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Life 13 02067 v2

This study assesses the drought tolerance of 93 upland cotton genotypes under various irrigation regimes during the seedling stage. The research identifies several drought-tolerant genotypes, including G56, G44, and G5, and suggests their use in breeding programs to develop drought-resistant cotton varieties. The findings highlight the importance of root and shoot systems in enhancing drought tolerance in cotton plants.

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0% found this document useful (0 votes)
14 views18 pages

Life 13 02067 v2

This study assesses the drought tolerance of 93 upland cotton genotypes under various irrigation regimes during the seedling stage. The research identifies several drought-tolerant genotypes, including G56, G44, and G5, and suggests their use in breeding programs to develop drought-resistant cotton varieties. The findings highlight the importance of root and shoot systems in enhancing drought tolerance in cotton plants.

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life

Article
Assessing Drought Tolerance in a Large Number of Upland
Cotton Plants (Gossypium hirsutum L.) under Different
Irrigation Regimes at the Seedling Stage
Sadettin Çelik

Department of Forestry, Genç Vocational School, University of Bingol, Bingol 12500, Turkey;
[email protected]

Abstract: The cotton plant is important since it provides raw materials for various industry branches.
Even though cotton is generally drought-tolerant, it is affected negatively by long-term drought stress.
The trial was conducted according to the applied experimental design as a completely randomized
design (CRD) with three replications to determine a panel of 93 cotton genotypes’ genotypic responses
against drought under controlled conditions in 2022. All genotypes were watered with 80 mL−1 of
water (100% irrigation, field capacity) until three true leaves appeared, and then water stress was
applied at a limited irrigation of 75% (60 mL−1 ), 50% (40 mL−1 ), and 25% (20 mL−1 ) of the field
capacity. After the trial terminated at 52 days, the cv. G56, G44, G5, and G86 in RL; G1, G56, G44,
G86, G51, and G88 in RFW; advanced line G5, followed by the cv. G56, advanced line G44, G75,
and the cv. G90 in RDW; G44, followed by G86, the cv. G56, and elite lines G13 and G5 in NLRs
were observed as drought-tolerant genotypes, respectively, while G35, G15, G26, G67, and G56 in
SL; G15, G52, G60, G31, and G68 in SFW; G35, G52, G57, G41, and G60 in SDW show the highest
drought tolerance means, respectively. In conclusion, the commercial varieties with high means in
roots, namely G86, G56, G88, and G90, and the genotypes G67, G20, G60, and G57 showing tolerance
in shoots, are suggested to be potential parent plants for developing cotton varieties resistant to
drought. Using the cultivars found tolerant in the current study as parents in a drought-tolerant
variety development marker-assisted selection (MAS) plant breeding program will increase the
Citation: Çelik, S. Assessing Drought chance of success in reaching the target after genetic diversity analyses are performed. On the other
Tolerance in a Large Number of hand, it is highly recommended to continue the plant breeding program with the G44, G30, G19,
Upland Cotton Plants (Gossypium G1, G5, G75, G35, G15, G52, G29, and G76 genotypes, which show high tolerance in both root and
hirsutum L.) under Different shoot systems.
Irrigation Regimes at the Seedling
Stage. Life 2023, 13, 2067. https:// Keywords: drought; cotton; root; shoot; tolerance; water scarcity
doi.org/10.3390/life13102067

Academic Editors: Daria Sanna


and Fabio Scarpa
1. Introduction
Received: 14 September 2023
Revised: 11 October 2023
Cotton (Gossypium spp.) provides raw materials to many industries with its various
Accepted: 12 October 2023
economic features, such as natural fiber for the textile sector, cotton seed for the oil sector,
Published: 16 October 2023 and pulp for the livestock sector [1–4]. This industrial plant, which directly and indirectly
affects the livelihood of many people around the world, is drought-tolerant and is cultivated
by about 30 countries, primarily China, India, the USA, Pakistan, and Brazil due to their
warm climate [5,6]. However, intensive cultivation also leads to the emergence of various
Copyright: © 2023 by the author. biotic diseases and pests and abiotic stress factors. Abiotic stress factors, such as drought
Licensee MDPI, Basel, Switzerland. and salinity, prevent the water intake of the cotton, and biotic stress factors, such as
This article is an open access article Verticillium and Fusarium, cause xylem vessels to be clogged and the plant to not receive
distributed under the terms and
sufficient water, which causes a decrease in the turgor pressure that provides the verticality
conditions of the Creative Commons
of the plants and causes the leaves to wilt and the fruits to be produced [7]. Drought
Attribution (CC BY) license (https://
means that plants suffer from water shortages for a long period on a global scale because
creativecommons.org/licenses/by/
of the greenhouse gas effect as a result of human and non-human activities. Drought is
4.0/).

Life 2023, 13, 2067. https://doi.org/10.3390/life13102067 https://www.mdpi.com/journal/life


Life 2023, 13, 2067 2 of 19

one of the most important abiotic stress factors that cause 73% of product loss in cotton [8].
In particular, in the USA, which is one of the leaders in world cotton production, the
production loss due to drought stress in the last 50 years is approximately 67% [9], while it
is 34% in Pakistan [10].
Although cotton has a higher tolerance to drought compared to other cultivated indus-
trial field crops, when it is exposed to long-term drought stress, undesirable effects such as
a decrease in yield, lower biomass, and stem weight, a slowdown in plant development,
a decrease in fiber quality, and small bolls may occur [11]. Drought adversely affects
Gossypium spp., along with other plants, by limiting plant height, leaf weight, the number
of nodes, transpiration rate, photosynthesis rate, and stomatal conductivity [11–15]. In
addition, when it is exposed to long-term drought stress, it closes its stomata, rolls its leaves,
tends to have osmotic regulation, and tries to reach water/moisture in the depths of the
soil [16], and the rate of photosynthesis is reduced [17].
A great deal of research is being conducted to understand the physiological, morpho-
logical, and metabolic reactions of plants against drought in roots [18,19]. Under drought
stress, the roots of the plants tend to go deeper and aim to reach underground water, and
these behaviors of the roots also play an important role in activating other mechanisms
related to drought [20]. Anjum et al. [21] indicated that a strong root system plays a very
critical role in physiological functions, carbohydrate storage, uptaking, and the absorption
of water and nutrients from the soil. In the first stage of drought, plants slow down the
elongation of the stem and develop the root system to reach deeper water [22].
The responses of the cotton plant to stress factors vary during different stages of
development. The seedling stage is a period of high sensitivity to environmental conditions
for many plant species, including cotton [23]. The seedling stage is not only adversely
affected by biotic stress factors but also negatively affected by abiotic stress factors, such
as drought [24] and salinity [25]. Brand et al. [26] discovered that low temperatures
at the seedling stage in cotton under chilling conditions are strongly associated with
low temperatures of the seedling stage diseases such as dumping off, which causes root
deterioration. Low temperatures at the seedling stage of cotton have a big effect on the
damage thresholds of pathogens such as Rhizoctonia and Pythium [27]. Furthermore,
seedling emergence rates and development under controlled conditions can determine
the sensitivity of varieties to cold weather [28]. When the cotton seeds emerged and were
exposed to sub-optimal temperatures in late spring, seedling growth was adversely affected,
and significant decreases were observed in yield [29]. When soil moisture tension generally
exceeds 30–50 centibars, drought stress occurs, depending on soil type [30].
To understand the root system responses under water scarcity conditions: a short-
term drought during the seedling period increased the cotton roots ability to reach deep
water but decreased the diameter of the roots [31]. It is revealed that the cotton roots were
affected by drought more than the root distribution [32]. Insufficient soil moisture reduces
the elongation of the roots [33,34], and a shortened root system is developed approximately
42–70 days after the germination of the cotton [32]. Root growth is a critical process to
be grown under sustainable temperatures [35,36]. Zahid et al. [37] stated that 22–30 ◦ C
is an optimal temperature for cotton root and shoot growth and an optimal root/shoot
(R/S) ratio, but a temperature between 32 and 40 ◦ C will restrict the distribution and
growth of the root/shoot system. A similar finding was declared by Koevoets et al. [38]
that the optimal temperature promotes an increase in the root/shoot ratio (R/S ratio);
nevertheless, a temperature above optimal temperature will decrease water absorption and
plant nutrients in the cotton plant root system, and as a results, the root system get weaker
against drought [39]. A decrease in soil water content by evaporation significantly reduces
root activities, but hydraulic lifting and transpiration by roots can increase groundwater
levels to levels close to the soil surface (to 0–40 cm soil depth) [40].
Various studies have been conducted to understand cotton plants’ genotypic responses
against drought. Studies such as developing cultivars that are tolerant to drought through
crossbreeding [41], hormonal regulation against drought and salinity stress [42], under-
Life 2023, 13, 2067 3 of 19

standing the drought tolerance mechanism of cotton and integrating it into variety de-
velopment breeding programs [43], drought stress generation and a determination of
tolerant genotypes in the cotton plant using Polyethylene Glycol (PEG) [44], determining
drought-stress-related traits in the seedling period [45], strategies for coping with drought
stress in cotton and their application to crop improvement programs [45], increasing the
drought tolerance of cotton genotypes by exposing them to salinity during the seedling
period [46], understanding the allelic genetic diversity of drought-resistant cotton geno-
types [47], and identifying cultivars carrying drought genes using Trait-assisted selection
(MAS) in cotton [48] have been carried out.
The research conducted shows that the seedling stage is the period during which
plant reactions can be observed the best [49]. This study aims to investigate the drought-
tolerant level of some advanced lines and some genetic stock cultivars of the cotton
(Gossypium hirsutum L.) species under artificial drought stress with limited irrigation meth-
ods during the seedling stage. In this study, unlike many studies, not only are cultivars
evaluated under drought stress conditions, but also advanced lines’ genetic potential
against drought is investigated.

2. Materials and Methods


2.1. Plant Materials
All of the 93 cotton genetic materials used in the experiment are a panel of both
accessions from advanced lines and commercial varieties of Gossypium hirsutum L. with
allotetraploid (2n = 4x = 52) chromosomes set with AD1 genome groups (Table 1). The
genotypes were provided by the East Mediterranean Transitional Zone Agricultural Re-
search Institute, East Mediterranean Agricultural Research Institute, GAP International
Agricultural Research and Training Institute, SET seed company STARseed, MAY BASF,
and Progen Inc., seed companies. The commercial cultivars’ properties, such as cotton seed
yield, resistance to some technological properties, and climate requirements, are known.
Due to continuous segregation, the economic properties of advanced breeding lines are
still not clear, so this experiment provides information about the individual properties of
advanced lines, including reactions to drought.

Table 1. Upland cotton genotypes properties and origins.

Genotype Genome Genotype Genome Genotype Genome


Code Species Origin Code Species Origin Code Species Origin
Name Group Name Group Name Group

G1 14 AD1 G. hirsutum L. Türkey G32 ARAS10 AD1 G. hirsutum L. Türkey G63 Fiona AD1 G. hirsutum L. Türkey

G2 15 AD1 G. hirsutum L. Türkey G33 ARAS11 AD1 G. hirsutum L. Türkey G64 Furkan AD1 G. hirsutum L. Türkey

G3 17 AD1 G. hirsutum L. Türkey G34 ARAS12 AD1 G. hirsutum L. Türkey G65 Gaia AD1 G. hirsutum L. Türkey

G4 18 AD1 G. hirsutum L. Türkey G35 ARAS13 AD1 G. hirsutum L. Türkey G66 Kartanesi AD1 G. hirsutum L. Türkey

G5 19 AD1 G. hirsutum L. Türkey G36 ARAS14 AD1 G. hirsutum L. Türkey G67 Lydia AD1 G. hirsutum L. Türkey

G6 21 AD1 G. hirsutum L. Türkey G37 ARAS15 AD1 G. hirsutum L. Türkey G68 Maraş 92 AD1 G. hirsutum L. Türkey

G7 22 AD1 G. hirsutum L. Türkey G38 ARAS16 AD1 G. hirsutum L. Türkey G69 May 344 AD1 G. hirsutum L. Türkey

G8 23 AD1 G. hirsutum L. Türkey G39 ARAS17 AD1 G. hirsutum L. Türkey G70 Maysos AD1 G. hirsutum L. India

G9 24 AD1 G. hirsutum L. Türkey G40 ARAS18 AD1 G. hirsutum L. Türkey G71 Nihal AD1 G. hirsutum L. Türkey

G10 26 AD1 G. hirsutum L. Türkey G41 ARAS19 AD1 G. hirsutum L. Türkey G72 SC-125 AD1 G. hirsutum L. Türkey

G11 27 AD1 G. hirsutum L. Türkey G42 ARAS20 AD1 G. hirsutum L. Türkey G73 SC-21 AD1 G. hirsutum L. Türkey

G12 28 AD1 G. hirsutum L. Türkey G43 ARAS23 AD1 G. hirsutum L. Türkey G74 SC-22 AD1 G. hirsutum L. Türkey

G13 30 AD1 G. hirsutum L. Türkey G44 ARAS24 AD1 G. hirsutum L. Türkey G75 SC-24 AD1 G. hirsutum L. Türkey

G14 31 AD1 G. hirsutum L. Türkey G45 ARAS26 AD1 G. hirsutum L. Türkey G76 SC-25 AD1 G. hirsutum L. Türkey

G15 37 AD1 G. hirsutum L. Türkey G46 ARAS27 AD1 G. hirsutum L. Türkey G77 SC-28 AD1 G. hirsutum L. Türkey

G16 69 AD1 G. hirsutum L. Türkey G47 ARAS31 AD1 G. hirsutum L. Türkey G78 SC-29 AD1 G. hirsutum L. Türkey

G17 ADN 513 AD1 G. hirsutum L. Türkey G48 ARAS32 AD1 G. hirsutum L. Türkey G79 SC-30 AD1 G. hirsutum L. Türkey

G18 ADN123 AD1 G. hirsutum L. Türkey G49 ARAS33 AD1 G. hirsutum L. Türkey G80 SC-36 AD1 G. hirsutum L. Türkey

G19 ADN413 AD1 G. hirsutum L. Türkey G50 ARAS34 AD1 G. hirsutum L. Türkey G81 SC-37 AD1 G. hirsutum L. Türkey

G20 ADN710 AD1 G. hirsutum L. Türkey G51 ARAS35 AD1 G. hirsutum L. Türkey G82 SC-38 AD1 G. hirsutum L. Türkey

G21 ADN741 AD1 G. hirsutum L. Türkey G52 ARAS43 AD1 G. hirsutum L. Türkey G83 SC-39 AD1 G. hirsutum L. Türkey

G22 ADN811 AD1 G. hirsutum L. Türkey G53 ARAS48 AD1 G. hirsutum L. Türkey G84 SC-41 AD1 G. hirsutum L. Türkey

G23 ARAS1 AD1 G. hirsutum L. Türkey G54 ASOS AD1 G. hirsutum L. Türkey G85 SC-42 AD1 G. hirsutum L. Türkey
Life 2023, 13, 2067 4 of 19

Table 1. Cont.

Genotype Genome Genotype Genome Genotype Genome


Code Species Origin Code Species Origin Code Species Origin
Name Group Name Group Name Group

G24 ARAS2 AD1 G. hirsutum L. Türkey G55 Aşkabat 71 AD1 G. hirsutum L. Turkmenistan G86 STV373 AD1 G. hirsutum L. USA

G25 ARAS3 AD1 G. hirsutum L. Türkey G56 BA119 AD1 G. hirsutum L. Türkey G87 STV468 AD1 G. hirsutum L. USA

G26 ARAS4 AD1 G. hirsutum L. Türkey G57 BA151 AD1 G. hirsutum L. Türkey G88 STV498 AD1 G. hirsutum L. USA

G27 ARAS5 AD1 G. hirsutum L. Türkey G58 Beren AD1 G. hirsutum L. Türkey G89 SG 125 AD1 G. hirsutum L. USA

DeltaPine
G28 ARAS6 AD1 G. hirsutum L. Türkey G59 AD1 G. hirsutum L. USA G90 Text AD1 G. hirsutum L. USA
332

DeltaPine
G29 ARAS7 AD1 G. hirsutum L. Türkey G60 AD1 G. hirsutum L. USA G91 TMN 199 AD1 G. hirsutum L. Türkey
396

G30 ARAS8 AD1 G. hirsutum L. Türkey G61 Erşan92 AD1 G. hirsutum L. Türkiye G92 TMN108 AD1 G. hirsutum L. Türkey

G31 ARAS9 AD1 G. hirsutum L. Türkey G62 Famosa AD1 G. hirsutum L. USA G93 TMS 108 AD1 G. hirsutum L. Türkey

2.2. Method
The pattern of the trial is the applied experimental design as a completely randomized
design (CRD) with 3 replications. The experiment was conducted at Bingol University,
Genc Vocational School (Coordinates: 38 ◦ 442’58” N and 40 ◦ 320 11” E), climate chamber.
The soil used is prepared with peat, perlite, and soil in a ratio of 3:1:1. All three components
were sterilized with an autoclave at 121 ◦ C for 15 min and transferred to 200 mL plastic
pots. Triangular holes were made at the bottom of the plastic pots for the excess water
to be drained. four seeds were sowed in each pot at a depth of about 2 cm, and potting
soil was brought to the field capacity. For control purposes, STV373 (G86), BA119 (G56,
White gold), and TEX (G93) varieties adapted to the Southeastern Anatolia Region, where
the experiment was established, were used. In the experiment, three pots were used for
each genotype as the 3 replications. The soil temperature was raised to 15 ◦ C for seeds to
germinate, and since the experiment was established in winter, the cotton seeds, which are
likely to be dormant, were kept in a hot water bath device at 65 ◦ C for 15–30 min before
sowing to break the dormancy. Then, the ambient temperature was adjusted to be between
28 and 38 ◦ C, and the humidity was between 57 and 67% [50].
LED lights were adjusted to provide daylight at 2.500 lux for 14–15 h a day, and
each pot was given 0.2 g of pure nitrogen-containing urea fertilizer for effective growth
14 days after planting. From the starting point of the experiment, cotton genotypes were
irrigated with 100% irrigation every 10 days until true leaves bloomed. All genotypes
were fully watered with 80 mL-1 of water (100% irrigation, field capacity) until three true
leaves appeared. As soon as all the genotypes produced true leaves, the limited irrigation
was started. Then, water stress was applied at limited irrigations of 75% (60 mL−1 ), 50%
(40 mL−1 ), and 25% (20 mL−1 ) of the field capacity. The experiment was terminated on the
52nd day.
The cotton plants were taken out of their pots intact, the soil was washed using tap
water without damaging the roots, and parameters such as the Root Length (RL), Root
fresh weight (RFW), Root dry weight (RDW), Shoot length (SL), Shoot fresh weight (SFW),
Shoot dry weight (SDW) and the number of lateral roots (NLRs) of one plant from each
replication genotype were measured. RL was calculated by a direct measurement of fresh
taproots, RFW by direct weighting of taproots, RDW was measured after drying in the oven
for 24 hours at 60 ◦ C, NLRs by direct counting of roots before drying, SL by measuring with
a ruler, SFW by calculation with a scale with 0.5 mg precision, and SDW was measured
after drying in the oven for 24 hours at 60 ◦ C [50]

2.3. Data Analysis


An analysis of variance (ANOVA) using data from these drought-related parameters
was carried out using the statistical software JUMP 17.0 (JMP® , Version <17>. SAS Institute
Inc., Cary, NC, USA, 1989–2021) [51]. The frequency charts were created by Microsoft Office
Excel 2016 ver. The correlation diagram, cluster analysis, principal component analysis
(PCA) analysis, and charts were obtained via JMP 17.00 ver. [51]. The Scatter plot charts
were created using Minitab 19 [52], and the significance of the difference between the means
Life 2023, 13, 2067 5 of 19

of the parameters was investigated (p < 0.05). The least significant difference (LSD) was
used to compare the means.

3. Results
3.1. Analysis of Variance (ANOVA)
In the experiment, the RL, RFW, RDW, NLRs, SL, SFW, and SDW of the cotton
germplasm panel were measured. As a result of the statistical analysis, it was observed that
there was a highly significant difference between the means for RL, RFW, RDW, NLRs, SL,
and SFW (p < 0.01). The statistical analysis conducted at a 95% confidence level (p < 0.05)
revealed that, according to the mean squares, there is a significant difference among the
genotypic means in terms of the shoot dry weight trait. Table 2 indicates the significance of
the population mean differences for each trait.

Table 2. One-way ANOVA of drought traits.

Mean Squares (Water-Stressed)


Source df NLRS RL RFW RDW SL SFW SDW
Genotypes 92 122.330 ** 65.75 ** 0.0147 ** 0.0014 ** 19.27 ** 0.2067 ** 0.0099 *
Replication 2 21.552 12.14 0.0054 0.0003 16.48 0.2593 0.0076
Error 184 16.711 12.44 0.0035 0.0004 6.29 0.0590 0.0071
* Significant at p = 0.05 level, ** Significant at p = 0.01 level. NLRs: number of lateral roots, RL: root length,
RFW: root fresh weight, RDW: root dry weight, SL: shoot Length, SFW: shoot fresh weight, SDW: shoot
dry weight.

3.2. Genotypic Performance under Drought


In this study, the genotypes with the longest roots (the ones with the highest tolerance
against drought) were G56 (27 cm) and G44 (25,6), followed by the GG5 and G86 cultivars
with 24.3 and 23.6 cm root lengths. G76 and G41 had the shortest RL (5.3 cm), followed by
the G35 and G14 genotypes with a 5.7 cm root length (Supplementary Table S1). The mean
RL of the population was 14.27 cm (Figure 1A). Figure 1A shows the mean distribution
frequency of the genotypes regarding the root length. The developed root system turns in
the direction where soil moisture is intense and solves the water problem of the plant, and
the roots disperse deeply and to the sides to enable the plant to hold onto the soil more
strongly under harsh weather conditions (phenotypic observation).
The mean RFW of the entire population in the study was 0.26 g. The lowest root fresh
weight was 0.123 g, and the RFW recorded in the G76 and G41 genotypes was 0.136 g. The
advanced genotypes were followed by G35 (0.140 g), and the highest root fresh weight was
recorded in the G1 (0.446 g) advanced line, followed by the cv. G56 (0.427 g), G44 (0.424 g),
commercial cultivar G86 (0.411 g), G51 (0.397 g), and G88 (0.384 g) (Supplementary Table S1). Ac-
cording to Figure 1B, the RW–frequency histogram graph, the number of genotypes with a mean
root weight above the mean of the whole population (0.260 g) is 57 (Supplementary Table S1).
This may be an indication that most of the individuals in the population can activate their
tolerance mechanisms against drought stress.
Root dry weight (RDW) is also one of the phenotypic traits used to see the reactions
of the cotton against drought. Accordingly, the mean RDW of the population, consisting
of 93 genotypes, was 0.06 g. The advanced genotype G5 recorded the highest RDW value
(0.127 g), followed by the cv. G56 (0.112 g), the advanced breeding line G44 (0.099 g),
G75 (0.092 g), and the cv. G90 (0.091), and this value was followed by the G85 (0.088 g) and
G7 (0.085 g) breeding genotypes. It is possible to say that the drought resistance genes show
themselves in the phenotype of the G5, G56, and G44 cultivars. The control cultivar G56,
which is well adapted to the Southeastern Anatolia and Mediterranean regions of Turkey,
where cotton cultivation is most common, and shows tolerance above the general mean, is
used as a control variety in the research (Supplementary Table S1). In Figure 1C, concerning
the RDW values, the mean of the genotypes was 0.0569 g. There were 50 genotypes with
Life 2023, 13, 2067 6 of 19

Figure 1
means above the overall mean and 43 genotypes with means below the overall mean. The
genotype with the highest frequency was 24.

A B

C D

E F

Figure 1. (A) Rooth length (RL), (B) root fresh weight (RFW), (C) root dry weight, (D) shoot length
(SL), (E) shoot fresh weight (SFW), (F) shoot dry weight (SDW), and (G) number of lateral roots
(NLRs) Frequency charts.
Life 2023, 13, 2067 7 of 19

Accordingly, the mean total shoot length (SL) of the genotypes was 12.47 cm. G35 (18.33 cm)
had the longest SL, and it was followed by the G15-G29 (16.67 cm) and G76 (16.33 cm)
breeding genotypes, respectively. The commercial cultivar G65 (6.33 cm) showed the
lowest SL means, followed by the elite lines G72-G32 (7.33 cm) and the local cotton variety
G22 (7.33) (Supplementary Table S1). Regarding the SL (Shoot length) trait, 31 genotypes
had means below the overall mean, and 62 genotypes had means above the overall mean
(Figure 1D).
The mean SFW of 93 cotton genotypes was 1.3280 grams. The highest SFW values were
recorded in the following genotypes: the advanced line G15 (1.853 g), G52 (1.815 g), and
the commercial variety G60 (1.805 g), respectively. These were followed by G31 (1.752 g),
G41 (1.736 g), G47 (1.724 g), G14 (1.700 g), and the commercial local variety G68 (1.691 g).
The lowest SFW value was measured in the advanced genotype G83 (0.686 g), followed
by G2 (0.716 g), G75 (0.739 g), and G91 (0.740 g), respectively (Supplementary Table S1).
Mostly of the cotton genotypes showed the expected responses under water scarcity. For
another drought-related trait, SFW (shoot fresh weight), 24 genotypes had the highest
means, while 68 genotypes had means above the population mean of 1.3280 g (Figure 1E).
The SDW mean of all the genotypes was recorded as 0.163 g. The highest SDW
value was recorded in G35 (0.349 g) and the lowest in the G65 (0.060 g) genotype. While
commercial cultivars such as G57 (0.337 g), G60 (0.294 g), and G66 (0.289 g) showed high
mean SDW values, the advanced lines G52 (0.345 g) and G41 (0.314) showed very high SDW
values. The second lowest SDW value was found for the commercial varieties G91 (0.078 g)
and G4 (0.078 g) and G20 (0.078 g), followed by G22 (0.081 g) and the cv. G90 (0.087 g)
(Supplementary Table S1). In the case of SDW (shoot dry weight), 34 genotypes had the
highest frequency, and the majority of the population had SDW values below the population
mean of 0.163 (Figure 1G).
In the current study, the G44 (38) RIL genotype had the greatest number of lateral roots
(NLRs), and it was followed by G86 (33), the commercial cv. G56 (32), and the advanced
lines G13 (30), G5 (29.33), and G49 (29). The mean of number of lateral roots of all the
genotypes was 19.16 (Supplementary Table S1). Regarding the NLRs (number of lateral
roots) characteristic, 21 genotypes had a mean of 15.556, 22 genotypes had a mean of 19.037,
and 26.778 was the mean for 18 genotypes. The population NLR mean was 19.159, with
28 genotypes having means below it and 68 genotypes having means above it (Figure 1G).
The scatter plot analysis was carried out to determine the relationship and relationship
type between every two variables of drought indicators. Yi [53] emphasized that the
closer they get to the linear line, the more the relationship between parameters increases.
According to the Figure 2 scatter plots and correlation diagram, while the strongest positive
correlation is seen between RL and RFW (0.823), followed by NLRs and RL (0.759), RFW and
NLRs (0.679), RFW–RDW (0.642), RL–RDW (0.568), and NLRs–RDW (0.494), the strongest
negative correlation took place between SL–RDW (−0.446), followed by RFW–SL (−0.327),
NLRs–SL (−0.318), NLRs–SFW (−0.219), and SDW–RFW (−0.143) (Table 3, Figure 2).
Similar correlations between the root–shoot traits of the cotton plant were identified by [54].
A principal component analysis (PCA) is a powerful multivariate statistical technique
widely used for identifying patterns of data, expressing the data, and highlighting their
similarities and dissimilarities [55]. A PCA simplifies complex datasets by transforming
them into a new coordinate system, where the data’s variance is maximized along the
principal components. This dimensionality reduction retains most of the relevant informa-
tion while eliminating redundant or noisy features, thus aiding in data compression and
visualization [56].
The graph (Figure 3) is the loading plot from the PCA analysis. The results of the
PCA are given in Figure 3. The PCA was conducted based on the morphological drought
parameters, and in the correlation circle, the first axes (F1) and the second axes (F2) represent
the discriminant function analysis (DFA), respectively, 47.6% and 21.4% of the variable.
Among the drought measurement parameters of the cotton plant’s aboveground portion,
the strongest positive correlation was found between shoot dry weight (SDW) and shoot
Life 2023, 13, 2067 8 of 19

fresh weight (SFW), while shoot length (SL) showed a positive correlation with both SFW
and SDW (Figure 3). Accordingly, as the SL increases, it appears that the SFW and SDW
values increase positively.

Figure 2. Scatter (XY) plot analysis of drought morphological traits using Minitab 19 [52] and
correlation diagram in JMP 17.00 [51]. version.

Table 3. Correlation between studied traits.

Traits NLRs RL RFW RDW SL SFW SDW


Number of lateral roots **** 0.7591 0.6795 0.4942 −0.3188 −0.2193 −0.1242
Root length 0.7591 **** 0.8232 0.5681 −0.2684 −0.1105 −0.0932
Root fresh weight 0.6795 0.8232 **** 0.6420 −0.3274 −0.0866 −0.1433
Root dry weight 0.4942 0.5681 0.6420 **** −0.4465 −0.1015 −0.1284
Shoot length −0.3188 −0.2684 −0.3274 −0.4465 **** 0.4774 0.2154
Shoot fresh weight −0.2193 −0.1105 −0.0866 −0.1015 0.4774 **** 0.4189
Shoot dry weight −0.1242 −0.0932 −0.1433 −0.1284 0.2154 0.4189 ****
Life 2023, 13, 2067 9 of 19

Figure 3. Correlation circle obtained by principal component analysis (PCA) of the variables factor
space of F1 and F2 using JMP 17.00 ver. [51] statistical analysis software.

In terms of the underground drought measurement morphological parameters, a very


close positive correlation between them is observed. The strongest positive correlation was
found between root length (RL) and root fresh weight (RFW), while a significant positive
correlation was also found between the number of lateral roots (NLRs) and root dry weight
(RDW). Generally, there is a negative correlation between aboveground drought traits and
underground traits in cotton plants. This situation can be explained by the increase in the
aboveground parts being hindered due to strengthening the roots of cotton plants under
water stress, transferring photosynthesis products from the aboveground parts to the roots
to draw water from the soil more effectively (Figure 3).
In Figure 4, generally, the most tolerant genotypes to drought are clustered at the
top, while the most sensitive genotypes are clustered at the bottom. Based on tolerance–
susceptible properties, ten clusters were obtained, with five being major and the other
five being minor. The majority of the genotypes, in response to drought stress, exhibited
different reactions in various organs, and a clear distinction was not observed (Figure 4).
The genotypic reactions of cotton genotypes under drought stress have been correlated
and clustered based on their sensitivity and tolerance. Simultaneously, shoot traits and root
traits have also been clustered according to their relationships with each other (Figure 4).
Accordingly, in root traits, the closest relationship was observed between the NLRs
and RL, while in shoot traits, the closest relationship was observed between the SL and
SDW. Although the root traits and shoot traits clustered together, a significant correlation
was observed between them. The SFW, in particular, was found to be associated with
all the other traits. The shoot fresh weight (SFW) may be concluded to have a very
close relationship with all the other organs of the cotton plant due to its possession of
chlorophyll pigments that enable photosynthesis and its production of glucose through the
photosynthetic pathway.
Life 2023, 13, 2067 10 of 19

Figure 4. Cluster analysis based on genotypes genotypic responses under drought stress using JMP
17.00 ver. [51] statistical analysis software.

4. Discussion
The responses of plant roots under drought stress have become the focus of researchers
in recent years [57,58]. The first plant organ that is exposed to water stress is the roots,
and by transmitting this stress to the rest of the plant, it goes through morphological,
Life 2023, 13, 2067 11 of 19

physiological, and metabolic changes, and the plant starts to activate its mechanisms to
cope with the water stress [18]. Root length (RL) growth has a great impact on the uptake
of nutrients and root system architecture [59,60]. It is a current trend in plant breeding
to select genotypes with the longest roots for drought tolerance [9,61]. It was seen in
this study that several advanced breeding lines had a high tolerance to drought in some
drought measurement parameters. The cv. G56 had the longest RL (27 cm), followed by
G44 (25.67 cm), G5 (24.33 cm), and G86 (23.67 cm) in this study (Supplementary Table S1).
Similar results were found by [50], which reported that taproots had a root length between
47.9 and 86.6 cm under full irrigation and between 48.3 and 82.5 cm under restricted
irrigation in their experiment. The higher root length means of [50] compared to our results
are believed to be dependent on the experimental conditions, trial materials, origins of the
plant varieties used, amount of water used, and duration of the experiment. In the study,
although the humidity and temperature in the greenhouse can increase excessively during
the day, the amount of soil mixture used was parallel to our study, and a longer taproot
system was observed.
Research has shown that immersing roots in depths (root length) where the moisture
content is high can compensate for the moisture losses that occur through evapotranspira-
tion [62]. In the current study, the G76 and G41 advanced lines produced a 5.33 cm RL as the
lowest RL, followed by G35, G14 (5.7), G79, G73, and G7 (7.3 cm) (Supplementary Table S1).
Our results are similar to [63], with the RL trait varying between 17.10 cm and 30.80 cm.
Similar root length (RL) values were obtained by Pawar and Veena [64], with the highest
root length at 12.72, 12.17, and 12.88 cm and the lowest at 9.8, 9.85, and 10.5 cm with the
PEG 6000 drought application. At the same time, Pawar and Veena [64] revealed that the
root length increased when the PEG 6000 application was increased by 10% and decreased
after that level. This situation can be explained by the plant root tolerance mechanism of the
plant: the plant sends more photosynthesis products to the root in order to strengthen the
roots and uptake more water from the soil [65]. In a study, Zahid et al. [66] found a highly
significant correlation between genotypes and watering, and they found the root length
to be 5.161 cm. According to the current study, with mean root length of 27 cm, 5.161 cm
is highly low, and the irrigation period, the amount of water given in each irrigation, soil
mixture, infiltration and germination ability, and speed can be shown as the reasons why
it is found to be lower than our study. Wang et al. [67] reported that the distribution of
photosynthetic products in the roots of beans increased under drought stress, which did not
last long, but the photosynthetic products in the production organs decreased significantly.
It was observed that the plant tries to strengthen its root system under long-lasting drought
stress, thereby severely reducing its yield and economic added value as a result of giving
all its weight to the roots. Studies reported that a situation similar to the growth period of
wheat can be observed in cotton: moderate water stress causes photosynthetic products to
be transported to the roots by vascular bundles and has a similar positive effect on buds
and bolls [68]. It was reported that moderate water stress during the grain-filling period of
the wheat plant increases the transport of photosynthetic products to the roots, and this
provides a significant benefit to the wheat, as the wheat will have developed roots and thus
take more moisture and therefore more nutrients from the soil [69].
A higher root weight enables the uptake of more plant nutrients from the soil and
plays a key role in vegetative and generative development [70]. In the current study, the
RFW trait values changed between 0.123 g (G76) and 0.446 g (G1), with a population
mean of 0.260 g. The genotypes that were tolerant according to the root fresh weight
trait were the cv. G56 (0.427 g), G44 (0.424 g), the commercial variety G86 (0.411 g),
G51 (0.397 g), and G47 (0.148 g), respectively (Supplementary Table S1). Our findings are
similar to Iqbal et al. [71], Mvula et al. [63], and Jaafar et al. [72]. Similarly, Shah et al. [73]
obtained a root weight ranging from 0.049 to 0.155 g under water stress in a trial conducted
under normal and water-stressed conditions for 52 days. In a similar study, Akbar and
Hussain [74] obtained fresh root weights ranging from 0.222 g to 0.394 g under different
drought stresses. In Figure 1B, it is observed that 22 genotypes have a mean RFW of 0.267,
Life 2023, 13, 2067 12 of 19

which is slightly higher than the population mean. The fact that 57 genotypes produce a
mean higher than the population suggests genetic diversity within the population, with
some genotypes exhibiting superior root growth in terms of fresh weight. The genotypic
mean for the RDW is 14.27 (Figure 1B). The research showed that there is a positive
correlation between root weight and drought and that the genotypes with quantitatively
higher root weights had a higher tolerance to drought stress than the others. While the
specific context or objectives of the study are not provided, this trait likely relates to the
ability of genotypes to allocate resources to root growth.
Under long-term drought stress, the root dry weight increased and the leaf area
decreased in Chinese spinach [75]. In the current study of drought response measurement
root trait, root dry weight, the highest value was recorded in the advanced line G5 (0.127 g),
followed by the control variety G56 (0.112 g), G44 (0.099 g), G75 (0.092 g), and control variety
G90 (0.091 g), while lowest RDW values were recorded in the advanced line G76 (0.016 g),
followed by genotypes G35 (0.021 g), G20 (0.024 g), G61 (0.026 g), G70 (0.028 g), G59-G15
(0.029 g), and G67 (0.030 g) (Supplementary Table S1). The RDW varied from 0.79 g to
1.18 g [63]. Istiqomah et al. (2021) [70] found a very significant difference between the
applications in terms of dry root weight or total dry weight in corn plants. Similarly,
in their study investigating the responses of the germination stage root drought trait
to drought in cotton, Fathi-Sadabadi et al. [54] obtained dry root weights ranging from
0.00342 to 0.00685 g. In Figure 1C, the genotypes exhibit a wide range of RDW values, with
50 genotypes having means above the overall mean and 43 below. This variation could
have practical implications for breeding programs, as it suggests the presence of genotypes
with different root biomass production potentials. The variation in genotypic means for
RDW (Figure 1C) indicates that some genotypes are more efficient at producing dry root
biomass than others.
As the importance of NLRs traits observed in this current study indicates, the num-
ber and development of lateral roots are important indicators of drought tolerance. The
development of the root system, which allows plants to stand, hold on to the soil, give
yield, and grow and develop, plays an active role in the fight against agricultural drought.
Wang et al. [76] stated that many previous studies related to the crop–root system improve-
ment suggest that increasing the number of lateral roots (NLRs) could characterize plant
development and increase crop yield. Lateral root numbers and development are regulated
by many diverse hormones and their interactions [77,78]. Lateral root initiation (LRI) and
development is coordinated by many genes. In Arabidopsis thaliana, there are more than
seven genes associated with lateral roots [79,80]. The population mean of the number
of lateral roots (NLRs) changed from 5 to 38. The advanced line G44 (38) showed the
highest NLRs, and this value was followed by G86 (33), the cv. (control variety) G56 (32),
G13 (30), and G5 (29.33). The lowest NLR values ranged from 5 to 10. In the current
study, the lowest NLRs were recorded in G47 (5), followed by G76 (6.33), and G79 (7)
(Supplementary Table S1). Similarly, the NLR value showed variability between 29.67 and
47.10 [63]. McMichael et al. [81] obtained similar NLR values ranging from 4 to 50 at 30 ◦ C
within 7 days after germination. The reason for the higher LRN values may be associated
with not applying drought stress in the first 7 days and plenty of water and plant nutrients.
In a study with 120 exotic cotton genotypes provided, the NLRs mean changed from 3.5,
17.5, 25.97, and 49.75, respectively [81]. The genotypic means for NLRs vary significantly
(Figure 1G), with 28 genotypes below the population mean and 68 genotypes above it. This
trait might be related to drought tolerance or stress response, and the observed variation
suggests potential candidates for further research or breeding programs. Lateral roots
and numbers play a key role in plants reaching and absorbing water [82] and uptaking
plant nutrients such as phosphorus [83]. Hund et al. [84,85]. Gallardo et al. [86] reported
that enough water in soil accelerates root development, and Luo et al. [87] reported that
moderate water stress in the middle and upper layers of the soil strengthens the root
system in the early stage of cotton plant development and contributes significantly to roots
reaching deep waters. The number, development, and growth of lateral roots are critical for
Life 2023, 13, 2067 13 of 19

the development and yield of field crops [88,89]. In another study, Forde and Lorenzo [90]
stated that the lateral roots, which are important in plants with a taproot system, initially
act as taproots and take water and nutrients dissolved in water. To increase tolerance to
drought stress, it was seen that researchers should focus on the development of genotypes
of lateral root systems. It was seen that the drought-resistance RIL genotypes G44 (38),
G86 (33), and G56 (32) varieties, which have good adaptation in the Southeast Anatolia
region of Turkey, where cotton is mainly cultivated, develop better lateral root systems
under drought stress. Even though the current study breeding lines have developed good
taproot systems, they may not show high resistance against high temperatures compared
to commercial cultivars adapted in warm regions. It has been put forward that lateral root
numbers can have positive effects of vigorous growth under low temperatures in maize
plants. Considering the studies in the literature, it is seen that the number of lateral roots is
a drought trait that increases the tolerance of plants against drought stress.
Shoot length (SL) can be used as a criterion for the selection of drought-tolerant
plants [91,92]. As a result of this study, it has been observed that shoot length (SL) dur-
ing the seedling stage is one of the organs most affected by drought in cotton, and the
population mean obtained was 12.47, while the genotype means ranged from 18.33 cm
(G35) to 6.33 cm (G91) (Supplementary Table S1). The shoot length averages ranging from
35.20 cm to 43.37 cm, as reported by [63], resemble our findings. During the initial period
that drought stress is observed, it has been noted that the shoot length remains constant,
but in the advanced stages of drought, the shoot length starts to increase. This situation
can be explained by the fact that the plant absorbs more water from the soil, leading to in-
creased photosynthesis and the production of C6 H12 O6 (glucose). Similarly, Iqbal et al. [71]
reported that drought during the seedling stage negatively affects both the shoot and root
morphology of cotton, reducing the shoot length by 29%. A study indicated that drought
stress causes a significant reduction in shoot elongation, and the percentage of decreasing
SL ranged from 97.14 to 35.1 [66]. A reduction in the shoot or in the root length may be
caused by an imbalance of water [93]. Genotypes with a mean SL of 12.47 (Figure 1D) show
significant variability, with 31 genotypes below the population mean and 62 above it. This
trait may be important in the context of plant height and overall shoot development, which
can impact crop yield and plant architecture.
For shoot fresh weight (SFW) drought traits, an advanced line, G15, has the weight
with a 1.853 g value, followed by G52 (1.815 g) and the commercial variety G60 (1.805 g),
respectively. (Supplementary Table S1). Similar SFW trait results were obtained by Akbar
and Hussain [74], Lund and Elsayed [94], and Iftikhar et al. [95]. In Figure 1E, 24 genotypes
exhibit the highest means for SFW, with 68 genotypes exceeding the population mean of
13.280. This suggests that there are genotypes with a higher capacity for shoot growth and
biomass production, which could be advantageous in terms of yield potential.
In Figure 1G, the majority of genotypes have SDW values below the population mean.
This indicates that, in general, genotypes tend to have less dry shoot biomass compared to
the population mean. However, the presence of genotypes with higher SDW values could
be of interest for further study or breeding efforts. The best SDW value was recorded in
G35 (0.349 g) and the lowest in the G65 (0.060 g) genotype. While commercial cultivars
such as G57 (0.337 g), G60 (0.294 g) had lower SDW values. (Supplementary Table S1). In a
study, Ahmad et al. [96] obtained similar results while investigating the root–shoot traits
responses under water deficiency. The genotype G35, which shows the highest value in
shoot dry weight (SDW), also exhibits the highest value in the shoot length (SL) parameter
as a drought trait. Genotypes G22 and G91 have low means in the SL, SFW, and SDW
parameters, indicating their low tolerance to drought stress (Supplementary Table S1).
Basal et al. [50] highlighted traits such as SFW and SDW, due to their ease of measurement
and reliability, that could be used as selection criteria in measuring drought tolerance. The
genotype G35 has emerged as the most tolerant genotype in both traits, with a mean SL
of 18.33 cm and SDW of 0.349 g. Meanwhile, genotype G15 registers the second-highest
mean SL, and the same genotype gave the highest SFW mean (Supplementary Table S1).
Life 2023, 13, 2067 14 of 19

These values indicate that an increase in SL does not always correspond to the highest
SFW; however, there is a positive correlation between an increase in SL and SFW. A similar
situation is observed in the tolerant genotype G31 for the SL and SFW traits. On the contrary,
the genotype G91 has the lowest SL value. Generally, genotypes with higher SL means
have higher SFW means, and genotypes with lower SL means have lower SFW values
In the experiment, among plants that shade each other, those receiving less light
exhibit higher shoot length but lower shoot fresh and shoot dry weights compared to plants
receiving full light. This situation can be explained by the hypothesis that as the amount of
light increases, the plant enhances its photosynthetic rate and consequently increases its
biomass. Based on the conducted phenotypic observations, it has been observed that under
greenhouse conditions, cotton genotypes exhibit shorter shoot lengths compared to the
same duration of laboratory trials. However, it is evident that the experiment established in
greenhouse conditions has significantly longer root lengths and a higher lateral root count
compared to the laboratory drought trial. This suggests that when the plant experiences
actual water stress, it strengthens its roots and engages in coping mechanisms to deal with
water scarcity. This response aims to minimize the impact of drought and indicates the
activation of defense mechanisms to mitigate the effects of water stress.
The provided information presents valuable insights into the performance of vari-
ous genotypes concerning SFW and SDW traits, as well as their comparison to control
genotypes. Genotypes G60, G52, and G41 stand out for their high mean in both SFW and
SDW, indicating their potential as superior performers in terms of biomass production. A
noteworthy observation is made with genotype G52, which exhibits a positive correlation
between SFW and SDW. As the SFW increases, the SDW also increases proportionally. This
correlation suggests that as these plants put on more fresh weight, they also accumulate
more dry weight, indicating an efficient conversion of water and nutrients into biomass.
This is a desirable trait in agriculture as it signifies robust growth and resource utilization
efficiency (Supplementary Table S1). However, the genotype G60 presents an interesting
contrast. While it boasts the highest SFW value among the genotypes, it ranks sixth in
SDW. This discrepancy suggests that in the case of G60, an increase in fresh weight does
not necessarily lead to a corresponding increase in dry weight. This could be due to various
factors, such as differences in water content or the allocation of resources within the plant.
Further investigation is warranted to understand this specific genotype’s growth pattern
better. (Supplementary Table S1).

5. Conclusions
In this current study, the genotypes G44 and G56 exhibit common high values in all
four drought traits (NLRs, RL, RFW, and RDW). The genotypes G13 and G5 share high
values in NLRs and RL, while the genotype G5 coincides in terms of NLRs, RL, and RDW.
The genotypes G1, G56, G51, and G88 show the highest values in RFW, and the genotypes
G5, G56, G44, G75, and G90 consistently yield the highest means in the RDW traits. The
genotype G35 shows the highest mean values in both SL and SDW; the genotype G15
shares high values in both SL and SFW; and the genotypes G41, G52, and G60 have high
values in both the SFW and SDW parameters, demonstrating high drought tolerance. Using
the tolerant cultivars found in the current study as parents in drought-tolerant variety
development in the marker-assisted selection (MAS) plant breeding program will increase
the success of reaching the target after genetic diversity analyses are performed. It is also
possible for the genes responsible for this tolerance to transfer from the donor plants to the
recipient plants through hybridization.

Supplementary Materials: The following supporting information can be downloaded at


https://www.mdpi.com/article/10.3390/life13102067/s1, Table S1: Drought morphological markers
mean in Upland cotton (Gossypium hirsutum L.) at the seedling stage.
Funding: The authors declare that no funds, grants, or other support were received during the
preparation of this manuscript.
Life 2023, 13, 2067 15 of 19

Institutional Review Board Statement: Not applicable.


Informed Consent Statement: Not applicable.
Acknowledgments: I am thankful for the support of the East Mediterranean Transitional Zone Agri-
cultural Research Institute, East Mediterranean Agricultural Research Institute, GAP International
Agricultural Research, and Training Institutes SET seed, MAY, BASF, and Progen Inc. seed companies,
which provided me with cotton seed, and to the Bingol University, Genç Vaccaional school personnel
and students for assistance during all processes in the experiment.
Conflicts of Interest: The authors declare no conflict of interest.

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