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
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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).
Muhammad Sohail Saddiq
2006-ag-1540
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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
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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
Muhammad Sohail Saddiq
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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
3.2.5 Statistical analysis 36
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
3.3.10 Statistical analysis 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
3.4.8 Statistical analysis 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
Sr. No. Title Page No.
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
Sr. No. Title Page No.
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
Comparison between two groups of wheat genotypes, Low Na+ accumulators
(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
Comparison between two groups of wheat genotypes, Low Na+ accumulators
(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
200mM). PC1 and PC2 explained total variation among genotypes. See Table
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
3.2.1 for codes of the genotypes.
59
4.2.4
Vector view of the genotype-by-trait biplot of showing the interrelationships
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
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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
Vector view of the genotype-by-trait biplot of showing the interrelationships
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
Vector view of the genotype-by-trait biplot of showing the interrelationships
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.
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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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200mM). PC1 and PC2 explained total variation among genotypes. See Table
3.2.1 for codes of the genotypes.
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).
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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).
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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).
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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
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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).
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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).
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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).
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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
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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
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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)
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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).
- 9 -
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
- 10 -
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
- 16 -
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)
- 18 -
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).
- 19 -
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…
Sr. No Genotype Sr. No Genotype
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…
Sr. No Genotype Sr. No Genotype
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…
Sr. No Genotype Sr. No Genotype
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…
Sr. No Genotype Sr. No Genotype
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.
3.3.6 Gas exchange parameters
Following the same protocol descried earlier in experiment 2.
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.
3.3.10 Statistical analysis
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
T= Tolerant S= Salt Sensitive
- 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:
Following the same protocol descried earlier in experiment 1.
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.
3.4.8 Statistical analysis
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
T= Tolerant S= Salt Sensitive
- 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
Fig 4.1.4 Comparison between two groups of wheat genotypes, Low Na+ accumulators (square
marks) and High Na+ accumulators in leaf blade (triangle mark). Fresh weight of 3rd leaf (a)
and dry weight of 3rd leaf (b).
52
Fig 4.1.5 Comparison between two groups of wheat genotypes, Low Na+ accumulators (square
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
Fig 4.2.4 Vector view of the genotype-by-trait biplot of showing the interrelationships 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
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
Fig 4.2.7 Vector view of the genotype-by-trait biplot of showing the interrelationships 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.
64
Fig 4.2.8 Vector view of the genotype-by-trait biplot of showing the interrelationships 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.
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
Fig 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 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).
4.3.6 Gas exchange parameters
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.
Error bars indicate S.E (n=3).
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
indicates check genotypes. Error bars indicate S.E (n=3).
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
indicates check genotypes. Error bars indicate S.E (n=4).
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.
Error bars indicate S.E (n=4).
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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