EFFECT OF SALICYLIC ACID AND GIBBERELLIC ACID ON ...

EFFECT OF SALICYLIC ACID AND GIBBERELLIC ACID ON

MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES IN

DROUGHT-STRESSED WHEAT (TRITICUM AESTIVUM L.)

CROP

ANEELA ULFAT

Regd. No. 2004-Gkig-630

Session (2012-2015)

Department of Botany

Faculty of Sciences

University of Azad Jammu and Kashmir, Muzaffarabad, Pakistan

ii

EFFECT OF SALICYLIC ACID AND GIBBERELLIC ACID ON

MORPHOLOGICAL AND PHYSIOLOGICAL CHANGES IN

DROUGHT-STRESSED WHEAT (TRITICUM AESTIVUM L.)

CROP

By

ANEELA ULFAT

(Regd. No: 2004-Gkig-630)

A Thesis

submitted in partial fulfillment of the requirement for the degree of

Doctor of Philosophy

in

Botany

(Session 2012-2015)

Department of Botany

Faculty of Sciences

University of Azad Jammu and Kashmir Muzaffarabad, Pakistan

vii

TO MY LOVING PARENTS

Whose devotion and inspiration towards knowledge served me as ray of light, who

always pray for my success and prosperity. Whose encouragement, sacrifices and

generous support both morally and financially enabled me to achieve this goal.

viii

CONTENTS

Page No.

LIST OF CONTENTS vii-xii

LIST OF ABBREVIATIONS xiii

ACKNOWLEDGEMENT xv

ABSTRACT xx

1. INTRODUCTION 01

1.1CLIMATE CHANGE AND ITS EFFECT ON AGRICULTURE 02

1.2 PAKISTAN STATUS IN TERM OF CLIMATE CHANGE 03

1.3 ABIOTIC STRESSES 03

1.4 STRATEGIES TO OVERCOME STRESSES 04

1.5 JUSTIFICATION OF THE STUDY 05

2. REVIEW OF LITERATURE 07

2.1 WORLD WHEAT MARKET 07

2.2 WHEAT STATUS IN PAKISTAN AND AZAD JAMMU

AND KASHMIR 07

2.3 CLIMATE CHANGE AND ITS IMPACT ON AGRICULTURAL

CROPS 08

2.4 DEMAND AND AVAILABILITY OF WATER 08

2.5 DROUGHT STRESS 10

2.5.1 Problems during Drought 11

2.5.2 Strategies to Cope Drought Stress 13

2.5.2.1 Priming 14

2.5.2.2 Hormonal seed priming 15

2.5.2.2.1 Role of salicylic acid 15

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2.5.2.2.2 Role of gibberellic acid 16

2.5.2.3 Priming effect on biochemical, morphological and

physiological processes 17

2.6 TRANSGENERATIONAL EFFECT OF EFFECT OF ELEVATED

CO2 19

2.7 CONSIDERATIONS FOR THE FUTURE 19

3. MATERIAL AND METHOD 21

3.1 STUDY 21

3.2 PLANT MATERIAL 22

3.3 SEED PRIMING TREATMENTS 22

3.4 EXPERIMENTAL SET-UP 22

3.5 SEED BIOCHEMISTRY ATTRIBUTES 22

3.5.1 Oxidative Enzyme Assays 22

3.5.1.1 Protease activity 22

3.5.1.2 Estrase activity 23

3.5.1.3 Amylase activity 23

3.5.1.4 Superoxide dismutase activity 23

3.5.1.5 Peroxidase activity 23

3.5.1.6 Catalase activity 24

3.6 MORPHOLOGICAL ATTRIBUTES 25

3.7 BIOCHEMICAL ATTRIBUTES 25

3.7.1 Hydrolytic antioxidant 25

3.7.2 Enzymetic antioxidant 26

x

3.8 PHYSIOLOGICAL ATTRIBUTES 26

3.8.1 Malondialdehyde Contents 26

3.8.2 Total Oxidant Status 25

3.8.3 Relative Water Contents 26

3.8.4 Cell membrane thermostability 27

3.8.5 Pigments Analysis 27

3.8.5.1 Chlorophyll a,b, Carotenoid and Anthocyanin 27

3.9 METABOLITIES ACCUMULATION 28

3.9.1 Total soluble Sugars 28

3.9.2 Total Proteins 28

3.9.3 Proline accumulation 28

3.10 Mineral Elements 28

3.10.1 Potassium and calcium ratio 28

3.11 Seed Quality attributes 29

3.11.1 Wet Gluten (%) 29

3.11.2 Gluten Index (%) 29

3.11.3 Falling Number (Sec) 29

3.11.4 Proteins, Moisture and Starch (%) 30

3.12 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE

GELS 30

3.13 TRANS GENERATIONAL EFFECT OF ELEVATED CO2

ON WHEAT AT ANTITHESIS DROUGHT STRESS 30

3.13.1 Gaseous Exchange and Water Relations 30

3.13.2 Yield and Yield Components 31

xi

3.13.3 Key Enzyme of Carbohydrate Metabolism (C Enzymes) Assays 32

3.13.4 Key Enzyme of Carbohydrate Metabolism (A Enzymes) Assays 34

3.14 Statistical Analysis of Data 35

4. RESULTS AND DISCUSSION 36

4.1 SEED BIOCHEMISTRY 36

4.1.1 Oxidative enzymes assay 36

4.1.1.1 Protease activity 36


4.1.1.3 Esterase activity 39


4.1.1.5 Peroxidase activity 40


4.1.1.7 Cluster analysis based on seed biochemistry attributes 42

4.1.2 Conclusion 43

4.2 MORPHOLOGICAL ATTRIBUTES 44

4.2.1 Cluster analysis based on Morphological attributes 66

4.2.2 Conclusion 67

4.3 BIOCHEMICAL AND PHYSIOLOGICAL ATTRIBUTES 67

4.3.1 Oxidative Enzymes 67

4.3.1.1 Estrases activity 67


4.3.1.3 Protease Activity 70


4.3.1.5 Peroxidase Activity 72

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4.3.1.7 Ascorbate peroxidase activity 75

4.3.2 Cluster Analysis based on oxidative enzymes attributes in

flag leaf of wheat 76

4.3.3 Conclusion 77

4.4 PHYSIOLOGICAL ATTRIBUTES 79

4.4.1 Malondialdehyde Contents 79

4.4.2 Total oxidative status 79

4.4.3 Relative water contents 80

4.4.4 Cell membrane thermostability 81

4.4.5 Photosynthetic pigments 82

4.4.6 Conclusion 85

4.5 METABOLITE ACCUMULATION AND MINERAL ELEMENTS 85

4.5.1 Sugar contents 85

4.5.2 Protein contents 87

4.5.3 Proline accumulation 87

4.5.4 Potassium ratio 88

4.5.5 Calcium ratio 89

4.5.6 Cluster Analysis Based on Biochemical and Physiological

Attributes 91

4.5.7 Conclusion 92

4.6 WHEAT GRAIN QUALITY 93

4.6.1 Wet gluten contents 93

4.6.2 Gluten index 94

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4.6.3 Falling number 96

4.6.4 Seed Storage Protein 98

4.6.5 Moisture Contents of seed 100

4.6.6 Starch content of seed 101

4.6.7 Conclusion 104

4.7 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE

GELS 104

4.8 TRANSGENERATIONAL EFFECT OF ELEVATED

CARBONDIOXIDE ON METABOLISM OF WINTER WHEAT

EXPOSED TO ANTHESIS DOUGHT 107

4.8.1 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on Yield

Attributes 107

4.8.2 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on physiological

attributes 109

4.8.3 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on Invertases

and Susy activity 111

4.8.4 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on the key

enzymes for Carbohydrate (C) Metabolism 116

4.8.5 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on (a) key

enzymes activities for Carbohydrate Metabolism 124

4.8.6 Conclusion 132

SUMMARY 136

LITERATURE CITED 138

APPENDIX 168

xiv

List of Abbreviation

SA Salicylic acid

GA Gibberellic acid

SOD Superoxide dismutase

POD Peroxidase

CAT Catalase

APX Ascorbate Peroxidase

GR Glutathione reductase

RWC Relative Water Contents

TOS Total Oxidant Status

MDA Malondialdehyde

CMT Cell Membrane Thermostability

K+ Potassium

Ca+ Calcium

CO2 Carbon dioxide

(a[CO2]) Ambient Carbon dioxide

(e[CO2]) Elevated Carbon dioxide

Tr Transpiration rate

An Photosynthesis

Gs Stomatal exchange

PVP Polyvinylpolypyrrolidone

CWInv Cell wall invertase

VACInv Vacuolar invertase

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CYTInv Cytoplasmic invertase

SUSY Sucrose synthase

AGPase ADP-glucose pyrophosphorylase

UGPase UDP-glucose pyrophosphorylase

PGM Phosphoglucomutase

PGI Phosphoglucoisomerase

G6PDH Glucose-6-phosphate dehydrogenase

Ald Aldolase

HXK Hexokinase

FK Fructokinase

PFK Phosphofructokinase

TAP Total antioxidant potential

xvi

ACKNOWLEDGEMENTS

Words are bound and knowledge is limited to praise ALMEIGHTY ALLAH,

the Lord of the world, the Omnipotent, the Beneficent, who gave me the requisite

potential and diligence for the successful accomplishment of this task. Special

praise to HAZRAT MUHAMMAD (PBUH), who is forever the source of

knowledge for whole mankind.

I am grateful for the financial assistance by Punjab Government (fee

reimbursement scheme) and Higher Education Commission (HEC) Islamabad

Pakistan, under the IRSIP PhD Fellowship Scheme.

I’m appreciative to my parents for all their love, prayers, sacrifices,

sympathies, guidance and encouragement which served as “beacon of hope” all

along my work. I offer my richest and heartiest gratitude to them.

I confess here that thesis would not be completed without the principal

contribution, affection, untiring help, invigorating encouragement and moral

support of my affectionate supervisor Prof. Dr. Syed Abdul Majid. I have never

seen such a polite and humble person. It is a great pleasure for me to be his student.

I owe special thanks and pray for his long life.

Here, I would like to express my gratitude to all my respected teachers of the

Botany Department specially, Dr. Ghulam Murtaza, Prof. Dr. M. Qayyuam Khan,

Dr. Rehana kausar, Dr. Hamayun shaheen, Dr. Sidiqa Firdous, Mis. Sidra Qayyum,

Dr. Rizwan Taj, Dr. Tariq Habib and Dr. Ejaz Dar for their direction, meaningful

suggestion and helpful attitude during the course of this degree. I extend my cordial

and profound thanks to my M.sc supervisor Dr. Altaf Hussain for his support and

guidance.

xvii

I am extremely grateful to Dr. Hamayun Shaheen, my teacher and member of

my supervisory committe for his continues guidance and support throughout my

Ph.D research till the completion mission impossible. I feel extremely privileged

for his cooperation, valuable suggestion, sympathetic attitude, words of

encouragement and advice over the course of my study. I’m also obliged to my co-

supervisor, Dr. Amjad Hameed, Principal Scientist (NIAB) for his help, guidance

and cooperation to achieve this task. I am thankful the services he provide me. He

is a supervisor whom I learned that “you cannot go wrong when research activities

are planned’.

I offer my cordial and profound thanks to Dr. Javeed Ahmad, Dr. Abrar, Dr

Ghulam Subhani, Miss Sadaf Afzal, Miss Hira Shair, Awais and Amir Hameed in

wheat research (AARI) Faislabad. They have been providing me the good facilities

and time during exhaustive moments of my work. I do not have words at my

command to express my heartiest thanks and gratitude to my external foreign

supervisors, Dr. Fulai Liu (Principal supervisor), Prof. Dr. Thomas Georg Roitsch

(Co-supervisor) and Xiagnan Li (Post doc fellow) at University of Copenhagen,

Denmark. Their visionary research activities made my investigations more fruitful

and I hope it will reshape my upcoming research directions.

I particularly wish to acknowledge the help rendered by my research fellows

in University of Copenhagen especially, Sajid Shokat Senior Scientist (NIAB) for

his input in my research works. The effectiveness of research work in Denmark

could not have been achieved without his help and cooperation.

xviii

I really have no words to express my cordial and sincere indebtness to my

brothers, Naveed, Nafees and Awais and beloved sisters, Nabila and Tayyeba for

their cooperation and deep love who supported me during the odd hours of life. I

would like to record my sincerest thanks to my uncles (Zahid and Shahid), all other

relatives and cousins for their cooperation during my studies. I wish to express my

profound gratitude and pray to my loving legends my grandmothers, grandfather

and uncle (late), though they are no more with me to see that their dream have

comes true. I love you and I will forever remember you.

I am highly indebted to my beloved rommates, Anila, Maria, Shamsa, Zara,

Safina, Nosheen, Hafsa, Amnah, Saba and Shaista. I wish to acknowledge my

everlasting friends and fellows, Khadija, Asia, Saima, Ghazala, Safina, Ammara,

Shazia, Sidra, Sidra and Anum. I can’t forget the love, cooperation and colorful

moments with my friends during my Ph.D. Special thanks to my friends from

NIAB, Fozia, Mehak, Sidra, Anum and Misbha. I extend my thanks to my fellows

at University of Copenhagen Rabecca, Milan, Lian, Sichen, Lamis, Shehnaz,

Rumana, Shumaila, Rizwan and especially Rehina for their love and they make my

time very special in Denmark

I cannot forget all those peoples who provided me spiritual and moral support

and they always make a silent prayer for me. I have only one sentence for all of

you, I love you all and your love led me every step to fruition. I would like to

acknowledge all the clerical staff and lab staff at all research institute and

universities for their help and good behavior. May the Almighty Allah shower his

blessing to all those who assisted me at different stages during my academic career.

ANEELA ULFAT

xix

ABSTRACT

Global warming and uneven climatic change have augmented the drought

prevalence. These dilemmas are enforcing the agri- scientist to develop some long

term future policies. The aim of present study was to examine the method for

improved growth and development of wheat under the premises of drought. Five

wheat cultivars were used to investigate the consequence of drought on plants and

it was also investigated that how hormonal priming can be helpful to cope drought.

Seed of these wheat cultivars were primed in 10-4 M Gibberellic acid and Salicylic

acid concentration. The response of antioxidant enzymes was variable among the

non primed and primed seeds for all studied genotypes. Shahkar had the highest

protease activity in primed seeds while AARI-11 had the highest amylase activity.

Similarly, AARI-11, Shahkar and Chakwal-50 had the highest Superoxide

dismutase activity while Shahkar and Pakistan-13 had the highest peroxidase

activity. Cell membrane thermo stability, proline and relative water contents were

decreased under drought stress. Hormonal priming with Gibberellic acid and

Salicylic acid improved the physiological response and antioxidant enzymes

activities in some genotypes under both conditions.

Yield and its contributing traits yield components were lessened under the

effect of drought. FSD-08 and Pakistan-13 showed maximum grain yield during

control and drought condition. Priming increased the grain yield in all varieties.

Grain quality characters were noticeably affected under drought stress. Hormonal

seed priming was able to maintain the grain quality by minimizing the adverse

effects of drought. FSD-08 was able to maintain the grain quality under normal and

stress conditions.

xx

Drought is unavoidable under changing climatic scenario however famine

can be avoided. The second experiment was conducted in order to know the

mechanism of trans-generational effect of elevated carbon dioxide on winter wheat.

Seeds obtained from the previous generations of ambient and elevated carbon

dioxide were regrown under ambient (400ppm) and elevated (800ppm) within the

green house. Drought stress was imposed for 4 days during anthesis stage and then

plants were re-watered. Flag leaves were used to analyze the activities of enzymes

involved in carbohydrate metabolism and antioxidant enzymes. This

transgenerational effect and its enzymatic basis were not investigated previously.

Results showed that glycolytic intermediates and antioxidants were enhanced under

elevated carbon which ultimately increased yield. Trans-generational effect

indicated that seeds had stress memory and thus maintained the effect of previous

exposure. We found within source (leaf) cytoplasmic invertases, sucrose synthase,

catalase and ascorbate peroxidase activities were decreased while the activity of

cell wall invertases was increased under drought and elevated carbon. Similarly,

the activities of glucose-6-phosphate dehydrogenase, superoxide dismutase,

ascorbate peroxidase and dialyzed peroxidase were decreased under drought and

elevated carbon within the sink (spike). Likewise, the activities of sucrose synthase

in the source and aldolase in the sink were increased upon re-watering indicating

that water is playing an important role to activate these enzymes. Similarly, lower

yield was recorded under ambient carbon dioxide. These results indicate that high

metabolism of sucrose synthase within the source; aldolase and glucose-6-

phosphate dehydrogenase within the sink can be helpful to mitigate the drought

stress under elevated carbon dioxide.

1

Chapter 1

INTRODUCTION

Wheat (Triticum aestivum L.) is most widely grown food crop across the globe

belonging to family poaceae, sub family pooidceae and tribe triticeae. This crop has been

improved extensively throughout the world and more than 5,000 cultivars of this species are

being used. It is estimated that more than 35,000 cultivars were developed in the past but

most of them were not able to get commercial fame among the farming community

(Feldman and Levy, 2015) and consequently disappeared quickly. Wheat is grown under

diverse climatic conditions from higher elevation to equator and this crop is well

acclimatized to from 30° and 60°N and 27° and 40°S latitudes (Walter and Breckle, 2013).

Across the world, wheat is being harvested anywhere during whole year due to its versatile

nature. Wheat is not only the oldest cultivated crop but also the staple food of European,

West Asian and North African civilizations for the last 8000 years. This king of cereals has

the highest consumers demand and it has biggest cultivation area among all crops, including

rice, maize and potatoes. Globally, wheat trade is higher than any other cereal (FAO, 2016).

Wheat is considered as world most imperative crop due to reason that it contains

many calories, vitamins, proteins and minerals. Its significance is consequent from the

properties of its gluten; a cohesive network of tough endosperm, proteins that stretch with

the expansion of fermenting dough. Wheat is used for making bread, unleavened bread,

used in pastry products, and for semolina products. Most of these uses, pooled with its

nutritive value and storage quality, have made wheat a staple food for more than one-third

of the world’s population. Among cereal, most of the food stuff were made with wheat

(Council, 2010). Being the staple diet of most of dominates all crops in acreage and

2

production. Global cropped area of wheat is around 240 million ha while world wheat

production during 2016 was 733.8, utilization 715.7, supply 945.0 and trade 164.9 (FAO,

2016).

In Pakistan, wheat is prime food crop in terms of area and consumption. Pakistan is

categorized among top ten wheat producers and consumers. Wheat is a major diet of whole

population and 3/5th of the daily dietary requirements are fulfilled by wheat and per capita

consumption is 125 kg. It also has key importance in country’s policy about food security.

In Pakistan wheat shares 2% in total GDP and around 10% in the value added products of

agriculture. It is estimated that around 0.6% increase in wheat cropped was observed during

2016 and area was increased from 9.204 to 9.260 million. Likewise, overall yield was

increased from 25.086 to 25.482 million tons and 1.6% yield increase was calculated (GoP,

2016).

1.1 CLIMATE CHANGE AND ITS EFFECT ON AGRICULTURE

Global warming and climate change influenced the socio-economic sector on one

hand and agriculture sector on another hand globally. Different plants have diverse

requirements for germination and for better growth. One important variable is plant habitat

and its ecology (Raven, 2008). Environmental change likewise conveys vulnerabilities to

the possibilities of development of wheat production.

As per the (International Maize and Wheat Improvement Center) (CIMMYT, 2016),

environmental change may influence wheat production through the immediate impacts on

yield by means of physiological process, through changes in sowing dates or expanded

precipitation, and through changes in the zones under production, as areas turn out to that

is not much reasonable for wheat. Increased carbon dioxide (CO2) focuses can possibly

build plant development and yield, fundamentally through extended photosynthesis. Before

the industrial revolution the global atmospheric CO2 was 35% less than today’s

3

amount. Although, current amount of atmospheric CO2 is less than 400 part per million yet,

it is expected to reach at 970 part per million at the end of this century (IPCC 4th report).

It is expected that poor people (almost 1.2 billion) have dependency on wheat but it is also

found that this crop is affected by environmental factors more. It is estimated (in South

Asia) that may be in near future about 2050, there is decline in wheat production. In

developing countries wheat demand increased 60% by 2050. In parallel, global change

increases temperature which is more effective in developing world to decline wheat

production by 20–30% (Wheat CRP).

1.2 PAKISTAN STATUS IN TERM OF CLIMATE CHANGE

Since 1947, Pakistan is prone to water shortage and drought conditions during

wheat grown cycle while on the other hand scarce rains coupled with high temperature

resulted in lower wheat production in both irrigated and rainfed regions. The most

economical solution to cop this problem is the development of drought and heat tolerant

varieties. Developing countries similar to Pakistan are also in front of troubles such as

glacier melting, flash floods, drought and heat index. May be we face melting of Pakistan’s

glaciers in 2035. It will bring a major terrible effect on fresh water flows (Stolton et al.,

2006). These climatic changes affect the economy of Pakistan because Pakistan is an

agriculture country and contributes 21% to GDP. Pakistan is 3rd among those countries that

are affected by climate change and stand in 135th number in terms of Co2 emissions (De

Vries, 2010). Among various climatic factors low water availability for irrigation is a

threatening issue and going to be increase as the time flows in Pakistan.

1.3 ABIOTIC STRESSES

All type of stresses weather biotic or abiotic affect the wheat growth and yield.

Environmental stresses appear in several forms, plant water status is badly affected by all

these stresses. It may be understood that all plants have the encoded capacity to response

4

to stress by signaling. Amid the diverse abiotic stresses, salinity, chilling, heat, and drought

stress affects the yield and growth of wheat crop (Shinozaki and Yamaguchi, 2000). Water

shortage reduces and affects the production of food crops up to seventy percent all over the

world (Akram and Ashraf, 2013). Plants adapt to drought stress in versatile way like

adaptations in morphology, physiology and metabolism (Moghadam et al., 2011). Drought

stress results in stunted growth of plants (Sairam and Saxena, 2000). Khan et al, (2010)

reported that growth and yield of wheat crops is affected by shortage of water (Khan et al.,

2010). When it occurs it acts as a limiting factor for the final produced crop. Serious water

stress in wheat amid the vegetative stages brings about diminished leaf region and this thus

influences tillering and spike measure (Denčić et al., 2000).

Grain yield has been found to be correlated with drought stress at critical growth

stages of wheat (Malik et al., 2010). Water scarcity results in changes in physiology of

wheat crop as well changes also occur at biochemical level. Many defense mechanisms i.e.,

ion homeostasis, osmoregulation, hormonal systems and antioxidant enzymes production

were occur in tolerant species which enable to survive them and develop properly before

reproductive stages (Ashraf, 2010).

1.4 STRATEGIES TO OVERCOME ABIOTIC STRESS

One of the most important steps is seed priming to overcome water shortage. By

this water is absorbed by seeds and metabolic processes start but radical does not emerge

from seed (Farooq et al., 2006b). Primed seeds frequently showed better results regarding

sprouting uniformity, germination rate and germination percentage (Kaya et al., 2006).

This technique has been applied to overcome the water shortage effects in many crop

species. Primed seeds during germination pass from different phases like imbibitions and

lag phase and are ready to grow under every condition (Eisvand et al., 2010). Certain

5

efforts have made to know the increase of yield under drought stress conditions when

grown under elevated CO2. Photosynthetic rate going to be increase with increase in CO2

level and it results in more photosynthate production along with enhancement of

antioxidants enzymes with increasing reducing power. This may improve the resistance

against environmental stresses, like drought (Hassanein et al., 2009). However, crop

improvement in context of mitigating the climate change would be an ideal strategy to

move forward for certain crop improvement schemes.

In Himalayan region of Pakistan like Azad Kashmir wheat cultivation and

production is almost neglected, although area has very good potential for the production of

spring as well as winter wheat varieties. Wheat is the staple food of the people of this

region.

1.5 JUSTIFICATION OF THE STUDY

Keeping in view the importance of climate change and to know about the plant’s

positive approach during this change especially in relation to the elevated CO2 and priming

the project was designed. It will probably help us to know the real mechanism of plants

stress tolerance. The rise in CO2 concentration has direct as well as indirect consequences

on agricultural production. Among many parameters growth of plant, physiology and

productivity are directly influenced by global climate change due to increase in

concentrations of CO2. Proper regulation of plant machinery under these environmental

changes is extremely important. Consequently, the plants have to create a balance from

source to sink along with stress tolerance (Godt and Roitsch, 2006). Plant source tissues

yieldsurplus of assimilates and these are either elated to the growing tissues or stored in the

form of different sugars. However, partitioning of these sugars can be estimated by the sink

strength (relatively) and by different abiotic along with biotic stress factors (Keunen et al.,

2013).

6

This study was designed to investigate the following myths;

To use the hormonal seed priming as shot gun approach to manage with drought

stress

To improve the wheat yield under drought stress grown under elevated CO2

To study the metabolic changes associated with drought stress, priming and

elevated CO2

To investigate the stress memory of trans-generational seed re-grown under

elevated CO2

To determine the effective ways to promote sustainable agriculture and to promote

the wheat production in Himalayan region of Pakistan like Azad Kashmir

7

Chapter 2

REVIEW OF LITERATURE

Wheat (Triticum aestivum L.) is a cereal grain, originated from Levant region of the

near East but now cultivated globally. Wheat is grown in most parts of the world, from

near-arctic to near-equator.

2.1 WORLD WHEAT MARKET

During the year 2011-12 wheat production was 697.4 million tons, supply 896.6

million tons, utilization 694.3 million tons, trade 148.5 million tons and Stock-to-

disappearance ratio was 18.2. In the year 2012-13 production of wheat was decreased as

compared to previous year. Wheat production increased upto 711.5 million tons during

2013-14, while during 2014-15 wheat production was 730.5 million tons, supply 913.7

million tons, utilization 703.6 million tons, trade 156.6 million tons and Stock-to-

disappearance ratio was 16.7. There was a significant increase during 2015-16 in

production 733.8 million tons, supply 945.0 million tons, utilization 715.7 million tons,

trade 164.9 million tons and Stock-to-disappearance ratio was 16.6. During 2016-17

production 742.4 million tons, supply 968.2 million tons, trade 730.5 million tons and

Stock-to-disappearance ratio 17.4 estimated (FAO, 2016).

2.2 WHEAT STATUS IN PAKISTAN AND AZAD JAMMU AND KASHMIR

Pakistan is ranked at sixth position among top wheat producing countries. Pakistan

is producing 25 million tons of wheat yearly though Punjab contributed its share of 19

million tons wheat to total production. Pakistan is self-sufficient nation in wheat production

as wheat is sown on more than 20 to 25 million acres of land in the country every year

consistently. In Azad Jammu and Kashmir (AJK) wheat is grown on around 92

8

thousand hectares with a yearly production of around 113 thousand tons. Azad Jammu and

Kashmir with a normal yield of 1226 kg for every hectare is for behind the normal yield of

Pakistan. The nearby wheat production can't satisfy household require and about 350

thousand tons of wheat were imported to AJK from Pakistan (PARC, 2016). The

atmosphere of Azad Kashmir is temperate to sub-tropical with a normal annual

precipitation of 1300mm. There is variation of height (sea level) ranges from south (360

meters) to north (6325 meters). The snow line (from ocean) varies from 1200 meters

(winter) to 3300 meters (summer) as reported in AJK Bureau of statistics (2015).

2.3 CLIMATE CHANGE AND ITS IMPACT ON AGRICULTURAL CROPS

Energy constraints, water availability, climate change and ecological degradation

are the largest threats that were facing agriculture (Kirschenmann, 2011). With every

passing year, the horticultural framework is affected by additional climate change

(IPCC).The ascent in CO2 fixation has immediate and circuitous impact on agriculture.

Plant development, profitability and physiology are straight forward affected by worldwide

environmental change by expanding in concentration of CO2. Subsequently, the best

possible direction of sugar production and appropriation is fundamental for plant

advancement and stress reaction. Critical variations in mRNA articulation levels and

actions of compounds associated with sugar digestion happen amid plant improvement,

separating starch leaf and spike tissues, that are essential in deciding the last bio-yield and

henceforth edit final yield and quality of grain (Godt and Roitsch, 2006).

2.4 DEMAND AND AVAILABILITY OF WATER

The expansion rate of human population of Pakistan requires an elevated food

progress while less water resources are provided for agriculture. This alarming condition

9

can only be resolved proficiently if water is managed more, so that crop produce per unit

of water utilization boosts. Agriculture is the major consumer of water generally in most

countries. On top of that, agriculture sector encounters the enormous obstacle of

development of crop, as almost 50% more food will be needed by 2030 and development

must be doubled by 2050. These goals should be achieved with less water, due to the fact

of growing stresses from urbanization, industrialization and environmental change (OECD,

2010).

Water is a generally critical constituent of the metabolism of every single alive

being, encouraging numerous imperative natural processes because water is good solvents,

medium of transportation and retain the property to impart cooling property by evaporation

(Mundree, et al.,2002). In all photoautotrophs including plants, water assumes the extra

part of giving the vitality important to initiate the process of photosynthesis. Water atoms

are split, in a procedure called autolysis, to yield the electrons that are utilized to drive the

vitality yielding photosystem II reaction center. It goes about as medium dissolvable in

which numerous biochemical procedures takes place. The proteins in the Calvin cycle and

Kreb's cycle are all skimming around in the stroma, which is only a fluid arrangement of

stuff. Numerous different pathways additionally include catalysts, substrates and item that

take part in whole processes. Water goes about as dissolvable bearer for mineral

nourishment. Water help to move the supplements in plants and those supplements move

upward in the xylem. Water creates turgor weight, which give strength to leaves and stems.

At the point when plants lose water, they lose turgor and shrink (Taiz and Zeiger, 2002).

Water constitute a fundamental prerequisite for germination. Growing seeds are

frequently dry and need to retain,a process of imbibition, a critical amount of water to avoid

desiccation through frequently drying according to seeds dry weight. Plants seeds require

in general require almost 35% to 45% of water contents for germination. Wheat plant needs

10

water at two basic stages, first at tillering that begins a week after rise so water system

ought to be connected not later than 20-25 later subsequent to seeding. The second water

system is essential amongst anthesis and grain arrangement if irrigation water is accessible.

For various developmental stages water supply is necessary in plants. For the most part 4

to 6 irrigations are required amid the entire yield cycle (Acevedo-Opazo et al., 2010).

2.5 DROUGHT STRESS

Drought is very important amongst the most widely recognized natural stresses that

influence growth and metabolism of plants. Drought stress keeps on being a critical

challenge to plant breeders and agricultural researchers. It is expected that by the year 2025,

around 1.8 billion individuals will confront supreme water lack and 65% of the total

population will live under drought stress situations (Xiong et al., 2006). It is estimated that

up to 2050 most of the arable land will face much problems due to drought (Vinocur and

Altman, 2005). Water shortage, restrict the development and profitability of crops and

damage more than other stresses. Drought is an overall issue, obliging worldwide less yield

production and environmental change has made this circumstance more genuine (Pan et

al., 2006).

Drought is multidimentional push influencing plants at different levels of their

growth. Dry season influences morphological, physiological and biochemical processes in

plants bringing about development hindrance, closure of stomata with back to back

lessening of transpiration, diminishing in chlorophyll substance and restraint of

photosynthesis (Demirevska et al., 2008) making it the major single component for yield

decrease all around the world (Narusaka et al., 2003). The reaction of plants to water

11

relies on a few components, for example, developmental stage, severity and length of stress

and cultivar hereditary qualities (Beltrano and Marta, 2008, Din et al., 2011).

2.5.1 Problems During Drought

Wheat is one of the essential grain crops on the planet. It can be developed in an

extensive variety of agrarian situations. Water accessibility is the most constraining

component for wheat production, drought stress antagonistically influences plant

development and advancement, seed germination, (Dash et al., 2010; Almaghrabi, 2012),

seedling development, enzyme action (Seckin et al., 2009), DNA, RNA as well as synthesis

of protein (Anuradha and Rao, 2001) and mitosis (Tabur and Demir, 2010). In wheat, most

delicate to drought stress stages are tillering, reproductive and germination (Passioura,

2007). Heat stress is the significant limitation to wheat in dry, semiarid, tropical and

subtropical areas of the world (Ashraf and Foolad, 2005). It influences the accessibility and

translocation of photosynthates to creating seed and starch combination, along these lines

antagonistically influencing the grain weight and quality (Mohammadi et al., 2004).

Better implementation of crops relies on accessibility of water. Among different

abiotic stresses, water deficiency is most important because it effect up to 70% of yield and

production of crops (Akram and Ashraf, 2013). Diverse abiotic components influence the

development and yield of the crop plants. Among these components, water condition is

most important because it decreases the yield and also effects its development (Kusvuran,

2012; Souza et al., 2004; Saensee et al., 2012).

12

Figure 2.1: Drought Stress effect on cell physiological processes

A few agents have portrayed the impact of water shortage on different physiological

traits of development in wheat. All in all, dirt water shortage brings about decline in relative

water contents (Tas and Tas, 2007), leaf succulence (Qi et al., 2009), chlorophyll content,

cell membrane stability index (Farooq and Azam, 2006; Tas and Tas, 2007), number of

grains per spike and weight of grains and grain yield (Sanjari Pireivatlou and Yazdansepas,

2010). Relative water contents identified with water take-up by the roots and water

misfortune by transpiration (Anjum et al., 2011). Cell membrane stability measured as rate

damage of leaf tissues of wheat cultivars, can be utilized for screening for drought stress.

The water stress diminishes in membrane stability index of all the wheat assortments.

Under water stress, tolerant cultivar showed higher membrane stability index support of

high RWC under dry season because roots develop more than the shoots and abscisic acid

actuated diminishment in stomatal opening has a tendency to keep up cell turgidity and

chlorophyll content (Keyvan, 2010).

Constrained water supply generally causes a decrease in chlorophyll content being

decidedly associated with yield (Zaharieva et al., 2001). Generally high chlorophyll

contents may add to the plant efficiency under stress conditions. Photosynthesis is

13

amongst the most delicate process to overcome the stress caused by drought (Chaves et al.,

2009). The inhibitory impacts of drought on photosynthesis might be connected with low

CO2 accessibility because of low stomata and mesophyll conductance (Flexas et al., 2008),

and/or impedances in carbon cycle (Peeva and Cornic, 2009). Stomata closure is an early

reaction to drought stress and an effective approach to lessen water availability in water-

restricting situations. Biochemical confinement of photosynthesis additionally assumes a

critical part under delayed times of drought stress (Flexas et al., 2008).

Water stress and high temperature are the major natural components influencing

wheat grain quality. It has been accounted for that times of heat stress with temperatures

higher than 35 °C may change flour quality. These impacts have been identified with an

expanded gliadins/glutenins proportion (Daniel and Triboi, 2000) and decreased the extent

of the bigger sub-atomic size glutenins (Wardlaw et al., 2002). It is realized that yield

diminishment that for the most part happens under drought stress is for the most part

connected due to protein content expansion (Rharrabti et al., 2003; Guttieri et al., 2005 ;

Garrido-Lestache et al., 2005; Pompa et al., 2009).

2.5.2 Strategies to Cope Drought Stress

These days different strategies are utilized to create plants that can withstand these

stresses. As of late, seed priming has been created as a basic strategy to produce tolerant

plants against different stresses (Ashraf et al., 2008). Drought stress impacts on seed

germination and seedling development of numerous plants. Seed priming could be utilized

to overcome the depressive impacts of drought. The enhancing impacts are affected by

numerous elements including priming strategies, plant species and drought stress intensity

(Farooq et al., 2009).

14

2.5.2.1 Priming

Diverse sorts of priming medications were recorded to upgrade drought resistance

in numerous plants. Scientists characterized priming as a technique utilized by

agriculturists in an extensive variety of plants, including wheat and chickpea. Seeds are

soaked with solution for around 6 h, 12 h, 24 h, then dried till to attain original weight that

was before soaking. Seed priming techniques includes hydro-priming (Farooq et al., 2013),

osmo-priming (Ghiyasi and Tajbakhsh, 2013), hardening with plant growth inducers

(Eivazi, 2012), and hormonal priming (Khan et al., 2009). Various priming agents

including ascorbic acid, salicylic acid, kinetin, CaCl2, abscisic acid are frequently reported

in literature for chemical priming of seeds (Jafar et al., 2012; Farooq et al., 2013).

Germination process can be mediated by soaking the seeds into water that helps in

imbibitions to break the dormancy as enactment of definite catalyst and so forth (Ajouri et

al., 2004). Various process animating germination are initiated by seed priming and hold

on taking after the redesiccation of the seed (Asgedom and Becker, 2001). The germination

procedure can be partitioned into stages: (i) quick imbibition (ii) beginning seed metabolic

process and (iii) consequent radical rise and carrying on the process growth (Fig 2.2).

Figure 2.2: Schematic diagram showed the effect of seed priming viz normal on seed germination process

(Source Rajjou et al.,2012)

15

2.5.2.2 Hormonal Seed Priming

During the most recent 20 years, phytohormones, drew the consideration of

researchers because of their capacity to initiate systemic acquired resistance (SAR) to

plants to various kinds of stresses (Tuna et al., 2007). Phytohormones can be used to

overcome the stress by priming of seeds and growth rate can be enhanced by foliar

application. During water deficiency, plants adapt the changes by different growth

hormones like salicylic acid (SA), Gibberellic acid (GA), cytokinase (CKS), absisic acid

(ABA) and Indole acetic acid (IAA) (Farooq et al., 2009).

2.5.2.2.1 Role of salicylic acid

Salicylic acid (SA) is regarded to persuade exclusive physiological and

biochemical activities of plants.

Salicylic acid take part in enhancing their activities and performance (Hayat et al.,

2010). SA helps in regulation of different physiological processes because it act as a

endogenous regulator due to phenolic nature (Hayat et al., 2010) furthermore gives security

against biotic as well as abiotic stresses, for example, stress induced by salt (Kaya et al.,

2006). SA prompted increment of the resistance in seedlings of wheat against stress induced

by salt (Shakirova et al., 2003).

Salicylic acid required in the growth regulation, development and advancement of

plants also their interaction to biotic as well as abiotic stress (Khan, 2013; Miura and Tada,

2014). SA is included in the regulation of different essential physiological processes for

example photosynthesis, nitrogen metabolism , proline, production of glycinebetaine

16

Antioxidant defense system and plants water relations during the condition of stress along

these lines gives insurance in plants against abiotic stresses (Khan, 2013).

It has been demonstrated that salicylic acid mitigate low-temperature stress in maize

and wheat plants during winter (Taşgín et al., 2003), or modulate different responses in

plants due to the stresses induced by salt (Borsani et al., 2001), ozone or ultraviolet light,

drought and herbicides (Ananieva et al., 2002). SA induces the defense process in plants to

mitigate salt stress (Afzal et al., 2011). SA helps a large number of crops against salt stress

for example, tomato (Tari et al., 2002) bean (Azooz, 2009), and maize (Gunes et al., 2007).

2.5.2.2.2 Role of Gibberellic Acid

Gibberellins (GA) are diterpenoids, regulating plant growth and development.

They are ordinarily utilized in present day in agriculture and were initially obtained from

pathogens especially on rice during 1938 known as Gibberella fujikuroi (Santner et al.,

2009; Yamaguchi, 2008).

They act all through the plant during cell division, help in cell during multiplication;

promote transitions mediated by developmental stages especially during breaking the seed

dormancy and germination during the adolescent and after that during developmental stages

also helps in reproduction improvement. In spite of the fact that GA activity is vital for

typical development and improvement, seedlings without the ability to incorporate or see

GAs will experience constrained advancement, even during the light conditions mediate

flowering (Griffiths et al., 2006; Ueguchi-Tanaka et al., 2005).

Gibberellins (GAs) are for the most part mandatory in progress and improvement.

They control germination of seed, leaf expansion, stretching of stem and flowering

(Magome et al., 2004). Gibberellic acid (GA) amasses quickly under all type of stresses.

17

Some researchers give more importance to Gibberellins (Hisamatsu et al., 2000).

Hydrolases synthesis and production can be enhanced by GA especially alpha amylase

helps in seeds germination. Gibberellins activates amylases especially proteases, Beta-

glucanases and alpha amylase (Ueguchi-Tanaka et al., 2005). During the production of

hormones especially gibberellins in plants, seed aleurone determine various transduction

pathways (Penfield et al., 2005). Drought stress alone delayed growth and elongation of

the hypocotyl, while use of gibberellic acid switched this impact. For this situation,

gibberellic acid somewhat expanded the water status of the seedlings and in part managed

protein synthesis (Taiz and Zeiger, 2006).

2.5.2.3 Priming effect on biochemical, morphological and physiological processes

To adapt stress plants prompts assorted biochemical and physiological mechanisms

for survival (Tas and Tas, 2007). Salicylic acid regulate the antioxidant enzyme activities

for example in tomato plants were sprayed with catalase and super oxide dismutase under

stress condition during drought (Hayat et al., 2010) or under the response of stress due to

presence of excess salt (Szepesi et al., 2009) and (Yusuf et al., 2008).

To lessen the unfavorable impact of drought stress plants have advanced some

protective systems, for example, a rise in the ROS (reactive oxygen species) (Miller et al.,

2010; Huang et al., 2012).

Reactive oxygen species (ROS), most of time produced in chloroplast also to found

to be in mitochondria, bringing on oxidative stress. Real ROS particals are leads Production

of singlet oxygen, anion radical, hydrogen peroxide and radicals of hydroxyl results in

ROS. Plants develop some defense mechanisms to protect themselves from harmful results

of oxidation especially during drought. The ROS scavenging mechanism is among the

18

common defense response against abiotic stresses (Vranová et al., 2000). To protect

themselves from ROS, plants can inherently create distinctive sorts of antioxidant that help

the plants to overcome the drought stress and face less harms due to oxidation. Peroxidase,

catalase and superoxide dismutase are important scavengers of free redicals (Khan et al.,

2008). The catalase (CAT) has potential to detoxify ROS in peroxisomes by dismutasing

the hydrogen peroxide into H2O and O2 (Prochazkova et al., 2001). Peroxidase and catalase

help in disposing of the H2O2 that is formed by SOD by detoxifying superoxide anion (O2-

) (Hasheminasab et al., 2012). Drought stress influences the development and yields of

wheat genotypes which brings about harmed development of the plants (Raza et al., 2014).

Under drought stress, different biochemical, physiological and molecular changes happen

in plants during drought stress condition (Arora et al., 2002). Consequently, amplified

superoxide dismutase transform in plants is the confirmation of pressure tolerance (Pan et

al., 2006; Hameed et al., 2011). Salicylic acid and its related compounds upon priming

cause initiation and inhibition in plants (Gill and Tuteja, 2010).

Salicylic acid advanced morphological trends that make contributions closer

to yield enhancement but, it depends on plant species, development and usage technique

(Arfan et al., 2007). Gibberellins (GAs) were for the most part required in development

and improvement. Wheat grain yield was increased by GA3-priming, it initiated regulation

of ion uptake and hormones homeostasis under salinity (Iqbal and Ashraf, 2013).

The enhancement in total protein in rice plants under drought stress by extrinsic use

of plant hormones might be because of their conceivable addition in water stress adjustment

(Tuna et al., 2008). The plants build proteins which are included in purification of free

radicals and along these lines assume very important for adjustment during the condition

of stress (Witzel et al., 2009) (Bandehagh et al., 2011;). Gluten

19

proteins are among the most complex protein arranges in nature because of ivarious

distinctive parts and diverse size brought about due to genotype, technological processes

and developing conditions (Wieser, 2007). They assume a key part in deciding the

interesting properties in relation to rheological dough and also used in baked products.

Bread wheat quality of assessment is utilized a considerable measure of quality analysis.

Some investigation strategies require a lot of test and result is acquired in the long time.

Quality properties in this study impact measuring the quality performance of genotypes.

These quality criteria can be utilized for quality evaluation as a part of early era breeding

programs.

2.6 TRANSGENERATIONALEFFECT OF ELEVATED CARBONDIOXIDE

Pre-exposure of plant to mild stress may activate the ‘stress memory’ that facilitate

safest protective response to the consequent stress events and happening (Boyko and

Kovalchuk, 2011). Stress memory can be defined as physiological changes at genetic level

and even epigenetic during stress conditions and to overcome this stress adjusts reactions

from generations to generations (Boyko and Kovalchuk, 2011). Stress memory in the

following generation supposed to be linked with improved tolerance in numerous species

regarding biotic as well as abiotic stresses of plants. Based on above literature survey, the

current study came up with the following major goals. There is need to develop drought

resistant cultivars and CO2 responsive genotypes to cope with coming environmental

issues.

2.8 CONSIDERATIONS FOR THE FUTURE

Due to the lack of good cultivars besides the inadequacy of related research, we are

not able to achieve mutual variation among wheat varieties and environments. Outcome

technical innovations to progress quality of wheat are the main challenge for whole world.

Additional studies must be directed towards the physiological, biochemical, and molecular

20

levels to reach a suitable conclusions about high yield and good wheat quality. There is

need to select special verities to achieve goals with respect to growth in special locations

and this in turn relate to increasing the probability of recognizing and predicting species

with maximum quality of grains in various environments. Similarly, genotypes that have

best response to elevated CO2 should be promoted. Water management and soil

conservation and irrigation techniques need to be improved. Promote agriculture in Azad

Jammu and Kashmir by using agricultural land technologies and strengthening of research

and technical services.

21

Chapter 3

MATERIAL AND METHOD

3.1 STUDY

This study was carried out in the field of University of Azad Jammu and

Kashmir Muzaffarabad. Azad Jammu and Kashmir consists of 13,297 Square Kilometer

area with latitude 33o–36o and longitude 73o–75o. It is a mountainous region and climate of

this state ranges from sub-tropical to alpine. Normal highest temperature is 45.2°C while

minimum temperature may go down to -2.6 °C (Anon, 2007). Study area is pointed out in

the map as shown below.

Fig 3.1 Map of the study area

3.2 PLANT MATERIAL

Seeds of wheat genotype AARI-11, Chakwal-50, Shahkar, Pakistan-13 and

Faisalabad-2008 were obtained from National Agricultural Research Center (NARC)

Islamabad Pakistan.

22

3.3 SEED PRIMING TREATMENTS

Priming was carried out by presoaking the seeds of each genotype in10-4M solution

of salicylic acid (SA) and gibberellic acid (GA) for eight hours. In addition, continuous

aeration was supplied by aquarium pumps, kept for drying and retried to original weight.

Half of the seeds were not primed which served as control.

3.4 EXPERIMENTAL SET-UP

The experiment was performed by split plot design by dividing the main plot into

two sub-plots i.e., well watered and drought followed via similarly cut up of sub-plots

into three sub-sub-plots appeared as replicates. Sub-sub-plots were supplied with

priming remedies and cultivars were completely randomized. Sub-sub-plots were provided

with priming treatments and cultivars were completely randomized. About fifteen sub-plots

were considered as control by supplying enough water while other fifteen sub-plots were

marked stress group without well watered condition and roofed with water proof sheet.

Sowing was done by hand drill, keeping seed to seed 1.5 cm and row x row distance of 20

cm.

3.5 SEED BIOCHEMISTRY ATTRIBUTES

3.5.1 Oxidative Enzyme Assays

3.5.1.1 Protease activity

Protease activity was estimated by the casein digestion assay established by

(Drapeau, 1974). Casein solution was prepared by mixing 6.5 mg/ml of casein in the 50mM

potassium phosphate buffer. For protease activity, seeds were homogenized in 50mM

potassium phosphate buffer (pH 7.8). One unit is that amount of enzyme, which releases

acid soluble fragments equivalent to 0.001 A280 per minute at 370C and pH 7.8. The

absorbance was recorded at wavelength of 660nm. Enzyme activity was expressed on fresh

weight basis.

23

3.5.1.2 Esterase activity

To measure the non–specific esterase namely α-esterase (alpha esterase) activity

(Van Asperen, 1962) method was used. The assay solution was prepared by taking 0.03 M

α or β–naphthyl acetate (substrate solution), 0.04 M phosphate buffer with pH 6.8 and

sample extract in each test tube separately. The absorption was recorded at 590nm by

spectrophotometer (HITACHI U-2800) when color developed for alpha esterase.

3.5.1.3 Amylase activity

To measure the α-amylase inhibition activity (Giancarlo § et al., 2006) method were

used. Starch solution (1%, w/v) was prepared by taking 1g of soluble starch, dissolved in

0.02M sodium phosphate buffer and sodium chloride (0.006 M) with pH 6.9. Dinitro

salicylic acid (DNS) color reagent was prepared by mixing 96 mM DNS solution with

sodium potassium tartrate solution (30.0g of SOD. K.) and absorbance was recorded at

540nm. Potassium phosphate buffer (pH 6.8) was used as blank.

3.5.1.4 Superoxide dismutase activity

The method of (Dixit et al., 2001) was followed for the estimation of Superoxide

dismutase (SOD) activity. Leaves were homogenized in a medium composed of 50mM

potassium phosphate buffer (pH 7.0), 0.1 mM EDTA and 1 mM dithiothreitol (DTT). The

SOD activity was evaluated by measuring its potential to inhibit the photochemical

reduction of nitroblue tetrazolium (NBT) as described by (Giannopolitis and Ries, 1977).

One unit of SOD activity was defined as the enzyme concentration that caused 50%

inhibition of NBT photochemical reduction.

3.5.1.5 Peroxidase activity

Peroxidase (POD) activity was determined by following the method of (Chance and

Maehly, 1955). The reaction was initiated by adding 0.1 ml enzyme extract in 50 mM

24

phosphate buffer (pH 7.0), 40mM H2O2 and 20mM guaiacol followed by recording in

absorbance at 470 nm after every 20s. One unit POD activity was defined as an absorbance

change of 0.01 units min_1.

3.5.1.6 Catalase activity

Catalase activity was determined by following the method of (Beers and Sizer,

1952). CAT activity was measured in assay solution (3mL) containing 50 mM phosphate

buffer (pH 7.0), 5.9mM H2O2 and 0.1 ml enzyme extract. After every 20 sec, decrease in

absorbance was recorded at 240 nm and absorbance change of 0.01 units min_1 was defined

as one unit CAT activity.

3.6 MORPHOLOGICAL ATTRIBUTES

Morphological attributes such as plant height (cm), spike length (cm), spikelets,

number of tillers, peduncle, extrusion length (cm), grains in pikes, total yield (kg/ha),

biomass yield (kg/ha), thousand grain weight (g) and harvest index (%) were recorded from

ten randomly selected plants at maturity. Yield and harvest index were calculated by.

Grain yield (kg /ha) = Grain Yiled

Sampled Area× 1000m−2

Harvest index = Grain Yield

Biological Yield× 100

While;

Stress tolerance index = values uder stress

Values under control×100

3.7 BIOCHEMICAL ATTRIBUTES

Fully emerged flag leaves were contribute grain yield directly up to 75% so fully

emerged flag leaves were collected for biochemical assay.

25

3.7.1 Hydrolytic antioxidant

Protease, esterase and amylase activities were done by using similar method as

already described for seeds.

3.7.2 Enzymatic antioxidant

Similarly, Superoxide dismutase (SOD), Peroxidase activity (POD), Catalase

(CAT) and Ascorbate Peroxidase (APX) activity was determined in leaf by using the same

method as used by seeds.

3.8 PHYSIOLOGICAL ATTRIBUTES

3.8.1 Malondialdehyde Contents

Malondialdehyde (MDA, a product of lipid peroxidation) contents from leaf tissues

were determined by method the method of (Heath and Packer, 1968) with some changes as

suggested by (Dhindsa et al., 1981). Samples were homogenized in 5 mL of 0.1% TCA and

centrifuged for 5 mints at 10,000rpm. In 1 mL aliquot of the supernatant, 4 mL of 20%

TCA containing 0.5% TBA were added. The mixture was heated at 950C for 30 min and

then quickly cooled in an ice-bath. The absorbance was recorded at 532 nm and the non-

specific absorption at 600 nm was subtracted. Extinction coefficient of 155 mM-1 cm-1 was

used to calculate MDA contents.

3.8.2 Total Oxidant Status

Total Oxidant Status (TOS) was evaluated by a novel automated method developed

by (Erel, 2005). Two types of reagent R1 and R2 were used and the results were expressed

in μmol H2O2 equivalents/L. Reagent R1, assay mixture contained (stock xylenol orange

solution (0.38g in 500μL of 25mM H2SO4), 0.49g NaCl, 500μL glycerol and volume up to

50mL with 25 mM H2SO4), sample extract and reagent R2

26

(0.0317g θ-anisidine, 0.0196g ferrous ammonium sulphate (II)). The absorption of each

assay mixture was measured at wavelength 560 nm after 5 minutes nm with the

spectrophotometer. The term μmoL H2O2 equivalents/L was used to express the results.

3.8.3 Relative Water Contents

According to (Weatherly, 1950), relative water contents (RWC) of the flag leaf

sample were estimated by measuring fresh weight, turgid weight and dry weight.

Relative water content = [Fresh weight–dry weight/turgid weight–dry weight] ×

100.

3.8.4 Cell Membrane Thermo stability

Plant material were placed into two sets of test tubes alongwith de-ionized water

and then put in a refrigerator at 10°C for 18 h. After that with de-ionized water plant sample

were washed and 15mL deionized water was added in the same test tube. Thereafter, one

set of the test tubes was kept at 45°C and the other half at 25°C for 1h. Now, to get

stabilization both samples sets were placed in a refrigerator for 18hr at 10°C. To take the

readings, conductivity meter was used for both sets, heat treated (T1) and control (C1). Now

samples were boiled for 1 hour samples. After cooling the samples second conductivity

reading (C2 and T2) was taken at 25°C. Cell membrane thermo stability was calculated by

the equation of (Blum et al., 2001).

MTS (%) = [1-(T1/T2)] x100

27

3.8.5 Pigments Analysis

3.8.5.1 Chlorophyll a, b, Carotenoid and Anthocyanin

Leaf samples were homogenized into 80% acetone for chl a, chl b and carotenoids,

while for anthocyanin methanol/HCl/water in a ratio of 90:1:1 instead of 80% acetone was

used. Centrifuged and measured optical density at the wavelength of 537, 647 and 663nm

(Sims and Gamon, 2002).

3.9 METABOLITIES ACCUMULATION

3.9.1 Total soluble Sugars

According to (Dubois et al., 1956) sugar content was measured. Leaf samples were

homogenized using a clean mortar in distilled water and centrifuged at 3000 rpm for 5 min.

Then in 0.1ml supernatant, 1ml phenol (5 % v/v) was added and left for 1 hr. incubation

was done by the addition of concentrated H2SO4. Concentration of unknown sample was

calculated by using standard curve of glucose. The absorbance was recorded at 420nm.

3.9.2 Total Proteins

The method of (Bradford, 1976) was used for the estimation of protein contents.

The Bradford’s reagent was made by mixing (25ml of 95% ethanol 50 with 50mg

Coomassie Blue G250 dye), after that mixture was added in 50mL of 85 % o-phosphoric

acid to make total volume of 500mL with distilled water. By using the solution (1 mg ml-

1) of BSA (Bovine Serum Albumin) a standard curve was made. Absorbance was observed

at 595nm on spectrophotometer (Boyer, 1993).

28

3.9.3 Proline Accumulation

Proline was determined from the sample by following the method of (Bates et al.,

1973). 0.15g, leaf sample was homogenoized in 10mL sulphosucilic acid solution. Solution

was prepared by taking 3g of sulphosucilic acid in 100mL of water. Then 2mL glacial acetic

acid and 2mL acidic ninhydrine prepared. 2.5 ninhydrine, 60mL glacial acetic acid, 30 mL

distilled water and 10mL orthophosphoric acid were added in the reaction mixture. After

boiling, cools the mixture and added 6mL toluen by shaking it thoroughly and then poured

it into separating funnel for proline extraction. Then proline was assayed at 520nm by using

spectrophotometer.

3.10 MINERAL ELEMENTS

3.10.1 Potassium and Calcium Ratio

Potassium (K+) and calcium (Ca+) were estimated by (Szabo-Nagy et al., 1992)

method. Suspension was prepared by boiling one gram of flag leaves in 10 mL of perchloric

acid for 30 minutes and then de ionized water was added to make the total volume one liter

in volumetric flask. Potassium and calcium contents were assayed with the help of

JENWAY PFP 7 Flame photometer and a standard curve was also made.

3.11 SEED QUALITY ATTRIBUTES

3.11.1 Wet Gluten (%)

Glutamate instrument ICC standard no 155 and 158 and AACC method 38-12 were

used for Glutametic test. About 10g sample was placed into glutametic washing chamber

on the top of polyester screen. Mixed and then washed the sample with a 2% NaCl salt

solution for 5 min. After washing, wet gluten was subjected for centrifugation and then

weight by using weighing balance.

29

3.11.2 Gluten Index (%)

The percentage of gluten that remained on the sieve during centrifugation is defined

as gluten index, which indicates gluten strength. Gluten index was calculated by separating

and weighting gluten on sieve and gluten that was passed from sieve.

3.11.3 Falling Number (Sec)

Alpha amylase activity was measured by using falling number instrument 1310ICC

standard no. 107/1 (1995) and AACC method 56-81B (1992). Flour sample of 7g with

25mL distilled water was added in the viscometer tube, shaking well with the help of

shaker, and then tube was placed in the water bath. After 5 sec automatic stirring started.

The total time in sec from the start of instrument until the stir has fallen. Time was

registered by instrument.

3.11.4 Proteins, Moisture and Starch (%)

Omega kernel analyzer was used to determine protein, moisture and starch contents.

500g sample placed in sample holder and set software accordingly and find all readings in

percentage.

3.12 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE GELS

For extraction of soluble proteins, leaves (0.5g) were ground in 50 mM phosphate

buffer (pH 7.8) and centrifuged in a micro-centrifuge machine for 10min at 14,000 rpm.

Protein concentration of extracts was measured by a dye binding assay as described by

(Bradford, 1976).The supernatant was decanted and used for protein profiling. Protein

profiling of samples was performed using SDS-polyacrylamide gels as described by

(Laemmli, 1970). The process of SDS-PAGE was repeated thrice. Gels were photographed

using UVIpro-platinum gel documentation system (UVItec UK). Computerized gel

analysis was performed using UVI pro Platinum 1.1 Version 12.9).

30

3.13 TRANSGENERATIONAL EFFECT OF ELEVATED CARBONDIOXIDE (CO)2 ON

WHEAT AT ANTHESIS DROUGHT STRESS

Another experiment was carried at crop science section of Plant and Environmental

Department (PLEN) at University of Copenhagen, Denmark. The experiment was carried

out on winter wheat (Triticum aestivum L. var. Lianmai 6). Grains harvested from three

successive previous generations were further exposed to two different levels of CO2 i.e.

ambient CO2 concentration (a[CO2], 400 mmol L _1) and elevated CO2 concentration

(e[CO2], 800 mmol L _1). These seed were sown in 4 L pots; pot size was 17 cm in diameter

and 16.5 cm in height with 4 drainage holes along with four replicates. Pots were filled

with peat material (Sphagnum, 32% organic matter, pH = 5.6–6.4 and EC = 0.45 ms cm

_1). The CO2 enrichment was achieved by emission of pure CO2 from a bottled tank,

released in one point and distributed in the greenhouse cells through internal ventilation.

The CO2 concentration in the greenhouse cells was monitored every six seconds by a CO2

Transmitter Series GMT220 (Vaisala Group, Helsinki, Finland). The climate conditions in

the greenhouse were set at: day/night temperature 20/16 0C, photoperiod 16h, relative

humidity 70%, supplemental light 400 mmolm _2s _1 was maintained by sunlight plus meta-

halide lamps. A and E was seeds from previous generation while a and e was current level

of (CO2). Experiment was carried in three series. Three sets were made before drought,

after drought and after recovery.

3.13.1 Gaseous Exchange and Water Relations

Photosynthesis (An), stomatal conductance (gs) transpiration rate (Tr) and leaf

water potential (Yl) was measured with LI6400 apparatus and pressure chambers.

31

3.13.2 Yield and Yield Components

Harvesting was done at the physiological maturity of the crop and data of different

parameters like, number of spike number, grain per spike, thousand kernels weight, grain

yield, biological yield and harvest index were measured by using similar method as already

described for first experiment.

3.13.3 Key Enzyme of Carbohydrate Metabolism (C Enzymes) Assays

A series of c-enzyme and a-enzyme involved in carbohydrate metabolism were

measured from flag leaf and spikes of wheat. Frozen plant material was used and grounded

in liquid nitrogen, also add 0.1% polyvinyl polypyrrolidone (PVPP). Homogenized

material centrifuged, pallet and supernatant was dialysed overnight against 20mM

potassium phosphate buffer (pH 7.4) at 4°C. Dialysed and cell wall both extract were shock

freeze in liquid nitrogen and stored at –20 °C.

For all kinetic enzyme activity assays UV-transmissive single-use microcuvettes

(Plastibrand®;Brand, Wertheim, Germany) was used in a total reaction volume of 200μl in

a spectrophotometer (U-3000; Hitachi, Tokyo, Japan). Assays were consequently made to

a 96-well micro titre plate format. For all measurement, aliquots (up to 25 μl) of the

different protein extracts were incubated in a plate reader (Ascent Multiskan; Thermo

Fisher Scientific) at 30°C for 40 min in UV-transmissive flat bottom 96-well plates (UV-

Star;Greiner Bio One, Kremsmünster, Austria) in a total reaction volume of 160μl with a

mixture of buffer components, substrate (s), auxiliary substance (s), and auxiliary enzymes

and absorbance at 340 nm was monitored throughout the entire period of incubation. All

assays were carried out in triplicate. For control reactions, substrate was neglected. The

change in absorbance per second during the linear phase of substrate conversion was used

as the basis for the calculation of specific enzyme activity in nkat g FW–1.

32

The activity of three types of invertases i.e cytoplasmic (CytInv), cell wall (CWInv)

and vacuolar (VacInv) invertases were examined based on the method of (Sung et al.,

1989). A flat bottom 96 well plate was selected (Sarstedt, Nümbrecht, Germany) and

extract up to 20μl were used. To calibrate curve, glucose standards (0–50 nmol) were used.

Sucrose was eliminated for the control reactions and all the measurements were carried out

in triplicate. The value of liberated glucose was determined by measuring the absorbance

at 405 nm in a plate reader (Ascent Multiskan; Thermo Fisher Scientific, Waltham, MA,

USA). Specific activities were expressed as nkat g FW–1.

Sucrose synthase (Susy) activity was determined by (Pelleschi et al., 1997) method.

Two reactions were performed; one was carried out by using 1mM UDP that detect the

cytInv and susy background activity. Second reaction was performed without 1mM UDP

to detect the cytInv background activity only. Susy activity was calculated by subtracting

cytInv background activity (2) from total activity (1).

For determination of UDP-glucose pyrophosphorylase (UGPase) and ADP-glucose

pyrophosphorylase (AGPase) activity, method of (Pelleschi et al., 1997) and (Appeldoorn

et al., 1999) were used respectively. For UDP-glucose, dialysed extract were used along

with 100 mM TRIS-HCl at pH 8.0, 5 mM MgCl2, 0.44 mM EDTA, , 0.1% BSA, 1.5 mMPPi,

2 mMUDPGlc, 1 mM NADP, 2 mM 3-PG, 0.432 U of PGM, and 1.28 U of G6PDH. For

AGPase activity dialysed extract and 100 mM TRIS-HCl at pH 8.0, 0.44 mM EDTA, 1.5

mMPPi, 5 mM MgCl2, 0.1% BSA, 2 mMADPGlc, 1 mM NADP, 2 mM 3-PG,0.432 U of

PGM, and 1.28 U of G6PDH.

Aldolase (Ald) activity was determined by method (Schwab et al., 2001). Dialysed

extract were used with 1 mMF1,6bisP, 1 mM EDTA, 5 mM MgCl2, 0.15 mM NADH, 0.48

U of TPI, and 0.8 U of GPDH in 50 mM TRIS-HCl at pH 8.0. For control reactions, F1,6

bisP was not used. For determination of fructokinase (FK) and hexokinase (HXK) standard

33

methods were used (Appeldoorn et al., 1999; Petreikov et al., 2001). For FK dialysed

extract with 5 mM fructose, 5 mM MgCl2, 2.5 mMATP, 1 mM NAD, 0.8 U of PGI, and

0.8 U of G6PDH in 50mM BisTris at pH 8.0. For HXK activity, similar method was used

as used for Fk. Only 5 mM glucose was used instead of 5 mM fructose.

For determination of phosphofructokinase (PFK) activity, method of (Klotz et al.,

2006) was used. Dialysed extract were taken in 50mM TRIS-HCl , pH 8.0, 5 mM MgCl2,

1 mM F6P, 1 mM EDTA, 0.2 mM ATP, 0.16 U of aldolase, 0.15 mM NADH, 0.48 U of

TPI, and 0.8U of G6PDH. For phosphogluco isomerase (PGI) activity, dialysed extract with

4 mM MgCl2, 4 mM DTT, 2 mM F6P, 0.25 mM NAD, and 0.32 mM G6PDH and 100 mM

TRIS-HCl with pH 8.0 (Zhou and Cheng, 2008). Phosphogluco mutase (PGM) activity

was measured by using (Manjunath et al., 1998) method. For it, ,4 mM DTT, 0.1 mM

G1,6bisP, 10 mM MgCl2, 1 mM G1P, 0.25 mM NAD, and 0.64 U of G6PDH with plant

extract in 20 mM TRIS-HCl at pH 8.0.

For determination of glucose-6-phosphate dehydrogenase (G6PDH) activity,

(Deschepper, 1982) method were used. Dialysed extract were used with, 1 mM G6P, 0.4

mM NADP in 100 mM TRIS-HCl at pH 7.6 and 5 mM MgCl2. The increase in absorbance

at 340 nm in all kinetics except phosphofructokinase (PFK) and Aldolase (Ald) due to

conversion of NADP to NADPH was monitored.

34

3.13.4 Key Enzyme of Carbohydrate Metabolism (A Enzymes) Assays

Activity of SOD was measured by method (Beauchamp and Fridovich, 1971).

Dialysed extract were incubated with buffer (50mMKPO4 PH 7.8 and 0.1mM EDTA)

along with 0.05 Cytochrome c and 10mM Xanthine. For control Xanthine were omitted.

Activity of CAT was determined by (Aebi, 1984). Dialysed extract were incubated with

(50mM buffer, AF 2040.11% and 9.8mM H2O2). For control H2O2 were not used. Activity

of APX were determined by (Nakano and Asada, 1981). Dialysed extract were incubated

with (50mM buffer, 50 mM ascorbate and 10mMH2O2). For control reaction H2O2 were

eliminated. Glutathione reductase (GR) activity was measured by using method (Edwards

et al., 1990). Dialysed extract were incubated (100Mm Thris Hcl pH 7.8, 0.2mM NADPH

and 0.6mM Glutathione oxidized (GSSG). For control GSSG were omitted.

The grounded material was put into a 10 ml centrifuge tube, where 5 ml of 80%

ethanol was added. The pellets were extracted two more times with 80% ethanol.

Supernatants were retained, combined and stored at − 20 °C and further soluble sugar were

determined. For starch determination ethanol-insoluble pellet was used. Glucose was used

as a standard. Concentration of soluble sugars and starch was expressed on a dry matter

basis. Total soluble sugar and starch concentration were sum up and then concentration of

non-structural carbohydrates was obtained.The analysis was made on HPLC with aminex

87H column at 37oC and 600 ml/min.

3.14 STATISTICAL ANALYSIS OF DATA

Microsoft Excel 2002 (Microsoft Corp., Redmond, WA, USA) was used for

statistical calculations and descriptive statistics were applied to organize and analyze the

data. Triplicate data were used; Factorial analysis were used to analyze data significance of

data was tested y Tucky’s test (Tukey, 1949). Values presented in table, graphs are mean

35

±SE; bars with different alphabets differ significantly from each other. For second

experiment four replications were used and microsoft excel was used for analysis. Three

ways ANOVA was used to see the differences.

36

Chapter 4

RESULTS AND DISCUSSION

Five wheat cultivars viz. AARI-11, Chakwal-50, Shahkar, Pakistan-13 and FSD-08

have been used to have a look at the impact of drought and to manage drought with

hormonal seed priming. Priming was done by exposing seeds of five genotypes in 10-4 M

aerated solution of SA and GA for 8h, non-primed seeds was also used. Following findings

were observed about seed biochemistry, yield attributes, leaf biochemical and

Physiological attributes and seed quality attributes.

4.1 SEED BIOCHEMISTRY

In this section of study, pre sowing treatments with plant growth hormones induced

biochemical changes in wheat seeds were investigated with main emphasis on different

oxidative enzyme modulations i.e. protease, amylase, esterase, superoxide dismutase,

peroxidase, catalase. Further we can use these biochemical markers for screening against

different stresses.

4.1.1 Oxidative Enzymes Assay

4.1.1.1 Protease activity

All genotypes depicted significant variation under normal and primed condition

regarding protease activity (Fig. 4.1). The highest Protease activity was found in Shahkar

(5965±285µM/min/g f.wt.) and the lowest Protease activity (3395±115µM/min/g f.wt.)

was found in AARI-11 genotypes in absence of any priming treatment. Shahkar also

expressed the highest Protease activity when primed with SA while AARI-11, Pakistan-13

and FSD-08 genotypes were lowest protease activity. Significant increase in protease

activity (7275±75µM/min/g f.wt.) was observed in Chakwal-50 and Shahkar

(9110±20µM/min/g f.wt.) on SA priming. FSD-08 and Pakistan-13 had the highest

37

protease activity on GA priming while other genotypes remain unaffected. Seed priming

with GA showed that Shahkar (10325±345µM/min/g f.wt.) performed better results than

AARI-11 (4015±25µM/min/g f. wt.) in ranking of enzyme activity.

Figure 4.1: Effect of priming on protease activity in wheat seeds; Np-non primed, SA-Salicylic acid and

GA-Gibberellic acid

Under water stress many antioxidant enzymes were induced among them proteases

were also induced (Bray, 2002; Carvalho et al., 2001). When plants were exposed to stress

intracellular proteases have an ability to degrade injure or unwanted proteins, metabolism

reorganization and also help in remobilization of nutrient (Feller et al., 2008). It was crucial

for the researchers to recognize the linking mechanism among proteolysis and plant concert

in water stress and remedies from stress. It was still not understandable that under stress

high proteolytic activity is beneficial for the plant to help in restructuring of protein model

or it leads to cell breakdown. Some investigational facts suggest that proteolytic activity

was maximum in drought sensitive species and varieties compared to resistant ones (Hieng

et al., 2004).


All tested genotypes depicting the significant variations under normal and primed

condition (Fig. 4.2). In control seeds was present in FSD-08 had (11.69±1.50mg/g. f.wt.)

fgef

de

gfg

g

c

b

g gg

cd

a

cdde

0

2000

4000

6000

8000

10000

12000

AARI-11 Chakwal-50 Shahkar Pakistan-13 FSD-08

Pro

teas

e (µ

M/m

in/g

f.

wt) NP SA GA

38

the highest amylase activity followed by Pakistan-13 (9.24±0.943) and AARI-

11(9.24±0.189mg/g. f.wt.) genotypes. When priming with SA applied it enhanced amylase

activity in AARI-11, Shahkar and Pakistan-13 along with unaffected biochemical changes

in FSD-08 and Chakwal-50 genotypes was observed. Priming with GA also increased

amylase activity in all genotypes and double increased of enzymatic activity in Shahkar,

Pakistan-13 and AARI-11.

Figure 4.2: Effect of priming on amylase activity in wheat seeds

Literature in support of this study exhibited that when α-amylase activity increased

in the seeds accountable to analogous increase in non-reducing sugar level in the seeds by

chitosan priming (Farooq et al., 2006a). Previous findings showing that amylase and sugar

contents directly increased in primed rice kernels confirmed that seed priming either

induced the de novo synthesis or increases the activities of existing enzymes (Lee et al.,

2007). Seed germination is result of enzyme generation or enzyme activation essential for

the mobilization and degradation of seeds reserves (Subramani et al., 2011). During this

process α-amylase and proteases control starch digestion and protein digestion respectively

while the hydrolysis of different types of esters was done by esterase enzyme (Subramani

et al., 2011).

cdd d cd

cd

a

cdcd

ab

cd

bc

cd

abab

cd

0

10

20

30

40

50


Am

yla

se (

mw

g/g

. f.

wt)

NP SA GA

39

4.1.1.3 Esterase activity

An increased esterase activity in priming with GA hormone was observed. Esterase

might be take part in metabolic process during seed germination and maturation of plants.

For growing embryos, esterase is responsible to release and provide stored food material

(Subramani et al., 2011). In recent study, among all genotypes under control and primed

seeds treatment esterase activity was slightly increased (Fig. 4.3). FSD-08 showed

significantly maximum esterase activity (1001± 7.86µM/min/g f.wt.) when primed with

GA. Seed germination is result of enzyme generation or enzyme activation essential for the

mobilization and degradation of seeds reserves (Subramani et al., 2011).

Figure 4.3: Effect of priming on esterase activity in wheat seeds


Superoxide dismutase (SOD) activity showed significant variability in tested

genotypes depicted the Shahkar at higher rank regarding SOD activity (18.41

±0.16units/buf used) while other genotypes had the same magnitude when non primed

seeds were observed (Fig. 4.4).Under seed priming with SA the highest SOD activity

(24.81±0.30units/buf used) showing genotype was Chakwal-50. Increasing trend in SOD

activity under treatment with SA was reported in Chakwal-50, AARI-11and Pakistan-13

while a decreased SOD activity was observed in FSD-08 and Shahkar genotypes. Under

b b bb bb b b b bb

b b ba

0

500

1000

1500


Est

rase

(µ

M/m

in/g

f.

wt) NP SA GA

40

GA mediated seed priming AARI-11 genotype showed leading trend related to SOD

activity among all tested genotypes showing enhanced SOD activity.

Figure 4.4: Effect of priming on SOD activity in wheat seeds

To protect cell against oxidative and environmental stress SOD is consider as an

important enzyme. These findings support the previous research on seed priming and

increased superoxide dismutase activity in rice seeds (Xiaohuan et al., 2009). Due to seed

priming in Victoria and Victor seedlings, antioxidant enzymes i.e. Catalase, Superoxide

dismutase and Peroxidase showed increased activities (Zhang et al., 2007a).

4.1.1.5 Peroxidase activity

Significant variation was present in all tested genotypes; the highest peroxidase

(POD) activity (33533± 699 units/g f.wt.) was observed in FSD-08while the lowest POD

activity (9823± 1032 units/g f.wt.) was present inAARI-11 in non primed seeds(fig. 4.5).

The SA priming significantly leading position related to POD activity was enhanced in all

genotypes except FSD-08 which remain unaffected. In case of GA priming, POD activity

increased in all tested genotypes except FSD-08.

f ef

de

eff

abc ab

ef

abc

g

a

cd

abc

cd bcd

0

5

10

15

20

25

30


SO

D (

unit

s/b

uf

use

d)

NP SA GA

41

Figure 4.5: Effect of priming on POD activity in wheat seeds

Increasing POD could significantly enhance seed tolerance to abiotic conditions

(Ansari and Sharif-Zadeh, 2012). The same results were reported on Berseem (Trifolium

alexandrinum L.) that depicted the improved activities of antioxidant enzyme like

superoxide dismutase, peroxidase and catalase in primed seeds as compare to those

untreated seeds (Rouhi et al., 2012).


Catalase resists the cell towards the oxidative harm by using elimination of

unfastened radicals or ROS and consequently also quality of seed was improved (Yeh and

Sung, 2008). Under control condition, AARI-11 (3340±300units/g. f.wt.) and Shahkar

(3320±180units/g. f.wt.) had the same magnitude of CAT activity. Salicylic acid enhanced

the CAT activity in all genotypes (Fig. 4.6). In study of CAT activity AARI-11 genotype

had the leading position under both SA and GA mediated seed priming analysis while FSD-

08 had the lowest rank on CAT activity in SA seed priming analysis. In case of GA priming,

CAT activity significantly increased in all tested genotypes. (Ahmed et al., 2012)

investigated that catalase (CAT), protease, amylase and superoxide dismutase (SOD)

activates were improved when different priming agents were used. The ascorbate

peroxidase improvement in wheat is also associated with the seeds primed with salicylic

f fef

f

ab

abc bcd

ab a

bcd

defcde cde

cdef

cd

0

10000

20000

30000

40000


PO

D (

Unit

s/g f

. w

t)

NP SA GA

42

acid and gibberellins in comparison to the non treated seeds (Tabatabaei, 2013).The

worldwide demand of wheat is increasing rapidly and may be exceed till 2050 up to 750

million tons (Mujeeb-Kazi, 2006).

Figure 4.6: Effect of priming on CAT activity in wheat seeds

Seed germination enhancement through priming is linked with incentive of

antioxidant enzymes activities (Afzal et al., 2011). The numerous valuable sound effects

of priming have been reported in various field crops including, sunflower, maize, soybean,

sugar beet and wheat (Kusvuran et al., 2014). Physiological feature of plants were enhanced

by seed priming through growth hormones under both drought and balanced conditions

with the increased activity of antioxidants i.e. CAT, POD, SOD and that protect the cell

adjacent to creation of free radical and prevent from oxidative stress (Eisvand et al., 2010).

4.1.1.7 Cluster analysis based on seed biochemistry attributes

Clusters analysis (CA) showed that genotypes possess similarity to each other form

main two groups (Fig 4.7). Group one and two further forms a similar cluster within group.

It was observed that the genotypes within group may be similar to each other while those

out of groups were not similar.

bcd

ef

ef

bcd

ef

f

ef

a bcd

e

ab bcd

e def

bc

bcd

e

bcd

e

ab

cd cdef

0

2000

4000

6000

8000


CA

T (

Unit

s/g.

f. w

t)

NP SA GA

43

Figure 4.7: Dendogram derived from hierarchical cluster analysis of combined seed biochemical attributes

4.1.2 Conclusion

Priming with SA and GA increased the activity of oxidative and antioxidant

enzymes such as amylase, protease, catalase, superoxide dismutase and peroxidase.

However, genotypes response varied in term of different antioxidant enzymes. Shahkar had

the highest protease activity in primed seeds while AARI-11 had the highest amylase

activity. Similarly AARI-11, Shahkar and Chakwal-50 had the highest Superoxide

dismutase activity while Shahkar and Pakistan-13 had the highest peroxidase activity. By

using SA and GA as a priming agent abiotic stress of wheat plant can be overcome by

modulating the activities of antioxidants.

44

4.2 MORPHOLOGICAL ATTRIBUTES

This study was designed in order to discover the phenomenon for better wheat

growth under water deficit conditions. Significant variability was noticed for plant height

(cm) under optimum and water deficit growing conditions (Table 4.1). More height was

observed for AARI-11 (102.7±1.27cm) while Chakwal-50 was short stature (91.1±1.15cm)

under normal conditions in absence of priming. AARI-11, Chakwal-50 and Pakistan-13

respond more to plant height by SA priming while slight in case of GA priming, more

height was observed for Pakistan-13, Chakwal-50 and FSD-08.

Similar response was noticed for plant height under drought. Highest value of plant

height was recorded for AARI-11 (95.0±5.21cm) and lowest for Chakwal-50 (78.8±1.42

cm) in the absence of priming. Both SA and GA were helpful to maintain the plant height

under irrigated conditions however, under drought stress height was significantly reduced.

As lower plant height is suitable to obtain desirable yield.

Under both growing conditions a reasonable variability was observed for the

number of tillers (

Table 4. 2). In the absence of priming, tillers was increased in Shahkar under normal

and for Pakistan-13 under drought conditions (6.4±0.78 and 3.8±0.33) respectively.

Similarly, the lowest number of tillers was observed in drought conditions. By the

application of SA and GA slight increase in tillers number was observed for Pakistan-

13under normal with their individual effect (6.4±0.65) and (5.8±0.99) respectively under

while their combined effect was (5.9±0.4). Likewise, more number of tillers were recorded

for Pakistan-13 under drought conditions after their treatment with SA and GA (4.8±0.60

and 4.2±0.26) respectively and their combined effect was (4.2±0.26). The combined

analysis revealed that more effect of drought was observed during 2014.

45

Substantial variation for spike length (cm) was measured. Pakistan-13 expressed

more spike length under non-primed normal conditions (13.0±0.21cm) followed by AARI-

11, Chakwal-50 and FSD-08 (12.6±0.25cm), 11.2±0.15cm) and (11.0±0.27cm)

respectively while small spike length (10.3±0.22cm) was observed in Shahkar. SA caused

improvement in spike length in Shahkar, Pakistan-13 and AARI-11. Similarly, GA also

caused improvement in spike length but non-significant differences were observed in

Pakistan-13.Interestingly, Pakistan-13 showed the highest spike length (10.8±0.15cm)

under drought conditions without any priming (

Table 4. 3). However, increased spike length was observed for FSD-08

(10.8±0.21cm) and AARI-11 (11.2±0.19cm) by treatment with SA and GA hormones.

Conversely, spike length was significantly decreased under Drought however this effect

was overcome by their treatment with SA and GA. AARI-11 (11.950cm) was observed to

be tolerant line based upon its mean values under both conditions. The mean analysis of

both years a decreasing trend of spike length. Conversely, spike length was significantly

decreased under Drought however this effect was overcome by their treatment with SA and

GA. AARI-11 (11.950cm) was observed to be tolerant line based upon its mean values

under both conditions. The mean analysis of both years a decreasing trend of spike length.

46

Table 4. 1: Effect of seed priming and drought on plant height (cm) of wheat during 2014 and 2015; Np-non primed, SA-Salicylic acid and GA-

Gibberellic acid

Genotypes Control Mean Drought Mean Overall

mean NP SA GA NP SA GA

AARI-11 102.7±1.27g-I

103.9±0.81i

101.1±0.83g-i

102.6±0.60e

84.9±1.27a-c

85.4±2.04a-c

90.7±1.52cd

87.0±1.09c

94.92

Chakwal-50 91.1±1.15cd

94.6±0.92d

95.4±0.86

93.7±0.70d

78.8±1.42a

82.03±0.64a

82.3±1.32a

81.0±0.74a

87.40

Shahkar 95.6±1.44d-g

96.4±1.11d-h

96.0±1.44d-g

96.0±0.73d

79.0±1.07a

84.4±1.12a-c

80.6±0.44a

81.3±0.75a

88.70

Pakistan-13 101.0±0.72f-I

103.5±0.79hi

103.5±0.77i

102.6±0.50e

82.9±1.12ab

90.1±1.79b-d

85.8±1.24bc

86.2±1.05bc

94.47

FSD-08 96.5±0.56d-h

97.3±0.78ef-i

98.7±1.17fg-i

97.5±0.52d

80.7±2.07a

82.6±1.04a

84.6±1.08a-c

82.6±0.88ab

90.10

Years

2014 97.64 100.08 100.18 94.69 80.56

85.40 86.41 84.12 91.53a

2015 96.14 98.28 98.46 97.96 82.02

83.47

84.28

83.59 90.67a

Mean 96.89

99.18

99.27

98.61

81.29

84.94

85.32

83.69

91.15

47

Table 4. 2: Effect of seed priming and drought on number of tillars of wheat during 2014 and 2015



AARI-11 3.0±0.68a 4.3±0.84a 3.8±1.01a 4.7±0.48ab 3.1±0.48ab 3.3±0.22a 3.5±0.66a 3.3± 0.26a 4.067

Chakwal-50 4.6±0.36a 4.9±0.85a 4.7±0.51a 3.7±0.35a 2.9±0.22a 3.2±0.11a 3.5±0.71a 3.2±0.24a 3.519

Shahkar

6.4±0.78a 5.9±0.82 5.4±0.89a 4.7±0.45ab 3.5±0.15a 3.4±0.38a 2.9±0.28a 3.3±0.16a 4.039

Pakistan-13

5.7±0.62a 6.4±0.65a 5.8±0.99a 5.9±0.43b 3.8±0.33a 4.8±0.60a 4.0±0.37a 4.2±0.26ab 5.089

FSD-08 5.3±0.76a 4.9±0.76a 3.1±0.93a 6.02±0.45b 3.3±0.22a 3.9±0.65a 3.8±0.78a 3.7±0.33a 4.875

Years

2014 5.59 5.81 5.64 5.68 3.45 4.31 3.96 3.90 4.588a

2015 4.48 4.52 4.58 4.52

3.31 3.56 3.28 3.38 4.048a

Mean 5.04 5.15 5.11 5.10 3.38 3.94 3.62 3.64 4.37

48

The highest number of spikelets was recorded for Pakistan-13 under non-primed

(22.0±0.64), SA (22.3±0.47) and GA (22.8±0.41) hormones (Table 4.4). However, lowest

number spikelets were counted for Chakwal-50 under primed and non-primed conditions.

In contrast, FSD-08 showed the highest number of spikelet under drought non-

primed (18.8±0.27) and SA (19.6±0.20) treatment while Shahkar respond more to GA

under drought conditions (19.8±0.32). An increasing trend in number of spikelet was

observed by the application of priming except in FSD-08. Surprisingly, GA treatment

decreased the number of spikelets in comparison to control. Mean avalue of both growing

situations manifested that highest spikelet (20.52) was found in Pakistan-13 while the

lowest mean was recorded for Chakwal-50 (18.70). Like most of the studied traits have

same trend for both consecutive years (2014 and 2015).

Extrusion length (cm) also showed significant variation for all the genotypes under

normal and drought conditions. FSD-08 showed highest extrusion length (13.8±0.81cm)

under normal non-primed condition. For this trait Pakistan-13 exhibited higher extrusion

length (16.0±0.68cm and 16.5±0.40cm) for SA and GA treatments respectively. FSD-08

performed well under drought conditions and highest extrusion length (12.1±0.66cm and

2.6±0.68cm) were recorded for non-primed and GA treatment respectively. However,

Shahkar was more responsive to SA application and highest extrusion length was observed

in this genotype.

49

Table 4. 3: Effect of seed priming and drought on spikelength of wheat during 2014 and 2015



AARI-11

12.6±0.25e-h 13.7±0.35h 13.2±0.23h 13.1±0.19d 10.6±0.18bcd 10.3±0.25a-d 11.2±0.19de 10.7±0.14b 11.950

Chakwal-50

11.2±0.15de 11.0±0.22cd 11.7±0.37defg 11.3±0.16bc 8.8±0.23a 9.3±0.15ab 9.6±0.15abc 9.2±0.12a 10.300

Shahkar

10.3±0.22bcd 11.7±0.08d-g 11.6±0.26def 11.2±0.19bc 9.3±0.18ab 9.7±0.29abc 9.3±0.15ab 9.4±0.12a 10.350

Pakistan-13

13.0±0.21gh 13.3±0.20h 12.8±0.35fgh 13.0±0.15d 10.8±0.15cd 10.6±0.29bcd 10.71±0.14bcd 10.7±0.11b 11.900

FSD-08

11.0±0.27cd 11.5±0.24def 12.8±0.27fgh 11.8±0.23c 10.3±0.24bcd 10.8±0.21cd 10.8±0.30cd 10.6±0.14b 11.236

Years

2014 11.66 12.40 12.41 12.16 9.89 10.36 10.46 10.23 12.19a

2015 11.68 12.12 12.46 12.08 10.12 9.96 10.23 10.10 11.09a

Mean 11.67

12.26 12.44 12.12 10.00 10.16 10.34 10.17 11.14

50

Table 4.4: Effect of seed priming and drought on spikelets of wheat during 2014 and 2015

Genotypes Control Mean Drought Mean Over

all


AARI-11 20.5±0.42d-

i 20.9±0.32e-i 21.5±0.32ghi 21.0±0.21b 18.5±0.46a-e 18.8±0.45a-f

18.9±0.38a-

f

18.7±0.23

a 19.88

Chakwal-50

17.9±0.41ab 19.8±0.73b-h 20.5±0.14c-i 19.4±0.37a 16.4±0.41a 18.6±0.52a-e

18.9±0.21a-

f

18.0±0.34

a 18.72

Shahkar 21.0±0.69e-

i 21.5±0.40ghi 21.4±0.42ghi 21.3±0.29b 18.0±0.47abc 19.1±0.26b-g

19.9±0.16b-

h

19.0±0.26

a 20.20

Pakistan-13 22.0±0.64e-

i 22.3±0.47f-i 22.8±0.41hi 21.4±0.302b 18.2±0.29a-d 18.7±0.18a-e

19.8±0.36b-

h

19.0±0.17

a 20.52

FSD-08 21.5±0.40gh

i 21.8±0.55hi 22.6±0.32i 21.9±0.26b 18.8±0.27a-f

19.6±0.20b

—h

18.6±0.32a-

e

18.9±0.22

a 20.20

Years

2014 20.92 21.74 21.88 21.51 18.74 19.96 20.11 19.94 20.05

a

2015 19.78 19.78 21.12 20.22 17.94 19.02 20.40 19.78 19.76

a

Mean 20.35

20.76 21.5 20.87 18.34 18.99 19.25 18.86 19.86

51

It was also observed that water deficiency significantly reduced the extrusion length

in all varieties. Mean value of drought and irrigated conditions exhibited that FSD-08 has

highest (13.61cm) extrusion length while Chakwal-50 had only 11.88cm. All the studied

genotypes showed non-significant variation for peduncle length under drought and normal

conditions. Pakistan-13 showed highest (34.5±1.16cm) while Chakwal-50 exhibited lowest

(30.5±0.80cm) peduncle length under normal and non-primed conditions. SA priming

slightly increased the peduncle length in all genotypes except in Shahkar. However, GA

Priming increased the peduncle length in all studied genotypes. However, FSD-08 and

Pakistan-13 performed well for peduncle length (31.7±0.49cm) under drought while lowest

length was recorded in Chakwal-50 (28.5±0.40cm). Even with the application of SA and

GA this trait was unaffected for all the genotypes. A small increase for peduncle length was

observed for AARI-11 and Chakwal-50 in non-primed genotypes. A significant reduction

in length of peduncle was found in all genotypes without water condition. Based upon the

mean data analysis for both

52

Table 4.5:Effect of seed priming and drought on extrusion length of wheat during 2014 and 2015



AARI-11 13.0±0.84a-e 15.1±1.03cde 14.9±0.77cde 14.3±0.53bc 10.1±0.56ab 11.5±0.58a-d 12.0±0.66a-e 11.2±0.38a 12.82

Chakwal-50

11.7±1.05a-d 13.1±0.72a-e 13.8±0.57a-e 12.9±0.48abc 9.7±0.35a 11.0±0.40abc 11.7±1.13a-d 10.81±0.43a 11.88

Shahkar

12.8±0.90a-e 14.6±0.75b-e 15.4±0.69c-e 14.32±0.50bc 11.5±0.56a-d 12.0±0.44a-e 12.4±0.71a-e 11.9±0.32ab 13.16

Pakistan-13 13.0±0.98a-e 16.0±0.68de 16.5±0.40e 15.20±0.54c 11.6±0.71a-d 11.9±0.83a-d 12.1±0.76a-e 11.9±0.42a 13.55

FSD-08 13.8±0.81a-e 15.3±1.14cde 15.9±0.63de 15.03±0.52c 12.1±0.66a-e 11.8±0.39a-d 12.6±0.61a-e 12.18±0.32ab 13.61

Years

2014 12.973 14.24 15.06 14.09 10.83 11.360 12.06 11.42 13.16a

2015 13.20 14.39 15.21 14.27 11.22 11.360 13.25 12.33 12.84a

Mean 13.09

14.31 15.13 14.18 11.03 11.36 12.30 11.75 12.96

53

Table 4.6: Effect of seed priming and drought on peduncle length (cm)of wheat during 2014 and 2015

Genotypes Control

Mean Drought

Mean Overall


AARI-11 57.2±1.16f-j 59.3±0.71h-k 59.3±1.21g-k 58.6±0.62de 49.5±1.23 52.1±1.07 56.0±1.01 52.5±0.88bc 55.59

Chakwal-50 50.7±0.61b-f 55.3±0.82d-h 55.4±0.85d-h 53.839±0.67bc 43.6±0.68 48.3±0.99 49.4±0.92 47.1±0.77a 50.49

Shahkar 57.2±1.31f-j 56.5±0.62f-j 62.8±1.21jkl 58.8±0.90def 47.9±0.78 52.0±0.62 55.7±0.83 51.8±0.87b 55.37

Pakistan-13 62.3±1.24h-l 60.3±1.02h-l 64.9±1.12kl 62.5±0.76ef 55.6±1.07 54.6±1.52 59.4±1.71 56.5±0.93cd 59.82

FSD-08 60.7±1.99h-l 62.4±1.17i-l 66.0±1.09l 63.0±0.96f 53.1±0.59 57.2±0.87 59.3±0.86 56.5±0.75cd 59.55

Years

2014 59.47 59.36 62.84 60.56 50.38 53.98 57.10 53.16 57.86a

2015 55.82 58.24 60.58 58.21 49.58 53.74 54.89 52.73 55.47a

Mean 57.65 58.80 61.71 59.39 49.98 52.86 56.00 52.94 56.16

54

drought and irrigated Pakistan-13 was with highest mean value (33.39cm) and Chakwal-50

had lowest (31.90cm).

Also, significant variation was observed for grain numbers under normal and

drought conditions. Highest number of grains (62.3±1.24) was recorded for Pakistan-13

whiles the lowest for Chakwal-50 (50.7±0.61) under non-primed normal condition. SA and

GA improved this number in all studied genotypes, Pakistan-13 (55.6±1.07) showed

highest number of grains while Chakwal-50 (43.6±0.68) exhibited lowest value under

drought non-primed conditions. SA priming increased the grain number in all genotypes

except Pakistan-13. However, GA priming increased this number in all genotypes.

Significant reduction in grain number was observed under drought. However, mean data

analysis for drought and irrigated condition suggested that Pakistan-13 and FSD-08 are

superior varieties for this trait (59.0) while lowest value was recorded for Chakwal-50

(50.0).

55

Table 4.7: Effect of seed priming and drought on number of grains of wheat during 2014 and 2015



AARI-11 37.3± 0.95b-i 40.0±0.66f-i 39.8±1.22e-i 39.0±0.60cd 32.9±0.67abc 34.5±0.57a 34.3±0.74a-e 33.9±0.39a 36.52

Chakwal-50

35.3±0.78a-g 38.1±1.00b-i 38.3±1.32c-i 37.2±0.66bc 31.7±0.75a-f 32.7±1.04ab 33.5±0.76a-d 32.6±0.505a 34.98

Shahkar

40.6±0.82ghi 39.0±0.70d-i 39.3±1.44e-i 39.7±0.58cd 31.5±0.71a 34.9±0.67a-g 35.0±1.01a-f 33.8±0.59a 36.78

Pakistan-13

39.6±1.29e-i 41.0±0.82hi 41.6±0.60i 40.7±0.55d 34.3±1.04a-e 35.0±0.70a-h 35.6±0.82a-h 35.0±0.48ab 37.90

FSD-08

39.3±1.05e-i 39.7±1.10ghi 40.9±0.76hi 39.9±0.56cd 34.6±0.33a-f 36.9±0.91a-e 35.0±1.26a-g 35.2±0.51ab 37.62

Years

2014 40.01 40.99 42.44 41.55 34.65 36.45 37.43 36.51 37.25a

2015 35.50 37.35 38.95 37.27 31.17 33.86 34.64 33.89 36.27a

Mean 37.76

38.271 39.20 38.41 34.41 35.16 36.04 35.20 36.80

57

Significant variation was found for 1000-grain weight under both drought and normal

conditions (Table 4.). Shahkar and Chakwal-50 showed highest (40.6±0.82g) and lowest

(35.3±0.78g) 1000-grain weight respectively under normal and non-primed condition.

Priming with SA caused slight increase for this trait in AARI-11, Chakwal-50 and Pakistan-

13 while GA triggered more 1000 grain weight for Shahkar. In non primed samples under

drought 1000-grain weight was higher in Pakistan-13 (34.6±0.33g) and lowest in Shahkar

(31.5±0.71g). Treatment with both primers increased the thousand grain weight of all the

studied germplasm.

Like many agronomic traits Pakistan-13 exhibited highest (1910.1±30.79kg/ha)

grain yield under normal non-primed conditions while FSD-08 showed high output

(1583.5± 76.21kg/ha) under drought and non-primed conditions (Table 4.9). Lower grain

yield was recorded for Chakwal-50 under irrigated (1289.3±40.98kg/ha) and drought

(936.1± 26.70kg/ha) in non-primed conditions. However, SA priming increased the grain

yield in all genotypes under normal conditions. Likewise, priming with this growth

regulator was also effective for all genotypes under drought

Also GA priming was effective to increase the grain yield under irrigated conditions

but under drought it was effective, however FSD-08 showed non-significant differences.

Mean values of normal and drought conditions suggested that Pakistan-13 performed well

(1764.77kg/ha) while Chakwal-50 was more severely affected (1189.66kg/ha).

58

Table 4.8: Effect of seed priming and drought on thousand grain weight of wheat during 2014 and 2015



AARI-11 37.3± 0.95b-i 40.0±0.66f-i 39.8±1.22e-i 39.0±0.60cd 32.9±0.67abc 34.5±0.57a 34.3±0.74a-e 33.9±0.39a 36.52

Chakwal-50 35.3±0.78a-g 38.1±1.00b-i 38.3±1.32c-i 37.2±0.66bc 31.7±0.75a-f 32.7±1.04ab 33.5±0.76a-d 32.6±0.505a 34.98

Shahkar 40.6±0.82ghi 39.0±0.70d-i 39.3±1.44e-i 39.7±0.58cd 31.5±0.71a 34.9±0.67a-g 35.0±1.01a-f 33.8±0.59a 36.78

Pakistan-13 39.6±1.29e-i 41.0±0.82hi 41.6±0.60i 40.7±0.55d 34.3±1.04a-e 35.0±0.70a-h 35.6±0.82a-h 35.0±0.48ab 37.90

FSD-08 39.3±1.05e-i 39.7±1.10ghi 40.9±0.76hi 39.9±0.56cd 34.6±0.33a-f 36.9±0.91a-e 35.0±1.26a-g 35.2±0.51ab 37.62

Years

2014 40.01 40.99 42.44 41.55 34.65 36.45 37.43 36.51 37.25a

2015 35.50 37.35 38.95 37.27 31.17 33.86 34.64 33.89 36.27a

Mean 37.76

38.27 39.20 38.41 34.41 35.16 36.04 35.20 36.80

59

Table 4.9.: Effect of seed priming and drought on grain yield(kg/ha) of wheat during 2014 and 2015



AARI-11 1696±42.2i-l 1636±48.31-l 1632±59.7h-l 1654±28.3d 1220±30.13a-e 1227±31.6 a-f 1388±18.7e-i 1278±23.9b 1466.8

Chakwal-

50 1289±40.9b-h 1368±42.2c-l 1458±68.3d-i 1372±32.8bc 936±26.7a 987±27.3 ab 1097±21.1abc 1007±21.3a 1189.6

Shahkar 1533±56.7e-j 1641±46.6i-l 1659±63.1i-l 1611±33.1d 1146±42.7 a-d 1174±75.1a-d 1278±48.6a-g 1199±34.0b 1405.5

Pakistan-13

1910±30.7kl 1917±87.2kl 1911±60.5kl 1912±34.6e 1463±144.8d-i 1579±35.5 g-k 1648±35.1i-l 1616±29.8d 1764.7

FSD-08 1849±26.3g-l 1947±36.7l 1935.1±37.2l 1910±21.1e 1583±76.2 g-k 1618±40.0g-l 1571±108.2f-k 1537±59.1cd 1724.1

Years

2014 1719.7 1754.2 1742.8 1738.9 1384.1 1374.0 1475.8

1411.3 1566.2

2015 1531.6

1563.6

1595.5

1563.5

1108.9

1227.2

1311.1

1215.7 1454.1

60

The highest biological yield was recorded in FSD-08 under non-primed normal

(5873.3±119.44kg/ha) and drought (5120.1±95.15kg/ha) conditions however, all the

genotype varied significantly. Lowest biological yield was recorded for Chakwal-50 under

irrigated and drought conditions (4983.0±64.11kg/ha and 4579.8±75.74kg/ha respectively)

without any treatment. Priming with SA and GA revealed the increase in biological yield

in most of genotypes. All the genotypes were responsive to SA priming under drought

except FSD-08 however; GA priming was effective for all genotypes. Combined results for

stress and without stress exhibited that FSD-08 was with more biological yield

(5498.11kg/ha) and Chakwal-50 was with less yield (4859.83kg/ha).

Non-primed seed of Pakistan-13 performed well under well watered (31.8±1.18%)

and drought (27.0±2.32%) situations while Chakwal-50 was again at bottom for irrigated

drought conditions (24.4±0.74%) and (18.8±0.41%) respectively. Priming with SA

increased in AARI-11and Chakwal-50 under both conditions. Mean analysis of drought

and irrigated condition suggested that FSD-08 (29.350%) is a superior variety while

Chakwal-50 could not perform well (23.24%). Based upon all yield contributing traits 2014

was better year as compared to 2015though the results were non-significant. Likewise,

drought affected these entire traits more severely during 2015.

Amongst testing cultivars in non primed samples, AARI-11 had the highest

plant height under stress and without stress conditions. The consequences suggested on

this experiment also matched with the findings in literature that when GA3 was applied on

fenugreek as a foliar spray it significantly

61

Table 4.10: Effect of seed priming and drought on biological yield (kg/ha) of wheat during 2014 and 2015

Genotypes Control Mean Drought Mean Overa

ll


AARI-11 5827±87.3h-i 5709±128.5ghi 5949±125.5i 5829±66.9f 4709±66.8a-c 4762±97.2a-d 4896±59.8a-f 4789±45.6ab 5309.1

Chakwal-50 4983±64.1a-f 5166±46.5e-g 5487±186.3f-i 5212±81.0cd 4579±75.7a-c 4610±58.0a 4631±48.5ab 4507±37.7a 4859.8

Shahkar

5325±81.3d-h 5456±57.0f-i 5345±80.0d-h 5375±42.3de 4846±77.2a-e 5031±78.5b-f 5068±76.1b-f 4981±48.1bc 5178.8

Pakistan-13

5789±71.1hi 5813±132.5hi 5426±143.4e-i 5676±77.9ef 5076±103.1b-f 5134±89.3c-g 5225±62.6b-g 5111±47.4cd 5394.1

FSD-08 5873±119.4hi 5948±189.2i 5864±109.1hi 5895±78.4f 5120±95.1b-g 5078±59.9b-f 5103±69.0b-f 5100±41.5cd 5498.1

Years

2014 5670.80 5753.13 5706.00 5709.9 5094.80 5180.00 5272.53 5115.77 5303.9

2015 5288.93 5184.80 5363.26 5279.0 4810.93 4853.66 4930.93 4831.84 5192.0

Mean

5479.86

5468.96 5534.63 5494.4 4831.84 5215.77 4951.73 4954.86 5224.6

62

Table 4.11: Effect of seed priming and drought on harvest index (%)of wheat during 2014 and 2015



AARI-11 27.0±0.57d-h 28.6±0.69fh 27.7±0.29d-h 27.8±0.33c-e 22.5±0.52 24.2±0.43b-f 20.8±0.27a-c 22.5±b0.40 25.20

Chakwal-50 24.4±0.74c-h 25.2±1.20d-h 25.0±0.34c-g 24.9±0.46bc 18.8±0.41a 20.5±0.24a-c 21.3±0.06ab 19.5±0.23a 23.24

Shahkar

28.5±0.29e-f 28.8±0.10f-h 28.7±0.62f-h 28.6±0.22d-f 20.8±0.27b-f 22.0±0.17a-c 24.4±0.50a-c 22.1±0.43ab 25.40

Pakistan-13 31.8±1.18h 28.9±0.70f-h 29.2±1.06 29.9±0.62ef 27.0±2.32d-h 28.3±0.85a-e 30.1±0.54gh 26.9±0.79cd 29.35

FSD-08

30.9±1.10h 31.2±1.22g-h 31.5±0.99 30.9±0.61f 23.3±0.85d-h 27.3±1.78d-h 28.9±1.10fgh 27.7±1.0cde 28.48

Years

2014 27.87 29.24 30.31 29.14 21.34 24.27 25.6 23.7 26.61a

2015 26.27 28.52 29.61 28.13 20.47 23.28 23.79 22.52 25.66a

Mean 27.07 28.88 29.96 28.64 20.91 23.783 24.70 23.13

25.88

63

increased plant height, branches and grain yield. The present work revealed a lessening in

the number of tillers in one plant, length of the spike, spikelets and a reduced thousand

kernel weight due to drought stress in comparison to the control, illustrating the effect of

water deficiency on wheat cultivars.

Preceding researchers discovered enormous decreased in spike length (m-2),

grain/spike and thousand kernel weight in a study on twelve wheat cultivars under drought

strain.Phloem translocation relies upon the turgor pressure and the water potential. It has

been observed that water deficiency reduce the capability of phloem which leads to reduced

grain yields. Furthermore, water pressure at the anthesis affected the grain filling rate that

induced reduction of grains per spike in comparison to different treatments. Literature

supported our findings that Plant regulators, specifically GA

have crucial function in improvement of wheat yield (Bari and Jones, 2009),

photosynthesis capacity, delay in leaf senescence and elevated seed quantity in wheat under

stress (Zheng et al., 2011).Drought stress remarkably affected the grain weight like plant’s

other morphological characteristics. Present study found that the wheat variety Shahkar

had the highest thousand kernels weights among all the tested varieties of wheat, which

shows it’s less sensitivity to drought strain (Table 4.8). But AARI-11, Chakwal-50 and

Pakistan-13 showed improvement of the trait under priming with SA and GA.

Decreased grain weight is caused by the moisture shortage at grain filling stage, as it

leads to unusual grain formation. Decline in Photosynthesis, reduced rate and duration of

grain development under drought is a leading cause of yield reduction (Pandey et al., 2001).

64

Keeping in picture the depth and duration of drought stress, a

pronounced drop in 1000-grain weight in wheat was recorded owning to the shortening of

grain filling period. The period of grain filling was shortened by water insufficiency after

anthesis, leading to endosperm desiccation and constraining embryo quantity (Gooding et

al., 2003). Similar behavior was observed by other researchers, reporting the reduced grain

weight due to drought at grain filling stage (Sharafizad et al., 2013).

Priming with SA and GA magnified the wheat potential in producing a better TKW.

Results were accordance to (Dawood et al., 2012) who informed that in sunflower and

mung-bean1000-kernel weight was increased through utilizing salicylic

acid. Drought strain disorder the absorption and translocation of photosynthetic assimilates

and consequently alters and reduce the yield components (Moghadam et al.,

2011). Salicylic acid priming and extended yield are related to the early flower

development, maximum flowers and greater number of pods (Kulshrestha et al., 2013).

Primed plants are capable of producing better yields than untreated seeds (Harris et al.,

2005). Salicylic acid is capable of boosting up the growth and yield of crop in either case,

stress or without stress(Moghaddam et al., 2011). Wheat yields are boosted up by Salicylic

acid (Shakirova et al., 2003). SA influences the plants responses, increase the translocation

of assimilates from leaf to grain that boost in yield and parameters of yield (Dawood et

al., 2012). FSD-08 had a better straw yield representing its better tolerance to drought

than the other examined cultivars. The reduced biological yield of the other genotypes was

compensated by priming with SA or GA. It was well documented in literature that in wheat

more biomass reduction occur in stress (Shamsi and Kobraee, 2013).

The extended biological yield might be due to the promising effects of

hormonal priming on biomass, number of seedlings and on plant nutrition (Zhang et al.,

65

2007b). Primed seeds have greater biomass and dry weights as compared to non-primed

(Rashid et al., 2002). Biological yield seems increased under normal conditions is due to

the precise vegetative growth. The broad leaf surface is the cause of higher photosynthetic

activity and therefore better organic yield. The genotype (Pakistan-13 and FSD-08) with

better grain yield showed highest harvest index. This is in accordance to the study of

(Reynolds et al., 2009)who reported that grain yield and harvest index is highly affiliated.

It was found that harvest index is markedly affected by the water shortage (Galavi and

Moghaddam, 2012) at anthesis stage.Distribution of

photosynthetic assimilatesamongst plant elementsis determined with the aid of harvest

index, drought stress reduced assimilates translocation into the grains and results in

reduced harvest index. Many researchers confirmed the lowered photosynthetic activity

due to decreased soil moisture, and in turn decreased translocation of assimilates to the

grain. In water deficit soil photosynthesis reduction cause assimilate to remobilize from

source to sink (Asseng and Van Herwaarden, 2003;Plaut et al., 2004). Drought at

specific growth stages in wheat plant contributes to reduce grain yield, organic yield,

harvest index and other yield contributing parameters (Harris et al., 2005).

66

Table 4.12: STI % of wheat genotypes based on morphological parameters across both years and priming

Genotypes Seedpr

iming

Plant

height

Tiller

s

Spikeleng

th

Spikelet

s

Extrusi

on

Peduncle Grains Grain

weight

Grain

Yield

Bio

Yield

Harv

est index

AARI-11 NP

82.69

62.7

1 84.26 90.04 78.03 93.88 86.49

88.2

5 71.95 80.80

83.2

7

SA

86.57

55.7

9 79.20 80.52 83.38 93.56 80.12

87.0

1 67.38 81.88

77.0

1

GA

82.61

61.8

8 90.18 85.88 89.77 90.33 83.67

77.6

5 74.74 91.00

85.5

9

Chakwal-

50

NP

82.08

74.2

9 82.68 87.06 88.66 91.85 89.36

86.8

0 76.59 87.67

86.1

1

SA

83.66

57.5

1 93.83 87.76 87.27 94.07 87.59

88.2

8 85.64 87.18

87.2

6

GA

82.17

63.2

1 75.33 89.59 75.96 94.54 87.84

86.3

1 74.99 83.40

84.6

6

Shahkar NP

86.65

74.9

0 84.29 94.12 84.12 95.53 87.38

83.2

2 72.13 85.36

81.6

5

SA

87.62

69.7

3 82.70 88.79 82.23 93.69 92.07

89.4

7 71.52 92.21

73.0

2

GA

87.05

81.2

3 80.20 88.03 74.61 90.00 90.47

85.4

4 82.37 88.33

80.6

8

Pakistan-

13

NP

84.90

62.0

8 93.78 89.92 77.12 86.23 91.57

90.5

7 83.11 85.38

90.2

1

SA

89.73

71.8

1 84.87 87.91 80.89 89.38 94.44

86.0

5 85.06 82.30

75.1

9

GA

86.34

72.6

0 82.10 92.20 84.96 90.53 89.18

87.5

0 75.27 82.59

77.2

1

FSD-08 NP

83.97

62.4

6 80.34 92.94 80.09 88.26 88.65

89.0

0 77.08 94.81

72.6

9

SA

82.93

62.0

2 83.62 90.02 73.66 86.64 91.55

85.7

9 82.21 94.45

96.1

0

GA

85.69

70.9

4 84.53 82.47 79.48 92.88 89.90

85.6

3 85.16 87.02

91.7

0

67

Table 4.13: Combine STI of wheat genotypes based on morphological parameters across both years

4.2.1 Cluster analysis based on Morphological attributes

The CA sequestrates genotypes into clusters which exhibit high homogeneity within

a same cluster. Shahkar-50 and AAR1-11 form same cluster while Pakistan-13 and FSD-

08 form same cluster and Chakwal-50 varied for other form different cluster (Fig 4.8).

Figure 4.8: Dendogram derived from hierarchical cluster analysis of all combined morphological attributes

of five wheat genotypes

AARI-11 84.83 70.43 81.37 89.16 78.29 92.54 89.63 86.84 77.26 82.16 81.06

Chakwal-50 86.52 86.32 81.85 82.39 84.19 93.18 87.58 85.28 73.39 86.48 74.63

Shahkar 84.74 69.07 84.18 89.23 83.71 90.72 88.13 87.68 74.45 92.67 77.08

Pakistan-13 84.04 70.74 82.14 88.40 78.30 89.42 90.48 86.00 80.39 90.06 89.88

FSD-08 84.76 61.90 90.44 86.66 81.08 91.01 89.71 88.13 84.62 86.52 89.74

68

4.2.2 Conclusion

Among genotypes it was concluded that FSD-08 and Pakistan-13 were top ranking.

Based on above results and discussion it is cleared that some genotypes have good tolerance

under stress. On the basis of this experiment we conclude and can recommended that

priming along with growth regulator overcome the coming environmental issues such as

drought. Temperature, humidity and environmental fluctuations were also responsible for

reduction in yield.

4.3 BIOCHEMICAL AND PHYSIOLOGICAL ATTRIBUTES

The rationale of this section of study was to explore the physiological and

biochemical mechanism of wheat genotypes along with growth regulators and drought

stress, as a result we are able to screen the suitable genotype to drought stress. Completely

emerged leaves (flag) were selected for biochemical and physiological analysis. This phase

is mainly considerable for the reason that flag leaf contribute grain yield directly make up

around 75% of effective leaf area.

4.3.1 Oxidative Enzymes

4.3.1.1 Estrases activity

Under normal condition, Shahkar (535 ±24.72µM/min/g f.wt.) had the maximum

esterase activity while Chakwal-50 had the lowest esterase activity (416±11.23µM/min/g

f. wt.) in non primed samples (Fig. 4.9). Esterase activity was increased notably upon

priming with SA in FSD-08 and Pakistan-13. When samples with GA priming were

studied, esterase activity was amplified significantly in Pakistan-13and Chakwal-50.

Under drought stress, FSD-08 had the highest esterase activity (778 ± 22.47µM/min/g f.wt.)

while AARI-11 had the lowest esterase activity (501± 11.23 µM/min/g f. wt.) in non

primed samples. Esterase activity becomes low on SA priming in Pakistan-13. When

69

stress was imposed esterase activity significantly increased in all genotypes. Priming with

SA increased the esterase activity in stress condition than well watered in AARI-11 and

Chakwal-50 while lower activity in Pakistan-13. When priming with GA was observed,

esterase activity amplified under drought inPakistan-13 while decreased in Chakwal-50.

Figure 4.9: Estrase activity in flag leaves of wheat genotypes grown under control and drought stress


Under normal condition, Shahkar had the highest amylase activity

(17.73±2.26mg/g. f.wt.) while FSD-08 had the lowest amylase activity (2.45±0.18 mg/g.

f.wt.) in non primed samples (Fig. 4.10). Amylase activity was decrease considerably upon

priming with SA in AARI-11 and Chakwal-50 while increased in Pakistan-13, Shahkar and

FSD-08 under normal condition. When GA primed samples were analyzed, amylase

activity was increased appreciably in FSD-08 and Chakwal-50 while reduced in Shahkar.

During drought stress, FSD-08 had the maximum amylase activity (37.17±1.69 mg/g. f.wt.)

while Shahkar had the lowest amylase activity (4.52±0.75 mg/g. f.wt.) without any

priming. Amylase activity was significantly raised on SA priming only

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hi

defg

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ij ij

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0200400600800

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-50

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ahkar

Pak

ista

n-1

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D-0

8

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control Drought

Est

rase

act

ivit

y (

unit

/µM

/min

/g f

. w

t) NP SA GA

70

AARI-11and Shahkar decrease in Pakistan-13. Regarding GA priming samples,

increased amylase activity was present in all experienced cultivars except AARI-11.

Amylase activity was significantly increased under drought stress in Pakistan-13 while

lowered in Shahkar. Amylase activity was increases in SA treated samples under drought

in Pakistan-13, Chakwal-50 and AARI-11. Amylase activity also increased in term of GA

priming under drought except AARI-11.

Figure 4.10:Amylase activity in flag leaves of wheat genotypes grown under control and drought

stress

In cereal crops amylases take part in the breakdown of starch. It is clearly depicted

from literature that in primed wheat seeds amylase activity was uplifted as compared to

none primed. Previously, enhanced amylase activity has been reported in rice seeds that are

subjected to priming; however amylase activity varied among different priming treatments

(Farooq and Azam, 2006). Our findings were supported by previous literature. In many

areas of the world due to striking climate-change scenarios like rise in aridity main focus

of the researcher is on plant reaction to water stress. Several physiological and biochemical

changes have been extensively studied previously while, vegetation are expose to water

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def

bcd

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a

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hi

defg

hijk

bcd

efg gh

ijk

jk

cdefg

defg

hijk

a

a a

a

0

10

20

30

40

50

60

70

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control Drought

Am

yla

se a

ctiv

ity (

unit

/mg/g

. f.

wt)

NP SA GA

71

deficit (Passioura, 2007). The retort of plants to water stress is multifaceted and concerned

with alteration in their metabolism, physiology and morphology.

4.3.1.3 Protease Activity

Without stress condition, FSD-08 had the maximum protease activity

(6945±65.00g.f.wt.) while AARI-11 had the lowest protease activity (5385±75.00g. f. wt.)

in non primed samples (Fig. 4.11). Upon SA priming Protease activity significantly

decreased in Pakistan-13 while increased in Shahkar. When we see GA priming samples,

protease activity was improved notably in Shahkar while lessened in FSD-08. When stress

was imposed, Pakistan-13 had the maximum protease activity (6265±45.00g. f. wt.) while

other genotypes have same dimension in non primed samples. When SA priming was

applied protease activity was significantly higher only in Shahkar. Drought stress

significantly increased the protease activity in Pakistan-13 while other genotypes remain

unaffected. SA priming decreased the protease activity under drought in AARI-11 and

FSD-08. Priming with GA decrease protease activity in normal as well as when stress was

imposed in all tested genotype.

Figure 4.11: protease activity in flag leaves of wheat genotypes grown under control and drought stress

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ab

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ab

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hij hij

ab fg

hij

efg

bcd

cd

e

ab

bcd cd

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fgh

ij ij j

gh

ij

hij

02000400060008000

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control DroughtPro

teas

e ac

tivit

y (

Unit

s/g.

f.

wt)

NP SA GA

72

Under water stress many antioxidants are induced among them proteases was also

induced (Cruz De Carvalho et al., 2001; Bray, 2002). When plants are exposed to stress

intracellular proteases take part in the degradation of injured or unwanted proteins,

metabolism reorganization and also help in remobilization of nutrient (Feller et al., 2008).

It is crucial for the researchers to recognize the mechanism and relation between proteolysis

and plant concert in water stress and remedies from stress. It is still not understandable that

under stress high proteolytic activity is beneficial for the plant to help in reorganization of

protein pattern or it leads to cell breakdown. Some experimental facts suggest that

proteolytic activity was maximum in drought sensitive species and varieties compared to

resistant ones (Hieng et al., 2004). Therefore, it is important to study the mechanism of

drought resistance of plant species in order to improve their biochemical and physiological

characters to facilitate developing cultivar with increased resistance. SA and GA played a

significant role in the protective response to stresses in different plant species (Shakirova

et al., 2003; Srivastava and Srivastava, 2007).


Without stress, Shahkar and AARI-11 has same superoxide dismutase (SOD)

activity while FSD-08 and Pakistan-13 has same activity in non primed samples (Fig. 4.12).

SOD activity was non significantly affected by when priming agent were used. Under the

condition of drought stress, Chakwal-50 had the highest SOD activity

(20.51±3.40unit/gf.wt.) while FSD-08 had the lowest SOD activity (16.96± 1.349

unit/gf.wt.) in non primed samples. SOD activity was notably increased AARI-11 and FSD-

08 when SA priming was applied while there was no effect in other genotypes. SOD activity

was increased in AARI-11 and FSD-08 when GA priming was applied. When stress was

imposed SOD activity increased in all genotypes as compared to without stress. SOD

activity was increased upon priming with GA and SA under drought as compared to well

73

watered in all tested genotypes. The SOD activity is conscientious for scavenging free (O2–

) radical to produce H2O2, greater than before within three drought days at tillering stage in

both drought susceptible and drought tolerant cultivars (Bano et al., 2012).

Figure 4.12: SOD activity in flag leaves of wheat genotypes grown under control and drought stress

It is accepted that H2O2play an chief role in signal transmission networks. Lot of

stress reactions involve H2O2, due to catalase regulating functionsand scavenging,the

homeostasis of H2O2 is maintained. Therefore, SOD activation may be triggred by H2O2

accumulation. So, to tolerate stress for adaptation and survival the balance between ROS

and the antioxidative system is necessary. Under water stress, some cultivar showed higher

membrane stability index and SOD activity. Drought tolerant genotype has higher

membrane stability and SOD activity (Sairam and Saxena, 2000). It also demonstrates that

under drought stress physiological parameters like proline, RWC and SOD activity could

be used as criteria for ranking of varieties for drought tolerance.

4.3.1.5 Peroxidase Activity

Under normal condition, AARI-11 has the highest peroxidase activity

(30003±323unit/g f.wt.) while Pakistan-13 had the lowest POD activity (20146 ± 209

unit/g f.wt.) in non primed samples (Fig. 4.13). Peroxidase activity was decreased upon

priming with SA in AARI-11 though improved in Shahkar and Pakistan-13. In AARI-11

and Pakistan-13 with GA priming POD activity was increased. Under drought stress, FSD-

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bcd

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de

ab

c a

ab

cd

ab

ab

cd

bcd

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bcd

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ab

cd

bcd

e de

a a

ab

cd

ab

cd a

cde

cde

bcd

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e

bcd

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a

ab

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ab

cde

ab

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a

05

1015202530

AA

RI-

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Chak

wal

-50

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ahkar

Pak

ista

n-1

3

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D-0

8

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control Drought

SO

D a

ctiv

ity (

unit

/gfr

esh

wei

ght)

NP SA GA

74

08 had the highest POD activity (37529±123unit/g f.wt.) while Pakistan-13 has the lowest

POD activity (21078±183unit/g f.wt.) in non primed samples. Priming with SA increased

POD activity in Pakistan-13, and Chakwal-50 while lowered in FSD-08. POD activity

increased in Pakistan-13 and Chakwal-50 with GA priming.

Peroxidase (POD) takes part in decomposition of H2O2. Researchers proved that

under drought stress condition scavenging system of hydrogen peroxide was more

aggressively induced in different wheat genotypes (Hameed et al., 2011). Generally,

hormones take part in defense mechanism to overcome stress. Effect of salicylic acid under

salinity stress on peroxidase and catalase activity was studied previously. It is demonstrated

that peroxidase activity was significantly increased when treatments was made with 0.05

mM SA solution. It is also confirmed that the exposure to exogenous SA accelerated the

drought and salt stress resistance of plants (Deef, 2007). During severe water stress excess

levels of H2O2 have the ability to overcome or prevent the synthesis of antioxidant enzymes

(Selote and Chopra, 2006).

Figure 4.13: POD activity in flag leaves of wheat genotypes grown under control and drought

stress

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cdfg

bcd

bcd

bcd

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ab

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defg

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a

defg

bcd

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efg fg

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ab

c

ab ab

c

ab

c

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AA

RI-

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Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control Drought

PO

D a

ctiv

ity

(unit

/g f

resh

wei

ght)

NP SA GA

75


Without stress condition, the highest catalase activity was found in FSD-08

(2609±0.039units/g.f.wt.) while Chkwal-50 has the lowest catalase activity

(2171±0.008units/g. f.wt.) in non primed samples (Fig. 4.14). Catalase activity was

decreased extensively in AARI-11and FSD-08 while improved in Chakwal-50 and Shahkar

under normal condition alongwith SA priming. GA priming increased the catalase activity

in Chakwal-50 Pakistan-13 and FSD-08 while decrease in Shahkar and FSD-08.

In stressed samples, FSD-08 had the maximum catalase activity (3638±0.002

units/g.f.wt.) while Chakwal-50 had the lowest catalase activity (3292±0.001units/g.f.wt.)

in non primed samples. When SA priming were applied catalase activity was increased in

Pakistan-13 and Shahkar while decrease in other genotypes. Catalase activity was increased

on GA priming only in Shahkar while decreased in Chakwal-50. When stress was applied

catalase activity was increased in FSD-08 and Shahkar while minimum in Chakwal-50.

Catalase activity was increased on SA priming in stress condition in comparison without

stress in Pakistan-13while decreased in Chakwal-50 and AARI-11. Priming with GA

increased the catalase activity in stress in Shahkar while decreased in Chakwal-50 and

AARI-11.

Figure 4.14: CAT activity in flag leaves of wheat genotypes grown under control and drought

stress

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hi

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hij

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bcd

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Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control Drought

CA

T (

Unit

s/g.

f. w

t)

NP SA GA

76

Our results are in accordance with Zhang et al., 2007 who said that accelerated

activities of most important antioxidant enzymes i.e. CAT, SOD and POD in Victoria and

Victor seedlings after priming treatments were found. Catalase is among the most important

enzyme for elimination or detoxification of too much hydrogen peroxide in the seeds.

Indeed, high level of antioxidative enzymes saves the cell alongside the oxidative damage

by free radicals removal of ROS. During stress as well as normal condition seed priming

with growth regulators improved physiological activities of wheat seedling. Meanwhile,

enhanced the activities of several antioxidants i.e. CAT, POD and SOD as a result it protect

the cell against free radical production and cellular damage due to oxidation (Eisvand et

al., 2010).

4.3.1.7 Ascorbate peroxidase activity

Under normal condition, AARI-11 has the highest ascorbate peroxidase (APX)

activity (1288±0.034units/g.f.wt.) while Chakwal-50 had the lowest ascorbate peroxidase

activity (0216±0.001units/g.f.wt.) in non primed samples (Fig. 4.15). When SA priming

was applied ascorbate peroxidase activity was lower in Pakistan-13 and AARI-11 however,

increased in Shahkar and Chakwal-50. During GA priming, Ascorbate peroxidase activity

was found to be accelerated significantly in Shahkar, Chakwal-50 and FSD-08 while

reduced in Pakistan-13.

Under drought stress, Pakistan-13 had the highest Ascorbate peroxidase (APX)

activity (1710±0.018units/g.f.wt.) while Shahkar had the lowest APX activity

(0808±0.071units/g.f.wt.) in non primed samples. Upon priming with SA ascorbate

peroxidase activity was appreciably increased in AARI-11while there was no effect in other

genotypes. Ascorbate peroxidase activity was increased in AARI-11 and Chakwal-50while

decreased in Shahkar alongwith GA priming.

77

Drought stress notably amplified the ascorbate peroxidase activity in Pakistan-13,

Chakwal-50, and FSD-08. Ascorbate peroxidase activity along with SA priming increased

in stressed samples than those that are controlled in FSD-08, Pakistan-13 and Chkwal-50.

Ascorbate peroxidase activity improved under stress same as in devoid of stress in

Chakwal, Pakistan-13, and FSD-08 on GA priming. Our findings were similar to previous

researchers who have found that anti-oxidative peroxidases or glutathione reductase

activity is increased when subject to the drought stress (Miller etal., 2010). Therefore,

these enzymes are excellent biochemical stress markers and their improved activity may

demonstrate the prospective for remediation. Under control and under drought stress

genotypes respond differently.

Figure 4.15:APX activity in flag leaves of wheat genotypes grown under control and drought stress

4.3.2 Cluster Analysis based on oxidative enzymes attributes in flag leaf of wheat

Major two cluster were formed which further form three sub cluster. Our data

represents the affinity of every grouped variable in one bunch to relate strongly to each

other (Fig. 4.16).

bc

h

h

de

bcd

efgh

bc

bcd

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efgh

a ab

ccde

fgh

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h

bcd

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ab

c

abcd

e

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fg

h

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a a

efgh

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ab

c

0

1

1

2

2

AA

RI-

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wal

-50

Sh

ahkar

Pak

ista

n-1

3

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D-0

8

AA

RI-

11

Chak

wal

-50

Sh

ahkar

Pak

ista

n-1

3

FS

D-0

8

Control Drought

AP

X (

unit

s/g.

f. w

t)

NP SA GA

78

Figure 4.16: Dendogram derived from hierarchical cluster analysis of all combined antioxidant activities of

five wheat genotype

4.3.3 Conclusion

Our experiment signifies that hormones play critical roles in plant responses to

drought. GA and SA induced drought tolerance by enhancing the antioxidant enzymes

activities i.e. superoxide dismutase, peroxidase, amylase and ascorbate peroxidase.

79

Table 4. 6: STI % of wheat genotypes based on oxidative enzymesacross genotypes and priming treatments

4.4 PHYSIOLOGICAL ATTRIBUTES

Priming induced physiological attributes against stress (Makhmudova et al., 2011).

4.4.1 Malondialdehyde Contents

There was non significant effect on Malondialdehyde (MDA) contents in all tested

genotypes under well water and drought. Priming with SA and GA slightly increased the

MDA (Fig 4.17). Generally, in literature under low water condition lipid peroxidation

significantly improved in wheat cultivars. However, if water level was changed from low

to medium or high level of lipids peroxidation could also change and varies genotype to

genotype (Hameed et al., 2011).

Genotypes Seed

priming

Estrase Amylase Protease SOD POD CAT APX

AARI-11 NP 104.48 105.26 87.28 142.12 142.12 143.93 56.86

Chakwal-50 NP 103.41 129.51 84.46 169.76 169.76 151.65 35.39

Shahkar NP 109.85 25.53 85.46 132.34 132.34 149.72 20.43

Pakistan-13 NP 91.12 160.00 91.33 225.14 225.14 114.58 47.03

FSD-08 NP 100.66 1515.38 77.97 209.55 209.55 139.44 39.09

AARI-11 SA 101.49 841.18 64.46 177.11 177.11 177.49 114.70

Chakwal-50 SA 92.19 393.94 85.50 164.73 164.73 152.55 284.14

Shahkar SA 119.01 233.57 100.16 174.21 174.21 131.39 81.18

Pakistan-13 SA 99.60 60.00 83.55 180.71 180.71 84.04 413.69

FSD-08 SA 110.67 131.51 78.77 231.74 231.74 127.49 146.00

AARI-11 GA 104.91 92.31 91.70 226.33 226.33 103.69 135.57

Chakwal-50 GA 97.12 243.44 77.54 179.15 179.15 146.13 303.35

Shahkar GA 102.33 520.75 59.29 121.92 121.92 154.58 116.37

Pakistan-13 GA 95.51 1652.94 67.10 325.70 325.70 154.82 325.37

FSD-08 GA 97.77 251.67 72.31 183.31 183.31 143.48 185.88

80

Figure 4.17: MDA contents in flag leaves of wheat genotypes grown under control and drought stress

4.4.2 Total oxidative status

Under normal condition, all genotypes have the same dimension in total oxidative

status (TOS) except Chakwal-50 which had the lowest TOS in non primed samples (Fig.

4.18).TOS was increased in Chakwal-50 and Pakistan-13 on SA priming while non

significant effect on other genotypes. Alongwith GA priming, TOS was improved much in

Chakwal-50. Under drought stress, all genotypes have same dimension in non primed

samples. TOS increased in Shahkar and Pakistan-13 on SA priming. Priming with GA has

non significant effect. Drought stress not significantly decreased TOS under normal

condition as compared to control. Priming with SA and GA raised maintained TOS to a

normal level under drought as in normal condition.

Figure 4.18: TOS in flag leaves of wheat genotypes grown under control and drought stress

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Control Drought

MD

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NP SA GA

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TO

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NP SA GA

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4.4.3 Relative water contents

Under normal condition, AARI-11 had the highest RWC (82.690±0.887%) while

Chakwal-50 had the lowest RWC (57.72±0.103%) in non primed samples (Fig. 4.19). RWC

increased significantly on SA priming in Chakwal-50 while other genotypes were not

affected. In case of GA priming, relative water content was increased significantly in

Chakwal-50 and Pakistan-13. Under drought stress, Pakistan-13 had highest relative water

content (80.19±0.01%) while Chakwal-50 has lowest relative water content (61.58±1.77%)

in non primed samples. Relative water content was increased alongwith SA priming in

AARI-11 and Shahkar while lower in Chakwal-50. GA priming had no effect on all

experienced genotypes. Relative water content was decreased under drought stress in all

genotypes except Pakistan-13. SA priming raised the relative water content in stress

condition in comparison with non stress condition in AARI-11 and Shahkar. Relative water

content increased along GA priming under drought condition as well as normal condition

in AARI-11.To investigate the physiological responses of wheat against water stress

researchers reported that as a consequence of water stress, relative water content (RWC),

chlorophyll and carotenoid and membrane stability was decreased while proline

accumulation was increased (Chandrasekar et al., 2000) . Similar conclusions were

revealed by other authors (Azooz, 2009) they also proved that remarkable increase in RWC

when SA was applied under water deficient condition ultimately increased yield. Constant

reduction in RWC in retort to PEG-mediated water stress have been explored and reported

in wheat (Bajji et al., 2001) in Brassica (Swati and Ahmad, 2000) and in rice (Hsu et al.,

2003) Many scientists suggested that RWC and proline could be used as a tool to determine

stress induced in plants due to low amount of water (Strauss and Agenbag, 2000).

82

Figure 4.19: RWC contents in flag leaves of wheat genotypes grown under control and drought stress.

4.4.4 Cell membrane thermostability

Without stress, FSD-08 had the maximum cell membrane thermo stability (CMT)

(6.011±0.33%) while Chakwal-50 had the lowest CMT (3.604±0.236%) in non primed

samples. CMT was increased significantly on SA priming in Chakwal-50 and FSD-08

without stress (Fig. 4.20). CMT was increased notably along GA priming in AARI-11 and

FSD-08 while reduced in Pakistan-13. Under drought stress, Pakistan-13 had the highest

CMT (4.547±0.33%) while AARI-11had the lowest CMT (3.203± 0.33%) in absence of

any priming treatment. CMT was appreciably increased only in AARI-11 and Pakistan-13

while decreased in Chakwal-50 along SA priming. When GA Priming was applied CMT

increased in AARI-11 and FSD-08 while decreased in Chakwal-50. It is accepted that

plants facing drought showed a variety of changes in different processes to flourish under

water stress situations (Arora et al., 2002).

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Figure 4.20: CMT contents in flag leaves of wheat genotypes grown under control and drought stress

In preceding literature, numerous reports described a work on both the tolerant

genotypes and susceptible genotypes and concluded that under drought stress genotype

having low electrolyte leakage and H2O2 are more tolerant than sensitive genotypes.

Tolerant varieties showed more membrane stability than susceptible ones (Sairam and

Saxena, 2000). A series of adaptive responses of wheat plants to face drought like

membrane stability, oxidative damages to plant cells and antioxidant protection were

reported by many researchers (Chandrasekar et al., 2000; Hameed et al., 2011). Water

deficiency adversely affects the cell membrane stability by leakage of membrane in all

crops. However, stability of membrane can be prevented under water deficient condition

by using SNP treatment to wheat seedlings (Hao et al., 2008).

4.4.5 Photosynthetic pigments

Without stress condition, had the highest chlorophyll a contents (8.620±0.49) was

found in FSD-08 while lowest chl a contents was found in Chakwal-50 (6.677±0.120) in

non primed samples. Chlorophyll a contents was slightly increased in all genotypes under

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normal condition along SA priming. Chlorophyll a increased in all studied genotypes when

samples were primed with GA.

Under drought stress, Chl a contents decreased in all studied genotypes. SA and GA

Priming slightly increase the chl a in all tested genotypes. Chl a was decreasd in all

genotypes under drought in comparison with nomal condition. Many authors (Shakirova et

al., 2003) and (Iqbal and Ashraf, 2013) on wheat plants and (Amin et al., 2008) on maize

plants found that SA and GA results in significant increase in chlorophyll content. That

accretion of photosynthetic pigments may be due to exogenous application of SA that

results in photosynthetic efficiency increment as represented, by increasing in both chl a,

chlb and carotenoids content in the leaves of wheat plants grown under stressed condition.

Figure 4.21: Chl a contents in flag leaves of wheat genotypes grown under control and drought stress

In case of Chl b slight variation was present in all studied genotypes (Fig. 4.22).

Under drought stress, chl b decreased in all tested genotypes. Priming was able to increased

chl b under drought stress condition. Under balanced environmental condition, FSD-08 had

the uppermost carotenoid contents (3.233) while lowest in AARI-11 (2.092) in non primed

samples. Carotenoid contents were somewhat increased on SA and GA priming in all under

experiment genotypes (Fig. 4.23).

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Chla

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.

Figure 4.22: Chlb contents in flag leaves of wheat genotypes grown under control and drought stress

Under drought stress, carotenoid contents were slightly decreased in tested

genotypes while SA and GA have slightly increased carotenoid contents in all tested

genotypes. Overall drought stress had significant effect on carotenoid contents in all tested

genotypes. Findings of (Hassanein, 2009) reported that in stressed wheat showed stunted

growth, accumulation of hydrogen peroxide and lipid peroxidation under high salinity and

found decline in Chl a, b, carotenoid and total pigments.

Figure 4.23: Carotenoids contents in flag leaves of wheat genotypes grown under control and drought stress

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Non significant difference in all tested genotypes in case of anthocyanine under

normal and stress due to drought (Fig. 4.24). SA and GA priming showed non significant

effect on anthocyanine contents under normal and drought stress. Some researchers Khan

et al., (2003) showed that photosynthetic rate was increased in SA treated corn and soybean.

The pretreated seeds with SA solution (10-2

mol/L) revealed higher chlorophyll and

anthocyanine content. Similar findings were reported by many authors and reported that

SA significantly enhances the pigment content under salt stress conditions (El-Tayeb,

2006).

Fig: 4.24 Anthocyanin contents in flag leaves of wheat genotypes grown under control and drought stress

4.4.6. Conclusion

Results revealed that priming with Gibberellic and Salicylic acid improved the

physiological response in studied genotypes under both drought and control conditions.

Relative water contents and cell membrane stability maintained under stress in primed

seeds. Similarly photosynthetic pigments increased with priming. Ultimately all these

altered physiological factors improved plant growth and enhanced yield under stress

condition.

4.5 METABOLITE ACCUMULATION AND MINERAL ELEMENTS

4.5.1 Sugar contents

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Without stress, maximum sugar contents were found in FSD-08 (2.76±0.050g.f.wt.)

while Chakwal-50 had the lowest sugar contents (2.09±0.029g.f.wt.) in non primed

samples. SA priming depicted that there was significant reduction Sugar contents in AARI-

11, whereas improved in Chakwal-50 and Shahkar without stress (Fig. 4.25). Priming with

GA increased the sugar contents in Pakistan-13 and Chakwal-50 while other remains

unaffected. Under drought stress, FSD-08 had the highest sugar contents

(2.84±0.081g.f.wt.) while Ckakwal-50 had the lowest sugar contents (1.743±0.033g.f.wt.)

in absence of any priming treatment. Sugar accretion was notably increased in Shahkar and

FSD-08 along SA priming while reduction was seen in other genotypes. When GA priming

was applied sugar contents only increased in Shahkar at the same time as decreased in

AARI-11, Chakwal-50 and Pakistan-13. In Shahkar under stress sugar contents increased

whereas decreased in AARI-11 and Chakwal-50. Under drought sugar contents was more

than control in Shahkar due to SA priming. Priming with GA increases the sugar contents

under drought in Shahkar while decreased in Pakistan-13 and AARI-11. Some researchers

worked on flag leaf of different wheat cultivars and concluded that amount of soluble

carbohydrate varied among different genotypes. Different wheat cultivars perform

differently depends on their tolerance and susceptibility. Hereditary composition justifies

the patience of wheat crop against stress. It was observed that more soluble sugars were

accumulated in tolerant species as compared to susceptible species(Nayyar and Walia,

2003).

88

Figure 4.25: Sugar contents in flag leaves of wheat genotypes grown under control and drought stress

4.5.2 Protein contents

There was non significant effect on protein contents in non primed samples.

Similarly under drought stress non significant effect was present. Only Chakwal-50 has the

highest protein under drought. All other genotype has the non significant effect.

Figure 4.26: Protein contents in flag leaves of wheat genotypes grown under control and drought stress

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4.5.3 Proline accumulation

Without stress, FSD-08 had the maximum Proline accumulation

(2.945±0.025µmole/g f.wt.) while Chakwal-50 had the lowest Proline accumulation

(1.733±0.08 µmole/g f.wt.) in non primed samples (Fig. 4.27). Reduction in proline occurs

in FSD-08 and Shahkar whereas increased in Chakwal-50 along SA priming. More ever,

GA priming improved Proline accumulation in AARI-11 while decreased in Chakwal-50

and Pakistan-13. Under drought stress, Shahkar had the highest Proline accumulation

(3.540±0.055 µmole/g f.wt.) while Chakwal-50 had the lowest Proline accumulation

(2.220±0.036 µmole/g f.wt.) in lack of any priming treatment. Proline accumulation was

increased on SA priming in Chakwal-50 while decrease in AARI-11. There is significant

increased the Proline accumulation in all studied genotypes under drought conditions.

Priming with GA and SA significantly increase the proline accumulation under drought in

contrast to normal condition. Previously, a substantial enhance was found in proline levels

in the seedlings faced to saline stress and treated with SA in comparison with control salt

stressed seedlings. SA exogenously applied under salt and drought stress enhanced the salt

and drought tolerance of plants (Deef, 2007). We conclude from above results that these

cultivars attempt to put up with drought situation by the accumulation of relatively high

osmolyte accumulation. Researcher reported that proline significantly increased (p<0.01)

under drought stress (Keyvan, 2010).

90

Figure 4.27: Proline contents in flag leaves of wheat genotypes grown under control and drought stress

4.5.4 Potassium ratio

Without stress, Pakistan-13 had the maximum Potassium (K+) ratio

(28.123±0.248mg/g f.wt.) while Shahkar had the lowest K+ ratio (17.721±0.196 mg/g f.wt.)

in non primed samples (Fig. 4.28). SA priming reduced the K+ in Shahkar and FSD-08

while in Pakistan-13 ratio was increased. Priming with GA increased K+ ratio in AARI-11

and Pakistan-13 whereas reduction occur in FSD-08 and also in Shahkar. In drought

condition, maximum K+ ratio was observed in Chakwal-50 (23.403 ± 1.634 mg/g f.wt.)

while minimum K+ ratio was present in FSD-08 (13.549 ± 0.187 mg/g f.wt.) in nonprime

samples. Similarly, Potassium ratio decreased in FSD-08 and increased in Pakistan-13 and

Chakwal-50 along SA priming. GA priming was effective to improve the K+ ratio in AARI-

11 and Chakwal-50 although decreased in FSD-08. In many previous studies, under salinity

stress importance of K+ was widely studied, it was reported that significant diminution in

K+ was reported for wheat under saline condition. GA3 priming was effective to increase

the shoot K+ concentration in normal condition. It has been reported that sensitive cultivars

had more K+ than tolerant. Since, by reviewing the literature to increase K+ in wheat

cultivars under salinity stress (Nayyar and Walia, 2004). Normally under drought stress

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nutrients uptake was reduced. It was observed that exogenous application of Si was at

anthesis stage was responsible for accumulating maximum plant nutrients. Under drought

stress control plants has highest nutrient then those treated with Si. A controlled plant has

high nutrients like K, Mg and Zn contents than those treated with Si (Bukhari et al., 2015).

Figure 4.28: K+ratio in flag leaves of wheat genotypes grown under control and drought stress

4.5.5 Calcium ratio

High Ca+ ratio was present in Pakistan-13 (28.12±0.248 mg/g f.wt.) while Ca+ ratio

was lower in FSD-08 (15.11±0.059 mg/g f.wt.) in nonprime samples without drought (Fig.

4.29). Low ratio of Ca+ was recorded in AARI-11 and Pakistan-13 and high ratio was

recorded in FSD-08 without stress condition along SA priming. Priming with GA revealed

the increase in Ca+ ratio in Pakistan-13 although decreased in Shahkar. Results showed that

Chakwal-50 had the maximum Ca+ ratio (23.40±1.63 mg/g f.wt.) whereas FSD-08 had the

less Ca+ ratio (13.54±0.187 mg/g f.wt.) under drought in non primed condition. Priming

with GA was effective in improving ratio of Ca+ in FSD-08, AARI-11and Pakistan-13.

Drought stress significant reduction was observed under drought in Ca+ ratio

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in AARI-11 and Pakistan-13. Results revealed that SA priming increased the Ca+Pakistan-

13 under situation of drought in comparison with normal while in FSD-08 ratio was

declined. Priming with GA increased the Ca+ ratio under drought in Pakistan-13and AARI-

11 in comparison with control.

Figure 4.29: Ca+ ratio in flag leaves of wheat genotypes grown under control and drought stress

For predicting the tolerant plants against drought (Asghari et al., 2001)

premeditated in two wheat varieties mannitol content, ABA, K+ and Ca+possibally due to

the ratio of K+ / Ca2+. It is mainly depending on the interferingamong the aspects and

connection such as membrane permeability, membrane of Chloroplast and stomatal

membrane because it take part in stomatal aperture and regulation of guard-cell turgor.

However, on the position of stomata the real fact about the ratio of K+ / Ca2+ are still under

investigation (Asghari et al., 2001). Due to hyperosmotic stress Cytosolic Ca+ was

increased, this increased in cytosolic Ca+, under stress maintained the osmosensing

(Matsumoto et al., 2002).

It confirms from previous studies that seed priming induce many metabolic changes

in plants,as a result enhances stress tolerance (Jisha et al., 2013; Kubala et al., 2015).

Another study confirmed that, β-amino butyric acid seed priming in green gram enhanced

proline accumulation, amount of carbohydrate and total protein contents (Jisha and Puthur,

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2016). Under stress, all parameters of plant’s growth along with development was increased

significantly by the application of salicylic acid exogenously. Increased chlorophyll

contents and significant increase in net CO2 assimilation has been reported (Martel and

Qaderi, 2016). Under stress conditions, applied SA regulates the plant physiological

processes like photosynthesis and proline metabolism thereby providing protection to plant

(Miura and Tada, 2014). The function of SA in thermo tolerance is well reported.

Among growth regulators, SA in particular has been well documented in improving

plant’s stress-tolerance (abiotic) through controlling plant’s major metabolic processes

(Khan et al., 2009). According to (Hasanuzzaman et al., 2014), damaging effect of abiotic

stress in plants can be reduced by the exogenous application of SA. Electrolyte leakage is

correlated with chlorophyll content, sugar, proline and protein.Increased levels of these

compatible solutes resulted decreased electrolyte leakage (Zhang et al., 2013).

4.5.6 Cluster Analysis Based on Biochemical and Physiological Attributes

Genotypes respond differently in term of physiological attributes. Two main

clusters were further divided into three sub cluster (Fig. 4.30). Our data reflected the

tendency of each grouped variables in one cluster to relate closely to each other.

94

Figure 4.30: Dendogram derived from hierarchical cluster analysis of all combined physiological and

biochemical attributes of five wheat genotypes

4.5.7 Conclusion

It was concluded from this section of study that, genotypes respond differently

under water stress due to adoptive changes in antioxidant and in other metabolic processes.

Cell membrane thermo stability, relative water contents are decreased while antioxidants

increased. However, growth regulator increased the physiological response and antioxidant

activities in some genotypes under stress and without stress conditions. Presoaking of seed

enhanced some enzymes that scavenge free radical like superoxide dismutase, catalase,

peroxidase, amylase, soluble sugars and proteins and trigger synthesis of proline that is an

osmo-protectant. Accumulation of proline increased under stress condition while ion

accumulation response was varied among genotypes.

95

Table 4.7: STI% of wheat genotypes based on physiological and biochemical attributes across

genotypes

4.6 WHEAT GRAIN QUALITY

Adding together with yield, wheat grain quality is essential to the welfare of beings.

The purpose of this section of study was to appraise the consequence of drought stress and

hormonal priming on physio-chemical properties of wheat grains.

4.6.1 Wet Gluten Contents

Significant variations were found among tested genotypes in wet gluten contents.

Under normal condition, Shahkar had the highest wet gluten contents (32.2 ± 0.28%) while

Chakwal-50 had the lowest wet gluten (24.6 ± 0.29 %) without any prior application of

growth regulators (Fig 4.31). There was non-significant under drought stress, FSD-08 has

the highest gluten strength (29.5 ± 0.73 %) while Pakistan-13 had the lowest wet gluten

(27.0 ± 0.34 %) in absence of any priming treatment. The SA priming had non-significant

Seed

primin

g

MDA TOS RW

C

CM

T

Chl

a

Chl b Caro

tenoi

d

Anthoc

yanin

Sugar Protein Proli

ne

K+ Ca+

AARI-11 NP 139 100 83 65 62 60 106 95 86 107 112 111 73

Chakwal-

50

NP

110 133 106

10

1 56 58 103 93 83 110 128 111 86

Shahkar NP

106 93 90

10

5 88 80 100 95 104 108 141 92 91

Pakistan-

13

NP

113 92 94 75 65 98 90 94 94 99 110 127 63

FSD-08 NP 66 89 86 62 68 51 84 92 103 102 107 92 89

AARI-11 SA 67 105 99 83 69 72 99 99 85 103 107 107 99

Chakwal-

50

SA

99 83 76 82 58 60 102 93 66 93 130 96 93

Shahkar SA 75 120 111 80 63 73 94 96 105 113 147 126 102

Pakistan-

13

SA

94 121 96 81 74 63 78 93 107 101 110 103 131

FSD-08 SA 98 94 83 63 70 63 78 94 104 99 114 119 62

AARI-11 GA 87 94 77 77 64 99 75 94 64 99 102 107 126

Chakwal-

50

GA

132 109 78 89 75 86 100 94 62 98 148 107 92

Shahkar GA 86 107 86 85 71 101 100 95 114 101 144 113 128

Pakistan-

13

GA

126 95 84 87 77 107 90 101 93 97 122 109 115

FSD-08 GA 110 116 84 65 89 95 86 101 107 99 119 103 102

96

effect on wet gluten. With GA increased the wet gluten in FSD-08 under drought however;

other genotypes are not affected. Wet gluten was increased under drought stress in

Ckakwal-50 (28.5±0.46%), while decreased in Shahkar (28.4±0.32%). The SA and GA

priming had non-significant difference in tested genotype on wet gluten content under

drought stress. The effect of both year was non significant.

Figure 4.31: Wet Gluten contents in seeds of wheat genotypes

Similar findings have been reported by many authors in which water stress apply at

the post anthesis period considerably increased the grain gliadin ratio (Fan et al., 2004).

Wet and dry gluten are also increased under drought stress in wheat grains. For end-use

quality, proteins are the major and vital components of grain of wheat. To maintain the

quality of dough the gluten proteins play an important role, dough quality is mainly

maintained by glutenins and gliadins as compared to (Balla et al., 2010) albumins and

globulins which are important in term of nutritional point of view and have little effect on

the dough quality. Albumins and globulins contain all essential amino acids that are

important for human health. (Balla and Veisz, 2007).

4.6.2 Gluten Index

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Gluten strength can be measured by the by knowing about the gluten index. In our

finding gluten index increased in most of genotypes. Most of the quality analyses were

related to grain and grain flour including starch, hardness index , moisture percentage, wet

gluten protein percent, , falling number, , dry gluten, gluten index and sedimentation of

zeleny. These quality characters were varied from one genotype to other. Mostly quality

traits are affected by water stress during the grain filling period. In previous studies, it is

well studied gluten index and SDS sedimentation volume indirectly correlate with a

decrease in that when a terminal water stress applied an increase in protein percent, wet

and dry gluten, falling number, moisture content, bread volume, grain yield and 1000 grains

weight (Aslani et al., 2013).

In recent study significant difference was evaluated in all genotypes under both

treatments (Fig 4.32). Gluten index remained unaffected both the years. Without stress

condition, FSD-08 had the maximum gluten strength (90.5±0.27%) while Pakistan-13 had

the lowest gluten strength (72.2±0.05%) in non primed seeds. Gluten index decreased

significantly on SA priming in Chakwal-50(75.4±0.18%), and Shahkar (77.1±0.12%) while

increased in Pakistan-13(78.2±0.05%) without stress. When GA priming applied, gluten

index was improved drastically in AARI-11(88.6±0.10), Pakistan-13(83.4±4.22) and FSD-

08(94.0±0.02) while decreased in Chakwal-50(76.3±0.21).Under drought stress; FSD-08

had highest gluten strength (95.0±5.21%) while Shahkar had the lowest gluten strength

(80.4±0.17%) in non primed samples. Gluten index was significantly raised on SA priming

only AARI-11(84.3±0.05%) while other genotypes remained unaffected. GA Priming was

able to boost the gluten index only in Shahkar (83.4±0.11%) under drought.

Gluten index significantly increased under drought stress in AARI-11

(84.3±0.05%), Pakistan-13(89.0±0.02%) and FSD-08 (95.0±5.21%) while decreased in

Shahkar (80.4 ± 0.17). Priming with SA increased the gluten index in FSD-08

98

(98.0±0.02%), Pakistan-13(89.0±0.02%) and Shahkar (83.4±0.11%) under drought

stressed condition. GA priming increased gluten index under drought same as in without

stress in Chakwal-50 (84.2±0.07%), Pakistan-13(85.4±0.07%) while decreased in Shahkar

(83.4±0.11%).

Figure 4.32: Gluten index contents in seeds of wheat genotypes

4.6.3 Falling number (Sec)

In literature it is reported that under drought and salt stresses the amount of water

absorption by the flour causes a decline in the value of gluten, glutenin and increased

various attributes like falling number, grain protein, gliadin and grain hardness index

(Eyvazi et al., 2005). Some other researcher has worked on diverse cultivars of spring

wheat (red and white seeded) and found that germination index, falling number and

sprouting are mostly affected by genotypic variation then the environmental factors and it

is possible to develop genetically variant varieties (PHS resistant cultivars) than

environmental factors (Rasul et al., 2012).

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Under normal condition (Fig. 4.33). FSD-08 had the highest falling number

(362.6±5.03 sec) while Shahkar had the lowest falling number (294.6±3.05 sec) in non

primed samples. Falling number was decreased significantly inChakwal-50 (280.3±7.76

sec) while increased in Pakistan-13(340.6±10.20sec) with SA priming under normal

condition. There is non-significant effect on all other genotypes. Priming with GA showed

that falling number was decreased appreciably in FSD-08 while higher in Pakistan-

13(355.6±3.51 sec). Under drought stress, FSD-08 had the highest falling number

(369.3±10.28 sec) while Chakwal-50 had the lowest falling number (298.6±3.05sec) in non

primed samples. Falling number was significantly decreased on SA priming in Chakwal-

50(266.3±12.0 sec) and Shahkar (306.3±5.13 sec). GA priming decreased the falling

number only in Chakwal-50(275.3±13.01sec) while other genotypes remain unaffected

under drought.

Drought stress raised the falling number only in one genotype Shahkar

(322.61±0.26sec) other genotypes have intermediate effect. In case of GA priming

droughtincreased the falling number in Shahkar(318.0±8.0sec) while decreased in

Pakistan-13(309.3±15.53sec), while on Falling number effect of year was non-significant.

100

Figure 4.33: F/No in seeds of wheat genotypes

Quality performance of wheat genotypes during grain filling is strongly affected by

weather conditions. The quality criterion for wheat in bread making is Hagberg falling

number (HFN) but it has a negative association with a-amylase activity. During pre-harvest

sprouting (PHS) or on other case late-maturity excessive levels of a-amylase are produced

(Major et al., 2001). During the time of grain filling in the absence of visible sprouting high

a-amylase activity or low HFN has been linked with high soil moisture and low

temperatures (Gooding et al., 2010; Gooding et al., 2013). Hagberg falling number (HFN)

is strongly affected by genotype-environment (Gooding et al., 2010), and agronomy

interactions in genotype- (Kindred et al., 2005).

4.6.4 Seed Storage Protein

Under normal condition, no difference was observed in tested genotypes in non

primed condition (Fig 4.34). During both years’ proteins were non significantly varied.

Protein percentage was increased significantly in Chakwal-50 while decreased in Pakistan-

13 (12.7±0.15%) when primed with SA. In term of GA priming protein percentage

increased in FSD-08 while other genotypes remained unaffected. Under drought stress,

Chakwal-50 (15.6± 0.49%) and Shahkar have the highest protein (15.7± 0.89%) while

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other three genotypes have same percentage without any priming. Protein percentage was

significantly raised on SA priming Chakwal-50 (16.7±0.20%) and Pakistan-

13(15.2±0.40%) while decrease in AARI-11(13.2±0.10).When GA priming was used it

help to increase the protein percentage in Chakwal- 50 (16.7±0.20) while significant

decrease in Pakistan-13(12.9±0.68%) under drought.

Drought stress significantly increased the protein percentage in Chakwal- 50

(15.6±0.49%) while decreased in FSD-08(13.4±0.36%).The SA priming raised the protein

percentage in Pakistan-13(15.2±0.40%) while decreased in AARI-11(13.2±0.10%) in

drought stressed in comparison with normal. Priming with GA increase protein in Shahkar

(15.7±0.15%) while decreased in AARI-11(15.7±0.15%) and FSD-08(14.2±0.15%) as

compared to normal.

Figure 4.34: Protein percent in seeds of wheat genotypes

It is notable that yield production fall that heppens under dry spell stretch situation

is for the most part in connection to an ascent in protein content (Pompa et al., 2009). Water

stress completely climbed protein content (Rharrabti et al., 2003).

Protein percentage was increased significantly under drought in the Chakwal- 50

while decreased in FSD-08. The SA priming raised the protein percentage in Pakistan-13

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while decreased in AARI-11under drought. Proteincontents increased under SA priming in

Shahkar while decreased in AARI-11 and FSD-08 as compared to normal.

Proteins are the major constituent of grain since wheat is widely used in many

industries and foods due to its high concentration of grain protein. Now a day in crop

sciences, quality of grain is becoming a challenging and broadly discussed topic.

Environmental factors affecting wheat quality directly. Due to food security and market

demand the main concern has now progressively shifted to the upgrading of processing

quality. Therefore, main focus of breeding is on giving out quality and improvement of

grain contents of protein (He et al., 2004; Yong et al., 2004; Wang et al., 2005).

In relative to drought stress effect on grain quality, we examined that protein

contents almost increases, similar findings have been earlier reported in literature in

different environment (Guttieri et al., 2005). Under irrigated and non-irrigated treatments

studied gliadin/glutenin ratio has studied and accomplished that there is no considerable

distinction between two treatments they also concluded that fraction of polymeric proteins

was higher under non irrigated treatment (Panozzo et al., 2001). Similar findings were

found previously bread wheat, researchers observed that when water availability becomes

limited non-significant change was seen in rate of accumulation of soluble and insoluble

proteins per degree day in bread wheat even though the polymer in solubilization in

progress earlier (Daniel and Triboi, 2000).

4.6.5 Moisture Contents of seed

Under normal condition, non-significant difference was found in all tested

genotypes under non priming treatment (Fig 4.35). Under SA priming, moisture contents

were decreased notably in Pakistan-13 (8.5±0.01%), AARI-11(8.7±0.15%) and Chakwal-

50 (8.2±0.10%), while increased in Shahkar (9.6±0.10%) and FSD-08 (9.3±0.11%) under

103

normal condition. priming with GA increased moisture contents appreciably in AARI-

11(9.5±0.08%), while decreased in Shahkar (9.1±0.10%) and FSD-08 (8.6±0.05%). Under

drought stress, AARI-11 had the highest moisture contents (9.2±0.10%) while Shahkar had

the lowest moisture contents (8.2±0.10%) in non primed samples. Moisture contents were

significantly raised on SA priming only AARI-11(9.5±0.05%) while decreased in

Chakwal-50 (7.7±0.10%). When GA Priming samples were examined it seems that GA

increased the protein in Shahkar under control, while significant decrease in Chakwal-50

(7.7±0.01%). Drought stress significantly decreased the moisture in Chakwal-50 (8.50.10),

Shahkar (8.2±0.10%) and in Pakistan-13(8.0±0.10%). Priming with SA raised the moisture

contents in AARI-11(9.5±0.05%) while reduced in Chakwal- 50 (7.7±0.10%) under

drought form as compared to control condition. Under observation genotypes have less

moisture contents under drought as compared to normal. During both years moisture

contents remained same and non significantly varied.

Figure 4.35: Moisture percent in seeds of wheat genotypes

The estimation of moisture content in seeds is an important factor as it influences

the storage life and seed quality. For optimum milling yield moisture content is important

for starch development as well as ensuring the filling out of the endosperm. Low moisture

content is expected during post-harvest which is essential for the storage life of the grain

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prior to being milled for its future use. If moisture contents are in excess it can lead to

sprouting in storage, mold growth, toxin formation and insect infestation. In our study all

tested genotypes has good moisture range afterharvesting which is suitable criteria for seed

storage. Drought stress reduced the moisture contents in three genotypes while SA priming

enhanced moisture in some genotype.

4.6.6 Starch content of seed

Under normal condition, AARI-11 and Shahkar had the highest starch

(52.4±0.05%) while other genotypes have non-significant difference in non primed samples

(Fig. 4.36). Starch was increased significantly on SA priming in Shahkar (52.4±0.10%),

Chakwal-50 (52.2±0.10%), and FSD-08(51.6±0.05%) while decreased in AARI-11.

Protein was increased significantly in GA priming in Shahkar (52.1±0.15%) and FSD-

08(51.7±0.05%) while decreased in Chakwal-50 (50.0±0.10%) and AARI-

11(50.2±0.10%).Under drought stress, Chakwal-50(49.1±0.10%) had the lowest starch

contents in non primed samples. Starch was significantly decreased in maximum genotypes

on SA priming. Starch only increased in Pakistan-13(51.2±0.20%) while decreased in other

genotypes when GA priming was applied. Under the effect of drought stress starch was

decreased in all other genotypes except Pakistan -13 which remain unaffected. In case of

GA and SA priming the starch contents are declined in all studied genotypes under the

condition of drought in comparison to control condition. During both the years non

significant effect occurred on starch contents.

105

Figure 4.36: Starch percent in seeds of wheat genotypes

Due to reduction in starch content the yield losses are caused because starch occupy

65% of cereal grain (Barnabás et al., 2008). Sucrose of grains are related to the

accumulation of starch which is correlated with sucrose synthesis and rest enzymes which

are responsible for starch synthesis (Yan et al., 2008). Under stress it is suggests that

enzymatic activityreduced that are involved in synthesis of starch and accumulation sucrose

contents become low. According to (Labuschagne et al., 2009) high temperature is

responsible for starch synthase inactivation which is the main enzyme in the biosynthesis

of starch causingdecline the starch ratio in endosperm. Some researcher reported that when

heat stress applied on bread wheat flour the protein content in flour increasesed(Bencze et

al., 2004); (Balla and Veisz, 2007). Similar results are also noted by other authors (Daniel

and Triboi, 2000); (Hrušková and Švec, 2009). After flowering, heat and drought stress

coupled with yield losses because of decline in the starch synthesis however grain proteins

are increased (Fowler, 2003). The basic useful properties of starch, particularly starch or

flour has the high water absorption ability and the drought flexibility, are reliant on the ratio

of amylose-to-amylopectin and the starch granules size distribution (Labuschagne et

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al., 2009). Most of the time wheat grain quality mainly affected by water stress and high

temperature.

Figure 4.37: Dendogram derived from hierarchical cluster analysis of all combined quality attributes of five

wheat genotypes

4.6.7 Conclusion

The magnitudes in which drought stresses affect quality depend on the sensitivity

and tolerance of genotype. Wheat grain quality characters were noticeably affected under

drought stress. The highest wet gluten was observed in Shahkar under normal condition

while under drought wet gluten decreased in Shahkar that showed the sensitivity of cultivar.

FSD-08 had the same values for wet gluten under normal and drought stress. GA improved

wet gluten in FSD-08. Gluten index and falling number was highest in FSD-08 followed

by Pakistan-13 and AARI-11. In Shahkar, under drought; gluten index and falling number

decreased while SA priming improved it in shahkar genotype. Grain protein content were

increased under drought stress in the Chakwal-50. Starch and moisture percentage were

highest in the Shahkar and AARI-11. Under drought, moisture and starch contents

107

decreased in all genotypes except Pakistan -13 in which these attributes remained

unaffected under drought.

4.7 SEED PROTEIN PROFILINNG USING SDS-POLYACRYLAMIDE GELS

In the present study five genotypes of wheat alongwith priming treatment were

evaluated by using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-

PAGE) for the estimation of protein contents stored in seeds. Standard protocol was

followed to separate protein subunits on polyacrylamide gel (10%). SDS-PAGE technique

resulted in fifty eight bands of total scorable bands. Based on these bands, difference of

primed and non primed seeds of wheat remained screened. Among fifty eight polypeptide

bands, 37% were generally existing in all genotypes and called as monomorphic, whereas

58% revealed differences and reflected as polymorphic. The size of these bands ranged

from 10 to 98 kDa. A protein, marker (pre stained) with weight ranges from 14.4 to 97kDa

was used to calculate the weight of these obtained polupeptide bands.

Band size vary between non primed and primed samples and bands that were not

prominent in some samples that were not counted, some bands were missing, while some

new bands were recorded.

The bands of the wheat samples were in sequence of band number 1, 2, 5, and 6.

Band number 3 was present only in primed samples. In FSD-08, SA treated samples have

two band among 66-45 while GA has four bands that showed variation to other samples.

In SA and GA treated samples in FSD-08 band size ranged to almost 10. In Punjab and

AARI-11 genotypes many new bands were detected between 45-20. The most prominent

change that occur in seed protein profile due to priming was the induction of a new band

25, 30,33 kDa protein induced by SA priming. GA priming also induced a 28 and 42 kDa

protein which was changed from non priming treatments. Priming treatment also caused

108

disappearance of two peptides with approximate molecular weights of 25 and 27and 35

kDa from Pakistan-13and Shahkar. However these two peptides did not disappear in

nonprime seeds. Priming also induced a change in the expression of a 33, 38 and 25 kDa

peptide. This priming induced protein was expressed by all priming treatments with more

prominent expression in FSD-08 and AARI-11 and Pakistan-13. A 26 kDa protein appeared

after seedpriming with GA. Another 25 and 30 kDa protein was expressed in case of SA

priming treatments (Fig 4.38a-b).

The reported findings are in line with those of Din and Flower (2002) who reported

that under non-stress conditions 15, 18 and 23 kDa proteins in ABA treated seeds.

Likewise, Reddy et al., (1993) worked on rice callus treated with ABA and found 15 and

23 kDa proteins under conditions of water stress. The plants showed adaptations by making

few proteins to overcome the stresses induced by of biotic as well as abiotic factors, among

them some of these produced proteins deduct by phytohormones, for example, salicylic

acid that was reported earlier (Hussein et al., 2007). Plants forms proteins to with stand

abiotic stresses and a considerable lot of these produced proteins are prompted by action of

phytohormones, for example, ABA and SA showed same affect earlier (Jin et al., 2001).

Seed priming promotes the synthesis of some new Proteins while loss of some

protein in some samples. A few new proteins appeared in the seed protein profiles after

priming treatment in the present study. It can be hypothesized that this peptide may be

related to a defense mechanism or osmotic adjustment under stress conditions.

109

Figure 4.38a: Electrophorogram showing control and seed priming induced variations in five wheat seed

protein profiles.

Figure 4.38b: Electrophorogram showing control and seed priming induced variations in five wheat seed

protein profiles

4.8TRANSGENERATIONAL EFFECT OF ELEVATED CARBONDIOXIDE ON

METABOLISM OF WINTER WHEAT EXPOSED TO ANTHESIS DOUGHT

Our study supports the idea that during the 20th century that over a number of

regions, drought index has increased and climate changes have altered the breeding targets.

110

4.8.1 Effect of elevated e[CO2], ambient a[CO2] and Drought on Yield Attributes

Grain number was also significantly affected by CO2 and irrigation treatment.

Symbols denoted E and A was seeds from previous generation while a and e was current

levels of CO2.In drought stressed plants grain number was reduced to a significant level

and severe reduction was observed under Ea[CO2] and Ee[CO2] as compared to Aa[CO2]

and Ae[CO2]. Carbon dioxidetreatments significantly affected the thousand kernel weight

however, it was unaffected by irrigation treatment. Plants grown-up under Ae[CO2] and

Ee[CO2] had higher yield in well watered plants than drought stressed plants. Harvest Index

was significantly exaggerated by both CO2 and irrigation management. Plants grown-up

under Aa[CO2], Ae[CO2] and Ee[CO2] had higher HI in both controlled and drought

stressed plants (Fig. 4.39).

Drought stress effect different phonological stages of the crop. When drought stress

was imposed near the beginning of reproductive stage it cause spikelet and pollen abortion

which leads to decrease the number of grains in wheat and rice (Kato et al., 2008; Dolferus

et al., 2011). Pollen and ovary abortion occurred when plant face drought stress at anthesis

stage due to induction in poor dehiscence of anther and inhibits panicle exertion owing to

reduced peduncle length, which cause in superior spikelet sterility in crops especially rice,

maize and wheat (Powell et al., 2012; Rang et al., 2011; Aslam et al., 2013). Our results

were also in agreement with the previous reports and grain number was reduced under

drought condition even under e[CO2]. The fatal drought at grain filling stage in wheat and

barley results during early senescence by means of shorter grain-filling period and low

persistence of flag-leaf green area (Samarah, 2005). In previous studies, number of grains

per plant and HI was increased under e[CO2] (Dong and Liu, 2005). Similar findings were

observed in our study that drought had significant effect on yield and yield contributing

factors. It was observed that e[CO2] enhanced grain yield, in addition to biomass.

111

Under elevated carbon it was observed that grain number in wheat was increased.

These results were confirmed by (Pleijel et al., 2000). Our results were supported by some

previous studies that e[CO2] enhanced yield even when seeds of ambient generation was

put under e[CO2]. (Wu and Wang, 2000)shown that under elevated [CO2] bean yield was

higher as a result of increase in bean number.The idea behind it was that high carbon

assimilation fix more carbon that result in the full development of flowers and grains.

Regarding individual grain weight the effect of high [CO2] was not always significant with

increase or decrease (Heagle et al., 2000; Pleijel et al., 2000). Ther is need of introduction

of such genotypes that is responsive to CO2- (Ainsworth et al., 2008) especially under

drought and salinity with the intention of providing starting lines for the process of

breeding.

112

4.8.2 Effect of elevated [e(CO2)], ambient a[CO2] and drought on

physiologicalattributes

Symbols denoted E and A was seeds from previous generation while a and e was

levels of CO2. Plants grown under Ae[CO2] and Ee[CO2] had higher photosynthesis while

during drought seeds grown under Ae[CO2] had high photosynthesis. After recovery

photosynthesis was higher under Ee[CO2]. Statistical analysis indicated that slight variation

was observed in term of stomatal conductance. The effect of CO2 was statistically

significant on transpiration rate while, plants grown under Ea [CO2] and Ee[CO2] had more

transpiration rate than plants under Aa[CO2] and Ae[CO2]. However, in all drought

stressed plants transpiration rate was significantly reduced. Results indicate that plants

grown under Aa[CO2] and Ee[CO2] had more leaf water potential(LWP) as compared to

Ae[CO2] and Aa[CO2] . Interactive effect of CO2 with LWP was also significant (Fig. 4.40-

43).

113

Plants intellect and react to e[CO2] during improved photosynthesis and stomatal

conductance going to be decreased in large number of species under different stress

conditions (Ainsworth et al., 2008). The consequences from this learning specify that

photosynthetic rate; stomatal conductance and transpiration rate was higher under elevated

CO2 in all three series of experiment. After drought transpiration rate was dramatically

reduced under both level of CO2. High [CO2] compensate the effect of drought stress

(Bencze et al., 2014)by increasing the levels of carbohydrates for the new tissues

development or filling grain (Wall et al., 2006). Though, effect of elevated [CO2] is not

always positively correlated for the stress tolerance in some studies (Pleijel et al., 2000).

Usually, transpiration is reduced by high concentration of [CO2] (Morison, 1987)

however; larger leaf area is responsible to induce reduction in transpiration by doubled

[CO2] (Wu and Wang, 2000). It was examined in our work that leaf water potential

dramatically decreased under ambient and elevated level of CO2. It is well documented

114

in previous studies that the main cause for this lower potential is due to effect of

complimentary masking factors. The masking factors are may be the difference in term of

temperature gradient flanked by the ambient air and the leaf that will escort to a deviation

of internal vapor pressures of leaf. CO2 assimilation and WUE adversely affected by low

water potential of leaf because of variation in different reactions during the process of

photosynthesis (Chaves et al., 2003) and decreased conductance in mesophyll (Warren,

2004).

4.8.3 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on Invertases and

Sucrose synthase activity


levels of CO2. In case of (VacInv) plants under Ee[CO2] had high vacInv activity followed

by plants grown under Ae[CO2]. In spike vacInv activity was increased under Aa[CO2] and

Ae[CO2] . This activity uplifted in both spike and leaf in drought stressed plants under

Ae[CO2] and Ea[CO2] after recovery. Cytoplasmic invertase (CytoInv) activity was

significantly affected by CO2. Higher activity was observed under Ae[CO2] while under

Aa[CO2] activity was reduced. In spike, dramatic increased activity was observed under

Aa[CO2], while it was reduced in all other [CO2] treatments. Cell wall invertase (CWInv)

activity was significantly increased under Aa[CO2] and Ae[CO2]. However, in case of spike

non-significant variation was observed. It is reported in previous literature that under

drought conditions, in mature maize leaves, cell wall invertase activity was not affected but

an increase in vacuolar invertase activity and accumulation of high hexoses was recorded

in the leaves. Cell wall invertases are considered the key enzymes in sucrose unloading and

in the source/sink balance within the plants (Tang et al., 1999), by supplying carbohydrates

to tissues through apoplastic pathway. Sucrose synthase (Susy) activity in leaf was showed

similar trend under all level of CO2 before stress were applied, in spikes CO2 had significant

Fig-40 Effect of optimum and elevated [CO

2] on the photosynthesis (An) before drought (a), after drought (b) and after recovery

115

effect on Susy activity. Susy activity was highest under Ea [CO2] and Ee[CO2] treatment.

In drought stressed plants Susy activity was reduced in leaf while again uplifted after

recovery. In spikes, different results were observed for Susy activity and it was high for

both well wtered and drought stressed plants under Aa[CO2] (Fig. 4.44-47).

117

A and E= seeds from previous generation, a and e= current level of CO2. W= irrigation, D= drought,

L=Leaf. S=Spike

Figure: 4. 48 Key enzymes (C) activities of primary carbohydrate metabolism under drought and

well watered in both source (L) and sink (S).

118

4.8.4 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on the key enzymes for

Carbohydrate Metabolism


levels of CO2. Significant variations were observed regarding UDP-glucose

pyrophosphorylase (UGPASE) activity in flag leaf. UGPASE activity was significantly

increased under Aa[CO2],while in all other treatments similar trend was observed.

However, within spike UGPASE activity dramatically increased under Aa[CO2] and

Ee[CO2]. Phosphoglucomutase (PGM) activity was increased manifold under the exposure

of e[CO2] in the leaf. In contrast within spikes PGM activity was significantly affected and

seems higher under e[CO2] as well as a[CO2]. In drought stressed plants PGM activity

remained unchanged in leaf while activity reduced after recovery in all except Aa[CO2]

(Fig. 4.49-50).

120

High Phosphoglucoisomerase (PGI) activity was observed under Aa[CO2] and

Ae[CO2] in leaf. In spikes, PGI activity increased many folds under Aa[CO2]. Glucose-6-

phosphate dehydogenase (G6PDH) activity was higher under Aa[CO2] and Ae[CO2]. In

spikes G6PDH activity was increased under Ae[CO2], Ea[CO2] and Ee[CO2] treatments.

Plants grown under drought stress had high G6PDH activity under Ae[CO2] and Ea[CO2]

in leaf . While in spike high activity was observed under Ae[CO2] and Ee[CO2] treatment

(Fig. 4.51-52).

121

Frutokinase (FK) activity was significantly affected by [CO2] in flag leaf. The

activity of this enzyme was increased in the plants grown under Ea[CO2] and Ee[CO2] as

compared to Aa[CO2] and Ae [CO2]. However, in spikes this activity remained unaffected.

Within drought stressed plants FK activity increased under all levels of CO2. In flag leaf,

Hexokinase (HXK) activity was slightly higher under Ea [CO2] and Ee[CO2] than Ae[CO2]

and Aa[CO2]. In contrast, within spikes this HXK activity was increased under Ae[CO2]

and Ee[CO2], and it was decreased under Aa[CO2] and Ea[CO2]. In drought stressed plants

HXK activity enhanced manifolds under e[CO2] (Fig. 4.53-54).

122

In leaf the highest Phosphofrutokinase (PFK) activity was found under Ae[CO2]

and Ea[CO2] treatment as compared to Aa[CO2] and Ee[CO2]. In spikes, the highest activity

of PFK was recorded under Ae[CO2]. In drought stress plants PFK activity was

123

less than control plants. The activity of Aldolase (Ald) showed increasing pattern under

e[CO2] treatments. In spikes, enzyme activity was similar for different [CO2] treatments.

In Spikes this activity showed dramatic changes, Aldolase activity showed distinct pattern.

Significant variations were observed regarding AGPASE activity in flag leaf. Similarly, the

activity of ADP- glucose pyrophosphorylase (AGPASE) was increased under Aa[CO2] and

Ae[CO2] treatments. However, within spike AGPASE activity dramatically increased

under Aa[CO2] and Ee[CO2] (Fig. 4.55-57).

124

To reveal the accomplishment of enzyme activity within physiological phenotyping

approach, no previous studies were focused on trans-generational effect of winter wheat

and it is even more difficult to extrapolate from one generation to the next.

125

Our analysis was built upon the largest scenarios of climate change because the

understanding of climate direction is of outmost importance. Some authors consider

climatic change as positive one, combined with development in cultivation technologies,

may increase potential wheat yield by 37-101% by 2050(Pérez-Carrillo and Serna-Saldívar,

2007). In present experiment, the metabolism of enzymes was determined at different

stages from source and sink organs. We found that plants of even a[CO2] background had

the highest activity under e(CO2). However, we also observed some of the enzymes have

higher activities under a[CO2] in both the organs (leaf and spike).

The present work suggested that UDP- glucose pyrophosphorylase (UGPase)

showed highest activity under Ae(CO2), while ADP- glucose pyrophosphorylase (AGPase)

activity increased under both level of CO2. It may be due to the adaptive response to with

stand the drought stress by carbon portioning, it results in stunted growth due to glycolytic

enzymes decrease expression. Elevated [CO2] can increase the amount of carbohydrates

within the leaf, mainly in the form of starch.

In one of previous studies, in crops ADPG pyrophosphorylase resultant activity

under elevated condition [CO2] was 1.6-fold more than that of the ambient conditions.

Similarly manifold increased AGPase activity has been shown in our study. Probable

mechanisms could be that sucrose synthesis due to feed-back inhibition of or recycling of

sucrose due to the activity of invertase and stimulation of AGPase by an improved 3PGA:

phosphate ratio, and increased expression of AGPase result of sugar-mediated signaling

(Stitt and Krapp, 1999). The results of present experiment indicate that activities of G6PDH,

FK, PGM and HXK showed distinct pattern in both source and sink organs. Activity of

these enzymes increased under e [CO2] in irrigated and drought stressed plants. These

results are similar to (Nakamura et al., 1997) who reported that in pollen cells, FK was

chief component whereas; the activity of HXK was comparatively low. It has been

126

found that part of the HXK protein is most important particulate, principally in

mitochondrial membranes, and this outward appearance is strongly introverted by

micro molar ADP concentrations (Yang et al., 2001).FK and HXK in the anther of

flower that present near the pollen may also have a key role to determine the

development of pollen cells and in turn germination capacity, given that the anther

walls be in charge of sugar nutrition as was found in Lilium pollen grains.

In our experiment, the PGI, PFK activities enhanced under e[CO2] while aldolase

and PFK activities showed decreasing pattern. In some previous studies no significant

variations were found for the actions of aldolase or PGI between optimum [CO2] treated

cells, whereas PFK and PFP activities decreased more than 40% under high CO2. A decline

of PFK as well as PFP activities under elevated CO2 conditions could be effected from an

inhibition or inactivation of preexisting PFK and PFP. However, it was reported that

regulating glycolysis by formation of PFK affected to a great extent on pH (Turner et al.,

1980).

4.8.5 Effect of elevated [e(CO2)], ambient a[CO2] and Drought on key Enzymes

(a)activities for Carbohydrate Metabolism


levels of CO2. Activities of different antioxidant enzymes were shown in (Fig. 4.58).

127

A and E= seeds from previous generation, a and e= current level of CO2. W= irrigation, D= drought,

L=Leaf. S=Spike

Figure-4.58 Key (A) enzymes activities of primary carbohydrate metabolism under drought and

wellwatered within source (L) and sink (s).

In recent study, superoxide dismutase (SOD) activity was significantly affected

under the exposure of [CO2]. This activity was increased in plants grown under Ea[CO2]

and Ee[CO2] in leaves. In spikes SOD activity remained unaffected in all plants under all

CO2 treatments. Before drought treatment, in leaf higher catalase (CAT) activity was

observed under Ee[CO2] and similar trend was estimated under remaining treatments. In

terms of spike CAT activity was significant affected under CO2. In contrast for drought

stressed plants CAT activity increased under Aa[CO2] and Ae[CO2](Fig.4.59-60).

128

Ascorbate peroxidase (APX) activity was increased in various species grown under

Ae[CO2] while decreased under Ee[CO2] in leaf. APX activity was significantly affected

in spikes under e[CO2] had greater APX activity. Glutathione reeducates (GR) activity

129

increased under Aa[CO2] and Ea[CO2] in leaf and spike respectively. After drought in leaf

and spike GR activity increased under e[CO2] (Fig. 4.61-62).

130

Peroxidase, dialysed extract (DPOX) activity increased under Ea, Ee[CO2] in

leaves while DPOX activity remained unchanged in spike. ZPOX activity increased under

Ea[CO2], Ee[CO2] in leaves. Peroxidase, Z extract (ZPOX) activity increased under

Aa[CO2] and Ae[CO2] in spike. Similarly, in drought stressed plants ZPOX activity

increased in leaf under Aa[CO2] and Ae[CO2] while in spike increased under Aa[CO2]

(Fig. 4.63-64).

Total antioxidant potential (TAP) showed similar trend in all plants grown under all

level of [CO2]. However, in spike significant variation was observed. Total antioxidant

potential was high in plants species that grown under Ae[CO2]. Under drought stressed

conditions plants TAP was dramatically increased under Ea [CO2] and Ee[CO2].

131

However, in spikes higher TA value was calculated for the plants grown under

Ae[CO2] (Fig. 4.65).Previous literature supported our findings that catalase under stress

has the ability of many reversible proteins in leaf and its activity is reduced under drought

condition. Due to the inhibition of photorespiration under elevated [CO2] enzyme activity

and catalase gene expression have been decreased in wheat, however, under drought they

increased (Vicente-Suarez et al., 2015).In literature, SOD activity was increased after 4

days of water stress in barley (Acar et al., 2001), and wheat (Sairam and Tyagi, 2004). (Lin

and Wang, 2002) observed that during the activities represented by SOD and CAT were

specifically much higher in CO2 enriched wheat than its ambient level.

132

The APX genes present in mitochondria, chloroplast and cytosol showed

differential inflection by large number of abiotic stresses in plants (Caverzan et al., 2014).

The activity of GR was higher in leaf under a[CO2] while in spikes, reverse effect was

observed and this activity was significantly higher under e[CO2]. Similarly, D-POX and

Z-POX activity increased under e[CO2]. Few previous reports about the activity of

antioxidants suggest that the activities of Catalase, supeoxide dismutase, Ascorbate

peroxidase, Peroxidase and Glutathione reductase decreased, in leaves of soybean genotype

grown at e[CO2]. Studies of (Zinta et al., 2014) supported that antioxidant level or activities

increased under e[CO2]. Some Previous findings supported our results and suggested that

e[CO2] had little or no effect on antioxidant and sometimes it even decrease the level of

these enzymes (Mishra et al., 2013). In most studies, about 50% of the observations of

some key antioxidant enzymes (ASC, APX and CAT) were seems to remain unchanged

under stress and elevated CO2 or even decreased for 28% of the observation (Glutathione

peroxidase). This showed that the under stress-mitigating conditions, effects of elevated

CO2 cannot be found all around the world to attributed to enhanced antioxidant defenses.

133

The concentrations of sucrose, glucose and fructose and starch were examined. In

wheat, significant increases were found for fructose and glucose in the high-CO2 treatment.

The concentration of sucrose was increased and decreased during different treatments.

However, CO2 effects were also statistically significant on starch. As reported previously,

the increased starch accumulation under elevated CO2 conditions affects sucrose

metabolism and causes the decrease in glucose content (Walter and Schurr, 2005). The

present study find that under elevated CO2 starch increased while in drought decreased.

(Woodhams and Kozlowski, 1954) and found the similar results. During photosynthesis

leaf soluble sugars formed which export for plant growth from the region of source that are

in most conditions are leaves into the area of phloem (Dietze et al., 2014). Consequently,

due to the assimilation of large amount of carbon or low concentration of carbon export

these sugars are a temporary pool and starch is a short-term storage induced. Meanwhile,

drought may limit the sink action, and sore strain the non-soluble carbohydrates export

from leaves and use, primary growth reduction is linked with leaf starch accretion (Lemoine

et al., 2013). During drought, photosynthesis is often decreased which is compensate by

e[CO2] (Bencze et al., 2014), which ultimately improved the levels of carbohydrates for

new tissue formation and also grain filling (Wall et al., 2006). Though, all theprevious

studies was not achieved the positive effects of e[CO2] for stress tolerance (Pleijel et al.,

2000). (Bencze et al., 2014) reported that, in bread wheat drought along e[CO2] result in

enhancement of the antioxidant enzyme which led to a high level of oxidative stress.

Previous studies that was done on durum wheat confirmed the approachability to e[CO2]

(Aranjuelo et al., 2013) , drought (Aprile et al., 2013;Habash et al., 2014) and the

combination of elevated carbon and water stress(Erice et al., 2007) is genotype specific.

Furthermore, different growth stages of durum wheat respond differently to e[CO2]

(Vicente et al., 2015). Some studies have shown the positive effect ofe[CO2] on water stress

134

tolerance (Harnos et al., 2002)(Robredo et al., 2007)(Bencze et al., 2014). It can be

summarized that Increased CO2 concentration along with availability of water contents may

retard the photosynthetic process (Robredo et al., 2007). Tha resultant reduced rate of

transpiration may alter or sometimes enhances the affect of stress induced by drought

(Kirkham, 2016; Miranda‐Apodaca et al., 2015; Kadam et al., 2015).

At the germination and development stages of crop, drought stress was foundin

winter months in previous studies (Russo et al., 2015). Response of plant to e[CO2] or

drought are correlated with growth stage, as well as environmental factors duration, level

and the genetic variability.

Wheat is being grown at the diverse climatic regions of the world. Due to wider

adaptability this crop has to face certain stresses and drought is one of them. Numbers of

strategies are being utilized to mitigate this stress however; e[CO2] has proved more

helpful. Nevertheless, the exact mechanism of this stress tolerance is still unknown. All

these studied attributes have major contribution towards starch synthesis, accumulation and

utilization. Photosynthesis has major contribution towards starch synthesis through these

enzymes activities. Likewise, accumulation of high starch within plants is always

rewarding in term of yield. However, better quality is always necessary alongwith high

yield and in our later study from same experiment we will also focus on quality of grain.

4.8.6 Conclusion

The change in atmospheric CO2 concentration affects the balance of carbohydrate

metabolism. However, more detailed analysis about gene determination is necessary to

relate carbohydrate accumulation with changes in the photosynthetic apparatus. In this

study we found that elevated e[(CO2)] provokes different enzymes in source (leaf) and sink

(spike) which are helpful to tolerate drought stress. It was observed that in source

135

CytInvertase, Sucrose synthase (Susy) along with catalase and ascorbate peroxidase was

decreased however, the activity CWInvertase was increased under drought and high CO2.

Similarly, within the sink the action of Glucose-6 phosphate dehydrogenase (G6PDH),

superoxide dismutase, ascorbate peroxidase and dialysed peroxidase was decreased under

drought while the activity of aldolase and frutokinase were increased. An interesting path

of activity of G6PDH within the source was observed as it remained unchanged before

drought but its activity was reduced under drought and increased under re-watering.

Similarly, the grain number was reduced under drought stress which indicates that aldolase,

G6PDH and susy are actually responsible to lower this number. Our results suggest that

high activity of Susy within the source and high aldolase and G6PDH activity within the

sink may be helpful to mitigate the drought stress under high value of CO2.

Under elevated CO2 conditions alongwith drought physiological processes can be

inhibited and promoted by photosynthesis and water use efficiency. Dissolved organic

carbon in soil is retained for a long time under elevated CO2 as a result mineralization

improved water use. Microbial carbon (biomass) and C:N ratio was decreased under

drought but drought along with CO2 is not as significant as alone. Plant root exudationcan

be stimulated under elevated CO2 as a result in more activities by invertase and catalase

activities. Findings suggest that soil carbon concentration, activities mediated by soil

enzyme and plant physiology are directly correlated with drought however elevated

CO2 reduced negative effects of drought (Yuhui et al., 2017).

136

SUMMARY

Two experiments were conducted during current study. First experiment was

carried out in University of (AJ&K) Pakistan and effect of drought stress on five wheat

cultivars viz. AARI-11, Chakwal-50, Shahkar, Pakistan-13 and FSD-08 was studied.

Hormonal seed priming (SA, GA) were used as a short term approach to reduce drought

stress. Different morpho-physiological, traits were studied from flag leaf while quality as

well as seed bio physiological changes were after harvesting.

Priming with SA and GA improved the activities of oxidative and antioxidant

enzymes in seeds like amylase, protease, catalase, SOD and POD. Genotypic difference

varied in term of treatments. Priming with both the growth regulators increased yield under

normal and drought conditions and it was observed that higher yield was due to rapid

emergence and vigorous seedlings. Pakistan-13 and FSD-08 were ranked higher for overall

grain. The study of soluble compounds, lipid peroxidation, relative water contents,

membrane stability, minerals, high proline, antioxidant and maintenance of cell membrane

integrity contributed toward osmotic adjustment which increased the yield under stress

conditions. These findings suggest that adverse effect of water stress on wheatcan be

overcome by using GA and SA. Wheat grain quality attributes were also studied and it was

observed that drought had significant effect. However, hormonal seed priming also help in

maintaining the undoing adverse effects of drought. The FSD-08 was maintainedquality of

grain under normal and stressed conditions. Seed protein profiling indicated that many new

peptides were appeared after priming and it can be hypothesized that these proteins may be

helpful to cope stress.

137

Second experiment was conducted at University of Copenhagen, Denmark to find

out the transgenerational effect of elevated e[CO2] in winter wheat at anthesis drought

stress. It was observed that, photosynthesis and activities of antioxidant enzymes was

increased under e[CO2]. Resultantly, all these traits contributed to increase the yield

especially under e[CO2]. Trans-generational effect indicated that seeds had stress memory

and thus maintained the effect of previous generation. Activities of all enzymes varied

within source and sink. UDP- glucose pyrophosphorylase (UGPase) activity was increased

under Ae[CO2], while ADP- glucose pyrophosphorylase (AGPase) activity increased under

both level of CO2.

Elevated [CO2] can increase the carbohydrates within the source, mainly in the form

of starch. These results indicate that high metabolism of Sucrose synthase, aldolase and

Glucose- 6 phosphate dehydrogenase might be helpful to mitigate the drought stress under

elevated CO2. Based on these results we can report that enzymes involved in carbohydrate

metabolism are affected under elevated CO2 and drought. Hence, studied cultivar had very

good yield under e[CO2] even in drought stress.

138

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APPENDIX

APPENDIX 1

Description of wheat genotypes used in current study

S.

No

Genotypes Properties

1 FSD-2008

Stem rust resistant; Smut resistant; recommended for irrigated

areas but also have very good performance in drought areas.

2 AARI-2011

For irrigated areas / Punjab

3 CHAKWAL-

50

For arid climate, drought and heat tolerant

4 Pakistan-13 For arid climate, drought and heat Resistant

5 SHAHKAR

2013

Bold seed, high yielding variety for irrigated areas

169

APPENDIX 2

Multiple ANOVA for seed biochemistry attributes

Estrase Protease Amylase SOD POD CAT

R² 0.929 0.990 0.950 0.855 0.933 0.913

F 13.944 106.768 20.567 6.322 14.966 11.206

Pr > F < 0.0001 < 0.0001 < 0.0001 0.001 < 0.0001 < 0.0001

Geotypes 9.311 252.468 21.260 2.614 9.225 14.449

0.001 < 0.0001 < 0.0001 0.077 0.001 < 0.0001

Treatment 28.004 24.617 29.029 6.715 55.108 8.320

< 0.0001 < 0.0001 < 0.0001 0.008 < 0.0001 0.004

Geotypes*Treatment 12.639 50.715 17.699 7.070 6.150 9.671

< 0.0001 < 0.0001 < 0.0001 0.001 0.001 0.000

170

APPENDIX 3

Multiple ANOVA for oxidative behavior

Protease Estrase Amylase SOD POD Cat ASP

R² 0.861 0.528 0.785 0.857 0.840 0.479 0.837

F 11.169 2.023 6.620 10.838 9.490 2.973 16.596

Pr > F < 0.0001 0.029 < 0.0001 < 0.0001 < 0.0001 0.000 < 0.0001

Genotype 5.830 2.664 2.909 2.562 1.957 7.404 8.879

0.001 0.047 0.034 0.054 0.121 < 0.0001 < 0.0001

Stress 20.898 0.689 13.705 0.858 3.794 11.042 278.997

< 0.0001 0.508 < 0.0001 0.432 0.031 0.001 < 0.0001

Treatmet 94.233 0.001 41.412 188.379 99.774 0.856 0.794

< 0.0001 0.979 < 0.0001 < 0.0001 < 0.0001 0.429 0.456

Genotype*Stress 8.390 0.654 3.704 1.506 4.615 3.401 6.401

< 0.0001 0.728 0.003 0.188 0.001 0.014 0.000

Genotype*Treatmet 0.447 2.680 1.079 2.630 7.989 0.741 0.565

0.774 0.046 0.380 0.049 < 0.0001 0.656 0.803

Stress*Treatmet 2.520 7.884 9.516 0.458 4.421 0.266 1.144

0.094 0.001 0.000 0.636 0.019 0.767 0.325

171

APPENDIX 4

Multiple ANOVA for physiological parametere

TOS MDA CMT RWC Chla Chlb Cartnid Antho protn Sugar Prol K+ Ca+

R² 0.384 0.462 0.837 0.862 0.733 0.567 0.593 0.514 0.514 0.857 0.960 0.956 0.870

F 1.127 1.551 16.594 20.200 8.906 4.243 4.709 3.422 3.423 19.447 77.501 69.641 21.636

Pr > F 0.365 0.117 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Geotypes 1.794 0.978 48.187 61.631 8.248 1.889 7.029 8.689 11.327 78.552 251.630 219.610 68.670

0.150 0.431 < 0.0001 < 0.0001 < 0.0001 0.122 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Stress 0.534 2.252 96.596 105.298 96.550 32.325 3.000 6.645 3.619 9.086 418.111 109.580 2.260

0.590 0.119 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.088 0.012 0.061 0.004 < 0.0001 < 0.0001 0.137

Treatmet 1.404 0.006 0.312 4.473 17.820 17.499 31.329 13.646 2.014 4.960 8.447 3.153 14.630

0.243 0.940 0.733 0.015 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.141 0.010 0.001 0.049 < 0.0001

Geotypes*Stress 1.509 1.269 11.835 3.643 0.340 0.328 0.619 0.104 1.208 5.649 33.940 14.583 2.306

0.187 0.288 < 0.0001 0.010 0.850 0.858 0.651 0.981 0.316 0.001 < 0.0001 < 0.0001 0.067

Geotypes*Treatmet 0.398 1.370 1.378 3.307 0.808 0.723 0.244 0.274 1.238 5.786 5.922 48.915 12.819

0.809 0.262 0.222 0.003 0.598 0.671 0.981 0.972 0.291 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Stress*Treatmet 0.173 4.073 0.072 11.197 7.011 3.565 0.343 0.283 2.093 3.147 1.428 9.236 18.186

0.842 0.025 0.931 < 0.0001 0.002 0.034 0.711 0.755 0.131 0.049 0.247 0.000 < 0.0001

172

APPENDIX 5

Multiple ANOVA for Morphology Parameters

Plant

height Tillar Spiklength Spiklts

Extrusin

length

Peduncl

length Grains

Grain

weight Grainyield Bioyield

Harvest

Index

R² 0.892 0.331 0.829 0.699 0.514 0.467 0.817 0.641 0.829 0.781 0.751

F 62.328 3.728 36.404 17.513 7.966 6.596 33.644 13.410 36.411 26.758 22.641

Pr > F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Geotypes 44.773 6.242 60.298 16.815 5.546 4.081 74.738 8.824 106.606 36.148 57.442

< 0.0001 0.000 < 0.0001 < 0.0001 0.000 0.004 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Stress 1075.433 41.100 445.663 225.630 103.541 83.735 267.451 227.914 313.053 363.827 193.977

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Treatmet 15.191 1.542 12.691 22.129 15.731 10.042 55.348 7.072 7.131 1.114 0.734

< 0.0001 0.217 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 0.001 0.001 0.331 0.482

Geotypes*Stress 1.916 1.544 6.560 2.608 0.784 0.675 0.234 0.523 0.880 8.794 4.138

0.110 0.192 < 0.0001 0.038 0.537 0.610 0.919 0.719 0.477 < 0.0001 0.003

Geotypes*Treatmet 1.536 0.348 2.621 2.434 0.240 1.588 2.950 0.267 0.661 1.944 4.124

0.149 0.945 0.010 0.016 0.983 0.132 0.004 0.976 0.725 0.057 0.000

Stress*Treatmet 2.014 0.082 2.522 0.355 2.523 1.480 2.447 0.018 1.041 0.276 0.354

0.137 0.921 0.084 0.702 0.083 0.231 0.090 0.982 0.356 0.760 0.703

173

APPENDIX 6

Multiple ANOVA for Quality parameters

Wglutn GlIndx F No Protein Moistr Strch

R² 0.654 0.867 0.942 0.543 0.830 0.802

F 6.116 21.040 52.828 3.849 15.768 13.137

Pr > F < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Geotypes 8.809 47.491 209.443 9.805 25.768 7.493

< 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001 < 0.0001

Stress 2.119 87.321 1.582 3.429 112.591 90.171

0.150 < 0.0001 0.213 0.068 < 0.0001 < 0.0001

Treatmets 1.912 10.836 30.195 3.563 3.765 17.050

0.156 < 0.0001 < 0.0001 0.034 0.028 < 0.0001

Geotypes*Stress 17.617 13.146 36.133 3.369 11.771 5.502

< 0.0001 < 0.0001 < 0.0001 0.014 < 0.0001 0.001

Geotypes*Treatmets 1.603 4.419 7.397 2.052 6.297 8.904

0.140 0.000 < 0.0001 0.053 < 0.0001 < 0.0001

Stress*Treatmets 1.981 27.472 2.962 0.584 5.241 14.200

0.146 < 0.0001 0.058 0.560 0.008 < 0.0001

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