FINAL REPORT

By

Ayesha Aziz FA19C1BA407

Muhammad Asim FA19C1BA
Muhammad Sajid Iqbal FA19C1BA313
Session 2019-2023
Supervised By

Dr. Abdul Rehman Sb

A research project submitted in partial fulfillment

Of the requirement for the degree of

BACHELOR OF SCIENCE (HONS.)

DEPARTMENT OF AGRONOMY
Faculty of Agriculture & Environmental Science

The Islamia University of Bahawalpur

In The Name of Allah,
The most Beneficent,
The Most Merciful,
 DECLARATION

I hereby declare that the research work entitled ……….is the product of our on work and
no part has been copied from any published source (except the references, standard
mathematical or genetic models/ equations/formulas/protocols, etc.). We further declare
that this work has not been submitted for the granting of any diploma/degree. If the
information submitted is determined to be inaccurate at any time, the institution may
take action. In case of any fault, the scholar shall be prosecuted by HEC's plagiarism
policy.

Ayesha Aziz.
Muhammad Asim.
Muhammad Sajid Iqbal.
 ACKNOWLEDGMENT

Above all, it is our responsibility to convey our extreme regard to the Most Merciful and the
Most Beneficent, ALLAH Almighty, who always awards us with His blessings and power to
achieve this duty, we also appeal to our extreme love to the Holy Prophet Hazrat Muhammad
(PBUH), the considerable social reformer and a touch of guidance for the whole humanity. I take
this opportunity to express our gratitude to the people who have been instrumental in the
successful completion of our research. A part from our effort, there are many other reasons that
depend on the success of students by providing them opportunity to gain knowledge and use this
in there practice and the success of this training largely depends on the encouragement and
guideline of many others. I take this opportunity to express our gratitude to the people who have
been instrumental in the successful completion of our research.
I express my sincere and deep sense of gratitude to our respected Supervisor Dr. Abdul
Rehman (Assistant professor) from the Department of Agronomy, for his dynamic supervision,
skillful guidance, and valuable comments, scholastic and constructive suggestions throughout the
session with him. This scholarly endeavor would have been futile without the cooperative
attitude and constructive minded and aluminous guidance of respected Dr. Aown Sammar Raza
(Chairman Department of Agronomy). Also thankful to laboratory staff those help us in
instrumental work in different purpose of our research work.

Ayesha Aziz.
Muhammad Asim.
Muhammad Sajid Iqbal.
 Dedication

This work is dedicated to my beloved Father, who taught me the best kind of
knowledge to have that which is learned for its own sake and devoted his whole
life to my study (I love u so much baba). It is also dedicated to my mother, who
taught me that even the largest task can be accomplished if it done on at a time.
 Table of Contents

Item Description Page

Declaration

Acknowledgement

Abstract

Chapter 1 Introduction

Chapter 2 Review and Literature

Chapter 3 Material and Methods

 Chapter 1

Introduction

Maize (Zea Mays. L.) is a C4 plant that enjoys a vital position in the existing cropping systems
of Pakistan. It ranks third after wheat and rice in Pakistan for its grain production. Maize is
grown in almost all the provinces of the country, but Punjab and KPK are the main areas of
production. Maize is one of the major Kharif cereal crops in Pakistan holds an area under
cultivation of 1083 thousand hectares and showed a 15.2 percent positive production change last
year (Agri. Statistics, 2010). Production of Maize has increased from 1185 thousand tons during
1990-91 to 4271 thousand tons in 2011-12 (GOP, 2012). The Production shows an increase of
13.7 percent over the previous year, which is due to a corresponding increase in area, favorable
weather conditions, and more area converted by Hybrid varieties. The average yield productivity
of Maize is 3944 kg/ha, which is the highest among all cereals grown in the country. Maize
contributes 4.88% of the value-added crops which hold 64.6% of all major crops which is the
third highest share after wheat and rice (GOP, 2012). Maize accounts for 4.8% of the total
cropped area and 3.5% of the value of agricultural output. The upward trend in yield is mostly
due to the adaptation of high-yielding varieties and hybrids, which is likely to continue (GOP,
2011).

It is not only consumed by human beings in the form of food grain but it is also used as
feed for livestock and poultry besides being a good scavenge crop. It is also gaining importance
due to being a commercial crop, where a large number of products are being manufactured out of
its grain. The chemical components of maize include carbohydrates, protein, fats, moisture
content, and crude fiber. Carbohydrates are the major chemical component of maize grains. The
second largest component is a protein. The crude fiber was found fourth largest chemical present
in maize grains. Maize grain contains about 72% starch, 10% protein, 4.8% oil, 5.8 % fiber,
3.0% sugar, and 1.7% ash (Chaudhary), 1993. Its grains are important for the production of oil,
starch, and glucose ( Krishnamurthi, 1969; Niaz and Dawar, 2009).

It is a staple food for millions of people around the globe, particularly in sub-Saharan
Africa, Latin America, and parts of Asia. It is an important food source for millions of people,
especially in developing countries. By producing maize, farmers can help ensure that local
populations have access to nutritious food and improve their food security. It is a highly valuable
crop that can be sold for a profit in local and international markets. Maize farming can provide a
source of income for farmers, creating jobs and boosting local economies. It is used as feed for
animals like cows, pigs, and chickens. This makes maize production important for the livestock
industry, which is crucial for many rural communities. It is a potential source of biomass, which
can be converted into biofuels such as ethanol. This makes maize an important crop for countries
seeking to reduce their dependence on fossil fuels. It has cultural significance in many countries,
particularly in indigenous communities in the Americas. It is an important part of traditional
cuisines and is often used in cultural ceremonies. Overall, maize production has significant
impacts on food security, economic development, and cultural practices around the world.

Drought stress is one of the most important environmental factors in the reduction
of growth, development, and production of plants. It can be said that it is one of the most
devastating environmental stresses. Iran, with an annual rainfall of 240 mm, is classified as one
of those dry regions (Jajarmi, 2009). According to Hayat and Ali (2004), Moisture stress is a
limiting factor for crop growth in arid and semi-arid regions due to low and uncertain
precipitation. Water stress due to drought is probably the most significant abiotic factor limiting
plant and also crop growth and development (Hartmann et al., 2005). Maize plays a great role in
human nutrition (20-25%) (Emam, 2004), Water shortage is a critical problem limiting maize
growth through its impact on anatomical, morphological, physiological, and biochemical
processes. Drought is one of three abiotic factors, most responsible for limiting maize production
and productivity in the developing world other two are the problems of waterlogging and low
soil fertility (Zaidi, 2002).The severity of drought damage depends on stress duration and crop
growth stage (Setter et al., 2001). Drought occurs when moisture around the roots is so reduced
that a plant is not able to absorb enough water, or in other words with transpiration of water
absorption (Benjamin, 2007).

Salt stress can significantly affect maize production and yield. Maize is a crop that
is highly sensitive to salt stress, and high soil salinity can cause a reduction in its growth,
development, and productivity. salt stress has become a serious threat to crop production in arid
and semi-arid regions of the world (Flowers and Yeo 1995; Munns 2002). Although the general
perception is that salinization only occurs in arid and semi-arid regions, no climatic zone is free
from this problem (Rengasamy 2006). More than 800 million hectares of land worldwide are
affected by either salinity (397 million hectares) or sodicity (434 million hectares) (FAO 2005;
Munns 2005). Seed germination is the most critical stage in seedling establishment which
determines the success of crop production on salt-affected soils. Generally, salt stress during
germination delays the start reduces the rate, and enhances the dispersion of germination events
(Ashraf and Foolad 2005; Farsiani and Ghobadi 2009). Germination and early seedling growth
are more sensitive to salinity than later developmental stages (Goldsworthy 1994). Salt stress
influences seed germination primarily by sufficiently lowering the osmotic potential of the soil
solution to retard water absorption by seeds, by causing sodium and/or chloride toxicity to the
embryo, or by altering protein synthesis. Hyper-osmotic stress and toxic effects of sodium and
chloride ions on germinating seeds in a saline environment may delay or inhibit germination
(Hasegawa et al. 2000; Khaje-Hosseini et al. 2003; Farsiani and Ghobadi 2009). However, in
maize, it is sodium toxicity and not chloride toxicity that is the major problem in the second
phase of salt stress.

Maize is an important crop that requires proper management strategies to achieve

optimal growth and yield. One key strategy is soil preparation, which involves removing weeds,
loosening the soil, and adding organic matter. Fertilization is also essential, and a balanced
fertilizer program that includes nitrogen, phosphorus, and potassium is recommended. Planting
density should be carefully considered to avoid competition among plants, and irrigation may be
necessary to supplement rainfall. Pest and disease management practices, such as crop rotation
and timely application of pesticides, can help prevent or reduce damage. Harvesting should be
done when the kernels are mature and dry to avoid yield losses. In addition to these strategies,
priming can be employed to improve germination and seedling vigor.

Seed priming is the control hydration of seeds in water or a solution of low osmotic
potential to initiate the germination metabolism without radical protrusion. Many studies have
reported that seed priming improves the stand establishment and productivity of field crops. Seed
priming is a very promising, efficient, and low-cost approach to increasing the germination,
growth, as well as productive capacity of crops. Water, inorganic salts, sugars, solid medium
with water and nutrients, beneficial microbes, micronutrients, hormones, rhizobacteria, and
organic sources are used as priming agents for seeds. Seed priming is not only  used for
improving plant growth and yield but it also increases abiotic stress tolerance.

Seed priming technique has been practiced in many countries including Pakistan,
China, and Australia and more than a thousand trials had been conducted to evaluate the
performance of priming in a variety of crops. Fifty-three farmers tested maize seed priming in
the kharif season in 1996 in tribal areas of Rajasthan, Gujarat, and Madhya Pradesh; India
(Harris et al., 1999). Almost all farmers thought that primed crops grew more vigorously,
flowered and matured earlier, and produced bigger cobs and higher yields. Abnormal condition
and also developed resistance to stress conditions (Pouramir Dashtmian and Khajeh Hosseini,
2014). Environmental stresses, such as salinity, drought, and disease induce changes in gene
expression and enzyme activities in crop plants which reduce growth and productivity.

Compatible osmolytes such as proline have been thought to accumulate in plants

under stressful environments (Kalsoom et al., 2016). Proline induces salt stress tolerance in crop
plants by regulating gas exchange characteristics (Ali et al., 2007), chlorophyll fluorescence
(Deivanai et al., 2011), enhances antioxidant defense system (Ben Rejeb et al., 2014), protects
protein turnover machinery and up-regulates stress-protective proteins (Ismail, 2014). Proline
concentration has been shown to prove effective in inducing salt stress tolerance in various
studies. Pre-sowing seed treatment with different concentrations of proline particularly 10 mM
increased salinity stress tolerance in tomatoes (Kaur & Gupta, 2018). However, the effect of
proline on different genotypes of the same species may vary significantly (Garg, 2003). It has
been reported that seed treatment with some chemicals can improve abiotic stress tolerance in
maize plants (Anosheh et al., 2011). Due to protective functions against abiotic stresses proline
has been used exogenously as a foliar spray.

Ascorbic acid, also known as Vitamin C, is an essential nutrient for plant growth
and development. Researchers have studied the role of ascorbic acid in maize (corn) and have
found several benefits. the role of ascorbic acid in maize plants is vital for their growth,
development, and stress tolerance. Researchers continue to study the effects of ascorbic acid on
maize plants to develop new strategies for improving crop productivity and sustainability. The
importance and changes in ascorbic acid (AsA) levels in plant cells in response to varying
environmental stress conditions are well established (Noctor and Foyer, 1998; Venkatesh and
Park, 2014). Functionally, it is an essential metabolite that operates as an antioxidant and acts as
a significant protagonist of several plant species for salt tolerance (Hameed et al., 2012; Ozgur et
al., 2013). Actually, it associates with H2O2 metabolism as well as lipid hydroxyl peroxidase
and also reacts with various sorts of biotic actions in plants as a patron or receptor in electron
transportation system and also as an enzyme co-factor (Conklin, 2001), this approach effectively
minimizes the stress impact of salinity and encourage the plant growth.

Iron sulfate, also known as ferrous sulfate, has been studied by researchers for its role
in promoting the growth and development of maize. Maize is an important crop for human and
animal consumption, and iron is an essential nutrient for its growth and development. Iron sulfate
is often added to soil to correct iron deficiencies that can cause chlorosis and other growth
problems in maize. Iron sulfate can significantly increase the yield of maize crops, particularly
in soils with low levels of available iron. Iron sulfate supplementation has been shown to
increase the number of maize ears per plant, as well as the number of kernels per ear. In addition,
iron sulfate has been found to increase the concentration of essential nutrients, such as
phosphorus and nitrogen, in maize plants. Iron sulfate has also been found to improve the quality
of maize crops. Studies have shown that iron sulfate supplementation can increase the levels of
protein and amino acids in maize kernels, which are important for human and animal
consumption. In addition, iron sulfate has been found to improve the taste and aroma of maize,
making it more appealing to consumers. Overall, the role of iron sulfate in maize is to promote
growth and development, increase yield, and improve the quality of the crop.

This study is carried out to check the impact of seed priming with proline, ascorbic acid, and
iron sulfate on maize germination rate or other biochemical traits. Nowadays, salinity in the soil
is a major issue, so in order to check the impacts of proline, ascorbic acid, and iron sulfate on
maize plants against salinity by providing salt stress.
Chapter 2

Review of Literature

2.1 Global Trends in Maize Production:

Maize (Zea Mays L.; corn) has played an increasingly diverse role in global agri-food systems.
Global maize production has surged in the past few decades, propelled by rising demand and a
combination of technological advances, yield increases, and area expansion. Maize was
domesticated more than 9,000 years ago in southern Mexico/Meso America (Awika, 2011;
Kennett et al., 2020), maize has quickly disseminated across the globe since then and has become
the leading global staple cereal in terms of annual production exceeding 1 billion metric tons
(García-Lara & SernaSaldivar, 2019).

The global maize area (for dry grain) amounts to 197 Mha, including substantive
areas in sub-Saharan Africa (SSA), Asia, and Latin America (FAOStat, 2021). It is an
established and important human food crop in a number of countries, especially in SSA, Latin
America, and a few countries in Asia, where maize consumed as human food contributes over
20% of food calories (Shiferaw et al., 2011). Compared to wheat and rice, maize is a more
versatile multi-purpose crop. In the developed economies it is primarily used as a livestock feed
crop with a varied role as an industrial and energy crop. With economic development (including
income growth and urbanization), the consumption of animal-source foods is accelerating and
propelling the demand for maize as feed, Asia is a prime example (Erenstein, 2010). Maize
thereby plays a diverse and dynamic role in global agri-food systems and food/nutrition security
(Grote et al., 2021; Poole et al., 2021; Ranum et al., 2014; Shiferaw et al., 2011). There has been
an increased interest in agri-food systems over the last decade (Brouwer et al., 2020; Fanzo et al.,
2021; HLPE, 2017; IFAD, 2021; Townsend, 2015).

Maize for dry grain2 is annually cultivated on an estimated 197 M ha of land

globally, making it the second most widely grown crop in the world after wheat. In terms of (dry
grain) annual production, maize’s 1,137 million tons (M t) globally (TE2019) is markedly higher
(+50%) than both rice and wheat (757 M t each; Table 1). The divergence reflects the
substantially higher maize grain yields (5.8 tons/ha), mostly linked to widespread hybrid
cultivation and complementing input use. Over the last quarter century, maize production more
than doubled (+118% over TE1995) supported by both substantive yield increases (+50%) and
area expansion (+46%). Of the three cereals, maize had a yield increase of nearly 2 tons over the
25-year period (up from 3.9 tons/ha, i.e., an increase of 76 kg/ha/yr or a simple average of 2.0%
per annum [pa]), compared to increases of 1 ton for rice and wheat (increases of 39 and
40 kg/ha/yr, or simple averages of 1.1 and 1.6% pa respectively).

The maize production dynamics over the last quarter century build on earlier trends.
Since 1961, the global maize area under maize production nearly doubled, up from 106 M ha
(TE1963) to the current 197 M ha (+87%), with an acceleration of area expansion since the early
2000s (Fig. 1). On current trends, and with wheat area relatively stagnant, maize is set to
overtake wheat as the most widely grown crop by 2030 (Erenstein et al., 2021). The global maize
yields nearly tripled since 1961, up from 2 tons/ha (TE1963) to the current 5.8 tons/ha.

Maize production faces several challenges, including pests and diseases, climate
change, and limited access to markets and finance for small-scale farmers. Climate change is also
a major challenge, as it can lead to changes in rainfall patterns and temperatures, which can
affect crop yields and quality. Climate change is set to gradually shift rainfed maize mega-
environments, including increased cultivation prospects in the northern and southern latitudes
and higher altitudes, but with increased frequency of abiotic (e.g., heat, drought, waterlogging)
and biotic stresses (e.g., diseases and insect-pests) in the (sub-)tropical environments (Cairns
et al., 2013; Jones & Thornton, 2003; Prasanna et al., 2021; Tesfaye et al., 2015). Over time new
virulent pests and diseases have emerged in previously unaffected geographies with far-reaching
consequences for maize. In Africa, in the past decade, maize lethal necrosis (MLN), a
devastating transboundary disease has emerged (Boddupalli et al., 2020; Marenya et al., 2018).
Since 2016, maize crops in over 40 countries in Africa have been adversely impacted by the
invasion of fall armyworm (Spodoptera frugiperda, Prasanna, et al., 2018; De Groote et al., 2020;
Kassie et al., 2020). Small farms predominate (84%) among global farms (Lowder et al., 2021).
In the case of maize, these comprise resource-constrained smallholders in Asia, Africa, and
South America, with many often dependent on maize for their food security and livelihoods. On
the other end of the farm size spectrum are large commercial mechanized maize producers, for
example, in the USA and Brazil. These systems combine highly productive new maize genetics
with intensive crop management

2.2 Salt Stress and Maize Productivity:

Soils with excess soluble salts or exchangeable sodium in the root zone are termed
salt-affected soils. Owing to limited rainfall and high evapotranspiration demand, coupled with
poor soil and water management practices, salt stress has become a serious threat to crop
production in arid and semi-arid regions of the world (Flowers and Yeo 1995; Munns 2002).
Although the general perception is that salinization only occurs in arid and semi-arid regions, no
climatic zone is free from this problem (Rengasamy 2006). High osmotic stress due to low
external water potential, ion toxicity by sodium and/or chloride, or imbalanced nutrition due to
interference with the uptake and transport of essential nutrients are three potential effects of salt
stress on crop growth. According to the biphasic model of salinity-induced growth reduction
(Munns 1993), osmotic stress during the first phase and ion toxicity during the second phase is
responsible for reduced growth in cereals, specifically wheat (Fig. 2). The same model for
salinity-induced growth reduction in maize was confirmed by Fortmeier and Schubert (1995),
but ion toxicity and the associated growth reduction can occur, to a small extent, in the first
phase in maize (Sümer et al. 2004).

The sensitivity of maize to salinity is associated with higher accretion of Na+ in the
leaves (Fortmeier and Schubert 1995). Osmotic stress is linked to ion accumulation in the soil
solution, whereas nutritional imbalance and specific ion effects are connected to ion buildup,
mainly sodium, and chloride, to toxic levels which interfere with the availability of other
essential elements such as calcium and potassium (El–Bassiouny, and Bekheta 2001; Munns et
al. 2006; Hussain et al. 2013). Toxic levels of sodium in plant organs damage biological
membranes and subcellular organelles, reducing growth and causing abnormal development
before plant mortality (Davenport et al. 2005; Quintero et al. 2007). Several physiological
processes such as photosynthesis, respiration, starch metabolism, and nitrogen fixation are also
affected under saline conditions leading to losses in crop productivity.
 A saline level of more than 0.25 M NaCl damages maize plants and may stunt growth
and cause severe wilting (Menezes-Benavente et al. 2004). Moreover, salt stress also induces
oxidative damage to plant cells with the overproduction of reactive oxygen species in maize (de
Azevedo Neto et al. 2006). Salt stress influences seed germination primarily by sufficiently
lowering the osmotic potential of the soil solution to retard water absorption by seeds, by causing
sodium and/or chloride toxicity to the embryo, or by altering protein synthesis. Hyper-osmotic
stress and toxic effects of sodium and chloride ions on germinating seeds in a saline environment
may delay or inhibit germination (Hasegawa et al. 2000; KhajeHosseini et al. 2003; Farsiani and
Ghobadi 2009). However, in maize, it is sodium toxicity and not chloride toxicity that is the
major problem in the second phase of salt stress. Salinity reduces shoot growth by suppressing
leaf initiation and expansion, as well as internode growth, and by accelerating leaf abscission
(Rios-Gonzalez et al. 2002; Akram et al. 2010a; Qu et al. 2012). Salt stress rapidly reduces leaf
growth rate (Munns 1993) due to a reduction in the number of elongating cells and/or the rate of
cell elongation (Szalai and Janda 2009).

Salt stress affects the growth and development of maize; however, the response of
plants varies with the degree of stress and crop growth stage. Short-term exposure of maize
plants to salt stress influences plant growth owing to osmotic stress in the first phase of salt stress
(Sümer et al. 2004) without reaching toxic sodium concentrations (Fortmeier and Schubert
1995). Seed germination is the most critical stage in seedling establishment which determines
the success of crop production on salt-affected soils. Generally, salt stress during germination
delays the start, reduces the rate and enhances the dispersion of germination events (Ashraf and
Foolad 2005; Farsiani and Ghobadi 2009). It is important to note that germination and early
seedling growth are more sensitive to salinity than later developmental stages (Goldsworthy
1994). Salinity reduces shoot growth by suppressing leaf initiation and expansion, as well as
internode growth, and by accelerating leaf abscission (Rios-Gonzalez et al. 2002; Akram et al.
2010a; Qu et al. 2012).

Salt stress rapidly reduces leaf growth rate (Munns 1993) due to a reduction in the
number of elongating cells and/or the rate of cell elongation (Szalai and Janda 2009). Although
the root is the first organ exposed to salt stress, shoots are more sensitive to salt stress than roots
(Munns and Sharp 1993). In salt-affected soils, excessive buildup of sodium and chloride ions in
the rhizosphere leads to severe nutritional imbalances in maize due to strong interference of these
ions with other essential mineral elements such as potassium, calcium, nitrogen, phosphorus,
magnesium, iron, manganese, copper, and zinc (Hasegawa et al. 2000; Karimi et al. 2005; Turan
et al. 2010).

For maize, sodium is the principal toxic ion (Fortmeier and Schubert 1995; Sümer et
al. 2004) interfering with potassium uptake and transport leading to disturbance in stomatal
modulations and causing water loss and necrosis. Increased sodium accumulation also disturbs
calcium nutrition (Hu et al. 2007; Kaya et al. 2010; Shahzad et al. 2012) during the first phase of
salt stress; calcium transport to the youngest leaves of maize is impaired (Fortmeier and Schubert
1995). Reduced leaf expansion with reduced calcium contents in expanding shoot tissues in
maize is due to reduced transport in a saline environment; some calcium is required to uphold
cell membrane integrity for proper functioning (Hu et al. 2007). Salt stress in maize, during the
reproductive phase, decreases grain weight (Abdullah et al. 2001; Kaya et al. 2013) and number
(Abdullah et al. 2001; Schubert et al. 2009; Kaya et al. 2013), resulting in substantial reductions
in grain yield (Abdullah et al. 2001; Schubert et al. 2009; Kaya et al. 2013). Salinity-induced
reductions in photosynthesis and sink limitations are the major causes of poor kernel setting and
reduced grain number (Hiyane et al. 2010; Schubert 2011)

2.3 Salt tolerance in maize:

Salt tolerance is a critical trait for maize production in many parts of the world. High
soil salinity can limit maize growth and development, leading to reduced yields and poor crop
quality. This paper will provide an overview of salt tolerance in maize, salt tolerance is a critical
trait for maize production in many parts of the world. High soil salinity can limit maize growth
and development, leading to reduced yields and poor crop quality. This paper will provide an
overview of salt tolerance in maize

Maize plants have developed several mechanisms to tolerate salt stress. One
mechanism is to exclude salt from the root system, which involves the active transport of ions
out of the root cells and into the soil. This can maintain a low salt concentration in the plant,
reducing the toxic effects of salt on plant growth and development. Another mechanism is to
compartmentalize salt within the plant, which involves the sequestration of salt in vacuoles
within the plant cells. This can reduce the concentration of salt in the cytoplasm, reducing the
toxic effects of salt on cellular metabolism and function. Also maintain ion homeostasis, which
involves the selective uptake of ions, such as potassium and calcium, which can counterbalance
the toxic effects of sodium ions in the plant.

2.4 Role of Proline in Maize:

Proline induces salt stress tolerance in crop plants by regulating gas exchange characteristics (Ali
et al., 2007), chlorophyll fluorescence (Deivanai et al., 2011), enhances antioxidant defense
system (Ben Rejeb et al., 2014), protects protein turnover machinery and up-regulates stress-
protective proteins (Ismail, 2014). Proline concentration has been shown to prove effective in
inducing salt stress tolerance in various studies. Pre-sowing seed treatment with different
concentrations of proline particularly 10 mM increased salinity stress tolerance in tomatoes
(Kaur & Gupta, 2018). However, the effect of proline on different genotypes of the same species
may vary significantly (Garg, 2003). Proline has improved salt stress tolerance in various plant
species. Under high-salt conditions, proline application enhances plant growth with increases in
seed germination, biomass, photosynthesis, gas exchange, and grain yield. Proline also alleviates
salt stress by improving antioxidant activities and reducing Na+ and Cl− uptake and
translocation while enhancing K+ assimilation by plants. Proline is the most common
endogenous osmolyte accumulated under various abiotic stresses including salinity (Szabados
and Savouré, 2010; Slama et al., 2015). When applied as an exogenous compound to crops,
proline can improve salt tolerance (Heuer, 2010).

Under Salt Stress Seed germination is one of the most critical stages in the plant life
cycle (Hubbard et al., 2012) because it is very sensitive to abiotic stress. In particular, salt stress
causes osmotic stress that limits seed water absorption and ion toxicity due to the high
accumulation of Na+ and Cl− (Murillo-Amador et al., 2002; Farissi et al., 2011). In recent years,
there have been numerous papers about the effect of exogenous compounds like hormones,
mineral elements, and amino acids in alleviating salinity stress during seed germination (Atia et
al., 2009; Dallali et al., 2012; Rizwan et al., 2015; Coskun et al., 2016). However, the effect of
exogenous proline on seed germination under salt stress is poorly understood as only a few
studies have been published.
 Deivanai et al. (2011) demonstrated that exogenous proline had a positive concentration-
dependent effect on seed germination under salt stress. The application of 1 mM proline
alleviates the negative effect of 400 mM NaCl, but 100 mM proline did not have a significant
effect. Similarly, 50 mM proline treatment improved seed germination of two cultivars of S.
bicolor under salt conditions (Nawaz et al., 2010). Therefore exogenous proline application at
suitable concentrations may alleviate the negative effect of salt stress by regulating cellular
osmotic balance, but detailed studies behind these data are still needed to better understand the
molecular mechanisms involved.

2.5 Role of Ascorbic Acid in Maize:

Also known as Vitamin C, is an essential nutrient for plant growth and development. Researchers
have studied the role of ascorbic acid in maize (corn) and have found several benefits. The role
of ascorbic acid in maize plants is vital for their growth, development, and stress tolerance.
Researchers continue to study the effects of ascorbic acid on maize plants to develop new
strategies for improving crop productivity and sustainability. The importance and changes in
ascorbic acid (AsA) levels in plant cells in response to varying environmental stress conditions
are well established (Noctor and Foyer, 1998; Venkatesh and Park, 2014). Functionally, it is an
essential metabolite that operates as an antioxidant and acts as a significant protagonist of several
plant species for salt tolerance (Hameed et al., 2012; Ozgur et al., 2013). It associates with H2O2
metabolism as well as lipid hydroxyl peroxidase and also reacts with various sorts of biotic
actions in plants as a patron or receptor in the electron transport system and also as an enzyme
co-factor (Conklin, 2001), this approach effectively minimizes the stress impact of salinity and
encourage the plant growth.

2.6 Role of Iron Sulphate in Maize:

It is also known as ferrous sulfate and has its role in promoting the growth and development of
maize. Maize is an important crop for human and animal consumption, and iron is an essential
nutrient for its growth and development. Iron sulfate is often added to soil to correct iron
deficiencies that can cause chlorosis and other growth problems in maize. Iron sulfate can
significantly increase the yield of maize crops, particularly in soils with low levels of available
iron. Iron sulfate supplementation has been shown to increase the number of maize ears per
plant, as well as the number of kernels per ear. In addition, iron sulfate has been found to
increase the concentration of essential nutrients, such as phosphorus and nitrogen, in maize
plants and also improve the quality of maize crops. Studies have shown that iron sulfate
supplementation can increase the levels of protein and amino acids in maize kernels, which are
important for human and animal consumption. Iron sulfate has been found to improve the taste
and aroma of maize, making it more appealing to consumers. Overall, the role of iron sulfate in
maize is to promote growth and development, increase yield, and improve the quality of the crop.
Chapter: 3
Material and Methodology

The current experiment was conducted to evaluate the seed priming with water and proline on
maize.

3.1. Experimental Area:

This experiment was carried out at the Lab of Agronomy Department of Faculty of
Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur.

3.2. Growing conditions:

The germination test was conducted in the biology laboratory having an average temperature
range of 25-30°C and a relative humidity ranging from 60 to 70 %. The experiment was laid out
in pots. Seeds were sown in pots. Seeds were sown in pots filled with Sandy-Loam soil
separately as a growth medium.

3.3. Treatments:

Treatments Seed priming concentration

T0 Treatment with distilled water
T1 Treatment with proline 1.15g/mol
T2 Treatment with proline 2.30g/mol

3.3. Maize Varieties:

V1= Maize p1429
3.4. Soil texture:

S1=Sandy-Loam soil

3.6. Date of Seed Priming:

On Boxes: 17 February 2023
3.7. Date of Sowing:
On pots: 18 February 2023
3.8. Planting material:
Clean and healthy seeds of maize were taken and the seeds were divided into 3 groups. The first
group is considered as a control and only distilled water is used as a priming agent. In the
second group, 1.15g/mol proline is used as a priming agent. In the third group, 2.30g/mol proline
is used as a priming agent.

3.9. Experiment detail:

This experiment was carried out at the Warehouse of the Agronomy Department of the
Faculty of Agriculture and Environment, The Islamia University of Bahawalpur, Bahawalpur.
Ten seeds were sown in each pot. Pots were filled with fallow, sandy loam soil, separately as
a growth medium. One variety of maize was selected. Label the boxes with prominent
markers or with sticky papers. The Boxes were set in the Laboratory, Islamia University of
Bahawalpur at room temperature ranging from 28-30°C. A seed was considered germinated
when its radical emerged. Take the pots and filled the pots with the fallow and sandy loam
soil and placed the pots according to design with three replication.

For our study, we have selected one variety of maize p1429. Seed from selected
maize genotype was used to grow in 30 pots, filled with 30kg of fallow soil and sandy loam soil
separately. To carry out our research work we have used the following sets for the treatment of
Proline:
1. Control only water priming
2. 1.15g/mol proline in 10 mm solution
3. 2.30g/mol proline in 20mm solution
The data was statistically analyzed and the following results were reported for our
study. The data was collected for different morphological and physiological traits, viz., leaf area
(LA), root length (RL), and shoot length (SL).