Effect of Silicon on Superoxide Dismutase in Wheat

(Triticum aestivum L.) Cultivars of Balochistan Grown

in Saline Condition

Maheen Baloch

M.Phil. Research Scholar

(2014 – 2016)

Registration No.2008/SBK(wu)/M.Phil-16/832

Supervisor

Saima Maher

Chemistry Department

Sardar Bahadur Khan Women’s University Quetta

Date .
 Supervisory Committee

Start with Beautiful Name of Allah Pak

The Most Merciful, The Most Beneficent
Effect of Silicon on Superoxide Dismutase in Wheat
Cultivars of Balochistan Grown under Salt Stress
 Abstract

Wheat (Triticum aestivum L.) is a sensitive plant for both biotic and a biotic stress and directly
losses the production rate under these stress. Salinity , Temperature and drought effect the plant
growth and production and excess of these can even lead to cell death. To overcome in this
situation plant develops a defense mechanism that is either enzymatic or non enzymatic methods.
A search study on Two wheat cultivar Umeed ( salt resistant ) Zarghoon (salt sensitive) were
grown under salt stress in control and silicon treatment The both wheat cultivars were compared
by determining the SOD activity and total protein quantification .The both wheat cultivars were
provided same conditions and same concentration of salt and silicon. The control treatment
showed protein was decreasing with increase in salinity in leaf and was decreasing in the
regulation of stomata. When silicon was provided in salt stress to plant it showed improvement
in protein quantity. The application of silicon was improving the defense mechanism of plant
was increasing the SOD quantity which was suppressed by salt in control treatment. Umeed
showed more improvement as compared to Zarghoon variety. Silicon has improved the tolerance
of wheat plant against salt stress.

Dedication

I would like to dedicate my research work to my loving Father

Mr Haramkhor
 Acknowledgment

Start with the beautiful name of Allah Almighty who is the creator of this Universe. The Most
Merciful and Beneficent. He is the One with all powers and enables human being to unrevealed
the secretes of His created Universe.
I want to express my sincere thanks to Almighty Allah who gave me life and courage to
accomplish my work. I am really grateful for his un countless Blessing.
I would like to pay my thanks to my Vice-chancellor Dr. Rukhsana Jabeen for providing us the
opportunity in Balochistan for getting higher education. I would also like to thanks Prime
Minister Youth scheme for granting us the scholarship and the platform to achieve higher
education.
I really feel happy to utter gratefulness from core of my heart to my Supervisor Saima Maher for
being so helpful during my whole work. She supported me guided me and gave me her pervious
time from her busy schedule.
I would also like to appreciate the cooperation of my head of department Dr. Uzma Jabeen and
Dr. Shaiza Iqbal , Dr. Saima Mehr and Dr. Saleh Sulmain all other staff of chemistry
department.
I don’t want to forget to appreciate the hard work and support of my loving family my Father,
Mother, Aunt and my Brothers. They always supported me encouraged me. Thanks for giving
me strength to carry out my work.
.
THANK YOU ALL

List of Abbreviations

Symbols Abbreviations

1. Si Silicon
2. SOD Superoxide Dismutase
3. CAT Catalase
4. POX Per oxidase
5. ROS Reactive Oxygen species
6. DNA ,RNA Deoxyribonucleic acid, Ribonucleic acid
7. SA Salicylic acid
8. ( GOX) Gultathione
9.(NBT) Nitro blue tetazolium
10. Ml Mili liter
11.Ul Micro liter
12.W Watts
13.Chl a Chlorophyll a
 Contents

Chapter Titles Page

A Abstract
B Acknowledgment
C List of Abbreviation
1 Introduction 1
1.2 Main Objectives
1.3 Significance of study
2 Literature Review
2.1 Toxicity of salt on Growth of Wheat

2.2 Impact of salt stress on Germination

2.3 Lipid per oxidation in germination of plant

2.4 Silicon

2.5 Silicon as bio fertilizer

2.6 Salicylic acid

2.7 Exogenous silicon supplementation

2.8 Change in Antioxidant Activity in Salt stress

2.9 Superoxide Dismutase

3 Material and Method

3.1 Phase I. Wheat grown

3.2 Phase II. Activity of antioxidant enzymes

3.3 Phase III. Protein Quantification

4 Results and Discussion

4.1 Superoxide Dismutase (SOD)

4.2 Protein Quantification

5 Conclusion

6 References

7 Appendices

List of figures

Figure Description Page

1. Changes in the activity of antioxidant
enzymes SOD in both cultivars of Wheat in
saline and Non saline condition.
1.2 Effect of silicon on activity of Antioxidant
enzymes SOD in both wheat cultivars
under saline and non saline condition.
2. Total protein quantification in two wheat
cultivars Umeed and zarghoon under saline
and non saline condition.
2.2 Protein quantification in 2 wheat cultivars
with control treatment in saline and non
saline condition.
2.3 Protein quantification in 2 wheat cultivars
with silicon application in saline and non
saline condition.

List of tables

Figure Description Page

1. Application of silicon on two
wheat cultivars under saline
and non saline conditions.

2. Effect of silicon on protein

quantification of two wheat
cultivars grown under saline
and non saline conditions.
Introduction Chapter 1

Pakistan a country situated in South Asia. It is having 769,092 sq kilometers area with four
provinces and most populated country of world. Among most populated countries it is having
sixth rank with about 207,77,4520 population according to the latest census. Every year its
population is increasing about 1.94% which is very alarming situation.(Jamil et al.,2017)
Balochistan a largest province of Pakistan via area having 347,091 sq kilometers is
located in the Southwest, with border connecting two major countries Iran and Afghanistan on
North and Northwest. The land of Balochistan is unfertile with hills and Rocky Mountains. Most
of the land here is unfertile and due to lack of water resources the land remains un cultivated. It
is tough for the inhabitants to fulfil needs of food and they totally depend upon the food imported
from the rest of provinces or to cultivate with hard efforts. Most of the crops grown in
Balochistan are Wheat, Bajra and Jowar but due to hard conditions there production is less to
complete the requirements of people. ( Ghulum Murtaza 2014).
Wheat (Triticum aestivum L.) Family (Poaceae) is a grain which was first grown in the
region named Levant (North East) but due to its health benefit now being grown all over the
world. It is a type of cereal whose head possess its fruit with many seeds which are edible. The
cultivation of wheat plant is spread in all part of the world since ancient times because of its easy
cultivation and large production. Pakistan is tenth most wheat producing countries of world.
Wheat production has increased about 3.33 to 23 million tons during last 2 decades.
These plants are sensitive to natural and external factors and when are unprotected from
temperature and water stress which decrease their production (Gillham and Dodge., 1987). The
main environmental factors that damage the production of crops are
Salinity of soil , Temperature , Unfertile soil , Lack of water recourse
But research is underway to overcome these conditions and grow crops in better way with
efficient production to fulfill the need of people living in area.
Salt stress is an environmental threat for all type of food worldwide according to (FAO
2010) the 800 square kilometers of land is affected by salt stress. Salinity causes ion toxicity in
plant and decreases the production of plant. This is caused by the loss of chloroplast activity,
metabolic changes such as increased in ROS decreased in the photosynthetic reactions. (Parida
and Das 2005). The (ROS) reactive oxygen species are highly reactive and causes damage to
plant metabolism, including damage of lipids, protein, and nucleic acid (Ashraf ., 2009).
The salt stress can be defined as the intake of Na+ & Cl- ions through water in soil that
causes the production of free ions formation of O (ROS) (Joshi, Mishra, Chatrath, Ferrara, &
Singh, 2007). Osmotic pressure, Oxidative stress and increase in the ions these are all well
known problems in wheat plant. Among all these parameters osmotic stress can even be caused
at lower quantity of salt that leads to dehydration of plant cell and lesser accumulation of water.
Another important damage of osmotic stress is to expand of cell size, smaller the size of
stomata and lesser carbon digestion. Ion stress increasing of Na and Cl ion in plant slowly
decreases the activity of plasma membrane and change in ions intake and effect homeostasis.
Plant germination period is a complicated biological phenomena and salinity in this stage can
either cause lack of water intake or lack of mineral resources.
Salinity directly affects the plant morphologically and damages the growth parameter.
(Ramagopal, 1990)It reduces the quantity of both Chlorophyll-a and chlorophyll-b and break up
protein to improve all these factors plants develop a mechanism a defense system that include
both enzymatic and non-enzymatic aspects.
Oxidative stress can lead to cell death, or can cause mutation of DNA, RNA, protein
oxidation and even sometimes enzymes inhibition (Tanou, Molassiotis, & Diamantidis, 2009).
The water stress and the salinity stress have negative effect on the gas intake and reduces CO2
for the formation of plant food (photosynthesis) and transportation of electron cycle and causes
formation of reactive oxygen species (ROS).
To overcome salt stress the plant has adopted defense mechanism to minimize the loss.
Various strategies are develop in wheat plant to reduce the toxic effect of salt stress, that can be
either enzymatic or bio fertilizer (Timmutus & Wagner, 1999). Numerous macronutrients are
being used to reduce the salt stress such as Salicylic acid, Selenium, Nitrogen, Silicon,
Phosphorus, Potassium and Zinc Waseem ,2006). All these treatments silicon is preferred the
most ( Hattori et al .,2005).
Silicon is the second most common element of Earth atmosphere after nitrogen and as much
as important as nitrogen though it is not essential for the growth of plant but it can increase the
growth especially in salt stress. (Esptain ,1994). It can either be used as fertilizer or can be
sprayed on plant it’s beneficial for wide variety of crops. (Bonifacio et al., 2011).
Silicon has shown many positive effect on the wheat plant in many studies (Ahmad et
al.,1992) Silicon can be basically used to increase plant mechanism against drought and salinity
stress with an increase of antioxidant enzymes such as Super oxidase (SOD), Peroxidase (POX)
and Catalase (CAT) and reduce the formation of free radicals by the recent research on silicon it
has suggested that silicon fertilizer can improve the soil and wheat production.
Free radicals in plants are also responsible for loss of production. The word free radical
can be defined as the elements with free electron and can occur in alone with independent but
these electrons are highly reactive. They are present in plant to built defense mechanism against
the bacterial growth, but the high percentage of the free radicals can destroy cell growth and
even can cause the death of cell. To face all these problems plants have developed antioxidants
defense system which neutralize the free Radical formation and prevents the plant death. This
can be either enzymatic or non enzymatic such as:

 Enzymatic system : Superoxide Dismutase (SOD), Peroxidase (POX) ,Cataslase (CAT),

Gultathione ( GOX)
 Non- Enzymatic system : Phenols , Silicon , Nitrogen

Antioxidants can be classified as Primary antioxidants, Secondary antioxidants.

1. Primary elements are used as terminating agent for the formation of free radical by donating
them H atom to form stable products.
2. Secondary element are used as chelating agent that help plant from oxidative damage.

These all helps the plant to prevent the buildup of (ROS).( Elstner., 1982). Superoxide
Dismutase , CAT, POX are most important enzymes and detoxify the cell.
 (POX) is basically an enzyme that have heme portion so mostly known as heme enzyme
which is usually presenting the cell wall of all living species like higher plant animals and some
smaller organisms. Peroxidase is important for the plant life and it controls the damage of H 2O2
by decomposing itself by oxidation of phenolic groups.(Dionisio-Sese & Tobita, 1998)
Peroxidase also play many other vital role in plants such as it prevents plant from deposit of
lignin of cell wall and it’s also prevents from microbial and it has different types which are still
being studied .(Miesle, Proctor, & Lagrimini, 1991)
(CAT) is also important enzymes functioning in higher plant species cell but it will be absent
in chloroplast. It works similar as peroxidase helping the plant from the formation of free
radicals and decomposing of H2O2.
Including all enzymes Superoxide dismutase (SOD) is very powerful antioxidant and first and
enzyme that works against the plant damage that can work against H2O2 and O2 formation.
(SOD) is an enzyme which is metallic in nature so mostly known as “Metalloenzyme” its
basic work is to catalyze the dismutase of hydrogen radicals peroxidase and molecular oxygen
from the plant. This free radical generation is increased in plant due to environmental stress
(Schooner and Krause., 1990).
(SOD) is well known for three different types of enzymes based on their cofactors that are
metal 1, (Fe SOD) Iron superoxide dismutase 2, (Mn SOD) Manganese superoxide dismutase 3,
copper superoxide dismutase (Cu SOD). The increase of these enzymes activities and plant
recovery are closely related to each other, and SOD is highly related with plant response to
environmental stress such as water stress, salinity stress. This enzyme is not only present in plant
but also in bacteria and yeast.
Aim and objectives

The aim of this study is that to determine the concentration of antioxidant enzyme SOD
(Superoxide dismutase) in the effect of silicon under salt stress and the total protein
quantification in the wheat plant.

Objectives:

The principle objectives of this research work are:

1. To determine the effect of salt stress on wheat.

2. To determine the activity of superoxide dismutase on wheat grown under silicon and salt
stress.

3. To quantify total protein in the wheat grown under the salt stress.

Significance of the study:

The significance of this search work is to understand the mechanism of salt stress and various
other factors effecting the growth of plant (Wheat) grown under salinity stress and the role of the
enzyme Superoxide dismutase (SOD) in alleviating salt stress. Wheat is very sensitive to plant
and all these factors like water stress salinity and high temperature directly affect the growth and
production of plant. To recover these damages plant develop a Defence mechanism a mechanism
of antioxidant enzymes that increase their quantity to recover the damage in plant as soon as the
plant recovers these enzymes reduce their concentration.
Literature Review Chapter 2

2.1 Toxicity of salt on Growth of Wheat:

Salinity is a biotic stress for the plant and effect the growth of plant .The ion formation of Na+
reduces the development of seed. According to UN salt stress is affecting about 55% of land
from which 20% of land is affected in Continent Asia. (Yokoi et al.,2002). To overcome this
problem number of studies has been carried out and a large number of proposals have been
suggested such as:
A search was conducted by Asgari and his college on wheat grown under salt stress. How
salt was effecting the growth, causing mineral deficiency and effect on production of wheat. For
this experiment the planted wheat in pots under control conditions and different levels of salts
were provided such as 8, 12, and 16 dsm-1 from the root and the leaf compartment. The salt
(NaCl) quantity was provided by mixing the salt with soil before the pots were filled and then
water was given to plant at 70% that pot size can bear and allowed the plants to grow. The
wheat Varity used for this experiment were Kouhdast , Tajan , Atrak , Rasoul, it was observed
that all varieties were having different kind of effect. The one Tajan was having highest and
kouhdast the lowest production of of grain among the four, but the leafs of both varieties
weren’t effected by salinity. Finally it was resulted that that among all four wheat crops the
Atrak and Tajan were capable of to grow under salinity. (Asgari et al., 2012)
 Another search was carried out by the University of Tando Jam in Pakistan for salt stress on
the production of wheat plant. By which it was concluded that if plants were being grown in salt
stress in pots with proper nutrition with level of salt ( 2.0, 4.0 , 6.0 , 8.0 and 10 dms-1). Salts were
of different types such as MgCl2 , CaCl2, and NaSo4. These salts were mixed with soil and placed
in pots. The plants were allowed to grow after which results were collected. The result showed
there was increase in Na+ , Ca- ,Mg+2 and Cl-. Presence of these ions were directly effecting the
growth including the height, number of grains, weight. ( kalhoro et al., 2016)

2.2 Impact of salt stress on Germination:

Plants have complicated growth mechanism from seedling till adulthood and salt stress affects
its growth throughout the period and even can cause of death of plant cells. This was shown in
work of ( Sourour et al., 2014) at agricultural and research office in North West of Tunisia
conducted a research on the wheat. To measure the effect of salt on wheat the grew plant in pots
under control treatment. Different levels ( 0 , 50 , 100 , 150 , 200 m mol ) were supplied to ten
genotype of wheat. There plants were kept for three months after which results were collected
including all parameters such as length of shoot, root, leaves and dry weight of shoot and root.
By all these data it was finally concluded that salt stress was decreasing the growth, but it was
different in two genotypes Maali and Derbassi in levels of 150 and 200 m mol.
Another search work was conducted by the molecular biology and genetics department
of Istanbul on the growth of Barley roots in salt stress. This experiment was basically done to
check out the effect of salt under different concentration 150mM and 250mM during which
plants were kept in dark for about 72 hours. The plant should direct decrease in length of roots an
shoots and mitotic abnormalities were also seen and these major impact of salt stress, and to
reduce these affects homobrassinosteriod HBR showed positive effect on both length of root and
shoot as well as production of plant. (Marakli, Temel, & Gozukirmizi, 2014)

2.2 H2O2 accumulation in water stress

 The biotic stress not only involves salinity but drought, temperatures are also important
factor. Drought is also harmful situation for plant growth it leads to oxidative damage in plant.
To examine drought in plant a research was carried out on five different wheat varieties to
examine the antioxidant defense mechanism and H2O2n collection. The wheat varieties Kw ,
UP2725, RPW343 , SO, LV were grown under salt stress and the samples for experiment were
done on every 3rd , 6th 9th day of salt stress. The growth was less in Kw , UP2725 and RPW343
as compare to the LV and SO. The enzymes POX , APOX and GR increased from the initial
level and it was greater as the time was increased. While it was seen that H 2O2 was also reducing
as per increase in time. Final work suggested that in five varieties of wheat Kw , UP2725 and
RPW343 was having more tendency for water stress the SO, LV. ( Chakraborty and Pradhan
2012).
Honold and Astahman (1968) did work quantitative and qualitative examination of these redox
enzymes in five different types of wheat which were grown in different culture two were grown
in winter and spring seasons. Different methods were used for the both quantitative and
qualitative measurements. Such as electrophoresis and trophotometric nonmetric. In
electrophoresis it was noticed that pattern were having same position for the enzymes like
catalyze and peroxidase but it was also concluded that wheat samples are consisting of 8
different types of peroxidase enzymes and 2 catalyze enzymes but the quantity of these enzymes
were different in these season. The ratio was lesser in winter as compare to spring and barley was
having catalyze concentration more than peroxidase

2.3 Lipid per oxidation in germination of plant

A research was conducted by the department of Agronomy and Plants breeding on enzymes
activity in wheat plant which activity on wheat plant which were grown under salt stress. For this
experiment to local types of seed( Alvand and sardari ) were grown in hydroponic technique
where NaCl solution (50,100, 150 and 200 mM ) and plants were allowed to grow in this
solution for maximum ten days. On the eleventh day the green leaf of both plant were collected
to study the activity of enzyme SOD . The local type Sardari was having larger quantity of SOD
in all salt stress quantity while the enzyme was having same quantity at all level of salt stress.
Catalase remained same in both type of wheat. (Esfandiari et al., 2007)

2.4 Aseorbic acid and phenols activity in salt stress

The soil salinity clearly decreases the plant growth and causes damage in plant mechanism to
study this damage an experiment work was carried by agricultural in Szczecin where were grown
in laboratory under 1% humus. The plant pots were filled about 1 kg soil with about 10, 30 and
50 mM kg_1 NaCl. Each pot was having seven seeds of Bean and they were allowed to grow
about 14, 21, and 28 days. Then result were collected to determine the activity of Superoxide
Dismutase, catalase , flavonoids , Phenols, ascorbic acid. After which result collected showed
that chloride was increasing due to salinity and there was also increase in both enzymes as
chloride was increasing. ( Nowak, 2008).

2.5 Silicon

Silicon is an important element for the growth of plant in biotic and a biotic stress. It
overcomes the salt drought and temperature stress and improves the growth of plant. This was
shown in research work carried out on seedling of Canola in salt stress. The effect of silicon on
the growth of Canola seeds which were already under salt stress. For carrying this experiment a
complete block was designed in which different quantity of silica were prepared and provided to
plants in pots (0 ,1 ,2, 3 , 4 g/L) and the quantity of NaCl (0, 60 , 90 , 120 , 150 mM) . The plants
were in pot or more then 3 months after which results were collected and it was observed that
length of radical and dry weight were affected by salinity but the rate of germination where
silicon was in higher quantity was better than rest of pots. Especially those pots which were
having 2 g/L was having high germination rate. (Solatni, Shekari, Jamshidi, Fotovat, &
Azimkhani, 2012)
Silicon is 2nd most found element after oxygen on earth and mostly used as fertilizer. An
agricultural university was working to check out the effect of silicon on the production of rice in
Faisalabad Pakistan. For this experiment work the selected some genotype of rice and designed a
complete block in which 3 replica of plants were planted in pots in which silicon was provided as
control treatment (0.25% , 0.50% , 1.00%) kept under aerobic conditions. The fertilizer was
also provided such as phosphate, potassium and nitrogen with amount of 100 66 and 67 kgh-1.
The data was collected and statical analysis was carried out and then final result showed that
silicon was not giving huge difference in height of plant and quantity of kernels. The plants with
0.50% of silicon as having larger grain and the protein level were also increased. The plant with
1.00% was over lapping from the 0.50% result. (Ahmed & Hussain, 2013)

2.7 Improves the Growth of plant

A study was carried out to check how the silicon was improving the characteristic of wheat
plant which was kept under salinity. For this experiment two samples of wheat were grown such
as durum wheat and bread wheat in pots. The plants were having control treatment with complete
nutrition supplement. During this experiment the level of NaCl was ( 0, 100 mM). After which
plant showed less effect on those plants that were having 100mm but silicon in both 0.25 and 0.5
mM were having less effect of salinity that showed the improvement of silicon on plants. There
was also increase in calcium and potassium in plants having higher silicon.(Tuna et al., 2008)

2.8 Silicon as bio fertilizer

Under drought stress the plant losses its capability to prevent the growth against (ROS) and
leads to cell death this was shown by Khalilzadeh reported the study of the role of antioxidant
enzymes with three different experimental conditions which included control water supply. The
first sample was given as limited water second normal and third average water supply were
collected and it was noticed that those plants with lacking in chlorophyll where on other hand the
antioxidant enzyme ratio was increased but when the mechanism were seen in plant and was
concluded that bio fertilizer can increase the production of wheat plant. ( Kalilzadeh et al.,2015)
A research work was carried out to germinate seed of wheat under sodium silicate to improve
plant growth under salt stress. The experiment showed that sodium silicate increase capability of
plant .Seeds of wheat were soaked in water and silicate for about 8 hours in 20 , 40, and 60 mM
sodium silicate solution in a Petri dishes. That increased the growth of wheat plant and the
highest growth was shown by 40 mM and the growth time was also decreased. The less time was
recorded in 60mM. (Hameed, Sheikh, Jamil, & Basra, 2013)
2.9 Impact of micronutrients on plant

Fouly and his college carried out an experiment at researcher of National Research center
Cairo on metal such as Fe , Mn and Zn in wheat were changing due to salinity. The wheat seeds
were planted in pots and the metals suspension were sprayed in two different concentration such
as (0.10 and 0.15%) of Fe , Mn and Zn with equal ratio and NaCl was supplied with water
sources (0- 1000 – 2000 -5000ppm) The final result showed that salt was decreasing all
parameters of Growth and the heavy metals were recovering that damage of plant and was also
increasing its potential to overcome the salt stress. (El-Fouly, Mobarak, & Salama, 2011)

2.10 Salicylic acid

Salicylic acid is also a common growth factor used under salt stress. It prevents the damages
such as leaf growth, area and stomata regulation. A search work was carried out by Islamic
Azad University in Iran on the beans in salinity. In this experiment the bean seeds were planted
in pots under salt stress and allowed to grow about ten days. On the 11 day samples were
collected then after 5 days again samples were collected. The result showed that chemical SA
salicylic acid in plant growth under stress condition. In salt stress where plant was being
damaged by its height, leaf growth. SA is on the other hand improving these damages and was
effective for plant under salt stress. (Sadeghipour & Aghaei, 2012)
A research was conducted on beans grown under salt stress and the result was compared by
two different treatment of Salicylic acid. It was observed that SA is an important molecule for
the environmental stress such as water and salt stress. This experiment was conducted by
providing seeds 4 different concentration ( 0, 0.1, 0.5,1.0 mM) that showed that 0.1 was the best
treatment of SA provided to plant while the 0 and 1 was not having any positive effect on plant.
(Hadi et al., 2014)

2.11 Exogenous silicon supplementation

 A research was carried out by the agricultural university Faisalabad on wheat grown under salt
stress. Providing the plant silicon treatment. For this experiment two wheat varieties were taken
and where NaCl was mixed with salt and silicon was also provided the plant were grown with
normal and water stress condition in plastic pots. The result showed that there was decrease in
plant height and minerals intake (phosphorus, potassium and zinc ). The silicon was responsible
to active the enzyme such as Catalase , Peroxidase and Superoxidase these enhance the growth of
plant under salt stress. This was concluded that silicon should be used as fertilizer for
maintenance of growth of wheat. ( Bukhari et al., 2015)
Silicon is a good fertilizer for the plant growth can recovered the production of wheat under
salt stress. A research was carried out for two wheat genotypes under salt stress. One was
provides normal water and other was under water stress. Calcium silicate was provided at
different levels. The salt was decreasing growth of plant which were under salt stress were being
treatment of silicate and that was showing positive result on growth and production. The plant
with salt were having more Na uptake where as the silicate was decreasing Na uptake. ( Tahir et
al., 2006)
A research work was carried out for the synthesis of nano particles by using silica for the
better growth of wheat under salt stress. For synthesis of these nano particles a special advanced
technique was used to overcome the stress of salt in wheat. The nano particles were prepared by
using spherical silica by Stober preparation method in which tretraethyl orthosilicate was use
as precursor. ( 50- 100 nm). The two methods were used called as T1 and T2 in which 50 nm
,100 nm nano particles were used that increased the growth of plants from 68 to 81 in T1 88 in
T2. The salt stress was decreasing all the characteristics of plant where as the silica nano particles
were having positive result and directly increasing the growth of wheat. (Mushtaq et al., 2017).

2.12 Effect of Antioxdiant enzymes (SOD), (POX) and (CAT)

The antioxidant enzymes improves the activity of plant under salt stress are very beneficial for
the growth of plant. This was experimentally proved by ( Mahmoudi et al., 2012) . In which they
grew Lettuce seed for 15 days in 100 and 200 mM NaCl.
 The enzymes such as Superoxidase ( Fe-SOD , Mn-SOD , CuZn-SOD. The enzymes
concentration was increasing with increase in salinity. Fe-SOD and Mn-SOD was limited but
CuZn- SOD was consistent in all level during all periods. Other enzymes Catalase was increased
in salinity in roots and leaves both. Peroxidase was lower than SOD. In clear ways the enzymes
was recovering the plant damage.
A research was conducted by the department of Agronomy and Plants breeding on enzymes
activity in wheat plant which activity on wheat plant which were grown under salt stress. For this
experiment to local types of seed( Alvand and sardari ) were grown in hydroponic technique
where NaCl solution (50,100, 150 and 200 mM ) and plants were allowed to grow in this
solution for maximum ten days. On the eleventh day the green leaf of both plant were collected
to study the activity of enzyme SOD. The local type Sardari was having larger quantity of SOD
in all salt stress quantity while the enzyme was having same quantity at all level of salt stress.
Catalase remained same in both type of wheat. (Esfandiari, Shekari, Shekari, & Esfandiari, 2007)
A research was carried out using high technique Near Infrared spectroscopy to determine
the activity of enzymes (superoxide Dismutase) in barley leaves. For this experiment they
compared seven different spectra methods were used to analyze. Among which 4 methods were
being used is Partial least Square (PLS), Multiple linear regression (MLR), Successive projection
algorithm (SPA) and regression coefficient (RC) these all methods were used for better result of
wavelength. After all this final result was made that multiplicative scatter correction (MCR) is
the most preferable method among the eight methods. The spectroscopy with multivariate is the
fastest method for the analysis of SOD activity in barley. (Kong et al .,2012)

2.13 Change in Antioxidant Activity in Salt stress

Sanjib Kumar Panda and Mohammad Hassain Khan (2004) worked on the changes of growth
and superoxide dismutase activity in hydrillah vertiiata L. under abiotic stress. The research was
based on the role of various metal effect of NaCl, water stress and the SOD activity in the plant
in which it was concluded that due to NaCl and water stress growth was reduced but the SOD
activity was inhibiting by some heavy metal like (Cu,Cd). The salinity was getting increased by
SOD and water stress was getting decreased and thermo stability was high at 450C.
 Another research was carried on the activity of catalase peroxidase and polyphenloxidase in
the leaves of rice it was observed that in the leaves of rice the catalase activity was decreased but
polyphenol oxidase was increased in rice leaves during senesces of both leaves attached and
detached leaves of rice. It was also noticed that kinetic benzimestase was delaying the senesce of
detached rice leaves. They noticed that chemical was increasing the quantity of peroxides and
polyphenol oxidase activity. They concluded that phenolic concentration of leaf was not present
in plant which were kept in dark but was different in leaves present in leaf senesces which were
in light these had no presence of phenolic.( Kar and Mishra .,2007)

2.13 Cadmium and Lead stress on Antioxidants

The research was carried on the antioxidant enzymes in wheat plant which were already under
Cadmium and Lead stress. Wheat seeds were grown in pot with soil mixed with CdCl2 and
Pb(No3) about (0 -200µM). The 7 days seed were examine for the antioxidant enzymes. The
results were showing the superoxide Dismutase, Catalase and Guaiacol Peroxidase were
increased in root and shoot of plant. The SOD quantity was very low at concentration of
Cadmium. The Catalase was more than SOD where the quantity of POX was 9 times greater than
rest enzyme. The result suggested that heavy metal Cd and Pb was responsible for oxidative
effect. (Dey et al., 2017).

2.14 Enzymatic and Non Enzymatic activity of antioxidant enzymes

An research work was done on enzymatic and non- enzymatic characteristic of Tylophora. The
work was done to determine the change of free radical and their damage to plant. How plant
response to recover these damages. The Tylophora was having strong mechanism which was
being studied throughout the world. The plant tylophera have strong resistance for oxidative
stress and free radical damage. With no time the antioxidant enzymes were improving the plant
growth. Among these Superoxide Dismutase was the first enzymes in recovering the salt stress
but other than these enzymes Phenolic system was also observed in performing strong action
against biotic stress. (Starlin et al., 2013)

2.15 Superoxide Dismutase

Cornelius carried out a research work on the spruce bark and deuterium depleted water to
extract polyphenolic and the activity of some enzymatic action which were involved for the
metabolism of maize and they determined peroxidase catalase and SOD activity in the plant. By
their work they concluded that the activity was getting decreased due to peroxidase in the root of
the plant but was increased with polyphenolic extract with deuterium depleted water and it was
also observed that the increase due to catalase activity when was being treated with polyphenolic
extract from spruce bark.
Another research was carried out by Syed Mohammad on the Super oxidase dismutase
performance in colza (Brassica napus L.) with salt stress and salt tolerance. By their work they
found that when plant was introduced to salt condition it will reduce the production of lipids,
proteins and other macro molecule in cell and this lead to death of cell in plants. To survive in
developing defense system of antioxidant enzymes like SOD, catalase, peroxidase in this work
different genotype were grown in four salt concentration and the enzyme activity was determined
by the inhibition in shoots, roots and the reduction of nitro blue tetrazolium in both genotype
plants and the result were same that got increased with increased in salt concentration. But
overall it was concluded that in shoot the increase in salt concentration will automatically
decreased in SOD activity but it was different in the plant with reduced salts condition.
(Mohammed et al., 2006).

Sypehalla and Desborough had reported a research on the antioxidant enzymes such as SOD
and catalyse and α tocopherol quantity in stored potato tubers they concluded that while plant is
under stress causes damage due to the free radical oxygen and the active form of oxygen while
the potatoes were stored at different temperature for different time. They noticed that the damage
to potatoes couldn’t be seen from the plant tissue directly but can be detected by the presence of
antioxidant enzymes so they carried out the test of these potatoes for 40 weeks in store at
different temperature such as 30C and 90C and then they examine the quantity of SOD and
catalyse and it was concluded that with the low temperature the activity was increasing of
enzymes and also time was effecting the quantity of these enzymes.

SOD is very active in wheat plant and perform key role in regulating the growth of wheat
plant. Gnanam and Jayapragasam carried out a research on the enzymes in the leaves of Sorghum
bicolor L. that was having stress of heat. There basic work was to examine the presence of
antioxidant enzymes in heat stress. The redox enzymes were damaging plant progress and that
was increasing the quantity of antioxidant enzymes in stress of heat but later on it was decreasing
on the reducing of heat stress but on the same time it was also increasing the quantity of catalyse
enzymes and decreasing on recovery and the phenomena was similar for SOD enzymes.

Andreia did an experimental work on the response of antioxidant enzymes of wheat plant
under stress. The work was basically done to improve the cultivation of wheat as it is good
source of protein and carbohydrate for human and as per demand the cultivation decreased by
their work it was concluded that the crop is being damaged due to different environmental stress
and formation of (ROS) species which were damaging plant and reducing the growth but they
also came to know that where plant is being damage it is also developing a mechanism of
recovering called antioxidant enzyme. When plant was under stress there was increased in this
enzyme production to recover plant.
Materials and Methods Chapter 3

Experimental procedure of this research is based on the following methodology.

Wheat of two genotypes (Umeed and Zarghoon) were grown in Quetta city for conducting
research work. Which were grown about 3 months under control treatment of silicon in normal
and salt stress condition. Samples of wheat leaves for the research were collected from pots.
About 40 samples of flag leaves of two different varieties were examined for antioxidant
enzymes and total protein count. About 3 replicates of each variety were examined. The work
was designed into three phase.

Phase I : To grow wheat seeds under normal and salt stress with control treatment of silicon.
Phase II : The flag leaves were examined for the antioxidant enzyme. (Superoxide
Dismutase).
Phase III: The total Protein quantity was measured.

The layout of this search work is as follow:

Phase I :
Germination period of Wheat cultivars
In the first step the Wheat seeds were grown in earthen pots with normal and salt stress. The salt
resistant Umeed and salt sensitive Zarghoon were allowed to grow for three months till the flag
leaves were appeared with silicon treatment. The wheat cultivars were obtained from Quetta
research Institute. The water and minerals were checked properly. The wheat were cold
resistance according to the weather of Quetta, Pakistan. The NaCl was mixed in pots while the
soil was filled in pots. Growth parameters was regularly checked.

A: Varieties
T1 = Umeed ( salt resistant )
T2 = Zarghoon (salt sensitive)

Figure: Wheat plant cultivation and collection of flag leaves

B : Silicon levels
Treatment I. = 5 mL-1
Treatment II. = 10 mL-1
Treatment III. = 15 mL-1

C : salt stress
Normal = 2 dSm-1
Salt stress = 5 dSm-1

Phase II :
Activity of antioxidant enzymes

As soon as the flag leaves were brought to lab, they were cared gently and were kept in small
packing bag. Prior to treatment the leaves were weighed on digital balance. About 0.5 grams of
each flag leaf were used as sample. For the enzyme extraction (antioxidant enzymes) fresh leaves
of the plant of wheat were ground in pestle and motor (0.5 g) which was cooled in refrigerator
about an half an hour. Then in presence of 50 mM cooled phosphate buffer having PH (7.8)
leave were slowly and carefully grinded. After this solution was centrifuged at 15000 x g for
about 20 min at 40 C and the supernatant was used for enzyme assays.
Figure: Flag leaves sample collection and packing for the experiment work.

Superoxide Dismutase (SOD):

Method of Giannopolitis and Ries (1977)

Principle:

For the (SOD) activity the method of Giannopolitis and Ries (1977) was used for monitoring
the inhibition of photochemical reduction of (NBT) Nitro blue tetazolium at 560 nm.
Figure: Test tubes containing enzyme assay and reaction mixture for SOD activity

Reagents:

1. Nitro Blue Tetazolium (50 µM)

2. Phosphate Buffer (pH. 7.8)
3. Ethanol ( 5 µM)
4. Riboflavin (1.3 µM)
5. Methionine 13 mM
6. EDTA (75 nm)
Figure : Preparation of Reagents for the determination of SOD enzyme activity

Procedure:

The activity of (SOD) was determined by taking the solution that contains 50 ul of the
extracted enzyme and 50 ul of NBT (that should be already dissolved in ethanol). About 1.3
ul riboflavin, 13 mM of methionine 75 mM of EDTA and at last was added 50 mM of
phosphate buffer (7.8). Now this solution was kept in fluorescent lamp of 30 W and was
taken out after 5 mints. Another solution which was not having only the extraction of the
plant but was having the reaction mixture was also kept in dark and now the absorbance of
irradiated solution was read in UV-Vis spectroscopy (IRMECOU-2020) which is available in
SBKW University. The one unit of SOD was used to cause 50 % inhibition of the rate NBT
reduction at 560 nm by comparing the extracted enzyme solution with the second tube
lacking the plant extract.
 Fig: Flag leave after grinding and centrifuge Enzyme assay .

Phase III :
Protein Quantification by Bradford method (1976)

Principle:
For total protein quantification from the wheat leaf extract Bradford method (1976) will be used
and the bovine serum albumin (BSA) will be used as standard

Reagents:
1.Coo massive Blue (0.1 g)
2. Ethanol (50 ml)

3. Distil water (200 ml)

4. Bar ford Reagent (10 ml)

5. Albumin serum ( Different dilution )

Fig: Enzymes assay after centrifuge for determining SOD activity.

Preparation of reagents

1. Coo massive Blue.

The solution was prepared by dissolving 5 gm of brilliant coo massive blue in 50 ml of

Ethanol then it was diluted by 200 ml of distill water.

2. Buffers 7.0 pH

The buffer solution was prepared by mixing 100 ml of KH2PO4 solution with 100 ml of
K2HPO4 and the pH was maintain by using pH meter (7.0).

3 KH2PO4:

About 6.968 gm of KH2PO4 was dissolved in 200 ml of distill water to prepare 0.2 M stock
solution of KH2PO4.
4 K2HPO4:

About 5.44 gm of K2HPO4 was dissolved in 200 ml of distill water to prepare 0.2 M stock
solution of KH2PO4.

5 Serum Albumin

To prepare stock solution of albumin 1 ml of serum was dissolved in 10 ml of NaCl. And then
further dilutions were made for standard curve. (0.8, 0.6, 0.4, 0.2, 0.1 and 1 ml )

Fig: Cooled enzymes Assay for protein quantification.

Procedure:

The Bradford reagent was prepared by 0.1 g of Coo massive Blue was dissolved in 50 ml of
ethanol 50 ml of distil water and was stirred for 10 mints and then was diluted with 200 ml distil
water and filtered. After reagent preparation 10ml of Bradford concentrate solution was diluted
1:5 to prepare the working solution .Then dilution of Albumin serum (BSA) for standard curve
of 0.8, 0.6, 0.4, 0.2, 0.1 and 1 ml from (BSA) stock solution. Some more test tubes were labeled
for the leaf extract and one for the blank. These test tubes was containing 100 ul of protein
sample and 5 ml of Bradford reagent while the blank was having 100 ul of distil water and 0.1
ml (BSA) solution and was incubated 20 mints in dark and then absorbance was measured at 595
nm by spectrometer. Standard graph was plotted by dilution.
Fig: Prepration of test tubes having enzyme assay along with co massive blue for protein
determination.

UV-Vis Spectroscopy used for the determination of protein quantity in wheat cultivars.
Results and Discussion Chapter 3

The experiment which was conducted on two different varieties of wheat, Umeed and Zarghoon
in silicon under salt stress. The plant were grown about 3 months till the flag leaves were
appeared after which were used to determine the activity of SOD and Protein quantification.

1. Superoxide Dismutase (SOD)

The data showed that two varieties which were grown under control treatment of silicon were
showing different values in salt and salt free condition.

Superoxide Dismutase Activity In Control

30.000
SOD (Unit /mg of protein)

25.000

20.000

15.000

10.000

5.000

0.000
C1 C2
Control(C1= without salt stress, C2= with salt stress)

V1 Umeed V2 Zarghoon

Figure .1 Changes in the activity of antioxidant enzymes SOD in both cultivars of Wheat in
saline and non saline condition.
Figure 1.2 Effect of silicon on activity of Antioxidant enzymes SOD in both wheat cultivars
under saline and non saline condition.

The SOD activities in the two wheat cultivars were showing difference in silicon and control
condition. Salt stress was having negative impact on plant and was decreasing the growth in both
varieties. But when compared both treatments it was observes that under non saline conditions
the SOD remained same in both wheat cultivars. But under saline conditions the SOD activity
was highly decreasing. On the application of Si in saline stress it was having adverse effect the
plant was recovering the damages. In both wheat cultivars Zarghoon was more effect in both set
of condition as compared to Umeed. The SOD activity was higher in Umeed.
Table.1. Application of silicon on two wheat cultivars under saline and non saline
conditions.
Discussion:

Salt stress decreases the growth of plant and the reduction may involves a series of
Physiological response which may involves the ion concentration, mineral deficiency, regulation
of stomata. (Greenway & Munns, 1980). Beside these changes oxidative stress also increases
due to saline condition (Goltsev, Zaharieva, Lambrev, Yordanov, & Strasser, 2003). SOD is the
first enzyme to get active in the salt stress.(Tester & Davenport, 2003). When salt stress
increases it causes increase in Na+ ion concentration in flag leaves of Wheat plant of both
cultivars. When the results were compared of both cultivars in saline and non saline condition, it
was clear that in non saline condition the SOD activity were seen normal in both wheat cultivars
but as soon plant was introduced the activity was decreasing.
Among Wheat cultivars Umeed (salt tolerant) was having more resistant for Na+ concentration
but the Zarghoon (salt sensitive was weak to salt stress and high concentration on Na+.
These ionic stress was responsible for the generation of ROS (Zhang, Shen, & Xu, 2003). H2O2
can be defined as non -radical of ROS which was formed when O molecule reduced 2 electrons,
this may take place on mitochondria or chloroplast. According to the new search biotic stress are
mainly responsible for the formation of H2O2, this leads toxicity in plant. (Prakash, Suri,
Upadhyay, & Singh, 2007)
The figure 1 was showing that activity of SOD was decreasing in saline condition as
compared to non saline condition that was indicating that SOD was being suppressed by salt
stress and caused weak defense mechanism. Antioxidant enzymes formation in both wheat
cultivars was decreased and ionic stress with ROS level were increased in both cultivar. This was
similar to the work of (Tester & Davenport, 2003).
Silicon though it is not essential element in development of wheat but its application is
beneficial for the growth of plant. It increases the potential of plant against salt stress.(V. Singh,
Pallaghy, & Singh, 2006). The effect of silicon can be observed in fig 1.2 under saline and non
saline condition. When silicon was applied in pots of plant it was improving the SOD activity
which was suppressed due to salt stress. SOD is an important antioxidant enzyme for the plant in
biotic stress. But it was decreasing in saline condition as compared to silicon application.
Among both Cultivar Umeed was having more SOD quantity then Zarghoon. From the research
it was conducted that wheat cultivars were affected in presence of salt but silicon was
significantly improving the growth and Umeed was more improved then Zarghoon in saline and
non saline condition. Similar result were observed in findings of (Tahir et al., 2006)

6 Total Protein Quantification:

When total protein quantification was carried out in two wheat cultivars. The following results
were concluded.

35.000
30.000
25.000
20.000
15.000
10.000 V1 Umeed
5.000 V2 Zarghoon
0.000
C1 C2 S1 S2
control control+ salt Silicon Silicon+ salt
units Units units Units
CAT IU mg of protein

Fig 2.1 Total protein quantification in two wheat cultivars Umeed and zarghoon under
saline and non saline condition.

From the above table it was observed that the total protein quantity was normal in non saline
condition but as the plant was introduced to the salt stress the level of protein was decreasing as
per increase in salt stress. As par silicon application the protein quantity was significantly
improving in non saline condition. On salt stress among both cultivars Umeed was showing
better result than Zarghoon.
 Soluble Proteins (mg/g Fresh Wt) Protein in flag leaves of Control
1.57
1.56
1.55
1.54
1.53
1.52
1.51
1.5
C1 C2
Control(C1= without salt stress, C2= with salt stress)

V1 Umeed V2 Zarghoon

Fig 2.2 Protein quantification in 2 wheat cultivars with control treatment in saline and non
saline condition.

Protein in flag leaves with Silicon

Soluble Proteins (mg/g Fresh Wt)

supplementation
1.65
1.63
1.61
1.59
1.57
1.55
1.53
1.51
S1 S2
Silicon supplementation (S1= without salt stress, S2= with salt stress)

V1 Umeed V2 Zarghoon

Fig 2.3 Protein quantification in 2 wheat cultivars with silicon application in saline and
non saline condition.
 Protein in the flag leaves
C1=control (Without salt
stress) C2=control (With salt stress)
S.No Variety Absorbance Concentration Absorbance Concentration
1 V1 Umeed 3.311 1.49 3.316 1.502
2 V1 Umeed 3.321 1.51 3.314 1.499
3 V1 Umeed 3.312 1.50 3.309 1.492
4 V1 Umeed 3.324 1.51 3.306 1.488
5 V1 Umeed 3.323 1.51 3.304 1.485
Average 3.3182 1.504913495 3.3098 1.493287197
Std Error 0.00278209 0.003850637 0.002289105 0.003168311
1 V2 Zarghoon 3.312 1.50 3.282 1.45
2 V2 Zarghoon 3.318 1.50 3.289 1.46
3 V2 Zarghoon 3.315 1.50 3.301 1.48
4 V2 Zarghoon 3.314 1.50 3.304 1.49
5 V2 Zarghoon 3.313 1.50 3.306
Average 3.3144 1.499653979 3.2964 1.474740484
Std Error 0.00102956 0.001425001 0.004654031 0.006441565
S1=Silicon supplementation (Without salt S2=Silicon supplementation (With salt
stress) stress)
S.No Variety Absorbance Concentration Absorbance Concentration
1 V1 Umeed 3.365 1.57 3.3324 1.52
2 V1 Umeed 3.364 1.57 3.3323 1.52
3 V1 Umeed 3.349 1.55 3.327 1.52
4 V1 Umeed 3.358 1.56 3.323 1.51
5 V1 Umeed 3.362 1.57 3.3324 1.52
Average 3.3596 1.562214533 3.32942 1.520442907
Std Error 0.00290861 0.004025755 0.001912172 0.002646604
1 V2 Zarghoon 3.362 1.57 3.3219 1.51
2 V2 Zarghoon 3.363 1.57 3.3321 1.52
3 V2 Zarghoon 3.339 1.53 3.312 1.50
4 V2 Zarghoon 3.341 1.54 3.316 1.50
5 V2 Zarghoon 3.343 1.54 3.317 1.50
Average 3.3496 1.548373702 3.3198 1.507128028
Std Error 0.0053066 0.007344775 0.003455575 0.004782803

Table.2 Effect of silicon on protein quantification of two wheat cultivars grown under
saline and non saline conditions.
Discussion:

Crops which are usually grown in saline conditions may suffer under various problems that
may include toxicity, loss of production, decrease in height of roots and shoots.(Perez-Alfocea et
al.,1996). Increase in salt stress directly decreases the minerals contents which are necessary for
plant growth. ( Graftan and Grieve.,1996). The salt stress is also responsible for reduced
chlorophyll and damages the photosynthesis reaction in flag leaves.( Parida and Das., 2005). On
the other hand it damages osmotic potential of plant by increasing ion concentration and solute
synthesis. Among these organic solute synthesis sugar is well known for reducing osmotic
damage (P. Ahmad, Jaleel, Azooz, & Nabi, 2009; Ashraf & Harris, 2004).
Excess amount of salt can even cause problem in germination of seed and can lead to cell
death. To solve all these problems various studies have been carried out by different scholars so
far to understand the actual effect on wheat crop. And to determine the concentration of salt that
damages the growth and development of plant, but till now no study has confirmed the précised
concentration of salt effecting the wheat cultivar (Nowak et al., 2008). But all these researchers
have concluded that salt affects the plant depending on its species and biotic factors.
Wheat has wide Variety of species both salt tolerant and salt sensitive which are grown
worldwide with different environmental conditions. But the production varies in presence of
biotic stress especially in presence of salt concentration. This research work was basically
conducted to measure those effects of salt on two wheat cultivars in saline and non saline
condition with silicon presence and control treatment. (Dionisio & Tobita, 1999)
Table 2.1 is showing the concentration of protein in two wheat cultivars in control and silicon
application. When plants were grown in saline and non saline condition with control treatment it
was observed that in non saline condition the total protein was normal with stable condition, but
as the plants were brought to saline condition the protein concentration was decreasing even
below average condition that was an alarming situation for plant growth. When these plants were
provided silicon treatment the result was surprising there was huge change in protein
concentration in saline condition which was decreasing in control condition.
Silicon was having positive effect on total protein quantification and recovery of plant from the
damages which was caused by salt stress. That was an important fact for the plant growth and
production. The similar finding of silicon improvement can be seen by (Al-aghabary et al.,2004)
in tomato, (Zhu et al.,2004 ) worked on cumber grown in saline condition with silicon
application. Silicon basically increase the production of antioxidant enzyme SOD , CAT , POX
that help the plant to recovery under salt stress ( leeet et al., 2001) . among both wheat cultivars
Umeed was showed high concentration of protein than Zarghoon in leaf under saline condition
with silicon application.
The study was showing that application of silicon can enhance the production of plant. (Tahir et
al., 2006). Silicon can be applied to wheat plant by different methods. That may either involve
by soil treatment or exogenous application. When the plant receives silicon treatment under
saline condition it starts to recover and maintain the growth and production. Its major function is
to remove the toxicity of cell by increasing the membrane function.(Ashraf & Harris, 2004)
Salt stress directly reduces the water content in flag leaves due to increase in transpiration rate.
( Munns , 2005) . The same results were seen in both wheat cultivars Umeed and zarghoon. In
control treatment the plant were showing better result unless the plant were exposed to salt stress.
This exposure leads to reduce in water content and si-application was minimizing the stress and
this was a positive sign for the plant production and development. This experiment confirmed
that Umeed performed better than Zarghoon. And Umeed was salt tolerant among both cultivars.
Salt stress decrease the pigments in Wheat flag leaves and decreases the photosynthesis
process. In young plant the salt stress completely damage the germination rate and stops the
growth process of plant from seedling period. As compared to young plant when a mature plant
is introduced to salt stress the damages are more visible , it can major problem such as decrease
in total protein, soluble carbohydrates, and reduced in chlorophyll content. (R. Ahmad, Zaheer,
& Ismail, 1992).
Plant in control treatment were affected more as compared to silicon application. (Handa,
Bressan, Handa, Carpita, & Hasegawa, 1983; Hernandez, Olmos, Corpas, Sevilla, & Del Rio,
1995). The decrease in total protein quantity in fresh leaves is mainly caused because of
insufficient amino acid into making of protein or impropriate site attachment of amino acid. This
stress leads to increase in enzymes quantity such as proteolytic or formation of monosome or
polysome and it can also form absiccic acid which may involve in RNa inhibition and stop
protein synthesis (N. Singh & Shepherd, 1985).
Protein play vital role in the cell structure and transportation along the cell. (Handa et al., 1983;
Hasegawa, Bressan, Zhu, & Bohnert, 2000).
Damage in the protein structure or protein inhibition effects cell membrane structure and stop
nutrition intake that reduce the plant growth.(Hanson & Trewavas, 1982).
Silicon application may increase the protein formation in stress condition of plant which leads to
nutrition transport and may also involve in protein synthesis in cell which will help the cell
membrane to defend the cell under saline condition. Silicon can also improve the chlorophyll
content in leaves that will have positive effect on photosynthetic reaction and will also increase
the water transportation this can be seen in finding of many workers (Seemann & Critchley,
1985). It was also concluded by other researchers that silicon can enhance the rate of carbon
dioxide intake and production of carbohydrates in flag leaves.(Liang, Chen, Liu, Zhang, & Ding,
2003). The current study was showing that with application of silicon the plant was improving
under saline condition and these was similar as (Hattori et al., 2003)
 Conclusion

Agriculture plays strategic role in the progress and development of country’s economy and a
country like Pakistan mostly depend on its agriculture sector. Approximately 28 percent of
country economy depends on production of crops like Wheat, Bajwra and Jawar. Most of crops
are cultivated in Punjab and Sindh. Province like Balochistan is having largest area but the land
is barren and unfertile with diversified soil and limited natural resources. The cultivation here is
also effected by physical factors such as cold temperature, salinity and drought. These all factors
effect the production of crops. In order to overcome all these factors and increase the production
many research work has been carried out at different levels. This research was also carried out on
the following objectives:

7 To determine the effect of salts stress on two wheat cultivars Umeed and zarghoon.
8 Change in the growth and production of both wheat cultivars after application of silicon.
9 Effects of silicon on activity of antioxidant enzymes SOD and total protein quantification.

In this experiment two wheat cultivars Umeed (salt tolerant) and Zarghoon (salt sensitive) were
grown with control and silicon treatment in both saline and non saline conditions. The plants
were grown in pots where salt was provided by mixing with soil. The pots were divided into two
main groups
 Control treatment with saline and non saline condition.
 Silicon treatment with saline and non saline condition.

Flag leaves were collected to determine the effect of silicon on the activity of antioxidant
enzymes SOD for recovering of plant under the stress of salt stress. From this research it was
concluded that Umeed was improving under saline stress better then Zarghoon on silicon
application and under control treatment it was normal and in saline condition the plant was
decreasing its production rate. This was showing the positive impact of silicon on wheat cultivars
under salt stress.
Silicon can be used for the betterment and better production of wheat cultivars.
References Chapter4

Ahmad, P., Jaleel, C. A., Azooz, M., & Nabi, G. (2009). Generation of ROS and non-enzymatic
antioxidants during abiotic stress in plants. Bot Res Int, 2(1), 11-20.

Ahmad, R., Zaheer, S. H., & Ismail, S. (1992). Role of silicon in salt tolerance of wheat
(Triticum aestivum L.). Plant science, 85(1), 43-50.

Ahmed, M., & Hussain, F. (2013). Chemical composition and biochemical activity of Aloe vera
(Aloe barbadensis Miller) leaves. Int. J. Chem. Biochem. Sci, 3, 29-33.

Ashraf, M., & Harris, P. (2004). Potential biochemical indicators of salinity tolerance in plants.
Plant science, 166(1), 3-16.

Bonifacio, A., Martins, M. O., Ribeiro, C. W., Fontenele, A. V., Carvalho, F. E.,
MARGIS‐PINHEIRO, M., & Silveira, J. A. (2011). Role of peroxidases in the
compensation of cytosolic ascorbate peroxidase knockdown in rice plants under abiotic
stress. Plant, cell & environment, 34(10), 1705-1722.

Dionisio-Sese, M. L., & Tobita, S. (1998). Antioxidant responses of rice seedlings to salinity
stress. Plant science, 135(1), 1-9.

Dionisio, S., & Tobita, S. (1999). Antioxidative responses of shoots and roots of wheat to
increasing NaCl concentration. J. Plant Physiol, 155, 274-280.

El-Fouly, M. M., Mobarak, Z. M., & Salama, Z. A. (2011). Micronutrients (Fe, Mn, Zn) foliar
spray for increasing salinity tolerance in wheat Triticum aestivum L. African Journal of
Plant Science, 5(5), 314-322.
Esfandiari, E., Shekari, F., Shekari, F., & Esfandiari, M. (2007). THE EFFECT OF SALT
STRESS ON ANTIOXIDANT ENZYMES'ACTIVITY AND LIPID PEROXIDATION
ON THE WHEAT SEEDLING. Notulae Botanicae Horti Agrobotanici Cluj-Napoca,
35(1), 48.

Goltsev, V., Zaharieva, I., Lambrev, P., Yordanov, I., & Strasser, R. (2003). Simultaneous
analysis of prompt and delayed chlorophyll a fluorescence in leaves during the induction
period of dark to light adaptation. Journal of Theoretical Biology, 225(2), 171-183.

Greenway, H., & Munns, R. (1980). Mechanisms of salt tolerance in nonhalophytes. Annual
review of plant physiology, 31(1), 149-190.

Hameed, A., Sheikh, M. A., Jamil, A., & Basra, S. M. A. (2013). Seed priming with sodium
silicate enhances seed germination and seedling growth in wheat (Triticum aestivum L.)
under water deficit stress induced by polyethylene glycol. Pak. J. Life Soc. Sci, 11, 19-24.

Handa, S., Bressan, R. A., Handa, A. K., Carpita, N. C., & Hasegawa, P. M. (1983). Solutes
contributing to osmotic adjustment in cultured plant cells adapted to water stress. Plant
Physiology, 73(3), 834-843.

Hanson, J., & Trewavas, A. (1982). Regulation of plant cell growth: the changing perspective.
New Phytologist, 90(1), 1-18.

Hasegawa, P. M., Bressan, R. A., Zhu, J.-K., & Bohnert, H. J. (2000). Plant cellular and
molecular responses to high salinity. Annual review of plant biology, 51(1), 463-499.

Hattori, T., Inanaga, S., Tanimoto, E., Lux, A., Luxová, M., & Sugimoto, Y. (2003). Silicon-
induced changes in viscoelastic properties of sorghum root cell walls. Plant and Cell
Physiology, 44(7), 743-749.
Hernandez, J., Olmos, E., Corpas, F., Sevilla, F., & Del Rio, L. (1995). Salt-induced oxidative
stress in chloroplasts of pea plants. Plant science, 105(2), 151-167.
Joshi, A., Mishra, B., Chatrath, R., Ferrara, G. O., & Singh, R. P. (2007). Wheat improvement in
India: present status, emerging challenges and future prospects. Euphytica, 157(3), 431-
446.

Liang, Y., Chen, Q., Liu, Q., Zhang, W., & Ding, R. (2003). Exogenous silicon (Si) increases
antioxidant enzyme activity and reduces lipid peroxidation in roots of salt-stressed barley
(Hordeum vulgareL.). Journal of plant physiology, 160(10), 1157-1164.

Marakli, S., Temel, A., & Gozukirmizi, N. (2014). Salt stress and homobrassinosteroid
interactions during germination in barley roots. Notulae Botanicae Horti Agrobotanici
Cluj-Napoca, 42(2), 446.

Miesle, T., Proctor, A., & Lagrimini, L. (1991). Peroxidase activity, isoenzymes, and tissue
localization in developing highbush blueberry fruit. Journal of the American Society for
Horticultural Science, 116(5), 827-830.

Prakash, D., Suri, S., Upadhyay, G., & Singh, B. N. (2007). Total phenol, antioxidant and free
radical scavenging activities of some medicinal plants. International Journal of Food
Sciences and Nutrition, 58(1), 18-28.

Ramagopal, S. (1990). Inhibition of seed germination by salt and its subsequent effect on
embryonic protein synthesis in barley. Journal of Plant Physiology, 136(5), 621-625.

Sadeghipour, O., & Aghaei, P. (2012). Impact of exogenous salicylic acid application on some
traits of common bean (Phaseolus vulgaris l.) under water stress conditions. Int J Agric
Crop Sci, 4(11), 685-690.
Seemann, J. R., & Critchley, C. (1985). Effects of salt stress on the growth, ion content, stomatal
behaviour and photosynthetic capacity of a salt-sensitive species, Phaseolus vulgaris L.
Planta, 164(2), 151-162.

Singh, N., & Shepherd, K. (1985). The structure and genetic control of a new class of disulphide-
linked proteins in wheat endosperm. Theoretical and Applied Genetics, 71(1), 79-92.

Singh, V., Pallaghy, C. K., & Singh, D. (2006). Phosphorus nutrition and tolerance of cotton to
water stress: II. Water relations, free and bound water and leaf expansion rate. Field
Crops Research, 96(2-3), 199-206.

Solatni, Z., Shekari, F., Jamshidi, K., Fotovat, R., & Azimkhani, R. (2012). The effect of silicon
on germination and some growth characteristics of salt-stressed canola seedling. Int J
Agron Agric Res, 2, 12-21.

Tahir, M. A., Rahmatullah, T., Aziz, M., Ashraf, S., Kanwal, M. M., & Maqsood, M. A. (2006).
Beneficial effects of silicon in wheat (Triticum aestivum L.) under salinity stress.
Pakistan Journal of Botany, 38(5), 1715-1722.

Tanou, G., Molassiotis, A., & Diamantidis, G. (2009). Hydrogen peroxide-and nitric oxide-
induced systemic antioxidant prime-like activity under NaCl-stress and stress-free
conditions in citrus plants. Journal of Plant Physiology, 166(17), 1904-1913.

Tester, M., & Davenport, R. (2003). Na+ tolerance and Na+ transport in higher plants. Annals of
botany, 91(5), 503-527.

Tuna, A. L., Kaya, C., Higgs, D., Murillo-Amador, B., Aydemir, S., & Girgin, A. R. (2008).
Silicon improves salinity tolerance in wheat plants. Environmental and Experimental
Botany, 62(1), 10-16.
Zhang, H., Shen, W.-B., & Xu, L.-L. (2003). Effects of nitric oxide on the germination of wheat
seeds and its reactive oxygen species metabolisms under osmotic stress. ACTA
BOTANICA SINICA-CHINESE EDITION-, 45(8), 901-905.