Exploring Physiological Mechanism of Salt Tolerance in ... · 3.3.1 Physical and chemical characteristic of soil used in pot study 39 3.3.2 Selected twenty genotypes from experiment

i

“Exploring Physiological Mechanism of Salt Tolerance in Wheat

Germplasm”

By

Muhammad Sohail Saddiq

M.Sc. (Hons.) Agronomy

2006-ag-1540

A thesis submitted in partial fulfillment of the requirement for the degree of

Doctor of Philosophy

in

Crop Physiology

DEPARTMENT OF AGRONOMY,

FACULTY OF AGRICULTURE,

UNIVERSITY OF AGRICULTURE, FAISALABAD

(PAKISTAN)

2017

ii

Declaration

I hereby declare that the contents of the thesis, “Exploring Physiological Mechanism of Salt

Tolerance in Wheat Germplasm’’ are product of my own research and no part has been

copied from any published source (except the references, standard mathematical and genetic

models/ equations/ formulae/ protocols etc.). I further declare that this work has not been

submitted for award of any other diploma/degree. The University may take action if

information provided is found inaccurate at any stage. (In case of any default the scholar will

be proceeded against per HEC plagiarism policy).


2006-ag-1540

iii

To,

The Controller of Examinations,

University of Agriculture,

Faisalabad.

We, the Supervisory Committee, certify that the contents and form of thesis submitted

by Mr. Muhammad Sohail Saddiq, Regd. No. 2006-ag-1540 have been found satisfactory

and recommend that it may be processed for evaluation of External Examiner (s) for the award

of degree.

SUPERVISORY COMMITTEE

Chairman _________________________

DR. IRFAN AFZAL

Member _________________________

DR. SHAHZAD M.A BASRA

Member _________________________

DR. ZUFIQAR ALI

iv

Oh, Allah Almighty open our eyes,

To see what is beautiful,

Our minds to know what is true,

Our heart to love what is good.

Dedicated to MY

Grandfather

Master Muhammad Sadiq (late)

Who provides me opportunities, resources and a way to get success in all

spheres of life.

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ACKNOWLDGEMENTS

All worships and praises are only due to the Lord of creation, the most

beneficent, merciful and compassionate, Whose blessings and exaltation

flourished my thoughts and thrived my ambitions to have the cherish fruit of my

modest effort in the form of this manuscript.

I offer my humblest thanks and countless salutations to the Holy Prophet

Muhammad (PBUH), who is forever, a torch of guidance for the entire

humanity.

I owe my deepest gratitude to my great supervisor Dr. Irfan Afzal, Associate

Professor, Department of Agronomy, University of Agriculture Faisalabad, who

in spite of his busiest tiring routine work provided his dexterous and valuable

suggestions throughout research efforts.

Thanks are extended to the members of my supervisory committee Dr. Shahzad

M.A. Basra, Dr. Zulfiqar Ali and my IRSIP supervisor Dr. Amir Ibrahim

(USA) for their sincere cooperation and invigorating encouragement during the

course of present investigation. I am also very thankful to my dear lab mates

Muhammad Shahid Iqbal, Muhammad Kamran, Amir Bakhtavar,

Muhammad Idrees Faisal, Muhammad Bilal Hafeez, Numan Ali and my

cousin Jahazaib for their valuable suggestions and guidance during my research

activities and thesis write up.

I can’t forget prayers of my beloved Nani Amma, my sweet mother and great

supportive Khala, Miss Fareeda Kusar, Miss Amber my sisters Robina Khan,

Esha Maihiq, Heera Baloch and my brothers for the strenuous efforts done by

them in enabling me to join the higher ideals of life and also their financial and

moral support, patience and prayers they had made for my success.

Financial support from International Foundation for Science (IFS) under the

project “Improvement of salinity tolerance in bread wheat by identifying novel

salt tolerant germplasm” is highly acknowledged and appreciated


vi

Contents

Chapter Title Page No.

1

Introduction

1

2

Review of literature

4

3

Material and method

26

4

Results and Discussion

45

5

Summary

105

Literature Cited

109

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Table of Contents

Sr. No. Title Page No.

Chapter 1: Introduction 1

Chapter 2: Review and Literature 4

2.1 Salinity scenario in the world 4

2.2 Salinity spread in Pakistan 5

2.3 Salinity stress effect, salt tolerance mechanism and wheat production 6

2.4 Effects of salt stress on wheat physiology and growth 7

2.5 Ionic and oxidative consequences of salinity 10

2.5.1 Osmotic effect 10

2.5.2 Ion-specific effect: 11

2.5.3 Oxidative burst 11

2.6 Salt tolerance 12

2.6.1 Osmotic adjustment 12

2.6.2 ROS detoxification 13

2.6.3 Ion regulation and compartmentalization 14

2.6.4 Na+ Exclusion 14

2.7 Molecular basis of salt tolerance in wheat 15

2.8 Approaches to utilize salt affected soils for crop production 18

2.9 Result of Breeding 18

2.10 Selection/Screening Criteria for Salt Tolerance 19

2.11 Selection through hydroponic 20

2.12 Selection criteria followed in hydroponic culture. 20

2.12.1 Morphologically traits 21

2.12.2 Early seedling traits 21

2.12.3 Cell or tissue damage related traits 21

2.12.4 Physiological traits 22

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2.12.5 Ionic traits 23

Chapter 3: Materials and methods 26

3.1 Experiment 1: Screening of wheat germplasm against high salinity

stress 27

3.1.1 Plant materials 27

3.1.2 Experimental details 27

3.1.3 Determination of Na+ and K+ concentrations 27

3.1.4 Morphological attributes 28

3.1.5 Statistical analysis 28

3.2 Experiment 2: Investigation of physiological and biochemical bases of

salt tolerance in selected wheat germplasm 35

3.2.1 Experimental details 35

3.2.2 Na+ and K+ determination 35

3.2.3 Growth parameters 35

3.2.4 Gaseous exchange parameters 35


3.3 Experiment 3: Identification of physiological markers associated with

salinity tolerance of wheat genotypes in saline sodic soil 38

3.3.1 Plant material 38

3.3.2 Pot experiment details 38

3.3.3 Leaf Na+ and K+ determination 39

3.3.4 Water relation attributes 39

3.3.5 Biochemical analysis 39

3.3.6 Gas exchange parameters 40

3.3.7 Chlorophyll fluorescence 40

3.3.8 Cell membrane injury 40

3.3.9 Yield related attributes 40


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3.4 Experiment 4: Agronomic and physiological performance of selected

wheat genotypes on saline-sodic soil 42

3.4.1 Experimental location of field trial 42

3.4.2 Plant material and design 42

3.4.3 Crop husbandry 42

3.4.4 Leaf Na+ and K+ determination 43

3.4.5 Physiological attributes 43

3.4.6 Stand establishment 43

3.4.7 Biomass and grain yield 43


Chapter 4: Results and discussions 45

4.1 Experiment 1: Screening of wheat germplasm against high salinity

stress. 45

4.1.1 Response of wheat germplasm against salinity stress (200 mM NaCl) 45

4.1.2 Salinity stress response on the basis of ion accumulation 45

4.1.3 Comparison between low Na+ and high Na+ genotypes 46

4.2 Experiment 2: Investigation of physiological and biochemical bases of

salt tolerance in selected wheat germplasm 53

4.2.1 Response of screened genotypes against different salinity levels 53

4.2.2 Biplot Na+ and K+ concentration in leaf and root 53

4.2.3 Biplot for growth rate (root and shoot length) and relative growth rates 54

4.2.4 Genotypes response against salinity levels based on gaseous exchange

parameters 55

4.2.5 Absolute tolerance at different salinity levels 55

4.3 Experiment 3: Identification of physiological markers associated with

salinity tolerance of wheat genotypes in saline sodic soil 71

4.3.1 Response of screened genotypes against different salinity levels 71

4.3.2 Leaf Na+ and K+ contents 71

4.3.3 Leaf water relation attributes 71

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4.3.4 Relative water contents 72

4.3.5 Cell membrane injury 72

4.3.6 Gas exchange parameters 72

4.3.7 Biochemical analysis 72

4.3.8 Non enzymatic antioxidants 73

4.3.9 Chlorophyll fluorescence 73

4.3.10 Yield related attributes: 73

4.3.11 Stress susceptibility index (SSI) based on grain yield 74

4.4 Experiment 4: Agronomic and physiological performance of selected

wheat genotypes on saline-sodic soil 89

4.4.1 Na+ and K+ content 89

4.4.2 Biochemical attributes 89

4.4.3 Non-enzymatic antioxidants 89

4.4.4 Crop stand establishment 90

4.4.5 Yield and yield related attributes 90

4.4.6 Biplot of yield related attributes 91

General Discussion 102

Summary 105

Limitations of Study 107

Future Need 108

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List of Tables


2.1 Total geographical, cultivated and salt affected area of Pakistan (M ha) 5

2.2 Salinity consequences on wheat phenology 9

2.3 Genes/QTLs involved in salt tolerance mechanisms in wheat in different

species of wheat 16

2.4 Improvement in salt tolerance of wheat using conventional breeding

approach 19

2.5 Selection outputs in control conditions 24

3.1.1 Total number of wheat genotypes screened at 200mM 29

3.1.2 Selected forty two genotypes (25 tolerated and 15 salt sensitive) from total

400 wheat genotypes screened at 200mM 34

3.2.1 List of selected forty two genotypes for GGE-biplot 37

3.3.1 Physical and chemical characteristic of soil used in pot study 39

3.3.2 Selected twenty genotypes from experiment 1 & 2 41

3.4.1 Selected twenty genotypes and their GGE-biplot codes for yield attributes 44

4.1.1

Mean squares from analysis of variance for ionic content (leaf Na+ and K+

in “mg g-1 dry weight”), growth traits (root and shoot length in “cm”; fresh

and dry weight in “g”) and chlorophyll index of 400 wheat genotypes

grown at 200 mM NaCl salinity at seedling stage

47

4.2.1

Mean squares from analysis of variance of ionic, seedling growth and

gasses exchange parameters of 42 wheat genotypes grown at three

different NaCl salinity levels.

56

4.2.2 Relationship between ionic and seedling growth traits. 56

4.3.1 Mean plant height, spike length and number of spikelet spike-1 and number

of fertile tillers/plant, of 20 wheat genotypes grown in different salinities 83

4.3.2 Mean number of grains/spike, 100-gain weight/plant and grain yield/plant

of 20 wheat genotypes grown in different salinities 84

4.4.1 Mean number of tillers, plant height, spike length and number of spikelet

spike-1 of wheat genotypes grown on saline-sodic soil. 96

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4.4.2 Mean number of grains/spike, 1000-gain weight and grain yield and

biological of 20 wheat genotypes grown on saline-sodic soil. 97

List of Figures


2.1

Salt tolerance diversification of various species, shown as increases in shoot

dry matter after growth in solution or sand culture containing NaCl for at least

3 weeks, relative to plant growth in the absence of NaCl.

8

2.2 Mechanistic model of salt tolerance in cereals 17

4.1.1

Mean performance of 400 wheat genotypes scaled from lower to higher values

at 200 mM NaCl salinity: a) Na+ and K+ concentration; b) root length (RL),

shoot length (SL); c) fresh (fw) and dry weight (dw) of 3rd leaf and d)

chlorophyll content index (CCI). Round circle is overall mean of genotypes,

and square marker is mean of the check variety (Lu26S).

48

4.1.2

Salt tolerance percentage by comparison of check, LU26S and average mean

value of 400 genotypes. Tolerant (genotypes best performance than the check

variety), Moderate tolerant (genotypes between check and the average mean

value of 400 genotypes), salt sensitive (genotypes, poor performance than

mean value of 400 genotypes/check).

49

4.1.3

Comparison between two groups of wheat genotypes, Low Na+ accumulators

(square marks) and High Na+ accumulators in leaf blade (triangle mark).

K+/Na+ ratio (a) and chlorophyll index (b).

50

4.1.4


(square marks) and High Na+ accumulators in leaf blade (triangle mark). Fresh

weight of 3rd leaf (a) and dry weight of 3rd leaf (b).

51

4.1.5


(square marks) and High Na+ accumulators in leaf blade (triangle mark). Root

length (a) and shoot length (b).

52

4.2.1

A “Which is best for what” genotype by traits biplot of Na+ (reciprocal) and

K+ concertation of 42 genotypes in leaf at various salinity stress (0, 100 and

200mM). PC1 and PC2 explained total variation among genotypes. See Table

3.2.1 for codes of the genotypes

57

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4.2.2

A “Which is best for what” genotype by traits biplot of Na+ (reciprocal) and

K+ concertation of 42 genotypes in root at various salinity stress (0, 100 and


3.2.1 for codes of the genotypes.

58

4.2.3

Vector view of the genotype-by-trait biplot of showing the interrelationships

among ionic traits measured in leaf at various salinity stress (0, 100 and

200mM).PC1 and PC2 explained total variation among genotypes. See Table


59

4.2.4


among ionic traits measured in root at various salinity stress (0, 100 and

200mM).PC1 and PC2 explained total variation among genotypes. See Table

3.2.1 for codes of the genotypes

60

4.2.5

“Which is best for what” genotype by traits biplot of relative growth rate of

shoot length (RGR-SL) and relative growth rate of root length (RGR-RL) of

42 genotypes in root at various salinity stress (0, 100 and 200mM). PC1 and

PC2 explained total variation among genotypes. See Table 3.2.1 for codes of

the genotypes.

61

4.2.6

A “Which is best for what” genotype by traits biplot of growth rate of shoot

length (SL) and root length (RL)of 42 genotypes in root at various salinity

stress (0, 100 and 200mM). PC1 and PC2 explained total variation among

genotypes. See Table 3.2.1 for codes of the genotypes

62

4.2.7


among growth rate of shoot length (SL) and root length (RL) at various salinity

stress (0, 100 and 200mM).PC1 and PC2 explained total variation among

genotypes See Table 3.2.1 for codes of the genotypes.

63

4.2.8


among relative growth rate of shoot length (RGR-SL) and relative growth rate

of root length (RGR-RL) at various salinity stress (0, 100 and 200mM).PC1

and PC2 explained total variation among genotypes. See Table 3.2.1 for codes

of the genotypes.

64

4.2.9

A “Which is best for what” genotype by traits biplot of leaf photosynthetic rate

(A), transpiration rate (E) and stomatal conductance (gs) of 42 genotype

evaluated in various salinity stress (0, 100 and 200mM). PC1 and PC2

explained total variation among genotypes. See Table 3.2.1 for codes of the

genotypes.

65

4.2.10 Vector view of the genotype-by-trait biplot of showing the interrelationships

among leaf photosynthetic rate (A), transpiration rate (E) and stomatal

conductance (gs) of 42 genotype evaluated in various salinity stress (0, 100 and

66

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4.2.11

Mean fresh shoot weight (a) and fresh root weight (b) of 42 wheat genotypes

grown at three different NaCl salinities at seedling stage.

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4.3.1

Influence of salt stress on (a) Na+ concentration in leaf, (b) K+ concentration

leaf and (c) leaf K+/Na+ in leaf of wheat genotypes. S and G indicate salinity

treatments and genotypes respectively and SxG indicates the interaction. Error

bars indicate s.e (n=3).

75

4.3.2

Influence of salt stress on (a) leaf osmotic potential (-MPa), (b) leaf turgor

potential (-MPa) and (c) leaf water potential of wheat genotypes. S and G

indicate salinity treatments and genotypes respectively and SxG indicates the

interaction. Error bar indicate S.E (n=3).

76

4.3.3

Influence of salt stress on (a) leaf photosynthetic rate (An), (b) leaf

transpiration rate (E) and (c) stomatal conductance (gs) of wheat genotypes. S

and G indicate salinity treatments and genotypes respectively and SxG

indicates the interaction. Error bars indicate s.e (n=3).

77

4.3.4

Influence of salt stress on (a) leaf chlorophyll a, (b) leaf chlorophyll b and (c)

leaf total chlorophyll contents of wheat genotypes. S and G indicate salinity

treatments and genotypes respectively and SxG indicates the interaction. Error

bars indicate S.E (n=3).

78

4.3.5

Influence of salt stress on (a) leaf phenolic, (b) leaf proline and (c) leaf

carotenoid of wheat genotypes. S and G indicate salinity treatments and

genotypes respectively and SxG indicates the interaction. Error bars indicate

S.E (n=3).

79

4.3.6

Influence of salt stress on chlorophyll fluorescence of wheat genotypes. S and

G indicate salinity treatments and genotypes respectively and SxG indicates

the interaction. Error bars indicate S.E (n=3).

80

4.3.7

Influence of salt stress on (a) relative water content, (b) cell membrane injury

of wheat genotypes. S and G indicate salinity treatments and genotypes

respectively and SxG indicates the interaction. Error bars indicate S.E (n=3).

81

4.3.8

Stress susceptibility index (SSI) based on grain yield of different wheat

genotypes at high salinity level in pot culture. Grey bars and white bars

indicates salt tolerant genotypes and salt sensitive genotypes respectively,

selected from Experiment 1 and 2 while black bars indicates check genotypes.

Error bars indicate S.E (n=3).

82

xv

4.4.1

Leaf Na+ concentration (a), leaf K+ concentration (b), leaf K+ use efficiency

(c) and leaf K+/Na+ ratio (d) of wheat genotypes grown on salt affected soil.

Grey bars and white bars indicates salt tolerant genotypes and salt sensitive

genotypes respect, selected from Experiment 1 and 2 while black bars indicates

check genotypes. Error bars indicate S.E (n=4).

92

4.4.2

Leaf chlorophyll a (a) leaf chlorophyll b (b) and leaf total chlorophyll contents

(c) of wheat genotypes grown on salt affected soil. Grey bars and white bars

indicates salt tolerant genotypes and salt sensitive genotypes respectively,

selected from Experiment 1 and 2 while black bars indicates check genotypes.


93

4.4.3

Leaf phenolic (a), Leaf proline (b) and Leaf carotenoid (c) of wheat genotypes

grown on salt affected soil. Grey bars and white bars indicates salt tolerant

genotypes and salt sensitive genotypes respectively, selected from Experiment

1 and 2 while black bars indicates check genotypes. Error bars indicate S.E

(n=4).

94

4.4.4

Percentage of crop density of wheat genotypes grown on salt affected soil.

Grey bars and white bars indicates salt tolerant genotypes and salt sensitive

genotypes respectively, selected from Experiment 1 and 2 while black bars

indicates check genotypes. Error bars indicate S.E (n=4).

Percentage crop density (m-2): 1 = 90 % Emergence or more (very good); 2 =

80–89 % (good); 3=70–79 % (acceptable), 4 = 60–69 % (poor)

95

4.4.5

Yield relating attributes plant height (PH), grain yield (GY), no of grain spike-

1 (NOG), Biological yield (BY), 1000 grain weight (THGW) and spike length

(SL) grown on salt effected soil. See Table 3.4.1 for codes of the genotypes

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4.4.6

Vector view showed relationship among yield relating attributes of plant height

(PH), grain yield (GY), no of grain spike-1 (NOG), Biological yield (BY),

1000 grain weight (THGW) and spike length (SL) grown on salt effected soil.

See Table 3.4.1 for codes of the genotypes

99

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Abbreviation Full

% Percent

cm Centimetre (s)

CAT Catalase

CGR Crop growth rate

d Day (s)

DAS Days after sowing

g Gram (s)

g m-2 Gram per square meter

ha-1 Per hectare

K Potassium

kg Kilogram

kg ha-1 Kilogram per hectare

m meter

m-2 Per square meter

ml Millilitre

mm Millimetre

mM Milli Molar

MPa Mega Pascal

M ha Million Hectare

MINFAL, Pakistan Ministry of Food, Agriculture and Livestock, Pakistan

Na Sodium

POD Peroxidase

ROS Reactive oxygen species

SOD Superoxide dismutase

SSRI Soil Salinity Research Institute

UAF University of Agriculture Faisalabad

https://www.google.com.pk/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&sqi=2&ved=0ahUKEwi905XV4I3SAhXBlxoKHUAuDXAQFggjMAI&url=http%3A%2F%2Fwww.acronymfinder.com%2FMinistry-of-Food%2C-Agriculture-and-Livestock-(Pakistan)-(MINFAL).html&usg=AFQjCNG-eVL2DH7_TpAOzqzOA8xj2qt4pw&bvm=bv.146786187,d.d24

xvii

Abstract

Salt stress is one of the major leading threat which affects growth and development of wheat

plant as salinization of cultivated land is increasing globally. In order to return from stressful

environment it is urged to use such strategies through which maximum crop stand could be

achieved under saline conditions. Therefore, the proposed study aims to identify novel germplasm

in exotic cereal landraces with high salt tolerance by using different approaches. First study

elucidates the identification of novel salt tolerant germplasm from very large diverse pool (four

hundred accessions of different origin) at 200 mM NaCl using fast and efficient

physiologically-based screens in hydroponic culture. Forty genotypes (25 salt-tolerant and 15

salt-sensitive genotypes) out of 400 were selected on the basis of Na+ exclusion in leaf blade.

Genotypes that accumulated low Na+ in their leaves had also more K+/Na+ ratios, leaf

chlorophyll content index and leaf dry mass as compared to salt sensitive genotypes. Selected

wheat lines from hydroponic experiment were further evaluated at different salinity levels (0,

100, 200 mM NaCl) hydroponically. GGE biplot analysis indicates that genotypes TURACO,

V-03094, V0005, V-04178, Kharchia 65 and V-05121 were the most salt-tolerant and declared

winners as depicted by improved gas exchange relations such as photosynthesis rate (A),

stomatal conductance (gs) and transpiration rate (E) and growth rate which was highly linked

with proper Na+, K+ discrimination in leaf and root zones. Genotypes PBW343*2, NING MAI

50, PGO, PFAU, V-04181, PUNJAB 85, KIRITATI, TAM200/TUI and TAM200 were poor

performer due to higher Na+ accumulation in leaf and root ultimately retarded growth. After

smart secerned from hydroponic studies, fourteen salt tolerant, four salt sensitive and two check

LU26S, Kharchia65 were further tested in pots and saline sodic field to explore physiological

mechanisms of salt tolerance in selected genotypes. Among low Na+ accumulators, V-03094,

V0005, V-04178, and V-05121 genotypes gave maximum seed yield in saline soil which were

highly linked with higher K+ accumulation and better biochemical and gas exchange attributes.

After very smart selection from hydroponic, pot and field studies it concluded that V-02156,

V-03094, V0005, TURACO, PVN identified as the best Na+ excluders genotypes, had better

performance with improved physiological and yield attributes in salt stress which can be used

in breeding programs to introduce the low Na+ trait in commercial hexaploide wheat cultivars.

- 1 -

Chapter 1

INTRODUCTION

The world human population will reach to 8.0 billion in 2025 (FAO, 2010). It is

projected that till 2025, there is need to increase world food production in order to feed these

people. Due to abiotic stresses such as drought, salinity, heat, chilling and other factor; the

estimated potential yield loses are 17%, 20%, 40%, 15% and 8% respectively (Ashraf and

Harris, 2005). Unfortunately, the damaging effects of these stresses on crop yield are

increasing due to anthropogenic contributions thus threatening global food security (Savvides

et al., 2016). Among abiotic stresses, salinity is the major obstacle for good crop production.

Salts occur naturally in water and land and salinization arises, when amount of salt exceeds

becomes unsuitable for production, environmental and aesthetic needs. Primary salinity refers

to weathering of natural materials, while secondary salinity may occur due to anthropogenic

activities. In low rainfall arid and semi-arid areas improper irrigation practices are main

sources of secondary salinization (Aslam and Prathapar, 2006). All over the world, 6% area is

salt affected, which is equal to 800 million hectares area of total globe (FAO, 2008). Most

serious risk due to salt stress are occurring in arid and semi-arid area including million-hectare

salt affected soils of Pakistan (10 Mha), Iran (23.8 Mha), Egypt (8.7 Mha) and Argentina (33.1

Mha) (FAO, 2008).

Salinity damages the soil beyond economic repair (Munns et al., 2006) by ion

imbalance, ion toxicity and production of reactive oxygen species (ROS) (Munns and Tester,

2008). Negative gradient of saline soil solution limits availability of water from soil that leads

cell plasmolysis, stomatal closure (Passioura and Munns, 2002; Munns and Tester, 2008),

tissue chlorosis, necrosis and premature senescence of older leaves (Munns, 2002; Tester and

Davenport, 2003; Munns et al., 2006). In general, cereal plants are very sensitive in both

vegetative and reproductive stages of development under salt stress, which result the stunted

growth and development of entire wheat plant and ultimately significant loss in grain yield

(Husain et al., 2003; Munns et al., 2006). Flowering time is another key factor that determines

yield of wheat in salt affected soils and thus early flowering wheat genotypes have more

advantages in saline fields (Setter et al., 2016).

- 2 -

In field, salinity affects germination (Soltania et al., 2006; Bhutta and Hanif, 2010),

productive tillers count, dry weight and fresh weight of roots and shoots and arrest nutrient

uptake (Afzal et al., 2006). Salt stress reduces wheat phenology such as leaf expansion and

number of leaves (El-Hendawy et al., 2005; Zheng et al., 2010), root and shoot growth rates

and root to shoot ratio, total dry weight and grain yield (El-Hendawy et al., 2005; Ruan et al.,

2008).

There are two main approaches to mitigate the salinity stress. One is short-term

approach which includes seed priming, coating, pelleting and exogenous application of

different organic, inorganic compound or plant growth hormones (Munns and Tester, 2008;

Ashraf and Akram, 2009). While the long term and permanent strategy is to develop and

introduce the salt tolerant germplasm in breeding program by genetic engineering (Ruan et al.,

2010).

Plants shielded from salinity can keep stomatal conductance and expansion of leaf (Hu

et al., 2007) by osmotic adjustment with compatible solutes such as glycine betain, K+

accumulation and proline to keep cell turgidity. Onset of salinity plant upgrades tissue

tolerance by Na+ exclusion from tissues (Tester and Davenport, 2003; Munns and Tester, 2008)

and compartmentalization in vacuole (Pardo et al., 2006; Munns and Tester, 2008) to avoid

Na+ toxicity. To keep the low Na+ in mesophyll cell of leaf by its effective Na+ exclusion

transporter is therefore an important character for salt tolerance in cereals such as durum and

bread wheat (Munns and James, 2003; Cuin et al., 2009; 2010). Bread wheat is more tolerate

than durum wheat due its efficient and effective Na+ exclusion (Munns and James, 2003).

Wheat is a source of staple food and important cereal crop for many countries. Salt

stress induces inhibitory effect on wheat growth and development by altered the biochemical

and physiological processes (Munns et al., 2006; Arslan and Ashraf, 2010). About 10 %

cultivated area of wheat is significantly affected by salt in South Asia (Mujeeb-Kazi and Leon,

2002). This is due to the limiting canal irrigation water and the fact that people are using tube

well water which resulted in reduced yield potential of wheat crop due to accumulation of

soluble salt in soil. Total cultivated area of wheat in Pakistan is 21 Mha, in which 32% is salt

affected. Quayyum and Malik (1988) reported about on average 65% yield loses of wheat on

moderately salt affected lands in the country.

- 3 -

In plant breeding program to develop the salt tolerant genotypes, the genetic diversity

is a prerequisite tool. Currently plant scientists are attempting to identify suitable physiological

modulations helpful in salt tolerance in available wheat germplasm (Sairam et al., 2002; Munns

et al., 2016). Low rate Na+ transport and high uptake of K+ selectivity in wheat is linked with

salt tolerance (Munns et al., 2006). Low influx of Na+ and enhanced K+ to Na+ discrimination

in bread wheat is controlled by Knal located on 4D chromosome (Dubcovsky et al., 1996).

Correlation between Na+ exclusion and grain yield has been shown in bread wheat which

linked enhanced K+ to Na+ discrimination (Munns et al., 2006).

Physiological attributes such as chlorophyll content, efflux of Na+, influx of K+, and

K+ to Na+ ratio were found very important parameters to screen the salt tolerant germplasm in

bread wheat (Munns et al., 2016). Flowers et al. (1995) reported crop sensitivity or failure

against salt stress due to low efflux of Na+ and Cl- from transpiration stream (Hollington,

1998). High concentration of NaCl inhibited growth and reduced the photosynthetic pigments

(Chlorophyll a and b), relative water content, osmotic potential and K+ to Na+ ratio in wheat

seedling (Sairam et al., 2004; James et al., 2008). Na+ exclusion or efflux of Na+ from shoot is

a reliable trait to develop the salt tolerance in cereals e.g. wheat germplasm (Poustini and

Siosemardeh, 2004; Din et al., 2008). Munns et al. (2006) reported that wheat genotypes,

which accumulated the low Na+ in their leaves, produced more dry mass as compared to high-

accumulated genotypes.

Aims and objectives:

The aim of this study was to identify novel wheat germplasm in exotic and local

landraces with high salt tolerance, using fast and efficient physiologically based screens.

Specifically, the research described in this thesis explored the physiological basis of sodium

exclusion in the bread wheat population. The objectives include:

Screen the salt-tolerant and salt-sensitive wheat germplasm

Study the physiological mechanisms of salt tolerance

Develop better physiological markers of salt tolerance for wheat germplasm

- 4 -

Chapter 2

REVIEW OF LITERATURE

Human population reach to 8.0 billion in 2025 and there is need to increase double

production of world food in order to feed these people (FAO, 2010). It is predicted in future

may be 800 million hectares will be affected by salinity stress, which is a major threat to future

food security (Waisel, 2001; Mckee et al., 2004). All over the world land degradation occur

gradually; in which soil salinity is a major contributing factor, about 7% of world land surface

is affected by salt stress, whereas sodium affected soil is more widespread (Panta, et al., 2014).

Land clearing for urbanization and industrialization in those saline regions aggravates the

problem farther. Moreover, continuous utilization of saline underground water in irrigation

causes globally deforestation and salt-salinization of previously productive land (Rengasamy,

2006). The estimation of potential yield loss by salinity is 20% (Ashraf and Harris, 2005).

Mainly salt stress occurs in semi-arid and arid areas where the annual precipitation exceeds

due to evapotranspiration, whereas irrigation with salt affected water disturb sustainable crop

production (Rengasamy, 2010). Increasing salinity tolerance in crops is one of the most

sustainable approaches to ensure food security in those affected areas (Blumwald et al., 2004;

Yilmaz et al., 2004; Iqbal et al., 2007).

2.1 Salinity scenario in the world

Increasing the concentration of soluble salt in water or soil caused the salt stress.

Exceed salinity progression due to weathering of rocks (primary salinization) or anthropogenic

activities (secondary salinization, Panta et al., 2014). Barrett- Lennard (2002) reported that

there are following factors which cause secondary salinization; by use brackish ground water

for irrigation which is salt contaminant, moreover cutting the deep rooted forest for pastures

and crop production. It is estimated that by increasing the salinization about on an average 100

million hectares land is converted into salt affected land, which is approximately 11% irrigated

area of total world (FAO, 2012). Expansion of salt affected land is very most alarming for

developing and dared countries i.e. 6.8 million hectares land is salt affected in Pakistan

(Ghafoor et al., 2004; Qureshi et al., 2008), 7 million hectares in India (Vashev et al., 2010)

http://jxb.oxfordjournals.org/content/62/8/2939.full#ref-36

http://jxb.oxfordjournals.org/content/62/8/2939.full#ref-36

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and 1 million hectares in Bangladesh (Hossain, 2010) which is severe threat to sustainably crop

production

2.2 Salinity spread in Pakistan

Pakistan is located between 30.37° N, 69.34° E on the south from Himalayan

Mountains. Salinity stress has been well known as major risk for sustainable agriculture and

yield production in Pakistan. Due to salt stress, about 25% yield reduction has been reported

in major crop (Kahlown and Azam, 2002). Near about 1.4 million hectares agriculture land is

affected due to salt stress/sodicity problems (World Bank, 2006). Salinity stress mostly occurs

in semi-arid and arid area of all province. While coastal areas of Baluchistan and Sindh

provinces are also salt affected (Schleiff, 2003).

In Pakistan, agriculture sector is totally depend on IBIS (Indus Basin Irrigation

System). Indus Basin Irrigation System adds more than 90% to GDP (agricultural gross

domestic product) of the country. Improper drainage system of IBIS caused salinity and

waterlogging problems. Lot of country revenue had been spent for the improvement of

drainage system but failed to achieve the success due to absence of proper maintenance and

operation as well as non- existence linkages between secondary or tertiary and main drains.

Which result shortage of canal water, that made force the famer to use the brackish water of

tube well, which causing sodification or secondary salinization (Murtaza et al., 2009; Sumia

and Shahid, 2009). Status of salt affected area in Pakistan is given in table 2.1.

Table 2.1 Total geographical, cultivated and salt affected area of Pakistan (M ha)

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2.3 Salinity stress effect, salt tolerance mechanism and wheat production

Salinity stress is a leading threats to sustainable agriculture (Waisel, 2001), which is

causing decline in growth, yield and production of agriculture crops and land (Tester and

Devenport, 2003; Munns and Tester, 2008). Soil damage by salinity stress is a big loss and not

possible to repair or fill this gap (Munns et al., 2006; Ashraf, 2009). Salinity causes very severe

effect on plants such as ion balance, ion toxicity and production of ROS (reactive oxygen

species). The fast and rapid effect of salinity that observed is osmotic stress, which resulted in

closed stomata, impaired cell expansion and cell division (Passioura and Munns, 2000; Munns,

2002; Munns and Tester, 2008). Accumulation of ion concentration in leaf causes ion toxicity,

which results in necrosis, chlorosis and also cause senesce of older leaves (Tester and

Davenport, 2003; Munns, 2006).

Plants have three main mechanisms to cope the salinity stress. First one is osmotic

tolerance, plants have ability to tolerate the salt stress by maintaining osmotic adjustment and

production of osmolytes (Hu et al., 2007). The other two mechanisms of salinity tolerance,

plant have capability to mitigate ionic effect by diminishing the amount of Na+ that accumulate

in the cytosol cell or in the transpiring leaves. The first mechanism indicate the exclusion of

Na+ from leaf blade while second shows the tissue tolerance/compartmentalization of salt

inside vacuole or in older leave where the damage is minimum (Pardo et al., 2006; Munns and

Tester, 2008).

Wheat is third main cereal in the world and staple crop of Pakistan. Around the globe,

it provides 20% calories of human food. In developing countries human quality of life directly

depends on wheat production and productivity. The actual yield of wheat in South Asia

including Pakistan is less than the potential one. Sometimes, the yield gap between actual and

potential yield may reach up to 60% which is quite alarming and need serious attention of

scientists and the policy makers. Reasons for this wide yield gap includes biotic stresses (pests

and diseases) and abiotic e.g. heat, drought and salinity stresses. Among abiotic stresses e.g.

drought, salinity, heat stress and cold stress, salt stress is the major threat for good agricultural

crop production (Ashraf and Harris, 2005).

About 10% cultivated area of wheat is salt affected in Pakistan, India, Iran, Mexico,

Egypt according to research data of CIMMYT (Mujeeb-Kazi and Diaz de Leon, 2002). In

Pakistan’s moderately salt affected area the average loses of yield is 65% (Quayyum and

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Malik, 1988). Average yield loss of wheat on moderately saline area of Pakistan is 65%

(Quayyum and Malik, 1988). There are twenty plant species including wheat that play a very

key role in human’s nutrition throughout world. Pakistan is included in top ten world wheat

producing countries with 10.1% contribution and added 22.2% to GDP in agriculture. During

2012 and 2013 the wheat production remained at 24.2 million tones but increase world

population in very swift pace. It is main alarming threats for present and future food supply

due to reduction in agriculture land for agriculture production (Allakhverdiev et al., 2000).

However, its production is seriously affected due to salt stress (Yıldız and Terzi, 2008; Ashraf

et al., 2010; Mehta et al., 2010). In field, rice will die, where level of salt stress may goes to

10 dS m-1, while wheat tolerated this salinity level and produced less yield (Munns et al., 2006).

2.4 Effects of salt stress on wheat physiology and growth

Salinity enforces the sound effects on wheat’ growth in the form of changed

biochemical and physiological processes (Munns et al., 2006; Arslan and Ashraf, 2010).

Physiological attributes such as efflux of Na+ and influx of K+, K+ to Na+ ratio and chlorophyll

contents were found important parameters to screen the salt tolerant germplasms in bread

wheat (Munns et al., 2016).

Plants grown on salt affected soil, show stunted growth due salt induced osmotic effect,

ion toxicity and oxidative burst due to production of ROS and alteration in level of endogenous

hormones (Ashraf, 2004; Ashraf and Foolad, 2007). Flowers et al. (1995) and Hollington,

(1998) reported that sensitivity of crops to salt stress is due to failure of salt exclude (Na+ and

Cl-) from transpiration stream (Hollington, 1998). NaCl reduced relative water content,

osmotic potential and K+ to Na+ ratio, (James et al., 2008) and chlorophyll pigments i.e.

carotenoids and Chl a and b in seedling of wheat crop (Sairam et al., 2004).

Under saline environment water potential and osmotic potential become more negative,

while tugar potential increased in contrast to water and osmotic potential under salinity stress

(Romeroaranda et al., 2001; Khan, 2001; Meloni et al., 2001). By increasing the salt stress in

medium the xylem tention and leaf osmotic potential increase in Rhizopora (Aziz and Khan,

2001). High leaf Na+ concentration reduced Rubisco activity, stomata conductance

intercellular CO2 concentration, sucrose accumulation and chlorophyll contents (Mittler, 2002;

Candan and Tarhan, 2003; Vaidyanathan et al., 2003) that resulted mark reduction in net

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photosynthetic pigments, transpiration rate, leaf water potential (Arslan and Ashraf 2012) and

carbon assimilation (Husain et al., 2003) in wheat at the reproductive phase.

According to USDA-ARS, (2005) grain yield of wheat started to drop when level of

salt stress reaches to 60-80 mM. However among the genotypes, salt tolerance my also occur

at various growth stages (Zheng et al., 2010; Ali et al., 2008; Munns et al., 2006).

Fig 2.1 Salt tolerance diversification of various species, shown as increases in shoot dry matter

after growth in solution or sand culture containing NaCl for at least 3 weeks, relative to plant

growth in the absence of NaCl (Munns and Tester, 2008).

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The earliest response of salinity is the reduction of leaf surface expansion rate leading

to stop expansion due to increased concentration of salt (Wang and Nil, 2000). Extensive

decrease was found in dry and fresh weight of leaves, stem and root under salinity stress

(Chartzoulakis and Klapaki, 2000). Cereals plants are most sensitive at vegetative and

reproductive stages to salt stress which result the impaired growth and development of whole

plants and ultimately loss in grain yield (Husain et al., 2003; Munns et al., 2006). Furthermore,

flowering and grain filling stage is less affected to salinity (Mass and Poss, 1989). In field

salinity affects germination percentage (Soltania et al., 2006; Bhutta and Hanif, 2010),

productive tillers count, dry weight and fresh weight of roots and shoots and arrest nutrient

uptake (Afzal et al., 2006). Salt stress has inhibitory effects on wheat phenology such as leaf

expansion and number of leaves (El-Hendawy et al., 2005; Zheng et al., 2010) root to shoot

ratio, shoot growth rate and root growth rate (El-Hendawy et al., 2005), total grain yield and

dry weight (Ruan et al., 2008). Flowering time is another key factor that determines yield of

wheat in salt affected soils and thus early flowering wheat genotypes have more advantages in

saline fields (Setter et al., 2016).

Table 2.2 Salinity consequences on wheat phenology

Stage Number of

Days

Events Hazards Effect

Germination 7-10 From sowing to

first leaf

Salinity + soil

crust

Weak root

development low

germination

Tillering-crown

root initiation

15-20 From first leaf

to third leaf

Salinity+++

water stress+++

Weak roots and

shoot growth

Jointing 30-35 1 cm long spike

and end of

tillering

Lower density

Booting/heading 15-20 From plant

growing up to

fecundation

Water stress+++ Spike abortion

Flowering 10-15 From flower

apparition to

grain growth

Flower fading

Grain filling 15-20 Soft juicy grain Grain abortion

Dough ripe 10-15 Tough grain

Total 102-135 Harvest

Source; Pintus, 1997

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Tolerance scoring against salinity related to physiological behavior of wheat plants like

leaf chlorophyll contents as well as high K+ to Na+ is the indication of plant preferring K+

accumulation instead of Na+ (Din et al., 2008). Hollington (1998) found that sensitive plants

to salinity was due to poor Na+ exclusion from transpiration stream. Salinity stress reduced K+

to Na+

ratio, osmotic potential, relative water content and photosynthetic or chlorophyll

pigments i.e. carotenoids and Chl a and b in seedling of wheat crop (Sairam et al., 2004;

Akbarimoghaddam et al., 2011).

2.5 Ionic and oxidative consequences of salinity

Normally, wheat and other cereal plants grow slowly and die swiftly when exposed to

salt conditions. Under moderate salinity levels the osmotic effect is more pronounced than

ionic stress, which affect at later stage on crop growth rate (Munns and Tester, 2008).

2.5.1 Osmotic effect

In soil solution, accumulation of salt make difficult for plant to absorb water and the

nutrient for growth. Hence decrease water potential, turgor potential and solute potential results

in cell dehydration and ultimately cell dies (Munns, 2002). In wheat, against salinity two

responses occur consequently i.e. osmotic and ionic effects. In the first phase under salt stress

the plants have face the most rapid effect is osmotic stress, in which plants unable to get water

from the soil due to more negative water potential of soil, which is also called physiological

drought. In this phase when Na+ and Cl ions reach in xylem, these accommodated in growing

tissue e.g. vacuoles. These ions neither accumulate in excess amount that inhibit the growth

rate. Salt are excluded affectively in the case of meristematic tissues, which are commonly

served in phloem (Munns, 2002; Munns et al., 2006). In cereals particularly wheat, rice and

barley, the main phenotyping effect of osmotic stress is a reduction in total leaf area and

number of tiller (Munns et al., 2006).

Particularly interesting is the greater sensitivity of shoot growth to salt as compared to

roots, which are primarily exposed to the saline soil (Munns, 2002). In addition to reduction in

leaf growth and shoot development by osmotic stress, the reproductive development like

reduced the number of flowers and early flowering have been affected significantly under salt

stress (Munns and Tester, 2008). Osmotic stress also affect stomatal conductance by reducing

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water uptake which results in reduction of CO2 assimilation and ultimately detrimental to crop

yield.

2.5.2 Ion-specific effect:

In the 2nd phase, the wheat plant is mostly affected by accumulation of excess amount

of Na+ and Cl- ions inside the plant, resulting leaves senescence or leaf die because of gradual

decrease in enzyme activity. This injury may be caused by accumulation of Na+ in cytoplasm

and which result the ion toxicity of salts. Alternatively, they cause dehydration if accumulated

near the cell wall. The death proportion of leaves is important for plant survival. If growth rate

of new emerging leaves is slower than the death rate of leaves, the plant are not able to

produced seed yield due to reduction in photosynthetic efficiency of plant and thus result the

stunted growth rate (Munns, 2002; 2005). In comparison to Cl-, Na+ ion is most responsible

for damage caused to wheat and other cereal plants (Munns and Tester, 2008). Excess of CI-

and Na+ concentrations in the root zone inhibit the uptake of K+ and its deficiency ultimately

results in necrosis and chlorosis (Gopa and Dube, 2003). Potassium (K+) plays an important

role in synthesis of protein, cell membrane integrity and osmoregulation, K+ plays a vital role

(Wenxue et al., 2003), fixing cell turgor and stimulates the rate of photosynthesis (Ashraf et

al., 2011). The inhibitory effects of salinity on nutrient composition showed that increase in

Na+ accumulation would decrease the K+ content and K+ to Na+ ratio in shoot and roots of

wheat (Akbarimoghaddam et al., 2011). Salinity stress expressively increased the endogenous

levels of Na+, CI- and decreased Ca 2+, K+ cations and their Ca2+ to Na+ and K+ to Na+ ratios in

wheat genotypes at various growth phenology (Arslan and Ashraf, 2012).

2.5.3 Oxidative burst

When a plant faced a stressful condition, production of ROS (reactive oxygen species)

overcome quenching system and resulted oxidative burst of cell integral structure. ROS are the

main source of damaging the structure of macromolecules under abiotic and biotic stresses

(Candan and Tarhan, 2003; Vaidyanathan et al., 2003). ROS are reduced form oxygen (O2),

which is produced in vital processes of photorespiration, respiration and photosynthesis

(Mittler, 2002; Munns et al., 2006). Four electrons in these processes for complete reduction

of oxygen but reactive oxygen species result from transference of one or two and three electron,

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to O2 to form O2·- (superoxide), H2O2 (hydrogen peroxide) and HO· (hydroxyl radical; Mittler,

2002). ROS are very vital and highly reactive with biomolecules such as DNA, protein, lipids

and causing protein denaturing, lipid peroxidation and DNA mutation respectively. (Quiles

and Lopez, 2004) and disturbed the normal metabolic pathways (Sakihama et al., 2002). It has

been reported that NaCl damage the permeability of plasma membrane (Candan and Tarhan,

2003) because with unsaturated fatty acid the ROS can cause lipid peroxidation of plasma

membrane (Karabal et al., 2003). During salinity stress, somatically stressed plants reduced

CO2 assimilation due to closing of stomatal pores which generate ROS in the plant leaves (Roy

et al., 2012).

2.6 Salt tolerance

Salt tolerance enabled plant to adopt saline environment by avoiding high ion

concentrations or make the cells capable to perform normal function with high concentrations

of ion (Greenway and Munns, 1980). Levitt (1980) characterized these resistant mechanisms

as tolerance and avoidance. Plants have different mechanisms to survive with the toxic effects

of higher salt concentration by antioxidant system, osmotic tolerance and Na+ exclusion

(Hajlaouia et al., 2010). For example delayed maturity or germination until the favorable and

appropriate conditions prevails; salt exclusion at the root zone, salt compartmentalization into

vacuole and secretion through specialized organs such as salt hair/salt gland or stored in older

leaves where less damage is occur (Hasegawa et al., 2000). As both halophytes and

glycophytes cannot tolerate excess amount of salts in their cytoplasm (Zhu, 2003; Kumar et

al., 2005). In contrast, some salt tolerant halophytes and glycophytes have capability to

accumulate excessive amount of Na+ in their shoot and known as Na+ accumulators (includers)

species (Collander, 1941).

2.6.1 Osmotic adjustment

Immediate response of osmotic stress is to reduce germination potential and then the

growth rate of roots and shoots. The reduction in leaves development is due to salts

accumulation surrounding the roots, which relates to the osmotic effect. Generally, tolerant

wheat plants to osmotic effect exhibit higher stomatal conductance and leaf growth to fix CO2

efficiently (Roy et al., 2012). It is evident that tolerance to osmotic stress is associated to the

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ability of plants to continue production of new leaves (Munns and Tester, 2008). Measurements

for osmotic tolerance has been considered as time consuming and frequent destructive

sampling is required to estimate the growth rates of plants. However, non-destructive imaging

technologies or infrared thermography have been now utilized to measure plant biomass, leaf

temperature and stomatal conductance of plants in saline environment (Rajendran et al., 2009).

Osmotic adjustment has a potential defense against salt stress (Neocleous and

Vasilakakis, 2007; Hajlaouia et al., 2010) and necessary to maintain the uptake of water from

salt affected soil (Ottow et al., 2005). Plant age, organ type and intensity of stress rate also

effect the degree of OA (Alves and Setter, 2004). The rate of active osmotic adjustment can be

developed only by increasing the concentration of solute (Silveira et al., 2009).

2.6.2 ROS detoxification

Plant response against salinity stimulus is a multigenic trait, tolerant plants switch on

antioxidant defense system to cope with ROS and salinity (Hameed et al., 2008). Plants have

efficient defensive systems for quenching ROS that keep them from destructive oxidative

reactions by alterations in the protein profiles (Yıldız and Terzi, 2008) either increase or

decrease the level of soluble proteins by new synthesis of protein and complete damage of

present proteins in wheat (Yıldız and Terzi, 2008).

Transcribed of stress response, antioxidant enzymes are key element to defense

mechanism systems. These enzymes as CAT (catalase), GR (glutathione reductase), SOD

(superoxide dismutase) and GST (glutathione-S-transferase) and SOD (Superoxide dismutase)

have been listed by Garratt et al. (2002) subsequently. Ascorbate peroxidase, CAT and a

variety of peroxidases catalyze the subsequent breakdown of H2O2 into oxygen and water

(Garratt et al., 2002). Hamid et al., (2011) unlocked the antioxidant defense mechanism in

wheat as increased the level of salt stress, the significant change was found in activity of

antioxidant enzymes. In plant leaves GPX (glutathione peroxidase) decreased and CAT, and

APX (ascorbate peroxidase) increased with salinity. Phenolic contents in leaf are important

protective components (Parida et al., 2004) and prompt tolerance in wheat against salinity

(Arslan and Ashraf, 2010).

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2.6.3 Ion regulation and compartmentalization

Under salinity stress, excessive concentration of salt disturb and change the ion

homeostasis of plant cell, therefore ion compartmentalization and ion uptake is very critical

for normal plant growth under stress (Adams et al., 1992). In salinity stress both glycophytes

and halophytes cannot tolerate the excessive concentration of salt in their cytosol, so plants

have two main mechanism either they may exclude the salt through their salt glands or by

dumping in vacuole or various part of tissue such as older leaves to ensure the normal cell’s

metabolic activities (Reddy et al., 1992; Iyengar and Reddy, 1996; Zhu, 2003). Glycophytes

are more sensitive than halophytes so they limit the uptake of salt or confine in older tissue act

as storage compartment (Cheeseman, 1988).

Salt inducible enzyme Na+/H+ antiporter are located on tonoplast membrane of vacuole,

which is responsible to exclude the Na+ ion from cytosol or dump into the vacuole (Apse et

al., 1999). At the vacuolar membrane two pump; H+-ATPase (V-ATPase) and vacuolar

pyrophosphate (V-PPase), which help for exclusion of salt from cytoplasm (Dietz et al., 2001).

Under stress environments such as drought, salinity, cold and heavy metal stress, survival of

plant cell, expression and regulation activity of genes depend on V-ATPase activity on long or

short term bases (Dietz et al., 2001).

2.6.4 Na+ Exclusion

Munns (2002) explained the tolerance mechanism in plants on molecular basis. In

wheat salinity tolerance is owing by Na+ efflux from leaves (Husain et al., 2003). Durum wheat

are less salt tolerant than bread wheat due to have poor K+/Na+ discrimination and higher

accumulation of Na+ (Munns et al., 2000). Loci Nax1 and Nax2 located in chromosomes 2A

and 5A respectively controlling Na+ influx, has been found in bread wheat genotype (Lindsay

et al., 2004). This molecular marker is being used in wheat breeding program to develop the

low Na+ character wheat germplasm. In salt tolerant wheat and other cereals maintained high

K+ to Na+ ratio by slower Na+ transport than K+ or exclude Na+ from fresh tissues by partition

Na+ in older leaves that serve as storage compartment (Poustini and Siosemardeh, 2004;

Garthwaite et al., 2005). Potassium plays a vital role in membrane potential, turgor

maintainers, regulation osmotic potential, enzyme activation, stomata movement and tropisms

(DeVries and Toenniessen, 2004). Munns et al. (2006) reported that low Na+ wheat genotypes

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produced more dry matter as compared to high Na+ genotypes. Apse et al. (1999) reported that

compartmentalization in the vacuoles or exclusion of Na+ from the cytoplasm is done by a salt-

inducible antiporter (Na+/H+).

Tissue tolerance is a complex trait that cannot be separable from osmotic adjustment

(OA) where many factors contribute Na+ and Cl- sequestration in vacuoles and maintenance of

low Na+ and Cl- concentrations in the cytoplasm, which helps in functioning of tissues (Munns

et al., 2016). Thus, OA is an active process and must be differentiated from a passive increase

in solute concentration due to loss of water under drought or salt conditions (Brian et al., 1999).

In some salt sensitive species i.e. durum wheat, large portion of OA occurs with organic solutes

which is energy expensive process to utilize assimilates for tolerance rather than growth

process and therefore low yield has been observed in saline soil which paid energy cost for

lowered Na+ concentration in the leaf (Munns and Gilliham, 2015). Similar concentration of

Na+ was reported in the mesophyll and epidermis of wheat and barley; however, K+

accumulation was more in the mesophyll, which favored higher K+ to Na+ ratio in these cells

(James et al., 2006). Wheat and barley also considered as natural accumulators of compatible

solutes like proline and glycine betaine, like other species in the Poaceae (Arslan and Ashraf,

2012). Inorganic solutes such as Na+ and K+ (cations) and the Cl- (anion) make a significant

contribution to turgor maintenance and osmotic adjustment in wheat (Bayuelo-Jimenez et al.,

2003; Peng et al., 2004).

2.7 Molecular basis of salt tolerance in wheat

A QTL (Quantitative trait loci) are defined as genetic loci where various alleles

segregate functionally and cause vital effect on a quantitative trait (Salvi and Tuberosa, 2005).

Chromosome region that contains a gene of quantitative traits can identified through advanced

statistical and DNA marker selection methods (Flowers, 2004; Collard et al., 2005). Lindsay

et al. (2004) found that a locus (Nax1) that controlling the Na+ accumulation are located on 2A

chromosome in durum wheat. The research have mapped genes on chromosome 5B and 5D

for salt tolerance using QTL in bread wheat (Quarrie et al., 2005). Knal genetic locus for

controlling the accumulation of K+ and Na+ influx in shoot and was located on 4D chromosome

(Dubcovsky et al., 1996). Na+ exclusion genes Nax1 and Nax2 were mapped on 2A and 5A

chromosome respectively using in QTL and SSR marker selection technique. The SSR markers

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are closely linked to Na+ exclusion gene. MAS (marker assisted selection) can be used to

develop the salt tolerance line in hexaploid and tetraploid wheat by Na+ exclusion gene (James

et al., 2006).

Table 2.3 Genes/QTLs involved in salt tolerance mechanisms in wheat in different species of

wheat

Genes/QTL Function Species References

Kna1 Na+ and K+ accumulation T. aestivum Dubcovsky et al., (1996)

TaSnRK2.8 NaCl and cold stresses T. aestivum Zheng et al., (2010)

TVP1 H+ transport across vacuole T. aestivum Brini et al., (2005)

TaST CDPK pathway T. aestivum Huang et al., (2012)

TaSOS1 Na+ detoxification T. aestivum Xue et al., (2004)

Nax1 Na+/+H anitiporter at plasma

membrane

T.

monococcum

James et al., (2006)

Nax2 Na+ exclusion at tonoplast T. durum Lindsay et al. (2004)

Q.K1D,Q.K3B, Q.K3D Shoot K+ concentration T. aestivum Genc et al., (2010)

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Mechanistic model of salt tolerance in cereals

Genes and transcriptomes which regulates ionic homeostasis under salinity in different wheat

species are summarized in Table 2.3 and illustrated in Fig 2.2

Fig 2.2 In cereals, high-affinity K+ transporter1;5 (HKT1;5) facilitates Na+ exclusion

from root xylem vessels to reduce shoot accumulation, whereas HKT1;4 partitions

Na+ from the root xylem stream to leaf sheaths, reducing movement of the cytotoxic

ion to photosynthetically active leaves. Salt tolerance of these cereals is linked

to HKT1 locus integration and allelic differences in expression, activity and/or

Na+/K+ selectivity (Michael et al., 2015)

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2.8 Approaches to utilize salt affected soils for crop production

From last few decades, research has been focused on plant response against increasing

the salinity (Dajic, 2006). There are two main approaches and way to cope the salinity stress:

first is reclamation the salt affected soil which is very costly and time taking approach

(Blumwald et al., 2004; Akhtar et al., 2010), while the second one is to introduce salt tolerant

germplasm for these problematic soils. This approach is very reliable and farmer friendly

(Blumwald et al., 2004; Yilmaz et al., 2004). Make this approach more impressive by

improving selection criteria of salt tolerance (Iqbal et al., 2007). Plant species show the

different biochemical and physiological response and behavior against salinity stress.

Therefore for maintaining the efficient and balance ecosystem, there is need to understand the

physiological and biochemical mechanisms of plant tolerance to stress (Seemann and

Critchley, 1985; Mandre, 2002).

2.9 Result of Breeding

Different approaches have been employed targeting at enhancing salt tolerance in

cereal crops in the past. Even though wheat is a very imperative cereal crop, most work related

to breeding for salt tolerance was done in Pakistan, Australia and India (Munns et al., 2016).

In India, Kharchia 65 is obtained as a result of selections in the sodic-saline lands of farmers’

fields at Rajasthan and all the salt tolerant germplasm of wheat is derived from this line.

Successful released salt tolerant line i.e. KRL1-4 was attained from a cross of wheat cultivar

WL711 with Kharchia 65, lined developed performed well on northern India saline soils, but

remains unsuccessful in Pakistan due to soil texture and water logging (Hollington, 2000). On

saline soils of Pakistan, line LU26S performed well (Qureshi et al., 1980), but problem with

this line was that it was highly rust susceptible and not performed well to saline-sodic land

where water logging conditions also prevails. Later on two salt tolerant genotype, S36 and S24,

were offspring of a cross between LU26S and Kharchia 65 and can perform well at salinity

level of 36 and 34 dS m-1 respectively (Ashraf and Leary, 1996). S-24 was highly salt tolerant

as compared to its LU26S, Kharchia 65 and SARC-1 and reported possible mechanisms for

increased salinity tolerance was low Na+ accumulations in leaves (Ashraf, 2002). S2-24 also

yield higher than many other cultivars (Shahbaz et al., 2008; Perveen et al., 2012).

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Table 2.4 Improvement in salinity tolerance of wheat by using conventional breeding

approach

2.10 Selection/Screening Criteria for Salt Tolerance

Many probable criteria for salt tolerance screening of germplasm were suggested by

scientists (Ashraf, 2002; Munns, 2002; Munns et al., 2006). Plant does not tolerate high salt

levels at all stages of growth but relatively tolerate differently stage to stage because salinity

tolerance is a polygenic complex trait where species as well as varieties within species differ

for this trait (Ashraf, 2002).

In field condition breeding for quantitative traits with polygenic background is very

difficult, because field selection is very influenced and affected by environment conditions.

Furthermore, slightly improvement has been observed for desire character per selection cycle,

and it take easily 10-20 years to develop desire variety (Ahmad, 2008). Selection in saline filed

has faced many challenges and problems such as soil heterogeneity and rainfall variability

(Munns and James, 2003). The other is complex nature of salinity tolerance traits and it is very

difficult to assess in field screening because in field plant face many environment factors

(Munns and James, 2003). That is based on most of screening experiments for selection of

desire traits were performed in control environments i.e. pot or hydroponic study (Munns et

al., 2000; Dasgupta et al., 2008; Mohammadi-Nejad et al., 2008). Screening or selection in

control conditions has many benefits and advantages such as in control condition less space

- 20 -

and time is required to control and manage the large population as well as plants material can

be kept and prevent from insect pest and disease also (Patade and Suprasanna, 2008; Patade et

al., 2008).

2.11 Selection through hydroponic

Hydroponic is a technique of growing plants in solution culture containing all 17

essential mineral nutrients and it has been used on large scale for about 40 years back only.

However, in this short time duration, it has been considered to numerous conditions from

outdoor fields to green-house culture. Another advantage, hydroponic provides weed free plant

growth media (Sheikh, 2006) and it is most appropriate for salt stress relevant studies. However

there are some precautions to be observed. Salt addition e.g. NaCl should be made

incrementally to avoid osmotic shock, abrupt rise in salt concentration in growth media may

cause necrosis of plants. Usually, salt addition is made twice daily (morning-evening) with 25

or 50 mM increments (Shavrukov et al., 2010a, b), also described by other salt research

scientists (Munns and James, 2003; Boyer et al, 2008). Scientists have also reported suitable

salt stress levels (NaCl mM) for selection of tolerant plants of different crops, for bread wheat

100-150 mM (Munns and James, 2003; Dreccer et al., 2004), 150-200 mM for barley

(Shavrukov et al., 2010a), and 250-300 mM for wild emmer wheata tolerant cereal tolerant

and for halophytes, saltbush and Atriplex species (Flower et al., 1977). Overall salinity

tolerance is measured in terms of relative growth and physiologically traits (growth in non-

saline conditions relative to NaCl stress). The main advantage of soil less solution or

hydroponics solution is that treatments can be measured accurate and precisely as well as can

be determined the reproducible plant responses. Genetic tolerance to abiotic stress, both

between and within species, can be mediated with confidence. Furthermore, in hydroponic

solution, it is very easy to assess and examine the physiological and morphological traits e.g.

root, shoot etc.

2.12 Selection criteria followed in hydroponic culture.

Salinity tolerance evaluated by examining morphological/physiological traits is more

viable in controlled environments (Flowers and Yeo, 1995). Salinity research group scientists

had followed different criteria for screening and selection of salt tolerant plants.

- 21 -

2.12.1 Morphological traits

Salinity tolerance assessed by investigating morphological traits are more feasible in

controlled-environments (Flowers and Yeo, 1995; Akhtar et al., 2003). Total dry matter value

(Meneguzzo et al., 2000) and proportion of biomass accumulation (El-Hendawy, 2009;

Mahmood, 2009) suggested a rapid and more efficient screening technique at early growth

sages of wheat in hydroponics. Ashraf et al. (2006) found positive correlation between dry

matter and plant height at early seedling stage. This could be reliable trait for screening wheat

genotypes under salt stress.

2.12.2 Germination traits

Plant phenology germination is the very first stage at which seed may be encountered

by salt stress. High salt level reduces germination, seedling emergence and results effects stand

establishment (Ashraf and Foolad, 2005). Evaluation seed germination potential in saline

conditions is valuable as it also reflects enhanced salinity tolerance for later growth stages.

However, most investigators could not establish a clear relationship between germination

under salinity conditions and later phenology growth in most of species including durum wheat

and bread wheat (Almansouri et al., 2001). Although wheat and barley’s seeds can germinate

at very high salt regimes (more than 300mM NaCl) but developing radicle further cannot grow

at this extreme level of salt stress. Tolerance against salinity at germination stage could be

explained after physio-chemical investigations in emerging radicles and plumule (Munns and

James, 2003).

2.12.3 Cell or tissue damage related traits

Leaf injury which is estimated by solute leakage from leaf tissues (membrane damage),

assessing damage to premature chlorophyll or injury to photosynthetic machinery may be also

set as sorting criteria for salt-tolerance. These methods facilitates to determine discrimination

between low and moderate salinity tolerant genotypes ranging between 50-100 mM NaCl

(Mehta et al., 2010; Shahbaz et al., 2011). A major limitation to use this criterion to classify

salt-tolerant and sensitive germplasm is that in most of cases, causes of injuries are unknown.

The injury occurs might be due to osmotic stress, more Na+ or Cl− accumulation in leaf or to

- 22 -

Ca2+ and K+ deficiency (Ashraf, 2004; Parida and Das, 2004). Membrane stability is also

considered as reliable selection criterion to distinguish salt-tolerant and sensitive genotypes

(Demiral and Turkan, 2005; Sairam and Srivasta, 2002; Jain et al., 2001). The membrane

integrity is greatly vulnerable to reactive oxygen species induced lipid-peroxidation and the

product of this oxidation is malondialdehyde (MDA). MDA measurements in leaf tissue is a

best index of membrane stability (Meloni et al., 2003; Azevedo Neto et al., 2006). Generally,

sensitive genotypes to salt are more liable to peroxidation of lipid in membranes as compared

to salinity tolerant ones (Sairam and Srivastava, 2002). Therefore, MDA contents, reflects

membrane stability, which is greatly linked to plants efficient antioxidants system to detoxify

ROS and might be used as potential index of tolerance against salt stress (Demiral and Turkan,

2005).

2.12.4 Physiological traits

Salinity induced physiological disturbances had been widely reported in terms of

modulations in gaseous exchange relations (stomatal conductance, transpiration rate and

photosynthetic rate), water relations and alteration in pigment compositions (Munns et al.,

2002; Koyro, 2006; Nawaz et al., 2010). Koyro (2006) stated that intrinsic water use efficiency

(WUE) and net photosynthesis were influenced by NaCl salinity in hydroponic culture

experiment. Carotenoid/Chlorophyll ratio also affected which causes reduction in

photosynthetic efficiency.

Salt stress disturbs water relation i.e. water potential and also osmotic potential (Parida

and Das, 2005; Ashraf, 2004). The interest in osmotic potential related studies increased due

to its role in plant osmotic adjustment. The plants which encounters salt stress accumulates

high inorganic ions or de novo synthesizes low molecular weight organic solutes (amino acids,

soluble carbohydrates, organic acids and proline) to make osmotic adjustments (Serraj and

Sinclair, 2002; Ashraf and Harris, 2004, Singh et al., 2010). This adjustment in osmotic

potential is helpful in maintaining turgor pressure, which vita role for normal cell functioning

and growth. Ashraf and Harris (2004) had reviewed in details about potential of compatible

solutes to be selection criterion for tolerance against salt stress.

- 23 -

2.12.5 Ionic traits

Plants under salinity, adjust themselves osmosis by uptake and accumulation of

inorganic ions from growing media which leads to mineral toxicity or nutritional imbalance

(Munns and Tester, 2008). Iqbal (2005) carried out a hydroponic study and noticed increase

concentration of Na+ ions in all plant parts due to increasing salinity. Plants have adaptation to

accumulate more Na+ in older leaves as compared to their young full expanding leaves.

Furthermore, concentration of Na+ was less in stems than roots under all salt regimes.

Antagonistically, K+ concentration gradient was found maximum in young expanding leaves

and minimum in older leaves. Moreover, K+ concentration was also high in stem as compared

to roots. K+ is a major inorganic osmotica of plant cell and also required in many physiological

process and activation of enzymes (Meneguzzo et al., 2000). Therefore, K+ to Na+ ratio is very

important characteristic for salinity tolerance to develop and screen the salt tolerant germplasm

in wheat (Maria and Epstein, 2001; Houshmand et al., 2005) and durum wheat (Munns et al.,

2000). Same like K+, high Ca2+ concentration are required by plants to decrease the toxic

impact of Na+ (Houshmand et al., 2005). Under salinity high shoot Ca2+ concentrations were

found, So Ca2+/Na+ ratio should also be considered while selecting genotypes (Houshmand et

al., 2005).

- 24 -

Table 2.5 Selection outputs in control conditions

Line/cultivar selected Selection criteria In control conditions Reference

Banysoif 1 Ionic and physiological

relations

Pots Amel et al

2008

Pasban 90, accessions 10790,

10828,10823, 4098805

Sakha-92

Growth attribute

(Root/shoot length and

weights)

Hydroponics Shahzad et al

2011

Genotypes WN-150, STW-

135, DH-14, Chenab-2000,

DH13, DH-3, 9436, WN-174,

066284, and DH-2

Growth attribute

(Root/shoot and weights)

Hydroponics Babar et al .,

2015

Abadgharr, Chakwal-86,

Sarsabaz ,Bhakkar-2000, -26-

S, Margalla-99, , Kiran-95,

LU , Marvi Pak-81,

Physiological stress

tolerance indices

Petri plate growth

chamber

Zafar et al.,

2015

Line Arg and Sorkh (derived

from a cross between Roshan

and Falat)

Ionic traits Hydroponic system Bahram et al.,

2014

Kharchia-65, Shorawaki, N-7,

N-9 and N-13

Ionic

Physiological

Biochemical

Hydroponic system Gurmani et al.,

2014

BWN-75, PARC-

N1,PARCN2 Bakhtawar

Exclusion of Na+ and Cl- Hydroponic system Naseem et al.

2000

Kavir, Niknejad, Chamran

and Falat

Ionic traits (low Na+ and

higher K+ and K+: Na+

ratio)

Pot soil system Goudarzi and

Pakniyat 2008

- 25 -

In order to return from stressful environment it is urged to use such strategies through

which maximum crop stand could be achieved under saline conditions. Therefore, the saline

lands should be used efficiently for crop growth. It involves the development of salt tolerant

varieties and management practices to reduce the salinity effects (Ashraf and Akram, 2009;

Ruan et al., 2010). The proposed project aims to identify novel germplasm in exotic cereal

landraces with high salt tolerance, using fast and efficient physiologically-based screens. Na+

exclusion from the leaf blade is a desirable trait and the ability to maintain low leaf blade Na+

is a major determinant of Na+ tolerance within this cereal species (Poustini and Siosemardeh,

2004; Din et al., 2008; Munns et al., 2016).

- 26 -

Chapter 3

MATERIALS AND METHODS

A total of 400 wheat genotypes were collected from AARI (Wheat Research Institute,

Ayub Agricultural Research Institute Faisalabad Pakistan) and CIMMYT (International Maize

and Wheat Improvement Center), Mexico. In first phase, hydroponic study was conducted in

wire house (open natural environment), Department of Plant Breeding and Genetics,

University of Agriculture Faisalabad with simple complete randomized design. On basis of

Na+ exclusion, forty wheat lines out of four hundred germplasm were screened at high salinity

level (200 mM NaCl). Twenty five salt-tolerant and fifteen salt-sensitive genotypes, selected

from first phase were again evaluated next season by exploring more physiological indices to

verify screening criteria of Na+ exclusion set in hydroponics. In third phase, twenty genotypes

(14 salt tolerant and 4 salt sensitive with two check LU26S and Kharchia 65) after smart

selection from previous hydroponic studies were further evaluated in pots having saline soil to

validate results of previous studies by exploring more physiological and yield attributes. In

fourth phase, the selected twenty genotypes were also tested in saline-sodic field at Salinity

Research Institute (SSRI), Pindi Bhatian (190 m above sea level 31.8950° N, 73.2706° E), and

Central Punjab, Pakistan during 2015-2016.

- 27 -

Experiment 1

Screening of wheat germplasm against high salinity stress.

Plant material

A total of 400 wheat genotypes (Table 3.1.1) were collected from wheat section of

Ayub agricultural research institute Faisalabad Pakistan (AARI) and International Maize and

Wheat Improvement Center (CIMMYT), Mexico. Salt tolerant genotype of Pakistan (LU26S)

was used as check (Munns et al. 2006). Hydroponic study was conducted in wire house (open

natural environment), department of plant breeding and genetics, University of Agriculture

Faisalabad with following details.

Experimental details

Nursery (400 accessions) was raised in November 2012 in wire house. Fifty seeds of

each genotypes were sown in 8 × 6 cm polythene bags filled with sand. Fifteen plants of each

genotypes were transplanted at two-leaf stage in solution of hydroponics tubs (118 × 88 × 30

cm) , which had capacity of 200 litter volume of half-strength Hoagland solution. Fortnightly,

it was being changed (Hoagland and Arnon, 1950). After two days of nursery transplantation

in hydroponic culture, salt was added with 25 mM NaCl increment twice daily to maintain 200

mM NaCl salinity level to prevent osmotic shocks. Wheat seedlings were allowed to grow in

salt solution up to one month.

Determination of Na+ and K+ concentrations

For determination Na+ and K+, young fully expended leaves was detached and found

the fresh weight and oven dry weight. Dried leaf samples were digested in 1% HNO3 solution.

25 ml solution was taken from 1% HNO3 solution in falcon tubes and digested on hot plate for

4 hour at 85C. After digestion, got 1 ml from 25ml solution to dilute 10 ml volume with distal

water for determination of Na+ and K+ concentration in dry leaf. Samples were run on flame

photometer (Sherwood, UK, Model 360; Munns and James, 2003; Shavrukov et al., 2009).

- 28 -

Morphological attributes

After transplanting of one month, seedling performance of each genotypes was

assessed on saline environment. Morphological traits i.e. seedling shoot length (RL), root

length (SL), fresh weight (FW) and dry weight (DW) of leaf that emerged under stress

condition. Chlorophyll content index in seedlings were made with a chlorophyll meter (model

SPAD 502; Fanizza et al., 1991).

Statistical analysis

Observation of traits data were uploaded in SAS 9.1 software to evaluate the results in

the form of the analysis of variance (ANOVA) at 5% probability level with simple CRD design.

Hot plate for digestion

Hydroponic

Culture

Flame photometer

- 29 -

Table 3.1.1 Total number of wheat genotypes screened at 200 mM NaCl

Sr. No Genotype Sr. No Genotype

1 V-04178 46 INQALAB 91*2/KUKUNA//KIRITATI

2 MEHRAN-89 47 V-06140

3 YECORA- 70 48 TRAP#1

4 KAUZ'S' 49 CHILERO

5 PEWEE'S' 50 BYRSA-87

6 CHAM-4 51 KOHISTAN 97

7 FRONTANA 52 PBW 343*2/CHAPIO

8 V-8310 53 V-04009

9 PBW 343 54 V-010309

10 PVN 55 F60314.76

11 KAKATSI 56 V-056132

12 V0005 57 TAN/PEW//SARA/3/CBRD

13 V-1034 58 WBLL1*2/KIRITATI

14 V94195 59 INQ-91*2/KUKUNA

15 TURACO 60 PUNJAB 76

16 MAYA/PVN 61 V-04179

17 PB24862 62 KHIRMAN

18 BB # 2 63 VEE'S'/ALD'S'//HUAC'S'

19 TRAP#1 64 V-010317

20 V-02156 65 REH/HARE//2*BCN/3/CROC-1

21 V-03094 66 LU 26 (Salt Tolerant)

22 V-05121 67 LU26/KEA'S'

23 V-06129 68 MH 97

24 V-06034 69 NEELKANT'S'

25 V-09196 70 HARRIER 17.B

26 GAMDOW-6 71 INQ-91*2/TUKURU

27 SATLUJ 86 72 V-04048

28 PFAU/WEAVER*2//KIRITATI 73 WATAN

29 SONOITA=SNI 74 PBW 343=ATTILA

30 CM 75113-B-5M-1Y-5M-4Y-2B-0Y 75 V-4022'

31 V-066205 76 V-03144

32 FRET-2 77 V-96059

33 V-05115 78 PB. 18242-3A-0A

34 V-056037 79 PF 70402/ALD'S

35 CM 91575-28Y-0M-0Y-4M-0Y 80 PBW 450

36 ZAMINDAR 80 81 WAXWING//INQLAB 91*2

37 PAVON 76 82 SHAFAQ-06

38 V-97097 83 OPATA//SORA/AE.SQ. 323)

39 V-11164 84 GOSHAWK'S'

40 EAGLE 85 BHITTAI

41 PF 70402 86 PUNJAB 81

42 NL 750 87 PBW 343*2/KONK

43 TURACO/PRINIA 88 CM 75031-A-IM-IY-2M-1Y-2B-0Y

44 V-3158 89 UP 262

45 CAR ,22/ANA 90 WL 711

- 30 -

Continue…


91 ONIX/ROLF07 136 MIRAJ-08

92 CON.'S'/ANA 75//CON.'S' 137 SH-2002

93 BT 2549/FATH 138 FRET-1

94 PVN//CAR422/ANA/5/… 139 V-06018

95 SKD-1 140 V-85054

96 FAREED-06 141 KASYON//PVN'S'/SPRW'S'

97 PB-96/87094//MH-97 142 CHAKWAL 86

98 PFAU/WEAVER 143 LASANI-08

99 V-10287 144 V-05082 (Millat-11)

100 BOBWHITE'S' 145 V-010296

101 V-06067 146 TUC'S'/MON'S'

102 BOW'S'/SPT'S' 147 ZARLASHTA 99

103 MAYA 74'S'/MON'S' 148 INQ-91*2/KHVAKI

104 V-04188 149 V-11154

105 V-07178 150 SASSI

106 ACHTAR*3//KANZ 151 POTCH93/4

107 SOGHAT-90=PVN 152 V-02192

108 SANDAL/CMH912 153 OASIS F 86

109 TUKURU//BAV92 154 PRL/V-87094//TRAP/V-87094

110 CHAM-6 155 ZINDAD-2000

111 WATAN/2*ERA 156 CHENAB-2000

112 INQ-91*2/KONK 157 SARSABZ

113 NING 8319 158 PIRSABAK 2004

114 FAISALABAD-08 159 LAKTA-1

115 TW69019 160 KIRAN-95

116 INQLAB 91*2/KUKUNA 161 V-05100

117 T.D-1 162 NAEEM 82

118 V-95035 163 MARVI-2000

119 V-03BT007 164 SINDH 81

120 SHAHKAR 95 162 PAK-81/2*V-87094

121 PBW 343*2/KHVAKI 163 V=11168

122 V-06016 164 SEHER-06

123 V-07189 165 DAPHE#1*2/SOLALA

124 TRAP#1/PBW65/3/ 166 CROW"S"

125 HUW 234 + LR34 167 V-7194

126 76309 168 10-BT002

127 FRET2*2/KUKUNA 169 KARIEGA

128 BAU'S' = BAGULA 170 V-05BT006

129 PARULA=PRL 171 Pb-96/2* V-87094

130 MUNAL #1 172 V-09314

131 PVN/YACO/3/ 173 V-07100

132 CHAKWAL-50 174 FRET2*2/4/SNI/

133 V-8200 175 SNI/TRAP#1/3

134 WH542 176 BABAX/LR42

135 ZA 77 177 WBLL1/KUKUNA//TACUPETO

- 31 -

Continue…


178 V-10110 223 V-10193

179 WL-1 224 V-09194

180 PASBAN 90 225 MANTHAR

181 V-06068 226 V-08173

182 PVN/PBW65 227 ZARDANA 89

183 FAISALABAD 83 228 V-06111

184 V-11184 229 JAUHAR-78

185 V-06056 230 UFAQ

186 PBW 343*2/KUKUNA 231 V-11182

187 IQBAL2000 232 SA 75

188 SNI/PBW 65 233 GA-2002

189 PUNJAB 96 234 V-11189

190 V-08212 235 KRICHAUFF/2*PASTOR

191 BHAKKAR-2000 236 SONALIKA

192 V-06007 237 PARWAZ 94

193 SHALIMAR 88 238 NACOZARI F 76

194 SALEEM 2000 239 SULEMAN 96

195 CMSA00Y00810T-040M 240 V-08118

196 V-8243 241 MEXIPAK 65

197 V-11156 242 KARAWAN-2

198 YANG87-158*2//MILAN/SHA7 243 BOW'S'//URES/VEE'S'

199 BAYA'S' 244 PAURAQ*2/SOLALA

200 PASTOR 245 PBW343*2/KUKUNA*2//YANAC

201 V-10378 246 V-08082

202 FAISALABAD 85 247 TACUPETO F2001

203 CROC 248 V-06117

204 PIRSABAK 2005 249 V-08171

205 V-11160 250 V-8335

206 WHEAR/CHAPIO//WHEAR 251 AS-2002=WD-97603

207 LYP 73 252 V-03007

208 V-87094/2*FSD85 253 V-11186

209 CHENAB 70 254 V-11166

210 PRL'S'/PVN 255 KOHSAR 95

211 V-11172 256 V-09136

212 POTCH93/4/MILAN 257 V-07007

213 WHEAR/KUKUNA/3 258 KOHINOOR 83

214 ATTILA/3*BCN 259 BARS-09

215 HOOSAM-3 260 V-06103

216 V-05066 (Punjab-11) 261 BACANORA T88

217 PASINA 90 262 FRET2

218 INQILAB 91 263 T.J-83

219 V-07155 264 V-10025

220 V-10002 265 CMSS08Y01134T

221 SAAR 266 PB. 21299-C-2A-OA

222 V-11149 267 OASIS/SKAUZ//4

- 32 -

Continue…


268 KIRITATI//SERI/RAYON 311 V-8310

269 TWS7091 312 CMSS07Y01306T

270 V-07102 313 TD-2

271 PAURAQ*2/SOLALA 314 MTRW A92.161/PRINIA/5

272 WAXWING/6/ 315 PFAU/MILAN/5/CHEN/A

273 V-11179 316 ROLF07*2/KIRITATI

274 V-9087 317 PFAU/MILAN/3

275 FRET2/KUKUNA//FRET2/3/WH

EAR

318

KANCHAN//INQALAB 91*2/KUKUNA

276 CMH81.793/PVN 319 V-08164

277 V-11181 320 CMSS07Y01297T-099Y-30M

278 V-010306 321 V-8305

279 PBW343*2/KUKUNA*2//YANA

C

322

WBLLI*2/VIVITSI//T

280 V-11178 323 V-08064

281 VILLA JUAREZ F2009 324 NR381

282 V-07142 325 CMSS07Y01314T

283 ROELFS F2007 326 QG4.37A/4

284 TOBA97/PASTOR*2//T 327 WHEAR/VIVITSI//WHEAR

285 V-8308 328 09-BT043

286 WHEATEAR 329 V-11180

287 KIRITATI//2*SERI/RAYON 330 V-07067

288 V-11161 331 ZARGOON 79

289 V-07096 332 V-076422

290 NR 388 333 SA 42

291 KINDE*2/SOLALA 334 DOLLARBIRD

292 05BT014 335 Kingbird#2

293 V-09082 336 V-08081

294 V-088132 337 76317

295 WBLL1/KUKUNA 338 V-10355

296 V-08171 339 WHEAR/KUKUNA//WHEAR

297 FRET2*2 340 PBW343*2/KUKUNA*2//YANAC

298 PRL/2*PASTOR 341 V-08008

299 PARI 73 342 V-11176

300 V-09006 343 SUNCO//TNMU/TUI

301 CHAKWAL 97 344 V-07032

302 07BT007 345 SOKOLL*2/TROST

303 V-07200 346 V-09031

304 WBLL1//UP2338*2/VIVITSI 347 V-10104

305 TAM200/TUI 348 WEBLLI*2/TUKURU

306 V-09091 349 SUNCO/2*PASTOR//EXCALIBUR

307 KIRITATI//PBW65/2*SERI.1B 350 V-87094/2*ERA

308 CMSS08Y01024T 351 SHARP/3/PRL

309 V-10217 352 WHEAR/TUKURU//WHEAR

310 V-9452 353 HD 2169/C591//PBW343

- 33 -

Continue…


354 CMSS06B01033T 388 TAM200

355 V-11153 389 PBW343*2

356 V-07076 390 NING MAI 50

357 INQLAB 91*2 391 ROLF07*2

358 V-11177 392 Kingbird#1

359 V-86711TC/SH-88//CROW 393 FRET2

360 HUW234+LR34/PRINIA*2//KIRITATI 394 PGO

361 V-08068 395 V-87094

362 9272 396 KIRITATI

363 PRL/LU26//TRAP/LU26 397 CROC 1

364 TRCH//PRINIA/PASTOR 398 PFAU

365 WBLLI*2/VIVITSI/3/T. 399 TAM200/TUI

366 PGO/SERI//BAV92 400 TAM200

367 D-07663

368 V-08057

369 AS2002/WL711//SHAFAQ 370 V-09221 371 T.SPELTA P 372 KIRITATI/4/2*SERI 373 V-10031 374 Kingbird#3 375 CROC_1/AE.SQUARROSA 376 V-87094/CHK86//SHAFAQ 377 PBW343*2 378 D-07663 379 V-08057 380 AS2002/WL711//SHAFAQ 381 V-09221 382 T.SPELTA P 383 KIRITATI/4/2*SERI

384 V-10031

385 KIRITATI//PBW65/2*SERI.1B

386 V-04181

387 PUNJAB 85

Red colored (Salt-tolerant); Blue colored (Moderately-tolerant); Black colored (Salt-sensitive)

- 34 -

Table 3.1.2 Selected 40 genotypes (25 tolerant and 15 salt sensitive) from total 400 wheat

genotypes screened at 200 mM salinity level

Sr. No Tolerant Sr. No Salt Sensitive

1 V-04178 1 TAM200

2 MEHRAN-89 2 PBW343*2

3 YECORA- 70 3 NING MAI 50

4 KAUZ'S' 4 ROLF07*2

5 PEWEE'S' 5 Kingbird#1

6 CHAM-4 6 FRET2

7 FRONTANA 7 PGO

8 V-8310 8 V-87094

9 PBW 343 9 KIRITATI

10 PVN 10 CROC 1

11 KAKATSI 11 PFAU

12 V0005 12 TAM200/TUI

13 V-1034 13 KIRITATI//PBW65/2*SERI.1B

14 V94195 14 V-04181

15 TURACO 15 PUNJAB 85

16 MAYA/PVN 17 PB24862 18 BB # 2 19 TRAP#1 20 V-02156 21 V-03094 22 V-05121 23 V-06129 24 V-06034 25 V-09196

- 35 -

Experiment 2

Investigation of physiological and biochemical bases of salt tolerance in selected wheat

germplasm

Hydroponics study II

3.2.1 Experimental details

Twenty five salt-tolerant and 15 salt-sensitive genotypes, selected from study I were

again evaluated next season by exploring more physiological details to verify screening criteria

of Na+ exclusion developed in experiment I. Nursery of screened genotypes (including two

checks LU26S and Kharchia 65; Table 3.1.2) was grown and transplanted at three leaves stage

in hydroponic solution following CRD design. After two days of nursery transplantation in

hydroponic culture, commercial grade salt was added to develop three salinity levels (0, 100

and 200 mM NaCl). One month after the transplantation the response of each genotype against

salt stress was evaluated on Na+ exclusion basis from leaves and roots and measurement of

growth attributes, i.e., root and shoot lengths, fresh weights of root and shoot, relative growth

rates were done (Hoffmann and Poorter, 2002).

3.2.2 Na+ and K+ determination

Twenty days after transplantation Na+ and K+ concentration in leaves and root were

recorded following the same protocol as descried earlier in section 3.1.3.

3.2.3 Growth parameters

Measurement of growth attributes, i.e., root and shoot lengths, fresh weights of root

and shoot, relative growth rates were done after one month of nursery transplantation in salt

stress condition (Hoffmann and Poorter, 2002).

3.2.4 Gas exchange parameters

Net C02 assimilation-rate (A), Stomatal-conductance and transpiration-rate (E) were

measured from young fully expanded-leaf with moveable infrared-gas analyzer (Analytical

development company, Hoddeson, UK). Measurement were taken in sunlight between hours

of 10:00 am to 12: pm, apparatus was used with specification i.e. leaf-chamber gas flow-rate

- 36 -

(295 m/min), leaf-chamber molar gas-rate (399 µmole s-1), leaf-chamber temperature range

(24-26°C), PAR at leaf-surface up to 760 µmol m-2 and ambient CO2 (365 µmol/mol).

3.2.5 Statistical analysis

Data relevant to different response variables of experiment was analyzed in SAS 9.1

software to evaluate the results in the form of the analysis of variance (ANOVA) as well as in

GGE-biplot to dipict the polygon view to describe the genotype’s performance based on

interaction between the entries (genotypes) and testers (traits). Sum of principal components

(PC1 and PC2) of GGE-biplot, explained variation between the genotypes based on traits

(tester). Moreover the vector view of GGE-biplot shows correlation (positive or negative)

among testers (traits). The traits are positive correlated if the angel between the vector of two

trait is an acute angel (< 90°), while if angel is greater than 90°, then traits are negatively

correlated.

- 37 -

Table 3.2.1 List of selected forty two genotypes and their codes for GGE-biplot anlysis

Code Type Genotypes Code Type Genotypes

1 T V-04178 22 S TAM200

2 T Kharchia65 23 T V94195

3 T MEHRAN-89 24 S NING MAI 50

4 T YECORA- 70 25 T TURACO

5 T LU26S 26 S Kingbird#1

6 T KAUZ'S' 27 T MAYA/PVN

7 S PBW343*2 28 S PGO

8 T PEWEE'S' 29 T PB24862

9 T CHAM-4 30 S KIRITATI

10 S ROLF07*2 31 T BB # 2

11 T FRONTANA 32 S PFAU

12 T V-8310 33 T TRAP#1

13 T PBW 343 34 S KIRITATI//PBW65/2*SERI.1B

14 S FRET2 35 T V-02156

15 T PVN 36 T V-03094

16 S V-87094 37 S V-04181

17 T KAKATSI 38 T V-05121

18 S CROC 1 39 T V-06129

19 T V0005 40 T V-06034

20 S TAM200/TUI 41 T V-09196

21 T V-1034 42 S PUNJAB 85

T= Tolerant S= Salt Sensitive

- 38 -

Experiment 3

Identification of physiological markers associated with salinity tolerance of wheat

genotypes in saline soil (pot study).

3.3.1 Plant material

Twenty genotypes (14 salt tolerant and 4 salt sensitive along with two checks LU26S

and Kharchia 65, table 3.3.2) out of four hundred, from hydroponic studies were selected and

further evaluated in saline soil in pots to validate results of previous hydroponic studies by

exploring more physiological details at vegetative stage and yield potential.

3.3.2 Pot experiment details

Twenty genotypes were assessed for their salinity tolerance in a pot study. Chemical

and physical characteristic (Table 3.3.1) of soil were analyzed according to standard protocol

described by U.S. Salinity Laboratory Staff (1954). Calculated amount of NaCl was added in

each pot containing 12 kg soil and mixing was done. Two levels of salinity was developed in

pots i.e. 1.41 (control) non-saline and 15 dS m-1 (saline). Ten seeds of each genotype were

sown with four replications of each treatments. Pots were put in appropriate wire cage under

proper ambient light and temperature with CRD design. At time of sowing recommended dose

(90: 75 kg ha-1) of P and K was applied in each pot respectively and half dose of N (50 kg ha-

1) as urea source at anthesis stage. P and K fertilizers source were diammonium phosphate and

sulfate of potash respectively. Tap water was used for irrigation according to requirement.

After emergence thinning was done to maintain five plants in each pot. Data for ionic (leaf Na+

and K+) traits, gas exchange parameters, water relations, biochemical analysis and yield

components were recorded at various stages as given below.

- 39 -

3.3.3 Leaf Na+ and K+ determination

Following the same protocol descried earlier in experiment 1.

3.3.4 Water relation attributes

After 45 days of sowing leaf water relation traits i.e. water potential (Ψw), osmotic

potential (Ψs) and turgor potential (Ψp) were recorded in leaf. At 6:00 am early in the morning,

young fully expended leaf was used to record leaf water potential with Scholander type water

potential apparatus. The same leaves were put into biomedical freezer for one week. Sap was

extracted from frozen leaves to measure osmotic potential (Ψs) with osmometer (VAPRO,

Model 5520, USA). Leaf turgor potential (Ψp) was determined by using following equation.

Ψp = Ψw – Ψs

3.3.5 Biochemical analysis

Full expended young leaves were taken after sixty days of sowing from each pot and

were kept in biomedical freezer at -30°C. Within one week following biochemical parameters

were recorded.

- 40 -

Chlorophyll contents in leaves were determined by a methods as described by the

Nagata and Yamashita (1992). While total phenolic contents and free proline in leaf samples

were measured by using a methods as described by Waterhouse, (2002) and Bates et al. (1973)

respectively.



3.3.7 Chlorophyll fluorescence

Using fluorescence meter (Multimode chlorophyll fluorimeter, OPTI Sciences, OS5P)

chlorophyll florescence traits were recorded following Strasser et al. (1995) method.

3.3.8 Cell membrane injury

Cell membrane injury or membrane thermostablity was measured by method described

by Yildirim et al., 2009.

3.3.9 Yield related attributes

Three plants were tagged in each pot to record the yield related attributes plant height,

Spike length, number of spikelet spike-1, fertile tillers (FT) per plant, grain spike-1, 100-grain

weight, grain yield and biological yield at maturity.


Recorded observations were uploaded in SAS 9.1 software to evaluate the results in the

form of the analysis of variance (ANOVA) at 5% probability level. Means are represented in

bar graphs provided with standard error bar values.

- 41 -

Table 3.3.2 Selected twenty genotypes from experiment 1 and 2

Sr. No Type Genotypes Sr. No Type Genotypes

1 T T V-04178 11 T BB # 2

2 T MEHRAN-89 12 T V-02156

3 T YECORA- 70 13 T V-03094

4 T PEWEE'S' 14 T V-04181

5 T CHAM-4 15 check LU26S

6 T FRONTANA 16 check Kharchia 65

7 T PVN 17 S TAM200/TUI

8 T V0005 18 S FRET2

9 T TURACO 19 S PUNJAB 85

10 T MAYA/PVN 20 S PBW343*2


- 42 -

Experiment 4

Agronomic and physiological performance of selected wheat genotypes on saline-sodic

soil.

3.4.1 Experimental location of field trial

The field trial was conducted at experimental site of Soil Salinity Research Institute

(SSRI), Pindi Bhattian (190 m above sea level 31.8950o N, 73.2706o E) in central Punjab

Pakistan during 2015-2016. Extent of salinity/sodicity is (ECe 9.5 dS/m; SAR 24 mmol/L and

pH 8.7). The climate of area under study is semi-arid with more than 1600 mm evaporation,

average annual rainfall is 325 mm and temperature is 32°C.

3.4.2 Plant material and design

This experiment examined the responses to salinity of genotypes under saline sodic

field. The genotypes were selected from previous screening work for Na+ exclusion in

hydroponic studies. In this experiment twenty bread wheat genotypes (including four high Na+,

fourteen low Na+ genotypes and two salt tolerant checks i.e. LU26S and Kharchia 65) with

contrasting to their sodium accumulation were used to evaluate the effect of Na+ exclusion

while growing on saline-sodic soil. Source, and origin of genotypes is mentioned in table 3.4.2.

The experimental design was RCBD with four replications. Size of each experimental unit was

13 m2 with 22.5 cm row to row distance.

3.4.3 Crop husbandry

Soil (0–30 cm depth) samples were collected before sowing and analyzed according to

methods cited in Soil Survey Staff, U. S. D. A (Anon, 1960). Salt affected soil was of saline-

sodic nature. Soil was prepared by two ploughings (depth 12 cm) followed by planking to

conserve moisture suitable for germination. Seeds were sown with drill in 22 cm row to row

distance at seed rate 125 kg/ha in November 2015. Soil nutrient supplementation was done @

100:90:75 N: P: K kg ha-1 using Urea, DAP and sulfate of potash as fertilizer source. Whole

dose of P and K and half dose of N were applied as basal dose during soil preparation while

half dose of N was applied during anthesis stage. Three irrigations were done equating 10

deltas of water. No major climatic hazard happened during crop growth duration.

- 43 -

3.4.4 Leaf Na+ and K+ determination:


3.4.5 Physiological attributes

After sixty day of sowing, five young fully expended leaves were collected from each

plots. Samples collection was done early in morning at 5:00 am. Leaf samples were put in

plastic zipper bags and immediately transferred in biomedical freezer (-80°C) for further

biochemical analysis. Chlorophyll contents in plant leaves were determined by the method

described by Nagata and Yamashita (1992). Non-enzyme antioxidants e.g. total leaf phenolic

contents (TPH) were measured by Waterhouse (2002) method The leaf proline content was

determined in fresh sample by Bates et al. (1973) method using spectrophotometer (UV 4000).

3.4.6 Stand establishment

Twelve days after sowing emergence was evaluated from 2 m2 area providing a

percentage of crop density. 1 = 90 % emergence or more (very good); 2 = 80–89 % (good);

3=70–79 % (acceptable), 4 = 60–69 % (poor).

3.4.7 Biomass and grain yield

Data were documented from ten randomly selected plants for plant height and spike

length, on centimeter scale while number of spikelet/spike and number of grain/spike were

counted manually. Plants from 1 m2 were harvested manually just above ground and left for

one week in open field for sun drying later on grain and biological yields were determined.


Each treatment was replicated four time and data were statistically analyzed by

ANOVA. Significance levels of treatments were computed using software “SAS” (version

9.1). Statistic 8.1 package was also used to find correlation between grain yield and crop

density parentage. Graphs are presented with standard error bars, also provided with p value.

Data regarding yield is presented in table with critical values to compare treatment means. GE

software was also used to describe best the performer genotypes for yield related attributes on

salt affected soil.

- 44 -

Table 3.4.1 Selected twenty genotype and their GGE-biplot codes for yield attributes

Code Type Genotypes Code Type Genotypes

1 T T V-04178 11 T BB # 2

2 T MEHRAN-89 12 T V-02156

3 T YECORA- 70 13 T V-03094

4 T PEWEE'S' 14 T V-04181

5 T CHAM-4 15 check LU26S

6 T FRONTANA 16 check Kharchia 65

7 T PVN 17 S TAM200/TUI

8 T V0005 18 S FRET2

9 T TURACO 19 S PUNJAB 85

10 T MAYA/PVN 20 S PBW343*2


- 45 -

Chapter 4

RESULTS AND DISCUSSION

The results concerning to the experiments conducted on physiologically based

screening of wheat germplasm during year 2013-2016 are presented and discussed below.

Experiment 1: Screening of wheat germplasm against high salinity

The present study elucidates the identification of novel salt tolerant germplasm from

very large diverse pool (four hundred accessions of different origin; Table 3.1.1). Identification

was done on Na+ exclusion basis from leaf blade along with other essential growth traits for

salinity tolerance. The results obtained from this experiment are given below.

Response of wheat germplasm against salinity stress (200 mM NaCl)

Significant differences (P ≤ 0.001) were found between 400 genotypes for Na+

exclusion from leaf blade and seedling growth at high salinity level (Table 4.1.1). Genotype

V-04178 accumulated less Na+ in leaf blade (6.28 mg g-1 dw; Fig 4.1.1a) while genotype TAM

200 accumulated substantial amount of Na+ in leaf (199.20 mg g-1 dw; Fig 4.1.1a).

Furthermore, maximum K+ concentration in leaf was observed in V-06129 (38.56 mgg-1 dw;

Fig 4.1.1a) and minimum in genotype MEXIPAK 65 (3.89 mg g-1 dw; Fig 4.1.1a). Regarding

seedling growth, Pb-96 had the longest root length (25.83 cm), while shortest one found at

CHENAB-70 (12.47 cm; Fig 4.1.1b). Maximum shoot length was found for KAUZ'S' (29.67

cm) and minimum for genotype V-11177 and V-08064 (both 13.00 cm; Fig 4.1b). Maximum

chlorophyll content index was observed in genotype V-056132 (29.17; Fig 4.1.1b) and

minimum was observed in genotype SINDH 81 (14.13; Fig 1b). Highest and lowest fresh leaf

weights were recorded in V-066205 (0.200 g) and V-08064 (0.019 g) respectively (Fig 4.1.1c).

Maximum dry weight of leaf was recorded in V-7194 genotype (0.060 g) and minimum was

noted in PVN/YACO (0.005 g) genotypes when compared with check LU26S (Fig 4.1.1c).

Salinity stress response on the basis of ion accumulation

About 17 % (Fig 4.1.2) genotypes were low Na+ accumulators as compared to check

LU26S (20.89 mg g-1 dw) and declared as Na+ tolerant, 36% were Na+ sensitive as depicted by

- 46 -

more Na+ accumulation in leaf when compared with mean leaf Na+ value of total genotypes

(53.73 mg g-1 dw). Forty seven genotypes (11%) accumulated more K+ in leaf even than that

of the check and considered as tolerant. Furthermore, when comparison was made among

genotypes and check on the basis of leaf Na+/K+ ratio, 8% genotypes were salt tolerant and

36% genotypes were considered as salt-sensitive. In the light of above, Na+ exclusion looked

reliable characteristic. Therefore, a criterion was set using mean value of leaf Na+ of all

genotypes (G) and leaf Na+ value of check LU26S (C). Genotypes had values below C were

declared as tolerant, genotypes had values between G and C were considered as moderately

tolerant while sensitive genotypes were those having value more than G.

4.1.3 Comparison between low Na+ and high Na+ genotypes

Twenty five most tolerant selected by above mentioned criterion, performed better

regarding physiological (chlorophyll index, K+ to Na+ ratio Fig 4.1.3a-b) and growth attributes

i.e. leaf fresh weight, dry weight (Fig 4.1.4a-b), root length and shoot length (Fig 4.1.5a-b) as

compared to salt sensitive group.

Finally, 25 most tolerant, 15 most sensitive and two checks (LU26S and Kharchia 65)

genotypes were advanced for study II (next season) with the objective to validate criterion by

explore further physiological modulations in response to salt stress.

47

Table 4.1.1 Mean squares from analysis of variance for ionic content (leaf Na+ and K+ in “mg g-1 dw”), growth traits (root and shoot

length in “cm”; fresh and dry weight in “g”) and chlorophyll index of 400 wheat genotypes grown at 200 mM NaCl salinity at

seedling stage

** Highly significant

SOV DF Na+ in leaf K+ in leaf Shoot

length

Root length Fresh leaf

weight

Dry leaf

weight

Chlorophyll

index

Genotypes 399 5411.69** 173.02** 22.21** 20.38** 0.00390** 0.000137** 29.63**

Residual 800 24.06 17.382 3.8636 4.3871 0.00040 0.0000387 6.1208

48

Fig 4.1.1 Mean performance of 400 wheat genotypes scaled from lower to higher values at

200 mM NaCl salinity: a) Na+ and K+ concentration; b) root length (RL), shoot length (SL);

c) fresh (fw) and dry weight (dw) of 3rd leaf and d) chlorophyll content index (CCI). Round

circle is overall mean of genotypes, and square marker is mean of the check variety (Lu26S).

49

Fig 4.1.2 Salt tolerance percentage by comparison of check, LU26S and average mean value

of 400 genotypes. Tolerant (genotypes best performance than the check variety), Moderate

tolerant (genotypes between check and the average mean value of 400 genotypes), salt

sensitive (genotypes, poor performance than mean value of 400 genotypes/check).

66 47 31

192

124

227

142

229

142

17% 11%8%

48%

31%

56%

36%57%

36%

Na+ K+ Na+/K+

Sa

linity s

tre

ss (

% a

nd

co

un

t)

Tolerant Moderate tolerant Sensitive

50

Fig 4.1.3 Comparison between two groups of wheat genotypes, Low Na+ accumulators (square

marks) and High Na+ accumulators in leaf blade (triangle mark). K+/Na+ ratio (a) and

chlorophyll index (b)

51


marks) and High Na+ accumulators in leaf blade (triangle mark). Fresh weight of 3rd leaf (a)

and dry weight of 3rd leaf (b).

52


marks) and High Na+ accumulators in leaf blade (triangle mark). Root length (a) and shoot

length (b).

53

Experiment 2: Investigation of physiological and biochemical bases of salt

tolerance in selected wheat germplasm

Genotypes, selected from first study were again evaluated next season by exploring

more physiological details to verify screening criteria of Na+ exclusion, developed in

experiment I. The results obtained from experiment 2 are given below.

4.2.1 Response of screened genotypes against different salinity levels

Forty-two genotypes (25 salt-tolerant, 15 salt-sensitive and two check LU26S;

Kharchia 65: Table 3.2.1) grown at various salt levels showed significant (P ≤ 0.001)

differences for ionic (in leaf and root), gas exchange and growth attributes were chosen for this

study (Table 4.2.1).

4.2.2 Biplot Na+ and K+ concentration in leaf and root

Biplot analysis for Na+ and K+ accumulation of forty-two genotypes showed distinct

variation in leaf (Fig 4.2.1) and root (Fig 4.2.2), under varying salt levels (0, 100 and 200 mM

NaCl). Genotypes were divided into seven and four sectors based on performance of Na+

reciprocal and K+ concentrations in (Fig 4.2.1) and root (Fig 4.2.2) respectively. For the biplot

analysis of Na+ and K+ accumulation in leaf (Fig. 4.2.1), genotypes placed on corners (vertex

or most responsive genotypes) were V-03094 (36), TURACO (25), PGO (28), CHAM-4 (9),

TAM200 (20), TAM200/TUI (22) V-8310 (12) and KAKATSI (17). The first sector represents

Na+ concentration at 0 mM, K+ concentration at 200 mM and showed genotype TURACO (25)

as the best and the most promising for above ionic traits followed by V0005 (19). The second

sector expressed Na+ concentration at 100 and 200 mM as well as K+ concentration at 0 and

100 mM with genotype V-03094 (36) as the most reliable followed by genotype CHAM-4 (9).

The remaining genotypes on other vertex, TAM200/TUI (22), TAM200 (20), PGO (28), and

KAKATSI (17) showed the poorest performance for the ionic traits (Fig. 4.2.1) as they were

located away from marked ionic traits on the biplot, while V-8310 (12), V0005 (19), PEWEE'S'

(8) and TAM200/TUI (22) were the most responsive for Na+ and K+ concentration in root (Fig.

4.2.2). The first sector depicts concentration of Na+ and K+ at 0, 100 and 200 mM NaCl with

V0005 (19) genotype as the best performing and the most favorable followed by V-03094 (36)

and TURACO (25). The remaining genotypes on other vertex, TAM200/TUI (22), V-8310

54

(12) and PEWEE'S' (8) were the poorest performers for K+ and Na+ and concentration in root

(Fig 4.2.2). They were located away far from all marked traits on the biplot.

The vector view of the GGE-biplot (Fig 4.2.3 and Fig 4.2.4) shows the correlation

between Na+ and K+ traits measured at different salinity levels in leaf (Fig 4.2.3) and root (Fig

4.2.4). Principal components sums (PC1 and PC2) explained 84.2% and 87.7% variation

among genotypes based on ions concentration in leaf (Fig 4.2.3) and root (Fig 4.2.4)

respectively under different salinity stress. Across 42 tested genotypes, positive correlation

was found in ionic traits. Na+ and K+ concentration at different salinity levels were positively

correlated for leaf (Fig 4.2.3) and root (Fig. 4.2.4) except Na+ concentration at 0 mM is positive

but weak correlated with ionic traits for root (Fig 4.2.4).

4.2.3 Biplot for growth rate (root and shoot length) and relative growth rates

The biplot (Fig 4.2.5) for root length (RL) and shoot length (SL) is divided into eight

sectors and genotypes V-05121 (38), V0005 (19), V-04178 (1), NING MAI 50 (24), TAM200

(20), YECORA- 70 (4) and PUNJAB 85 (42) as the vertices were most reactive for relative

growth of shoot length and root length (Fig 4.2.5). Sector one showed V-03094 (36) genotype

as the winner for RGR-SL at different salinity levels while MEHRAN-89 (3) and CHAM-4 (9)

successively were chasing. The sector two displayed V-04178 (1), V0005 (19) and V-05121

(38) genotypes as winners while genotype TURACO (25) and FRONTANA (11) genotypes

were lagging behind for RGR-RT at various stress levels. Remaining genotypes on other

sectors i.e. NING MAI 50 (24), TAM 200 (20), PBW343*2 (7) and PUNJAB 85 (42)

genotypes showed poorest performers for RGR-RL. While (Fig. 4.2.6) displays five sectors for

root and shoot length. The corner genotypes Kharchia 65 (2), V0005 (19), TURACO (25), V-

05121 (38), PFAU (32), NING MAI 50 (24), PBW343*2 (7) and PUNJAB85 (42) were more

reactive at various salinity levels. Sector five grouped genotypes Kharchia 65 (2), V0005 (19),

and TURACO (25) as winner followed by V-03094 (36) on basis of all traits expect RL that

showed maximum measurement for genotype V-05121 (38) at 0 mM falls in sector one. The

remaining other vertex genotypes PFAU (32), NING MAI 50 (24), PBW343*2 (7) and

PUNJAB85 (42) were the poorest performance for RL and SL against all levels of salinity (Fig

4.2.6).

55

Vector view of GGE-biplot (Fig 4.2.7 and Fig 4.2.8) depicted the positive correlation

between the growth rate (RL and SL, Fig 4.2.7) parameter and relative growth rate (RGR-RL

and RGR-SL, Fig 4.2.8) at all salt levels. The angel between all tested traits vector is an acute

angle (< 90) and showed positive correlation for growth rate RL and SL (Fig 4.2.7) and relative

growth rate (RGR-RL and RGR-SL, Fig 4.2.8). Vector view of bipolt depicts huge variation

observed among genotypes based on growth rate (RL and SL, Fig 4.2.7) and relative growth

rate (RGR-RL and RGR-SL, Fig 4.2.8) and it was found 85.6% for growth rate (Fig 4.2.7) and

85.3% variation for relative growth rate (Fig 4.2.8).

4.2.4 Genotypes response against salinity levels based on gaseous exchange

parameters

Polygon view of the GGE-biplot (Fig 4.2.9) for leaf net CO2 assimilation rate (A),

stomatal conductance (gs) and transpiration rate (E) of forty two genotypes were divided into

seven sectors. The vertex genotypes Kharchia 65 (2), TURACO (25), V-05121 (38),

TAM200/TUI (20), V-87094 (16), CROC 1 (18), V-02156 (35) and BB # 2 (31) were most

responsive and favorable at various salinity levels (0, 100 and 200 mM NaCl). Sector seven

showed gs, E and A traits with genotypes Kharchia 65 (2), TURACO (25) performing best

followed by V0005 (19), V-05121 (38) at all salt levels while E was displayed in sector two

with PBW 343 (13) genotype as best performer at 0 mM salinity level. TAM 200/TUI (20), V-

87094 (16), CROC 1 (18), V-02156 (35), and BB # 2 (31) genotypes on remaining sectors

were the poorest performers for the gas exchange parameters in leaf. PC1 and PC2 sums

explained 78% variation among the genotypes based on gas exchange parameters. All vectors

traits showed positive and strong correlation but transpiration rate (E) at 0 mM and stomatal

conductance (gs) at 0 mM and 200 mM had weak correlation as compared to reaming traits

(Fig 4.2.10).

4.2.5 Absolute tolerance at various salinity levels

Absolute tolerance (Fig 4.2.11a-b) by maximum fresh root weight (FRW, Fig 4.2.11b)

measurement of all genotypes at all salinity level showed Kharchia 65 as the most tolerant,

while lagging behind genotypes were V005, TURACU and V05121. Similarly maximum fresh

56

shoot weight (FSW, Fig 4.2.11a) was exhibited by Kharchia 65 followed by TURACO, V005,

V05121 and PBW343 at various salinity levels.

Table 4.2.1. Mean squares from analysis of variance of ionic content (leaf Na+ and K+ in “mg

g-1 dw”) and growth traits (root and shoot length in “cm”; fresh weight in “g”) and gasses

exchange parameters (net CO2 assimilation rate “μmol m–2 s–1”; Transpiration rate and

Stomatal conductance in “mmol m–2 s–1”) of 42 wheat genotypes grown at three different NaCl

salinity levels.

Traits Treatment (T) Genotypes (G) G*T Residual

DF 2 41 82 250

Na+ in leaf 24825.6** 550.4* 104.5* 9.2

Na+ in root 7135.83** 151.9* 30.75* 6.95

K+ in leaf 4411.43** 192.29* 19.63* 7.93

K+ in root 1797.58** 34.50* 3.25* 1.32

Fresh shoot weight 2.89** 0.47* 0.022* 0.014

Fresh root weight 0.32** 0.13* 0.0084* 0.0039

Shoot length 1900.46** 52.99* 5.51* 2.69

Root length 2418.46** 25.93* 3.37* 1.7

RGR-RL 0.0052** 0.00017* 0.00001* 0.00001

RGR-SL 0.0047* 0.00019* 0.00002* 0.00001

Photosynthesis rate 60.554** 5.711** 0.377** 0.0011

Transpiration rate 26.175** 0.849** 0.158** 0.00016

Stomatal conductance 0.245420** 0.006553** 0.006001** 0.00188

Relative growth rate of root length (RGR-RL), Relative growth rate of shoot length (RGR-SL)

*Significant; ** Highly significant

Table 4.2.2 Relationship between ionic and seedling growth traits.

Na-L Na-R K-S K-R FWP FWS FWR RGR-P SL

Na-R 0.8431**

K-S -0.7269** -0.7486**

K-R -0.8166** -0.8074** 0.8116**

FWP -0.5039** -0.5263** 0.5122** 0.6054**

FWS -0.4947** -0.5110** 0.5000** 0.6041** 0.9774**

FWR -0.4503** -0.4821** 0.4635** 0.5211** 0.9024** 0.7909**

RGR-P -0.5215** -0.5352** 0.6061** 0.6141** 0.6011** 0.5785** 0.5607**

SL -0.7772** -0.7794** 0.7443** 0.8290** 0.6937** 0.6944** 0.5926** 0.5853**

RL -0.8412** -0.8413** 0.7720** 0.8613** 0.5807** 0.5756** 0.5075** 0.5837** 0.8048**

Na+ content in leaf and root (Na-L, Na-R respectively), K+ content in leaf and root (K-L, K-R

respectively), Fresh weight of plant (FWP), Fresh weight of shoot (FWS), Fresh weight of root (FWR),

Shoot length (SL), Root length (RL), Relative growth rate of Plant (RGR-P).

**Highly significant difference at P ≤ 0.001.

57

Fig 4.2.1 A “Which is best for what” genotype by traits biplot of Na+ (reciprocal) and K+

concertation of 42 genotypes in leaf at various salinity stress (0, 100 and 200mM). PC1 and

PC2 explained total variation among genotypes. See Table 3.2.1 for codes of the genotypes.

58

Fig 4.2.2 A “Which is best for what” genotype by traits biplot of Na+ (reciprocal) and K+

concertation of 42 genotypes in root at various salinity stress (0, 100 and 200mM). PC1 and

PC2 explained total variation among genotypes. See Table 3.2.1 for codes of the genotypes.

59

Fig 4.2.3 Vector view of the genotype-by-trait biplot of showing the interrelationships among

ionic traits measured in leaf at various salinity stress (0, 100 and 200mM).PC1 and PC2

explained total variation among genotypes. See Table 3.2.1 for codes of the genotypes.

60


ionic traits measured in root at various salinity stress (0, 100 and 200mM). PC1 and PC2

explained total variation among genotypes. See Table 3.2.1 for codes of the genotypes

61

Fig 4.2.5 A “Which is best for what” genotype by traits biplot of relative growth rate of shoot

length (RGR-SL) and relative growth rate of root length (RGR-RL) of 42 genotypes in root at

various salinity stress (0, 100 and 200mM). PC1 and PC2 explained total variation among

genotypes. See Table 3.2.1 for codes of the genotypes.

62

Fig4.2.6 A “Which is best for what” genotype by traits biplot of growth rate of shoot length

(SL) and root length (RL)of 42 genotypes in root at various salinity stress (0, 100 and 200mM).

PC1 and PC2 explained total variation among genotypes. See Table 3.2.1 for codes of the

genotypes.

63


growth rate of shoot length (SL) and root length (RL) at various salinity stress (0, 100 and

200mM).PC1 and PC2 explained total variation among genotypes See Table 3.2.1 for codes

of the genotypes.

64


relative growth rate of shoot length (RGR-SL) and relative growth rate of root length (RGR-

RL) at various salinity stress (0, 100 and 200mM).PC1 and PC2 explained total variation

among genotypes. See Table 3.2.1 for codes of the genotypes.

65

Fig 4.2.9 A “Which is best for what” genotype by traits biplot of leaf photosynthetic rate (A),

transpiration rate (E) and stomatal conductance (gs) of 42 genotype evaluated in various

salinity stress (0, 100 and 200mM). PC1 and PC2 explained total variation among genotypes.

See Table 3.2.1 for codes of the genotypes.

66


leaf photosynthetic rate (A), transpiration rate (E) and stomatal conductance (gs) of 42

genotype evaluated in various salinity stress (0, 100 and 200mM). PC1 and PC2 explained

total variation among genotypes. See Table 3.2.1 for codes of the genotypes.

67

Fig 4.2.11 Mean fresh shoot weight (a) and fresh root weight (b) of 42 wheat genotypes grown

at three different NaCl salinities at seedling stage.

68

Discussion (Experiment 1 and 2)

The present study elucidates the identification of novel salt tolerant germplasm from

very large diverse pool (four hundred accessions of different origin) of bread wheat. Low Na+

genotype expressed maximum tolerance against the salt stress by increased the influx of K+

over the Na+ and improved the chlorophyll index (Fig 4.1.3a-b). This low influx of Na+ has

been associated with Na+ exclusion from leaf blade (Din et al., 2003; Husain et al., 2003).

Furthermore, tolerant genotypes may also have sophisticated K+ regulation system as Shabala

and Pottosin (2014) described two-pore K+ channels and shakertype; non-selective cation

channels which aids permeability of K+ and transporters (HKT, KUP/HAK/KT and K+/H+). It

is also reported that if K+ to Na+ ratio is higher than the variety is salt-tolerant, if it is narrow

then the variety is called salt-sensitive (Tester and Davenport, 2003). Higher K+ to Na+ ratio

could also be attributed to an efficient efflux of Na+ from the cell or more influx of K+ over

Na+ (Munns et al., 2006). This efflux of Na+ is due to Na+/ H+ antiporter that located on plasma

membrane (Munns and Tester, 2008). Loci Nax1 and Nax2 located in chromosomes 2A and

5A respectively, controlling influx of Na+, have been found in wheat genotype (Lindsay et al.,

2004) and this molecular marker usually used in wheat breeding program to develop varieties

with low Na characteristics.

Genotypes with low Na+ accumulation resulted in maximum root length, shoot length,

fresh weight and dry weights of leaves (Fig. 4.1.4a-b; Fig. 4.1.5a-b). The effective efflux of

Na+ and control of its transport from the mesophyll cell of leaves is a thrust need for salt

tolerance. Removal of Na+ from the leaves is linked with salt tolerance in wheat (Cuin et al.,

2009; 2010). Salinity affects wheat growth markedly when ECe value of root zone equal or

exceeds 5.7 dS/m (Ali et al., 2008; Zheng et al., 2010). Low growth occurs due to osmotic

shock as well as Na+ is cytotoxic that directly affects physiological processes and biochemical

processes by inhibiting uptake other nutrients such as Mg2+, K+, Ca+2, Mg2+, H2PO4, HPO4,

NO3, K+ to Na+ and Ca2

+to Na+ ratios in wheat genotypes at various growth stages with

devastating effect at seedling stage (Munns et al., 2006; Afzal et al., 2007).

Selected forty two genotypes (25 tolerant, 15 sensitive, and two check LU26S and

Kharchia 65) from above phase were further analyzed in a subsequent year to validate previous

results and inferences using GGE biplot by exploring physiological indices. The biplot analysis

identified the best performing genotypes under salt stress (Yan, 2001).

69

According to sum of principle components (Fig 4.2.1; Fig 4.2.2) shows 84.2% and

87.7% variation among genotypes based on concentration of ions in leaf and root respectively.

In this analysis, the genotypes TURACO (25), V0005 (19), V-03094 (36) and CHAM-4 (9)

were the best performers while PGO (28), TAM 200/TUI (22), TAM2 00 (20), KAKATSI (17)

and PEWEE'S' (8) showed poor performance for ionic traits in leaf and root, respectively (Fig

4.2.1; Fig 4.2.2) under salinity stress. The main mechanism of salinity tolerance in wheat is

Na+ exclusion or decrease the influx of Na+ and increased efflux of K+ in cell to maintain

balance discrimination Na+ and K+ in the shoot (Tester and Davenport, 2003). Very strong

positive correlation has been reported between leaf K+ content and salt-tolerance in plant (Chen

et al., 2005, Garthwaite et al., 2005), and root’s ability to keep K+ was proven to be one of the

important traits consulting salt tolerance in wheat (Cuin et al., 2010) and barely (Chen et al.,

2005, 2007). While another study reported that more sequestration of Na+, signaling and high

tissue specificity of Na+ in root is also linked with salt stress tolerance in bread wheat (Wu et

al., 2015).

Genotypes that accumulated higher Na+ in their leaf had limited growth (shoot, root

lengths), lower growth rate (Fig 4.2.5; Figure 4.2.6), and lower fresh shoot and root weight

(Fig 4.2.11) which resulted in poor performance as compared to those genotypes that

accumulated low Na+ in their leaves and roots (Fig 4.2.1; Fig 4.2.2). This reduction in growth

could be attributed to three types of effects created by saline environment i.e. osmotic stress

(Munns and Tester, 2008) Na+ toxicity and Na+ induced K+ deficiency (Munns et al., 2006),

which is also evident from present findings (Fig 4a-b). Shabala et al. (2016) reported that

potassium deficiency under saline conditions is caused by two major mechanisms. One of them

is membrane depolarization by salt that prompts the opening of the depolarization-activated

K+ efflux (GORK) channels resulting in a massive K+ loss. The second pathway is via reactive

oxygen species (ROS) activated K+-permeable non-selective (NSCC) cation channels.

Significant variation (76%) was observed among the genotypes for mentioned

physiological traits such as net CO2 assimilation rate, stomatal conductance and transpiration

(Fig 4.2.9). Kharchia 65 (2), TURACO (25), V-05121 (38) and V0005 (19) have improved

their physiological traits, which were strongly linked the stamp out of Na+ from plant (Figure

4.2.9). Enzyme activities increase in response to ROS production to save the photosynthetic

machinery by detoxify ROS (Apel and Hirt, 2004). Furthermore, Arslan and Ashraf et al.

70

(2012) proposed that K+ plays a very key roles in protein synthesis, stimulates photosynthesis,

osmoregulationand maintains cell turgor. It is also stated that salinity tolerance is linked with

K+ content (Ashraf, 2004) because of its key role in osmotic adjustment and competition with

Na+ (Ashraf and Foolad, 2007).

Physiological traits were negatively affected in salt-sensitive genotypes that

accumulated high Na+ in their leaves (Fig 4.2.9). TAM 200/TUI (20), V-87094 (16), CROC 1

(18), V-02156 (35), and BB # 2 (31) reflected the worst performance due to high concentration

of Na+ in their leaves (Fig 4.2.1). High concentration of Na+ can cause leaf senescence and loss

activity of photosynthetic system in wheat (El-Hendawy et al., 2005; Arslan and Ashraf, 2012)

that resulted in reduced carbon assimilation rate and ultimately low growth of seedling (Munns

et al., 2006). It has been well reported that NaCl causes increase the permeability of plasma

membrain and enhanced the production of reactive oxygen species (ROS) in wheat. ROS are

main source of damage to cells macromolecules such as DNA, protein and chloroplast under

salinity stress as a result of growth arrest (Gara et al., 2003).

Conclusions

Based on hydroponic studies, at seedling stage significant genetic variation for salinity

tolerance was detected in wheat genotypes. Forty genotypes out of 400 were selected as salt

tolerant on basis of low Na+ accumulation in leaf. Genotypes that accumulated low Na+ in their

leaves had also more K+/Na+ ratios, leaf chlorophyll content index and leaf dry mass. By using

biplots, based on physiological parameters the genotypes PVN (15), V0005 (19), V94195 (23)

TURACO (25), MAYA/PVN (27), PB24862 (29), BB # 2 (31), V-06129 (39), V-02156 (35),

V-05121 (38) and V-03094 (36) were screened as the best salt-tolerant genotypes and showed

good performance including the check genotypes LU26S and Kharchia 65. Biplot analysis is

good statistical way to measure level of salinity tolerance in wheat due to its advantages over

approaches such as relative or absolute salinity-tolerance indices. Sodium exclusion, higher

potassium uptake and improved K+/Na+ ratios were found to be reliable traits and selected

genotypes can be used in future wheat breeding program for the achievement of salt tolerant

in bread wheat.

71

Experiment 3: Identification of physiological markers associated with

salinity tolerance of wheat genotypes in saline soil (pot study).

Some of genotypes (fourteen salt tolerant, four sensitive and two check) were selected

from previous hydroponic experiments for further to explore detailed physiological responses

on normal and saline soil in pots.

4.3.1 Response of screened genotypes against different salinity levels

After smart selection in hydroponic studies wheat genotypes were further evaluated on

salt affected soil. Twenty genotypes (14 tolerant, 4 sensitive and two check LU26S; Kharchia

65; Table 3.3.2) were grown in pots at different salinity levels that expressed significant

differences (P ≤ 0.001) among genotypes (G), salinity levels (S) and their interaction (G*S)

for ionic, physiological, biochemical and yield related attributes.

4.3.2 Leaf Na+ and K+ contents

Significant variation was observed among genotypes for Na+ accumulation in leaf (Fig

4.3.1a). Under saline condition Na+ content increased in the leaves of all genotypes as

compared to plants grown on non-saline environment (Fig 4.3.1a). Among genotypes, V-

03094, V0005, V-02156 and V-04181 accumulated low Na+ content in their leaves as

compared to checks LU26S and Kharchia65 while TAM200/TUI, FRET2, PUNJAB 85 and

PBW343*2 had high Na+ concentrations in their leaf blade and considered as salt sensitive

genotypes (Fig 4.3.1a). K+ concentration and K+: Na+ ratio were found higher in control (non-

saline) plants (Fig 4.3.1b). Moreover, in saline environment V-03094, V0005, V-02156 and

V-04181 genotypes had higher K+ and K+: Na+ ratio as compared to checks and those

genotypes that accumulated high Na+ concentrations in their leaf blades (Fig 4.3.1b-c).

4.3.3 Leaf water relation attributes

Turgor potential (Ψp), osmotic potential (Ψs) and water potential (Ψw) were affected

due to exposure of plant to salinity stress in pots (Fig 4.3.2a-c). Leaf Ψw and Ψs of all

genotypes decreased (more negative) under saline regimes (Fig 4.3.2c and 4.3.2a). Maximum

reduction in leaf Ψw was recorded in V-04178, MAYA/PVN, V-04181 and Kharchia65 under

saline conditions (Fig 4.3.2c). However, leaf turgor potential (Ψp) was found higher (more

72

positive) in non-saline as compared to salt regimes (Fig 4.3.2b). Among genotypes Lu26S and

Kharchia65 had higher leaf Ψp followed by V-03094, V0005, V-02156 and V-04181 (Fig

4.3.2b). While minimum reduction in leaf water potential (Ψw) were observed in

TAM200/TUI, FRET2, PUNJAB 85 and PBW343*2 genotypes.

4.3.4 Relative water contents

Relative water content was significantly decreased in all genotypes under salt stress as

compared to control. Lowest relative water content was found in PBW343*2, PUNJAB 85 and

TAM200/TUI genotypes (Fig 4.3.7a) in saline regime.

4.3.5 Cell membrane injury

A cell membrane injury increased under stress as compared to non-stress plants (Fig

4.3.7b). Maximum injury was found in salt sensitive genotypes TAM200/TUI, FRET2,

PUNJAB 85 and PBW343*2 as compared to rest of genotypes (Fig 4.3.7b).


All leaf gas exchange parameters were significantly influenced by salt stress (Fig

4.3.3a-c). net CO2 assimilation rate (A), stomatal conductance (gs) and transpiration rate (E)

were found higher in control and expressively decreased in all genotypes in saline regime (Fig

4.3.3a-c). Genotypes Kharchia65, BB # 2, V-03094, V-02156 and V-04181 showed improved

performance at saline environment while genotype PBW343*2 PUNJAB 85 FRET2

TAM200/TUI had lower values of gas exchange parameters (Fig 4.3.3a-c).

4.3.7 Biochemical analysis

Leaf biochemical analysis indicated several modulations (Fig 4.3.4a-c). Chlorophyll

contents (a, b and total) decreased in the leaves of all genotypes during salt stress (Fig 4.3.4a-

c). Maximum chlorophyll a, b, and total chlorophyll content were observed in CHAM-4,

LU26S V-04181, PVN, and V-02156 and V-03094 genotypes in saline pots as compared to

salt sensitive genotypes (Fig 4.3.4a-c)

73

4.3.8 Non enzymatic antioxidants

Under saline regime non-enzymatic antioxidant, leaf phenolic and carotenoid were

decreased in all genotypes as compared to non-saline pot (Fig 4.3.5). In contrast, leaf proline

accumulation was higher at salt regime as compared to control in all genotypes (Fig 4.3.5b).

Maximum total phenolic and carotenoid pigment were observed in CHAM-4, LU26S V-04181,

PVN, V-02156 and V-03094 genotypes in saline pots. While genotypes V-03094 PEWEE'S'

V-04181 showed maximum proline accumulation in leaf (Fig 4.3.5a-b) under salt stress.

4.3.9 Chlorophyll fluorescence

Exposure of wheat genotypes against salinity stress marked decrease was recorded in

quantum yield of primary photochemical reaction (Fv/Fm) as compared to non-saline

environment (Fig 4.3.6b). Maximum quantum yield was recorded in Kharchia 65 followed by

LU26S, V-02156, and V-04181 in saline culture (Fig 4.3.6b). Salt stress induced slightly

increase electron transport rate (ETR) as compared plants grown on non-saline pots (Fig

4.3.6c). The highest values of ETR were recorded in Kharchia 65 followed by V0005, V-02156

and V-04181 (Fig 4.3.6c). Constant increase was measured in NPQ (non-photochemical

quenching) of all genotypes grown under salt stress condition (Fig 4.3.6a). Overall, salt stress

induced increase in NPQ. Maximum NPQ value was recorded in Kharchia 65 followed by

LU26S, V-02156, and V-04181(Fig 4.3.6a).

4.3.10 Yield related attributes

Significant variations (P < 0.001) were found among genotypes, salinity levels and their

interaction for the yield related components (Table 4.3.1 and Table 4.3.2). Under salt stress

significant decrease was recorded in plant height, spike length and number of spikelet spike-1

as compared to control (Table 4.3.1). Maximum plant height, spike length and number of

spikelet spike-1 were recorded in V0005, V-04181, V-02156 and V-03094 than checks (Table

4.3.1). Less production of fertile tillers and grains spike-1 were observed in plants matured in

salt medium as compared to control (Table 4.3.2). Genotypes V0005, V-04181, V-02156 and

V-03094 produced maximum fertile tillers and grains spike-1 than checks LU26S and Kharchia

65 (Table 4.3.2.). In contrast to grains spike-1, 100-grain weight found more in salt stressed

plants as compared to control. Minimum 100-grain weight was recorded in TAM200/TUI,

74

FRET2, FRONTANA and PBW343*2 genotypes (Table 4.3.2.) during salt stress. Moreover,

genotypes TAM200/TUI, FRET2, PUNJAB 85 and PBW343*2 harvested less grain and

biomasses yields as compared to check Lu26S and Kharchia65 (Table 4.3.2).

4.3.11 Stress susceptibility index (SSI) based on grain yield

Significant difference were found between the genotypes for SSI against the salinity

stress (Fig 4.3.8). Among genotypes YECORA-70 indicated the lowest SSI values (0.47)

followed by Lu26S (0.56) and V-04178 (0.58). TAM200/TUI presented the highest SSI value

(1.81) followed by FRET2 (1.68), PUNJAB 85 (1.52) and PBW343*2 (1.21) genotypes as

compared to checks Kharchia 65 and LU26S (Fig 4.3.8).

75

Fig 4.3.1 Influence of salt stress on (a) Na+ concentration in leaf, (b) K+ concentration leaf

and (c) leaf K+/Na+ in leaf of wheat genotypes. S and G indicate salinity treatments and

genotypes respectively and SxG indicates the interaction. Error bars indicate S.E (n=3).

76

Fig 4.3.2 Influence of salt stress on (a) leaf osmotic potential (-MPa), (b) leaf turgor potential

(-MPa) and (c) leaf water potential of wheat genotypes. S and G indicate salinity treatments

and genotypes respectively and SxG indicates the interaction. Error bar indicate S.E (n=3).

77

Fig 4.3.3 Influence of salt stress on (a) leaf photosynthetic rate (An), (b) leaf transpiration rate

(E) and (c) stomatal conductance (gs) of wheat genotypes. S and G indicate salinity treatments

and genotypes respectively and SxG indicates the interaction. Error bars indicate S.E (n=3).

78

Fig 4.3.4 Influence of salt stress on (a) leaf chlorophyll a, (b) leaf chlorophyll b and (c) leaf

total chlorophyll contents of wheat genotypes. S and G indicate salinity treatments and

genotypes respectively and SxG indicates the interaction. Error bars indicate S.E (n=3).

79

Fig 4.3.5 Influence of salt stress on (a) leaf phenolic, (b) leaf proline and (c) leaf carotenoid

of wheat genotypes. S and G indicate salinity treatments and genotypes respectively and SxG

indicates the interaction. Error bars indicate S.E (n=3).

80

Fig 4.3.6 Influence of salt stress on chlorophyll fluorescence of wheat genotypes. S and G

indicate salinity treatments and genotypes respectively and SxG indicates the interaction.


81

Fig 4.3.7 Influence of salt stress on (a) relative water content, (b) cell membrane injury of

wheat genotypes. S and G indicate salinity treatments and genotypes respectively and SxG

indicates the interaction. Error bars indicate S.E (n=3).

82

Fig 4.3.8 Stress susceptibility index (SSI) based on grain yield of different wheat genotypes at

high salinity level in pot culture. Grey bars and white bars indicates salt tolerant genotypes

and salt sensitive genotypes respectively, selected from Experiment 1 and 2 while black bars


83

Table 4.3.1 Mean plant height, spike length and number of spikelet spike-1 and

number of fertile tillers/plant, of 20 wheat genotypes grown in different salinities

Genotypes

Plant height (cm)

Spike length (cm)

No of spikelet spike-1

Fertile tiller plant-1

Control 15 dS m-1 Control 15 dS m-1 Control 15 dS m-1 Control 15 dS m-1

V-04178 53c-i 45.4k-o 15.43a-d 10.60jkl 18.6d-e 13.1i-n 3.67a-f 2.33e-i

MEHRAN-89 52.2e-j 40.9no 13.57e-h 7.73no 17.5efg 12.5lmn 3.67 a-f 2.33e-i

YECORA- 70 56.3a-f 47.6h-n 14.40c-g 10.47kl 15.4g-j 11.5m-p 3.67 a-f 2.33e-i

PEWEE'S' 58.8a-e 44.86k-o 14.77b-f 11.53ijk 15.2g-k 13.7h-m 3.67 a-f 2.00f-i

CHAM-4 52.8d-i 42.6l-o 14.23c-g 10.20klm 14.7h-l 11.0n-q 4.00a-e 2.00f-i

FRONTANA 59.74abc 43.6k-o 13.00ghi 8.00no 15.7gh 10.6n-q 3.67 a-f 1.67ghi

PVN 58.4a-e 45.8j-n 14.40c-g 10.80jkl 20.8a-d 12.0mno 3.67 a-f 2.67d-i

V0005 61.6a 48.7g-l 16.23ab 13.20fgh 21.3abc 15.2g-k 5.00ab 2.67d-i

TURACO 59.8ab 46.4i-n 14.03d-g 10.13klm 20.6a-d 13.0j-n 3.33b-g 1.33hi

MAYA/PVN 52.2e-j 49.94f-k 13.03ghi 8.80mno 15.9fgh 12.3lmn 4.00a-e 1.33hi

BB # 2 59.32a-d 56.1a-f 13.37fgh 9.87lm 19.3cde 12.8k-n 4.00a-e 1.67ghi

V-02156 59a-d 53.52b-h 16.53a 13.30fgh 21.6abc 14.7h-l 5.33a 3.00c-h

V-03094 52.2e-j 48.26h-m 16.40a 13.00ghi 23.1a 15.1g-k 5.33a 2.67 d-i

V-04181 55.4a-g 49.66f-k 15.83abc 12.97ghi 22.2ab 15.6ghi 4.67abc 3.33b-g

LU26S 56.4a-f 43.6d-i 15.07a-e 12.10hij 18.4def 13.9h-m 4.67abc 1.67ghi

Kharchia 58.6a-e 52.6k-o 15.60a-d 12.20hij 19.9b-e 13.8h-m 4.67abc 2.33 e-i

TAM200/TUI 55.8a-f 33.8p 15.20a-d 7.97no 17.5efg 8.8q 4.33a-d 1.00i

FRET2 60.8a 39op 15.50a-d 9.30lmn 16.0fgh 9.1pq 4.00 a-e 1.33hi

PUNJAB 85 56a-f 40.9no 14.57ghi 9.93mno 15.9fgh 9.1lmn 3.67 a-e 1.00hi

PBW343*2 57.2a-e 41.8mno 14.73b-f 7.67o 15.5g-j 9.6opq 4.33a-d 1.00i

CVCs 6.7790 1.6253 2.5752 1.9961

84

Table 4.3.2 Mean number of grains/spike, 100-gain weight/plant and

grain yield/plant of 20 wheat genotypes grown in different salinities

Genotypes

No of grain spike-1

(g)

100-Grain wt

(g)

Grain Yield

(g)

Shoot biomass

(g)

Control 15 dS m-1 Control 15 dS m-1 Control 15 dS m-1 Control 15 dS m-1

V-04178 32.2b-f 17.2l-p 1.43jkl 2.48efg 1.77c-h 1.30g-o 6.93a-d 2.97e-i

MEHRAN-89 31.4c-f 15n-r 1.39jkl 2.54d-g 1.90a-g 0.93k-p 6.10cd 2.89e-i

YECORA- 70 30.6d-g 15.4n-r 1.29jkl 3.26abc 1.70d-j 1.33f-n 6.23cd 3.37e-i

PEWEE'S' 30.8c-g 18k-p 0.91klm 2.64d-g 1.67d-k 0.87l-p 6.03d 3.28e-i

CHAM-4 33.2b-e 13.2o-r 1.72ij 2.21ghi 1.97a-g 1.23g-o 5.83d 3.36e-i

FRONTANA 24.4g-k 16.6m-q 1.49jk 1.66ij 1.93a-g 0.97j-p 6.23cd 2.53f-i

PVN 26f-j 15.2n-r 1.66ij 2.18ghi 1.87b-g 1.37e-m 6.80e-i 2.63 e-i

V0005 39ab 23i-m 2.67d-g 3.35abc 2.10a-e 1.30g-o 8.03e 4.17e

TURACO 28.4e-i 19k-o 1.27jkl 3.67ab 2.07a-f 1.30g-o 7.67e 4.13e

MAYA/PVN 22.6i-m 15.2n-r 1.26jkl 2.98cde 1.67d-k 1.10h-p 6.77a-d 3.73e-h

BB # 2 31.8c-f 14.4n-r 1.42jkl 2.91c-f 1.73c-i 1.00i-p 6.13cd 3.80e-h

V-02156 36.2a-d 26f-j 2.35fgh 3.79a 2.47abc 1.43e-m 7.83efg 4.00efg

V-03094 42.6a 23.6h-l 2.14ghi 3.45abc 2.63a 1.44e-m 8.03ef 4.03ef

V-04181 35b-e 17.8k-p 1.79hij 2.10ghi 2.30a-d 1.35f-m 6.27bcd 3.20 e-i

LU26S 33.8b-e 20.8j-m 1.38jkl 3.64ab 1.97a-g 1.43e-m 6.57a-d 3.83e-h

Kharchia65 37.6abc 23.6h-l 2.14ghi 3.10bcd 2.57ab 1.60d-l 6.00d 3.37 e-i

TAM200/TUI 30.8c-g 9.8qr 0.41m 1.70ij 2.27a-d 0.43p 6.07d 1.97i

FRET2 30.4d-h 12.8o-r 1.23jkl 1.72ij 2.27a-d 0.56op 6.50a-d 2.43ghi

PUNJAB 85 35b-e 11.8pqr 0.85lm 1.21jkl 1.90a-g 0.60nop 6.80a-d 2.10i

PBW343*2 32.2b-f 9.6r 1.74ij 2.21ghi 1.73c-i 0.80m-p 6.87a-d 2.33hi

CVCs 6.9071 0.5857 0.7479 1.5837

85

Discussion:

Selective genotypes (fourteen salt tolerant, four sensitive and two check) from

previous hydroponic experiments were further studied in normal and saline environments

developed in pots under wire house natural condition of Faisalabad. The main objective of

this experiment was to explore detailed physiological responses of selected wheat

germplasm under salinity stress.

Growth and yield of all genotypes decreased under saline regimes as compared to

control (normal soil) but all genotypes survived and contributed to grain yield (Table 4.3.2

and Table 4.3.2). Findings of this study reveal that all wheat genotypes produced yield

under saline regime and showed significant different yield potential. Significant yield

differences can also be associated with plant height, dry biomass, spike length, number of

spikelet per spike, number of grains per spike and thousand grain weight. Genotypes which

were screened as salt tolerant produced more yield and shoot biomass as compared to

sensitive ones. Sensitive genotypes, might be linked with high salt susceptibility index

(SSI) and cell membrane injury due to Na+ toxicity in growing embryo (Ashraf, 2004; Raza

et al., 2006; Afzal et al., 2007). Yield and biomass production are closely associated with

leaf ionic contents of wheat genotypes under salt stress (Ashraf et al., 2005) as discussed

earlier. The genotypes with maximal Na+ accumulation have been recognized as salt

sensitive in the past by many scientists and also confirmed by results of this study due to

low yield and biomass production (Ashraf et al., 2005).

These reductions might be associated with salinity induced decline in

photosynthesis capacity (Ashraf, 2004; Raza et al., 2006; Afzal et al., 2007). Moreover,

findings of this study are also evident of this decline as low Na+ and high K+ accumulator

genotypes V-02156, V-03094 and V-04181 also exhibited higher net CO2 assimilation rate

(A) as compared to genotypes TAM200/TUI, FRET2 and PUNJAB 8 which were high Na+

accumulators under salt stress. Likewise, salt stress of 15 dS m-1 adversely affected

photosynthetic efficiency of wheat genotypes especially in TAM200/TUI, FRET2 and

PUNJAB 8 which was linked to stomatal conductance (Fig 4.3.3a-c). Under salt stress

closures of stomata leads to reduction of CO2 assimilation to carboxylation sites. While

other most important factor of stomatal closer is due to alteration in cytosolic K+ to Na+

ratio (Ozgur et al., 2013). Because K is cofactor of more than 50 enzyme including enzyme

catalyzing chlorophyll biosynthesis (Shabala, 2003). Chlorophyll reductions might be due

to deficiency of K+ as depicted in this study (Fig 4.3.4a-c). Thus, a low cytosolic K+ to Na+

ratio in leaf tissues of wheat genotypes, might be responsible to reduce capacity of

86

photosynthesis in plant. In addition depletions of K+ from mesophyll cell, active the

processes of cell death (Shabala, 2000; Shabala et al., 2005; 2006) by increasing the

senescence of leaf due to increase the number of proteases (Shabala, 2009). In addition,

salinity induced, production of reactive oxygen species, destroys membranes and

macromolecule i.e. DNA, proteins (Mittler 2003; Vaidyanathan et al., 2003; Gara et al.,

2003; Candan and Tarhan 2003). Being membrane bounded and protein in nature more

chlorophyll destruction in Na+ includer genotypes is linked with these ROS attack due to

absence of appropriate antioxidant system (Ashraf et al., 2009). These destructions might

be also reasons of less photosynthesis and ultimately less growth and yield.

Another reason of reduction in growth could be attributed salinity induced

modulation in water relations of plant, as excess presence of salts in growth medium also

disturbs water uptake and cause osmotic stress to plants (Munns and Tester, 2008). For the

normal cell function and growth of plant, appropriate water relation is very important

salinity tolerance mechanism to maintain the turgor of plant cell (Jensen et al., 2000) as

confirmed by this study (Fig 4.3.2). Water relations have been directly associated with

gaseous relations and ionic contents (Adolf et al., 2012).

Under stress, ROS has also been reported to destroy photosynthetic machinery as

in Fig (4.3.6) There are two primary photosystems (PS I and PS II) exist in plant, of these

two, photosystem II (PS II) is more sensitive to adverse effects of salt stress (Mehta et al.,

2010). Chlorophyll fluorescence is a good indicator to quantify the salt induced destruction

in photosynthetic system (Mehta et al., 2010) Damage to photosystem II has been studied

using this technique. ROS degrades various protein, which are necessary for hooking in of

pycobilisomes to thylakoids (Ashraf, 2004; Nawaz et al., 2010). ROS burst destroys

thylakoid membranes, resulting into modulations in membrane protein profiles which leads

to deceased activity of OEC (oxygen evolving complex) of PS II and increased working of

PS I. Plants grown under salt regime down regulate the PS II for improving efficiency of

excitation energy (Fv/Fm) (Fig 4.3.6a). Which was reported significant effect of salinity on

Fv/Fm (Houimli et al., 2008). Damage of PS II using chlorophyll fluorescence could yield

meaningful result, which can be used good physiological indicator for screening of salt

tolerance germplasm at different growth stages (Mehta et al., 2010).

Leaf proline, phenolics and carotenoid contents increased significantly in Na+

excluder genotypes as compared to includers (Fig 4.3.5a-c) which can play their role

against salt stress. Although the level of non-enzymatic antioxidants was increased but not

significant enough to contribute to lower osmolality of cytosol thus these compounds might

87

be had role in osmo-protection rather than osmotic adjustments in order to safe cell from

salt induced oxidative destructions (Adolf et al., 2012). Furthermore, Cuin and Shabala

(2005; 2007) proposed that osmolytes such as K+ play a key role in osmotic adjustment

while proline and phenolic act as osmoprotectnat to mitigate effect of salinity caused by

Na+ toxicity.

The current study shows that genotypes TAM200/TUI, FRET2, PUNJAB 85 and

PBW343*2 were emerged as hyper accumulator of Na+ in leaf than check genotypes,

Kharchia 65 and LU26S (Fig 4.3.1) which seems that these wheat genotypes are salt

includers and sensitive to salts. In other case, genotypes which accumulated less Na+ in

their leaves, can be said as Na+ excluders which shows salt tolerance. This efflux of Na+

might be due to exclusion of Na+ from leaves (Hussain et al., 2003; Din et al., 2008), low

Na+ influx at root boundary, and controlled unloading in the stele (Munns, 2006; Tester and

Davenport, 2003). In the meantime, these genotypes had also high leaf K+ contents and

more K+/Na+ ratios (Fig 4.3.1). Additionally, Na+ and K+ uptake occurs via common protein

thus both ions compete to enter in the cell under salt stress conditions (Pervez et al., 2004).

Higher K+/Na+ ratios of tolerant genotypes show more selective uptake of K+ than Na+

(Munns et al., 2006). Excessive Na+ in the rhizosphere had negative impact on uptake of

K+ in plants (Ashraf, 2005). It is also stated that salt tolerance is linked with K+ content

(Ashraf and Fold, 2003) because of its role in osmotic adjustment and competition with

Na+ (Ashraf et al., 2009). Many researchers reported this salinity induced decrease in leaf

K+ and exposure of plants to salinity increases, cause more reduction in accumulation of

K+ ( Zhu et al., 2001).

The results of current study show that leaf K+/Na+ ratio decreased in all wheat

genotypes at saline regimes but drastic reductions were recorded in Na+ includer genotypes.

Researchers believed that one of the important characters of glycophytes to tolerate salt

stress is maintenance of appropriate K+/Na+ ratio (Tester and Davenport, 2003). High

accumulation of detrimental ion (Na+) in salt-sensitive genotypes also leads to abrupt

decrease in K+ concentration and K+/Na+ ratio, which indicates physiological damages due

to ion toxicity (Zheng et al., 2010). In plants salinity tolerance deepened on K+ to Na+ ratio,

carbohydrates, proteins contents, amino acid and activities of antioxidant enzyme (Hamada

and El-Enany, 1994). However, photosynthetic pigments e.g. chlorophyll contents (a and

b) were important physiological mechanism which was more strongly linked with salinity

tolerance in wheat (Munns et al., 2006. Higher K+ to Na+ ratio could also be attributed to

an efficient efflux of Na+ from the cell or more influx of K+ over Na+ (Munns et al., 2006).

88

This efflux of Na+ is due to Na+/ H+ antiporter that located on plasma membrane (Munns

and Tester, 2008). Loci Nax1 and Nax2 located in chromosomes 2A and 5A respectively,

controlling influx of Na+, have been found in wheat genotype (Lindsay et al., 2004) and

this molecular marker usually used in wheat breeding program to develop varieties with

low Na characteristics

Conclusion

Genotypes V-02156, V-03094, V0005, TURACO, PVN performed better

physiologically and produced more grain yield which was strongly linked with low Na+

accumulation in leaves and high K+ to Na+ ratio as compared to genotypes that were hyper

accumulators of Na+ in their leaves. Leaf proline and phenolic found higher in all genotypes

under salt regimes. Poor performance of sensitive genotypes was due to both osmotic and

ionic stresses.

89

Experiment 4: Agronomic and physiological performance of selected

wheat genotypes on saline-sodic soil

Twenty genotypes (selections from previous experimental screenings; Table 3.4.1)

were further tested on saline-sodic field in order to evaluate yield response and to establish

physiological and biochemical markers for salt tolerance in selected hexaploid wheat lines.

The results obtained from this experiment are given below.

4.4.1 Na+ and K+ content

Significant genetic variation was observed among genotypes for Na+ concentrations

when sampled from salt affected field. Genotypes TAM200/TUI, FRET2, PUNJAB 85 and

PBW343*2 were emerged as hyper accumulators of Na+ as compared to check Kharchia

65 and LU26S (Fig 4.4.1a.) Increased concentration of K+ was noted in the leaves of

Kharchia 65 followed by V-03094, V0005, Lu26S and V-02156, meanwhile highest leaf

K+/Na+ ratio recorded in V0005 genotype followed by Kharchia65 and V-03094 (Fig

4.4.1b-c). Maximum K+ use efficiency was measured in V0005 and Kharchia65 while other

genotypes showed intermediate response except genotypes TAM200/TUI, FRET2,

PUNJAB 85 and PBW343*2 that had lowest K+ use efficiency (Fig 4.4.1d).

4.4.2 Biochemical attributes

Substantial differences were observed for leaf chlorophyll contents of salt stressed

genotypes (Fig 4.4.2a-c). Maximum chlorophyll a was observed in V-02156 genotype

followed by LU26S, V-04181, MAYA/PVN and V-03094 while genotypes PBW343*2,

MEHRAN-89 and FRET2 had lower chlorophyll contents a as compared to check

Kharchia65 and Lu26S (Fig 4.4.2a). Differences were observed among genotypes for

chlorophyll content ‘b’ contents (Fig 4.4.2b). Genotypes V-04178, PEWEE'S' showed

highest value while PBW343*2, PUNJAB 85 and FRET2 had less chlorophyll content b as

compared to checks Lu26S and Kharchi65 (Fig 4.4.2b). Meanwhile genotypes V-03094,

V0005 and V-02156 had maximum values of leaf total chlorophyll contents (Fig 4.4.2c).

4.4.3 Non-enzymatic antioxidants

Significant variations were recorded for proline accumulation and total phenol in

leaf among the genotypes (Fig 4.4.3). Highest proline accumulation was recorded in

Kharchia 65 under saline regime followed by V-03094, FRONTANA, BB # 2 and V0005

90

while maximum leaf total phenol were observed in V-03094 followed by Kharchia 65, V-

04181, V0005 and V-02156. Meanwhile highest carotenoids were observed in Kharchia 65

and PEWEE'S' followed by MEHRAN-89, V-02156 and PVN (Fig 4.4.3c). Significantly

decreased level of non-enzymatic antioxidants (leaf total phenolic, leaf proline and

carotenoid) in leaves were recorded in genotypes TAM200/TUI, FRET2, PBW343* and

PUNJAB 85 as compared to Kharchia 65 and LU26S (Fig 4.4.3).

4.4.4 Crop stand establishment

Data regarding percentage of crop density is presented in (Fig 4.4.4). Plant

emergence on saline-sodic soil was observed significantly affected in wheat genotypes.

Genotypes which had more percentage density showed poor emergence as described in (Fig

4.4.4), while genotypes Kharchia 65 (names) exhibited more plant emergence followed by

V-04178 and CHAM-4.

4.4.5 Yield and yield related attributes

Statistically significant differences were found between the genotypes for yield

related attributes e.g. plant height, spike length and number of spikelet spike-1(Table 4.4.1

and 4.4.2). Significant variations were noted in all genotypes for plant height, spike length

and number of spikelet spike-1. Kharchia 65 had highest plant height followed by V0005,

CHAM-4 and V-03094 while maximum spike length was recorded in V-04181 which were

followed by BB # 2, MEHRAN-89 and V-03094. Maximum spikelet spike-1 were observed

in genotypes YECORA- 70, V-02156 and V-04181. Overall among genotypes

TAM200/TUI, FRET2, PBW343* and PUNJAB 85 had poor performance as compared to

checks Kharchia 65 and LU26S for plant height, spike length and no spikelet spike-1 (Table

4.4.1)

Substantial genetic variation was observed among genotypes for number of grains

per spike, thousand grain weight, grain and biological yields (Table 4.4.2). Maximum

number of grain per spike were recorded in V0005 which was followed by V-02156,

Kharchia 65 and V-03094 meanwhile genotypes TAM200/TUI, FRET2, PBW343* and

PUNJAB 85 had lowest value for this attribute (Table 4.4.2). Maximum thousand grain

weight was noted in genotypes MAYA/PVN and BB # 2 followed by MEHRAN-89 and

V0005. Overall, Kharchia 65 produced maximum grain and biological yields which were

followed by V0005, V-02156 and V-03094 meanwhile TAM200/TUI, FRET2, PBW343*

91

MEHRAN-89 and PUNJAB 85 produced less biomass and grin yield as compared to check

LU26S and Kharchia 65 (Table 4.4.2).

4.4.6 Biplot of yield related attributes

Polygon view of GGE-biplot (Fig 4.4.5) describe the performance of genotypes

based on interaction between the genotypes) and traits. The vector view of GGE-biplot (Fig

4.4.6) shows the interrelationship between traits. Principal components sums (PC1 and

PC2) explained total variation between genotypes based on traits. Two traits are positive

correlated if the angle between their trait vectors is less than 90°. If the angel is greater than

90 then it is negative correlation between the traits.

Biplot analysis for yield related traits of 20 genotypes showed distinct variation

under salt affected soil (Fig 4.4.4). Genotypes were divided into six sectors based on the

performance of traits. For biplot analysis of yield components, genotypes placed on corners

(vertex or most responsive genotypes) were Kharchia65 (16), CHAM-4 (4) FRONTANA

(6), BB # 2 (11), MEHRAN-89 (2), PUNJAB 85 (19) and TAM200/TUI (17). Most

responded sectors were sector one and six in which tester (traits) fall (Fig 4.4.4). The first

sector represents yield related traits such as plant height (PH), grain yield (GY), no grain

per spike (NOG) and biological yield (BY) and showed genotype Kharchia 65 (16) as the

best and the most favourable for above traits followed by V-03094 (13) V-02156 (12), V-

04181 (14), PEWEE'S' (8), LU26S (15), CHAM-4 (5), FRONTANA (6). The six sectors

expressed spike length (SL), number of spikelet per spike (NOS) and thousand grain weight

(THGW) with genotype BB # 2 (11) as the most favourable followed by MAYA/PVN (10),

YECORA-70 (3) and V-04178 (1). The remaining other vertex genotypes, TAM200/TUI

(17), PUNJAB 85 (19), FRET2 (18) and PBW343*2 (20) showed the poorest performance

for the yield related traits (Fig 4.4.4) as they were located away from marked traits on the

biplot.

The vector view of the GGE-biplot showed positive correlation between yield

related traits measured at salt affected soil (Fig 4.4.6). Principal components sums (PC1

and PC2) explained 82.6% variation between genotypes based on performance of yield

related traits. Across 20 tested genotypes, positive correlation was found in all yield related

traits (Fig 4.4.5). Thousand grain weight had positive but poor correlation with others traits

such as plant height (PH), grain yield (GY), number of grains per spike (NOG) and

biological yield (BY; Fig 4.4.5).

92

Fig 4.4.1 Leaf Na+ concentration (a), leaf K+ concentration (b), leaf K+ use efficiency (c)

and leaf K+/Na+ ratio (d) of wheat genotypes grown on salt affected soil. Grey bars and

white bars indicates salt tolerant genotypes and salt sensitive genotypes respect, selected

from Experiment 1 and 2 while black bars indicates check genotypes. Error bars indicate

S.E (n=4).

93

Fig 4.4.2 Leaf chlorophyll a (a) leaf chlorophyll b (b) and leaf total chlorophyll contents

(c) of wheat genotypes grown on salt affected soil. Grey bars and white bars indicates salt

tolerant genotypes and salt sensitive genotypes respectively, selected from Experiment 1

and 2 while black bars indicates check genotypes. Error bars indicate S.E (n=4).

94

Fig 4.4.3 Leaf phenolic (a), Leaf proline (b) and Leaf carotenoid (c) of wheat genotypes

grown on salt affected soil. Grey bars and white bars indicates salt tolerant genotypes and

salt sensitive genotypes respectively, selected from Experiment 1 and 2 while black bars


95

Fig 4.4.4 Percentage of crop density of wheat genotypes grown on salt affected soil. Grey

bars and white bars indicates salt tolerant genotypes and salt sensitive genotypes

respectively, selected from Experiment 1 and 2 while black bars indicates check genotypes.


Percentage density (m-2): 1 = 90 % Emergence or more (very good); 2 = 80–89 % (good);

3=70–79 % (acceptable), 4 = 60–69 % (poor)

0

1

2

3

4

5

Perc

enta

ge o

f cro

p d

ensity

Genotypes

96

Table 4.4.1 Mean number of tillers, plant height, spike length and number of spikelet spike-1 of

wheat genotypes grown in saline-sodic soil.

Genotype Plant height (cm) Spike length (cm) No of spikelet spike-1

V-04178 59 ef 8.28 a-e 14.8 a-e

MEHRAN-89 67.2 bcde 9.82 abc 14.2 b-e

YECORA- 70 58.2 ef 9.16 a-e 18 a

PEWEE'S' 60.8 ef 7.86 a-e 14 b-e

CHAM-4 75.2 bc 7.36 b-e 13.8 b-e

FRONTANA 64 def 8.16 a-e 13.6 b-e

PVN 58 ef 8.2 a-e 14 b-e

V0005 75.4 b 8.3 a-e 14.6 a-e

TURACO 62.2 ef 9.12 a-e 15.4 abc

MAYA/PVN 59.8 ef 8.08 a-e 14.6 a-e

BB # 2 61.4 ef 10.02 ab 14.8 a-e

V-02156 65.6 bcde 9.12 a-e 16.4 ab

V-03094 73.4 bcd 9.58 a-d 15.2 a-d

V-04181 67.2 bcde 10.36 a 15.8 ab

LU26S 65.2 cde 9.98 abc 15.8 ab

Kharchia65 94.2 a 9.92 abc 14.6 a-e

TAM200/TUI 42.6 g 6.68 e 9.2 f

FRET2 39.8 g 7.2 cde 11.4 ef

PUNJAB 85 36.6 g 6.94 de 11.6 def

PBW343*2 55 f 7.68 a-e 12.0 c-f

CVCs 10.119 2.8003 3.7059

97

Table 4.4.2 Mean number of grains/spike, 1000-gain weight and grain yield and biological of 20 wheat

genotypes grown in salt affected soil.

Genotype No of grain per spike

(g)

1000- Grain wt (g) Grain yield (kg ha-1) Biological yield (kg

ha-1)

V-04178 25.6 bcd 34.35 abc 2826 cde 5211 cd

MEHRAN-89 22.2 de 35.41 ab 1687.5 g 3690 i

YECORA- 70 22.8 de 34.00 a-d 2205 f 4207.5 g

PEWEE'S' 24.4 cde 33.98 a-d 2088 f 3978 gh

CHAM-4 29.2 abc 30.41 cd 2763 e 4630.5 f

FRONTANA 30.8 ab 29.65 d 2812.5 cde 4792.5 ef

PVN 21.6 def 31.74 bcd 2911.5 b-e 5026.5 de

V0005 34 a 34.75 abc 3010.5 bcd 5620.5 ab

TURACO 29.2 abc 32.16 bcd 2925 b-e 5220 cd

MAYA/PVN 26.6 cd 36.9 a 2781 de 5188.5 d

BB # 2 28.8abc 36.9 a 2977.2 b-e 5047.2 d

V-02156 34a 31.74 bcd 3105 ab 5445 bc

V-03094 33.6 a 32.25 bcd 3037.5 bc 5512.5 ab

V-04181 30.6 ab 32.04 bcd 2929.5 b-e 4999.5 de

LU26S 25 bcd 31.37 bcd 2956.5 b-e 5026.5 de

Kharchia65 33.8 a 31.88 bcd 3294 a 5701.5 a

TAM200/TUI 10.2 g 30.71 cd 697.5 i 2767.5 k

FRET2 18.6 ef 30.41 cd 1701 g 3771 hi

PUNJAB 85 16.2 f 33.06 abcd 1206 h 3276 j

PBW343*2 9.6 g 31.28 bcd 990 h 3060 j

CVCs 5.9626 4.4806 245.79 252.71

98

Fig 4.4.5 Yield relating attributes plant height (PH), grain yield (GY), no of grain spike-1

(NOG), Biological yield (BY), 1000 grain weight (THGW) and spike length (SL) grown on

salt effected soil. See Table 3.4.1 for codes of the genotypes.

99

Fig 4.4.6 Vector view showed relationship among yield relating attributes of plant height

(PH), grain yield (GY), no of grain spike-1 (NOG), Biological yield (BY), 1000 grain weight

(THGW) and spike length (SL) grown on salt effected soil. See Table 3.4.1 for codes of the

genotypes.

100

Discussion

Wheat germplasm with contrasting to their sodium accumulation was used to assess

the effect of Na+ exclusion on stand establishment, yield component, biochemical and

physiological traits in saline-sodic field. Crop stand establishment (in percentage crop

density) of wheat genotypes were markedly reduced by the salt stress as compared to check

variety Kharchia 65 (Fig 4.4.4). In this study, crop stand establishment and yield reduction

was less in those genotypes that accumulated low Na+ than high Na+ genotypes. Poor crop

stand establishment was observed in genotypes FRET2, PUNJAB 85 and PBW343*2

which might be due to salt-sensitivity of genotypes to saline-sodic soil characterized by

high Na+ (Fig 4.4.1). The mechanism by which salt stress affect the crop stand

establishment can be via its osmotic and its Na+ specific effect due to accumulation of salt

in leaves or to the salt outside the roots (Munns, 2002; Munns et al., 2006; Ali et al., 2008;

Zheng et al., 2010).

Significant genetic variation (86.2%) for yield component was detected between

wheat genotypes by using biplots (Fig 4.4.5), and the genotypes (8) V0005, (11) BB#2,

(12)V-02156, (13) V-03094 and (14) V-04181 were identified as good yield performer

genotypes including (16) Kharchia 65 (Fig 5a, b), these wheat genotypes are salt excluder

and most of salt excluder genotypes have been recognized as salt tolerant in the past by

many scientists and also confirmed from findings of this study due to reduce less yield and

biomass production as compared to high Na+ genotypes (Table 4.4.1; Table 4.4.2; Fig

4.4.5). This low Na+ might be due to efflux of Na+ from leaves (Hussain et al., 2003; Din

et al., 2008). Loci Nax1 and Nax2 located in chromosomes 2A and 5A respectively,

controlling influx of Na+, have been found in wheat genotype (Lindsay et al., 2004) and

this molecular marker usually used in wheat breeding program to develop varieties with

low Na+ characteristics.

Munns et al. (2006) found that low Na+ genotypes had also high grain yield (Table

4.4.2) which was strongly linked with high leaf K+ contents and K+/Na+ ratios as evident

of current study (Figure 4.4.1b, d). It is also stated that salt tolerance is linked with K+

content as evident of this work (Ashraf et al., 2002) because of its role in osmotic

adjustment and competition with Na+ (Ashraf and Foolad, 2007). Researchers believed that

one of the important characters of glycophytes to tolerate salt stress is the maintenance of

appropriate K+/Na+ ratio (Tester and Davenpor, 2003). The results of current study show

that leaf K+/Na+ ratio decreased in all wheat genotypes as compared to check Kharchia65

but drastic reductions were recorded in Na+ includer genotypes (Fig 4.4.1d). High

101

accumulation of detrimental ion (Na+) in high Na+ genotypes also leads to abrupt decrease

in K+ concentration and K+ to Na+ ratio, which indicates physiological damages may occur

from ion toxicity (Zheng et al., 2010). In plants salinity tolerance depended on K+ to Na+

ratio, carbohydrates, proteins contents, amino acid and activities of antioxidant enzyme

(Hamada and El-Enany, 1994).

However, photosynthetic pigments e.g chlorophyll contents (a and b) were

important physiological mechanism which was more strongly linked with salinity tolerance

in wheat (Munns et al., 2006). Significant difference were observed among genotypes for

chlorophyll contents (chlorophyll a, b; Fig 4.4.2a, b) and non-enzymatic antioxidant (leaf

proline, phenol and carotenoid; Fig 4.4.3a-c), when grown on saline-sodic soil. The extent

of salt stress, also caused reduction in the photosynthetic pigments such chlorophyll a and

b. This depends on salt-tolerance potential of plants (Hamada and El-Enany, 1994). In term

of photosynthetic pigments, low Na+ genotypes showed less reduction as compared to high

accumulator genotypes (Fig 4.4.2a-c). Possibly higher grain yield of low Na+ genotypes

might be due to more supply of assimilates from leaves to growing grains during grain

filling stage. This was linked with prolonged retention of chlorophyll in leaves of low Na+

accumulator genotypes which have ability to efflux more Na+ from leaf and delayed the

time at which toxicity level reached (Hussain et al., 2003). In response to reduce the rate

the photosynthesis and formation of reactive oxygen species plant increase the process of

biochemical and enzyme activity that protect the photosynthesis machinery and detoxify

the ROS (Apel and Hirt, 2004). Contrary to leaf chlorophyll contents (Fig 4.4.2a, b), leaf

proline, phenolic and carotenoid contents (Fig 4.4.3a-c) increased significantly in low Na+

genotypes as compared to high Na+ genotypes. Furthermore, Cuin and Shabala (2005;

2007) proposed that osmolytes such as K+ play a key role in osmotic adjustment while

proline and phenolic act as osmoprotectnat to mitigate effect of salinity caused by Na+

toxicity.

Conclusion

Taken together all physiological indices it can be concluded that salt tolerance in

wheat genotypes under saline-sodic regimes was linked with low Na+ accumulation

(exclusion), better K+/Na+ ratio and biochemical indicators proline, phenols. These

attributes are suggested as potential markers for salt tolerance. Under saline field overall

response of V-02156, V-03094, V-04181, V0005, V-04178 and PVN was as good salt

excluders which was linked with low Na+ accumulation in leaf and better non- enzymatic

antioxidants activities and have high yield.

102

GENERAL DISCUSSION

Most of previous studies on salinity tolerance mechanism are based on small scale

or limited number of varieties (Brugnoli and Lauteri, 1991). The small and limited number

is not enough to satisfy plant breeders to screen and develop salt tolerant varieties and the

outcome can be affected by genetic background of screened varieties (Brugnoli and Lauteri,

1991; Chen et al., 2007). Furthermore, Bhutta and Amjad (2015) revealed that there is

decreasing propensity of genetic diversity in wheat genotypes and it may have impact on

future plans of breeding for salt tolerance in Pakistan.

Therefore, to develop the salt-tolerant germplasm, genetic variation is perquisite for

any wheat breeding program. Currently plant scientists are attempting to identify suitable

physiological modulations helpful in salt tolerance in available wheat germplasm. Salinity

tolerance in wheat is linked with low transport rates of Na+ to shoots and high uptake of K+

over Na+ (Munns et al., 2006). This thesis investigates the screening of a large number of

wheat (Triticum aestivum L.) cultivars to find genetic variation for salinity tolerance across

local and exotic germplasm by performing two years hydroponic studies followed by field

experimentation. Identification was done on Na+ exclusion basis from leaf blade.

Significant genetic variation in salt tolerance was detected in wheat genotypes for growth,

ionic, and physiological traits. Genotypes that have low influx of Na+ in their leaves showed

the maximum tolerance against the salinity by improving K+ to Na+ ratio and chlorophyll

index. This low influx of Na has been associated with Na+ exclusion from leaves (Hussain

et al., 2006; Din et al., 2008). Furthermore, tolerant genotypes may also have sophisticated

K+ regulation system as Shabala and Pottosin (2014) described two-pore K+ channels and

shaker type; non-selective cation channels which aids permeability of K+ and transporters

(HKT, KUP/HAK/KT and K+/H+). It is also reported that if ratio K+ to Na+ is wide then

the variety is salt-tolerant, if it is narrow then the variety is called salt-sensitive (Tester and

Davenport, 2003). Higher K+ to Na+ ratio could also be attributed to an efficient efflux of

Na+ from the cell or more influx of K+ over Na+ (Munns et al., 2006). This efflux of Na+ is

due to Na+/ H+ antiporter that located on plasma membrane (Munns and Tester, 2008). Loci

Nax1 and Nax2 located in chromosomes 2A and 5A respectively, controlling influx of Na+,

have been found in wheat genotype (Lindsay et al., 2004).

Selected genotypes (from hydroponic studies) with contrasting to their sodium

accumulation were also used to assess the effect of Na+ exclusion in saline soil. Growth

and yield of all selected genotypes were decreased under saline soil but all survived.

103

Genotypes that produced maximum grain yield have also improved their osmoprotectant

attributes which was strongly linked with low Na+ and considers as salt tolerant genotypes,

While genotypes have poor performance for yield might be linked with high salt

susceptibility index (SSI) and cell membrane injury due to Na+ toxicity in growing embryo

as evident from this study (Ashraf, 2004; Raza et al., 2006; Afzal et al., 2007). Yield and

biomass production are closely associated with leaf ionic contents, better physiological and

biochemical traits under salt stress (Ashraf et al., 2005; Iqbal et al., 2007). The most of salt

excluder genotypes have been recognized as salt tolerant in the past by many scientists and

also confirmed by results of this study due to less reduction in grain yield and biomass

production (Ashraf et al., 2005).

Other key reason for reduced growth in saline regime was the modulation in water

relation attributes, as excess concentration of salt in growth medium disturbed the uptake

of water, which resulted into the imposition of osmotic stress (Munns and Tester, 2008).

The appropriate water relation is very important and crucial to maintain turgor for normal

cell functioning as vital mechanism of salt tolerance crops. Generally, salt tolerant

genotypes/cultivar maintained water relation attributes and exhibit higher water use

efficiency (WUE) than the salt sensitive genotypes (Jensen et al., 2000) as confirmed by

this study. Higher WUE is one of major adaptations in plants, which grown on salt regime

soil (Hsiao, 1973; Morgan, 1984; Ashraf, 2003). Increase in WUE may have been due to

reduction in transpiration rate by salinity stress (Pagter et al., 2009; Gorai et al., 2010).

Physiological attributes such as gas exchange traits e.g. net CO2 assimilation rate,

transpiration rate and stomata have been played a prime role in screening of salt tolerant

plants. Generally salinity caused the marked reduction in stomatal conductance, net CO2

assimilation rate and transpiration rate in crop as depicted in current study (Ashraf, 2004;

Nawaz et al., 2010). Present study elucidates that low Na+ genotypes had less reduction in

stomatal conductance, net CO2 assimilation rate and transpiration as compared to high Na+

genotypes. Reduction in gas exchange traits under salt stress due to lower water potential

of root resulted in the closure of stomata (Zheng et al., 2010). The extent of salt stress, also

caused reduction in the photosynthetic pigments such chlorophyll a and b, this depends on

salt-tolerance potential of plants (Hamada and El-Enany, 1994). In term of photosynthetic

pigments, low Na+ genotypes showed less reduction as compared to high accumulator

genotypes. Possibly higher grain yield of low Na+ genotypes might be due to more supply

of assimilates from leaves to growing grains during grain filling stage. This was linked with

prolonged retention of chlorophyll in leaves of low Na+ accumulator genotypes which have

104

ability to exclude more Na+ from leaf and delayed the time at which toxicity level reached

(Hussain et al., 2003). Under salt stress assessing the chlorophyll fluorescence is another

important character to quantify the salt induced destruction in photosynthetic apparatus.

Using this technique, damage to photosystem II has been studies. In saline regimes the

production of ROS in plant destroys the thylakoid membrane, which leads to reduce the

activity of oxygen evolving complex (OEC) of PS II and significant effect on Fv/Fm as

observed in present study (Houimli et al., 2008; Nawaz et al., 2010).

Conclusion

It is concluded that, the hexaploid inbred wheat that accumulated low Na+ content

in their leaves had good biochemical, antioxidant and high grain production under saline

environment. Proline accumulation in leaf is a good physiological indicator of salt tolerance

in hexaploid wheat under salt conditions, which was highly linked with low Na+. Similarly

increased phenolic concentration is better marker to screen the germplasm for salt

tolerance. Under salt stress, distinct variation was recorded among the genotypes for

chlorophyll fluorescence, which is used as physiological marker against salinity. Grain

yield can be used as screening criteria for salt tolerance because significance variations

were recorded among the genotypes for grain yield under salt regime which was strongly

linked with Na+ concentration in leaf. After very smart selection from hydronic, pot and

field studies it concluded that V-02156, V-03094, V0005, TURACO and PVN identified

as the best Na+ excluders genotypes and had better performance with better physiological

and have higher yield attributes under salt stress and can used in breeding programs to

introduce the character for low Na+ accumulation in commercial hexaploid cultivars

105

Chapter-5

SUMMARY

Salinity is a leading cause of low productivity and threat to burgeoning world population including Pakistan. Wheat (Triticum aestivum

L.) is a staple food of country which is salt sensitive. Screening of genotypes on physiological and biochemical bases is imperative pre-requisite

to develop salt tolerant germplasm for farmers holding salt affected lands. Therefore, wheat genotypes were tested for salt tolerance through

following wire house, pot and field trials under agronomy ecological conditions of Faisalabad and Pindi Bhatian, Punjab, Pakistan. The

summery table of research study is given in table S1.

Summery Table S1 of Research Study

Study Phase No. genotypes Type of study Salinity stress Traits measured Selected

T S Total

Phase I 400 Hydroponic 200 mM NaCl Ionic, growth 25 15 40

Phase II 40+2C Hydroponic 0 mM, 100 mM and 200

mM NaCl

Ionic, growth and physiological

traits

14 4 18

Phase III 18+2C Pot study Control (1.41 dS m-1) and

15 dS m-1

Ionic, growth, physiological,

biochemical, antioxidant,

chlorophyll fluorescent and yield

components

14 4 18

Phase IV 18+2C Field study

(Saline-sodic soil)

ECe 9.5 dS/m, SAR 24

mmol/L and pH 8.7

Yield and physiological traits 6 0 6

T (Tolerant); S (Sensitive); C ( Check varieties i.e. Kharchia 65 and LU26S)

106

First study elucidates the identification of novel salt tolerant germplasm from very

large diverse pool (four hundred accessions of different origin) using fast and efficient

physiologically-based screens in hydroponic culture. Forty genotypes (25 salt-tolerant and

15 salt-sensitive genotypes) out of 400 were selected on Na+ exclusion basis in leaf blade.

Genotypes that accumulated low Na+ in their leaves had also more K+/Na+ ratios, leaf

chlorophyll content index and leaf dry mass as compared to salt sensitive genotypes.

In second experiment, genotypes selected from first study were again evaluated next

season by exploring more physiological details to verify screening criteria of Na+ exclusion,

developed in study I. Forty-two genotypes (15 salt-sensitive, 25 salt-tolerant, and two

checks i.e. LU26S and Kharchia 65) were grown at various salinity stress levels that

expressed significant differences (P ≤ 0.001) for ionic (leaf +root), gaseous exchange and

growth characteristics were chosen for this study. By using biplots, based on physiological

and measured traits the genotypes PVN (15), V0005 (19), V94195 (23) TURACO (25),

MAYA/PVN (27), PB24862 (29), BB # 2 (31), V-06129 (39), V-02156 (35), V-05121(38)

and V-03094 (36) were screened as a tolerant genotypes and had good performance,

including checks genotypes LU26S and Kharchia 65. 84.2%, 87.7% and 76% of total

variation observed among genotypes based on concentration of ions in leaf and root and

physiological traits (net CO2 assimilation rate, stomatal conductance and transpiration

rate)Photosynthesis rate, transpiration rate and stomatal conductance) respectively.

Physiological traits were negatively affected in salt-sensitive genotypes that accumulated

high Na+ concentrations in their leaves and roots, TAM 200/TUI (20), V-87094 (16),

CROC 1 (18), V-02156 (35), and BB # 2 (31) reflected the worst performance due to hyper

accumulation of Na+ in their leaves and roots.

Selected twenty genotypes including two check Kharchia65 and LU26S from

hydroponic studies were further tested in pots and Soil and Salinity Research Institute

(SSRI) Pindi Bhatian.

During pot study, low Na+ accumulator’s genotypes (selected from previous

studies) also exhibited improved ionic, biochemical and physiological attributes and

produced high yield as compared to genotypes that accumulated high Na+ that which were

considered salt sensitive inbreeds in previous experiments 1 and 2. Among low Na+

accumulators V-03094, V0005, V-04178, and V-05121 genotypes gave maximum seed

yield per plant in pot study and saline field which were highly linked with high K+

accumulation and improved biochemical and gas exchange attributes as compared to

genotypes TAM200/TUI, FRET2 and PUNJAB 8 which were high Na+ accumulators under

107

salinity stress. Leaf proline, phenolic and carotenoid contents increased significantly in Na+

excluder’s genotypes as compared to salt includers. Results of this study shows that

genotypes TAM200/TUI, FRET2, PUNJAB 85 and PBW343*2 were emerged as hyper

accumulator of Na+ in their leaves than check genotypes, Kharchia 65 and LU26S and it

seems that these wheat genotypes are salt includers and the most of salt includer genotypes

have been recognized as salt sensitive genotypes. Under salt regime, distinct variations

were recorded among the genotypes for chlorophyll fluorescence, which is used as

physiological marker against salinity. It concluded that V-03094, V0005, V-04178 and V-

05121 hexapodies inbred lines that accumulated low Na+ content in their leaves exhibited

improved antioxidant activities and produced more yield.

The fourth experiment was conducted in open field trial, twenty-five genotypes

(selections from previous in hydroponic screenings) were further tested on saline-sodic soil

of Pindi Bhatian in order to evaluate yield response and to establish physiological and

biochemical markers for salt tolerance in selected hexaploid wheat lines. Findings of this

study reveal that all wheat genotypes contributed in grain yield, however, showed

significant yield potential. Genotypes which were screened as salt tolerant in previous

studies also produced higher yield as compared to sensitive ones, which could also be

linked with poor crop stand. Overall genotypes, V-02156, V-03094, V-04181, V0005, V-

04178 and PVN were good salt excluders which was linked with better non- enzymatic

antioxidants activities and also produced high yield in saline sodic field.

Limitations of Study

There were following limitation, which were faced during experimentations.

The wire house was not wind and rain proof, it may change performance of plants

Canal water was not available on field experimental site

Weather data of field experimental site was not available and did not document in

thesis

108

Future Need In proposed project, V-02156, V-03094, V0005, TURACO, PVN screened as good

Na+ excluders genotypes and had good performance due to improved physiological and

biochemical traits that ultimately contributed to higher yield under salt stress conditions.

However, following investigations are needed for the characterization of these genotypes

on molecular basis.

As V-02156, V-03094, V0005, TURACO and PVN wheat genotypes have been

identified for their salinity tolerance on Na+ exclusion basis. There is need to work on

quantitative expression of genes, which is responsible for Na+ exclusion and then

quantify weather theses finding could be well correlated or not to the Na+ exclusion

traits.

Furthermore, induced variation in pyrophosphate due salinity, should be studied. It

provide the energy for transport of ion across the plasma membrane. The variation in

these cellular mechanisms will guide to understand the salt tolerance basis at cellular

level.

This work needs to be extended by further crossings of selected low Na+ genotypes with

commercial cultivar and analysis in a wheat breeding programme to develop salt

tolerant wheat cultivar in Pakistan.

The economic, and thus social, benefits from the development of such varieties will be

increased yield and thus farmer’s income across more than half of all production areas

for bread wheat in these selected genotypes.

109

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Exploring Physiological Mechanism of Salt Tolerance in ... · 3.3.1 Physical and chemical characteristic of soil used in pot study 39 3.3.2 Selected twenty genotypes from experiment