PLOS ONE

RESEARCH ARTICLE

The impact of PEG-induced drought stress on

seed germination and seedling growth of
different bread wheat (Triticum aestivum L.)
genotypes
Shahzadi Mahpara1*, Aleena Zainab ID1*, Rehmat Ullah2, Salma Kausar3,
Muhammad Bilal2, Muhammad Imran Latif4, Muhammad Arif5, Imran Akhtar6,
Abdulrahman Al-Hashimi7, Mohamed S. Elshikh7, Marek Zivcak ID8*, Ali Tan Kee Zuan ID9*
a1111111111
1 Department of Plant Breeding and Genetics, Faculty of Agricultural Sciences, Ghazi University, Dera Ghazi
a1111111111
Khan, Pakistan, 2 Soil and Water Testing Laboratory for Research, Dera Ghazi Khan, Punjab, Pakistan,
a1111111111 3 Senior Scientist (Agri Chemistry), Soil and Water Testing Laboratory, Lodhran, Pakistan, 4 Scientific
a1111111111 Officer, Soil and Water Testing Laboratory, Narowal, Pakistan, 5 Scientific Officer (Lab), Soil and Water
a1111111111 Testing Laboratory, Layyah, Pakistan, 6 Senior Scientist (Ento), Regional Agriculture Research Institute,
Bahawalpur, Pakistan, 7 Department of Botany and Microbiology, College of Science, King Saud University,
Riyadh, Saudi Arabia, 8 Department of Plant Physiology, Slovak University of Agriculture, Nitra, Slovakia,
9 Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, Selangor, Malaysia

* szrikhan@gmail.com (AZ); smahpara@gudgk.edu.pk (SM); marek.zivcak@uniag.sk (MZ); tkz@upm.edu.

OPEN ACCESS my (ATKZ)
Citation: Mahpara S, Zainab A, Ullah R, Kausar S,
Bilal M, Latif MI, et al. (2022) The impact of PEG-
induced drought stress on seed germination and
seedling growth of different bread wheat (Triticum
Abstract
aestivum L.) genotypes. PLoS ONE 17(2):
Wheat is an important crop, used as staple food in numerous countries around the world.
e0262937. https://doi.org/10.1371/journal.
pone.0262937 However, wheat productivity is low in the developing world due to several biotic and abiotic
stresses, particularly drought stress. Non-availability of drought-tolerant wheat genotypes at
Editor: Shahid Farooq, Harran Üniversitesi: Harran
Universitesi, TURKEY different growth stages is the major constraint in improving wheat productivity in the devel-
oping world. Therefore, screening/developing drought-tolerant genotypes at different growth
Received: October 8, 2021
stages could improve the productivity of wheat. This study assessed seed germination and
Accepted: January 7, 2022
seedling growth of eight wheat genotypes under polyethylene glycol (PEG)-induced stress.
Published: February 11, 2022 Two PEG-induced osmotic potentials (i.e., -0.6 and -1.2 MPa) were included in the study
Copyright: © 2022 Mahpara et al. This is an open along with control (0 MPa). Wheat genotypes included in the study were ‘KLR-16’, ‘B6’,
access article distributed under the terms of the ‘J10’, ‘716’, ‘A12’, ‘Seher’, ‘KTDH-16’, and ‘J4’. Data relating to seed germination percent-
Creative Commons Attribution License, which
age, root and shoot length, fresh and dry weight of roots and shoot, root/shoot length ratio
permits unrestricted use, distribution, and
reproduction in any medium, provided the original and chlorophyll content were recorded. The studied parameters were significantly altered by
author and source are credited. individual and interactive effects of genotypes and PEG-induced osmotic potentials. Seed
Data Availability Statement: All relevant data are germination and growth parameters were reduced by osmotic potentials; however, huge dif-
within the manuscript and its Supporting ferences were noted among genotypes. A reduction of 32.83 to 53.50% was recorded in
Information files. seed germination, 24.611 to 47.75% in root length, 37.83 to 53.72% in shoot length, and
Funding: The current study was partially supported 53.35 to 65.16% in root fresh weight. The genotypes, ‘J4’, ‘KLR-16’ and ‘KTDH-16’, particu-
by Ghazi University, Dera Ghazi Khan, Pakistan. larly ‘J4’ better tolerated increasing osmotic potentials compared to the rest of the genotypes
The authors extend their appreciation to the
included in the study. Principal component analysis segregated these genotypes from the
Researchers Supporting Project number (RSP-
2021/219), King Saud University, Riyadh, Saudi rest of the genotypes included in the study indicated that these can be used in the future
Arabia. This work was supported by the project
APVV-18-0465 and VEGA 1/0589/19. The funders

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

had no role in study design, data collection and studies to improve the drought tolerance of wheat crop. The genotype ‘J4’ can be used as a
analysis, decision to publish, or preparation of the breeding material to develop drought resistant wheat genotypes.
manuscript.

Competing interests: The authors have declared

that no competing interests exist.

Introduction
Wheat (Triticum aestivum L.) belongs to Triticeae tribe and Poaceae family. It globally impor-
tant cereal supposed to be originated in the Middle East region of Asia [1, 2]. Tetraploid and
hexaploidy form of wheat has been domesticated since 10,000 years ago [3]. Hexaploid form is
modern day bread wheat and fulfills dietary needs of the global population. The Northern
India, Northern USA, and neighboring areas in Canada, Northern and Central Europe, South-
ern Australia, and South Africa are the major bread wheat producing areas in the world.
Global population is expected to reach10 billion by 2050, which would require double of the
current global food production. Expected climate changes would make the crop production
difficult because of sudden changes in temperature and rainfall [4]. Wheat contributes 2%
towards gross domestic product and 9.9% towards value added in agriculture. The area under
wheat production in the country fluctuates within 2–5% increase or decrease due to various
factors [5].
Seedling stage of crop plants is highly vulnerable to the water deficit. Seed germination is a
prerequisite and important transition stage for crop plants from seeds to seedlings. The semi-
arid regions of the world experience low moisture availability during seed germination of
wheat crop [6]. Low moisture availability during seed germination and subsequent growth
stages of wheat crop declines both production maturity time [7, 8]. The impacts of water stress
on seed germination and vegetative growth of different crops such as wheat [8], maize and bar-
ley [8–10] in earlier studies. The impact of drought stress on seed germination and seedling
stage of four bread wheat varieties have been evaluated and reduction in these traits was noted
with significant differences among tested varieties [6, 11].
The successful establishment of crop plants relies on microclimatic conditions of seedbed
and seed quality [12, 13]. Hence, seed germination of crop plants is tested under simulated
environments to infer their tolerance to adverse environmental conditions. Observing seed
germination under polyethylene glycol (PEG) induced drought stress is the most common
screening method used to test the drought tolerance of different crop varieties during seed ger-
mination and early stand establishment [14]. Inferring changes in root length or root depth of
the seedlings subjected to drought stress could provide valuable insights regarding these traits
[15, 16]. Higher tolerance to adverse environmental conditions during seedling stage results in
better crop production [15]. Screening a large pool of available genotypes under adverse envi-
ronmental conditions is a fundamental method to select the tolerant genotypes for improved
crop production. The use of osmotic substances of high molecular weight such as PEG is a
common method to test the drought tolerance of crop plants during seed germination and
seedling establishment [17, 18].
Seed germination and seedling emergence/establishment are important criteria for testing
the tolerance of wheat genotypes to various abiotic stresses, particularly, drought stress [8, 19].
Seed germination percentage and seedling establishment are significantly reduced when soil
osmotic potential reaches to -1.5 MPa [20]. Short-statured wheat cultivars have slower initial
growth and their coleoptile length and leaf index undergoes decline during early growth peri-
ods [21]. Reduced coleoptile length indicates low seed germination and subsequent low plant
height, whereas increased coleoptile length would result in larger initial leaf sizes and

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

accelerate seed germination [22]. Positive correlation has been reported among seed germina-
tion and radical, plumule, coleoptile length, and dry weight of radical and plumule [23, 24].
Plant breeding concentrated on the above-ground traits for a long time, while root traits
have been ignored due to several difficulties [25]. Root traits have gained significant attention
during the last decade [26, 27]. Screening genotypes for early drought tolerance and inferring
their root attributes at seedling stage has witnessed significant progress [28, 29]. The genotypes
with higher root volume combined with longer seminal and adventitious root length has been
suggested as useful candidates for increasing grain yield [30]. Plant growth, root to shoot ratio
and root length could also be useful characteristics for improving the yield under arid and
semi-arid climatic conditions [31, 32].
The PEG has been frequently used to for genotypes’ screening for drought tolerance at ear-
lier growth stage. The PEG reduces seed germination and growth by reducing water potential,
and the effect is observed more on the shoots compared to primary roots [13, 33]. Several stud-
ies indicated that in vitro screening using PEG is one of the reliable approaches to select
drought-tolerant genotypes based on germination indices [16, 32]. The PEG is involved in the
transfer of ions and nonionic compounds such as mannitol, raffinose and inulin [34, 35]. The
earlier study [35] proposed that PEG is a high molecular weight non-ionic substance that is
water soluble and anti-penetrable. The decrease in osmotic and water potential due to PEG
has a positive correlation with the accumulation of proline which leads to decrease in osmotic
stress and helps to maintain plant growth [36].
Although plenty of lines/genotypes of wheat crop are being developed on regional scales,
their testing for drought tolerance at seedling stage is rarely done. Therefore, current study
tested drought tolerance of eight recently developed wheat genotypes/lines in Pakistan through
PEG-induced osmotic stress. It was hypothesized that the tested genotypes will differ in their
drought tolerance and growth traits. It was further hypothesized that increasing negative
osmotic potential would reduce seed germination and seedling traits. The results will help to
select the most tolerant genotypes for breeding purposes to develop drought tolerant geno-
types in the future.

Materials and methods

Experimental site
The current study was conducted at Plant Breeding and Genetics Laboratory, Dera Ghazi
Khan, Pakistan. Eight different wheat genotypes (Table 1) with unknown drought tolerance
were included in the study. There was no permit needed required to conduct the study as it
did not involve any endangered species. Three different osmotic potentials, i.e., 0, -0.6 MPa
and -1.2 MPa were included in the study by using PEG-6000 [37]. The desired quantity of

Table 1. The codes, names and drought tolerance levels of different wheat genotypes included in the study.
Genotype Code Genotype Name Drought tolerance
G1 ‘KLR-16’ Unknown
G2 ‘B6’ Unknown
G3 ‘J10’ Unknown
G4 ‘716’ Unknown
G5 ‘A12 (Ujala)’ Unknown
G6 ‘Seher’ Mild
G7 ‘KTDH-16’ Unknown
G8 ‘J4 (9268)’ Unknown
https://doi.org/10.1371/journal.pone.0262937.t001

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

PEG-6000 was mixed in the distilled water to make the solutions of -0.6 MPa and -1.2 MPa,
whereas distilled water was used in the control treatment [37].

Seed germination experiment

Three replicates of 25 sterilized (with 5% sodium hypochlorite) seeds were germinated
between the two layers of Whatman No.1 filter paper in Petri dishes (150 × 15 mm). The 10 ml
treatment solution or distilled water was poured on the filter paper and afterwards the solution
or distilled water was given according to the needs. The Petri dishes were sealed with Parafilm
to prevent evaporation. Seeds were incubated at 20 ± 2˚C and 12 hours light dark period for 10
days. Seed germination percentage was observed every 24 hours for 10 days and then seed ger-
mination percentage was computed. The seed was considered as ‘germinated’ once the radicle
elongated to 1–2 mm.

Seedling growth experiment

Seedling growth experiment was carried out in plastic pots (97 × 165 × 90 mm) filled with a
mixture of the sand and peat (1:1). The pots were placed into growth cabinet with 3 replica-
tions and 10 seeds were planted in each replication. Seeds were sown 3 cm in depth and pots
irrigated with PEG-6000 solutions as to generate osmatic potentials of 0, -0.6 and -1.2 MPa.
Pots were incubated under 25˚C and 70–80% relative humidity for 20 days. A seed/seedling
was considered as emerged when the emerging radicle reached to soil surface. Different
growth traits such as root length, shoot length, fresh root weight, fresh shoot weight, dry root
weight, dry shoot weight, and root, shoot ratio were measured from three weeks old seedling.
The experiment was laid out according to randomized complete block design with split plot
arrangements. Genotypes were considered as main factor, whereas osmotic potentials were
regarded as sub-factor. The seedlings were taken off from the pots, rinsed with water to remove
the debris, measured for root, and shoot lengths, divided into roots and shoots and dried in an
oven (roots and shoots separately) to infer the dry weight. The chlorophyll index was measured
with SPAD meter and expressed as SPAD values.

Statistical analysis
The collected data were tested for normality, which indicated that data were normally distrib-
uted. Two-way analysis of variance (ANOVA) was then used to infer the significance in the
data. Least significant difference test at 5% probability was used to compare the means where
ANOVA indicated significant difference. Principal component analysis with Kaiser normaliza-
tion was used to better visualize the data. The principal components with >1 eigenvalue were
interpreted. Similarly, the variable having >0.60 factor loading was considered to significantly
affect the relevant principal component. All computations were made on XLSTAT add-in of
Microsoft Excel program. The minimal dataset of the study has been uploaded as S3 Table.

Results
Individual and interactive effects of genotypes and PEG-induced drought stress significantly
altered seed germination percentage, root and shoot length, fresh and dry weights of roots and
shoot, root:shoot ratio and chlorophyll index (S1 Table). Overall, the highest seed germination
percentage (78.11%) was recorded for genotype ‘J4’, whereas genotype ‘716’ resulted in the
lowest (64.33%) seed germination (Table 2). Similarly, the highest (16.02 cm) and the lowest
(11.72 cm) root length was noted for the genotypes ‘J4’ and ‘716’, respectively. The highest
shoot length (11.98 cm), root fresh weight (0.47 g), root dry weight (0.23 g), shoot fresh weight

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Table 2. The impact of different genotypes on their seed germination and growth traits grown under different osmotic potentials.
Genotypes GP RL SL RFW RDW SFW SDW R/S Chl
(%) (cm) (cm) (g) (g) (g) (g) (SPAD value)
J4 78.11 a 16.02 a 11.98 a 0.47 a 0.23 a 0.42 a 0.24 a 1.35 d 49.86 a
KTDH-16 73.22 b 14.58 b 10.58 b 0.43 b 0.22 a 0.36 bc 0.20 b 1.40 c 46.83 b
KLR-16 71.11 c 14.03 c 10.71 b 0.43 b 0.23 a 0.37 b 0.18 c 1.31 e 43.57 de
B6 70.55 c 13.37 d 9.62 d 0.43 b 0.23 a 0.35 cd 0.18 c 1.45 b 44.90 c
J10 71.22 c 12.43 e 10.05 c 0.40 c 0.20 b 0.34 d 0.16 d 1.26 f 44.13 cd
Seher 67.00 d 12.32 e 9.27 e 0.39 cd 0.18 c 0.34 d 0.17 d 1.34 d 41.02 f
A12 66.66 d 12.27 e 8.34 f 0.38 d 0.16 d 0.28 e 0.11 e 1.52 a 46.55 b
716 64.33 e 11.72 f 9.04 e 0.35 e 0.17 c 0.27 e 0.10 e 1.30 e 42.86 e
LSD 5% 1.01 0.22 0.25 0.09 0.013 0.015 0.01 0.24 0.85

Here, GP = seed germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight,
SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index. The means sharing same letters within a same column are statistically non-significant
(p > 0.05).

https://doi.org/10.1371/journal.pone.0262937.t002

(0.42 g), shoot dry weight (0.24 g) and chlorophyll index (49.86 SPAD value) was noted for the
genotype ‘J4’. However, the lowest shoot length (8.34 cm) was recorded for the genotype ‘A12’,
while the genotype ‘A12’ observed the lowest root fresh weight (0.35 g), shoot fresh weight
(0.27 g), and shoot dry weight (0.10 g). Nonetheless, the lowest root dry weight (0.16 g) was
recorded for the genotype ‘716’, whereas the genotype ‘Seher’ resulted in the lowest chlorophyll
index (41.02 SPAD value) (Table 2).
The highest values of seed germination percentage, root and shoot length, fresh and dry
weights of roots and shoot, and chlorophyll index were recorded for control treatment,
whereas the lowest values of these traits were noted for -1.2 MPa osmotic potential (Table 3).
Contrastingly, the highest root:shoot ratio was noted for -0.6 MPa osmotic potential, whereas
the lowest value was noted under control treatment of the study (Table 3).
Regarding genotypes by drought stress interaction, all genotypes resulted in 100% seed ger-
mination under control treatment; however, seed germination recorded a significant decrease.
The genotype ‘J4’ with control treatment recorded the highest values for seed germination per-
centage, root and shoot length, fresh and dry weights of roots and shoot, and chlorophyll
index, whereas the lowest values for these traits were noted for the genotypes ‘716’ an ‘A12’
germinated under -1.2 MPa osmotic potential (Table 4). The genotypes ‘J4’ and ‘KTDH-16’
better tolerated increasing level of drought stress compared to the rest of the treatments
included in the study, whereas genotypes ‘716’ and ‘A12’ proved as the most sensitive
genotypes.

Table 3. The impact of different osmotic potentials on seed germination and growth traits of different wheat genotypes included in the study.
Osmotic potential GP RL SL RFW RDW SFW SDW R/S Chl
(%) (cm) (cm) (g) (g) (g) (g) (SPAD value)
0 MPa 100.00 a 18.34 a 14.55 a 0.69 a 0.31 a 0.60 a 0.30 a 1.27 c 49.44 a
-0.6 MPa 70.08 b 13.97 b 9.79 b 0.37 b 0.21 b 0.30 b 0.15 b 1.43 a 45.53 b
-1.2 MPa 40.75 c 7.72 c 5.50 c 0.17 c 0.08 c 0.12 c 0.05 c 1.41 b 39.92 c
LSD 5% 0.62 0.13 0.15 0.015 0.008 0.009 0.006 0.013 0.52

Here, GP = seed germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight,
SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index. The means sharing same letters within a same column are statistically non-significant
(p > 0.05).

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Table 4. The impact of wheat genotypes by different osmotic potentials’ interaction on seed germination and growth traits of wheat genotypes included in the
study.
Interactions GP RL SL RFW RDW SFW SDW R/S Chl
(%) (cm) (cm) (g) (g) (g) (g) (SPAD value)
G1 × O1 100.00 a 17.20 d 13.73 d 0.54 e 0.28 ef 0.48 f 0.17 g 1.25 gh 47.50 de
G2 × O1 100.00 a 17.50 d 13.00 e 0.65 d 0.26 fg 0.53 e 0.23 e 1.34 ef 52.66 ab
G3 × O1 100.00 a 18.46 c 14.03 cd 0.72 b 0.32 bc 0.64 c 0.33 c 1.33 ef 49.40 c
G4 × O1 100.00 a 17.13 d 14.36 c 0.68 c 0.30 cd 0.61 d 0.30 d 1.19 i 51.66 b
G5 × O1 100.00 a 19.16 b 16.03 a 0.76 a 0.37 a 0.69 a 0.38 a 1.22 h 53.53 a
G6 × O1 100.00 a 19.46 ab 15.66 ab 0.72 b 0.33 b 0.62 cd 0.31 d 1.24 h 46.73 ef
G7 × O1 100.00 a 19.66 a 15.43 b 0.75 a 0.36 a 0.66 b 0.36 b 1.28 g 48.66 cd
G8 × O1 100.00 a 18.13 c 14.20 c 0.70 bc 0.28 de 0.62 cd 0.31 cd 1.28 g 45.40 fg
G1 × O2 59.66 g 11.50 i 8.73 i 0.34 jk 0.17 k 0.24 j 0.09 i 1.32 f 43.00 hi
G2 × O2 62.00 f 12.26 h 7.93 j 0.33 k 0.19 jk 0.24 j 0.07 j 1.58 b 46.40 ef
G3 × O2 70.66 d 13.46 g 9.36 h 0.36 ij 0.22 hi 0.29 i 0.16 g 1.45 d 45.66 f
G4 × O2 71.00 d 13.10 g 10.33 g 0.35 ijk 0.20 ij 0.29 i 0.13 h 1.25 gh 43.03 hi
G5 × O2 82.00 b 18.36 c 12.33 f 0.42 f 0.24 g 0.38 g 0.23 e 1.48 c 52.40 ab
G6 × O2 67.66 e 14.43 f 10.30 g 0.39 gh 0.24 gh 0.33 h 0.16 g 1.36 e 44.16 gh
G7 × O2 75.66 c 16.03 e 10.20 g 0.40 fg 0.25 g 0.34 h 0.20 f 1.57 b 50.00 c
G8 × O2 72.00 d 12.63 h 9.16 hi 0.37 hi 0.21 ij 0.33 h 0.17 g 1.41 d 39.63 k
G1 × O3 33.33 m 6.46 m 4.66 m 0.16 o 0.08 m 0.11 n 0.04 l 1.32 f 38.10 l
G2 × O3 38.00 l 7.06 l 4.10 n 0.17 o 0.03 n 0.06 p 0.02 lm 1.66 a 40.60 jk
G3 × O3 41.00 k 8.20 k 5.46 l 0.22 lm 0.15 l 0.13 m 0.06 jk 1.58 b 39.63 k
G4 × O3 42.66 jk 7.06 l 5.45 l 0.18 no 0.10 m 0.14 lm 0.07 j 1.34 ef 37.70 l
G5 × O3 52.33 h 10.53 j 7.60 j 0.24 l 0.08 m 0.19 k 0.11 h 1.35 ef 43.66 h
G6 × O3 45.66 i 8.20 k 6.16 k 0.19 mn 0.13 l 0.16 l 0.09 i 1.34 ef 39.83 k
G7 × O3 44.00 ij 8.06 k 6.13 k 0.13 p 0.05 n 0.10 no 0.04 kl 1.34 ef 41.83 ij
G8 × O3 29.00 n 6.20 m 4.46 mn 0.12 p 0.04 n 0.08 op 0.01 m 1.32 f 38.03 l
LSD 5% 1.76 0.39 0.44 0.027 0.022 0.023 0.017 0.037 1.48

Here, G1 = 716, G2 = A12, G3 = B6, G4 = J10, G5 = J4, G6 = KLR-16, G7 = KTDH16, G8 = Seher, O1 = control (0 MPa), O2 = -0.6 MPa, O3 = -1.2 MPa. Here, GP = seed
germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight, SDW = shoot dry weight, R/
S = root:shoot ratio, Chl = chlorophyll index. The means sharing same letters within a same column are statistically non-significant (p > 0.05).

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The decrease in seed germination percentage, root and shoot length, fresh and dry weights
of roots and shoot, and chlorophyll index, and increase in root:shoot ratio was significantly
altered by individual and interactive effects of genotypes and PEG-induced drought stress lev-
els (S2 Table). Overall, the highest decrease in seed germination percentage (53.50%) and root
length (45.75%) was recorded for genotype ‘716’, whereas genotype ‘J4’ recorded the lowest
decline in these traits compared to the rest of the genotypes included in the study (Table 5).
The genotype ‘A12’ observed the highest decrease in shoot length, root dry weight, shoot fresh
weight and shoot dry weight. The lowest decrease in these traits was recorded for the genotype
‘J4’. Similarly, genotypes ‘716’ and ‘Seher’ observed the highest increase in root:shoot ratio,
whereas the lowest increase was noted for genotype ‘A12’ (Table 5).
The highest decrease in seed germination percentage, root and shoot length, fresh and dry
weights of roots and shoot, and chlorophyll index was recorded for -1.2 MPa osmotic potential
compared to the control treatment of the study, whereas the lowest decrease was recorded for
-0.6 MPa osmotic potential (Table 6). The root:shoot ratio was not altered by the osmotic
potentials included in the study (Table 6).

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Table 5. The impact of different genotypes on percentage decrease in their seed germination and growth traits under different osmotic potentials.
Genotypes GP RL SL RFW RDW SFW SDW R/S � Chl
(%) (cm) (cm) (g) (g) (g) (g) (SPAD value)
716 53.50 a 47.75 a 51.21 b 53.35 c 54.19 b 62.82 de 61.67 c 6.05 a 14.59 bc
A12 50.00 b 44.75 b 53.72 a 61.19 b 56.37 ab 71.37 a 79.23 a 20.80 c 17.38 b
B6 44.16 c 41.33 c 47.15 c 59.64 b 42.21 d 66.40 bc 66.66 b 14.03 b 13.65 c
J10 43.16 c 41.14 c 45.05 c 61.03 b 50.05 c 64.22 cd 66.60 b 9.40 a 21.87 a
J4 32.83 e 24.61 e 37.83 d 56.09 c 55.37 ab 58.44 f 54.78 d 16.08 b 10.27 d
KLR-16 43.33 c 41.86 c 47.45 c 59.63 b 43.46 d 60.50 ef 58.59 cd 9.19 a 10.11 d
KTDH-16 40.16 d 38.73 d 47.07 c 64.35 a 58.30 a 66.80 b 66.199b 13.43 b 5.62 e
Seher 49.50 b 48.06 a 51.99 ab 65.16 a 54.60 b 67.04 b 69.51 b 6.667a 14.46 c
LSD 5% 1.54 1.76 2.45 2.87 3.63 2.35 4.05 3.48 2.91

Here, GP = seed germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight,
SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index. The means sharing same letters within a same column are statistically non-significant
(p > 0.05).
�
indicated that relevant trait was increased instead of decrease

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Regarding genotypes by drought stress interaction, the genotype ‘Seher’ with -1.2 MPa
treatment recorded the highest decrease in seed germination percentage, root and shoot
length, fresh and dry weights of roots and shoot, and chlorophyll index, whereas the lowest
decrease in these traits were noted for the genotypes ‘J4’ under -0.6 MPa osmotic potential
(Table 7). The genotypes ‘J4’ and ‘KTDH-16’ better tolerated increasing level of drought stress
compared to the rest of the treatments included in the study, whereas genotypes ‘Seher’, ‘716’
and ‘A12’ proved as the most sensitive genotypes.
Principal component analysis executed on germination and growth traits of different geno-
types yielded in two principal components (PCs) with eigenvalues >1 (Table 8). The first two
PCs collectively explained 91.86% variability in the dataset. The first PC was positively influ-
enced by all measured traits except root:shoot ratio, whereas the second PC was positively
affected by root:shoot ratio and chlorophyll index (Table 8).
The biplot of first two PCs divided the genotypes in two major groups. The first group had
three genotypes having similar seed germination percentage and growth-related traits, whereas
the second group was not influenced by any studied traits. The first group contained the geno-
types with higher drought tolerance while the second group included the genotypes with the
lowest drought tolerance recorded in the current study (Fig 1).
Principal component analysis executed on reductions in seed germination and growth traits
of different genotypes in three PCs with eigenvalues >1 (Table 9). The first three PCs

Table 6. The impact of different osmotic potentials on percentage decrease in their seed germination and growth traits under different osmotic potentials.
Osmotic potential GP RL SL RFW RDW SFW SDW R/S� Chl
(%) (cm) (cm) (g) (g) (g) (g) (SPAD value)
-0.6 MPa 29.91 b 24.05 b 32.92 b 45.96 b 30.59 b 49.47 b 50.00 b 12.80 7.88 b
-1.2 MPa 59.25 a 58.00 a 62.45 a 74.15 a 73.04 a 79.93 a 80.81 a 11.11 19.10 a
LSD 5% 0.77 0.59 1.22 1.43 1.81 1.17 2.02 NS 1.45

Here, GP = seed germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight,
SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index. The means sharing same letters within a same column are statistically non-significant
(p > 0.05).
�
indicated that relevant trait was increased instead of decrease

https://doi.org/10.1371/journal.pone.0262937.t006

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Table 7. The impact of wheat genotypes by different osmotic potentials’ interaction on decrease in seed germination and growth traits of wheat genotypes included
in the study.
Interactions GP RL SL RFW RDW SFW SDW R/S Chl
(%) (cm) (cm) (g) (g) (g) (g) (SPAD value)
G1 × O2 40.33 h 33.12 f 36.38 de 37.37 h 38.08 f 48.61 fg 47.04 gh 6.01 a-d 9.43 gh
G2 × O2 38.00 i 29.88 g 38.97 d 48.39 f 26.64 hi 54.57 e 70.03 e 17.81 fg 11.87 fg
G3 × O2 29.33 k 27.07 h 33.26 e 49.70 f 30.85 gh 54.15 e 51.45 fg 9.01 b-e 7.55 h
G4 × O2 29.00 k 23.54 i 28.05 f 48.53 f 33.73 fg 51.38 ef 56.59 f 5.64 abc 16.70 cde
G5 × O2 18.00 m 4.17 k 23.07 g 44.04 g 33.30 fg 44.25 h 40.00 i 21.46 gh 2.12 i
G6 × O2 32.33 j 25.85 h 34.26 e 46.32 fg 26.30 hi 46.80 gh 46.21 gh 10.13 cde 5.47 hi
G7 × O2 24.33 l 18.47 j 33.90 e 45.94 fg 30.31 ghi 48.67 fg 44.41 hi 22.14 gh -2.77 j
G8 × O2 28.00 k 30.33 g 35.44 e 47.39 fg 25.53 i 47.36 gh 44.24 hi 10.19 cde 12.71 efg
G1 × O3 66.66 b 62.38 b 66.04 a 69.33 de 70.29 c 77.04 bc 76.30 cd 6.10 a-d 19.76 bc
G2 × O3 62.00 c 59.62 c 68.46 a 73.99 b 86.11 a 88.18 a 88.44 b 23.78 h 22.90 b
G3 × O3 59.00 d 55.58 d 61.04 b 69.58 cde 53.57 e 78.65 b 81.86 c 19.05 gh 19.75 bc
G4 × O3 57.33 de 58.75 c 62.05 b 73.53 bc 66.36 c 77.06 bc 76.61 cd 13.16 ef 27.03 a
G5 × O3 47.66 g 45.04 e 52.59 c 68.14 e 77.43 b 72.62 d 69.56 e 10.70 de 18.42 cd
G6 × O3 54.33 f 57.86 c 60.63 b 72.94 bcd 60.61 d 74.20 cd 70.98 de 8.24 b-e 14.70 def
G7 × O3 56.00 ef 58.98 c 60.25 b 82.76 a 86.29 a 84.93 a 87.95 b 4.72 ab 14.02 ef
G8 × O3 71.00 a 65.80 a 68.54 a 82.92 a 83.67 a 86.72 a 94.78 a 3.14 a 16.20 cde
LSD 5% 2.18 2.52 3.46 4.06 5.14 3.32 5.73 4.93 4.12

Here, G1 = 716, G2 = A12, G3 = B6, G4 = J10, G5 = J4, G6 = KLR-16, G7 = KTDH16, G8 = Seher, O2 = -0.6 MPa, O3 = -1.2 MPa. Here, GP = seed germination percentage,
RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight, SDW = shoot dry weight, R/S = root:shoot ratio,
Chl = chlorophyll index. The means sharing same letters within a same column are statistically non-significant (p > 0.05).
�
indicates that the relevant trait was increased instead of decrease

https://doi.org/10.1371/journal.pone.0262937.t007

Table 8. Eigenvalues, variability and factor loadings of first two principal components of principal component
analysis executed on seed germination and growth traits of wheat genotypes included in the study.
Traits PC1 PC2
Eigenvalue 6.76 1.50
Variability (%) 75.10 16.75
Cumulative % 75.10 91.86
Factor loadings
GP 0.91 -0.03
RL 0.99 0.05
SL 0.94 -0.28
FRW 0.96 -0.05
DRW 0.89 -0.28
FSW 0.99 -0.05
DSW 0.96 0.08
R/S 0.11 0.93
CHL 0.61 0.66

Here, GP = seed germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root
dry weight, SFW = shoot fresh weight, SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index. The
bold values indicate that the relevant trait significantly affected the corresponding principal component

https://doi.org/10.1371/journal.pone.0262937.t008

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Fig 1. Biplot of the first two principal components of principal component analysis executed on seed germination
and growth traits of wheat genotypes included in the study. Here, GP = seed germination percentage, RL = root
length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight, SFW = shoot fresh weight, SDW = shoot
dry weight, R/S = root:shoot ratio, Chl = chlorophyll index.
https://doi.org/10.1371/journal.pone.0262937.g001

Table 9. Eigenvalues, variability, and factor loadings of first two principal components of principal component
analysis executed on percentage decrease in seed germination and growth traits of wheat genotypes included in
the study.
PC1 PC2 PC3
Eigenvalue 4.23 2.36 1.024
Variability (%) 47.09 26.25 11.37
Cumulative % 47.09 73.32 84.72
Factor loadings
GP 0.82 0.47 0.03
RL 0.84 0.46 -0.20
SL 0.89 0.18 -0.20
FRW 0.52 -0.69 -0.23
DRW 0.08 -0.67 -0.34
FSW 0.82 -0.53 -0.03
DSW 0.86 -0.36 0.25
R/S 0.17 0.70 -0.48
CHL 0.54 0.19 0.68

Here, GP = seed germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root
dry weight, SFW = shoot fresh weight, SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index. The
bold values indicate that the relevant trait significantly affected the corresponding principal component

https://doi.org/10.1371/journal.pone.0262937.t009

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Fig 2. Biplot of the first two principal components of principal component analysis executed on percentage
decrease in seed germination and growth traits of wheat genotypes included in the study. Here, GP = seed
germination percentage, RL = root length, SL = shoot length, RFW = root fresh weight, RDW = root dry weight,
SFW = shoot fresh weight, SDW = shoot dry weight, R/S = root:shoot ratio, Chl = chlorophyll index.
https://doi.org/10.1371/journal.pone.0262937.g002

collectively explained 84.72% variability in the dataset. The first PC was positively influenced
by seed germination percentage, root and shoot length and fresh and dry weight of root. The
second PC was positively affected by root:shoot ratio and negatively affected by fresh and dry
weight of root (Table 9) The third PC was only positively influenced by chlorophyll index.
The biplot of first two PCs divided the genotypes in two major groups. The first group had
three genotypes having similar values for decrease in seed germination percentage and
growth-related traits, whereas the second group was not influenced by the decrease studied
traits. The first group contained the genotypes with the lowest drought tolerance while the sec-
ond group included the genotypes with the highest drought tolerance recorded in the current
study (Fig 2).

Discussion
Different genotypes significantly differed for their tolerance to PEG-induced drought stress as
hypothesized. Similarly, the highest reduction in seed germination and growth traits was
recorded under -1.2 MPa osmotic potential level compared to the control treatment of the
study which supported our second hypothesis [14, 38]. Seed germination is an important tran-
sition stage from seeds to seedlings for crop plants and higher seed germination under stressful
and benign environmental conditions enable plants to thrive and produce higher yields under
adverse as well as benign environments [38]. Seed germination is controlled by the microcli-
matic conditions of the seedbed as well genetic potential of the crop plants. Genotypes by envi-
ronment interactions is significant for getting higher crop yields. The semi-arid regions of the
world experience low moisture availability during seed germination of wheat crop. Low

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

moisture availability during seed germination crop declines both production maturity time [7,
8]. The impacts of water stress on seed germination and vegetative growth of different crops
such as wheat [8], maize and barley [8–10] has been reported in earlier studies and a constant
decline in the germination was recorded. The impact of drought stress on seed germination
and seedling stage of four bread wheat varieties have been evaluated and reduction in these
traits was noted with significant differences among tested varieties [11].
Seed germination is controlled by several necessary enzymes and stored food for the grow-
ing embryo. The increasing negative osmotic potential disrupts the activities of these enzymes;
thus, seeds lose their germination potential [39]. The other major reason of decreased seed ger-
mination is lower imbibition of water and the moisture needs of the seeds required for seed
germination are not fulfilled. The reduced seed germination under higher negative osmotic
potential in the current study is linked with lower water imbibition and subsequently reduced
enzyme activities necessary for seed germination. Several earlier studies have reported that
increasing osmotic potential have lowered seed germination of crop plants and weed species.
The tested genotypes significantly differed for their drought tolerance and the genotype ‘J4’
proved the most tolerant one compared to the rest of the genotypes included in the study. The
differences among genotypes are owed to their genetic make-up as well as ability to uptake
moisture necessary for the seed germination. The genotype ‘J4’ is a potential candidate for
developing drought tolerant wheat varieties through conventional breeding [40].
Different growth traits of the tested genotypes were also significantly altered by the osmotic
potentials used in the current study. Like seed germination, genotype ‘J4’ better tolerated mois-
ture deficiency compared to the rest of the genotypes included in the study. The decreased
growth traits under higher drought stress level can be explained with the lower moisture avail-
ability and subsequent lower transport of photosynthate from source to the sink. The differ-
ences among genotypes are owed to their inherent genetic makeup [41, 42].
Earlier studies [43, 44] have reported under water deficit reduced root length, shoot length,
root weight, shoot weight, number of spike, number of grains number/spike, 1000-grain,
weight and grain yield of wheat genotypes. Under drought stress root growth is limited but
shoot growth is abruptly decreased [45]. The root:shoot ratio was increased in the current
study indicating that all the tested genotypes tended to increase their root length under low
moisture availability. However, the increased root length could not compensated the damaged
caused by low water availability to growth traits [46, 47]. Chlorophyll concentration has been
reported to decrease under drought stress [24, 48] and similar was recorded in the current
study.
Generally, G × PEG-induced drought stress interactions reduced germinations and seedling
characteristics of the studied genotypes. The PCA divided the genotypes into 2 distinct groups,
i.e., group 1 and group 2 according to seed germination growth traits and decrease in seed ger-
mination and growth traits were tolerant to drought stress compared to the rest of the geno-
types included in the study. Thus, the identified genotypes, particularly, ‘J4’ can be used for
improving drought tolerance of bread wheat genotypes [49].

Conclusion
Different genotypes significantly differed for their tolerance to PEG-induced drought stress as
hypothesized. Similarly, the highest reduction in seed germination and growth traits was
recorded under -1.2 MPa osmotic potential level compared to the control treatment of the
study which supported our second hypothesis. The genotype ‘J4’ better tolerated drought stress
compared to the rest of the genotypes included in the study. Therefore, ‘J4’ can be used as
breeding material to improve drought tolerance of wheat crop.

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PLOS ONE The impact of PEG-induced drought stress on seed germination and seedling growth of different wheat genotypes

Supporting information
S1 Table. Analysis of variance for seed germination percentage and growth traits of differ-
ent wheat genotypes grown under various PEG-induced drought stress levels.
(DOCX)
S2 Table. Analysis of variance for percentage decrease in seed germination percentage and
growth traits of different wheat genotypes grown under various PEG-induced drought
stress levels.
(DOCX)
S3 Table. Minimal dataset used in the study to build graphs and perform statistical analy-
sis.
(XLSX)

Acknowledgments
The authors extend their appreciation to the Researchers Supporting Project, King Saud Uni-
versity, Riyadh, Saudi Arabia.

Author Contributions
Conceptualization: Shahzadi Mahpara, Abdulrahman Al-Hashimi, Mohamed S. Elshikh,
Marek Zivcak, Ali Tan Kee Zuan.
Data curation: Aleena Zainab.
Formal analysis: Aleena Zainab, Salma Kausar, Muhammad Bilal, Muhammad Imran Latif,
Muhammad Arif.
Funding acquisition: Shahzadi Mahpara, Abdulrahman Al-Hashimi, Mohamed S. Elshikh,
Marek Zivcak, Ali Tan Kee Zuan.
Investigation: Muhammad Imran Latif.
Methodology: Rehmat Ullah, Salma Kausar, Muhammad Arif, Imran Akhtar.
Project administration: Shahzadi Mahpara.
Software: Salma Kausar, Muhammad Bilal, Imran Akhtar.
Validation: Aleena Zainab, Rehmat Ullah, Muhammad Imran Latif, Muhammad Arif, Imran
Akhtar.
Visualization: Aleena Zainab, Rehmat Ullah, Muhammad Bilal, Imran Akhtar.
Writing – original draft: Aleena Zainab.
Writing – review & editing: Shahzadi Mahpara, Rehmat Ullah, Salma Kausar, Muhammad
Bilal, Muhammad Imran Latif, Muhammad Arif, Imran Akhtar, Abdulrahman Al-Hashimi,
Mohamed S. Elshikh, Marek Zivcak, Ali Tan Kee Zuan.

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