Rehman Thesis

1
CHAPTER NO.1
INTRODUCTION
Wheat is considered a main staple food of Pakistan (Anjum et al., 2020). It belongs to
Poaceae family. Wheat is thought to have been the first crop produced around 10,000 to 8000
before Christ. It has been a significant source of calories (18%) and proteins (20%) for humans
(Kan et al., 2015; Muminjanov and Karagöz, 2018).
The wheat harvest was grown on 220.11 Mha and produced 765.43 million tonnes of
grain, or 3405 kg of grain per hectare on average (FAOSTAT, 2019). It grows all throughout the
world and is the second most widely planted crop after maize (Shewry, 2009). Wheat is a
required food crop in 43 nations and is a staple food (Raza, S.H. 2005). Pakistan produced 28.9
million tonnes of wheat on an area of around 31.18 Mha (Govt, 2018).
Wheat is also the most widely consumed food in the planet. Wheat flour contains gluten
protein, which aids in the production of roti. It has a sticky quality. China is the world's leading
wheat producer. Wheat production consumes 40 percent of Pakistan's land area. Wheat is farmed
on 6.97 million hectares in Punjab, accounting for 75% of total wheat production in Pakistan.
Wheat is farmed on 1.15 million hectares in Sindh, accounting for 12% of the total. Wheat
production is restricted to 0.73 million hectares in KPK, or 8%, and 0.38 million hectares in
Baluchistan, or 4% of total wheat production in Pakistan (Haider et al., 2019).
According to an agricultural report, the Faisalabad 2008 and Sehar 2006, wheat varieties
dominate Punjab, accounting for over half of the total area. Pakistan also boasts over 30 wheat
varieties, each with its specific water and nutrient requirements (Raza et al., 2012; Abid et al.,
2014; Mohsin et al., 2015).
There are two varieties of commercially farmed wheat: durum wheat and bread wheat,
which vary in genetic complication, adaptation, and application. Both forms of wheat are
currently used to make and consume a broad range of goods across the world. Wheat is mostly
consumed by humans and feeds roughly a third of the world’s population. It is nutrient-dense,
easy to store, andtransport, and may be transformed into a variety of foods. Wheat requirement is
predicted to outpace that of any other major agricultural product (Shaheen et al., 2004). Over one
billion people in both developing and developed countries rely on bread wheat for food and
livelihood (Metwali et al., 2011).
Due to many factors such as quality seed availability, irrigation water, fertilizers, farm
mechanization, and biotic and abiotic stress conditions, wheat productivity in Pakistan is lower
than in further wheat-growing countries throughout the world. Salt, drought, heat, cold and
heavy metal are the abiotic stress conditions that reduced wheat yield, production, and
overproduction in Pakistan (Charles et al, 2006; Raza et al., 2006; Kirkegaard et al., 2008).
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One of the most significant sources of stress affecting crop yield and yield in the
predictable future, along with drought, is salinity (Parida and Das, 2005). By 2050, it is expected
that 20 percent of agricultural fields across the world will be affected by salinity (Kang et al.,
2010). In dry and semi-arid places around the world, more than 6% of the total land area (or
roughly 800 million hectares) that may be used for agriculture is adversely impacted by saline
conditions (Sarkar et al., 2018). Pakistan has 6.63 million hectares of salt-influenced soils, which
is lowering the profitability of its agriculture (Malik et al., 2021).
One of the main issues disturbing plant diversity and agricultural output in our nation and
around the world is salinity. Poor rainfall and high evaporation, inadequate drainage, incorrect
agricultural methods, and soil potentials are the major reasons of salinity, which affect broad
areas, especially in dry and semi-arid climates. Soluble salts present in soil, provide the majority
of components required for plant development, but excessive amounts can be hazardous. In dry
and semi-arid regions where annual rainfall is insufficient to meet the evapotranspiration
requirements of the plants, saline soils are common. Soil salinization is one of the most
significant abiotic stressors, resulting in decreased crop growth and yield (Sairam et al., 2002).
Plants can readily absorb soluble salts. When a particular concentration of salt compounds enters
the plant structure, it might be damaging to the plant, depending on the type and amount.
Salinity is hazardous to plants because it disrupts their nutrition and metabolism. Three
factors influence plant growth; a) Physical impact: Because of the increased osmotic pressure,
water intake by plant and, as a result, nourishment slows or stops entirely. Water input is
problematic for the plant. The osmotic pressure effect is another name for this. b) Direct effect:
Some salts make it harder for plants to absorb nutrients and destroy their structure by
interrupting their metabolism. This is also known as particular ion toxicity. c) Indirect effect: The
effects of salinity or sodium on the soil have an impact on plant growth. For example, a decrease
in efficiency and the expenditure of metabolic energy to assure water intake. (Ekmekçi et al.,
2005; Munns and James, 2003). While osmotic effect creates disturbance in osmotic potentials,
specific ion effect results in the toxicity of distinct ions (Brady and Weil, 2002).
Salinity not only affects plant growth and productivity, but it also creates nutritional
imbalance and degrades grain quality. In wheat, salinity reduces grain protein, oil, and fibre
levels, as well as yield components (Abbas et al., 2013).
All important activities in a plant are affected during the initiation and development of
salinity stress, including lipid metabolism, energy production, protein synthesis, and
photosynthesis. Depending on the salinity, disruption of these processes results in loss of wheat
grain productivity and quality (Acevedo et al., 2002; Cicek, 2002) found that a decline in leaf
area is typically the first symptom of salinity stress. Root and root growth are negatively
impacted by salinity, although the shoot is typically more sensitive because salt inhibits cell
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division and expansion at the developing point, which impairs wheat growth and tiller viability,
reducing the number of main and secondary tillers (Parida and Das et al., 2005).
When the tiller first appears and wheat is germinating, it is most vulnerable to salinity.
Tillering capacity in wheat is also diminished as salinity concentrations rise. Under salinity
circumstances, effective ears per plant have been found to be revealed as the most significantly
damaged yield component in wheat (Munns et al., 2006).
Salinity also reduces number of leaves in main shoot and the number of spikelets in the
main spike, lowering seed set and grain production. Salinity stress is one of the most significant
restrictions to wheat production, and it has a bigger impact on durum wheat, which is less
salinity tolerant than bread wheat (Turki et al., 2012).
Salinity stress causes morphological and physiological alterations in wheat stem and leaf
cells, as well as tissues and organs. Salinity reduced stem and leaf diameter, wall thickness, the
size of the hollow pith cavity, the total number of vascular bundles, the proportion of large to
tiny vascular bundles, the length and width of the bundles, and phloem tissue thickness, among
other morphological features (Rania et al., 2020).
Excessive salt accumulation interferes with essential plant metabolites (Ashraf and
Akram 2009). In reaction to salt stress, plants develop osmotica/osmoprotectants/compatible
solutes, such as sugars like trehalose, to modify the osmotic potential of the cell (Shahbaz et al.,
2017).
Wiggers and others discovered trehalose, a non-reducing disaccharide, in organisms in

the 19th century from ergot in rye (Kosar et al., 2019). A few drought-tolerant plants, such
Myrothamnus flabellifolius, also have sufficient trehalose reserves to serve as osmolytes.
Trehalose is mostly utilised by plants as a signal molecule, where it controls plant growth,
development, and metabolism in response to adverse environmental conditions such high
temperatures, salinity, drought, and cold stress (Fernandez et al., 2010; Tournu et al., 2013;
Henry et al., 2015; Fichtner and Lunn, 2021). Trehalose-6-Phosphate was once believed to be a
signalling molecule that informed other pathways involved in plant growth, development, and
environmental reactions of the glucose state of the plant (Fichtner et al., 2021).
As a result, trehalose's numerous metabolic actions could be attributed to its participation

in sugar signal transduction. In plants, there is only one metabolic pathway i.e., trehalose-6-
phosphate synthetase - Trehalose-6 phosphate phosphatase, which produces trehalose by the
transitional T6P (Lin et al., 2017).
The role of trehalose metabolism in stress tolerance in plants is currently attracting a lot
of attention. Modification of the trehalose pathway in transgenic plants has been linked to stress
tolerance and recovery in recent research. Furthermore, exogenous trehalose treatment
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demonstrated promising results in crop movement. Lower concentrations of exogenous trehalose,

can diminish sodium ion buildup in plants, whereas higher concentrations of trehalose can
prevent chlorophyll loss in leaves and root damaged caused by excessive salinity, minimising
salt ion damage to plants (Lunn et al.,2014; Islam et al., 2019). Exogenous trehalose has also
been shown to reduce ROS outbreak, ion imbalance, and programmed cell death in Arabidopsis
thaliana seedlings (Garapati et al., 2015), Zea mays (Henry et al., 2015), Catharanthus roseus
(Chang et al., 2014), and Solanum lycopersicum L. (Feng et al., 2019). Moreover, salinity stress
induced OsTPP1 gene expression in Oryza sativa (Zhang et al., 2017), whereas AtTPPD
overexpressed plants are more tolerant to high salinity stress (Krasensky et al., 2014).
Trehalose is also utilized as a carbon source by animals, particularly insects, during flight
activity (Carpinelli et al., 2006). Mannitol and trehalose levels are greater in ascospores under
extreme stress (Dijksterhuis et al., 2002). Asexalual spores can germinate on a growing medium
with low water but enough nutrients, such as conidia with a high trehalose and mannitol content.
(Wang et al., 2012). Stress sensitivity has been documented in conidia lacking trehalose
(Sakamoto et al., 2008). Trehalose also reduces the toxicity of hydrocarbons like toluene and
ethanol (Wen et al., 2016).
Trehalose, to put it simply, is a natural disaccharide that is adaptable and functions as a

bio-preservative in metabolic machinery when faced with abiotic stress. Trehalose also
contributes to the development of flowers and embryos. It controls how plants use carbon as well.
Trehalose content also affects how a plant and microbes interact (Iturriaga et al., 2009).
Trehalose may therefore play the role of preservation in metabolic pathways. The extent to
which it might preserve various biomolecules involved in stress tolerance in various plant
species is not yet well understood.
So a study is planned based on above mentioned facts. The effect of salinity is studied on
wheat under two levels of salinity stress. To minimize the salinity stress, trehalose has been used
by foliar application.
The purpose of this study is to mitigate the salinity stress in wheat and its effect on
morphological and physiological structure of wheat plant.
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CHAPTER NO.2
REVIEW OF LITERATURE
Mohamed et al. (2018) determined that trehalose reduces the negative effects of salt stress
on wheat. Four different wheat cultivars were used in this experiment to determine how foliar
application of trehalose, either alone or in combination with salt stress, affected growth, grain
production, and several biochemical components (Giza 168, Gemiza-10, sids-12 and Sakha-94).
The findings showed that irrigating wheat plants with sodium chloride (200 mM) reduced all
growth parameters, yield components, and some anatomical features of the leaves, especially in
the most sensitive cultivars (Giza 168 & Sads 12). These parameters had no appreciable impact
on the other cultivars (Gemiza 10 & Sakha 94), which were thought to be tolerant cultivars.
Additionally, during salt stress, the amino acid and sugar fraction of wheat cultivars' shoots
dramatically increased in all cultivars. The tolerant cultivars showed the most substantial gains
(Gemiza 10 & Sakha 94). Wheat plants were treated with 10 mM trehalsoe to reduce the harmful
effects of salt stress, and both non-stressed and salt-stressed plants showed a clear increase in all
of the parameters listed as compared to control plants. According to the data, trehalose is a useful
osmoprotectant for reducing salt stress' detrimental effects on wheat plant.
Iqra et al., (2020) examined the salt stress effect on wheat cultivars. Salt stress affects
several morphological features in six of Pakistan's most common wheat cultivars. Two salt
solutions were employed in this study: 10 dSm-1 and 15 dSm-1 NaCl concentrations. According
to the data gathered throughout the study, salt treatments reduce all morphological features,
except for two traits; carotenoid content and root length. It was discovered that under the
influence of both salt and sugar, The amount of carotenoids in the leaves and roots grew
significantly, as did the length of the roots was also boosted. The results of the analysis of
variance revealed that genotype 5 (Ujala-16) had higher leaf carotenoids (998.32 mgg-1 of fresh
weight of leaf), followed by genotype 1 (Inqalab-91) (995.99 mgg-1 of leaf fresh weight) and
genotype 2 (Shafaq-06) had fewer carotenoids (825.65 mgg-1 of leaf fresh weight). Shafaq-06
had the highest root weight after being treated with 15dSm-1 NaCl. When all Pairwise
comparison tests were combined, genotype 4 had the longest root length (Galaxy-13).
Carotenoids appear to have the least impact on plant height, according to linear regression.
Except for carotenoids, photosynthetic pigments had the highest genetic heritability, with 99.99 %
for chlorophyll b. Fresh stem weight has a faster rate of genetic advancement (309.870 %).
According to our findings, better heritability and genetic progress suggested that selecting salt
stress wheat genotypes based on root length aid in the development of wheat genotypes having
salt stress tolerance with maximum grain yield.
Akher et al. (2018) examined that salt stress has a significant impact on plant growth and
development. The purpose of this study was to see if salicylic acid could help wheat to cope with
salinity stress. The treatment in this experiment included 4 different salinity levels: So= no salt
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(control), S1= 2.8 gkg-1 soil 3-4 dSm-1, S2= 6.0 gkg-1 soil 7-8 dSm-1, S3 = 9.0 gkg-1 soil 11-12
dSm-1, and three different levels of salicylic acid (SA): Ao= 0mM, A1= 0.2mM and A2= 0.4mM.
It was done by using a four-replication randomized complete block design (RCBD) with two
factors. There were a total of 12 therapy combinations (4x3). In terms of the primary metrics, the
results of the trial revealed a considerable difference between the treatments. Wheat yields were
lowered by varying degrees of salt. Salinity levels that were higher resulted in a lower yield.
S0A2 (Without Salt+0.4 mM SA) yielded the highest grain production (1.55 t ha-1). There was no
change between the S0A0 (Without Salt+ Without SA) and S0A1 (Without Salt+0.2mM)
treatment combinations. However, as the salt level increased, the grain yield declined
significantly. Salicylic acid is used to treat different amounts of salinity and boosted grain yield
in different ways. The yields were improved with SA(0.4 mM) application from 1.14 tha-1 to 1.32
tha-1, 1.07 tha-1 to 1.14 tha-1 and 0.26 tha-1 to 0.31 tha-1 at 3-4 dSm-1, 7-8 dSm-1, and 11-12 dSm-1
NaCl, respectively. The findings imply that salicylic acid can reduce the negative effects of salt
while also increasing wheat grain yield.
Kizilgeci et al. (2020) determined that plant tolerance to drought and salinity can be
enhanced by maintaining turgor by osmotic adjustment. The study was conducted to better
recognize the impacts of salinity and drought stress on many genotypes of bread wheat at an
early developing stage, specifically during the seedling stage, due to the critical role of PEG.
Five bread wheat genotypes were employed as seed material, including four kinds such as
'Ceyhan 99,' 'Dinc,' Empire Plus,' 'Tekirdag,' and a line known as 'DZ17-1'. To determine which
wheat genotypes are salinity and drought tolerant, all wheat genotypes were tested in 4 different
concentrations of NaCl for salinity stress (i.e., 0, 4, 8 and 12 dSm-1) and 4 different
concentrations of PEG-6000 for drought stress (i.e., 0, -0.2, -0.4 and -0.6 MPa). All treatments
were administered three times in a completely randomized design. Bread wheat cultivars 'Dinc'
and 'Ceyhan 99' were proven to be salinity and drought tolerant at the seedling stage after careful
study. The study also discovered that knowing seedling growth parameters such as mean
germination time, coleoptile length, seedling height, root length, and relative water content
allows for genotype selection for salinity and drought tolerance. The findings of the study will be
useful in determining which wheat genotypes are suitable for cultivation under osmotic stress
caused by both salinity and drought stress.
Borlu et al. (2018) examined that under salinity, the majority of plants are sensitive during
early growth phases such as germination and seedlings, resulting in a lower final yield. To thrive
under saline conditions, plants with a high germination rate and early intensive growth should be
favored. In this context, an investigation was conducted under controlled environmental
conditions to estimate the effectsof salinity on germination, early seedling growth, and
development of commercial Durum wheat cultivars, as well as to screen out salt tolerant wheat
cultivars that are suitable for growing and expanding wheat in the Central Anatolia Region of
Turkey, at various salinity levels. Six salt concentrations (0, 50, 75, 100, 150, and 200 mM NaCl)
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and five durum wheat genotypes, 'Akmak 79', 'Cultivar 1252', 'Kunduru 1149', 'Kzltan 91', and
'Selcuklu 97') were used in the study. The results demonstrated that increasing salinity had a
substantial effect on the value for the examined variables such as their elongation rate, fresh and
dry weight, root length, and shoot length, but that the effects differed depending on cultivars and
salinity levels. Plant sodium (Na+) concentration increased as salt levels increased, and K+
content declined, while other minerals varied according to genotypes. The wheat cultivar
'Selçuklu 97' has the highest fresh weight (FWSI), dry weight (DWSI), and salt tolerance index
when measured in % salt tolerance index. As a result, cultivar 'Selçuklu 97' is a salt tolerant
genotype that is suiTable for growing in Turkey's salinity region.
Ouhaddach et al. (2018) examined the influence of soil salinity in two wheat varieties,
Ashtar and Salama, on morpho-physiological and biochemical parameters in the early stage was
investigated using three different NaCl concentrations (50, 75, and 100mM). The aerial part's
relative water content, chl "a" and "b" concentrations, membrane stability index, Na+ content,
and dry weight all rose under salinity stress. Salt treatment has reduced the plant's height,
number of leaves, and specific leaf area as well as its fresh and dry weight, K+ content, and
K+/Na+ ratio. The results revealed that the variety Ashtar has a higher salt tolerance. The mild
decrease in shoot and root biomass demonstrated this tolerance.
Yassin et al. (2019) determined the impact of salinity stress on a few physiological and
biochemical traits of two bread wheat genotypes, and to clarify the mechanism by which these
wheat genotypes tolerate salt. Salt concentrations of 50, 100, and 150 mM NaCl were applied to
two wheat genotypes, "Sakha 95" and "Misr 2." Both genotypes differed considerably for all
variables at all levels of salinity stress, according to the findings. Among the genotypes, the 'Misr
2' genotype grew more faster than the 'Sakha 95' genotype, retaining a higher dry biomass. When
compared to genotype 'Sakha 95,' the genotype 'Misr 2' had higher levels of soluble sugars,
proline, and antioxidant enzyme activity such as glutathione reductase, peroxidase, and catalase;
however, it had low levels of lipid peroxidation as measured by the malondialdehyde
concentration. This indicates that the genotype 'Misr 2' is more tolerant of salt stress than the
genotype 'Sakha 95'. In both wheat genotypes, salt stress caused considerable changes in Ca++
concentration and the Na+ /Ca++ ratio. The relative decline in Ca++ concentration and the Na+
/Ca++ ratio was found to be larger in the genotype 'Sakha 95' thanin the genotype 'Misr 2',
resulting in damage symptoms. As a result, the genotype 'Misr 2' could be valuable in the future
breeding effort to generate salinity tolerant wheat types.
Saddiq et al. (2018) investigated that physiological seed improvements can help to reduce
salinity stress. In a pot research, the genotypes Kharchia 65 (salt tolerant) and PI.94341 (salt
sensitive) were examined under salt stress (20 dSm-1) using several priming treatments, including
hydropriming and halopriming (50 mM, KCl, and NaCl). The experiment was carried out in a
thoroughly randomized design with three replicates in a factorial layout. Priming agents
enhanced the final emergence percentage, emergence index, and decreased the mean emergence
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time of both wheat genotypes under normal and saline circumstances when compared to non-
primed seed. Priming treatments improved the efficiency of wheat seedlings in both normal and
salt-stress condiions by raising both Fv/Fm and Ft, which were closely related to low Na+ and
high K+.. Finally, by increasing seed emergence and stimulating the physiological features of
wheat,all three priming agents effectively negated the negative consequences of salt stress.
Alsahli et al. (2019) determined the effects of salicylic acid (SA; 0, 0.5, and 0.75 mM) on wheat
growth, antioxidant enzyme activity, and biochemical characteristics under salt stress. Salinity exposure
(0, 25, 75, and 125 mM NaCl) inhibited wheat growth by lowering the fresh and dry weight of the shoot
and root, inhibiting leaf development, and triggering necrosis on old leaves. With 125 mM NaCl
concentration, hydrogen peroxide and lipid peroxidation production rose by 3.37 and 2.54, respectively,
while membrane stability decreased, which was alleviated by the application of SA. When compared to
untreated controls, SA (0.5 mM) considerably boosted growth under normal circumstances. Salinity (125
mM) stress increased proline accumulation by 4.63 times, carbohydrates by 39.61 %, free amino acids
by 9.44 %, and protein content by 7.91 %, all of which were further boosted by SA, resulting in greater
stress adaptation. Application of SA to salt exposed cells increased the activity of antioxidant enzymes
like SOD, CAT, and APX by 1.76, 2.25, and 2.22 fold, respectively, resulting in better reactive oxygen
species removal and protection from oxidative stress. Furthermore, excessive Na uptake in salinity-
stressed plants inhibited K+ uptake and triggered leaf necrosis. The use of SA, on the other hand,
significantly reduced these unfavorable consequences. Finally, the salt stress has a negative impact on
wheat plant growth and development. Supplementing with a suitable dose of SA, while alleviated the
detrimental effects of salinity by modulating osmolyte levels, antioxidant enzyme activity, and critical
element intake.
Mushtaq et al. (2019) investigated that wheat is considered a staple food in Pakistan,
accounting for 60 % of a person's daily calories intake. Wheat production can be enhanced by
utilising salt affected areas and marginal lands, as well as enhancing fertilizers and silicon
supplementation. Because nanotechnology has revolutionized agriculture, silica nanoparticles
(SiO2 Nps) were used to alleviate salinity stress in this study. Seeds of four wheat cultivars were
grown, in hydroponics: Raskoh and Umeed (salt resistant) and Shahkar and Zarghoon (salt
sensitive). CRF (Controlled Release Factor), CRF with SiO2 Nps, sodium silicate, SiO2 Nps, and
silicic acid were employed as treatments, with Hoagland's Nutrient Solution (HNS) serving as
the control. A 100 mM NaCl salt stress was applied. For TDS, germination rate, root and shoot
length, fresh and dry weight, and chlorophyll content were all measured. At the 0.05 level, all of
the data were statistically significant, and SiO2 Nps and sodium silicate were discovered to
influence salinity. The use of CRF and CRF with SiO2 Nps yielded excellent results. It has been
determined that its CRF and SiO2 Nps can be used to combat drought and salinity stress when
growing wheat in Pakistan's marginal and salt affected soil.
Ibrahim et al. (2019) investigated that salinity is the main abiotic factors limiting
agriculture productivit, especially in dry and semi arid regions. The goal of this study was to
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explore if nitrogen fertiliser could help wheat seedling emergence, seedling growth
characteristics, and physiological responses to salinity. Two wheat types (Xumai 30 and Elnilein)
were planted in soil that had been treated with NaCl at concentrationsof 1.4, 2.5, 4.2, and 7.6 dS
m−1. The salty soil was fed with urea fertiliser at three different nitrogen levels (0, 86, and 210 kg
Nha-1). The interaction between the varieties, nitrogen, and salinity had a substantial impact on
emergence %, root and shoot length, and total dry weight. The seedling vigour index was
affected by the interplay of nitrogen and salt (SVI).Protein and superoxide dismutase (SOD)
levels increased when nitrogen levels increased, but dropped in salt stressed plants. In this
investigation, nitrogen was found to be effective in minimize the negative effects of NaCl.
Furthermore, the effects of 86 and 210 kg Nha-1 on wheat seedling, seedling growth, and
physiological responses were similar. Furthermore, the results demonstrated that nitrogen
fertiliser had a good effect on wheat plants at moderate salinity, but had a negative effect or was
ineffective at high salinity. As a result, fertiliser management is essential in salt affected areas in
order to maintain yield and reduce soil damage.
Cholewa et al. (2018) investigated how silicon affected spring wheat that had been
exposed to salt stress. On 10-day-old wheat seedlings, the experiment was carried out in
hydroponic settings. Salt stress was generated by adding NaCl to the nutritional medium at
concentrations of 70 and 100 mM. At concentrations of 1.0 and 1.5 mM, silicon (H4SiO4)
significantly enhanced the weight of wheat seedling shoots and roots, as well as the quantity of
photosynthetic pigments in leaves. It inhibited peroxidation of membrane lipids and minimised
the harmful effects of salt stress. As a result of the silicon addition, we noticed a higher
accumulation of nitrates and a decrease in malondialdehyde concentration in plant tissues.
Silicon had no effect on the sugar level of wheat shoots and roots when exposed to osmotic stress.
Silicon had no discernible effect on proline content. Overall, the findings suggest that silicon can
be employed to alleviate the negative effects of salinity on plant health.
Kanwal et al., (2017) determined that Salinity is a key abiotic stress that has a global
impression on crop yield. A soil conditioner made of activated carbon called biochar, help to
reduce salinity's detrimental effects. The study's objective was to determine whether adding 1%
and 2% biochar to wheat seeds would improve their ability to germinate and thrive while
exposed to salt. Both biochar levels, boosted germination and development under salt conditions,
although 2% biochar was more effective than 1%. With 2% biochar, root and shoot lengths
increased by 23% and 11%, respectively. 150 mM salt with 2% biochar, the greatest increase in
leaf water potential and osmotic potential was 16 % and 10%, respectively. At 2% biochar, the
concentration of proline and soluble sugar decreased by 51% and 27%, respectively. Under stress,
biochar reduced superoxide dismutase activity by 15.3% at a concentration of 2%. Wheat
productivity increases because of biochar's ability to lessen the impacts of salinity.
Chen et al., (2018) examined the rheological qualities of doughs made from hard wheat
using 5 different amounts of NaCl. To understand how salt alters the structure of gluten,
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researchers looked at its zeta potential, disulfide-sulfhydryl groups, secondary structure, surface
hydrophobicity, extractable glutenin and gliadin. Higher salt levels in doughs (2.0 and 2.4 %,
fwb) increased elongational viscosity, storage, and loss modulus as a result. With more salt, the
temperature of starch gelatinization increased. The presence of salt reduced the free sulfhydryl
concentration of gluten while increasing the -sheet structure. Salt increased the macromolecular
accumulation of gluten protein, according to RP-HPLC. The network structure and conformation
of the gluten molecule were altered by salt, and these changes considerably improved the
physicochemical properties of the dough. These results gives us well understanding of how salt
works in doughs and can help us find salt substitutes for bakery products.
Iqbal et al. (2018) studies that numerous abiotic stresses that plants are exposed to have an
adverse effect on their growth, metabolism, and yield. Salt stress is one of the most detrimental
elements for plant growth and productivity. Wheat crops have been studied for their
physiological and morphological characteristics in relation to salinity stress, including
photosynthesis, stomatal conductance, water relations, and chlorophyll content. Morphological
characteristics include plant height, root length, number of leaves, leaf area, and root hair count.
Additionally, anatomical, biochemical, and molecular factors have been taken into account.
Choosing the features that are most crucial for salt tolerance in a wheat system might be
challenging. To simplify this difficulty, the authors recommend using graphs to aid in displaying
correlations between features, particularly biochemical and molecular characterizations, which
are important to consider when modifying current or developing new kinds.
Sehar et al., (2020) examined the effects of salt stress (100mM NaCl) and nitric oxide (as
sodium nitroprusside, SNP) on glucose (Glu)-inhibited photosynthetic characteristics in wheat.
Plants inoculated with either NaCl or 6% Glu displayed decreased photosynthesis due to
increased oxidative stress. The increased levels of Na+ and Cl- as well as lipid peroxidation in
crops cultivated under salt stress were decreased with the injection of 50 M SNP. SNP-treated
plants with or without 6% Glu reported decreased Glu and ethylene susceptibility and oxidative
stress, which improved photosynthesis under salt stress involving activation of the antioxidant
system, proline storage, and N uptake. The results show that, in the lack of salt stress, SNP
enhances plant photosynthetic capability, and that, in the presence of salt stress, SNP reduces
glucose-mediated photosynthetic suppression.
Iqbal et al. (2021) investigated that Two wheat varieties, Anaj-2017 and Ujala-2016, can
be sprayed with foliar applications of the plant growth promoters gibberellic acid (GA3) and
salicylic acid (SA) to lessen the impact of soil salt. Salt was given at 150mM concentration after
the germination of seeds had taken place for two weeks. The eleven therapies listed below were
applied: To is the control; T1 is 150 mM NaCl; T2 is 0.5 mM SA; T3 is 1.0 mM SA; T4 is 100
mgL-1 GA3; T5 is 150 mgL-1 GA3; T6 is 150 mM NaCl with 0.5 mM SA; T7 is 150 mM NaCl
with 1.0 mM SA; and T8 is 150 mM NaCl with 100 mgL-1 GA3. Three doses of GA3 and SA
were given after a week of salinity stress. Morphophysiology, biochemistry, and yield
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characteristics were all recorded. In Ujala-2016, anthocyanin was increased by 0.0035% at 100
mgL-1 GA3, whereas glycinebetain was increased by 0.26% at 150 mgL-1. 1.0 mM SA and
150mgL-1 GA3 were found to considerably improve different growth metrics. Finally, a salt
stress treatment of 0.1mM SA and 150mgL-1 GA3 in combination with the variety Ujala-2016 is
advised for future cultivation to mitigates salinity stress and increases development and
production.
Ibrahim et al. (2018) examined that salinity is an increasing problem around the world,
and measures are needed to address it. The goal of this study was to see if adding nitrogen (N)
fertiliser to wheat may help alleviate the negative effects of salt on yield and productivity. Xumai
and Elnilein 30 different varieties of wheat were planted in soil that had been exposed to NaCl at
levels of 1.4, 2.5, 4.2, and 7.6 dS m-1. Three rates of urea-N (0, 86, and 210 kg N ha–1) were
applied to the NaCl-treated soils. Varieties' interactions with N, as well as interactions among
salinity and N, was influenced At 7.6 dS m-1 salinity, and 210 kg N ha-1 increased yield by 54.7%
regardless of spike length, number of spikelets, the amount of kernels for each spike, grain
weight per spike, and 1,000 kernel weight. The negative effects of NaCl were reduced by N in
this study. Furthermore, the effects of 86 and 210 kg N ha–1 on wheat yield were identical
through all N levels tested in this study. Under varied salinity and N levels, Elnilein had lower
yields than Xumai 30 across all N and salinity treatments. These results imply that cultural
activities can help to mitigate the effects of rising salt levels around the world.
Nahrawy et al. (2020) investigated that Salinity is a major limiting factor to agricultural
yield around the world, and it's only going to become worse. As a result, to address this issue, we
should devise techniques to increase salinity tolerance in various crops. plant growth
promoting rhizobacteria (PGPR), can aid plants in tolerating challenging environmental
conditions. Six Azotobacter sp. (Az1-Az6) isolates were examined in this work in laboratory for
growth, PGPR features such indole-3 acetic acid (IAA) generation and nitrification, and
germination signals for Misr 1, Gemmiza 12, and Sakha 95 wheat cultivars at varying NaCl
concentrations. The effectiveness of inoculating various wheat cultivars with two superior
isolates in a greenhouse experiment and Gnotobiotic Sand System to enhance growth dynamics,
physiological characteristics, nutrient uptake, and antioxidant enzymes under various salinity
levels of sandy soil was also tested (0, 4, 8, and 12 dS m-1). Out of the six examined isolates, two
(Az2 and Az6) demonstrated salt resistance, PGPR characteristics, and enhanced germination
assays. Both bacteria have three separate mechanisms for accelerating growth.
Zafar et al. (2019) determined that Melatonin has become a novel plant growth regulator
as a result of its advantageous role in the reduction of abiotic stress. Melatonin's impact on wheat
plants' antioxidant activities, development, and yield under 10 dSm-1 NaCl salinity stress was
examined using two genotypes Sarsabaz and S-24, which were cultivated under 10 dSm-1 salinity
stress. After 45 days of seeding, different levels of melatonin i.e., 50, 100, 300 and 500 µM were
administered by foliar application. Melatonin considerably increased the development and
12
productivity in both genotypes of wheat under salt stress, as compared to non treated wheat
genotypes.In both wheat cultivars, 50 µM melatonin raised yield by 5 and 11 %, respectively,
and 500 µM melatonin increased yield by 44 and 48% in Sarsabaz and S-24, respectively, as
compared to control (only salinity). Additionally, under stressful conditions, melatonin foliar
spray increased the catalase, peroxidase, and superoxide dismutase activities in wheat genotypes.
In terms of antioxidant enzyme activities, 500 µM melatonin was more efficient in decreasing
oxidative damage during salinity stress, with an alternate decrease in malondialdehyde level.
Improved antioxidant enzyme activities are favorably connected to increased biomass production
and yield. Melatonin's ability demonstrated through increasing the antioxidant defence system,
salt stress is reduced. The findings proposed that foliar application of melatonin could be a good
method for wheat plants to fight adverse circumstances and increase output.
Kosar et al. (2018) studied that trehalose is a naturally occurring non-reducing sugar
present in a diverse array of species, comprising bacteria, yeasts, crustaceans, and even plants. It
is a one-of-a-kind chemical in terms of its properties. In lesser creatures, it serves as a carbon
source, and in larger animals and plants, it serves as an osmoprotectant or a stabilising molecule.
Although it is present in small amounts in plants, its levels rise when they are exposed to abiotic
stressors. Trehalose is thought to protect against several abiotic stressors such as temperature
fluctuations, salt, and desiccation. In most plants, it also affects water use efficiency and stomatal
mobility. Endogenous trehalose levels that may be detected are critical for maintaining growth
under stressful conditions. Trehalose, when administered exogenously in small amounts, reduces
biochemical and physiological abnormality caused by diverse abiotic stimuli, delay leaf
abscission, and promotes blooming in plants. The stress sensitive genes in plants exposed to
environmental signals are likewise up-regulated when trehalose is applied externally. Plants that
have been genetically engineered with trehalose production genes have better stress tolerance.
Transgenic plants that overexpress genes involved in microbial trehalose production have been
found to have higher levels of trehalose. These transgenic plants, on the other hand, have
improved heat, cold, drought and salinity tolerance. This sugar has made significant progress in a
variety of disciplines due to its many bio-functions. Exogenous usage of this bio-safe sugar, on
the other hand, would only be practical in the field if low-cost trehalose manufacturing tactics
were adopted. In a nutshell, trehalose is a one-of-a-kind molecule that helps plants maintains
their vitality in adverse environmental conditions. Certainly, the recent discoveries on this
disaccharide will open up a slew of new possibilities.
Akcin et al. (2018) investigated that one of the most significant abiotic stresses is heavy
metal poisoning. Copper, manganese, cobalt, zinc, and chromium, for example, have the
potential to be harmful to plants when present in excessive amounts. The study looked into the
significant anatomical changes affected by chromium (Cr) in wheat variety i.e., "Ekiz." Plants
were given 0.3 and 0.4 mM Cr for 21 days to achieve this goal. Cr increased primarily in the
roots of plants treated with metal, according to microscopic examinations. The diameter of
13
parenchyma cells improved in the stems of Cr-treated plants, but sclerenchyma thickness
decreased. The thickness of phloem, xylem, and mesophyll tissue in leaves of Cr treated plants
was reduced as compared to control plants. Cr has a significant impact on Triticum aestivum cv.
"Ekiz" based on morphological alterations in the root, stem, and leaves of plants treated with Cr.
Zafar et al. (2021) determined that Zinc deficiency is a critical soil restriction for
agriculture, especially grains. Zinc is an essential element for a variety of physiological activities,
including glucose metabolism and plant production. The role of zinc grain priming in reducing
the negative effects of salinity stress was investigated in this study. Salt stress (100 mM) had a
deleterious impact on plant components, causing physiology and biochemistry to be disrupted in
wheat varieties Faisalabad-2008 and Galaxy-2013. ZnSO4 (0, 0.5, 1, 2, 5, and 10 mM)
preconditioning of Triticum aestivum L. wheat grains accelerated root and shoot expansion,
chlorophyll, K, and Zn contents in both wheat varieties. In roots and shoot Na+ ions are
favorably associated with each other, and adversely control photosynthetic pigments, ionic levels,
and plant development, according to correlation analysis, dendogram, and PCA analysis. Overall,
the data specified that priming with 5 mM ZnSO4 reduced salinity stress in both wheat genotypes.
Ahmed et al. (2021) examined that abiotic stressors have a negative effect on crop plant
growth and physiological processes, resulting in a significant drop in production. Based on
increasing dangers of saline and drought circumstances, the current study was conducted to
investigate the effects of foliar applied salicylic acid (SA) on the development, gaseous exchange
abilities, and leaf pigments in wheat under salinity and water stress. Under a CRD-factorial
method that was repeated three times, three commercial wheat varieties (Galaxy-2013, Punjab-
2011, and Millat-2011) were exposed to two concentrations of foliar SA (Control, SA @ 100
mg/L). The results showed that salt and drought conditions significantly affected all growth traits,
pigments, and gas exchange parameters (p0.01), and that foliar SA treatment greatly attenuated
the adverse effects of stresses and improved the attributes under study in all wheat cultivars.
Chlorophyll a (2.31 mg g-1 FW), Chlorophyll b (3.24 mg g-1 FW), and Total Chlorophyll Content
were all higher in SA-treated plants under both types of stresses (5.55 mg g-1 FW). Glaxy-2013
observed the highest photosynthesis rate (5.33 M CO2 m-2s-1), transpiration rate (3.47 mM H2O
m-2 s-1), and stomatal conductance under foliar applied SA (100 mg/L) (0.85 mM H2O m-2 s-1).
Finally, when it came to growth traits under salt and drought stress, the wheat cultivar Glaxy-
2013 fared better than any other cultivar.
Lou et al. (2021) examined that trehalose mitigate the salinity stress in wheat. Trehalose is
a non-reducing disaccharide that has been linked to controlling how plants react to different
stresses. The aim of this study was to analyse the biochemical signaling pathway of exogenous
trehalose in order to improve wheat heat tolerance using RNA-sequencing technologies and
physiological data. According to physiological data and RNA-seq, trehalose decreased the levels
of malondialdehyde and relative conductivity in wheat roots, changing the pathways for the
synthesis of phenylpropane, the metabolism of starch and sucrose, the metabolism of glutathione,
14
and other processes. Our research showed that exogenous trehalose, through recoding general
gene expression, coordinates the effect of wheat on heat tolerance by reducing oxidative damage
brought on by high temperatures. However, following trehalose pretreatment, two wheat
cultivars responded differentially to high temperatures. This research demonstrated the impact of
trehalose on the control of gene expression in wheat roots during high-temperature stress,
providing a foundation for further study.
Zhang et al. (2022) determined the importance of trehalose in Maize and wheat seedlings
under high temperatures. The goal of this research is to figure out how exogenous trehalose
improves maize and heat stress responses. Exogenously supplied trehalose upregulated the
transcript levels of key enzymes small subunit in maize seedlings. It was also able to improve the
activities of C4 pathway enzymes like Ribulose-1,5-bisphosphate carboxylase, PEPC, NADP-
MDH, and NADP-ME. Exogenously applied trehalose stimulated photosynthetic carbon
assimilation in wheat seedlings by increasing RUBPCase activity. Exogenous trehalose increased
photosynthetic carbon absorption capabilities in heat-stressed maize and wheat seedlings via
distinct pathways, according to these findings.
Kunika et al. (2019) studied that Potassium transporter (HKT) genes mitigate the salt
stress in wheat. Salinity One of the most frequent elements that affects plant development and
output is stress. Physiological and morphological features are both significantly influenced by
salinity. Examples of morphological attributes include things like root length, plant height, and
leaf count, whereas physiological qualities include things like photosynthesis, water relations,
and chlorophyll content. Because plants are less tolerant of salinity, saline land is not suiTable
for agriculture. Plant development is divided into two phases in response to salinity: rapid and
slower phase.Young leaves are inhibited in the quick (osmotic) phase, but adult leaves senesce in
the slower (ionic) phase. Osmotic stress tolerance, Na+ or Cl- exclusion, and tissue tolerance to
accumulated Na+ and Cl- are the three main forms of plant adaptations to salt. Overall control of
Na+ buildup and osmotic stress tolerance, is a biochemical mystery. However, molecular
genetics and functional genomics offer a fantastic chance to combine molecular and
physiological information in order to improve plant salinity resistance
15
CHAPTER NO. 3
MATERIALS AND METHODS
Present study was performed to investigate the importance of trehalose to minimize the
salt stress on wheat. Experiment was carried out in the Botanical Garden, University of
Education Lahore, Faisalabad Campus. The experiment was performed by using two varieties of
wheat i.e., Uqaab 2000 and Galaxy 2013 and three salinity stress conditions i.e., 0mM, 150mM
and 200mM. Seeds were collected from Department of Ayub Agriculture Research Institute
(AARI), Faisalabad.
The experiment with three replicates for each treatment was completely randomized. Mix
soil and sand 2:1 v/v and filled the 36 pots for the whole experiment. The size of plastic pots
used for sowing of seed was 14cm diameter and filled with 1.8kg of thoroughly sandy soil.
3.1. Surface sterilization of seed
For presowing treatments, seeds were washed with water and then put in beaker
containing ethanol (30%) for 30 minutes. This was done for both varieties. After 30 minutes,
seeds were taken out and washed with distilled water.
3.2. Sowing of seed
Seeds were sown in pots after surface sterilizing. Ten seeds were sown per pot. The sowing of
seeds was done on 22nd February, 2022.
Figure 2.1: Sowing of seeds

16
3.3. Seed Germination
Seeds germination was started on 28th Feb, 2022 and finalized the germination process in
almost 5-6 days.
3.4. Application of salinity stress
Salinity stress was applied on 2 leaf stage. Each variety replicates was divided into two
groups. For group A dose of 150mM of salinity was applied to 6 pots of both varieties and same
was done for 200mM stress. For group B distilled water was applied.
3.5. Hoagland Solution
Hoagland solution was applied to fulfill nutritional need of plants. About 70 ml of

200mM the solution was applied to each pot of both varieties on 4 leaf stage.
3.6. Trehalose application
Foliar application of trehalose was applied with 10mM concentration to 3 pots from each
stress level of both varieties on 5 leaf stage. Foliar application of distilled water was applied to
remaining pots.
3.7. Harvesting
Harvesting of all the pots was done of both varieties on 7 leaf stage after 28 days of germination.
The plants were then taken for further parameters. One plant from each of the three replicates
(pots) of all treatments was taken. Rest of the plants were stored in air tight zipper bag in freezer
for measuring following attributes:
Figure: Harvesting of Plants

17
3.8. Growth Parameters
The following attributes of growth were analyzed:
3.8.1. Shoot and Roots Fresh Weight (g)
The fresh weight of roots and shoots were recorded immediately after uprooting the plants with
electronic weighing balance (DM-305F).
3.8.2. Plant Height (cm)
Total height of each plant was measured with the help of a measuring tape.
Figure: Measuring of plant height
3.8.3. Shoot and Root Length (cm)
Roots and shoots were separated and measured their length with the help of a measuring tape,
and their data were recorded separately.
3.8.4. Shoot and Root Dry Weight (g)
After calculating the fresh weight of roots and shoots, plants were dried in brown envelopes for
two weeks at room temperature. Their dry weight calculating by using Electronic Weighing
Balance (DM series, DM-305F).
3.9. Moisture Content
Moisture content of shoot and root was recorded by subtracting the dry weight (g) from fresh
weight (g).
Moisture contents were determined following the equation given by Enders and Lehmann, 2017.
18
𝐹𝑟𝑒𝑠ℎ 𝑤𝑒𝑖𝑔ℎ𝑡−𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡

Moisture contents (%) = × 100
𝐹𝑟𝑒𝑠ℎ 𝑊𝑒𝑖𝑔ℎ𝑡
3.10. Electrolyte Conductivity (µS)
Leaves of 0.5g weight of plants per replicates of all treatments was taken, grind them into small
pieces and was added in the test tube. Then added 10ml distilled water in the test tube, and left it
overnight.
EC-1 (uS) was finded with the help of an EC meter and record their values. Now put the test
tubes in a burner bath containing water at 80oC for 20 minutes. After heating, find the EC-2 (uS)
and record their values (Dionisio-Sese and tobita, 1998).
Then final value of EC was recorded using the formula:

𝐸𝐶−1
Electrolyte Conductivity = × 100
𝐸𝐶−2
Figure: measuring of Electrolyte conductivity by EC meter
3.11. Chlorophyll Contents
Chlorophyll contents of leaves were examined by using Arnon (1949) and Davis (1946) method.
0.5g of leaves per replicates of all treatments was taken,grind them into small pieces and was
added in the glass test tube. Then added these pieces into 10ml of 70% ethanol solution in small
plastic bottles and left it overnight. Samples were centrifuged and supernatant was added in
Falcon tubes for an analysis of chlorophyll content. 70% ethanol solution was used as reference
in spectrophotometer. Put the samples one by one into Cuvette and then adjusted the ultraviolet
UV-VIS visual Beam at three wavelengths i.e., 480nm, 645nm and 663 nm for chlorophyll
19
analysis. Recorded their values and find out chlorophyll contents i.e., total chlorophyll,
chlorophyll a, chlorophyll b and chlorophyll a/b.
3.12. Statistical Analysis
Appropriate statistical software was used for analysis of variance of obtained data. A factorial
with three replications and three factors (variety, salinity stress, and trehalose), The CoStat
version Cohort 6.303 was used to perform an ANOVA. P ratios and significance levels are
presented in Tables and described in the findings section for various probabilities (0.05, 0.01,
and 0.001). The graphs were created using an Excel file.
20
CHAPTER NO.4
Results and Discussion
4.1. Whole plant length
The result showed that plant length was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Plant length was highly significantly decreased by NaCl (200mM) as
compared to control. A significantly increased was observed by foliar application of trehalose
(Figure 4.1). A significant difference was observed between stress and treatment while Non-
significant difference was observed between varieties and interaction between stress and
treatment, stress and variety, treatment and variety and stress, treatment and variety (Table 4.1).
The height of the plants significantly decreased as NaCl concentrations rose. Numerous plant
species have reported height reduction due to salinity stress, which has been primarily linked to
ion toxicity and osmotic stress, which slow down plant growth. Dune spinach's growth was
significantly hampered by high salt concentrations. This has been documented in multiple
halophyte investigations, where increased salinity has a negative effect on plant growth, resulting
in a decrease in biomass, leaf number, and plant height (Rahneshan et al.,2018, Guo et al.,2018,
Orlovsky et al.,2016). Tian et al.,2020) further stated that prolonged salt availability in the root
zone limits flow of water and plant nutrients, inflicting direct damage to cells through the
formation of harmful ions, which results in drop of plant growth. Trehalose treatment increased
all growth indicators in both wheat varieties. Similar findings have been stated by Shahbaz et
al.,(2017) who found that exogenous trehalose treatment minimized the harmful effects of
salinity on growth characteristics in rice plants.
Table 4.1: Variance analyzing for plant length of wheat grown under salinity treated with
exogenous application of trehalose
Source of variation Df P
NaCl 2 0.413*
Trehalose (T) 1 0.0000*
Variety (var) 1 0.2612 ns
NaCl×trehalose 2 0.2670 ns
NaCl×Variety 2 0.1338 ns
Tehalose×Variety 1 0.9975 ns
NaCl×trehalose×variety 2 0.7214 ns
ns= Non-Significant *, ** and *** significant, very significant and highly significant respectively
21
70
0mM 10mM
60
Plant length (cm)

50
40
30
20
10
0
Control NaCl NaCl Control NaCl NaCl
150mM 200mM 150mM 200mM
Uqaab 2000 Galaxy 2013
Treatments
Figure 4.1:Influence of foliar application of trehalose on plant length of wheat plant grown under
control and salinity stress
22
4.2. Shoot Length
The result indicated that shoot length was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Shoot length was highly significantly decreased by NaCl (200mM)
as compared to control. A significantly increased was observed by foliar application of trehalose
(Fig. 4.2). Highly significant difference was perceived between stress and treatment while non-
significant difference was observed in interaction between stress and variety and stress, treatment
and variety (Table 4.2). Earlier research, which found that salinity significantly decreased the
shoot length of Sesamum indicum (Koca et al., 2007), Vigna radiate (Saha et al., 2010), Cassia
angustifolia (Agarwal et al.2004), cabbage, sugar beet, Paniculate amaranth, Pak-choi (Jamil et
al., 2006), and Pisum sativum (Noreen et al., 2009), as well as Vigna radiate, Pak-choi, and
Paniculate amaranth. High salinity can induce water imbalances that might hinder osmotic
adjustment and inhibit plant development (Bohnert et al.1996). Trehalose treatment increased
shoot length in both wheat varieties.
Table 4.2: Variance for analyzing shoot length of wheat grown under salinity treated with
NaCl 2 0.0004 ***
Trehalose (T) 1 0.0000 ***
Variety (var) 1 0.0042 **
NaCl×trehalose 2 0.0119 *
Tehalose×Variety 1 0.0342 *
23
40
0mM 10mM
35
Shoot length (cm)

30
25
20
15
10
5
0
150mM 200mM 150mM 200mM
varieties
Figure 4.2: Influence of foliar application of trehalose on shoot length of wheat plant grown
under control and salinity stress
24
4.3. Root Length
The result showed that root Length was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Root Length was highly significantly decreased by NaCl (200mM) as
compared to control. A significantly increased was observed by foliar application of
trehalose(Fig. 4.3).Very significant alteration was observed between treatment while non
significant difference was observed between stress and variety and interaction between stress
and treatment, stress and variety, stress and variety and stress ,treatment and variety(Table 4.3).
Higher salt concentrations in the root zone cause a number of reactions, including root density,
root turgor pressure, and its development, as well as preventing water absorption (Maggio et al.
2004). Salinity of the nutrient solution has a significant impact on spinach and lettuce's
vegetative growth and root development (Kaya et al. 2001).
Table 4.3: Variance for analyzing root length of wheat grown under salinity treated with
NaCl 2 0.5594 ns
Trehalose (T) 1 0.0054 **
25
35
0mM 10mM
30
Root length (cm)

25
20
15
10
5
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.3: Influence of foliar application of trehalose on root length of wheat plant grown under
26
4.4. Plant Fresh Weight
The result showed that Plant Fresh Weight was significantly greater in wheat variety Uqaab 2000
as compared to Galaxy 2013. Plant Fresh Weight was highly significantly decreased by NaCl
(200mM) as compared to control. A significantly increased was observed by foliar application of
trehalose (Fig. 4.4). High significant difference was observed between treatment while non-
significant difference was observed between stress and varieties and interaction between stress
and variety, treatment and variety and stress, treatment and variety (Table 4.4). In this regard,
Azooz et al. (2009) observed that salinity stress decreased three cultivars of maize (Zea mays
L.growth )'s characteristics, particularly the fresh and dry weight of both shoot and root.
Additionally, El-Bassiouny and Bekheta (2005) discovered reductions in both fresh and dry
masses as a result of salt stress while working on a wheat plant. This reduction could be the
result of disturbed metabolic processes brought on by a reduction in water adsorption and/or
water balance. Furthermore, salinity stress is the only factor contributing to the rise in salt ion
concentrations. By reducing cellular osmotic potential, plants use osmotic adjustment systems to
counteract the osmotic impact. Our results provide significant support for past research that
shown the beneficial benefits of exogenous trehalose administration on crop development and
production (Zeid, 2009). This effect may be explained by the administration of trehalose, which
induces specific enzymes involved in the buildup of store carbohydrates in photosynthetic tissues
(Wingler et al.2012).
Table 4.4: Variance for analyzing plant fresh weight of heat grown under salinity treated with
Source of variation df P
NaCl 2 0.0872 ns
Trehalose (T) 1 0.0000 ***
NaCl×trehalose 2 0.0151 *
27
1.6
0mM 10mM
1.4
Plant Fresh Weight (g)

1.2
1
0.8
0.6
0.4
0.2
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.4: Influence of foliar application of trehalose on plant fresh weight of wheat plant
grown under control and salinity stress
28
4.5. Shoot Fresh Weight
The result showed that shoot fresh weight was significantly greater in wheat variety Uqaab 2000
as compared to Galaxy 2013.Shoot Fresh Weight was highly significantly decreased by NaCl
trehalose (Fig. 4.5). Highly significant difference was observed between stress and treatment
while non-significant difference was observed in interaction between stress and variety and stress,
treatment and variety (Table 4.5). Earlier research, which found that salinity significantly
decreased the fresh shoot weight and length of Vigna radiate (Saha et al.2010), Cassia
angustifolia (Agarwal et al.2004), cabbage, sugar beet, Paniculate amaranth, Pak-choi (Jamil et
al. 2006), as well as Vigna radiate, Pak-choi, and Paniculate amaranth. High salinity can induce
water imbalances that might hinder osmotic adjustment and inhibit plant development (Bohnert
et al.1996). Tre treatment gave plants that were under salt stress a better shoot fresh weight than
salt stress management.This effect may be explained by the administration of trehalose, which
induces specific enzymes involved in the buildup of store carbohydrates in photosynthetic tissues
(Wingler et al., 2012).
Table 4.5: Variance for analyzing shoot fresh weight of wheat grown under salinity treated with
NaCl 2 0.0720 ns
Trehalose (T) 1 0.0000 ***
Variety (var) 1 0.0381 *
NaCl×trehalose 2 0.0098 **
29
1.4 0mM 10mM
1.2
Shoot Fresh Weight (g) 1
0.8
0.6
0.4
0.2
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.5: Influence of foliar application of trehalose on shoot fresh weight of wheat plant
30
4.6. Root Fresh Weight
The result showed that Root Fresh Weight was significantly greater in wheat variety Uqaab 2000
as compared to Galaxy 2013.Root Fresh Weight was highly significantly decreased by NaCl
trehalose (Fig. 4.6). Highly significant difference was observed between stress and treatment
while non-significant difference between variety and interactions i.e., between stress and
treatment, stress and variety, variety and treatment and stress, treatment and variety (Table 4.6).
Salinity, which causes water stress and ultimately has a detrimental impact on photosynthesis as
well as the breakdown in assimilates translocation or the photosynthetic process, may be the
cause of the decrease in root dry matter (Satti and Lopez 1994). Trehalose treatment gave plants
that were under salt stress a better root fresh weight than salt stress management. This effect may
be explained by the administration of trehalose, which induces specific enzymes involved in the
buildup of store carbohydrates in photosynthetic tissues (Wingler et al., 2012).
Table 4.6: Variance for analyzing root fresh weight of wheat grown under salinity treated with
NaCl 2 0.0007 ***
Trehalose (T) 1 0.0000 ***
31
0.35 0mM 10mM

0.3
Root Fresh Weight (g)

0.25
0.2
0.15
0.1
0.05
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.6: Influence of foliar application of trehalose on root fresh weight of wheat plant grown
32
4.7. Plant Dry Weight
The result showed that Plant Dry Weight was significantly greater in wheat variety Uqaab 2000
as compared to Galaxy 2013. Plant Dry Weight was highly significantly decreased by NaCl
trehalose (Fig. 4.7). Highly significant difference was perceived between treatment while non-
significant difference between stress and treatment, stress and variety, variety and treatment and
stress, treatment and variety (Table 4.7). It is possible that salinity stress changed the normal
physiological and metabolic processes of the salinity exposed plants in the current study, which
led to a drop in DW, as a result of which biomass of plant in both genotypes reduced as the level
of salinity increased. Munns et al. (2006) and Rahman et al. (2017) both supported the current
study's premise that salinity has harmful impact on plant biomass. Similar to this, Dayiragije and
Lutts (2006) also demonstrated that plants that are vulnerable to salt stress continue to be
underdeveloped as a result of a decrease in cell elongation and devision as well as a restriction
on the synthesis of growth hormones (auxin), all of which result in a decrease in the total
biomass of the impacted plant. Trehalose treatment increased all growth indicators in both wheat
varieties. Similar findings have been stated by Shahbaz et al. (2017) who found that exogenous
trehalose treatment reduces the harmful effects of salinity on growth characteristics in rice plants.
Table 4.7: Variance for analyzing plant dry weight of wheat grown under salinity treated with
NaCl 2 0.0024 **
Trehalose (T) 1 0.0000 ***
33
0.4 0mM 10mM

Plant Dry Weight (g) 0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.7: Influence of foliar application of trehalose on plant dry weight of wheat plant grown
34
4.8: Shoot Dry Weight
The result showed that shoot dry weight was significantly greater in wheat variety Uqaab 2000
as compared to Galaxy 2013. Shoot Dry Weight was highly significantly decreased by
NaCl(200mM) as compared to control. A significantly increased was observed by foliar
application of trehalose(Fig. 4.8).Highly significant difference was preceived between stress and
treatment while non-significant difference in interaction between stress and variety and
stress ,treatment and variety(Table 4.8). It is possible that salinity stress changed the normal
physiological and metabolic processes of the salinity exposed plants in the current study, which
led to a drop in DW, as a result of which biomass of plant in both genotypes reduced as the level
of salinity increased. Munns et al. (2006) and Rahman et al. (2017) both supported the current
study's premise that salinity has harmful impact on plant biomass. Similar to this, Dayiragije and
Lutts (2006) also demonstrated that plants that are vulnerable to salt stress continue to be
underdeveloped as a result of a decrease in cell elongation and devision as well as a restriction
on the synthesis of growth hormones (auxin), all of which result in a decrease in the total
biomass of the impacted plant. Trehalose treatment increased all growth indicators in both wheat
varieties.
Table 4.8: Variance for analyzing shoot dry weight of wheat grown under salinity treated with
NaCl 2 0.2123 ns
Trehalose (T) 1 0.0000 ***
Variety (var) 1 0.0436 *
35
0.3 0mM 10mM
Shoot Dry Weight (g) 0.25
0.2
0.15
0.1
0.05
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.8: Influence of foliar application of trehalose on shoot dry weight of wheat plant grown
36
4.9. Root Dry Weight
The result showed that root dry weight was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Root Dry Weight was highly significantly decreased by NaCl(200mM)
as compared to control. A significantly increased was observed by foliar application of
trehalose(Fig. 4.9).Highly significant difference was preceived between stress ,treatment and
variety while Non-significant difference was observed in interaction between stress and
treatment, stress and variety, variety and treatment and stress, treatment and variety Table 4.9).
Salinity, which causes water stress and ultimately has a detrimental impact on photosynthesis as
well as the breakdown in assimilates translocation or the photosynthetic process, may be the
cause of the decrease in root dry matter (Satti and Lopez 1994). The highest level of tolerance
after which the weight of tomato roots decreases as salinity rises, was seen when salinity reduced
root biomass (Papadopoulos and Rendig 1983). Trehalose treatment increased all growth
indicators in both wheat varieties.
Table 4.9: Variance for analyzing root dry weight of wheat grown under salinity treated with
NaCl 2 0.0000 ***
Trehalose (T) 1 0.0000 ***
Variety (var) 1 0.0006 ***
37
0.25 0mM 10mM
0.2
Root Dry Weight (g)
0.15
0.1
0.05
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.9: Influence of foliar application of trehalose on root dry weight of wheat plant grown
38
4.10. Moisture Content
The result showed that moisture content was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Moisture Content was highly significantly decreased by
application of trehalose (Fig. 4.10). Highly significant difference was perceived between stress
and variety while non-significant difference in interaction between stress and treatment, stress
and variety, variety and treatment and stress, treatment and variety (Table 4.10). It is commonly
accepted that a decrease in RWC results in a decrease in photosynthetic rate. In leaf the drop
relative water content may be caused by root systems or by the inability of the plant to overcome
water loss by reducing the absorbing surface through transpiration when it is under stress
(Gadallah 2000).
Table 4.10: Variance for analysing moisture content of wheat grown under salinity treated with
NaCl 2 0.3719 ns
Trehalose (T) 1 0.0000 ***
39
1.4
0mM 10mM
1.2
Moisture content 1
0.8
0.6
0.4
0.2
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.10: Influence of foliar application of trehalose on moisture content of wheat plant
40
4.11. Electrolyte Conductivity
The result showed that EC (µs) was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. EC (µs) was highly significantly decreased by NaCl(200mM) as
compared to control. A significantly increased was observed by foliar application of
trehalose(Fig. 4.11). Non-significant difference between stress, treatment and variety and
interaction between stress and treatment, stress and variety, variety and treatment and stress,
treatment and variety (Table 4.11). Through the generation of ROS, salt stress typically results in
oxidative stress. Lipid peroxidation caused by increasing ROS is thought to be a possible
biomarkers of salinity induced oxidative damage to membranes that results in electrolyte leakage,
permeability loss, and dysfunctional membrane proteins and ion channels.
Table 4.11: Variance for analyzing EC (µS) of wheat grown under salinity treated with
NaCl 2 0.0676 ns
Trehalose (T) 1 0.7320 ns
41
120
0mM 10mM
100
EC 80
60
40
20
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.11: Influence of foliar application of trehalose on EC (µs) of wheat plant grown under
42
4.12. Chlorophyll a
The result showed that chlorophyll a was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Chlorophyll a was highly significantly decreased by NaCl(200mM)
trehalose(Fig 4.12). Non-significant difference was perceived between stress, treatment and
variety and interaction between stress and treatment, stress and variety, variety and treatment and
stress, treatment and variety (Table 4.12). Salinity reduced the amounts of carotenoid and
chlorophyll a in green gramme seedlings (Yasar et al.2008). Through the production of ROS, a
decline in chlorophyll content causes a drop in excited electrons throughout the photosynthetic
process (Djanaguiraman et al.,2018). Due to the reduction in chlorophyll content, has a
significant impact on photosynthetic processes and frequently has a negative impact on
membrane stability (Seemann et al,1985). A large decline in photosynthesis in plants exposed to
salinity has been demonstrated by other researchers (Kapulnik et al.,1991; Francois et al.,1994).
The amount of total chlorophyll in tomatoes was found to decrease (Tantawy et al.2009) as salt
levels rose (Roy et al., 2014, Mukami et al.,2020). Theerakulpisut and Gunnula (2013) and
Abdelgawad et al., (2014), reported that trehalose therapy improved rice plant photosynthetic
pigments under salinity stress.
Table 4.12: Variance for analyzing chlorophyll a of wheat grown under salinity treated with
NaCl 2 0.1421 ns
43
1
0.9 0mM 10mM
0.8
0.7
0.6
chl a
0.5
0.4
0.3
0.2
0.1
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.12: Influence of foliar application of trehalose on chlorophyll a content of wheat plant
44
4.13. Chlorophyll b
The result showed that chlorophyll b was significantly greater in wheat variety Uqaab 2000 as
compared to Galaxy 2013. Chlorophyll b was highly significantly decreased by NaCl(200mM)
trehalose(Fig. 4.13). A very significant alteration was perceived in interaction between stress and
treatment while Non-significant difference between variety and interaction between treatment
and variety and stress, treatment and variety(Table 4.13). Wheat leaves' concentrations of
chlorophyll b, carotenoids, and total pigment were all dramatically reduced by salinity stress
(Table 4.15). Some pigments are oxidized which harms other photosynthetic pigments and
impairs biosynthesis of pigments, was thought to be the cause of the diminished effect of salinity
on photosynthesis (Rady et al. 2015). The production of proteolytic enzymes like chlorophyllase,
which is in charge of degrading chlorophyll and/or harming the photosynthetic machinery, may
be the cause of these diminished salinity effects (Sadak and Ahmed 2016). The restrictive impact
of salinity induced stress on the chlorophyll content and photosynthetic activity could be
responsible for the reduction in growth criterion as a result of lowering the anabolic processes
caused by salinity stress. The photosynthetic pigments in stressed and control leaves responded
favourably to trehalose treatment.
Table 4.13: Variance for analyzing chlorophyll b of wheat grown under salinity treated with
NaCl 2 0.0178 *
Trehalose (T) 1 0.0225 *
NaCl×trehalose 2 0.0072 **
NaCl×Variety 2 0.0399 *
45
0.25
0mM 10mM
0.2
chl b 0.15
0.1
0.05
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.13: Influence of foliar application of trehalose on chlorophyll b content of wheat plant
46
4.14. Total Chlorophyll
The result indicated that total chlorophyll was significantly greater in wheat variety Uqaab 2000
as compared to Galaxy 2013. Total chlorophyll was highly significantly decreased by NaCl
(200mM) as compared to control. A significantly increased was observed by foliar application
of trehalose (Fig. 4.14) while Non-significant difference between stress, treatment and variety
and interaction between stress and treatment, stress and variety, variety and treatment and stress,
treatment and variety (Table 4.14). According to the findings, the chlorophyll concentration was
minor in the saline-stressed plants than it was in the corresponding unstressed plants. Lower
overall chlorophyll concentration may be caused by increased chlorophyllase enzyme activity
and decreased N absorption under salt stress (Van Hoorn et al., 2001). ROS degradation may be
the primary cause of the decrease in chlorophyll concentration (Sharifi et al., 2016).
Table 4.14: Variance for analyzing total chlorophyll of wheat grown under salinity treated with
NaCl 2 0.1528 ns
47
1.4
0mM 10mM
1.2
1
total chlorophyll
0.8
0.6
0.4
0.2
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.14: Influence of foliar application of trehalose on total chlorophyll contents of wheat
plant grown under control and salinity stress
48
4.15. Chlorophyll a/b
The result indicated that chlorophyll a/b was significantly greater in wheat variety Galaxy 2013
as compared to Uqaab 2000. Chlorophyll a/b was highly significantly decreased by
application of trehalose(Fig. 4.15). A significant difference was observed in stress while Non-
significant difference was observed between treatment and variety and interaction between stress
and treatment, stress and variety, variety and treatment and stress, treatment and variety (Table
4.15). In leaves, concentrations of chlorophyll a/b, carotenoids, and total pigment were all
dramatically reduced by salinity stress (Table 4.15).
Table 4.15: Variance for analyzing chlorophyll a/b of wheat grown under salinity treated with
NaCl 2 0.0397 *
49
8
7
6
Chl a/b 5
4
3
0mM
2
10mM
1
0
150mM 200mM 150mM 200mM
Treatments
Figure 4.15: Influence of foliar application of trehalose on chlorophyll a/b content of wheat
plant grown under control and salinity stress

Rehman Thesis

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1

Wiggers and others discovered trehalose, a non-reducing disaccharide, in organisms in

As a result, trehalose's numerous metabolic actions could be attributed to its participation

demonstrated promising results in crop movement. Lower concentrations of exogenous trehalose,

Trehalose, to put it simply, is a natural disaccharide that is adaptable and functions as a

MATERIALS AND METHODS

3.1. Surface sterilization of seed

3.2. Sowing of seed

Figure 2.1: Sowing of seeds

3.3. Seed Germination

3.4. Application of salinity stress

3.5. Hoagland Solution

Hoagland solution was applied to fulfill nutritional need of plants. About 70 ml of

3.6. Trehalose application

Figure: Harvesting of Plants

3.8. Growth Parameters

The following attributes of growth were analyzed:

3.8.1. Shoot and Roots Fresh Weight (g)

3.8.2. Plant Height (cm)

Figure: Measuring of plant height

3.8.3. Shoot and Root Length (cm)

3.8.4. Shoot and Root Dry Weight (g)

3.9. Moisture Content

𝐹𝑟𝑒𝑠ℎ 𝑤𝑒𝑖𝑔ℎ𝑡−𝐷𝑟𝑦 𝑤𝑒𝑖𝑔ℎ𝑡

3.10. Electrolyte Conductivity (µS)

Then final value of EC was recorded using the formula:

Figure: measuring of Electrolyte conductivity by EC meter

3.11. Chlorophyll Contents

3.12. Statistical Analysis

Trehalose (T) 1 0.0000*

Variety (var) 1 0.2612 ns

Plant length (cm)

4.2. Shoot Length

NaCl 2 0.0004 ***

Trehalose (T) 1 0.0000 ***

Variety (var) 1 0.0042 **

Shoot length (cm)

4.3. Root Length

Trehalose (T) 1 0.0054 **

Variety (var) 1 0.7432 ns

Root length (cm)

4.4. Plant Fresh Weight

Trehalose (T) 1 0.0000 ***

Variety (var) 1 0.1037 ns

Plant Fresh Weight (g)

4.5. Shoot Fresh Weight

Trehalose (T) 1 0.0000 ***

Variety (var) 1 0.0381 *

1.4 0mM 10mM

4.6. Root Fresh Weight

NaCl 2 0.0007 ***

Trehalose (T) 1 0.0000 ***

Variety (var) 1 0.2548 ns

0.35 0mM 10mM

Root Fresh Weight (g)

4.7. Plant Dry Weight

Trehalose (T) 1 0.0000 ***

Variety (var) 1 0.8633 ns

0.4 0mM 10mM

4.8: Shoot Dry Weight

Trehalose (T) 1 0.0000 ***

Variety (var) 1 0.0436 *