination of the L. scindicus seeds increased with the increasing NaCl
concentrations after transferring them to distilled water (Figure 3, Supplementary Table
S2). Aside from the fact that the seeds treated with the higher salinities, such as 200 mM
NaCl, did not germinate, large proportions of these seeds recovered after being trans-
ferred to distilled water. The highest recovery percentage was recorded in the seeds with
the highest level of salinity stress (200 mM) at the optimum temperature T1 (85%), fol-
lowed by the seeds treated with 150 mM NaCl stress at both T2 (83.5%) and T1 (82.2%).

Figure 3.
Figure 3. Effect
Effectof
oftemperature
temperatureandandsalinity
salinityonon
recovery
recoverygermination of L.
germination of scindicus seeds.
L. scindicus BarsBars
seeds. rep-
resent the mean values and nails on the top indicate standard error. Capital and small letters
represent the mean values and nails on the top indicate standard error. Capital and small letters in-
dexed on the top of the bars represent the significance of the temperature and salinity effect, respec-
indexed on the top of the bars represent the significance of the temperature and salinity effect,
tively, at p = 0.05, Duncan’s test.
respectively, at p = 0.05, Duncan’s test.

3.2. Seedling Growth

The aRL and aSL decreased significantly with the increasing salt concentrations (p =
0.0001, Supplementary Table S1 and Figure S2). The highest aRL and aSL were shown in
the seedlings germinated with the non-saline treatment and the lowest were recorded at
Sustainability 2022, 14, 8387 8 of 17

3.2. Seedling Growth

The aRL and aSL decreased significantly with the increasing salt concentrations
(p = 0.0001, Supplementary Table S1 and Figure S2). The highest aRL and aSL were shown
in the seedlings germinated with the non-saline treatment and the lowest were recorded at
the highest (150 mM) salinity level (Figure 4A,B). The temperature showed a significant
effect on the aRL at all salinity levels. The maximum aRL was shown by the seedlings
germinated at T1 (Figure 4A). However, in the case of the aSL, the temperature showed no
significance in the seedlings germinated under non-saline conditions, while the aSL of the
Sustainability 2022, 14, x FOR PEER REVIEW
seedlings germinated at T1 was significantly higher compared to seedlings at T2 with9 ofall
17

the salinity levels (50–150 mM NaCl, Figure 4B).

Figure4.4.Effect
Figure Effectof
oftemperature
temperatureandandsalinity
salinityon
onthe
theseedling
seedlinggrowth
growthparameters
parametersof ofL.L.scindicus.
scindicus.(A)
(A)
averageroot
average rootlength;
length;(B)
(B)average
averageshoot
shootlength;
length;(C)
(C)root
rootdry
dryweight;
weight;(D)
(D)shoot
shootdry
dryweight;
weight;(E) (E)seedling
seedling
biomass; and (F) specific leaf area. Bars represent the mean values (n = 5) and nails on the top indi-
biomass; and (F) specific leaf area. Bars represent the mean values (n = 5) and nails on the top indicate
cate standard error. Capital and small letters indexed on the top of the bars represent the significance
standard error. Capital and small letters indexed on the top of the bars represent the significance of
of the temperature and salinity effect, respectively, at p = 0.05, Duncan’s test.
the temperature and salinity effect, respectively, at p = 0.05, Duncan’s test.
3.3. Effect of Salinity and Temperature on Chlorophyll Content of L. scindicus Seedlings
The effects of temperature (A) and salinity (B), and their combination (A × B), on the
chlorophyll content of L. scindicus seedlings were found to be highly significant (p < 0.0001,
Supplementary Table S1). The results indicated that the salinity significantly decreased
the chlorophyll a (Figure 5A), chlorophyll b (Figure 5B), and total chlorophyll (Figure 5C)
content of the L. scindicus seedlings incubated at both T1 and T2. The optimum tempera-
ture, i.e., T1, displayed better chlorophyll pigment concentrations at all the studied salin-
Sustainability 2022, 14, 8387 9 of 17

The RDW and SDW of the L. scindicus seedlings showed a significant decline with the
increasing salinity (p = 0.0001, Figure 4C,D). A similar pattern was observed in the SB, as
far as salinity is concerned (Figure 4E). The temperature showed no significant effect on
the RDW and SDW at all the NaCl concentrations. The highest RDW was calculated in the
non-saline-treated seedlings germinated at T2, while the highest SDW was shown by the
50 mM-salinity-treated seedlings germinated at T1. The most reduction in both the RDW
and SDW was determined in the T2-incubated seedlings at the 150 mM salinity level. The
SB showed significant variation with the change in temperature (p = 0.0001). The SB of the
seedlings germinated at T2 was higher at all the salinity levels except at 150 mM NaCl,
where the SB of the seedlings incubated at T1 was higher (Figure 4E).
Salinity, temperature, and their combination showed a significant effect on the specific
leaf area (SLA) of the L. scindicus seeds (Supplementary Table S1). The SLA showed a con-
siderable decline with increasing NaCl concentrations, except for the seedlings incubated
at T1 and treated with 50 mM salinity, which showed an abrupt increase (Figure 4F). The
highest SLA was determined in the seedlings grown in T1 incubators at 50 mM salinity,
while the lowest was estimated in the seedlings at T2, treated with 150 mM NaCl stress.

3.3. Effect of Salinity and Temperature on Chlorophyll Content of L. scindicus Seedlings

The effects of temperature (A) and salinity (B), and their combination (A × B), on the
chlorophyll content of L. scindicus seedlings were found to be highly significant (p < 0.0001,
Supplementary Table S1). The results indicated that the salinity significantly decreased
the chlorophyll a (Figure 5A), chlorophyll b (Figure 5B), and total chlorophyll (Figure 5C)
content of the L. scindicus seedlings incubated at both T1 and T2. The optimum temperature,
i.e., T1, displayed better chlorophyll pigment concentrations at all the studied salinity levels.
The highest chlorophyll content accumulation was measured in non-saline-treated seedlings
at T1 and the largest decline was recorded in 150 mM NaCl-treated-seedlings at T1. It is
worthwhile to mention that there was not the required seedling material available for the
study of the chlorophyll content at T2 at the 150 mM salinity level.

3.4. Effect of Salinity and Temperature on the Shoot and Root Proline Content of
L. scindicus Seedlings
Increasing levels of NaCl application (0–100 mM) significantly enhanced the produc-
tion of proline in the shoots and roots of the L. scindicus seedlings at both the applied
temperatures T1 and T2 (Supplementary Table S1, Figure 6). The proline content of the
shoot and root systems was higher in the seedlings germinated at T2 at all the studied
salinity concentrations. The maximum proline buildup was recorded at the highest salinity
stress for the seedlings germinated at T2. It is pertinent to mention that there was not
enough material to investigate the proline content at the salinity level of 150 mM.
Sustainability 2022,14,
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1017of 17

Figure5.5.Effect
Figure Effectofoftemperature
temperatureandand salinity
salinity onon
thethe chlorophyll
chlorophyll a (A);
a (A); chlorophyll
chlorophyll b (B);
b (B); andand
totaltotal
chlorophyll(C)
chlorophyll (C)ofofL.L.scindicus
scindicusseedlings.
seedlings.Bars
Barsrepresent
representthe
themean
meanvalues
values(n(n== 5)
5) and
and nails
nails on
on the
the top
indicate standard error. Capital and small letters indexed on the top of the bars represent the signif-
top indicate standard error. Capital and small letters indexed on the top of the bars represent the
icance of the temperature and salinity effect, respectively, at p = 0.05, Duncan’s test.
significance of the temperature and salinity effect, respectively, at p = 0.05, Duncan’s test.

3.4. Effect of Salinity and Temperature on the Shoot and Root Proline Content of L. scindicus
Seedlings
Increasing levels of NaCl application (0–100 mM) significantly enhanced the produc-
tion of proline in the shoots and roots of the L. scindicus seedlings at both the applied
temperatures T1 and T2 (Supplementary Table S1, Figure 6). The proline content of the
shoot and root systems was higher in the seedlings germinated at T2 at all the studied
salinity concentrations. The maximum proline buildup was recorded at the highest salin-
ity stress for the seedlings germinated at T2. It is pertinent to mention that there was not
enough material to investigate the proline content at the salinity level of 150 mM.
Sustainability 2022,14,
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11 of 17
17

Figure6.6.Effect
Figure Effect of temperature
of temperature and salinity
and salinity on theon theproline
shoot shoot (µg/gFW)
proline (µg/gFW) and root
and root proline proline
(µg/gFW)
(µg/gFW) content of L. scindicus seedlings. Bars represent the mean values (n = 5) and nails on the
content of L. scindicus seedlings. Bars represent the mean values (n = 5) and nails on the top indicate
top indicate standard error. Capital and small letters indexed on the top of the bars represent the
standard error. Capital and small letters indexed on the top of the bars represent the significance of
significance of the temperature and salinity effect, respectively, at p = 0.05, Duncan’s test.
the temperature and salinity effect, respectively, at p = 0.05, Duncan’s test.

4.4.Discussion
Discussion
Thisstudy
This studyaimed
aimedtotoexplore
explorethe theeffects
effectsofof different
different salt
salt concentrations
concentrations on on
seedseed ger-
germi-
mination,
nation, seedling
seedling growth,
growth, chlorophyll
chlorophyll content,
content, and and proline
proline contentcontent
in L. in L. scindicus
scindicus seed-
seedlings
lings germinated at two different temperature levels. Salinity
germinated at two different temperature levels. Salinity and temperature are important and temperature are im-
portant factors affecting seed germination [3,4]. The temperature
factors affecting seed germination [3,4]. The temperature has been shown to modulate has been shown to mod-
ulate salinity
salinity tolerancetolerance
duringduring the germination
the germination stage, with stage,
thewith the tolerance
highest highest tolerance
found around found
around the optimal temperature [36]. In glycophytes and to a lesser
the optimal temperature [36]. In glycophytes and to a lesser extent in halophytes, increased extent in halophytes,
increased
salt salt been
stress has stressdemonstrated
has been demonstrated
to cause atoreduction
cause a reduction in the percentage
in the percentage of seed
of seed germi-
germination
nation as wellasaswell as a delay
a delay in theingermination
the germination processprocess [51,56–58].
[51,56–58]. Moreover,
Moreover, the the ability
ability of
of seeds
seeds to germinate
to germinate through
through a wide
a wide rangerange of salinities
of salinities and and temperatures
temperatures is critical
is critical for thefor
the production
production of plants
of plants in varying
in varying environments
environments [59]. Our[59].findings
Our findings
revealed revealed that ]in-
that increasing
creasing salt concentrations significantly decreased the GP,
salt concentrations significantly decreased the GP, GR, and SVI at both the incubating GR, and SVI at both the incu-
bating temperature
temperature levels T1 levels
and T1T2 and T2 (Figure
(Figure 1). The 1). The results
results also indicated
also indicated that thethat the germi-
germination
nationatspeed
speed at thesalinity
the highest highestlevels
salinity
waslevels
delayedwas(Figure
delayed 2). (Figure 2). These
These results wereresults were in
in agreement
agreement
with previous with previous
studies studies
on acacia on acacia
species [60],species
Acacia[60], Acacia ehrenbergiana
ehrenbergiana and Acaciaand Acacia
seyal [61], seyal
Fes-
[61],arundinacea
tuca Festuca arundinacea [58], Haloxylon
[58], Haloxylon ammodendron ammodendron
[62], Festuca [62], Festuca arundinacea,
arundinacea, Bromus inermis,Bromus andin-
Elymus
ermis, andbreviaristatus [40], and Lasiurus
Elymus breviaristatus [40], andscindicus
Lasiurus and Panicumand
scindicus turgidum
Panicum [42].
turgidum [42].
Additionally,
Additionally,the theresults
resultsindicated
indicatedthatthatthe seedsofofL.L.scindicus
theseeds scindicusincubated
incubatedatat35/30 35/30◦°C, C,
D/N
D/N temperature (T2) showed a decline in germination germination indices such as the the GP,
GP,GR,GR,and and
SVI
SVIcompared
comparedto tothe
theseeds
seedsgerminated
germinatedat atT1T1(optimal
(optimaltemperature
temperaturefor forthethegermination
germinationof of
L.L.scindicus).
scindicus). However,
However,the thegermination
germinationrate rateand/or
and/or thethe cumulative
cumulative germination
germination speed speed
were
werehigher
higherin inthe
thecase
caseof ofseeds
seedsincubated
incubatedatatT2 T2under
underthe thenon-saline
non-salineor orlower
lowersalinity
salinity
concentration,
concentration, which which then showed a decline under the highest salinity
showed a decline under the highest salinity levels. Similar re- levels. Similar
results werereported
sults were reportedby by [41,42,59],
[41,42,59], where
where thethe germination
germination of of Medicago
Medicago ruthenica;
ruthenica;Lasiurus
Lasiurus
scindicus, Panicum turgidum; and Sorghum bicolor decreased at
scindicus, Panicum turgidum; and Sorghum bicolor decreased at the highest temperaturethe highest temperature underun-
the
dervarying
the varyingconcentrations
concentrations of NaCl.
of NaCl.These
Theseresults
resultscancan
bebe linked
linked totothethecapacity
capacityofofhighhigh
temperatures
temperaturestotoimpede impedeseed seedgermination
germination bybyinducing
inducing thetheproduction
production of large levels
of large of en-
levels of
dogenous abscisic acid (ABA) via transcriptional activation of
endogenous abscisic acid (ABA) via transcriptional activation of ABA signaling genes ABA signaling genes [63,64].
Apparently, under higher
[63,64]. Apparently, under temperatures, salinity can
higher temperatures, affectcan
salinity germination by directly
affect germination byaltering
directly
seed
altering seed water imbibition due to the reduced osmotic potential of germinationand
water imbibition due to the reduced osmotic potential of germination media, the
media,
and the biochemical reactions that regulate the metabolism involved in seed germination
Sustainability 2022, 14, 8387 12 of 17

biochemical reactions that regulate the metabolism involved in seed germination [65,66].
Also, the effects of NaCl are increased on the osmotic potential and specific ion toxicity
(accumulation of an excess of Na+ and Cl− ions) under increasing temperatures [33,40,51].
Aside from that, the absorption of excess Na+ and Cl− ions from soils cause ionic stress
and toxicity, which contributes to the disruption in biochemical processes such as nucleic
and protein metabolism, energy production, and respiration [51,67]. Salinity also disrupts
nutrient and hormone balances during germination, particularly gibberellin (GA) and
abscisic acid (ABA). As a result, high salinity levels cause seed germination to be delayed or
even inhibited, depending on the salt tolerance of the plants [68]. High salinity stress may
disrupt the dynamic balance between the generation and scavenging of reactive oxygen
species (ROS) such as hydroxyl radicals (OH˙), superoxide (O2 •− ), and hydrogen peroxide
(H2 O2 ). ROS damage macromolecules such as proteins, carbohydrates, nucleic acids, and
lipids, as well as cellular structures such as membranes, resulting in seed germination
inhibition [69].
According to the results of the recovery germination, ungerminated L. scindicus seeds
from the two incubating temperatures and exposed to high NaCl concentrations for 20 days
germinated successfully after being transferred to distilled water (Figure 3). The results in
this study were in accordance with [70,71], where the results also indicated the recovery of
ungerminated Arthrocnemum macrostachyum, Arthrocnemum indicum, and Atriplex canescens
seeds from high salinity levels. Recovery of germination indicates the ability of seeds to
survive in harsh environments. Seed survival rather than germination is the more effective
method for plants to establish successfully under elevated salt stress conditions, because
germination occurs when high-saline conditions are alleviated, such as during a rainy
season [70].
In addition to seed germination, increased salt and temperature had detrimental
effects on seedling growth, as measured by the root length, shoot length, and seedling
biomass. The seedling growth parameters such as the aRL, aSL, RDW, SDW, SB, and
SLA of L. scindicus showed a significant decline with the increasing salinities at both the
incubating temperatures of T1 and T2 (Figure 4). This is a common phenomenon in plants
under salinity stress [72]. Many plants experience a reduction in seedling root and shoot
lengths under saline conditions. This could be due to the harmful effects of NaCl, as
well as a lack of water and nutrient intake [73]. Furthermore, salt can have a deleterious
impact on cells, tissue, and organ ultrastructure [8]. Secondly, salinity, through osmotic and
specific ion toxic effects, prevents the preservation of sufficient nutrient levels important
for plant growth, reducing root and shoot emergence, and seedling growth [20,21,74]. Our
findings are in line with the results of [51,58], where the seedling growth of Sorghum bicolor
and Festuca arundinacea showed a constant decline with the increase in salinity treatment.
Moreover, the exposure of plants to high temperatures can have morphological implications,
such as sunburn on the entire plant’s shoot, leaf senescence, and root and shoot growth
inhibition [75]. The results of our study indicated that the aRL, aSL, and SLA of L. scindicus
seedlings decreased when incubated at T2 along with all the varying salinity concentrations.
These results are in accordance with the findings of [59], where all the growth parameters
of Sorghum bicolor showed a significant decline at the highest temperature of 40 ◦ C. This is
due to the increased detrimental impact of high temperatures on enzymic activities, protein
synthesis, and ATP content, as well as the generation of reactive oxygen species (ROS) [27].
Plants’ responses to excessive salinity have been shown to have a negative impact
on photosynthetic activity, resulting in a decrease in chlorophyll concentration [76]. This
decrease in chlorophyll concentration could be attributed to the loss of grana stacking or to
changes in the thylakoid structure [77]. The results indicated a general declining trend of
chlorophyll a, chlorophyll b, and total chlorophyll in L. scindicus seedlings when exposed
to increasing salinity concentrations at two incubating temperatures T1 and T2 (Figure 5).
Also, the chlorophyll content of the seedlings showed an abrupt decline at the temperature
T2 compared to the seedlings at T1 with all the salinity levels. These results indicate that
the response to high salinity and temperature involves a drop in the chlorophyll content
Sustainability 2022, 14, 8387 13 of 17

of L. scindicus plants, as also shown in the previous studies on L. scindius [8], Sorghum
bicolor [59], and P. simonii [78].
Proline, a suitable osmolyte, is accumulated in plants in response to abiotic stress such
as salinity, and low and high temperature [79]. This molecule has a variety of protective
functions, including cellular structure stabilization and damage reduction in the photo-
synthetic apparatus, redox status balance, cytosolic pH maintenance, protein structure
stabilization, and participation in stress signaling [80]. It also acts as an antioxidant and
an ROS scavenger [81]. Moreover, proline metabolism plays an important role in seed
germination via the oxidative pentose phosphate pathway (OPPP), which relies on proline
metabolism to generate NAD(P)+ in the cytosol [82]. The proline content (for both the root
and shoot) in this study showed a significant increase with the increase in salinity stress and
temperature (Figure 6). Similar results were observed in Brassica napus and tobacco plants,
wherein salinity and temperature induced the accumulation of proline [83,84]. Proline
overproduction has been linked to halophytic behavior in several plant species with the
goal of providing salinity tolerance [81]. Furthermore, it has recently been discovered that
proline helps to alleviate the effects of heat stress [84].

5. Conclusions
In conclusion, this study was carried out to evaluate the germination and stability of L.
scindicus seeds incubated at two temperature levels and exposed to varying salinity stress.
The results in this study demonstrated that, under a low level of salinity stress, L. scindicus
seeds, incubated at two varying temperatures, can germinate and grow successfully, while
with the increasing salinity concentration, there is a consistent decline both in germination
and growth. The same was true for the incubation temperatures, as the L. scindicus seeds
at the highest temperature, T2, showed a progressive decline compared to at T1. For
example, at optimum incubation temperature T1, the seed germination under salinity level
100 mM was significantly higher (82%) than at the highest incubation temperature T2 (21%).
However, the ungerminated seeds did not lose their viability on exposure to high salinity
stress as they restored germination after being transferred to distilled water. The growth and
physiological parameters showed a similar trend and indicated that L. scindicus was more
resistant to salinity stress, especially when incubated under the optimum temperature. Seed
germination research is critical for establishing a scientific and theoretical foundation for
large-scale L. scindicus ecosystem restoration, enhancement, and protection under changing
environments. This can also help with sustainable development in dry and saline areas, as
well as vegetation restoration and ecosystem reconstruction, which is otherwise degrading
at an enormous pace with the increase in global warming and salinity.
However, this experiment has been conducted in completely controlled conditions.
The results in natural conditions may vary owing to various environmental factors. There-
fore, conducting germination trial experiments in natural habitats would provide us with a
better understanding.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/su14148387/s1, Table S1: Analysis of variance for different
germination and growth parameters of L. scindicus under increasing salinity and temperature levels;
Table S2: Analysis of variance for recovery germination of L. scindicus seeds under different salinity
levels and temperature; Figure S1: Monthly temperature data of Riyadh region, Saudi Arabia; Figure
S2: Showing seedlings of L. scindicus germinated at two temperatures T1 (A) and T2 (B), and exposed
to different salinity levels.
Author Contributions: Conceptualization, J.A.M. and B.A.D.; methodology, J.A.M., B.A.D. and
M.M.H.; software, B.A.D. and J.A.M.; formal analysis, J.A.M. and B.A.D.; investigation, J.A.M. and
M.M.H.; resources, A.A.A., M.N.A. and J.A.M.; data curation, J.A.M., M.M.H. and B.A.D.; writing—
original draft preparation, J.A.M.; writing—review and editing, M.N.A., A.A.A., B.A.D., M.M.H.,
S.N.S.I. and J.A.M.; visualization, J.A.M. and B.A.D.; supervision, A.A.A.; project administration,
A.A.A. and M.N.A. All authors have read and agreed to the published version of the manuscript.
Sustainability 2022, 14, 8387 14 of 17

Funding: The authors extend their appreciation to Princess Nourah bint Abdulrahman Univer-
sity Researchers Supporting Project number (Grant No. PNURSP2022R103), Princess Nourah bint
Abdulrahman University, Riyadh, Saudi Arabia.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Acknowledgments: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.

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