Zinc Nutrition and Microbial Allelopathy for Improving...

Zinc Nutrition and Microbial Allelopathy for Improving

Productivity, Grain Biofortification and Resistance against Abiotic Stresses in Wheat

BY

ABDUL REHMAN

M.Sc. (Hons.) Agronomy

A thesis submitted in partial fulfillment of the

requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

AGRONOMY

Faculty of Agriculture,

University of Agriculture, Faisalabad, Pakistan.

2017

OPENING

HE IS THE FIRST,

HE IS THE LAST

HE IS THE MANIFEST,

HE IS THE HIDDEN, &

HE KNOWS EVERYTHING

HE BRINGS THE NIGHT INTO THE DAY, &

BRINGS THE DAY INTO THE NIGHT, &

HE KNOWS THE THOUGHTS OF THE

HEART

S

A - HA - A3 - QUR

A

OH ALLAH,

OPEN OUR EYES,

TO SEE WHAT IS BEAUTIFUL,

OUR MINDS

T KNOW WHAT IS TRUE,

OUR HEARTS

TO LOVE WHAT IS GOOD.

vi

DEDICATED

T

MY FAMILY

SO MUCH OF WHAT I HAVE BECOME

IS BECAUSE OF YOU

AND I WANT TO TELL YOU

THAT I APPRECIATE YOU

THANK YOU AND LOVE YOU

vii

ACKNOWLEDGEMENTS

All worships and praises are only due to the Lord of Creation, the most beneficent, merciful, gracious

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. My prime thankfulness is for the

Almighty Lord who blessed me with a vision to accomplish the herculean task of dissertation which is a

definite mile stone with a noble mission for seeking knowledge and spreading its sparkle around.

I offer my humblest thanks, and countless salutations to the Holy Prophet Muhammad (PBUH), the bacon of

enlightenment, the fountain of knowledge, the messenger of peace, biggest benefactor of the mankind ever had and

forever torch of guidance for humanity.

I deem it utmost pleasure to avail the opportunity to express the heartiest gratitude and deepest sense of devotion

to my esteemed supervisor, Dr. Muhammad Farooq, Associate Professor, Department of Agronomy, University of

Agriculture, Faisalabad for his inspiring guidance, keen interest and unstinted help during my study period. He was

and is the ultimate source of tremendous assistance and extended his prodigious co-operation in compiling this research

work in my academic journey. Appreciations are due for Dr. Riaz Ahmad and Dr. Shahzad M. A. Basra, Professors,

Department of Agronomy, University of Agriculture, Faisalabad, and members of my supervisory committee for

valuable suggestions during the research endeavors.

I offer my sincere thanks to Dr. Levent Ozturk, Associate Professor, Faculty of Engineering and Natural Sciences,

Sabanci University, Istanbul, Turkey, for his inspiring guidance and unstinted help during my visit to his laboratory.

I am very thankful to Dr. Muhammad Naveed, Assistant Professor, Institute of Soil and Environmental Sciences

University of Agriculture, Faisalabad for guidance and valuable suggestions during my research.

I am truly indebted and thankful to my colleagues Dr. Ahmad Nawaz, Muhammad Asif, Esin Tunc, Faisal

Nadeem, Asif Nadeem, Amanullah, Ahmad Mehmood, Moeen-ud-din, Kamran Saleem and Numan Ahmad. I feel

pleasure to say gratitude to all the staff of Allelopathy Lab and Agronomic Research Area, University of Agriculture

for their assistance and cooperation during my research work.

I feel proudly privileged to mention the feelings of obligations towards my brothers Hafiz Muhammad Irfan,

Hafiz Muhammad Farhan, and Abdul Raheem, beloved sisters for their love, affection and prayers which enabled me

to accomplish this long-adhered goal and cherished dream. I must mention my nephew Muhammad Shaheer whose

smile makes me smile (may Allah keep them smiling).

I am thankful to Higher Education Commission, Government of Pakistan, for awarding me the indigenous

scholarship. I am also grateful to TUBITAK Turkey and International Plant Nutrition Institute for the financial

support through BIDEB-2216 fellowship and IPNI scholar award, respectively.

viii

Abdul Rehman

TABLE OF CONTENTS

Chapter Title Page

Acknowledgements i

Table of Contents ii

List of Tables vii

List of Figures xiii

List of Abbreviations xv

Abstract xvii

1 INTRODUCTION 1

2 REVIEW OF LITERATURE 5 2.1. Zinc in Wheat Biology 6

2.1.1. Protein synthesis 6

2.1.2. Enzyme activation and resistance to abiotic stresses 7

2.1.3. Structural and functional integrity of plasma membranes 8

2.1.4. Cell division and reproduction 9

2.1.5. Photosynthesis 10

2.2. Factors Affecting Zinc Availability to Wheat 11

2.2.1. Soil pH 11

2.2.2. Soil organic matter 14

2.2.3. Soil temperature, moisture and light intensity 14

2.2.4. Soil salinity and interaction of Zn with other elements 15

2.2.5. Zinc interaction with soil biota/mycorrhizal colonization 16

2.2.6. Zn efficiency in wheat 17

2.2.7. Value of intrinsic seed zinc for germinating wheat seedlings 19

2.3. Zinc in Soil and its Dynamics in Different Wheat-Based Cropping 20 Systems

2.3.1. Rice-wheat cropping systems 21

2.3.2. Cotton-wheat cropping systems 22

2.3.3. Zinc deficiency in other wheat-based cropping systems 22

2.4. Methods of Zinc Fertilizer Application 24

2.4.1. Soil Zn fertilization 24

2.4.2. Foliar zinc application 26

2.4.3. Seed treatments 26

2.4.3.1 Seed priming 30

2.4.3.2 Seed coating 30

2.5. Economics of Zinc Application 31

2.6. Agronomic Approaches to Managing Zn in Wheat-based Production 32

Systems

ix

2.6.1. Tillage 32

2.6.2. Crop rotations and intercropping 32

2.6.3. Manure application 33

2.7. Zinc Biofortification of Wheat 37

2.7.1. Agronomic approach 37 2.7.2. Breeding approach 38

2.7.2.1 Selection and conventional breeding 38

2.7.2.2 Molecular approaches 39

2.8. Conclusion and Future Research Needs 40

3 MATERIAL AND METHODS 44

3.1 Optimizing Zinc Seed Priming Treatments for Improving the Stand 43

Establishment Productivity and Grain Biofortification of Wheat

3.2.1. Experimental details 44 3.1.2. Observations 45

3.1.2.1. Seedling establishment 45

3.1.2.2. Seedling growth 45

3.1.2.3 Yield and yield components 45

3.1.2.4 Zinc and chlorophyll content determination 46

3.1.3. Data analysis 46

3.3. Optimizing Zinc Seed Coating Treatments for Improving the Stand 46

Establishment,Productivity and Grain Biofortification of Wheat

3.3.1. Experimental details 46

3.4. Characterizing Wheat Genotypes for Zinc Biofortification Potential 47 and

Genetic Diversity

3.4.1. Experimental treatments 47

3.4.2. Crop husbandry 47

3.4.3. Recording of data 48

3.4.3.1 Yield parameters 48

3.4.3.2 Grain mineral analysis 48

3.4.3.3 Phytate concentration and estimated bioavailable Zn 48

3.4.3.4 DNA extraction 49

3.4.3.5 Amplified fragment length polymorphism (AFLP) fingerprinting 49


3.5. Zinc Nutrition and Microbial Allelopathy for Improving 52

Productivity and Grain Biofortification of Wheat (Glass house experiment)

3.5.1. Experimental treatments 52

3.5.2. Crop husbandry 53

3.5.3. Recording of data 53

3.5.3.1 Photosynthetic and water relation traits 53


3.5.3.3 Grain elemental analysis and grain Zn localization 54 3.4.3.4 Phytate concentration

and estimated bioavailable Zn 55 3.4.3.5 Organic acid estimation in root exudates

55


3.6. Zinc Nutrition and Microbial Allelopathy for Improving Productivity 56 and

Grain Biofortification of Wheat (Field experiment)

x

3.6.1. Experimental details 56 3.5.2. Crop husbandry 57

3.5.3. Data Recording 58


3.6.1.2 Water relation traits 58

3.6.1.3 Grain quality analysis 59

3.6.1.4 Phytate concentration and estimated bioavailable Zn 59


3.7. Improving the Drought Resistance in Wheat through Zinc Nutrition 60

3.7.1. Experimental conditions 60

3.7.2. Chlorophyll and gas exchange traits 60

3.7.3. Enzyme extraction and analyses 60

3.7.4. Yield related traits 61

xi

3.7.5. Biomass production and mineral analyses 61

3.7.6. Phytate concentration and estimation of bioavailable Zn 62


3.8. Improving the Salt Resistance in Wheat through Zinc Nutrition

62

3.8.1. Experimental condition and treatments 62




3.8.5. Biomass production and Nutrient analyses 62

3.8.6. Phytate concentration and estimation of bioavailable Zn 62


3.9. Improving the Resistance against Cold Stress in Wheat through Zn

62 Nutrition





3.9.5. Biomass production and mineral analyses 63

3.9.6. Phytate concentration and estimated bioavailable Zn 63


3.10. Improving the Resistance against Heat Stress in Wheat through

63 Zinc Nutrition


3.10.2. Photosynthesis/biomass production and nutrient analyses/root traits 64

3.10.3. Enzyme extraction and analysis 64 3.9.4. Digestion and nutrient analyses 64


4 RESULTS AND DISCUSSION 65

4.1. Optimizing Zinc Seed Priming Treatments for Improving the Stand

65 Establishment Productivity and Grain Biofortification of

Wheat

4.1.1. Results 65

4.1.1a. Petri plate experiment 65

4. 1.1b. Sand-filled pot experiment 65 4.1.1c. Glass house experiment 69

4.1.2. Discussion 70

4.2. Optimizing Zinc Seed Coating Treatments for Improving the

Stand 72 Establishment, Productivity and Grain Biofortification of

Wheat 4.2.1. Results 72

4.2.1a. Petri plate experiment 72 4.2.1b. Sand-filled pot experiment 72

4.3.1c. Glass house experiment 73


xii

4.3. Charactering Wheat Genotypes for Zinc Biofortification Potential

79 and Genetic Diversity

4.3.1. Results 79

4.3.1.1 Yield related traits 79

4.3.1.2 Grain mineral concentration 83

4.3.1.3 AFLP primer combinations 84

4.3.1.4 Genotypic and genetic diversity within wheat genotypes

84 4.3.1.5 Genetic similarity and Analysis of molecular

variance 84

4.3.1.6 Principal component analysis 89


4.4. Zinc Nutrition and Microbial Allelopathy for Improving

Productivity 97 and Grain Biofortification of Wheat

4.4.1. Results (Glass house experiment) 97

4.4.1.1 Zn solubilization activity 97

4.4.1.2 Photosynthetic traits 97

4.4.1.3 Water relation traits 98


4.4.1.5 Grain quality 99

4.4.1.6 Organic acids 109

4.4.1.7 Concentrations of Zn, Fe and Ca in seed fractions 110 i.

Lasani-2008 110 ii. Faisalabad-2008 111

4.4.2 Discussion 111

4.5. Zinc Nutrition and Microbial Allelopathy for Improving 115

Productivity and Grain Biofortification of Wheat

4.5.1. Results (Field experiment) 115

4.5.1.1 Water relations 115


4.5.1.3 Grain analysis 121

4.5.1.4 Economic and marginal analyses 122


4.6. Improving the Drought Resistance in Wheat through Zinc

Nutrition 131

4.6.1. Results 131

4.6.1.1 Gas exchange, biomass and relative water content

131

4.6.1.2 Biochemical traits 132

4.6.1.3 Leaf mineral concentrations 132

4.6.1.4 Yield and yield related parameters 133

4.6.1.5 Grain quality and grain mineral concentration 133



144

4.7.1. Results 144

xiii

4.7.1.1 Gas exchange, water relation and biomass production

144

4.7.1.2 Biochemical traits 145

4.7.1.3 Leaf mineral concentrations 145 4.7.1.4 Yield and

yield related parameters 146 4.7.1.5 Grain quality and

grain mineral concentration 146


4.8. Improving the Resistance against Cold Stress in Wheat through Zn

157 Nutrition

4.8.1. Results 157

4.8.1.1 Gas exchange 157 4.8.1.2 Biochemical traits

157

4.8.1.3 Biomass and leaf mineral concentrations 158

4.8.1.4 Yield and yield parameters 159

4.8.1.5 Grain quality and mineral concentration 159


4.9. Improving the Resistance against Heat Stress in Wheat through

168 Zinc Nutrition

4.9.1. Results 168

4.9.1.1 Seedling growth traits 168

4.9.1.2 Gas exchange 169

4.9.1.3 Enzyme activities 172

4.9.1.4 Seedling mineral analysis 173 i. Seedling Zn

analysis 173 ii. Seedling potassium analysis 173 iii.

Shoot nitrogen (N) analysis 174 iv. Seedling

phosphorus (P) analysis 174 v. Seedling calcium (Ca)

analysis 182 vi. Zinc efficiency index and heat tolerance

index 182


4.10. General Discussion 185

5 SUMMARY 190

Project Conclusions 194

Future Rresearch Thrusts 194

LITERATURE CITED 195

xiv

LIST OF TABLES

Table Title Page

2.1 Effect of PGPRs and AMF on Zn uptake, grain yield and

organic acids/compounds production of wheat

23

2.2 Comparative performance of Zn application methods and

sources for grain yield and grain Zn concentration of wheat

27

2.3 Comparative performance of different Zn sources in wheat 29

2.4 Economics of Zn application in wheat 34

2.5 Effect of organic amendments/green manuring and crop

rotation on grain yield and grain Zn concentration of wheat

35

2.6 QTLs responsible for grain Zn concentration in wheat 42

2.7 Genes responsible for grain Zn concentration in wheat 43

3.1 Bread wheat genotypes of Pakistan used in the study 51

4.1 Influence of seed priming with Zn on the germination and

seedling growth of wheat cultivars Lasani-2008 (LS-2008)

and

Faisalabad-2008 (FSD-2008) (Petri plate experiment)

66

4.2 Correlations coefficients (r) among different germination stand

establishment and seedling growth traits of wheat as influenced

by seed priming with Zn

66

4.3 Influence of seed priming with zinc on the emergence and

seedling growth of wheat cultivars Lasani-2008 (LS-2008)

and

Faisalabad-2008 (FSD-2008) (Pot experiment)

67

4.4 Analysis of variance for influence of seed priming with Zn on

stand establishment, yield related traits, grain yield and grain

biofortification of bread wheat

67

4.5 Analysis of variance for influence of seed priming with Zn

on chlorophyll and shoot Zn concentration of bread wheat

67

4.6 Influence of seed priming with Zn on stand establishment,

yield related traits and grain biofortification of bread wheat

68

4.7 Influence of seed priming with Zn on chlorophyll and straw

Zn concentration of bread wheat

68

4.8 Influence of seed priming with Zn on grain yield and harvest

index of bread wheat

68

4.9 Analysis of variance for influence of seed coating with Zn

on germination and seedling growth of bread wheat

75

4.10 Influence of seed coating with zinc on germination and

seedling growth of bread wheat (Petri plate experiment)

75


on germination and seedling growth of bread wheat

75

4.12 Influence of seed coating with zinc on germination and

seedling growth of wheat cultivars (Sand filled pot

experiment)

76

xv

4.13 Analysis of variance for influence of seed coating with Zn on

stand establishment, productivity and grain biofortification of

wheat cultivars

76


on chlorophyll a, b and straw Zn contents of wheat cultivars

76

4.15 Influence of seed coating with Zn on stand establishment, 77 productivity and

grain biofortification of wheat cultivars (Glass house experiment)

4.16 Influence of seed coating with Zn on chlorophyll a, b and straw 77 Zn

contents of wheat cultivars (Glass house experiment)

4.17 Mean comparison of productive tillers and grains per spike of 80 wheat

genotypes under no and adequate Zn supply

4.18 Mean comparison of 1000 grain weight and harvest index of 81 wheat


4.19 Mean comparison of grain yield and grain protein concentration 82 in wheat


4.20 Mean comparison of grain Zn, Fe and Ca concentration of wheat 85


4.21 Mean comparison of grain phytate concentration, [phytate]:[Zn] 86 ratio and

bioavailable Zn of wheat genotypes under no and adequate Zn supply

4.22 Mean comparison of Zn, Fe and Ca concentration (mg kg-1) of 87 embryo and

aleurone of wheat genotypes under no and adequate Zn supply

4.23 Mean comparison of Zn, Fe and Ca concentration (mg kg-1) of 88 endosperm

in wheat genotypes under no and adequate Zn supply

4.24 Evaluation of 4 primer pair combinations for use in studying 89 genetic

diversity of wheat cultivars.

4.25 Population genetic analysis of 28 wheat genotypes using AFLP 90

Fingerprinting

4.26 Analysis of molecular variance (AMOVA) among wheat and 91 within

cultivars

4.27 Correlation coefficients of grain mineral concentrations with 94 embryonic,

aleurone and endosperm Zn concentration and bioavailable Zn under

no and Zn application treatments

xvi

4.28 Analysis of variance for effect of different Zn application method 100 or

without Pseudomonas sp. MN12 addition on photosynthetic tr

wheat cultivars

4.29 Effect of different Zn application methods with or without 100

Pseudomonas sp. MN12 addition on photosynthetic traits of wheat

cultivars

4.30 Interactive effect of different Zn application methods with or 101 without

Pseudomonas sp. MN12 addition on photosynthetic traits of wheat

cultivars

4.31 Analysis of variance for effect of different Zn application methods 101 with

or without Pseudomonas sp. MN12 addition on water

relation of wheat cultivars at anthesis


Pseudomonas sp. MN12 addition on water relation traits of wheat

cultivars

4.33 Analysis of variance for the effect of different Zn application 102 methods

with or without Pseudomonas sp. MN12 addition on yield

components of wheat cultivars


Pseudomonas sp. MN12 addition on yield components, grain

protein and phytate concentration of wheat cultivars


Pseudomonas sp. MN12 addition on grain yield (g plant-1) of wheat

cultivars

4.36 Analysis of variance for the effect of different Zn application 104 methods

with or without Pseudomonas sp. MN12 addition on grain minerals

composition of wheat cultivars


Pseudomonas sp. MN12 addition on phytate/Zn molar ratio, Fe, P

and Ca concentration in grains of wheat cultivars

xvii


Pseudomonas sp. MN12 addition on organic acid concentration of

bread wheat ± S.E.

4.39 Analysis of variance for effect of Zn application and 117

Pseudomonas sp. MN12 on water relation traits of wheat cultivars

4.40 Influence of Zn application and Pseudomonas sp. MN12 on water 117

relation traits of wheat cultivars

4.41 Influence of Zn application and Pseudomonas sp. MN12 on 118 osmotic

potential (2013-14) and water potential of wheat (201415)

4.42 Analysis of variance for effect of Zn application and 118

Pseudomonas sp. MN12 on yield parameters of wheat cultivars

4.43 Effect of Zn application and Pseudomonas sp. MN12 on yield 119

parameters of wheat cultivars

4.44 Analysis of variance for the effect of Zn application and 119 Pseudomonas

sp. MN12 on grain minerals composition of wheat cultivars

4.45 Effect of Zn application and Pseudomonas sp. MN12 on grain 120 mineral

composition of wheat

4.46 Influence of Zn application and Pseudomonas sp. MN12 on grain 120

protein (2013-14), phytate Concentration, phytate/Zn ratio and

bioavailable Zn of wheat cultivars (2014-15)

4.47 Influence of Zn application and Pseudomonas sp. MN12 on 126 economics

of wheat cultivars

4.48 Influence of Zn application and Pseudomonas sp. MN12 on 127 marginal

analysis of wheat cultivars

4.49 Effect of zinc (Zn) nutrition on chlorophyll density, 134

photosynthesis (A), transpiration rate (E), water use efficiency (WUE),

intercellular carbon dioxide (Ci), stomatal conductance (gs) and

quantum yield (QY), relative water content and biomass

production of wheat cultivars under drought and well-watered

conditions

4.50 Effect of Zn nutriton on buffer-extractable protein concentration 135 and

specific activities of antioxidative enzymes superoxide dismutase

xviii

(SOD), ascorbate peroxidase (APX) and glutathione reductase

(GR), melanodialdehyde content (MDA) and total soluble

phenolics (TSP) in wheat cultivars under drought and well-watered

conditions

4.51 Effect of zinc (Zn) nutrition on concentration and contents of Zn, 136 N, K

and Ca in wheat cultivars under drought and well-watered

conditions

4.52 Effect of zinc (Zn) nutrition on grains per spike (GPS), 100 grain 139

weight, biological yield, grain yield and harvest index of wheat

cultivars under drought and well-watered conditions

4.53 Effect of zinc (Zn) nutrition on concentration and contents of 140 protein and Zn in

whole grain, embryo, aleurone and endosperm Zn concentration, phytate

concentration, phytate/Zn ratio and bioavailable Zn in wheat cultivars under

drought and wellwatered conditions

4.54 Correlation coefficients of gas exchange traits, enzyme activities, 141

biomass production, relative water content, leaf Zn and K content

of wheat cultivars under well watered and drought stressed

conditions (n=4)

4.55 Correlation coefficients of grain yield, protein content, Zn 141 content,

endosperm Zn concentration, phytate and bioavailable Zn in wheat

cultivars under well watered and drought stressed conditions (n=4)

4.56 Effect of zinc (Zn) nutrition on chlorophyll density, 148

photosynthesis (A), transpiration rate (E), water use efficiency (WUE),

intercellular carbon dioxide (Ci), stomatal conductance (gs) and

quantum yield (QY), relative water content and biomass

proudcitoin of wheat cultivars under normal and salt stressed

conditions

4.57 Effect of Zn nutriton on buffer-extractable protein concentration 149 and

specific activities of antioxidative enzymes superoxide dismutase

(SOD), ascorbate peroxidase (APX) and glutathione reductase

(GR), melanodialdehyde content (MDA)and total soluble

xix

phenolics (TSP) in wheat cultivars under normal and salt stressed

conditions


and Na in wheat cultivars under normal and salt stressed conditions



cultivars under normal and salt stressed conditions

4.60 Effect of zinc (Zn) nutrition on concentration and contents of 155 protein

and Zn in whole grain, embryo, aleurone and endosperm Zn

concentration, phytate concentration, phytate/Zn ratio and

bioavailable Zn in wheat cultivars under normal and salt stressed

conditions

4.61 Correlation coefficients of gas exchange traits, enzyme activities,

156 biomass production, relative water content, leaf Zn and K

content

of wheat cultivars under normal and salt stressed conditions (n=4)

4.62 Correlation coefficients of grain yield, protein content, Zn 156

content, endosperm Zn concentration, phytate and bioavailable

Zn in wheat cultivars under normal and salt stressed conditions

(n=4)

4.63 Effect of zinc (Zn) nutrition and cold stress on chlorophyll 160

density, photosynthesis (A), transpiration rate (E), water use

efficiency (WUE), intercellular carbon dioxide (Ci), stomatal conductance

(gs) and quantum yield (QY), relative water content

(RWC) and biomass proudcitoin of wheat cultivars

4.64 Effect of Zn nutriton and cold stress on buffer-extractable protein 161

concentration and specific activities of antioxidative enzymes

superoxide dismutase (SOD), ascorbate peroxidase (APX) and

glutathione reductase (GR), melanodialdehyde content (MDA) and

total soluble phenolics (TSP) of wheat cultivars


and Na in wheat cultivars under optimal and and cold stressed

conditions

xx



cultivars under normal and cold stressed conditions

4.67 Effect of zinc (Zn) nutrition on concentration and contents of 165 protein

and Zn in whole grain, embryo, aleurone and endosperm Zn

concentration, phytate concentration, phytate/Zn ratio and

bioavailable Zn in wheat cultivars under optimal and cold stressed

conditions


biomass production, relative water content, leaf Zn and K content

of wheat cultivars under optimal and cold stressed conditions (n=4)

4.69 Correlation coefficients of grain yield, protein content, Zn 166 content,

endosperm Zn concentration, phytate and bioavailable Zn in wheat

cultivars under optimal and cold stressed conditions (n=4)

4.70 Effect of temperature regime and zinc (Zn) supply on total 170 biomass,

shoot and root dry matter production, shoot:root ratio, root length,

surface area, diameter and volume of two wheat cultivars LS-2008

and FSD-2008

4.71 Effect of temperature regime and zinc (Zn) supply on chlorophyll 171

density, photosynthesis (A), transpiration rate (E), water use

efficiency (WUE), intercellular carbon dioxide (Ci), stomatal

conductance (gs) and quantum yield (QY)in two wheat cultivars

LS-2008 and FSD-2008

4.72 Effect of temperature regime and zinc (Zn) supply on buffer- 175 extractable

protein concentration and specific activities of antioxidative

enzymes superoxide dismutase (SOD), ascorbate peroxidase (AP)

and glutathione reductase (in leaves and roots of two wheat

cultivars LS-2008 and FSD-2008

4.73 Effect of temperature regime and zinc (Zn) supply on 176

concentration, content and total uptake of Zn in two wheat cultivars LS-2008

and FSD-2008


xxi

concentration, content and total uptake of potassium (K) and shoot N

concentration and contents in two wheat cultivars LS-2008 and

FSD-2008


concentration, content and total uptake of phosphorus (P) in two wheat



concentration, content and total uptake of calcium (Ca) in two wheat cultivars

LS-2008 and FSD-2008.


biomass production, relative water content, Zn, K and Ca uptake

of wheat cultivars under optimal and heat stressed conditions (n=4)

22

List of Figures

Figure Title Page

2.1 Effect of Zn nutrition on salt stress tolerance of 30-day-old wheat

plants (T.

aestivum L. cv. Faisalabad-2008).

8

2.2 Effect of Zn nutrition on drought stress tolerance of 30-day-old wheat

plants (T. aestivum L. cv. Faisalabad-2008).

9

2.3 Effect of Zn nutrition on heat stress tolerance of 21-day-old wheat

plants (T.


10

2.4 Mechanism of Zn uptake from soil and its translocation into

developing grains.

13

4.1 UPGMA dendrogram illustrating Nei's (1978) genetic distances of 28

wheat cultivars based on AFLP fingerprinting analysis.

92

4.2 Polygon view of the GGE biplot show the relation of wheat genotypes

for (a) grain mineral, yield components and Zn bioavaiblity (b) Zn, Fe

and Ca localization in embryo, aleurone and endosperm of wheat

genotype under –Zn and +Zn conditions.

93

4.3 Effect of different Zn application methods with or without Pseudomonas

sp. MN12 addition on grain and seed fractions Zn concentration of wheat

cultivars (a) Lasani-2008 (b) Faisalaabad-2008 ±S.E. SP= Seed priming;

SC= Seed coating; SA= Soil application; FA= Foliar application; B=

MN12

106


sp. MN12 addition on grain and seed fractions Fe concentration of wheat



MN12

107


sp. MN12 addition on grain and seed fractions Ca concentration of wheat



MN12

108

4.6 Zinc localizaton in seed fractions of wheat using DTZ in wheat cultivar

Lasani-2008 as influenced by Zn application methods and MN12

inoculation (a) Control (b) Seed priming with Zn (c) Seed coating (d) Soil

application (e) Foliar spray (f) Control + MN12 (g) Seed priming with Zn

+ MN12 (h) Seed coating with Zn + MN12 (i) Soil application of Zn +

MN12 (j) Foliar application of Zn + MN12

109

23

4.7 Zinc localizaton in seed fractions of wheat using DTZ in wheat cultivar

Faisalabad-2008 as influenced by Zn application methods and MN12

inoculation (a) Control (b) Seed priming with Zn (c) Seed coating (d) Soil

application (e) Foliar spray (f) Control + MN12 (g) Seed priming with Zn

+

MN12 (h) Seed coating with Zn + MN12 (i) Soil application of Zn +

MN12 (j) Foliar application of Zn + MN12

109

4.8 Influence of Zn application and Pseudomonas sp. MN12 on grain and

seed fractions Zn concentration of wheat cultivars (a) Lasani-2008 (b)

Faisalaabad-2008 ±S.E. SP= Seed priming; SC= Seed coating; SA= Soil

application; FA= Foliar application; B= Pseudomonas sp. MN12

123

4.9 Influence of Zn application and Pseudomonas sp. MN12 on grain and

seed fractions Fe concentration of wheat cultivars (a) Lasani-2008 (b)

124

Faisalaabad-2008 ±S.E. SP= Seed priming; SC= Seed coating; SA= Soil application;

FA= Foliar application; B= Pseudomonas sp. MN12

4.10 Influence of Zn application and Pseudomonas sp. MN12 on grain and seed

125 fractions Ca concentration of wheat cultivars (a) Lasani-2008 (b)

Faisalaabad-2008 ±S.E. SP= Seed priming; SC= Seed coating; SA= Soil

application; FA= Foliar application; B= Pseudomonas sp. MN12 4.11 (a) Influence of temperature regimes on Zn efficiency index of wheat 180 cultivars

(b) Effect of Zn supply on heat tolerance index of wheat cultivars

24

LIST OF ABBREVATIONS

Abbreviation Description Abbreviation Description

% Percent FYM Farmyard manure

µg Micro gram GM Green manure

A Photosynthesis GR Glutathione

reductase

ABA Abscisic acid gs Stomatal

conductance

AFLP Amplified fragment length

polymorphism

h hours

AMF Arbuscular mycorrhizal fungi H2O2 Hydrogen

peroxide

AMOVA Analysis of molecular

variance

H2SO4 Sulphuric acid

ANOVA Analysis of variance K2SO4 Potassium

sulphate

AOSA Association of Ofiicial

Seed analysts

KH2PO4 Potassium

phosphate

APX Ascorbate peroxidase LP Leaf soluble

protein

AZRI Arid Zone Research

Institute, Bhakkar,

Pakistan

LSD Least significant

difference test

BARI Barani Agricultural

Research Institute,

Chakwal, Pakistan

M Molar

BCR Benefit Cost ratio m Meter

Bio Zn Bioavailable Zn MC Marginal cost

Ca Calcium MDA Melanodialdehyde

CA Carbonic anhydrase MET Mean emergence

time

Ca(NO3)2 Calcium nitrate MGT Mean germination

time

CaCO3 Calcium carbonate Mg Magnesium

Chl Chlorophyll mg Milli gram

Ci Intercellular CO2 Mg ha-1 Maga gram per

hactare

Cl- Chloride MGT Mean germination

time

CTAB Cetyltrimethyl ammonium

bromide

mL Milli liter

Cu Copper mm Mili meter

DAP Di ammonium phosphate mM Mili molar

25

DOC Dissolved organic carbon MNB Marginal net

benefits

DTPA Diethylene tri-amine

pentaacetic acid

Mo Molybdenum

E Rate of transpiration MPa Mega Pascal

EC Electrical conductivity MRR Marginal rate of

return

EDTA

Ethylenediaminetetraacetic

acid

N

Nitrogen

N Normal SDS

Sodium dodecyl

sulfate

OP Osmopriming SOD Super oxide

dismutase

P Phosphorus SPP Signal peptide

peptidase

PCR Polymerase chain reaction TSP Total soluble

phenolics

PGPR Plant growth promoting

rhizobacteria

WRI Wheat Research

Institute, Faisalabad,

Pakistan

PKR Pakistani rupees WUE Water use efficiency

ppm Parts per million ZnCl2 Zinc chloride

QTL Quantitative trait loci ZnCO3 Zinc carbonate

RARI Regional Agricultural Research

Institute

Bahawalpur, Pakistan

ZnSo4 Zinc sulphate

ROS Reactive oxygen species Ψp Pressure potential

RWC Relative water content Ψs Osmotic potential

RWCS Rice wheat cropping

systems

Ψw Water potential

26

ABSTRACT

Zinc (Zn) is vital for the plants and humans. Wheat (Triticum aestivum L.) is one of the

leading cereals and is consumed as staple by billions across the globe. However, wide scale

Zn deficiency has been observed in wheat growing regions. This situation necessities the Zn

nutrition in wheat and to improve its grain yield and grain Zn contents. This study was

conducted (i) to optimize the source and application of Zn through seed treatments, (ii) to

characterize the wheat genotypes for genetic diversity, Zn bioavailability and localization in

different seed fractions, (iii) to improve the productivity and grain biofortification of wheat

by combined application of Zn and Zn solubilizing plant growth promoting rhizobacteria

(PGPR), and (iv) to explore the potential of Zn nutrition in improving tolerance against abiotic

stresses (drought, salt, heat, chilling) at the University of Agriculture, Faisalabad, Pakistan

and Sabanci University, Istanbul, Turkey. For optimizing Zn seed treatments, Zn was applied

as seed priming and seed coating using ZnSO4 and ZnCl2 as sources. Seed priming with 0.5

M Zn and seed coating with 1.25 g Zn kg-1 seed, using ZnSO4 as source, were the best

treatments to improve the stand establishment, grain yield and grain Zn concentration of

wheat. Twenty-eight wheat genotypes, of diverse morphology, were characterized for genetic

diversity and grain biofortification potential. There was very less genetic diversity (0.0335-

0.0677) among the tested wheat genotypes of Punjab, Pakistan. However, there was

substantial variation for yield and grain mineral concentration. Maximum grain yield was

recorded for Chakwal-50 while highest Zn concentration was measured in Blue silver with

Zn application. Application of Zn also enhanced the Zn localization in embryo, endosperm

and aleurone along with high bioavailability of Zn. In glass house and field experiments, Zn

was applied as preoptimized seed priming and seed coating treatment, soil and foliar

applications. Zinc solubilizing bacterial strain viz. Pseudomonas strain MN12 was used in

combination with different Zn application methods. Zinc application, by either method,

improved the yield and the grain Zn accumulation. However, use of PGPR in combination

with Zn seed priming was the best in improving the grain yield by 44% followed by soil Zn

fertilization (41%). Application of Zn through soil and foliar methods in combination with

PGPR substantially increased the Zn concentration in grain (90%), germ (50%), aleuron

(98%) and endospermic fraction (80%) of wheat with high bioavailable Zn (70%) compared

to control. Zinc application (3ppm Zn kg-1 soil) proved helpful for improving wheat

performance under abiotic stresses viz. cold 10/7ºC day/night, drought (35% field capacity)

and salt (2500 ppm NaCl) stresses. Adequate Zn supply (1 µM Zn) also mitigated the

detrimental effect of heat stress (36/28ºC day/night). Abiotic stresses severely reduced the

growth and productivity of wheat and adverse effect of these stresses was escalated under Zn

deficient conditions. However, adequate Zn application improved the grain yield and quality

of wheat under abiotic stresses by increasing chlorophyll intensity, photosynthesis, enzymatic

activities, relative water contents, leaf Zn, nitrogen and potassium concentration and lower

lipid peroxidation. Adequate Zn supply increased the Zn and protein concentration and

contents and bioavailable Zn in whole grain and seed fractions by about 2-fold compared to

low Zn treatment. In conclusion; Zn application improved the productivity of wheat. Use of

Zn solubilizing PGPR in combination with foliage and soil Zn application enhanced the Zn

uptake and grain Zn bioavailability. However, seed priming in combination with PGPRs

produced more yield with maximum net economic return. Wheat genotypes of Punjab are

genetically diverse for grain yield and mineral concentration. However, genotypes Blue silver

had the maximum grain Zn concentration and Zn bioavailability. Furthermore, adequate Zn

supply can help ameliorating the adverse effect of abiotic stresses and improving the yield

and grain quality of bread wheat.

Keywords: Zinc, PGPRs, bread wheat, abiotic stresses, biofortification, seed fractions

27

CHAPTER 1

INTRODUCTION

Zinc (Zn) is an important microelement and is necessary for all the life forms (Stein,

2010). Its deficiency in humans causes premature birth, growth and weight loss in children

and infants (Hess and King, 2009). In plants, Zn is needed for the enzyme activity,

transcription and translation of DNA and it is integral component of Zn metalloproteins

(Broadley et al., 2012). Zinc deficiency is the most important micronutrient disorder

resulting in low crop productivity. The extent of Zn deficiency is widespread as half of the

arable land is Zn deficient (FAO, 2002). Wheat is staple for most of the world population

and is inherently low in Zn. Soils of major wheat growing areas are highly deficient in plant

available Zn (Alloway, 2008a) as 33% of Pakistani soils are Zn deficient (Hamid and

Ahmad, 2001; Kauser et al., 2001). In a recent survey, Maqsood et al. (2015) reported that

>75% of soil of cotton-wheat cropping system of Punjab, Pakistan are deficient in Zn with

very low bioavailability and average Zn concentration (20 µg g-1) in wheat grains. Zinc

deficiency is predominant in salt affected, waterlogged calcareous soils, with high P

content, highly weathered and coarse textured soils (Sillanpaa, 1982; Alloway, 2008a).

On the verge of changing climate, crop plants are exposed to different abiotic

stresses like drought, salinity and temperature (chilling and heat stress). Water scarcity for

crop production is a serious threat to meet the future food demands as changed precipitation

patterns, increasing population pressure and declining water resources are expected to

aggravate the effect of drought on crop production systems. Drought stress restricts the

plant growth by reducing the root proliferation, and canopy size thus resulting in reduction

in net rate of photo-assimilation (Farooq et al., 2009). Global warming is also another

challenge for future crop production systems as increase in temperature is also expected to

tax the wheat yield (Farooq et al., 2011). Increase in soil salinization, besides drought and

heat stresses, is another threat to the future world food security as poor emergence, growth

and yield are characteristic feature of crops raised on saline soils (Munns and Tester, 2008).

Poor performance of crop plants in saline soils is due to salinity induced osmotic stresses,

nutritional imbalance and specific ion toxicity (Munns, 2002; Zhu, 2007).

In this scenario, Zn nutrition can help alleviating the detrimental effects of drought, salt

and thermal stresses (Khan et al., 2004). Zinc nutrition helps in protecting the plants from

damage of ROS because Zn is structural part of Cu/Zn SOD which is located in the chloroplast

28

and plays key role in protecting the plant membranes from oxidative damages to the biological

membranes (Cakmak, 2000). Moreover, under saline stress, Zn limit the Na and Cl uptake by

maintaining root cell membrane integrity. Application of Zn has been found to reduce adverse

effect of heat stress through increase in K concentration and synthesis of reducing sugars

(Shahriaripour et al., 2010). Zinc improves the resistance against heat and light extremes by

regulating the gene expression (Cakmak, 2000). Application of Zn also improves the resistance

against abiotic stresses due to enhanced activity of total SOD, Cu-Zn SOD, carbonic anhydrase

(CA), ribonuclease, fructose 1, 6 bisphosphate aldolase and acid phosphatase (Pandey et al.,

2002). Zinc maintains the photosynthesis by regulating the photosynthetic machinery as it is

required for signal peptide peptidase activity and repairs the photo damaged proteins of

photosystem II (Hansch and Mendel, 2009).

Zinc plays several crucial roles in human body and its dearth is caused due to

inadequate Zn intake. Wheat is major source of daily food, calorie and protein intake in

developing countries (Cakmak, 2008). So, routine and excessive intake of wheat products

results in Zn malnutrition as wheat in genetically have higher concentration of anti-nutrient

like phytate and poor in Zn thus have low Zn bioavailability (Welch and Graham, 2004;

Cakmak et al., 2010a). Furthermore, cultivation of wheat on Zn deficient soil further

reduces Zn in wheat seed. High yielding wheat cultivars “a product of green revolution”

intensified the problem of Zn deficiency (Zhao and McGrath, 2009; Cakmak et al., 2010a;

Stein, 2010). Zinc malnutrition is a major health issue to human population worldwide as

about 1/3 of humans are suffering from Zn deficiency (Cakmak, 2008; White and Broadley,

2011). In Pakistan, 40% of the children and females are suffering from Zn malnutrition

(Ministry of Health, 2009)

The problem of Zn deficiency can be alleviated by enhancing the Zn concentration

in daily food intake using different approaches (White and Broadley, 2009; Stein, 2010).

Zinc concentration in food can be exalted through fortification and supplementation but

these approaches have endorsed a specific part of the society (Singh and Parasad, 2014). In

this scenario, biofortification of food crops is suitable option to overcome the issue of

micronutrient malnutrition (Bouis, 2003). Biofortification is a strategy to enhance the

bioavailable concentration of nutrients in edible parts of crop plants (Mayer et al., 2008). It

includes development of nutrient dense plant genotypes (genetic biofortification) through

plant breeding or biotechnology (Welch and Graham, 2004; White and Broadley, 2005);

and agronomic biofortification i.e. escalating the concentration of micronutrients in edible

plant parts through fertilizer applicatiomn (Graham et al., 2001). However, genetic

29

biofortification is very costly, time taking and may result in development of less yielding

cultivars. Moreover, development of genetically modified crops have restricted use due to

ethical issues (Prasad, 2012). In this scenario, agronomic approach is the most suitable

approach particularly for developing world (Welch and Graham, 2004).

Zinc concentration and its bioavailability can be increased by application of Zn

fertilizers as it is short term and harmonizing approach which builds up Zn pool for uptake

and translocation (Cakmak, 2008). To correct the Zn deficiency and increase the Zn

concentration in wheat, Zn can be applied through soil, foliage and seed treatment viz. seed

priming and seed coating (Johnson et al., 2005). Soil Zn application is the principal method

of Zn fertilization and has found effective in enhancing the grain Zn concentration (Hussain

et al., 2013). However, plant respond to applied nutrient about 5-6 days later if edaphic

conditions are suitable (Fageria et al., 2009). Likewise, cost of fertilizer, quality of fertilizer

and its uniform application on soil surface make this method difficult to practice by resource

poor farmers. Foliar Zn fertilization is another strategy and found effective in improving

grain Zn concentration (Yilmaz et al., 1997; Johnson et al., 2005). However, for effective

foliage application, higher leaf area is need to efficiently absorb nutrient solution.

Moreover, suitable climatic condition, nutrient source and solution concentration are

prerequisite of effective spraying (Fageria et al., 2009).

Seed treatment is another approach which is gaining popularity these days. It

includes seed priming, which involves dipping of soaking in aerated water or nutrient

solution for a defined time period (Farooq et al., 2012). Seed priming with Zn has been

found to improve the seedling establishment in wheat (Rehman et al., 2015). Moreover,

improvement in growth, grain yield and Zn concentration has been reported for maize

(Harris et al., 2007) and chickpea (Harris et al., 2008). In seed coating, different growth

substances like nutrient, plant hormones and pesticides are adhered to the seed surface using

some sticky inert material which augment the crop performance (Freeborn et al., 2001).

Delivery of nutrients through seed coating help in uniform distribution and improved

seedling growth (Silcock and Smith, 1982; Scott and Blair, 1988). Seed coating with Zn

improved the seedling emergence, grain yield and grain Zn concentration of wheat

(Rehman and Farooq, 2016).

Zinc availability in rhizosphere is also influenced through different natural processes like

allelopathy. It is an ecological process where plants and microbes release secondary metabolites

which influence the agricultural and biological system inhibitory or promotory way (Farooq et

al., 2011). The allelochemicals released by the interaction of plants and microbes enhance the

30

nutrient uptake by increasing the solubilization, mobilization and chelation. Thus, uptake and the

phytoavailability of Zn can be increased by use of beneficial rhizospheric microbes (He et al.,

2010) and may help reducing the Zn deficiency in human population as well (Mäder et al., 2010).

Zinc solubilizing plant growth promoting rhizobacteria (PGPR) reside in the rhizosphere

(Ahmad et al., 2008; Maheshwari et al., 2012). These PGPRs enhance the concentration of

soluble Zn by modulating rhizospheric pH through enhaced organic acid production and

mineralizing the CaCO3 and organic bound Zn (Ramesh et al., 2014). Recently, Sirohi et al.

(2015) found that use of Zn solubilizing PGPR impressively enhanced the grain Zn concentration

of wheat (Sirohi et al., 2015).

Although, soil and foliar fertilization are the most prevalent methods for Zn

application, seed treatment (seed priming and seed coating) is cost effective and alternate

to soil and foliar application. However, optimization of the source and rate of Zn application

through seed treatment is needed. Zinc solubilizing PGPRs proved effective in increasing

the Zn uptake of wheat, but their interaction with chemical Zn sources/methods has never

been studied. Moreover, information regarding genetic diversity of Pakistani bread wheat

genotypes for Zn bioavailability and localization in different seed fractions have never been

studied. Zinc is required for activity of enzymatic antioxidant and membrane function but

potential role of Zn nutrition in improving the tolerance against abiotic stresses tolerance

in wheat is yet to be explored. Therefore, this study was conducted with the following

objectives:

To optimize the source, method and amount of Zn through seed treatments.

To characterize the wheat genotypes for genetic diversity, Zn bioavailability and

localization in different seed fractions.

To improve the productivity and grain biofortification of bread wheat by combined

application of Zn and Pseudomonas.

To explore the potential of Zn nutrition in improving tolerance against abiotic (drought,

salt, heat, chilling) stresses in wheat.

CHAPTER 2

REVIEW OF LITERATURE

Zinc (Zn) deficiency in higher plants was first reported by Sommer and Lipman

(1926). In Pakistan, it was reported as Hadda disease by Yoshida and Tanaka (1969). Zinc

deficiency in plants and soils is a universal micronutrient disorder in more than 40 countries

31

(Alloway, 2004) with almost 30% of agricultural soils worldwide deficient in plantavailable

Zn (Alloway, 2008a).

Zinc deficiency is prevalent in cereal crops across the globe resulting in low yields

and poor quality grains (Cakmak et al., 1999). Cereals are inherently low in Zn, and the

cultivation of cereal crops on low Zn soils further reduces grain Zn concentrations

(Cakmak, 2008). For instance, grain Zn concentrations can decline by 80% when cereals

(e.g. rye, triticale, bread wheat and durum wheat) are cultivated on soils with low

plantavailable Zn (Cakmak et al., 1997a). Among cereal crops, wheat is most sensitive to

Zn deficiency. Wheat also has higher concentrations of fiber and phytate, which further

reduces Zn absorption by humans (Welch, 1993). The application of Zn to wheat grown on

Zn-deficient soils increased grain yield by 32% (Kalayci et al., 1999). In extremely

Zndeficient areas (e.g. DTPA-Zn: 0.11 mg kg–1 soil), where wheat could barely produce

grain yield, Zn fertilization alone increased wheat grain yield by 550% (Cakmak et al.,

1996a).

Zinc deficiency affects more than two billion people worldwide (Cakmak et al.,

2010a). It is the principal reason for malnutrition in humans resulting in retarded growth

and infectious syndrome vulnerability (Graham et al., 2012). In regions with Zn-deficient

soils (e.g., Pakistan, Iran, Turkey and India), the incidence of Zn deficiency in humans is

also high (Cakmak et al., 1999; Hotz and Brown, 2004). Zinc deficiency in populations has

been widely reported in South Asia and is linked with a high incidence of death, particularly

in children (Black et al., 2008). For instance, in Pakistan, about 40% of women and children

suffer from Zn deficiency (Ministry of Health, 2009).

Rice–wheat cropping systems (RWCS) occupy about 26 Mha in South and East

Asia, and 85% of this area is in the Indo-Gangetic Plains. Zinc deficiency is widespread in

RWCS (Alloway, 2008b) due to the sequential cropping of rice and wheat. Likewise, Zn

deficiency is common in cotton–wheat cropping systems (Rashid, 2005; Alloway, 2008b;

Ahmed et al., 2010) as well as other cropping systems across the globe (Katyal and Vlek,

1985; Nayyar et al., 1990).

Several reviews have reported on Zn biofortification in wheat (Cakmak, 2008,

2010a; Prasad et al., 2013) and edaphic factors influencing Zn uptake (Alloway, 2009;

Gupta et al., 2016). However, there is no comprehensive review of Zn dynamics and its

management in different wheat-based cropping systems. This review highlights the role of

Zn in plant biology, compares different Zn application methods, and discusses the

interaction of Zn with other mineral nutrients, and the role of plant growth promoting

32

rhizobacteria (PGPRs) and arbuscular mycorrhizal fungi (AMF) in improving Zn

availability for wheat. Advances in agronomic and breeding approaches for biofortification

of wheat with Zn are also highlighted.

2.1. Zinc in Wheat Biology

Zinc is an essential micronutrient for plants, which is involved in a wide variety of

physiological and biochemical processes (Sommer and Lipman, 1926; Marschner, 2012).

Wheat roots take up Zn predominantly as Zn2+, but also through the formation of Zn

complexes with organic chelating compounds, i.e. phytosiderophores, which enhance the

solubilization of Zn in alkaline soils (Welch, 1993). The critical Zn concentration for the

youngest emerged wheat leaves is 14 mg kg–1 dry weight (Brennan, 2001) while at tillering

and anthesis, this value is 16.5 and 7 mg kg–1 dry weight, respectively (Riley et al., 1992),

and in whole grain is 10 mg kg–1 (Riley et al., 1992; Rengel and Graham, 1995).

Zinc is crucial for many physiological processes such as enzyme activation, protein

synthesis, and nucleic acid and carbohydrate metabolism in wheat (Cakmak, 2000; Palmer

and Guerinot, 2009). It is also a structural component of Zn finger proteins, which control the

differentiation and proliferation of cells (Palmer and Guerinot, 2009).

2.1.1. Protein synthesis

Zinc modulates protein synthesis; plants with Zn deficiency have poor protein

biosynthesis due to the accumulation of amides and amino acids (Marschner, 2012). It

defines the function and three-dimensional structure of many proteins (Broadley et al.,

2007), e.g. Zn finger proteins (Feinauer et al., 2013). Moreover, Zn helps to bridge amino

acid residues (methionine and cysteine) as it is strongly bound to the –SH group and limits

disulfide bond peroxidation (Marschner, 2012).

Zinc fertilization increases the protein content and quality of wheat grains by

increasing the concentrations of albumin, glutenin, gliadins and globulin (Liu et al., 2015).

Moreover, Zn concentration is linked to the storage protein in wheat grain and is closely

associated with grain gluten content (Peck et al., 2008). Zinc also modulates the sodium

dodecyl sulfate (SDS) extractable polymeric protein fraction (Peck et al., 2008). Zinc

deficiency affects protein synthesis due to the decline in RNA biosynthesis and/or by

ribosomal deformation (Alloway, 2004). Zinc is thus needed for increased protein

biosynthesis and better quality wheat.

33

2.1.2. Enzyme activation and resistance to abiotic stresses

Zinc regulates the activity of several enzymes (Barak and Helmke, 1993). It acts as

a cofactor for more than 300 enzymes which are largely Zn finger proteins, and RNA and

DNA polymerases (Coleman 1998; Lopez-Millan et al., 2005). Zinc forms strong

complexes with polar group radicals consisting of oxygen (O), nitrogen (N) and sulfur (S)

(Brown et al. 1993). The expression and activity of carbonic anhydrase (CA) and

superoxide dismutase (SOD) depend on Zn availability. The expression of Cu/Zn SOD is

up-regulated in Zn-efficient genotypes of wheat, while Zn-inefficient genotypes have low

Cu/Zn SOD activity (Hacisalihoglu et al., 2003).

With the changing climate, crop plants are vulnerable to abiotic stresses, which may

substantially reduce yields. Nutrient deficiency may further exacerbate the stress-induced

damage. Zinc deficiency is widespread in water-limited environments especially in

semiarid and arid regions where soils receive less water (Bagci et al., 2007). Indeed, poor

root growth and reduced soil volume lead to poor nutrient uptake (especially Zn) in

waterdeficient soils (Marschner, 2012).

With the rising temperature, heat stress is a significant threat to crop production as

elevated temperature disturbs plant performance by altering the structure and function of

cellular membranes (Wahid et al., 2007; Barnabás et al., 2008), chloroplasts and

photosynthetic machinery (Al-Khatib and Paulsen, 1990). Similarly, under salt stress,

excess ROS production causes structural and functional damage to lipids, proteins and

nucleic acids, photosynthesis and water relations (Munns and Tester, 2008). However, Zn

supply to stressed plants (drought, salt and heat stress) can help to mitigate the adverse

effects of these stresses (Figs 2.1, 2.2 and 2.3) by sustaining SOD activity which

participates in the detoxification of ROS and helps to maintain photosynthetic performance

under high temperature (Cakmak, 2000). For instance, the application of Zn to wheat

enhanced the activities of SOD and acid phosphatase by 96.8 and 75.8%, respectively

(Bharti et al., 2014). Zinc is also needed to maintain and regulate gene expression to cope

with abiotic stresses such as light and temperature extremes (Cakmak, 2000). In conclusion,

Zn protects plants from the adverse effects of abiotic stresses by regulating gene expression,

maintaining root growth, and detoxifying ROS by regulating the activities of enzymatic

antioxidants (SOD, APX and CAT).

34

Fig. 2.1: Effect of Zn nutrition on salt stress tolerance of 30-day-old wheat plants (T.


Plants were cultured in a Zn-deficient soil (DTPA-Zn: 0.1 mg kg–1 soil) with low

(–Zn: 0.3 mg Zn kg–1 soil) or sufficient Zn (+Zn: 3 mg Zn kg–1 soil) supply and

under salt stress (2500 ppm) for 30 days.

2.1.3. Structural and functional integrity of plasma membranes

Zinc upholds the structure and function of biological membranes. As root exudates

are indicators of plant cell membrane stability, plants suffering from Zn deficiency have

higher concentrations of phosphorus (P) in their root exudates than plants with adequate Zn

(Welch et al., 1982). Zinc helps to protect membrane lipids, protein, DNA and other vital

cell components (Cakmak, 2000). It helps to maintain the integrity of biological membranes

by encapsulating protein and lipids from superoxide radicals and other ROS (Cakmak and

Marschner, 1988).

Zn deficiency alters the ionic transport pattern across the cell membrane (Rengel

and Graham, 1996; Rengel, 1999). For instance, roots of Zn-deficient wheat had leaky

membranes and low levels of the –SH group in cell membranes compared with those with

high levels of Zn (Rengel, 1995a). This suggests that Zn is involved in the maintenance and

regulation of ionic movement through membranes by preventing peroxidation of the –SH

group and proteins containing ionic channels of the root cell plasma membrane (Kochian,

1993; Welch, 1995). In crux, Zn maintains the stability and integrity of biological membranes

by regulating the level of the –SH group and protecting membrane lipids and proteins from

peroxidation.

- Zn

+ Zn

35

Fig. 2.2: Effect of Zn nutrition on drought stress tolerance of 30-day-old wheat plants (T.


Plants were pre-cultured in a Zn-deficient soil (DTPA-Zn: 0.1 mg kg–1 soil) with low

(–Zn: 0.3 mg Zn kg–1 soil) or sufficient Zn (+Zn: 3 mg Zn kg–1 soil) supply and

thereafter subjected to drought stress (35% field capacity) for 15 days.

2.1.4. Cell division and reproduction

Zinc is involved in plant reproduction as 40% of transcription factors are Znbinding

proteins required for gene regulation to start gametogenesis, flowering (Colasanti et al.,

2006) and floral development (Takatsuji et al., 1992). Zinc deficiency influences plant

growth, maturity, fertilization and pollen viability (Nautiyal et al., 2011). Plants suffering

from Zn deficiency have low concentrations of IAA possibly due to inhibition of IAA

synthesis (Cakmak et al., 1989). Moreover, degradation of IAA by superoxide radicals may

lead to low IAA levels under Zn-deficient conditions (Cakmak et al., 1989). Poor seed

setting under Zn deficiency is due to increased levels of abscisic acid (ABA) causing

premature flower shedding and disrupting the structure of pollen and anthers (Brown et al.,

1993).

- Zn

+ Zn

36

Fig. 2.3: Effect of Zn nutrition on heat stress tolerance of 21-day-old wheat plants (T. aestivum

L. cv. Faisalabad-2008).

Plants were pre-cultured in nutrient solution under controlled conditions (25/20°C

day/night) with low (–Zn: 0.1 μM ZnSO4) or sufficient Zn (+Zn: 1 μM ZnSO4) supply and

thereafter subjected to heat stress (36/28°C day/night) for 10 days.

Zinc deficiency alters the structure and function of the stigma and pollen grains and

is often linked to a reduction in grain set, which indicates its involvement in pollen function

and fertilization (Pandey et al., 2006). In the Easter lily (Lilium longiflorum), the zinc

concentration in the growing tips of pollen tubes can be as high as 150 mg g–1 compared

with the basal region with 50 mg g–1 Zn (Ender et al., 1983). An adequate supply of Zn to

plants triggers endogenous gibberellins (Sekimoto et al., 1997) which may promote plant

growth. In crux, Zn participates in cell division and reproduction in plants by regulating

flowering and pollination and maintaining IAA levels.

2.1.5. Photosynthesis

Zinc is required for chlorophyll synthesis; chlorophyll content declined under a limited

supply of Zn (Cakmak and Marschner, 1993) due to alterations in chloroplast structure (Brown

Control Heat stress

- Zn + Zn

- Zn

+ Zn

37

et al., 1993). Zinc regulates the functioning and development of chloroplasts. It is required for

signal peptide peptidase (SPP) activity, and it regulates the repair of photosystem II (PSII) by

transferring the photodamaged protein D1 (Hansch and Mendel, 2009). For instance, Graham

and McDonald (2001) reported that Zn application under heat stress enhanced photosynthesis by

improving the chlorophyll fluorescence ratio (Fv/Fm) in Zn-inefficient genotypes of wheat.

Zinc is required for carbonic anhydrase (CA) activity, a key enzyme in

photosynthesis. Impaired nutrition with Zn limits CA activity (Rengel, 1995b; Salama et

al., 2006). Carbonic anhydrase is in the mesophyll cell chloroplasts of glumes and flag

leaves in wheat. It is also present in the cytosol of mesophyll cells in paleas and lemmas

and in xylem (Li et al., 2004). A reduction in photosynthesis due to reduced CA activity

has been observed in wheat under Zn deficiency (Hacisalihoglu et al., 2003; Wolff et al.,

2013). For instance, Rengel (1995b) measured CA activity in two contrasting wheat

genotypes under Zn deficiency; on re-supply of Zn, CA activity increased two-fold in the

Zn-efficient genotype suggesting a higher photosynthetic rate than the Zn-inefficient

genotype.

In crux, Zn has a crucial role in plant biology as it regulates enzyme activity and protein

synthesis, maintains RNA biosynthesis, and maintains bio-membrane integrity by

protecting the peroxidation of membrane lipids. Moreover, it regulates photosynthesis by

maintaining CA activity, photosynthetic pigments, and the structural and functional

integrity of photosynthetic machinery.

2.2. Factors Affecting Zinc Availability to Wheat

Total Zn concentration in soils varies from 3 to 770 µg g–1 with an average of 64 µg

g–1 (Alloway, 2009). Soils with less than 10 µg g–1 Zn are considered deficient while soils

with >200 µg g–1 are considered contaminated. Availability of soil Zn to plant roots is

determined by physiochemical properties of soils (e.g., Zn content, pH, organic matter and

clay content, temperature, moisture) as well as root exudates and soil microorganisms (Fig.

2.4). In the following section, factors affecting Zn availability to wheat are discussed. 2.2.1.

Soil pH

Soil pH is the most critical factor influencing Zn availability to crop plants, as 90%

of the variability in Zn availability to crops depends on differences in soil pH. Zinc uptake

decreases significantly with increasing soil pH from 4.6 to 6.8 (Sumner and Farina, 1986;

Wilkinson et al., 2000; Fageria et al., 2002). Wheat grown on high pH/alkaline soils with

high clay contents may suffer Zn deficiency (Qadar, 2002) as Zn availability decreases on

38

calcareous soils due to increasing soil pH and high concentrations of CaCO3. CaCO3 is

highly adsorptive and retains Zn through chemisorption (Kiekens, 1995). Lindsay (1979)

reported that the ZnSO4 complex can be critical in soils and can contribute significantly to

total Zn in solution. Sorption/desorption experiments with Zn clearly indicated that the lack

of availability of Zn in these soils was due to chemisorption on CaCO3. In Zn-sufficient

soils, CaCl2 subsequently desorbed about 20% of the additional sorbed Zn, but in

Zndeficient soils, only 1% of additional sorbed Zn was desorbed (Cakmak et al., 1999;

Cakmak, 2008). For instance, in Turkey, 65% of the soils in Central Anatolia under wheat

cultivation had >20% CaCO3 with a pH from 7.5 to 8.1 and DTPA-extractable Zn of 0.29

mg kg–1. However, >90% of wheat-growing soils had Zn levels lower than the critical limit,

i.e. 0.5 mg kg–1 DTPA-extractable Zn (Eyupoglu et al., 1994). Others have shown that an

increase in soil pH from 8.0 to 8.3 doubled the Zn-binding strength to the CaCO3 mineral

calcite. The Zn-binding strength increased up to seven-fold with 0.05% Fe coating on

calcite (Uygur and Rimmer, 2000) which severely reduced Zn availability to plants

resulting in a higher incidence of Zn deficiency (Uygur and Rimmer, 2000). High soil pH

reduces Zn availability due to the formation of insoluble complexes with carbonates and

hydroxides (Rupa and Tomar, 1999). Loamy soils or heavily limed sandy soils may also

have high calcite content with pH>7, which can reduce plant Zn uptake (Fageria and Stone,

2008). However, soils with low pH and calcite are common in tropical regions and should

be limed to increase cereal production (Fageria and Stone, 2008). In crux, high soil

pH/alkaline soils are linked to Zn sorption on carbonates, hydroxides and clay minerals thus

limiting Zn uptake by plants.

39

Fig. 2.4: Mechanism of Zn uptake from soil and its translocation into developing grains.

Plant uptake zinc as Zn2+ by ZIP transporter or via secretion of phytosiderophores. Soil physiochemical

properties (soil pH, organic matter, moisture, soil microflora and root structure) influence the Zn

availability in soil. Moreover, wheat roots and soil microbes also release organic acids, such as citrate

and malate, to increase Zn solubility. Zinc moves through apoplastic and symplastic pathways followed

by loading in the xylem and transfer to the phloem. Xylem loading of Zn takes place through HMA

pumps. Some of the Zn may sequester in the vacuole during route to xylem from absorption form soil.

Zinc moves from xylem with the help of transport proteins to living xylem parenchyma cells of leaf

symplast; from here, it is transferred to leaf apoplast and then to phloem, which is the vascular route of

Zn translocation into developing seed. Zn transfers from ear to maternal tissue and then to endosperm.

Most of the Zn after uptake is stored in phytate bounded protein storage vacuoles, and very small portion

of Zn is present in the endosperm. YSL = Yellow stripe like proteins, MTP = Metal tolerance protein,

MFS = Major facilitator superfamily transporter, HMA= Heavy metal ATPase, PS= phytosiderophores

SP = Small protein, NA = Nicotinamine, NRAMP = Natural resistance-associated macrophage protein,

VIT = Vacuolar iron transporter

40

2.2.2. Soil organic matter

Soil organic matter plays an important role in Zn solubility and its availability to

growing plants (Harter, 1991). Zinc deficiency is prevalent in soils, which are naturally

high or low in organic carbon, waterlogged, and light-textured (Ahmad et al., 2012). In

soils with low organic matter content, increasing the amount of soil organic matter can

enhance the formation of soluble complexes, which may increase Zn uptake by plants

(Ozkutlu et al., 2006). The addition of organic matter to such soils enhances Zn

bioavailability to plants (Mandal et al., 1988). Soil organic matter itself can influence Zn

adsorption; soils with high organic matter content may exhibit higher Zn adsorption

compared to soils with low organic matter content (Gurpreet-Kaur et al., 2013). Moreover,

soil type affects Zn adsorption as peat soils are naturally deficient in Zn due to adsorption

and further liming of these soils reduces Zn availability to plants (Abat et al., 2012).

Alloway (2004) noted a significant positive correlation between soil extractable Zn

and soil organic matter content. Mandal et al. (1988) reported that the addition of organic

matter increased Zn bioavailability to rice plants. Clark (1982) stated that Zn deficiency is

most likely to occur in plants growing on calcareous soils with inherently very low organic

matter content. In general, soils with very low organic matter cannot maintain ample

reserves of available Zn and are more vulnerable to Zn deficiency. In mineral soils, organic

matter content rapidly decreases with increasing soil depth accompanied by a decline in

DTPA-extractable Zn (Alloway, 2008a). However, the addition of organic matter that

decomposes quickly, such as manure, forms soluble Zn complexes and thus enhances Zn

mobility and absorption by roots (Alloway, 2008a). In contrast, Zn availability can be

severely impaired in soils with high organic matter and low Zn content due to the formation

of insoluble complexes with organic matter (Alloway, 2008a). In crux, Zn availability in

soils is under the influence of soil organic matter. Furthermore, the nature and content of

organic matter are decisive factors for availability and thus uptake of Zn by plant roots.

2.2.3. Soil temperature, moisture and light intensity

Environmental factors influence Zn availability as Zn deficiency is most prevalent

in arid and semiarid regions where the topsoil is often low in plant-available water under

rainfed conditions (Cakmak et al., 1996a). Plants become more sensitive to Zn deficiency

under low water availability (Bagci et al., 2007). Under limited water supply, Zn movement

in the soil is limited resulting in poor root growth and reduced soil volume that restricts Zn

uptake (Marschner, 2012). Several studies concluded that growth of Zn-deficient plants was

poor under water-limited conditions and that sensitivity to Zn-deficiency stress became

41

more pronounced when plants were drought-stressed (Hajiboland and Amirazad, 2010;

Bagci et al., 2007; Ekiz et al., 1998). These studies also pointed out that, in drought-stressed

plants, the effect of irrigation on grain yield was maximized with adequate Zn fertilization.

Soil temperature also influences Zn availability as wet and cool seasons result in

reduced Zn availability (Moraghan and Mascagni Jr, 1991) due to the reduced rate of

mineralization (i.e. the liberation of Zn in organic matter by decomposition) in soil (Takkar

and Walker, 1993). Low temperature restricts not only organic matter decomposition but

also root growth, which further limits plant Zn uptake (Alloway, 2008a). Furthermore, low

root-zone temperatures reduce mycorrhizal colonization, root growth, and Zn uptake and

translocation to shoots (Moraghan and Mascagni Jr, 1991). Long day lengths and exposure

to high light intensity also induce the occurrence of Zn deficiency symptoms (necrosis and

chlorosis of leaves) and related physiological responses such as impairments in ROS

detoxification (Marschner and Cakmak, 1989). In crux; environmental factors such as low

soil temperature, less precipitation, long day lengths and high light intensity are linked to

Zn deficiency.

2.2.4. Soil salinity and interaction of Zn with other elements

Zinc deficiency is most common in arid and semiarid environments on saline soils.

Higher Ca together with high pH reduces Zn availability to plants (Alloway, 2008a). Zinc

uptake decreases under saline soils due to strong competition between Zn and salt cations

at the root interface (Tinker and Lauchli, 1984), For example, in saline–sodic soils, the

exchange sites are occupied by Na+ resulting in leaching of Zn, especially if the irrigated

water has high Na+ (Alloway, 2008a). The uptake of Zn declines in salt-affected soils

contaminated with Cd due to the negative interaction of Zn with Cd and the formation of

CdCl2, which is readily available to plants due to its high solubility (Khoshgoftar et al.,

2004). In addition, high soil electrical conductivity and pH, and a higher concentration of

Ca, Na, Mg and HCO3 are the principle reasons for low availability of Zn (Deckers et al.,

1998). The application of Zn to saline soil improves plant growth by limiting the uptake

and translocation of Na+, Cd2+ and Cl– (Abd El-Hady 2007).

Zinc is a cation that interacts with almost all plant nutrients present in the soil,

especially anions. For instance, Zn has a positive interaction with N in cereals (Lakshmanan

et al. 2005) as enhanced N supply increased the seed Zn concentration possibly by

influencing the abundance of Zn transporters and level of Zn-chelating nitrogenous

compounds (Kutman et al., 2010). For instance, increasing rates of N application to wheat

enhanced root uptake and shoot translocation rate of Zn by 300% (Erenoglu et al., 2011).

42

Increasing the rate of applied N also enhanced grain Zn concentration in wheat (Kutman et

al., 2011). In contrast, Zn has a negative interaction with P. For example, increasing P

application rates from 0 to 400 kg ha–1 reduced grain Zn concentration from 29 mg kg–1 to

13 mg kg–1 as higher rates of P application widened the P:Zn molar ratio thus reducing the

bioavailability of Zn (Zhang et al., 2012). Recently, the negative interaction of P on grain

Zn concentration was shown to be dependent on mycorrhizal infection as low or high P

supply had no effect on grain Zn when soil was sterile of mycorrhiza (Ova et al., 2015).

Nevertheless, Zn had a positive interaction with K as Zn maintains membrane integrity and

reduces leakage of K and amides (Cakmak and Marschner, 1988). Sulfur application

enhanced the Zn concentration in wheat (Cui and Wang, 2005). However, Zn suppressed

the availability of Ca (Kalyansundaram and Mehta, 1970). High Ca concentration reduced

Zn uptake and translocation (Kawasaki and Moritsugu, 1987), while Zn had a negative

interaction with Cu (Brar and Sekhon, 1978) and Mn (Gupta and Gupta, 1984). However,

Zn can help to overcome B toxicity (Mishra and Singh, 1996) as it reduces B uptake. The

Zn and Fe interaction is complex as Zn application either decreases, increases or does not

influence Fe status (Loneragan and Webb, 1993). Under Zn deficiency, reduced expression

of MTP3 and HMA3 in an Fe-deficient mutant showed that Fe deficiency is linked with Zn

accumulation (Colangelo and Guerinot, 2006) as stunted growth and chlorotic leaves are

visible at high levels of Zn suggesting Fe deficiency in plants (Gupta et al., 2016).

In crux, saline soils limit Zn uptake due to a high concentration of Na on

exchangeable sites together with other ions i.e. Ca+, Cd2+ and Cl–. Zn has a positive

interaction with some elements (N, K and Mg, and Ca) while it suppresses the availability

and uptake of others (P, Cu, Mn and B).

2.2.5. Zinc interaction with soil biota/mycorrhizal colonization

Soil microorganisms can increase Zn uptake in plants. For instance, mycorrhizal

colonization can effectively enhance the absorption of nutrients whose uptake is limited to

diffusion from soil solution to plant roots (Fageria et al., 2011). Arbuscular mycorrhizal

fungi (AMF) improved Zn uptake in several crops including wheat (Ryan and Angus,

2003). Increased Zn uptake is due to colonization of AMF on the plant root system, which

increases the surface area via a hyphal network beyond the nutrient-deficient zone of roots

(Smith and Read, 2008). Moreover, AMF help in Zn acquisition from pores and patches of

soil not accessible by plant roots (Bolan, 1991). In a meta-analysis of 33 field studies,

Pellegrino et al. (2015) reported that AMF increased grain yield and Zn concentration in

wheat.

43

Soil bacteria also help to enhance nutrient uptake. Among soil microbes, PGPRs are

the most important as they increase nutrient uptake by colonizing the root surface due to

signal transduction between the host plant and bacteria (Bianciotto et al., 2000). In one

study, He et al. (2010) identified Bacillus subtilis, Bacillus cereus, Flavobacterium spp. and

Pseudomonas aeruginosa as Zn-tolerant bacteria that help to enhance Zn availability in soil

and its uptake by plants. Bacillus aryabhttai also improved Zn accumulation in wheat grain

by solubilizing the Zn bound to organic complexes and CaCO3 and increasing soil

exchangeable Zn due to enhanced activities of soil microbes and increased redistribution of

available Zn in the rhizosphere (Ramesh et al., 2014). Naz et al. (2016) reported that

Znsolubilizing bacteria improved Zn uptake and partitioning in vegetative parts at different

growth stages when used in combination with N and P fertlizer, as shoot Zn concentration

increased with inoculation of Azospirillum; while inoculation with Rhizobium,

Pseudomonas and Azospirillum increased the grain Zn concentration.

AMF and PGPRs are effective for improving growth, grain yield and Zn availability

in wheat by directly enhancing Zn uptake or by releasing organic acids/compounds, which

help in Zn solubilization and its uptake by plants (Table 2.1). For example, Rana et al.

(2012a) reported that inoculation of seed with Providencia sp. PW5 strain + recommended

fertilizer increased protein and grain Zn concentrations by 18 and 24.3%, respectively,

compared with the control.

In conclusion, Zn uptake in plants is limited in soils with high pH, CaCO3 and P

concentrations, and low organic matter content. Soil temperature and moisture play a

significant role in Zn availability. AMF and PGPR are effective for improving wheat Zn

status and could be used along with chemical fertilizers to overcome Zn deficiency.

2.2.6. Zn efficiency in wheat

Zinc efficiency as a genetic trait is generally attributed to the ability of a cultivar to

grow and yield well in Zn-deficient conditions as compared to standard cultivars. Zinc

efficiency is a complex trait controlled by different physiological and molecular

mechanisms that can be divided into root- and shoot-based mechanisms. Root-based

mechanisms involve the efficient uptake of Zn from soil by modifying root morphology

and the chemistry of the rhizosphere. Shoot-based mechanisms include root-to-shoot

translocation, remobilization within the whole plant, and subcellular compartmentation of

Zn (Rengel and Graham, 1996; Hacisalihoglu and Kochian, 2003; Genc et al., 2006). In

this section, mechanisms affecting Zn efficiency in wheat are discussed.

44

Root Zn uptake plays a major role in Zn efficiency in wheat (Genc et al., 2006).

Increased root surface area, release of phytosiderophores into the rhizosphere, and

induction of polypeptides involved in Zn uptake and transport across membranes play a

major role in Zn uptake from the soil (Rengel and Graham, 1995; Cakmak and Braun,

2001). Root morphology may influence Zn uptake (Dong et al., 1995) as longer and thinner

roots with larger surface area will uptake more Zn (Rengel and Graham, 1995). Roots

change rhizosphere pH by releasing organic acids (Wang et al., 2006) which help to

solubilize the Zn present in inorganic and organic soil complexes (Hacisalihoglu and

Kochian, 2003). Moreover, roots release phytosiderophores which make chelates with Zn

and increase Zn availability (Cakmak et al., 1994). Grasses generally release

phytosiderophores under Fe (Marschner, 2012) and Zn (Zhang et al., 1991; Kochian, 1993)

deficient conditions. Phytosiderophores are also involved in mobilizing Zn from the root

apoplast of wheat plants (Zhang et al., 1991), and thus may influence Zn efficiency.

Znefficient bread wheats release more phytosiderophores under Zn deficiency as compared

to Zn-inefficient durum wheat cultivars (Cakmak et al., 1996b, 1998). However, Erenoglu

et al. (1996) found no correlation between phytosiderophore release and Zn efficiency in

Znefficient or Zn-inefficient wheat genotypes.

The role of root-to-shoot translocation of Zn may be important in Zn efficiency

(Rengel and Graham, 1995; Cakmak et al., 1996b). Higher Zn efficiency in rye as compared

to other cereals can be due to its higher capacity to uptake and translocate Zn from roots to

shoots (Cakmak et al., 1997b). Similarly, enhanced root Zn uptake and translocation to

shoots can influence Zn efficiency in chickpea (Khan et al., 1998). However, Zn

concentration was similar in shoots of Zn-efficient and Zn-inefficient wheat genotypes

which differ significantly in visual Zn deficiency symptoms (Cakmak et al., 1999;

Hacisalihoglu et al., 2003). In a study with 37 bread wheat and three durum wheat

genotypes differing in Zn efficiency, Kalayci et al. (1999) found no significant difference

in shoot Zn concentrations of different genotypes.

Shoot-mediated mechanisms, i.e. subcellular Zn compartmentation and/or efficient

biochemical utilization of cellular Zn, may be important in Zn efficiency determination

(Hacisalihoglu and Kochian, 2003). However, Hacisalihoglu et al. (2003) found that both

Zn-efficient and Zn-inefficient genotypes had similar Zn contents in the vacuole (83–85%)

and cytoplasm (9–11%) suggesting no role for subcellular compartmentation in Zn

efficiency. Zinc efficiency was correlated with biochemical utilization of Zn in superoxide

dismutase (SOD) and carbonic anhydrase in wheat and bean plants (Hacisalihoglu et al.,

45

2003). The activities of Cu/Zn superoxide dismutase and carbonic anhydrase significantly

increased under Zn-deficient conditions in Zn-efficient genotypes as compared to

Zninefficient genotypes (Rengel, 1995b; Cakmak et al., 1997c). When cultivated under low

Zn conditions, Zn-efficient genotypes perform better than Zn-inefficient genotypes by

efficiently using available Zn through the action of carbonic anhydrase and Cu/Zn

superoxide dismutase (Hacisalihoglu and Kochian, 2003). As a complex trait, Zn efficiency

warrants further attention at the physiological and molecular levels to expose the

mechanisms involved for better Zn utilization in wheat.

2.2.7. Value of intrinsic seed zinc for germinating wheat seedlings

Increasing grain Zn concentration through biofortification may impact not only

human and animal nutrition but also plant nutrition, at least in the early stages of plant life,

i.e. germination and seedling establishment in the field. The value of intrinsic seed Zn

reserves would be even more crucial when wheat is cultivated for intensive farming,

particularly in rice–wheat or maize–wheat cropping systems as well as in soils with low Zn

availability.

Previous studies with wheat showed that seed vigor (germination rate and seedling

height) and biomass production at the early vegetative stage are closely linked to intrinsic

seed Zn. For example, low seed Zn concentrations decreased the germination rate and

seedling growth in a Zn-deficient soil (Yilmaz et al., 1998). However, our knowledge on

the effects of Zn biofortification on germination and crop performance of progeny is scarce.

Welch (1999) and Cakmak (2008) discussed the possible roles of intrinsic seed Zn reserves

for improving crop productivity and proposed that Zn-enriched seeds can exhibit better seed

viability and vigor. Increasing seed Zn concentration by Zn fertilization of the maternal

plant was indeed reported to enhance biomass production and grain yield in progeny in sand

culture (Rengel and Graham, 1995) and the field (Yilmaz et al., 1998). Therefore, it is likely

that agronomic Zn biofortification could bring about additional benefits when Zn-

biofortified seeds are used, particularly in Zn-deficient soils and under stressful conditions

such as drought or salinity. In general, a high Zn content in seed may act as a starter fertilizer

(Rengel and Graham, 1995), although this effect may not match conventional Zn

fertilization in the field (Yilmaz et al., 1998). Nevertheless, under severe Zn-deficient

conditions, higher seed Zn concentration can significantly support field establishment of

seedlings and early vegetative growth.

Our knowledge on why low intrinsic seed Zn results in loss of seed viability and vigor is

also limited. Low levels of seed Zn may be associated with the existence of cellular defects or

46

damage in seeds, reductions in nutrient reserves (i.e. starch, protein, oil and mineral nutrients) or

disruption of critical biochemical processes during imbibition and germination (Ozturk et al.,

2006; Cakmak, 2008). In germinating wheat seeds, Zn is rapidly mobilized to axis organs (i.e.

roots and coleoptile) at very high concentrations (Bityutskii et al., 2002, 2004; Moussavi-Nik et

al., 1997, 1998; Ozturk et al., 2006). Extreme rates of cell division and expansion, protein

synthesis, enzymatic activity and gene expression could be reasons for the high Zn demand in

the axis organs of germinating seeds. Apparently, a high intrinsic seed Zn would ensure that all

Zn-dependent processes during imbibition and germination are accomplished for maximum seed

vigor and viability ((Moussavi-Nik et al., 1997; Ozturk et al., 2006). Moreover, low intrinsic

seed Zn can render seedlings more susceptible to biotic and abiotic stress factors such as

infectious diseases, pest damage, temperature extremes and drought, leading to depressions in

field stand and growth (reviewed in Cakmak, 2008). For example, SOD activity is highly

dependent on the Zn nutritional status in plants (Cakmak and Marschner, 1993) and associated

with Zn efficiency (i.e. ability to grow and yield under limited Zn supply) in wheat (Cakmak et

al., 1997a; Hacisalihoglu et al., 2003). In germinating wheat seedlings, ample Zn would be

required to sustain the activity of ROS scavenging enzymes, particularly that of SOD.

In conclusion, initial seed Zn reserves are crucial for the survival of germinating

seeds and the resultant seedlings, particularly when sown in Zn-deficient soils. High seed

Zn can also contribute to ROS scavenging by sustaining high SOD activity and thus CO2

fixation and biomass production. It is, therefore, proposed that a high intrinsic seed Zn

would not only serve for biofortification purposes but also greatly contribute to agricultural

productivity by enhancing seed vigor and seedling establishment particularly under

multiple stress conditions such as Zn deficiency and drought.

2.3. Zinc in Soil and its Dynamics in Different Wheat-based Cropping Systems

Zinc is present in different inorganic and organic forms in the soil that influence its

availability to plants. Zinc concentration varies depending on soil type. For instance, in

mineral and organic soils, the Zn concentration is about 50 and 60 mg Zn kg–1 of soil,

respectively. However, most agricultural land has a broad range from 10 to 300 mg Zn kg–

1 of soil (Barber, 1995). Zinc present in soil is not completely available to plants, and its

accessibility relies on soil physiochemical properties, soil microbes, plant root activity and

climatic factors (Alloway, 2008a). Some of the Zn in soil exists as adsorbed and

exchangeable forms or insoluble complexes, while some is water soluble and readily

available to plants. Plant roots assist in Zn uptake by ion exchange, and the release of

47

organic acids and phytosiderophores (Fig. 2.4; Cakmak et al., 1994; Gupta et al., 2016).

Zinc is present in soils as a water-soluble Zn pool, soil exchangeable sites, organically

bound complexes, and as primarily weathered minerals (Viets, 1962). Zinc present as

soluble fractions is readily available to plants due to desorption, but the chances of leaching

are also high for this fraction (Alloway, 2008a).

2.3.1. Rice–wheat cropping systems

The rice–wheat cropping system is a major cropping system around the globe,

particularly in South Asia where more than 75% of this cropping system occurs in the

IndoGangetic Plains of Pakistan (2.2 Mha), India (9.2 Mha), Bangladesh (0.4 Mha) and

Nepal (0.55 Mha) (Timsina et al., 2010), which is severely deficient in Zn (Alloway, 2009).

The Green Revolution helped in the development of new wheat and rice varieties of short

duration enabling the rotation of two crops per season (Timsina and Connor, 2001). The

rice–wheat cropping system results in Zn deficiency in rice due to increased soil P and

bicarbonate concentrations together with elevated soil pH owing to flooded conditions,

while chemisorption and high soil pH results in Zn deficiency in wheat (Alloway, 2009).

wheat after transplanted flooded rice changes the soil redox potential, thus affecting

micronutrient availability. Zinc availability declines under anaerobic conditions compared

with aerobic conditions. Most of the soils in RWCS are calcareous with a basic soil pH,

which reduces the availability of Zn (Alloway 2008a, b). Soil pH is the key factor

influencing Zn availability in the Indo-Gangetic Plains (Qadar, 2002). Additionally, Zn

translocation and uptake is restricted by the presence of high bicarbonate (HCO3) in soils

(Dogar and van Hai, 1980). Moreover, in RWCS, due to the high P availability, Zn uptake

and its translocation in plants are restricted (Lindsay, 1979). Flooding initially increases the

Zn concentration in soil solution, but this declines with time due to the formation of

insoluble compounds such as franklinite, zinc carbonate (ZnCO3) and zinc sulfide (ZnS).

The formation of these compounds occurs due to the decomposition of soil organic matter

(Brar and Sekhon, 1976). However, Zn fertilization improves yield and grain Zn

concentration in wheat in a RWCS (Table 2.2). Moreover, green manure crops enhance Zn

uptake and grain yield in wheat. For instance, in a basmati rice–durum wheat cropping

system, the incorporation of dhaincha (Sesbania culeate) increased grain yield and grain

Zn concentration in durum wheat (Singh and Shivay, 2013).

It is inferred that Zn deficiency is widespread in rice–wheat cropping systems due

to the alkaline nature of soils, the presence of CaCO3, and conventional production practices

48

for rice and wheat in RWCS. In addition, the use of green manure crops along with chemical

fertilizer can help to alleviate Zn deficiency in this cropping system.

2.3.2. Cotton–wheat cropping systems

Zinc deficiency is prevalent in cotton–wheat cropping systems because this system

is mostly practiced on alkaline calcareous soils in the Indo-Gangetic Plains (Rashid, 2005;

Ahmed et al., 2010). Rafique et al. (2012) linked Zn deficiency in a cotton–wheat cropping

system with Zn fixation in calcareous soil rather than with low soil Zn contents. They

further reported that Zn uptake was higher with organic Zn sources. Soil organic matter has

a significant role in the solubilization and uptake of Zn (Harter, 1991) as soils rich in organic

matter have higher uptake and solubility of Zn by plants due to the formation of Zn soluble

complexes (Ozkutlu et al., 2006). Maqsood et al., (2015) surveyed cotton–wheat cropping

systems in Punjab, Pakistan to find that 76% of the collected soil samples were deficient in

plant-available Zn. The wheat grains were also low in Zn with an average 20 µg g–1 Zn with

very low bioavailable Zn. They further reported that saline soils and low organic matter are

other factors that result in low Zn concentration in this cropping system. 2.3.3. Zinc

deficiency in other wheat-based cropping systems

Besides rice–wheat and cotton–wheat systems, Zn deficiency has been reported in

various other wheat-based cropping systems. For example, Kumar and Qureshi (2012)

evaluated the effect of Zn application on the succeeding maize crop after wheat and found

that Zn applied to the wheat crop was available to maize. They further reported a positive

interaction between plant Zn concentration and available Zn concentration applied from

two sequential Zn extractions (organically complexed and water soluble, and exchangeable

fractions). Zinc application also increased N and P concentrations in the plant. Moreover,

soil Zn fractions after harvest of wheat and maize had a positive and significant correlation

(Kumar and Qureshi, 2012).

Table 2.1: Effect of PGPRs and AMF on Zn uptake, grain yield and organic acids/compounds production of wheat

Soil microbe/AMF Organic compounds/acid

production Increase in Zn uptake in

vegetative tissues (%) Increase in grain

Zn concentration

(%)

Increase in

grain yield

(%)

Reference

Bacillus aryabhattai MDSR7 Dehydrogenase, β-Glucosidase,

Auxin content

Shoot 40.7

Root 28.7

– – Ramesh et al. (2014)


Auxin content

Shoot 26.4

Root 20.5



Auxin content

Shoot 67.9

Root 48.7


CW1

(Providencia sp PW5

(Anabaena sp CW1 + Providencia sp PW5

CW1 + Calothrix sp CW2 + Anabaena sp CW3

Fluorescein diacetate activity,

Dehydrogenase, Alkaline

phosphatase, Microbial biomass

carbon

–

–

–

–

14.9

32.1

26.0

19.0

– 14.1

17.4

12.3

Rana et al. (2012a)

Rana et al. (2012a)

Rana et al. (2012a)

Rana et al. (2012a)

Rhizophagus irregularis (isolate BEG140),

Rhizophagus irregularis, Funneliformis mosseae

(isolate BEG95), Funneliformis geosporum,

Claroideoglomus claroideum

– – 11.3 – Cabral et al. (2016)

P. indica Chlorophyll (a & b), carotenoid Shoot 34.2 – – Abadi and Sepehri (2016)

P. indica + A. Chroococcum Ascorbate peroxidase and

peroxidase activity Shoot 26.0 – – Abadi and Sepehri (2016)

S. liquefaciens FA-2 – – 31.3 14.3 Abaid-Ullah et al. (2015)

B. thuringiensis FA-3 – – 24.1 11.4 Abaid-Ullah et al. (2015)

S. marcescens FA-4 – – 39.8 14.3 Abaid-Ullah et al. (2015)

Consortium1 - - 71.6 21.4 Abaid-Ullah et al. (2015)

23

50

Sowing wheat in rotation with pigeon pea (Cajanus cajan L.) increased grain Zn

concentration in the succeeding wheat crop (Nawab et al., 2011). Growing wheat in

soybean (Glycine max L.)–green manure (GM) and soybean–fallow (FW) rotation

increased grain Zn and N concentrations in wheat. While the increases in Zn and N

concentrations were not significant during the first year, they increased by 26 and 12%,

respectively, in wheat grown in the soybean–GM rotation compared with 14 and 6% in the

soybean–FW rotation.

Zinc concentrations were significantly higher in wheat grains cultivated after

sunflower (Helianthus annuus L.) compared with the cotton–wheat rotation in salt-affected

soils of Iran (Khoshgoftarmanesh and Chaney, 2007). Recently, Habiby et al. (2014)

conducted a pot study using clover (Syzygium aromaticum L.), safflower (Carthamus

tinctorius L.), sunflower and sorghum (Sorghum bicolor L.) as the preceding crop to wheat.

They found that the incorporation of residues in all crops increased dissolved organic

carbon (C), organic matter and DTPA-extractable Zn. However, the addition of clover and

safflower residues resulted in the maximum increase in grain Zn concentration while the

addition of sorghum residues negatively influenced the grain yield of wheat. The increase

in Zn concentration in shoot and grain was due to dissolved organic carbon (DOC)

generated from residue decomposition which enhanced Zn uptake by wheat roots.

Zn deficiency is widespread in rice–wheat and cotton–wheat cropping systems due

to high soil pH and the formation of insoluble Zn compounds. However, growing wheat in

sunflower–wheat/legume–wheat rotations increased the availability of Zn to the succeeding

wheat crop.

2.4. Methods of Zinc Fertilizer Application

The aim of Zn application is to improve Zn uptake by plants either through soil,

foliage or seed treatments (Johnson et al., 2005; Farooq et al., 2012). In wheat, Zn is phloem

mobile as foliar-applied Zn moves to other leaves and root tips through phloem (Haslett et

al., 2001). Similarly, Zn translocation from leaves and stems to developing grains is

significant (Pearson et al., 1996) showing the possible involvement of phloem in Zn

translocation. Moreover, the selection of suitable Zn sources is imperative for improving

plant Zn status (Table 2.3). Different Zn application methods are discussed below.

51

2.4.1. Soil Zn fertilization

Zn soil fertilization is the most common strategy to cope with Zn deficiency. Soil

Zn fertilization is performed via broadcasting, fertigation or band placement. Application

of Zn through soil increases the Zn concentration in whole grain, embryo, aleurone and

endosperm of wheat. In wheat, soil application of ZnSO4 before sowing is the common

practice to overcome Zn deficiency (Cakmak, 2008). Soil fertilization not only increases

seed Zn concentration but also enhances crop growth and grain yield (Khan et al., 2003).

Recently, Gomez-Coronado et al. (2016) demonstrated that under low water availability,

plants tend to have high Zn concentrations, but lower grain yields and fertilization of soil

with Zn did not improve grain Zn concentration under water-limited conditions. However,

under such conditions, the application of Zn through soil + foliar spray increased grain Zn

accumulation. Soil application of Zn (6 mg Zn kg–1) increased the Zn concentration from

51.7 to 69.9% compared with the control (Maqsood et al., 2009). In another study, soil +

foliar application of Zn increased the grain Zn concentration by up to 80% while decreasing

the phytate concentration by 23.2% (Bharti et al., 2013). In a pot study by Hussain et al.

(2013), soil application of 7.4–10.8 mg Zn kg−1 soil increased yield, seed Zn concentration

and bioavailable Zn (>2.9 mg Zn in 300 g grains) and reduced the [phytate]:[Zn] ratio by

decreasing the phytate concentration.

Soil application of Zn increased grain yield in wheat by 29%. Furthermore, the Zn

concentration in wheat grain increased by 95% while the estimated bioavailable Zn

increased by 74% (Hussain et al., 2013). Higher rates of soil application increased the Zn

bioavailability in humans. However, a medium rate of soil application along with foliar

fertilization may be more beneficial for increasing grain Zn concentration where higher soil

application of Zn is not cost-effective. In some studies, soil application was not effective in

enhancing the grain Zn status at significant rates (Nattinee et al., 2009). For instance; soil

Zn application increased soil DTPA-Zn, but there was no significant increase in grain Zn

concentration (Zhao et al., 2014).

The application of Zn fertilizer is a quick solution for Zn deficiency and is deemed

necessary together with a breeding approach (Cakmak, 2008). Soil Zn fertilization is

generally adopted to overcome Zn deficiency (Rengel et al., 1999) thus increasing plant

growth and grain yield (Mortvedt and Gilkes, 1993; Rengel et al., 1999) and grain Zn

concentration (Rafique et al., 2006; Hussain et al., 2012). The most commonly used Zn

52

fertilizer is ZnSO4. Zn-EDTA fertilizer is more effective than inorganic Zn sources

(Mortvedt 1991) but it is not suitable for cereal farming due to its high cost.

In crux; soil application of Zn is the most common practice, and it effectively

corrects Zn deficiency and improves grain yield. However, higher rates of Zn application,

suitable Zn sources and uniform distributions remain the bottleneck for success with this

method.

2.4.2. Foliar zinc application

Crop micronutrient demand can be fulfilled by foliar fertilization. In several studies,

foliar application of Zn significantly increased seed Zn concentration (Yilmaz et al., 1997;

Cakmak et al., 2010b). However, repeated spraying, climate factors, and the high price of

foliar fertilizers make this strategy difficult for small resource farmers to practice (Johnson

et al., 2005). Foliar application of Zn increased grain Zn concentration more than soil Zn

fertilization when applied at later stages of growth (Zhao et al., 2014). Foliar Zn application

is effective for ameliorating plant Zn deficiency and increasing Zn content in grain;

although its effectiveness may depend on the time of application (Phattarakul et al., 2012;

Abdoli et al., 2014). Recently, Esfandiari et al. (2016) found that foliar Zn application

improved yield and yield-contributing traits in wheat. They also reported increases in grain

Zn concentration and ascorbic acid, more so when Zn was sprayed during early grain filling.

Li et al. (2015) observed that foliar Zn application during early grain filling increased grain

Zn concentration and Zn utilization efficiency by 82.9 and 49%, respectively, and the

phytate to Zn molar ratio decreased more when sprayed during early stages of growth (Xi-

wen et al., 2011). The application of Zn through foliar spray increased yield irrespective of

the source used (Ghasemi et al., 2013). However, use of Zn–amino acid chelates (ZnAAC)

as a Zn source increased grain Zn bioavailability by lowering the phytate concentration and

phytate:Zn ratio (Ghasemi et al., 2013).

Foliar Zn fertilization is an effective technique for improving grain Zn concentration

if applied at later stages of crop growth. The time of application, Zn source and its

concentration are key factors determining the success of this application method.

2.4.3. Seed treatments

Micronutrients can be delivered to plants through seed treatments which are an

economical and effective alternative to foliar and soil applications (Farooq et al., 2012).

Seed treatments are cost-effective as very small amounts of nutrient are used, and the

applied nutrient is directly available to the germinating seed (Singh et al., 2003).

53

54

Table 2.2: Comparative performance of Zn application methods and sources for grain yield and grain Zn concentration of wheat

Cropping

system Source Rate/crop Application method Increase in grain

Zn concentration

(%)

Increase

in grain

yield (%)

Reference

ZnSO4 5 kg ha–1/wheat Soil application – 14 Zoz et al. (2012)

ZnSO4.7H2O 23 kg Zn ha–1/wheat Soil application 80 265 Yilmaz et al. (1997)

Rice–wheat ZnSO4 25.0 kg ZnSO4 ha–1/wheat Soil application 10.9% – Dwivedi and Srivastva (2014)

Rice–wheat ZnSO4 25.0 kg ZnSO4 ha–1/wheat Soil application 29.2 – Dwivedi and Srivastva (2014)

Rice–wheat Zinc-enriched urea (ZEU) 3.5% (ZEC) i.e. 9.1 kg Zn ha–1/wheat Soil application 20.9 26.21 Shivay et al. (2008)

Rice–wheat Zinc-enriched urea (ZEU) 3.5% (ZEC) i.e. 9.1 kg Zn ha–1/wheat Soil application 30.8 19.8 Shivay et al. (2008)

Rice–wheat ZnSO4.7H2O (Zn 20%) 600 kg ha–1/wheat Soil application 143.6 – Wang et al. (2015)

Rice–wheat ZnSO4.7H2O (Zn 20%) 1500 kg ha–1/wheat Soil application 194.99 Wang et al. (2015)

ZnSO4 10 mg Zn kg−1 soil/wheat Soil application 92.4% – Nautiyal et al. (2011)

ZnSO4.H2O 18 mg Zn kg–1 soil/wheat Soil application 189.47 Hussain et al. (2013)

ZnSO4.H2O 18 mg Zn kg–1 soil/wheat Soil application 244.44 Hussain et al. (2013)

ZnSO4.H2O 4.5 mg Zn kg–1 soil/wheat Soil application 83 20.63 Hussain et al. (2012b)

ZnSO4.H2O 9.0 mg Zn kg–1 soil/wheat Soil application 154 34.44 Hussain et al. (2012b)

Cotton–wheat ZnSO4.H2O 5 kg ha–1/cotton Residual Zn/soil application 19.7 5.5 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 7.5 kg ha–1 Residual Zn/soil application 49.9 8.1 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 10 kg ha–1 Residual Zn/soil application 67.9 9.7 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 12 kg ha–1 Residual Zn/soil application 75.5 10.2 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 5 kg ha–1 Soil application 53.7 7.6 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 7.5 kg ha–1 Soil application 78.4 10.4 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 10 kg ha–1 Soil application 100.6 14.4 Abid et al. (2013)

Cotton–wheat ZnSO4.H2O 12 kg ha–1 Soil application 111 13.8 Abid et al. (2013)

ZnSO4.7H2O 23 kg Zn ha–1 Soil application 59.1 Torun et al. (2001)

55

ZnSO4.7H2O 50 kg Zn ha–1 Soil application 11.3 4.26 Zou et al. (2012)

ZnSO4.7H2O 23 kg Zn ha–1+ 440 g Zn ha–1 Soil + foliar application 250 250 Yilmaz et al. (1997)

ZnSO4.7H2O 50 kg Zn ha–1+ 0.5% Zn Soil + foliar application 78.8 2.13 Zou et al. (2012)

Zn arginine [Zn(Arg)2] 2 mmol Foliar application – 20.3 Ghasemi et al. 2013)

Zn glycine [Zn (Gly)2 ] 2 mmol Foliar application 25.6% 29.1 Ghasemi et al. (2013)

ZnSO4.7H2O 440 g Zn ha–1 Foliar application 170 124 Yilmaz et al. (1997)

ZnSO4.7H2O 0.44 g Zn L−1 Foliar application 134 – Esfandiari et al. (2016)

ZnSO4.7H2O 2.5 kg ha–1 Foliar application 50–84% – Xi-wen et al. (2011) ZnSO4.7H2O 0.5% Foliar application 75.2 4.3 Zou et al. (2012)

ZnSO4.7H2O 30% ZnSO4.7H2O solution Seed treatment + foliar 190 268 Yilmaz et al. (1997) + 440 g Zn ha–1 application

Zn glycine [Zn(Gly)2] 40 ppm Seed priming 46 Seddigh et al. (2016)

Zn glutamine[Zn(Gln)2] 40 ppm Seed priming 9 14 Seddigh et al. (2016)

Zn arginine [Zn(Arg)2] 40 ppm Seed priming 103 Seddigh et al. (2016)

Zn histidine [Zn(His)2] 40 ppm Seed priming 34 Seddigh et al. (2016)

Rice–wheat ZnSO4.H2O 0.004 M Seed priming 900 – Johnson et al. (2005)

ZnSO4.H2O 0.1 M Seed priming – 5.42 Nazir et al. (2000)

ZnSO4.H2O 0.1% Zn Seed priming – 34.87 Arif et al. (2007)




ZnSO4.H2O 1.25 g Zn kg–1 seed Seed coating 21.06 54.97 Rehman and Farooq (2016)

ZnSO4.H2O 1.50 g Zn kg-1 seed Seed coating 26.69 32.77 Rehman and Farooq (2016)

ZnCl2 1.25 g Zn kg-1 seed Seed coating 26.82 41.01 Rehman and Farooq (2016)

ZnCl2 1.50 g Zn kg-1 seed Seed coating 34.69 – Rehman and Farooq (2016)

ZEC= Zinc-enriched urea; in all cases, Zn was applied to wheat crop

56

Table 2.3: Comparative performance of different Zn sources in wheat

Source Findings Parameters studied Reference

Zinc oxide and ZnSO4 enriched urea ZnSO4 was slightly better than ZnO Grain yield, Zn uptake, grain and

straw Zn concentration, agronomic

and recovery efficiency

Shivay et al. (2008)

Zn glutamine, Zn glycine, Zn

arginine, and Zn histidine and

ZnSO4.7H2O

Zn glycine > Zn glutamine > ZnSO4.7H2O > Zn histidine >

Zn arginine

Zn arginine > Zn histidine > Zn glutamine > ZnSO4.7H2O

> Zn glycine

Grain yield

Grain Zn concentration and protein

content

Seddigh et al. (2016)

Seddigh et al. (2016)

Zn arginine, Zn glycine, Zn histidine,

ZnSO4.7H2O

Zn glycine > Zn histidine > Zn arginine > ZnSO4.7H2O

Zn arginine > Zn glycine > Zn histidine > ZnSO4.7H2O

Grain yield

Grain Zn concentration and protein

content

Ghasemi et al. (2013)

Ghasemi et al. (2013)

Zn arginine < Zn glycine < Zn histidine < ZnSO4.7H2O Grain phytate Ghasemi et al. (2013)

Dhaincha GM, Cowpea (Vigna

unguiculata L.) Walp. GM, Leucaena

(Leucaena leucocephala Lam.) GM,

Mungbean (Vigna radiata L.)

Wilczek. GM, Wheat straw and FYM

FYM > Sesbaniab GM, Cowpea GM > Leucaena GM,

Mungbean GM > Wheat straw

FYM > Leucaena GM > Sesbaniab GM, Cowpea GM,

Mungbean GM and wheat straw

FYM > Cowpea GM > Sesbaniab GM > Mungbean GM >

Leucaena GM > wheat straw

Grain yield

Grain Zn concentration

Zn uptake

Mishra et al. (2006)



ZnSO4.7H2O and Zn-EDTA Zn-EDTA > ZnSO4.7H2O Grain yield

Grain Zn concentration

Zhao et al. (2016)

Zn-EDTA, Zn-Lignosulfonate and

ZnSO4

Zn-EDTA > Zn-LS ≈ ZnSO4 Shoot and root Zn concentration Martin-Ortiz et al. (2009)

57

58

2.4.3.1 Seed priming

In nutrient seed priming, seeds are dipped into a nutrient to start pre-germination

metabolic activities and radical emergence. Primed seeds germinate earlier and more

uniformly than unprimed seeds (Farooq et al., 2009). Zinc seed priming is effective for

improving crop performance; rice seeds primed with ZnSO4 produced higher yields than

untreated seeds (Slaton et al., 2001). Harris et al. (2007) reported that nutrient seed priming

with Zn enhanced seed Zn concentration before sowing which improved germination and

seedling growth. In a study on chickpea, seed Zn concentration and grain yield increased

by 29 and 19%, respectively, by priming seeds with 0.05% Zn solution (Harris et al., 2008).

Likewise, seeds of maize primed with 1% ZnSO4 solution resulted in better growth, seed

yield and seed Zn content (Harris et al., 2007). Zn application through seed priming is a

more economical approach than soil Zn fertilization due to the low cost:benefit ratio (Harris

et al., 2007, 2008). In another study, Harris et al. (2005) found that seed priming of chickpea

with Zn solution (0.05%) generated a cost:benefit ratio of 1:1500 and yield increased by

10–122% compared to hydroprimed seeds. Seed priming with 0.5 M Zn solution improved

germination and seedling growth in wheat (Rehman et al., 2015). Seed priming with

synthetic ZnAACs, i.e. Zn glutamine and Zn glycine, improved grain yield in wheat by 14–

46% compared with soil Zn application through ZnSO4. In addition, seed priming with Zn

glycine, Zn histidine and Zn arginine improved protein content (Seddigh et al., 2016).

Furthermore, seeds primed with Zn histidine and Zn arginine had the highest seed Zn and

Fe concentrations, and lowest phytate to Zn molar ratio compared with soil Zn application,

thus increasing Zn bioavailability. Zinc application through seed priming is a cost-effective

technique for increasing grain yield with high economic return, but has little added value

for increasing grain Zn concentration compared with soil and foliar Zn applications. 2.4.3.2

Seed coating

Seed coating is another cost-effective and efficient method for delivery of growth

hormones, chemicals and nutrients by adhering them to the seed surface using a sticky agent

to enhance performance (Freeborn et al., 2001). Nutrient seed coating increases the

availability of the nutrient to the seed by forming a nutrient layer around the germinating

seed (Taylor and Herman, 1990). Seed coating is an effective strategy for improving the

crop stand and plant growth (Scott and Blair, 1988; Farooq et al., 2012) and an economical

approach due to the small amount of nutrient used (Baudet and Peres, 2004). Seed coating

has effectively improved the yield of several crops such as barley (Zelonka et al., 2005)

59

and rice (Tavares et al., 2012). In wheat, seed coating with Zn improved germination,

seedling growth and tissue Zn concentration compared with untreated seeds (Rehman and

Farooq, 2016).

Zinc application by soil, foliar and seed treatment improves plant Zn status (Table

2.2). While soil Zn fertilization is the most common method for meeting crop Zn demand,

the cost and source of fertilizer, and edaphic and climatic conditions need to be optimal for

effective soil application. Foliar sprays can effectively increase grain Zn concentration, but

the plant growth stage and time of application are important. Seed treatments are

costeffective for improving grain yield but less effective at increasing grain Zn

concentration.

2.5. Economics of Zinc Application

Zinc application methods and sources aim to improve Zn uptake by plants to

improve grain yield, grain Zn concentration and other net benefits (Table 2.4). In this

regard, Khan et al. (2008) applied ZnSO4 (5, 10, 15, 20, 25 and 30 kg ha–1) to wheat and

found that soil application of 5 kg ha–1 ZnSO4 was the most economical rate with the highest

marginal rate of return (185.8%). In a two-year field experiment with wheat, ZnSO4 was

applied at 7.5, 15, 22.5 and 30 kg ha–1, of which 22.5 kg ha–1 returned the lowest cost:benefit

ratio of 1:4.26 with an 18–21% increase in grain yield (Abbas et al., 2010). In another two-

year study, Zn fertilization increased the average grain yield of wheat by 31 and 32% with

cost:benefit ratios of 1:3.38 and 1:5.24 in each respective year (Kalayci et al., 1999). In a

RWCS study, Prasad et al. (2002) found that soil application of 25 kg Zn ha–1 increased

wheat yield by 38.1% with a cost:benefit ratio of 1:2.18.

Yilmaz et al. (1997) applied Zn to wheat using different application methods, all of

which increased grain yield compared with the control. However, foliar + soil fertilization,

soil application, and foliar + seed fertilization were the most effective methods, with

increases in average grain yield of 250, 265 and 268%, respectively. Similarly, grain yield

increased by 204% and 124% with seed and foliar applications of Zn, respectively. The

foliar-applied Zn treatment had the lowest cost:benefit ratio (1:53.7). Nazir et al. (2000)

reported that seed priming with 0.1 M Zn increased wheat yield by 6.83% compared with

the control, with a cost:benefit ratio of 1:11.5. Seed priming with 0.1% Zn solution as

ZnSO4 increased grain yield in wheat by up to 35% with a cost:benefit ratio of 1:60.6 (Arif

et al., 2007). Seed primed with 0.4% ZnSO4 fulfilled the wheat Zn requirement and

60

increased grain yield by 21%, which was more cost-effective than soil Zn application with

cost:benefit ratios of 1:360 compared to the control (Harris et al., 2005).

When wheat is cultivated in Zn-deficient soils, application of Zn improves the net

economic return by increasing the crop yield. Seed treatments are the most effective method

of Zn application to improve the net benefit with minimal cost.

2.6. Agronomic Approaches to Managing Zn in Wheat-based Production Systems

Zinc availability is influenced by crop management practices. Tillage practices

change the soil pH thus influencing Zn availability. Likewise, crop rotation/intercropping

change the Zn dynamics in soil (Table 2.5). The application of manures can increase Zn

availability (Table 2.5). In the following sections, agronomic practices for managing Zn

nutrition in wheat-based cropping systems are discussed.

2.6.1. Tillage

Soil cultivation and nutrient management practices can improve Zn availability to

plants. The addition of poultry manure to no-till soil increased the concentration of soil

extractable Zn (Shuman and McCracken, 1999). In South Asia, zero tillage is practiced in

wheat crops planted after flooded rice. However, conventional tillage is used in wheat

planted after aerobic rice (Bouman et al., 2007). This change in tillage practice may

influence soil pH and nutrient dynamics (Grant and Bailey, 1994), thus reducing Zn

availability. No-tillage improves the availability of nutrients viz. P, K, and Zn when

compared with conventional tillage (Franzluebbers and Hons, 1996). Several studies have

indicated that tillage had little or no effect on soil Zn status (Shuman and McCracken, 1999;

Grant et al., 2010). In conclusion; conservation soil practices are more beneficial than

conventional tillage for improving the soil Zn status.

2.6.2. Crop rotations and intercropping

The rice–wheat cropping system is a major cropping system worldwide; the

sequential growing of rice and wheat depletes nutrients from the soil creating nutrient

imbalance and reduces soil organic matter and quality (Alam et al., 2013). Crop rotation

and intercropping can improve soil Zn status. Crop rotation and the residual effect of the

previous crop are important for micronutrient availability and their uptake by the next crop

(Graham et al., 2001; Cakmak, 2008). Moreover, intercropping or crop rotation can

increase Zn uptake in wheat as organic acids released from roots of different plants form

complexes with Zn (Neumann and Romheld, 2001). Gunes et al. (2007) conducted a field

61

study on wheat and chickpea (Cicer arietinum L.) intercropping and revealed that Zn

concentration in both chickpea and wheat was higher under intercropping than

monocropping (Gunes et al., 2007). Zuo and Zhang (2009) found that intercropping

gramineous crops with dicot plants for Zn biofortification is a sustainable, effective and

practical practice in developing countries, as it benefits both dicot and gramineous plants

for better Zn uptake. The incorporation of legumes in RWCS improved crop productivity

by increasing soil organic matter, and N, P and Zn contents (Hossain et al., 2016). Soltani

et al. (2014) conducted a field study using safflower, sunflower, clover and Sudan grass as

preceding crops to wheat along with a fallow control. They reported that wheat after these

crops had higher grain Zn concentrations. In another study, the cultivation of wheat after

sunflower resulted in higher Zn density in grains than after cotton (Khoshgoftarmanesh and

Chaney, 2007).

In crux, sequential cropping results in Zn deficiency in wheat. Growing wheat in

rotation with sunflower, legumes and fodder crops improves Zn uptake in wheat.

Intercropping wheat with legumes enhances wheat Zn acquisition more than wheat

monocropping.

2.6.3. Manure application

The application of organic manures can help to meet crop Zn requirements by the

addition of Zn and its mobilization (Gao et al., 2012). Many studies demonstrated that

application of animal manure increased Zn availability in soil and thus plant Zn uptake in

a variety of crops including rice and wheat (Alok and Yadav, 1995; Egrinya et al., 2001).

Gupta et al. (1992) found that application of poultry manure alone or with ZnSO4 increased

grain Zn concentration and yield in wheat. Manure application in RWCS sustained

highlevel soil available Zn more than inorganic sources (Alok and Yadav, 1995). Aghili et

al. (2014) found that sole application of sunflower green manure improved grain Zn

concentration in wheat to 31 mg kg–1, and to 54 mg kg–1 when red clover green manure was

also added. Incorporation of green manure to soil enhanced DTPA-extractable Zn. The

application of farmyard manure (FYM) (10 t ha−l), piggery manure (2.5 t ha–1) and poultry

manure (5 t ha–1) was equally effective at adding 11.2 kg Zn ha–1 to fulfill crop Zn

requirements in a maize–wheat rotation (Nayyar et al., 1990).

Crop management practices i.e. zero tillage, crop rotation and incorporation of

legumes and fodder crops with wheat, can reduce Zn deficiency. Furthermore, the use of

62

organic manures can meet wheat Zn demand because they are rich in Zn, and improve soil

physiochemical properties and Zn uptake by forming soluble Zn complexes.

63

Table 2.4: Economics of Zn application in wheat

Application method Source/rate Increase in yield

(%)

Additional cost of Zn

application (USD) Increase in income

over control (USD) cost: benefit

ratio Reference

Soil application 23 kg ZnSO4.7H2O 265 47.5 412.6 1:8.68 Yilmaz et al. (1997)

Seed application 30% ZnSO4.7H2O solution 204 7.75 344.8 1:44.5 Yilmaz et al. (1997)

Foliar application 440g Zn ha–1 (ZnSO4.7H2O) 124 4.01 215.5 1:53.7 Yilmaz et al. (1997)

Soil + leaf application 23 kg ZnSO4.7H2O + 440g ha–1

(ZnSO4.7H2O)

250 51.6 381.3 1:7.40 Yilmaz et al. (1997)

Seed + leaf application 30% ZnSO4.7H2O solution +

440 g Zn ha–1

268 11.8 453.7 1:38.6 Yilmaz et al. (1997)

Soil application 23 kg ZnSO4.7H2O 30.8 47.5 160.8 1:3.38 Kalayci et al. (1999)

Soil application 23 kg ZnSO4.7H2O 31.7 47.5 248.9 1:5.24 Kalayci et al. (1999)

Soil application 7.5 kg ZnSO4 (33% Zn) 8.6 8.34 335 1:4.26 Abbas et al. (2010)

Soil application 15 kg ZnSO4 (33% Zn) 10.7 16.7 335.1 1:2.63 Abbas et al. (2010)

Soil application 22.5 kg ZnSO4 (33% Zn) 17.9 25.0 356.7 1:2.95 Abbas et al. (2010)

Soil application 30 kg ZnSO4 (33% Zn) 7.8 33.4 306.6 1:0.96 Abbas et al. (2010)

Soil application 5 kg ZnSO4 3.43 6.37 1:1.85 Khan et al. (2008)

Soil application 25 kg ZnSO4.7H2O 38.4 51.7 112.7 1:2.18 Prasad et al. (2002)

Seed priming 0.1 M ZnSO4 6.83 6.74 77.3 1:11.45 Nazir et al. (2000)

Seed priming 0.1% Zn (ZnSO4) 34.9 3.91 237.3 1:60.6 Arif et al. (2007)




In all cases, the price of Zn source and grain yield was calculated according to Pakistan price and expressed in USD

64

Table 2.5: Effect of organic amendments/green manuring and crop rotation on grain yield and grain Zn concentration of wheat

Organic amendments/green

manuring/crop rotation Experiment

duration Rate/method Increase in grain Zn

concentration (%) Increase in

Zn uptake/ha

(%)

Increase in

grain yield (% ) Reference

Sunflower 1 year Incorporation/4 g DM kg–1 dry

soil 55 – – Aghili et al. (2014)

Sunflower + Red clover 1 year Incorporation 140 – – Aghili et al. (2014)

Sheep manure 1 year 3 kt ha–1 17.2–21.6 – – Wang et al. (2016)

Dhaincha 2 years Incorporation 42 DAS 27.06 – 23.8 Singh and Shivay (2013)

Dhaincha 2 years Incorporation 42 DAS 31.3 – 21.72 Singh and Shivay (2013)

Sunhemp (Crotalaria juncea L.) 2 years Incorporation 42 DAS 21.76 – 18.41 Singh and Shivay (2013)

Sunhemp (Crotalaria juncea L.) 2 years Incorporation 42 DAS 24.23 – 16.75 Singh and Shivay (2013)

Cowpea 2 years Incorporation 42 DAS 12.05 – 12.46 Singh and Shivay (2013)

Cowpea 2 years Incorporation 42 DAS 18.4 – 10.73 Singh and Shivay (2013)

Rice–Cowpea–Wheat 2 years Cowpea straw incorporation – 31.02 19.77 Pooniya and Shivay (2011)

Rice–Mungbean–Wheat 2 years Mung bean incorporation – 26.44 16.27 Pooniya and Shivay (2011)

Rice–Dhaincha–Wheat 2 years Dhaincha incorporation – 34.90 19.77 Pooniya and Shivay (2011)

Safflower + Wheat 1 year Residue incorporation 86.80 – 58.43 Habiby et al. (2014)

Safflower–Wheat 1 year No residue incorporation 20.51 – 39.27 Habiby et al. (2014)

Clover + Wheat 1 year Residue incorporation 101.35 – 39.46 Habiby et al. (2014)

Clover–Wheat 1 year No residue incorporation 31.16 – 34.10 Habiby et al. (2014)

Sorghum + Wheat 1 year Residue incorporation 39.11 – – Habiby et al. (2014)

65

Sorghum–Wheat 1 year No residue incorporation 23.85 – 5.94 Habiby et al. (2014)

Sunflower + Wheat 1 year Residue incorporation 39.11 – – Habiby et al. (2014)

Sunflower−Wheat 1 year No residue incorporation 6.52 – – Habiby et al. (2014)

Dhaincha 3 years Incorporation 9–10 weeks after

sowing 18.5 25.8 Mishra et al. (2006)

Cowpea 3 years Incorporation 9–10 weeks after

sowing 19.4 25.8 Mishra et al. (2006)

Leucaena 3 years 2 t ha–1 yr–1 loppings

incorporation 15.4 22.6 Mishra et al. (2006)

Mungbean 3 years Incorporation after pod piking 18.5 22.6 Mishra et al. (2006)

Wheat straw 3 years 5 t ha–1 yr–1 incorporation 4.3 12.9 Mishra et al. (2006)

FYM 3 years 10 t ha–1 yr–1 6.1 20.9 38.7 Mishra et al. (2006)

66

2.7. Zinc Biofortification of Wheat

Hidden hunger caused by a deficiency of minerals and vitamins is affecting about

onethird of people around the globe. About two billion people suffer from Zn deficiency. Zinc

is an important mineral needed in small amounts by humans and plants for normal growth and

development, immune system function, neurotransmitter function and reproductive health

(Hotz and Brown, 2004). Wheat is a major source of daily food intake, which is poor in Zn.

The concentration of Zn can be enhanced in wheat by using agronomic or breeding/molecular

approaches to develop microelement-dense wheat genotypes (Velu et al., 2014). Zinc

deposition in wheat grains requires the translocation of Zn from leaves (Pearson and Rengel,

1995). The major bottlenecks in Zn biofortification are (i) storage of excess Zn in root vacuoles,

(ii) grain Zn concentration being dependent on leaf Zn translocation to leaves rather than Zn

uptake during seed filling, and (iii) discontinuous xylem at the base of each grain in cereals

being a major hurdle in the transfer of Zn (Palmgren et al., 2008). Agronomic and genetic

biofortification approaches are discussed below.

2.7.1 Agronomic approach

Growing wheat on low Zn soil is the principal reason for low Zn bioavailability to

humans (Alloway, 2009). The agronomic approach involves a fertilizer strategy; it is a rapid

solution to reduce Zn deficiency and increase grain Zn concentration (Cakmak, 2008). The

application of organic/inorganic Zn fertilizer increases grain Zn concentration (Tables 2.2, 2.5).

For effective Zn fertilization, information on the source of Zn (Table 2.3) and time of foliar

application is critical to increasing Zn accumulation in grains. Foliar application of zinc is more

effective than soil application at improving Zn accumulation in wheat seeds (Cakmak et al.,

2010a, b). For instance, Zn application through foliar spray increased seed Zn content beyond

breeding targets suggested by nutritionists (Velu et al., 2014).

Zinc bioavailability is more important than grain Zn concentration as most of the Zn is

present in the aleurone and embryo of wheat seeds (Cakmak et al., 2010b). Likewise, phytic

acid concentration is higher in the germ and aleurone and accounts for 75% of P stored in seeds

(Lott and Spitzer, 1980). For instance, foliar fertilization of Zn not only improved the whole

grain Zn concentration, but it also enhanced the endosperm Zn concentration (Cakmak et al.,

2010b) suggesting the high bioavailability of Zn. Moreover, application of Zn at later stages of

67

grain development increased the Zn concentration in endosperm as very low phytate is present

in this seed fraction (Cakmak et al., 2010b; Cakmak, 2012). The phytate to Zn molar ratio is

generally used to check Zn bioavailability (Gibson, 2006). In a study by Hussain et al. (2012b),

soil application of Zn improved Zn bioavailability to humans by reducing phytate content and

phytate:Zn ratio. Similarly, Bharti et al. (2013) also found an increase in grain Zn concentration

of wheat with lower phytate concentration and phytate:Zn ratio. Rosado et al. (2009) conducted

a study regarding Zn bioavailability in humans on Mexican women and reported that

absorption of Zn was higher for biofortified wheat flour than the control.

Agronomic biofortification is a time-saving and effective approach for improving Zn

bioavailability in wheat grains. Soil and foliar Zn fertilization improve the Zn concentration

and bioavailability to humans by reducing the phytate concentration and phytate:Zn ratio.

2.7.2. Breeding approach

2.7.2.1. Selection and conventional breeding

In genetic biofortification, plant breeding strategies are used to develop genotypes of

staple food crops that are rich in micronutrients and poor in nutrient inhibitors by enhancing

the concentration of substances which aid in nutrient uptake and absorption (Bouis, 2003).

Biofortification of wheat cultivars with Zn through conventional cross breeding represents an

alternative to the agronomic approach as a cost-effective and sustainable method for delivering

important microelements in food (Graham et al., 2007). Conventional biofortification includes

traditional crop breeding to exploit genetic variation in mineral micronutrient concentrations.

Gene discovery and marker assisted selection also fall in this category (Grusak, 2002).

Cereal crops have large variation in terms of sensitivity to Zn deficiency (Graham and

Rengel, 1993; Cakmak et al., 1997a); this genetic diversity can be used for the selection and

development of Zn-efficient genotypes. Amiri et al. (2015) reported that Zn concentrations in

bread wheat genotypes (Triticum aestivum L.) range from 31.64–55.06 mg kg–1 DW. Among

relatives of wheat, Triticum turgidum ssp. dicoccoides (wild emmer wheat) has a wide range

of variability for Zn (14–190 mg kg–1 DW) and Fe (15–109 mg kg–1 DW), and the highest

concentration of these two microelements surpassed those in modern wheat cultivars (Cakmak

et al., 2004). Moreover, grain Zn concentrations in the RILs population ranged from 44.4–95.6

mg kg–1, respectively (Roshanzamir et al., 2013). Chatzav et al. (2010) reported that wild

68

emmer wheat genotypes are genetically diverse for seed nutrient concentration. In fact, of the

studied populations of wild emmer wheat, Tabigha (Terra rossa) has great potential in breeding

programs with its high Zn, Fe and seed protein concentrations. The linkage between seed

protein, Fe and Zn concentrations provide an opportunity to develop these three traits

simultaneously (Chatzav et al., 2010). Furthermore, Zn-efficient wheat genotypes can be

developed by transferring the genes in rye responsible for Zn efficiency (Rengel, 1999).

Precision phenotyping is important for the development of high Zn wheat genotypes.

However, environmental factors, particularly soil physiochemical properties, influence

breeding for high Zn cultivars (Trethowan, 2007). This can be overcome by applying Zn

fertilizer and maintaining a homogenous Zn concentration in soil. In wheat, Zn and Fe are

quantitative traits (Trethowan, 2007). There is a strong positive correlation between modern,

wild and spelt wheat for grain Zn and Fe concentrations showing that genetic and physiological

factors responsible for Zn and Fe are similar (Cakmak et al., 2004; Morgounov et al., 2007).

In conclusion, genetic diversity among different wheat genotypes and wild and cultivated

relatives for grain Zn concentration should be exploited to develop Zn-dense wheat cultivars.

2.7.2.2. Molecular approaches

Molecular markers are useful for identifying genotypes with high mineral concentration

without the need for field testing. In this regard, QTL mapping is effective in the identification

of QTLs responsible for grain Zn concentration in wheat (Table 2.6). However, little

information is available on QTLs linked to Zn uptake in cereals. Xie and Nevo (2008) found

that wild emmer wheat has diverse alleles for Fe and Zn concentration. Peleg et al. (2009) were

successful in mapping 82 QTLs responsible for ten different nutrients. Moreover, QTLs

responsible for protein, Zn and Fe significantly overlap suggesting a positive correlation

between Zn, Fe and grain protein concentrations. Distelfeld et al. (2007) cloned the TtNAMB1

gene from Tritium dicoccoides, which influences seed Zn, Fe and protein concentrations.

Quantitative trait loci are mapped using two methods viz. association and linkage mapping

(Zhu et al., 2008; Myles et al., 2009).

In a recent study, Sadeghzadeh et al. (2015) identified QTLs for Zn in barley (Hordeum

vulgare L) and reported that two regions (2HL and 2HS) are linked with grain Zn concentration.

They found that 45% and 59% of the variation in seed Zn concentration and seed Zn content

69

was linked with these two regions. This QTL can help in the identification of genes, and their

transfer in plants for improving crop Zn status as transgenic wheat genotypes have high grain

Zn concentration with more bioavailable Zn (Table 2.7).

Molecular/transgenic approaches offer a more rapid solution than the conventional

breeding approach to identify and transfer genes and QTLs responsible for grain Zn

concentration or phytase activity (Table 2.7), and their transfer and expression in newly

developed wheat genotypes has proved effective in increasing grain Zn concentration and Zn

bioavailability.

2.8. Conclusion and Future Research Needs

Zinc deficiency is widespread across the globe. Zinc plays several crucial roles in plant

biology. It is important for protein synthesis and the activities of various enzymes within plants.

Among field crops, cereals are most affected by Zn deficiency and, among cereals, wheat is

most vulnerable to Zn deficiency. Growing wheat in rotation with cereals such as rice and

maize further increases Zn deficiency. However, wheat grown in rotation with legumes or

sunflower has better Zn uptake and grain Zn concentration. Zinc has positive interactions with

some elements, but it inhibits the uptake of other elements. Zinc deficiency can be corrected

by crop management practices as soils with low tillage have high Zn availability. Furthermore,

the application of organic Zn sources and farmyard manure are equally effective at meeting

crop Zn demand. Wheat grains can be fortified with Zn simply by fertilizing the crop with Zn

through foliar or soil applications (i.e. agronomic biofortification). The use of breeding

approaches to exploit the variation in grain Zn concentration and QTL mapping and molecular

approaches could be helpful in the development of Zn-efficient wheat genotypes with higher

grain Zn concentration.

There is a need for a geological survey to identify Zn-deficient areas around the globe

as this information is about a decade old. In addition to the application of Zn fertilizers, future

research should focus on cereal cultivation with legumes or other similar cropping systems to

enhance Zn availability to wheat. The use of organic sources of Zn should be encouraged as

they improve soil physiochemical properties and improve soil Zn status. The mechanism of Zn

availability using PGPRs and AMF needs to be evaluated. Fertilizer management should

70

include PGPRs and remote sensing technology for efficient utilization of Zn fertilizers.

Furthermore, QTL mapping should be undertaken for modern wheat and wild relatives of

wheat to identify loci responsible for the uptake and deposition of Zn into the grain.

Zinc transfer to seed via increased expression of genes is associated with

biosynthesis/exudation of Zn chelates, which is yet to be fully characterized. The genetic

variability among different wheat genotypes and their wild relatives for grain Zn concentration

should be exploited to develop wheat genotypes with better yield and grain Zn concentration

under specific environments.

Genes and transporters responsible for Zn uptake should be identified to improve wheat

Zn status. Furthermore, research should be undertaken to reduce the concentration of phytates

and other anti-nutrient compounds by identifying the genes/QTLs linked with these traits and

suppress their expression. Moreover, the use of functional genomics will help in understanding

the molecular basis of genotypic differences in Zn dynamics expressed in different wheatbased

cropping systems

Table 2.6: QTLs responsible for grain Zn concentration in wheat

Crop/species Trait Chromosome QTL İncrease in Zn

concentration (%) References

Triticum aestivum Grain Zn concentration 1A and 4A QZn.shu-1A

QZn.shu-4A

Roshanzamir et al. (2013)

Triticum aestivum (hexaploid)

Triticum sphaerococum (tetraploid)

Grain Zn concentration 1B and 6B QGzn.ada.1D

QGzn.ada.6B

Velu et al. (2016)

Triticum aestivum (hexaploid)

Triticum sphaerococum (tetraploid)

Grain Zn and Fe

concentration 2B QGZn.ada.2B Velu et al. (2016)

Ae. longissima Zn and Fe 1Sl, 1S2 Wang et al. (2011)

Ae. searsii Zn and Fe 1Sl, 1S2 Wang et al. (2011)

Ae. Umbellulata Zn and Fe 2U and 6U Wang et al. (2011)

Ae. Caudata B Wang et al. (2011)

Ae. Peregrina 4Sv Wang et al. (2011)

Ae. Geniculata 5 Mg Wang et al. (2011)

Aegilops kotschyi Zn and Fe 2S and 7U 136 Tiwari et al. (2010)

BC2F2 16-1-8-4 7Up 251.2 Neelam et al. (2011)

BC2F2 16-1-8-56 7Up 156.3 Neelam et al. (2011)

Ae. Peregrine 1155-1-1 153.6 Neelam et al. (2011)

T. aestivum–Thinopyrum

bessarabicum Zn and Fe 6D of T. aestivum

6Eb Thinopyrum

bessarabicum

27.9 Ardalani et al. (2016)

42

Table 2.7: Genes responsible for grain Zn concentration in wheat

Crop/species Gene Trait Mechanism of transfer

and gene expression

study

Increases in Zn

concentration/gene expression

References

T. dicoccoides NAM-B1 Protein, Zn and Fe Increased gene expression Waters et al. (2009)

Uauy et al. (2006)

Sickle alfalfa (Medicago

falcata L.) ferritin GluB-1 Zn and Fe PCR, RT-PCR and western

blotting 44% grain Zn Liu et al. (2016)

Aspergillus japonicus Phytase gene

(phyA)

Increased phytase

activity Agrobacterium

(AgL1)mediated wheat

transformation

40–99% increase in phytase

activity

4–115% bioavailable Zn

Abid et al. (2016)

Aspergillus niger

Phytase gene

(phyA)

Phytase-encoding gene Escherichia coli strain

DH5α

103% increase in phytase activity

Brinch-Pedersen et al.

(2000)

T. dicoccoides Gpc-B1 Protein, zinc, iron and

manganese 12% higher grain Zn

concentration Distelfeld et al. (2007)

Rice Nicotianamine

synthase 2 OsNAS2 Zn >40% Zn concentration in wheat

EAR

Singh et al. (2017)

43

74

CHAPTER 3

MATERIALS AND METHODS

3.1. Optimizing Zinc Seed Priming Treatments for Improving the Stand Establishment


3.1.1. Experimental details

This study consisted of Three independent experiments. First two experiments were

conducted in the Allelopathy Laboratory, University of Agriculture, Faisalabad, Pakistan

during 2012-13. Seeds of wheat cultivars Lasani-2008 and Faisalabad-2008 were obtained

from the Wheat Research Institute, Faisalabad, Pakistan. The initial germination was 90 and

88% for Lasani-2008 and Faisalabad-2008, respectively. For seed priming, 50 g seeds of both

cultivar at 12% moisture content were soaked in aerated solution of 0.01, 0.05, 0.1, 0.5 and 1

M Zn (ZnSO4 and ZnCl2) or water (hydropriming) for 12 h at 25 ± 2°C and then dried back to

their original weight. Aeration was provided with a simple aquarium pump. Dry seeds and

hydropriming were the controls. For the first experiment, nine seeds were sown in each Petri

plate (90 mm-diameter × 15 mm-deep) between two layers of moist filter paper. Petri plates

were placed at 25 ± 2°C throughout the experiment. Seeds were scored as germinated when

radicles reached 2 mm in length.

For the second experiment, nine seeds were sown in each pot (150 mm × 90 mm) filled with

river sand at depth of 35 mm. Plumule appearance above the sand was scored as seedling

emergence. Third study was conducted in 15 kg soil filled earthen pots (48 cm × 30 cm) placed

in glass house of Faculty of Agriculture, University of Agriculture, Faisalabad, Pakistan during

2012-13. Seeds of wheat cultivars Faisalabad-2008 and Lasani-2008 were primed with

preoptimized rate of 0.1, 0.5 M solution of Zn using ZnSO4 and 0.05 and 0.1 M solution of Zn

using ZnCl2 on thes basis of optimization studies. The crop was sown on November 21, 2012.

In each pot, twenty seeds were sown and after emergence, were thinned to maintain 5 uniform

seedlings per pot. The day temperature of glass house during the whole crop season was 24±3ºC

while it was 14±3ºC during night. Relative humidity varied from 35 to 80% from midday to

mid-night, respectively and the light intensity ranged from 300-1200 µmol photon m-2 s-1. The

experimental soil was sandy loam with electrical conductivity 1.61 dS m-1 (US Salinity

75

Laboratory Staff, 1954), pH 7.9 (US Salinity Laboratory Staff, 1954), organic matter 1100 µg

g-1 (Ryan et al., 2001), and nitrogen (N), phosphorus (P) and potassium (K) were 600, 7.2 and

133 µg g-1 while DTPA extractable Zn was 0.57 µg g-1 (Ryan et al., 2001). On the basis of soil

analysis, 100 mg kg-1 N was applied as Ca (NO3)2.4H2O, 75 mg kg-1 P as KH2PO4 and 25 mg

kg-1 K as K2SO4. Moreover, plants were irrigated to 70% of water holding capacity until

physiological maturity. In all experiments, treatments were arranged using a two factor

completely randomized design with four replications.

3.1.2. Observations

3.1.2.1 Seedling establishment

The germination/emergence count was taken according to AOSA (1990) until a

constant count was achieved. After attaining the constant count, four seedlings of uniform size

were maintained in each Petri plate and pot for measurement of seedling length and biomass.

Mean germination time (MGT) was calculated using the following formula (Ellis and Roberts,

1981):

Where n is the number of seeds germinated on day D and D is the number of days

counted from the beginning of germination. Mean emergence time (MET) was also calculated

using this equation substituting emergence for germination.

3.1.2.2 Seedling growth

Seedlings were harvested 12 days after sowing to record seedling shoot and root length.

Seedlings were oven-dried at 70°C for 48 h to determine seedling dry weight.

3.1.2.3 Yield and yield components

Crop was harvested at harvest maturity on April 13, 2013. Spike length and spikelets

per spike were noted from five primary spikes from each pot. Similarly, five spikes from each

pot were manually threshed to record the number of grains per spike. A sub-sample of hundred

grains was taken to record 100-grain weight using digital weighing balance. Wheat plants from

each individual treatment was harvested, dried, threshed manually, grains were separated and

weighed on digital weighing balance to record the grain yield per pot. Harvest index was

calculated as a ratio of grain yield to biological yield and was expressed in percentage.

76

3.1.2.4 Zinc and chlorophyll content determination

Shoot Zn concentration and leaf chlorophyll contents were determined before (BBCH

29) and after anthesis (BBCH 69). However, grain Zn contents were estimated at maturity.

Penultimate leave were harvested, and leaf chlorophyll a and b contents were estimated

following Arnon (1949). For determination of shoot and grain Zn contents, di-acid (HNO3:

HClO4, 2:1 v/v) was used to digest plants and grain samples. Shoot and grain Zn contents was

estimated in digested samples using Atomic absorption spectrophotometer (Thermo scientific

- SOLAAR S series AA Spectrometer).

3.1.3. Data analysis

Data were analysed using Computer Software “Statistics 8.1”. Tukey (HSD) test at 5%

probability level was applied to compare the treatment means. Germination and emergence

data were transformed before statistical analysis. Correlation coefficients among different

germination, stand establishment and seedling growth traits were calculated using Microsoft

Excel.

3.2. Optimizing Zinc Seed Coating Treatments for Improving the Stand Establishment,


3.2.1. Experimental details

This study was comprised of three separate experiments. The first two optimization

experiments were carried out in Allelopathy Lab, Department of Agronomy, while the third

experiment was consisted of optimized treatments from the first two experiments, and was

carried out in glass house of Faculty of Agriculture, University of Agriculture, Faisalabad,

Pakistan during 2012–2013. Zinc chloride (ZnCl2), and (ZnSO4.7H2O) with ≥ 98% and ≥

99.0% purity were used as Zn sources. For optimization study, seeds of both wheat cultivars

were coated with 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2 g Zn kg -1 seed using ZnCl2 and

ZnSO4 as Zn source, while untreated seeds were taken as control. For glass house experiments,

seeds of both wheat cultivars were coated with pre-optimized rate at 1.25 and 1.5 g Zn kg-1

seed. Arabic gum was used for adhering the Zn to the seed surface. All other experimental

procedures and details were similar as described in section 3.1.

77

3.3. Characterizing Wheat Genotypes for Zinc Biofortification Potential and Genetic

Diversity

3.3.1. Experimental treatments

This study was conducted for two growing seasons of 2013-14 and 2014-15 at the

Agronomic Research Area, University of Agriculture, Faisalabad, Pakistan (lattitude 31ºN,

longitude 73ºE and altitude 184.4 msl). Seeds of twenty eight bread wheat genotypes were

obtained from Wheat Research Institute, Faisalabad, Pakistan; Regional Agricultural Research

Institute Bahawalpur, Pakistan; Arid Zone Research Institute, Bhakkar, Pakistan and Barani

Agricultural Research Institute, Chakwal, Pakistan. The parentage and code of wheat

genotypes are given in Table 3.1. Zinc was soil applied at 10 kg Zn ha -1 as basal dose using

zinc sulphate as source while no application of Zn was taken as control. The mean maximum

temperature during 2013-14 was 28, 25, 22, 23, 30 and 37.5ºC while it was 30, 17.2, 23.6, 26.4,

33.6 and 42.5 during 2014-15. Mean minimum temperature during the crop season 2013-14

was 6.5, 3.0, 0.0, 4.5, 8.5 and 14ºC while it was 8.5, 1.5, 2.0, 7.2, 8.0 and 15.6ºC during second

year of experimentation.

3.3.2. Crop husbandry

Crop was sown with single row hand drill on November 26, 2013 and November 25, 2014

during first and second years, respectively in 22.5 cm spaced rows using seed rate of 125 kg

ha-1. The experimental soil was sandy loam with pH 8.2, EC, 0.37, organic matter 0.90% and

nitrogen 0.063% while phosphorus, potassium and Zn were 5.02, 1.72 and 0.71 ppm

respectively. On the base of soil analysis, fertilizers were applied at 100-90-75 N-P-K kg ha-1

using urea (46% N), diammonium phosphate (18%N, 46% P) and sulfate of potash (50% K)

as sources. Whole of the P, K and one third of the N were applied as basal dose. Remaining N

was applied with 1st and 2nd irrigation in equal splits. Selective broad-spectrum herbicide

(Atlantis (iodo+mesosulfuron) at 14.4 g a.i. ha-1) was applied as early post emergence on

January 13, 2014 and January 15, 2015 during first and second year of experimentation

respectively, to control weeds. In total, four irrigations (each of 308 m3) were applied to the

crop during the growth period in addition to soaking irrigation of 411 m3. Crop was harvested

on April 24, 2014 and April 26, 2015 during first and second years of the study, respectively

and was threshed to record the yield and other related traits.

78

3.3.3. Recording of data

3.3.3.1 Yield parameters

Total and productive tillers were counted from unit area (1 × 1 m) in each plot at final

harvest from two locations. From each plot, twenty spikes were randomly taken and were

threshed manually to separate the grain. The grains separated were counted to record number

of grains per spike. The crop was harvested, tied into bundles and sundried for a week in

respective plots. The crop was threshed manually. Three sub-sample of 1000 grains was taken

from each plot then weighed on a digital weighing balance to record 1000-grain weight. Grain

yield for each treatment was recorded on a digital weighing balance in kilograms and later was

expressed in mega gram per hectare (Mg ha-1). The values of grain yield were adjusted to 12%

moisture level. Harvest index was recorded as ratio of grain yield to biological yield.

3.3.3.2 Grain mineral analysis

At full maturity, grains were harvested to determine the grain yield and the grain

concentrations of Zn, Fe and Ca. Zn and Fe were determined by an inductively coupled plasma

optical emission spectrometer (ICP-OES; Vista-Pro Axial; Varian Pty Ltd., Mulgrave,

Australia). The grain samples were digested with concentrated H2O2 and HNO3 using a closed

microwave digestion system (MarsExpress CEM Corp., Matthews, NC). To estimate the grain

N, 0.2 g ground grain sample was used in a Leco Tru Spec CN analyzer (Leco Corp., St. Joseph,

MI)

Grain embryos were separated using a surgical blade from around 100 grains. The

endosperm fraction was obtained by milling the de-embryonated seeds in a vibrating agate cup

mill (Pulverisette 9, Fritsch GmbH, Idar-Oberstein, Germany) for 20 sec at 700 × g. The

resulting flour was sieved using a 100 μm mesh plastic sieve. The sieved particles were treated

as the endosperm, whereas the fraction remaining on the sieve was sieved again with a 1000

μm mesh plastic sieve to separate the aleurone from the shorts.

3.3.3.3 Phytate concentration and bioavailable Zn

Phytate concentration in the wheat grains was determined following the protocol of Haug

and Lantzsch (1983) with some modifications. Ground sieved sample (0.1 g) was added with

10 mL NaSO4 (10% solution dissolved in 0.4 M HCl) followed by shaking for 3 h. After

shaking, the samples were centrifuged at 4600 × g for 20 min. One mL of the centrifuged

79

samples was taken and added with ferric solution followed by heating in water bath at 95ºC for

30 min. After heating, the samples were immediately cooled down using ice cold water until

they reach room temperature. The samples were again centrifuged at 4600 × g for 20 min. After

centrifugation, a thin gelly like layer was precipitated at the bottom of tube. One mL of the

centrifuged samples was taken to the new tubes and added with 3 ml of 2.2mL biphyridine

solution. The formed pink colour was read at 519 nm to the standards (0, 25, 50, 100 and 200

ppm). The bioavailable Zn was determined by molar concentration ratios of Zn and phytate

([phytate]:[Zn] ratio) and trivariate model of Zn absorption (Miller et al., 2007).

3.3.3.3 DNA extraction

Grains (10) of each wheat genotype were germinated in potting media at 25oC. The leaf

tissue (2 g) from 5 different plants of the same genotype was ground separately into fine powder

in liquid nitrogen. DNA was extracted from 100 mg of the powder in a 1.5 mL microcentrifuge

tube following cetyltrimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1990).

The extracted DNA was maintained at -80ºC until used. 3.3.3.4 Amplified fragment length

polymorphism (AFLP) fingerprinting

AFLP fingerprinting was used to estimate genetic diversity and relatedness among the 28

wheat genotypes using 5 samples for each cultivar following protocol of Vos et al., (1995)

modified by Al-Sadi et al. (2012a).

The first step involved digestion of DNA using EcoRI (NEB, Frankfurt, Germany) and

MseI (NEB) enzymes for 90 min at 37ºC, which was followed by ligation using T4 DNA ligase

(NEB) and 100 mM of ATP-Lithium salt (Roche Diagnostics GmbH, Mannheim, Germany)

for 90 min at 37ºC. The restriction ligation product was checked on 1.5% agarose gel and the

remaining reaction was diluted at a ratio of 3 R/L: 1 Milli-Q water. The digested and ligated

DNA samples were subjected to pre-selective amplification using PuReTaqTM Ready-To-GoTM

PCR beads, and EcoRI+A (5'-GACTGCGTACCAATTCA-'3) and MseI-C (5'-

GATGAGTCCTGAGTAAC-'3) primers. This was followed by dilution of the pre-selective

amplification product with 210 µL of TE0.1. The selective amplification reaction was carried

out using FAM-6-labelled EcoRI-AGA and AGT selective primers and MseI-CTC, CTG, CAT

and CAA primers. Fragment analysis was carried out at Macrogen Inc. (Korea) using ABI

3730XL (Applied Biosystems, Carlsbad, CA).

80


Data were analysed using statistical software Statistics 8.1. Treatment means were

separated using HSD at p ≤ 0.05. Molecular data within the size range of 50-500 base pairs

(bp) generated from fragment analysis were scored as 0 for the absence and 1 for the absence

of each amplified locus. The level of genotypic diversity (G) within each cultivar was

determined as described by Stoddart and Taylor (1988) and Grunwald et al. (2003). The percent

polymorphic loci, genetic distance and the level of Nei's gene diversity (Nei, 1973) were

determined using POPGENE (v 1.32) (Yeh and Boyle, 1997). Mega 6 was used to edit the

constructed dendrogram (Tamura et al., 2013). Genetic variation among and within genotypes

was estimated using the analysis of molecular variance (AMOVA) (Arlequin v.3.1; Excoffier

et al., 2005).

Table 3.1: Bread wheat genotypes of Pakistan used in the study

Code Varieties Year of release Institute Parentage

1 Millat-11 2011 WRI Chenab-2000/Inqlab-91

2 FSD-2008 2008 WRI PBW62/2*PASTOR

3 SHAFAQ-2006 2006 WRI LU-26/HD-2179//2*//INQ-91

4 Potohar 1973 WRI URES/BOWS

5 LS-2008 2008 WRI LUAN/KOH-97

6 SH-02 2002 WRI INQLAB-91/FINK’S

7 UFAQ-2002 2002 WRI V.84/33/V.83150

8 IQBAL-2000 2000 WRI BURGUS/SORT-12-13//KAL/BB/3PAK-81

9 PUNJAB-11 2011 WRI AMSEL/ATTILA//INQ91/PEWS

10 Kohinoor-83 1983 WRI OREF1158-FDL/MEXFEN’S’-TIBA63)C0C75

11 Sehar-2006 2006 WRI CHILL/2*STAR/4/BOW/CROW//BUC/PVN/3/

12 GA-02 2002 WRI PBW343

13 Pasban-90 1990 WRI INIA66/A.DISTT//INIA66/3GEN81

14 FSD-83 1983 WRI FURY/KAL-BB

15 AARI-11 2011 WRI SHALIMAR88/V-90A204//MH-97

16 FAREED-06 2006 RARI PTS/3TOB/LFN//BB/HD832-5//ON/5/4 V/ALD’S’//HPO’S’

17 MAIRAJ-2008 2008 RARI SPARROW/INIA//V.7394/WL711//3BAU’S’

18 BAKHAR-2002 2002 AZRI P-20102/PIMA//SKA/3/TTR’S’

19 CHENAB-2000 2000 WRI CBRD

20 PUNJAB-96 1996 WRI SA42/3/CC/INIA//BB/INIA/4/CNO/HD832

21 MH-97 1997 WRI ND/VG69144/KOL/BB/3YACO’S/4 VEE# 5’S’

22 CHAKWAL-50 2009 BARI ATTILA/3/HUI/CARC//CHEN/CHTO/4/ATTILA

23 SANDAL-73 1973 WRI CNO//SN64/KL.REND/3/8156

24 PAK-81 1981 WRI KVZ/BUHO/KAL/BB

25 INQLAAB-91 1991 WRI WL711/CROW’S’

26 BLUE SILVER 1971 RARI II-54-388-AN(YT.54-N 10B/LR 64)

27 FSD-85 1985 RARI MAYA/MON/KVZ/TRM

28 AAS-11 2011 WRI PRL/PASTOR/2236(V.6550/SULLY-86)

WRI= Wheat Research Institute, Faisalabad, Pakistan; RARI= Regional Agricultural Research Institute Bahawalpur, Pakistan; AZRI= Arid Zone Research Institute, Bhakkar, , Pakistan; BARI=Barani Agricultural Research Institute, Chakwal, Pakistan

51

83

3.4. Zinc Nutrition and Microbial Allelopathy for Improving Productivity and Grain

Biofortification of Wheat (Glass house experiment)


This study was carried out for two growing seasons in glass-house (latitude N

31º26’6.9 and longitude E 73º4’16.3), Faculty of Agriculture, University of Agriculture,

Faisalabad, during 2013-14 and 2014-15. Seeds of two bread wheat cultivars (Triticum

aestivum L.), Lasani-2008 and Faisalabad-2008 were kindly provided by Ayyub

Agricultural Research Institute, Faisalabad. Zinc (in the form of ZnSO4·7H2O) was applied

as soil application (5 mg Zn kg-1 soil) before sowing, foliar application (0.025 M

ZnSO4·7H2O solution) at tillering stage (BBCH 23), seed priming (0.5 M ZnSO4·7H2O for

12 hrs.) (Rehman et al. 2015) and seed coating (1.25 g Zn kg-1 seed) (Rehman and Farooq

2016). Hydroprimed seeds without any Zn application were taken as control.

The Zn solubilizing PGPR strain Pseudomonas sp. MN12 (designated as MN12

hereafter) was previously isolated and evaluated for improving growth and yield of maize

(Naveed et al. 2014). Zinc solubilization activity of the selected strain MN12 was tested

following the method of Bunt and Rovira (1955). The isolate was inoculated into Bunt and

Rovira agar medium containing 0.1% of each insoluble zinc compounds viz zinc oxide

(ZnO), zinc sulphide (ZnS), zinc phosphate [Zn3(PO4)2] and zinc carbonate (ZnCO3)

incubated at 30ºC for 96 h. Appearance of halo zone around the colonies indicated the

potential to solubilize Zn.

Inoculum of Pseudomonas sp. MN12 was cultured in 750 mL tryptic soya broth

(TSB) in 1000 mL Erlenmeyer flasks and incubated at 28±2 °C for 48 h in an orbital shaking

incubator (Firstek Scientific, Tokyo, Japan) at 180 rev min-1. Bacteria were collected by

centrifugation (4,500 × g, 15 min) and washed twice with normal saline buffered. The

concentration of the inoculum was then adjusted to approximately 6 × 108 CFU mL-1 with

distilled water, based on the optical density at 600 nm using a spectrophotometer (Evolution

300 LC, Cambridge, UK), and was confirmed by plate counting as described by Pillay and

Nowak (1997).

For hydropriming and osmopriming, 30 mL microbial culture was added in solution

and seeds were primed as described above. Likewise, for seed coating, for 30 g seed, Zn

was mixed with 30 mL microbial culture and 30 mL of solubilized Arabic gum. The Arabic

gum (30 g) was solubilized in distilled water by heating for 1 h at 95±5ºC and its coating

was done on seeds. The seeds were shade dried and used for sowing. For soil application

84

of MN12, 30 mL of microbial culture per pot was mixed with Zn, and was applied to soil

at the time of sowing. In foliar application, for each pot 30 mL microbial culture (mixed in

30 mL distilled water) was sprayed on leaves at tillering stage (BBCH 23). Bacterial

population in the final dilution was 3 × 108 CFU mL-1 measured as described above differed

non-significantly from the original population.


Seeds of selected wheat cultivars were sown on November 29, 2013 and November

26, 2014. Fifteen seeds per pot were sown in plastic pots (25 cm × 21 cm) filled with 10 kg

of soil. Seedlings were thinned to maintain five plants after uniform emergence. The

experimental soil used in this study was sandy loam with electrical conductivity (EC) 0.36

dS m-1, pH 8.17, organic matter 0.95%, nitrogen 0.06%, available phosphorus 5 mg kg-1,

extractable potassium 167 mg kg-1 and Zn concentration 0.70 ppm. Nitrogen was estimated

by the method of Bremner and Mulvaney (1982), while available P, exchangeable K, DTPA

extractable Zn and organic matter were estimated according to Olsen et al. (1954), Richard

(1954), Lindsay and Norvell (1978), and Walkley and Black (1934) respectively, as detailed

in the USDA Handbook (U.S. Salinity Lab. Staff, 1954; Volume 60). Nutrients were

applied to the experimental soil as 100 mg kg-1 N as Ca(NO3)2.4H2O, 75 mg kg-1 P as

KH2PO4 and 25 mg kg-1 K as K2SO4. Plants were irrigated to maintain 70% water holding

capacity. The day temperature of glass-house during the whole crop season was 25±3ºC,

while it was 14±3ºC during night. Relative humidity varied from 35 to 80% from mid-day

to mid-night, respectively and the light intensity ranged from 300-1200 µmol photon m-2 s-

1.

3.4.3. Recording of Data

3.4.3.1 Photosynthesis and water relation traits

Photosynthetic traits of penultimate leave were recorded from two plants at anthesis

stage in the morning using an infrared gas analyzer (IRGA) LCi (ADC, Bio scientific,

Germany). Water relation traits were recorded at the anthesis stage using same leaves as

were used for photosynthetic traits. Sampling was done early in the morning. The samples

were kept in liquid N containing cylinder. Relative water contents were measured following

the protocol developed by Barrs and Weatherly (1962). Fresh leaves were weighed (Wf)

and then these leaves were kept in water for four hrs to get saturated weight (Ws). The

saturated leaves were dried in electric oven at 95±5oC until a constant weight to get dry

weight (Wd). Relative water contents (RWC) were measured using formula:

85

RWC=(Wf-Wd)/(Ws-Wd) × 100

Leaf water potential (ψw) was determined using pressure chamber (Model 1000

pressure chamber instrument, PMS Instrument Company). For leaf osmotic potential, leaf

tissues used for ψw were kept in -20ºC for 48 h, thawed and sap was extracted. The extracted

sap was centrifuged at 5000 × g and leaf osmotic potential (ψs) was determined using

osmometer (Osmomat 030 Gonotec). Leaf water potential was calculated as difference of

ψw and ψs.


From each pot, five main spikes were taken and were threshed manually to separate

the grain. The grains separated were counted to record number of grains per spike and a sub

sample of 100-grains was weighted on an electric balance to calculate the 100-grain weight.

The plants were harvested and dried, tied into bundles and sundried for a week. The crop

was threshed manually. Grain yield for each treatment was recorded by an electric balance

in g plant-1.

3.4.3.3 Grain elemental analysis and grain Zn localization

Fully matured seeds were used to determine the grain concentrations of N, Zn, Fe

and Ca. To separate the seed fractions from whole seeds initially, whole embryos (i.e., the

embryo, including the scutellum) of about 100 seeds were severed using a surgical blade.

Endosperm fraction was separated by milling the de-embryonated seeds with a vibrating

agate cup mill (Pulverisette 9, Fritsch GmbH, Idar-Oberstein, Germany) for 120s at 700

rpm followed by sieving of resulting flour with a 100 μm mesh plastic sieve. The sieved

fraction was treated as endosperm, whereas the fraction left on the sieve was sieved again

using a 1000 μm mesh plastic sieve to separate the bran fraction from the shorts.

Iron, Zn and Ca were measured in the whole grain and the respective grain fractions

viz. embryo, aleuron, endosperm by an inductively coupled plasma optical emission

spectrometer (ICP-OES; Vista-Pro Axial; Varian Pty Ltd., Mulgrave, Australia) after

digesting the whole grain and fractionated grain samples in a closed microwave digestion

system (MarsExpress CEM Corp., Matthews, NC) in the presence of concentrated HNO3

and H2O2. The grain N concentration was measured by a LecoTru Spec CN analyzer (Leco

Corp., St. Joseph, MI) using 200 mg of ground whole grain sample. Grain protein

concentration was calculated by multiplying the N concentration using the conversion

factor of 5.36 (Mosse, 1990).

86

To study the Zn localization, wheat seeds were stained with DTZ (1,5-diphenyl

thiocarbazone) which from Zn–dithizonate complex of red color (McNary, 1954); by

following Ozturk et al. (2006). Seeds were initially cut in half through the crease using a

surgical blade and incubated at room temperature for 30 min in 500 mg L -1 dithizone (1,

5diphenyl thiocarbazone) dissolved in pure methanol. The stained seeds were then rinsed

with distilled water and analyzed qualitatively by using a reflectance light microscope

(Nikon SMZ1500, Melville, NY) with a high-resolution digital camera (Diagnostic

Instruments Inc., Sterling Heights, MI).

3.4.3.4 Phytate concentration and estimated bioavailable Zn

Phytate concentration in the seeds of wheat was determined following the protocol

of Haug and Lantzsch (1983) with some modifications. For phyate determination 0.1 g

ground was added with 10 mL Na2SO4 (10% solution dissolved in 0.4 M HCl ) followed

by shaking for 3 h. After shaking, the samples were centrifuged at 4600 × g for 20 min.

Centrifuged samples (1 mL each) were taken and added with ferric solution followed by

heating in water bath at 95ºC for 30 min. After heating, the samples were immediately

cooled down to room temperature in ice-cold water. The samples were centrifuged at 4600

× g for 20 min and one mL of supernatant was taken to the new tubes and added with 3 mL

of 2.2 mL bipyridine solution. The formed pink color was read at 519 nm against phytic

acid standards. Bioavailable Zn was determined both by molar concentration ratios of

phytate and Zn ([phytate]:[Zn] ratio) and trivariate model of Zn absorption (Miller et al.,

2007).

Where, TAZ= total daily absorbed Zn (mg Zn day-1); AMAX= maximum absorption;

TDP= total daily dietary phytate (mmol phytate day-1); KR= equilibrium dissociation

constant of Zn-receptor binding reaction; TDZ= total daily dietary Zn (mmol Zn day -1).

This model has independable variables i.e. TDP and TDZ (predictor), while TAZ is the

dependent variable (response). The parameters KR, KP and AMAX relate to Zn homeostasis

in human intestine and have constant value of 0.680, 0.033 and 0.091, respectively

(Hambige et al., 2010)

3.4.3.5 Organic acid estimation in root exudates

In 2nd year experiment, four replicates were harvested fifty days after sowing. The

seedlings along with root were gently removed from the substrate. Roots were washed

87

thoroughly under tap water. In each treatment 12 plants were used for the extraction of root

exudates. The root exudates were extracted in acetate buffer (25 mM, pH = 5.5) for 6 h as

modified from Hage-Ahmed et al. (2013). The final concentration of the root exudates was

adjusted with acetate buffer to 10 ml/g of root fresh weight. The exudates were filtered

through 0.22 µm sterile filters (Steriflip, Millipore, ROTH, Karlsruhe, Germany) and stored

at -40°C till further use.

Sterilized root exudates of each treatment were analyzed using HPLC system

(Agilent 1200, USA) with an XDB-C 18 column (4.6 mm 6250 mm, Agilent, USA). The

root exudates profiles were determined by following HPLC conditions in which 9 standards

of organic acids (pyruvic acid, citric acid, malliec acid, chlorogenic acid, oxalic acid, caffeic

acid, fumaric acid, succinic acid and salicylic acid) could also be completely separated. In

these conditions, the mobile phase consisted of 0.1% trifluoroacetic acid (A) and

acetonitrile (B) with a gradient elution of 0 min; 95% A and 5% B at a flow rate of 0.5

mL/min R 10 min; 95% A and 5% B at a rate of 1 mL/min R 20 min; 90% A and 10% B at

a rate of 1 mL/min R 40 min; 65% A and 35% B at a rate of 1 mL/min for 50 min; and 65%

A and 35% B at a rate of 1 mL/min. The UV detector wavelength was set at 280 nm. The

column temperature was maintained at 40ºC. The standard compounds were

chromatographed alone and in mixtures. Retention times for the standard compounds and

the major peaks in the extracts were recorded. The organic compounds from each fraction

were identified by their retention times and the addition of standards to the samples

(Banwart et al., 1985).


The experimental treatments were executed in randomized complete block design

in factorial arrangement with four replications during year one and eight replications in year

two. In second year, four replications were used for estimation of organic acid production.

Data were analyzed using statistical software Statistix 8.1. Treatment means were separated

using HSD Tukey test at p ≤ 0.05. Graphs were developed using Microsoft Excel (2013).

Standard errors were also calculated using Microsoft excel.

88


Biofortification of Wheat (Field experiment)


This experiment was conducted for two growing seasons at Agronomic Research

Area, University of Agriculture, Faisalabad (latitude 31.7 °N, longitude 73.98 °E) during

2013-14 and 2014-15. Seeds of two bread wheat cultivars, LS-2008 and FSD-2008 were

obtained from Wheat Research Institute, Ayyub Agricultural Research Institute (AARI)

Faisalabad. Zinc was applied as soil application (10 kg Zn ha-1), foliar application (0.025 M

Zn solution); seed priming (0.5 M ZnSO4 for 12 hrs) (Rehman et al., 2015) and seed coating

(1.25 g Zn kg-1 seed) (Rehman and Farooq, 2016). Hydroprimed seeds were taken as control.

Zinc solubilizing endophytic bacteria Pseudomonas sp. MN12 was also used with each

treatment. Pseudomonas sp. MN12 was previously isolated and evaluated for improving

growth and yield of maize (Naveed et al., 2014) under controlled and natural soil conditions.

Zinc solubilization activity of the selected strain was performed following the method of Bunt

and Rovira (1955). The strain showed Zn solubilization activity as was observed the halos on

the selected petri-plates.

Inoculum of strain MN12 was prepared in TSA broth in 1000 mL Erlenmeyer flasks

and incubated at 28±2°C for 48 h in the orbital shaking incubator (Firstek Scientific, Tokyo,

Japan) at 180 rev min-1. The optical density of the broth was adjusted to 0.5 measured at

600 nm using spectrophotometer (Evolution 300 LC, Cambridge, UK) to obtain a uniform

population of bacteria (108–109 colony-forming units (CFU) mL-1) in the broth at the time

of application. For one hectare area, 5 L microbial culture was used in each of the

application method. For hydropriming and osmopriming, 5 L microbial culture was added

in the solution and seeds were primed with similar to above mentioned procedure. Likewise,

for seed coating, for 125 kg seeds ha-1, Zn was mixed with 5 L microbial culture and Arabic

gum, and the resulting slurry was coated on seeds. Moreover, in soil application for one

hectare area, 5 L microbial culture was mixed with Zn and was mixed into soil before

sowing. Similarly, in foliar application, 5 L microbial culture was added with spray water

and was sprayed on leaves at tillering stage.


Seeds of wheat were sown on November 29, 2013 and November 26, 2014 using

single row hand drill in 22.5 cm spaced rows using seed rate of 125 kg ha-1. The

experimental soil was sandy loam with pH 8.18, EC, 0.34, organic matter 0.92% and

89

nitrogen 0.065% while phosphorus, potassium, Zn, B and iron were 5.02, 1.68, 0.7, 0.55

and 6.78 ppm, respectively. Based on the soil analysis, 100-90-75 N, P2O5 and K2O kg ha1

was applied using urea (46% N), diammonium phosphate (18% N, 46% P2O5) and sulfate

of potash (50% K2O) as sources. Whole of the P, K and one third of the N was applied as

basal dose. Remaining N was applied with 1st and 2nd irrigation in equal splits. Selected

herbicide (Atlantis (iodo+mesosulfuron) at 14.4 g a.i. ha-1) was applied as early post

emergence on January 13, 2014 and January 15, 2015 respectively, to control weeds. In

total, four irrigations (each of 3 acre inches) were applied to the crop during the growth

period in addition to soaking irrigation of four acre inches. Crop was harvested on April 24,

2014 and April 26, 2015 during first and second years of the study, respectively and was

threshed to record the yield and other related traits. The mean maximum temperature was

26.2, 19.5, 17.9, 21, 24.6 and 32.7ºC while mean minimum temperature was 11.6, 7.2, 6.5,

10.0, 13.6 and 19.7ºC from November to April during both years, respectively. Moreover,

rain fall was 0.5, 0.0, 0.0, 14.3, 41.7 and 28.2 mm during 2013-2014, while it was 10.0, 0.0,

12.2, 20.5, 67.9 and 32.8 mm during year 2014-15 from November to April, respectively.

3.5.3. Data recording


Total and productive tillers were counted from unit area (1 × 1 m) in each plot (1.8

× 6 m) at final harvest from two locations. From each plot, twenty main spikes were

randomly taken and were threshed manually to separate the grain. The grains separated

were counted to record number of grains per spike. Three sub-sample of 1000 grain was

taken from each plot then weighted on an electric balance and average 1000grain weight

was calculated. The crop was harvested, tied into bundles and sundried for a week in

respective plots. Total wheat biomass (biological yield) of sun-dried samples was recorded

for each treatment by using a spring balance. The crop was threshed manually. Grain yield

for each treatment was recorded by an electric balance in kilograms and later was expressed

in mega gram per hectare (Mg ha-1). The values of grain yield were adjusted to 12%

moisture level. 3.5.3.2 Water relation traits

To measure the water relation traits, sampling was done early in the morning. The

samples were kept in N containing cylinder. Relative water contents were recorded using

protocol of Barrs and Weatherly (1962). Weight of fresh leaves (Wf) were taken and then

these leaves were floated on water to get saturated weight (Ws) for 4 h. Then the leaves

90

were dried till constant weight to get dry weight (Wd). Relative water contents (RWC) were

measured using formula:

RWC=(Wf-Wd)/(Ws-Wd)×100

Penultimate leaves were harvested at tillering, booting and anthesis to determine

water relations traits. Leaf water potential (ψw) was determined using pressure chamber

(Model 1000 pressure chamber instrument, PMS Instrument Company). To calculate the

leaf osmotic potential, the leaf tissues used for determining of ψw were frozen for 48 h,

thawed and sap was extracted. The extracted sap was centrifuged at 5000 × g and leaf

osmotic potential (ψs) was determined using osmometer (Osmomat 030 Gonotec). Leaf

water potential was calculated as difference of ψw and ψs.

3.5.3.3 Grain quality analysis

Fully matured grains were used for protein and nutrient analyses. Protein was

calculated in wheat flour by analyzing the total N concentration of flour samples using a

Leco TruSpec CN analyzer (Leco Corp., St. Joseph, MI) and multiplying the total N with

conversion factor of 5.36 (Mosse, 1990). Moreover, grain concentrations of Zn, Fe and Ca

were measured by an inductively coupled plasma optical emission spectrometer (ICP-OES;

Vista-Pro Axial; Varian Pty Ltd., Mulgrave, Australia) after digesting the grain samples in

a closed microwave digestion system (MarsExpress CEM Corp., Matthews, NC) in the

presence of concentrated HNO3 and H2O2.

Initially, whole embryos (i.e., the embryo, including the scutellum) of about 100

seeds were severed using a surgical blade. To separate the endosperm, de-embryonated

seeds were milled with a vibrating agate cup mill (Pulverisette 9, Fritsch GmbH, Idar-

Oberstein, Germany) for 20s at 700 rpm, and the resulting flour was sieved with a 100 μm

mesh plastic sieve. The sieved particles were treated as the endosperm, whereas the fraction

remaining on the sieve (bran and shorts) was sieved again with a 1000 μm mesh plastic

sieve in order to separate the bran from the shorts.

3.5.3.4 Phytate concentration and estimated bioavailable Zn

Phytate concentration in grains were estimated as detailed in section 3.3.3.3


Data were analysed using statistical software Statistics 8.1. Treatment means were

separated using HSD at p ≤ 0.05. Graphs were developed using Microsoft Excel (2013).

Standard errors were also calculated using Microsoft excel.

91

Economic analysis was performed following the procedure of Byerlee (1988), on

the basis of variable cost in different treatments. Gross income of each treatment was

calculated. The benefit cost ratio (BCR) for all individual treatments was calculated by the

ratio of gross income and the total cost. Net field benefit was calculated by subtracting the

total variable cost from the total benefits of each treatment combination. Input and output

cost for each treatment combination was converted into USD. Marginal rate of return

(MRR) was calculated by the following formula (CIMMYT, 1988). Marginal net benefits

refer to change in net field benefits, calculated by subtracting net benefits of treatments,

whereas marginal cost is the cost that varies among the treatments and calculated by

subtracting cost of different treatments.

3.6. Improving the Drought Resistance in Wheat through Zinc Nutrition

3.6.1. Experimental conditions

This study was conducted in the growth chambers of Plant Physiology Laboratory,

Sabanci University, Istanbul, Turkey. The crop was sown on December 20, 2015. Fifteen

seeds of two bread wheat genotypes Lasani-2008 and Faisalabad-2008 were grown in soil

brought from central Anatolia. The plants were thinned to ten after completion of

germination. The experimental soil have pH 8.06, EC 0.21 mmhos cm -1, organic matter

1.08% and CaCO3 12%. Moreover, phosphorus, potassium concentration as 3.63 and 340

mg kg-1 respectively. The soil was highly Zn deficient with Zn concentration of 0.17 mg

kg-1, while, Fe, Mn, Cu concentrations were 4.11, 4.65 and 1.04 mg kg-1 respectively.

Organic matter content of the experimental soil was 1.08% with 12% CaCO3. On the basis

of soil analysis, fertilizer were applied as 150 mg kg-1 N as Ca (NO3)2.4H2O, 100 mg kg-1

phosphorus KH2PO4 and S as 25 mg kg-1 as K2SO4. Zinc was applied as low Zn (0.3 ppm/kg

soil) while adequate Zn plants received 3 ppm Zn kg/soil using ZnSO4.7H2O as source.

The temperature of the growth chamber was 20/15°C day/night during the whole

experimental period. Photosynthetic photon flux density in growth chamber was about 400

μmol m−2 s−1 at the level where plants were grown. After 15 days of sowing, the water stress

was imposed. Drought stressed plants were maintained at 35% field capacity, while well-

watered plants were maintained at 70% field capacity during the whole crop season.

3.6.2. Chlorophyll and gas exchange traits

Chlorophyll intensity was measured using SPAD. From each pot three reading of

SPAD were taken and their average was used thereafter. Moreover, rate of photosynthesis

and other gas exchange traits were recorded using LI-6400 XT (LI-COR Biosciences, Inc.

92

Lincoln, Nebraska, USA). Two fully expanded leaf were used for each replication of

treatment and then their average was worked out. Relative water contents were recorded

using the protocol described in section 3.5.4.2.

3.6.3. Enzyme extraction and analyses

After 30 days of sowing, a known weight of frozen leaf samples of wheat were used

for enzyme and protein assay. Leaf and root samples were homogenized in 10 ml of 50 mM

potassium phosphate buffer (K-P); while liquid N2 and quarts was used to ease the

homogenization. The buffer was prepared by mixing of 50 mM K2HPO4 and 50 mM

KH2PO4 with final pH of 7.6. After preparation of KP buffer 0.1mM EDTA Titriplex-III

was added before samples homogenization. Homogenized samples were then centrifuged

at 15000g for 20 min at 4ºC. The supernatant after centrifugation were used for protein and

enzyme assay. Protein in the supernatants of root and leaf samples was estimated following

the protocol of Bradford (1976). For SOD, estimation method of Giannopolitis and Ries

(1977) was used. This method uses the SOD, and inhibits the photochemical reduction of

p-nitro blue tetrazolium chloride (NBT) and its spectroscopic measurement at 560nm. The

reaction mixture contained 2.9 mL K-P buffer, 0.0.5 mL 50 mM Na2CO3, 0.5 mL 50 mM

L-methionine, 0.5 mL 75 μM NBT and 500 μL 2 μM riboflavin with 100 μL supernatants.

After addition of Riboflavin, the tubes containing reaction mixture were placed under light

in a growth room for 8 min, then absorbance was taken at 560 nm.

For ascorbate peroxidase (APX), activity was estimated using the protocol of

Nakano and Asada (1981) by observing the decline in absorbance at 290 nm. For APX the

reaction mixture contained 50mMK-P 650-800 µL K-P buffer (pH 7.6) containing 0.1 mM

Na-EDTA, 100 μL of 12 mM H2O2, 50- 150 µL sample and 100 μL of 0.25 mM ascorbic

acid.

Glutathione reductase activity was estimated according Foyer and Halliwell (1976)

with some modifications by monitoring the nicotinamide adenine dinucleotide phosphate

(NADPH) oxidation at 340 nm. The 1 mL reaction mixture to assay the GR activity

contained 650–750 μl of 50 mM K-P buffer (pH 7.6) with 0.1 mM Na-EDTA, 100 μL of

0.5 mM oxidized glutathione (GSSG), 50–150 μL of the enzyme extract and 100 μL of 0.12

mM NADPH.

3.6.4. Yield related traits

Yield related traits were estimated as described in section 3.4.3.2

93

3.6.5. Biomass production and mineral analyses

Three plants from each replications were harvested for biomass production and

nutrient analyses. Leaf samples were ground to fine powder using an agate vibrating mill

(Pulverisette 9, Fritsch GmbH, Germany). A known weight of sample (0.2g) was acid

digested in 2mL H2O2 and 5 ml of 65 % HNO3 using a closed vessel microwave reaction

system (MarsExpress; CEM Corp., Matthews, NC, USA). After sample digestion sample

were diluted to 20 mL using double deionized water. Inductively coupled plasma optical

emission spectroscopy (ICP-OES; Vista-Pro Axial; Varian Pty Ltd, Mulgrave, Australia)

was used for measurement of Zn and other elements concentration. For leaf N concentration

(%) grounded samples were analyzed using automated N analyzer (TruSpec CN, LECO

Corp., Michigan, USA).

3.6.6. Phytate concentration and estimation of bioavailable Zn

Phytate concentration and estimation of bioavailable Zn was done as detailed in

section 3.3.3.3 3.6.7. Data analysis

Experimental data were analyzed using analysis of variance technique (Steel et al.,

1996) through statistical software Statistix 8.1. For mean separation Tukey Honesty

significant difference test was used.


3.7.1. Experimental condition and treatments

In this study salt stress was applied as 2500 ppm NaCl/kg soil while no salt

application was taken as control. Wheat cultivars, Zn levels and all other experimental

conditions were similar as described in the section 3.6.1.

3.7.2. Chlorophyll and gas exchange traits

Chlorophyll and gas exchange traits were recorded as detailed in section 3.6.2.


Enzymes extraction and analyses were done as detailed in section in section 3.6.3

3.7.4. Yield related traits

Yield related traits were recorded as detailed in section 3.4.3.2

3.7.5. Biomass production and mineral analyses

Biomass production and mineral analyses were done as detailed in section 3.6.4.

94

3.7.6. Phytate concentration and estimation of bioavailable Zn


section 3.3.3.3.


Data analysis was done as detailed in section 3.6.5.

3.8. Improving the Resistance against Cold Stress in Wheat through Zn Nutrition 3.8.1

Experimental condition and treatments

In this study, plants were grown under optimal temperature 20/15ºC day/night

(control) for 15 days after sowing. After 15 days of sowing half of the pots were transferred

to other growth chamber with low temperature i.e. 10/7ºC day/night. Cold stress was

imposed till booting stage. Wheat cultivars, Zn levels and all other experimental conditions

were similar as described in the section 3.8.1

3.8.2 Chlorophyll and gas exchange traits

Chlorophyll and gas exchange traits were recorded as detailed in section 3.6.2.

3.8.3 Enzyme extraction and analyses

Enzymes extraction and analyses were done as detailed in section in section 3.6.3

3.8.4 Yield related traits

Yield related traits were recorded as detailed in section 3.4.3.2

3.8.5 Biomass production and mineral analyses

Biomass production and minerals analyses were done as detailed in section 3.6.4.

3.8.6 Phytate concentration and estimated bioavailable Zn


section 3.3.3.3.



3.9. Improving the Resistance against Heat Stress in Wheat through Zinc Nutrition

3.9.1. Experimental condition and treatments

Two bread wheat genotypes Lasani-2008 and Faisalabad-2008 were grown in

hydroponic solution culture in growth rooms under controlled environmental conditions.

The temperature of the growth chamber was 25/18°C day and night for control while for

heat stress 36/28°C day and night temperature was maintained. Photosynthetic photon flux

95

density in growth chamber was about 400 μmol m-2 s-1 at the level where plants were grown.

Seeds of wheat were sown in wetted perlite steeped with CaSO4.2H2O solution and were

allow germinating at room temperature for 5 days and then transferred to pots with nutrient

solution.

All the pots were kept at 25/18°C day/night temperature for 5 days; latter half of the

pots were transferred to the heat stress chamber viz. 36/28°C day night temperature. The

nutrient solution used in this study was composed of 2 mM Ca(NO3)2.4H2O, 0.2 mM

KH2PO4, 0.85 mM K2SO4, 0.1 mM KCl, 1 mM MgSO4.7H2O, 100 μM Fe-EDTA, 1 μM

MnSO4.H2O, 0.2 μM, 1 μM H3BO3, CuSO4.5H2O, 0.14 μM (NH4) 6Mo7O24.4H2O.

Moreover, high Zn pots were supplied with 1 μM ZnSO4.7H2O while the low Zn pot

contained 0.1 μM ZnSO4.7H2O. Nutrient solution were aerated continuously till harvesting

and were changed after every 3 days throughout the experimental period

3.9.2. Photosynthesis/ biomass production and nutrient analyses/root traits

Data regarding photosynthesis was taken 10 days after heat stress. Photosynthetic

traits were measured using LI-6400 XT (LI-COR Biosciences, Inc. Lincoln, Nebraska,

USA). Two fully expanded leaves were used for each replication of treatment and then their

average was worked out. After 15 days of transplanting, plants were harvested. Four plants

from each replications were harvested for biomass production and nutrient analyses while

two plants were used to measure root related traits. Moreover, root related traits were

measured using image equiring EPSON flatbed scanner Perfection V700 and Win RHIZO

2013 software was used for measurement of root traits.


Enzymes extraction and analyses were done as detailed in section in section 3.7.3.

3.9.4. Digestion and mineral analyses

Digestion and mineral analyses were done as detailed in section 3.7.4.



96

Chapter 4

RESULTS AND DISCUSSION



4.1.1. Results

4.1.1a. Petri plate experiment

All seed priming treatments, including hydropriming, significantly improved

germination and seedling growth. However, seed priming with 0.5 M ZnSO4 was more

effective than the other treatments in improving the germination rate and final germination

(Table 4.1). Increase in root length, shoot length and seedling dry weight was observed 12

days after sowing with seed priming of 0.5 M ZnSO4 as a result of more rapid germination.

Seed priming with higher concentrations than 0.5 M Zn with ZnSO4 or 0.1 M Zn with ZnCl2

did not improve the germination and seedling growth further. Seedling dry weight was

positively correlated with seedling root and shoot lengths, and was negatively correlated

with mean germination time (Table 4.2).

4.1.1b. Sand-filled pot experiment

In the sand-filled pot study, hydroprimed seeds showed improvement in seedling

emergence and seedling growth (Table 4.3). Addition of Zn in the priming solution further

improved performance. Cultivar Lasani-2008 showed better emergence percentage in

response to seed priming with 0.05 and 0.1 M ZnSO4 while cultivar Faisalabad-2008 had

better emergence following priming with 0.5 M Zn solution of ZnSO4 and 0.1 M Zn

solution of ZnCl2. Seed priming with 0.5 M Zn solution of ZnSO4 enhanced the rate of

emergence as indicated by low MET. As a result of rapid and uniform emergence,

improvement in shoot length, root length and dry weight of seedlings was also observed

after sowing. However, seed priming beyond 0.5 M Zn solution using ZnSO4 and 0.1 M Zn

solution of ZnCl2 inhibited seedling emergence and growth. Seedling dry weight was

positively correlated with seedling shoot and root lengths, and negatively correlated with

seedling dry weight (Table 4.2).

97

Control 88.89

87.50 88.19 AB

3.44 3.64

3.54 BC 71.8

67.4 CD 69.6

71.5 74.5

73.0 D 35.00

38.75 36.88 CD

Table 4.1: Influence of seed priming with zinc on the germination and seedling growth of wheat cultivars Lasani-2008 (LS-2008) and

Faisalabad-2008 (FSD-2008) (Petri plate experiment)

Final Germination (%) Mean germination time (days) Shoot length (mm) Root length (mm) Seedling dry weight (mg) Seed priming LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T)

HP 94.44 91.67 93.06 A 3.19 3.20 3.19 CDE 80.1 79.5 79.8 ABC 82.1 86.3 84.2 BCD 43.75 40.00 41.88 BC

ZnSO4 0.01 M 94.44 88.89 91.67 AB 3.18 3.06 3.12 CDE 74.0 78.8 76.4 A-D 89.3 86.0 87.6 ABC 50.00 46.25 48.13 B

0.05 M 94.44 97.22 95.83 A 3.09 3.02 3.05 DE 77.3 76.3 76.8 A-D 83.0 87.5 85.3 BCD 47.50 50.00 48.75 AB

0.1 M 97.22 97.22 97.22 A 3.03 3.08 3.06 DE 85.5 78.8 82.1 AB 92.0 97.5 94.8 AB 48.75 50.00 49.38 AB

0.5 M 97.22 100.0 98.61 A 2.69 2.89 2.79 E 85.5 85.8 85.7 A 100.8 99.8 100.3 A 60.00 58.75 59.38 A

1.0 M 83.34 80.56 81.95 B 3.33 3.37 3.35 CD 70.0 71.3 70.6 CD 74.0 85.8 79.9 CD 32.50 31.25 31.88 CD

ZnCl2 0.01 M 94.44 86.11 90.28 AB 3.20 3.87 3.53 BC 72.0 62.8 67.4 D 89.0 78.3 83.6 BCD 32.50 38.75 35.63 CD

0.05 M 97.22 94.44 95.83 A 3.02 3.58 3.30 CD 73.8 71.0 72.4 BCD 91.0 88.8 89.9 ABC 32.50 32.50 32.50 CD

0.1 M 100.00 97.22 98.61 A 2.97 3.09 3.03 DE 77.0 76.3 76.7 A-D 93.3 95.8 94.5 AB 42.50 36.25 39.38 BC

0.5 M 63.89 72.22 68.06 C 3.98 4.04 4.01 A 56.5 50.0 53.3 E 61.5 52.8 57.1 E 23.75 32.50 28.13 DE

1.0 M 41.67 44.44 43.06 D 4.03 3.90 3.96 AB 49.5 43.0 46.3 E 50.5 51.8 51.1 E 21.25 18.75 20.00 E

Mean (C) 87.27 86.46

3.26 B 3.39 A 72.7 A 70.1 B

81.5 82.0 39.17 39.48

Means not sharing the same letter, for a parameter, don’t differ significantly at p ≤ 0.05. T

= Treatment; C = Cultivar; HP = Hydropriming; ; M= Molar

Table 4.2: Correlations coefficients (r) among different germination stand establishment and seedling growth traits of wheat as

influenced by seed priming with zinc (n =12)

Traits

Seedling dry weight Petri plates Pots

Mean germination time - 88.12* - 88.12*

98

Root length Shoot length

*= significant at p ≤ 0.05

Table 4.3: Influence of seed priming with zinc on the emergence and seedling growth of wheat cultivars Lasani-2008 (LS-2008) and Faisalabad-

2008 (FSD-2008) (Pot experiment) Final emergence (%) Mean emergence time (days) Shoot length (mm) Root length (mm) Seedling dry weight (mg) Seed priming LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T)

HP 93.06 ab 88.89 ab 90.97 A 4.72 a-f 4.72 a-f 4.72 BC 184.6 cde 183.5 cde 184.0 C 185.9 190.6 188.3 BC 80.00 a-f 77.50 a-f 78.75 B-E

ZnSO4 0.01 M 90.28 ab 83.33 abc 86.81 AB 4.83 abc 5.10 a 4.96 AB 176.3 def 179.3 de 177.8 CD 183.8 188.0 185.9 BC 86.25 a-d 68.75 c-h 77.50 C-F

0.05 M 97.22 a 88.89 ab 93.06 A 4.38 c-f 5.03 ab 4.70 BC 181.8 cde 171.5 efg 176.6 CD 184.3 193.8 189.0 BC 86.25 a-d 87.50 a-d 86.88 ABC

0.1 M 97.22 a 87.50 ab 92.36 A 4.29 ef 4.90 ab 4.59 CD 203.0 bc 172.3 efg 187.6 BC 190.3 195.5 192.9 ABC 93.75 ab 92.50 abc 93.13 AB

0.5 M 88.89 ab 94.44 a 91.67 A 4.26 f 4.26 f 4.26 E 226.3 a 221.0 ab 223.6 A 223.3 203.5 213.4 A 95.00 a 95.00 a 95.00 A

1.0 M 66.67 d 77.78 bcd 72.22 CD 5.01 ab 4.78 a-d 4.90 AB 151.0 gh 156.0 fgh 153.5 E 151.0 148.3 149.6 DE 48.75 hi 77.50 a-f 63.13 FG

ZnCl2 0.01 M 91.67 ab 88.89 ab 90.28 A 4.85 ab 4.75 a-e 4.80 ABC 171.5 efg 181.0 cde 176.3 CD 184.0 175.5 179.8 BC 66.25 d-i 75.00 a-g 70.63 DEF

0.05 M 88.89 ab 86.11 ab 87.50 AB 4.56 b-f 4.96 ab 4.76 ABC 178.9 de 185.0 cde 181.9 CD 192.8 184.5 188.6 BC 68.75 c-h 60.00 e-i 64.38 EFG

0.1 M 91.67 ab 94.45 a 93.06 A 4.33 def 4.37 c-f 4.35 DE 196.5 cd 202.0 bc 199.3 B 200.8 190.8 195.8 AB 85.00 a-d 82.50 a-e 83.75 A-D

0.5 M 77.78 bcd 77.78 bcd 77.78 BC 4.82 abc 5.00 ab 4.91 AB 167.0 efg 170.8 efg 168.9 D 169.8 171.0 170.4 CD 51.25 ghi 57.50 f-i 54.38 GH

1.0 M 69.44 cd 63.89 d 66.67 D 4.95 ab 4.92 ab 4.93 AB 142.7 h 136.5 h 139.6 E 154.8 134.8 144.8 E 50.00 hi 42.50 i 46.25 H

Mean (C) 86.81 84.84

4.67 B 4.81 A 179.1 177.4

182.7 179.1 72.50 73.85

Means not sharing the same letter, for a parameter, don’t differ significantly at P ≤ 0.05. T = Treatment; C = Cultivar; HP = Hydropriming; ; M= Molar

Control 88.89 ab

86.12 ab AB 87.50

5.09 a 4.98 ab

5.04 A 169.8 efg

efg 169.8 169.8 D

172.1 173.0

172.6 C 58.75 e - i

70.00 b - h 64.38 EFG

80.96* 83.92* 88.75* 80.8*

99

Treatment (T) 4 1.47** 1.37 ns 141.84** 0.45** 2.28* 94.15**

Table 4.4: Analysis of variance for influence of seed priming with Zn on stand establishment, yield related traits, grain yield and grain

biofortification of bread wheat

Variety (V) 1 0.0004ns 15.63ns 0.52ns 10.30** 40.00* 0.03ns 0.08ns 18.59ns 15.07ns T × V 4 0.192ns 148.44ns 0.57ns 0.84ns 19.06ns 0.11ns 0.46* 48.32* 3.30ns Error 30 0.145 62.29 0.61 1.17 8.67 0.05 0.16 12.81 9.10 Total 39

*= p≤ 0.05; **= P≤ 0.01; ns= Non-significant; SOV= Source of variation; DF= Degree of freedom; MET= Mean emergence time; EP= Emergence percentage;

SL= Spike length; Spikelet per spike; GPS= Grains per spike; HGW= 100 grain weight; GY= Grain yield; HI= Harvest index; GZn= Grain Zn concentratıon

Table 4.5: Analysis of variance for influence of seed priming with Zn on chlorophyll and shoot Zn concentration of bread wheat Pre-anthesis Post-anthesis SOV DF

Chlorophyll a Chlorophyll b Shoot Zn concentratıon Chlorophyll a Chlorophyll b Shoot Zn concentratıon Variety (V) 1 0.04ns 0.11ns 3.94ns 2.57** 2.91* 4.73ns T × V 4 0.05ns 0.20ns 2.17ns 0.08ns 0.48ns 1.16ns Error 30 0.16 0.57 14.74 0.09 0.59 12.14 Total 39

*= p≤0.05; **= P≤0.01; ns= Non-significant; SOV= Source of variation; DF= Degree of freedom

Table 4.6: Influence of seed priming with Zn on stand establishment, yield related traits and grain biofortification of bread wheat

ZnSO4 0.1 M 5.96 BC 87.50 AB 7.09 AB 13.29 A 26.50 BC 2.88 AB 28.01 AB 0.5 M 5.90 CD 91.88 AB 7.48 A 14.00 A 31.38 A 2.98 A 30.81 A ZnCl2 0.05 M 6.34 AB 84.38 B 6.83 AB 12.00 B 25.00 CD 2.69 B 26.78 B 0.1 M 5.56 D 94.38 A 7.25 A 13.46 A 29.25 AB 2.68 B 28.33 AB

Means sharing the same letter, for a parameter, don’t differ significantly at p ≤ 0.05; T= Treatment; M=Molar

SOV DF MET EP SL SPS GPS HGW GY HI GZn Treatment (T) 4 1.422** 517.81** 1.27* 8.64** 104.21** 0.43** 10.48** 100.45** 83.74**

Control 6.65 A C 73.75 B 6.44 11.54 B 22.13 D 2.38 C 22.03 C

100

MET= Mean emergence time; EP= Emergence percentage; SL= Spike length; SPS= Spikelet per spike; GPS= Grains per spike; HGW= 100 grain weight; GZn= Grain

Zn concentratıon

Table 4.7: Influence of seed priming with Zn on chlorophyll and straw Zn concentration of bread wheat Pre-anthesis Post-anthesis

Treatments Chlorophyll a Chlorophyll b Shoot Zn concentratıon Chlorophyll a Chlorophyll b Shoot Zn (mg g-1) (mg g-1) (µg g-1) (mg g-1) (mg g-1)

concentration (µg g-1)

ZnSO4 0.1 M 4.70 AB 7.01 39.84 AB 3.36 A 5.77 A 35.73 AB 0.5 M 4.98 A 7.43 43.02 A 3.57 A 6.14 A 37.69 A ZnCl2 0.05 M 4.55 B 6.86 37.44 B 3.40 A 5.52 A 34.09 B 0.1 M 4.75 AB 6.98 40.02 AB 3.47 A 5.49 A 35.60 AB

Means sharing the same letter, for a parameter, don’t differ significantly at p ≤ 0.05; T= Treatment; M=Molar

Table 4.8: Influence of seed priming with Zn on grain yield and harvest index of bread wheat

Control 5.05 e 4.40 f 34.35 b 28.19 c

ZnSO4 0.1 M 6.91 cd 7.53 ab 37.36 ab 41.73 a

0.5 M 7.55 a 7.86 a 38.11 ab 41.58 a

ZnCl2 0.05 M 6.40 cd 6.34 d 40.33 a 36.60 ab

0.1 M 6.76 cd 6.96 bc 38.55 ab 33.77 b

Means sharing the same letter, for a parameter, don’t differ significantly at p ≤ 0.05

LS-2008 = Lasani-2008; FSD = Faisalabad-2008; M=Molar

Control 3.97 C 6.28 31.77 C 2.96 B 4.69 B 28.65 C

LS - 2008 FSD - 2008 LS - 2008 FSD - 2008

101

4.1.1c. Glass house experiment

Seed priming with Zn significantly affected the seedling emergence, yield related traits,

grain yield, and grain biofortification of wheat cultivars (Table 4.4). However, interaction of

seed priming treatments and wheat cultivars was only significant for grain yield and harvest

index (Table 4.4). Moreover, leaf chlorophyll a contents and shoot Zn contents before and after

anthesis; while chlorophyll b contents after anthesis were significantly affected by Zn seed

priming (Table 4.5).

Seed priming with Zn resulted in early and synchronized seedling emergence compared

to dry seeds; as maximum EP and less MET was noted from seeds primed with 0.1 M Zn

solution (ZnCl2) (Table 4.6). Moreover, seed priming with Zn improved the spike length of

wheat than untreated dry seeds. Maximum increase in spike length was recorded from seeds

primed with 0.1 M Zn solution (ZnCl2) and 0.5 M Zn solution (ZnSO4) (Table 4.6). Likewise,

seed priming increased the number of spikelets per spike than control, however, maximum

spikelets per spike were recorded from seed primed with 0.1 M Zn solution (ZnSO4, ZnCl2)

and 0.5 M Zn solution (ZnSO4) (Table 4.6).

Seed priming with Zn also increased the grain weight and number of grains per spike

of wheat than untreated seeds. In this regard, maximum 100-grain weight and number of grains

per spike were observed from seeds primed with 0.5 M Zn using ZnSO4 as source (Table 4.6).

Furthermore, seed priming with Zn enhanced the grain Zn concentration than untreated control,

and maximum increase was recorded from seed priming with 0.5 M Zn solution (ZnSO4)

(Table 4.6).

Seed priming with Zn also enhanced the leaf chlorophyll a and b contents before and

after anthesis compared to untreated seeds. Before anthesis maximum increase in leaf

chlorophyll a contents was recorded from seed priming with 0.5 M Zn solution (ZnSO4) (Table

4.7). Likewise, after anthesis, seed priming with Zn using either source/concentration improved

the leaf chlorophyll a and b contents of wheat (Table 4.7). Zn seed priming also increased the

shoot Zn concentration than untreated dry seeds before and after anthesis. However, maximum

increase in shoot Zn contents was noted from seed priming with 0.5 M Zn solution (ZnSO4)

(Table 4.7).

102

Grain yield and harvest index were also improved by Zn seed priming than untreated

dry seeds. In this regard, maximum increase in grain yield was recorded from seeds primed

with 0.5 M solution of ZnSO4 in both cultivars. Nevertheless, maximum harvest index was

noted in seeds of cultivar Faisalabad-2008 primed with 0.1 and 0.5 M Zn solution (ZnSO4);

while seed primed with 0.05 M Zn solution (ZnCl2) had the maximum harvest index for cultivar

Lasani-2008 (Table 4.8).

4.1.2. Discussion

Seed priming with Zn improved the germination and seedling growth of bread wheat.

Hydropriming also improved germination and seedling growth, but to a lesser extent. Seed

priming stimulates the activities of enzymes like α-amylase (Kaur et al., 2002; Farooq et al.,

2006b), which then accelerate the breakdown of food reserves and supply of energy to growing

embryos (Kaur et al., 2002; Farooq et al., 2006a, b). Seed priming promoted the seedling

emergence possibly due to accumulation of germination promoting metabolites (Farooq et al.,

2006a) which accredited in metabolic repair in early phase of water uptake (Burgass and

Powell, 1984). Moreover, use of Zn in priming medium further enhanced the seedling

emergence as was observed in our previous study (Rehman et al., 2015); suggesting the role

of Zn in early stage of seedling emergence as Zn concentration is high during early stages of

radicle and coleoptile development (Ozturk et al., 2006). Moreover, increase in seedling

growth is possibly due to participation of Zn in protein synthesis, membrane functioning, cell

elongation and resistance against environmental stresses (Cakmak, 2000).

Seed priming improves crop performance by triggering physiological, molecular and

biochemical changes (Farooq et al., 2006b; Chen et al., 2012). However, addition of Zn in

priming solution further improved emergence and seedling growth, possibly due to the

involvement of Zn in the early stages of coleoptile and radicle development (Ozturk et al.,

2006). Seed priming with Zn increased the seedling dry weight due to uniform and early

germination, which improved the seedling growth as affirmed by strong correlations of mean

germination / emergence time, seedling shoot and root lengths with seedling dry weight (table

4.1). Seed priming in solution beyond 0.5 M Zn was toxic as germination and seedling growth

were suppressed (Tables 4.2 and 4.3). Higher Zn concentration may restrict the root growth as

Zn toxicity suppresses cell division (Prasad et al., 1999). Moreover, higher concentration of

103

Zn may diminish root and leaf development owing to substantial decrease in NADPH

production in chloroplasts (Mousavi, 2011). During priming, use of ZnSO4 as a Zn source was

a better option than ZnCl2 (Tables 4.1, 4.3). Poor seedling growth in seeds primed with ZnCl2

indicates the possible toxic effects of Cl- on photosynthesis and cellular respiration (Stankovi

et al., 2011).

Seed priming with Zn augmented the yield related traits, grain yield, leaf chlorophyll

contents, straw and grain Zn concentration of wheat as evident from increase in spike length,

spikelet per spike, 100-grain weight, grain yield and harvest index of wheat. These

improvements in wheat growth due to seed priming with Zn was might be due to role of Zn in

stabilization of biological membranes and synthesis of auxin (Alloway, 2008a). Moreover,

increase in seed yield might be due to more grains per spike and 100 grain weight for seed

priming with Zn than untreated seeds. The seed yield followed the order 0.5 M (ZnSO 4) > 0.1

M (ZnSO4) > 0.1 M (ZnCl2) > 0.05 M (ZnCl2) > Control. Furthermore, improvement in spike

length, spikelet per spike, number of grains per spike and 100-grain weight was noted for Zn

seed priming owing to improved grain setting, suggesting the role of Zn in pollination and

fertilization possibly by improving the anther (Sharma et al., 1987) and pollen development

(Sharma et al., 1990); prompting the pollen tube formation (Pandey et al., 2006).

Seed priming with Zn maintained the stay green character of wheat by increasing the

leaf chlorophyll contents possibly due to adequate supply of Zn as poor chlorophyll

biosynthesis and chlorophyll disruption has been observed under Zn deficiency (Chen et al.,

2007). Zn plays key roles in regulation of enzyme activity and proteins synthesis during

pigments biosynthesis (Balashouri, 1995), which possibly resulted in more chlorophyll

contents of wheat before and after anthesis. Furthermore, increase in grain yield of wheat seeds

primed with Zn was might be due to improvement in stay green character of wheat; as plant

with functional stay green character sustain photosynthetic activity for longer time period

resulting in higher grain weight and seed yield (Spano et al., 2003).

Seed priming with Zn increased the Zn concentrations in shoot and grains of wheat

before and after anthesis following order 0.5 M Zn (ZnSO4) > 0.1 M (ZnSO4, ZnCl2) > 0.05 M

(ZnCl2) > Control. The increase was higher for seed priming in Zn solution of higher

concentration, which might have triggered the uptake and translocation of Zn in vegetative

104

parts and seed. Moreover, seed priming with Zn increased the seed and shoot Zn concentration

directly by supplementing the nutrient and possibly by promoting of root growth which helped

in nutrient acquisition (Imran et al., 2013) from soil leading to higher Zn accumulation in shoot

and seed

In conclusion, seed priming with 0.5 M Zn solution using ZnSO4 performed better than

other treatments for improving the yield and grain biofortification of wheat. However, seed

priming with Zn solution beyond 0.5 M Zn using ZnSO4 and 0.1 M Zn solution using ZnCl2

proved toxic.



4.2.1. Results

4.2.1a. Petri plate experiment

Seed coating with Zn significantly affected the seedling germination, seedling growth

and seedling dry weight of wheat (Table 4.9). Moreover, wheat cultivars differed significantly

for germination percentage (GP); however, interaction of wheat cultivars and seed coating

treatments was only significant for seedling root and shoot length (Table 4.9). Seed coating

with 1.50 g Zn kg-1 seed (ZnCl2) took less time to complete mean germination time (MGT)

(Table 4.10). Moreover, seed coated with 1.25 g Zn kg-1 seed using ZnSO4 increased the GP.

However, seed coating beyond 1.5 g Zn kg-1 seed caused a significant reduction in seed

germination and seedling growth (Table 4.10). Nonetheless, seed coated with 1.25 and 1.5 g

Zn kg-1 seed (ZnSO4) produced longest shoots in Faisalabad-2008 and Lasani-2008,

respectively (Table 4.10). Moreover, lengthiest roots were observed for seed of Faisalabad2008

coated with 1.25 and 1.5 g Zn kg-1 seed (ZnSO4). Seed coating with 1.25 g Zn kg-1 seed (ZnSO4)

improved the seedling dry weight. Moreover, for wheat cultivars, Lasani-2008 showed higher

GP and seedling dry weight (Table 4.10).

4.2.1b. Sand-filled pot experiment

Seed coating with Zn significantly affected the seedling germination, seedling growth

and seedling dry weight. Moreover, wheat cultivars differed significantly for mean emergence

time (MGT), emergence percentage (EP) and seedling dry weight (Table 4.11). Seed coated

105

with 1.25 g Zn kg-1 seed (ZnSO4) took less time to complete MET. Seed coating with Zn

enhanced emergence of wheat seeds as increase in EP was noted for seeds coated with 1.25

and 1.5 g Zn kg-1 seed using ZnSO4, and it was statistically similar to seed coating with Zn

form 0.25–1.00 g Zn kg-1 seed (Table 4.12). Contrary to this, seed coating beyond 1.5 g Zn kg1

seed caused significant decrease in seedling emergence (Table 4.12). In case of seedling

growth, highest shoot length was observed by seed coating with 1.5 g Zn kg-1 seed (ZnSO4).

Moreover, maximum seedling root length was observed for seed coated with 1.25 g Zn kg-1

seed (ZnSO4). However, smallest seedling shoot and roots were recorded for seeds coated with

2 g Zn kg-1 seed using ZnCl2 (Table 4.12). Highest seedling dry weight was recorded for seed

coated using ZnSO4 with 1.5 g Zn kg-1 seed, and it was statistically similar to 1.25 g Zn kg-1

seed. However, minimum seedling dry weight was observed for seed coating with 2.0 g Zn kg1

seed using ZnCl2 (Table 4.12). For wheat cultivars, EP and seedling dry weight were maximum

for Faisalabad-2008, while minimum MET was recorded for Lasani-2008 (Table

4.12).

4.3.1c. Glass house experiment

Seed coating with Zn significantly affected the stand establishment, yield and related

components and grain Zn concentration of wheat (Table 4.13). Moreover, seed coating with

Zn significantly affected the chlorophyll a, b and straw Zn concentration before and after

anthesis (Table 4.14). However, wheat cultivars differed only for MET, while interaction

remained non-significant for all studied traits (Tables 4.13, 4.14).

Seed coating with Zn improved the seedling emergence as minimum MET and high EP

were recorded for seed coated with 1.25 g Zn kg-1 seed (ZnCl2). Moreover, seed coating with

1.25, 1.5 g Zn kg-1 seed (ZnSO4) and 1.25 g Zn kg-1 seed (ZnCl2) produced tallest plant and

increased the harvest index of wheat cultivars. However, seed coating with 1.5 g Zn kg-1 seed

(ZnCl2) did not improve the plant height and HI of wheat, and was similar to control (Table

4.15). On the other hand, more number of grains per spike was recorded for seed coating with

1.25 g Zn kg-1 seed using both sources of Zn. An increase in 100-grain weight of wheat was

recorded for seed coating with 1.25, 1.5 g Zn kg-1 seed using ZnSO4 and 1.25 g Zn kg-1 seed

using ZnCl2. Seed coated with 1.25 g Zn kg-1 seed improved the grain yield of wheat. However,

seed coating with 1.5 g Zn kg-1 seed using ZnCl2 did not improve the grain yield and was

106

similar to control (Table 4.15). Seed coating with Zn increased the grain Zn concentration of

wheat cultivars (21–35%) compared to untreated seeds, and more Zn content was recorded for

seed coated with 1.5 g Zn kg-1 seed (ZnCl2), and it was statistically similar to seed coating with

1.25 and 1.5 g Zn kg-1 seed using ZnCl2 and ZnSO4, respectively (Table 4.15).

Seed coating with Zn improved the chlorophyll contents of wheat cultivars, before and

after anthesis. In this regard, chlorophyll a contents before and after anthesis were maximum

with seed coating of 1.25 g Zn kg-1 seed using either source of Zn (Table 4.16). Moreover,

maximum chlorophyll b contents before anthesis were recorded for seed coating with 1.25 g

Zn kg-1 seed using either source of Zn, while seed coating with 1.25 g Zn kg-1 seed (ZnSO4)

enhanced the chlorophyll b contents of wheat after anthesis. Moreover, seed coating with 1.5

g Zn kg-1 seed did not enhance the chlorophyll concentration before and after anthesis (Table

4.16). Zinc contents in the straw were maximum before anthesis, while there was a decrease in

straw Zn concentration after anthesis. However, Zn application through seed coating increased

the straw Zn concentration, and maximum Zn contents were recorded with 1.5 g Zn kg-1 seed

using either Zn source before and after anthesis, while minimum straw Zn concentration was

recorded for untreated seeds (Table 4.16).

4.2.2. Discussion

The present study enlightened the potential of seed coating for Zn application as it

improved the germination, early seedling growth, chlorophyll, grain yield and grain Zn

contents of wheat. Seed coating with Zn fostered the seedling germination as coating forms a

nutrient layer in the vicinity of the emerging seedling, hence making the nutrient available

during initial phase of seedling growth (Taylor and Herman, 1990). Application of Zn through

seed coating increased the seedling shoot and root length of wheat owing to participation of Zn

in metabolism of germinating seeds, because Zn is present in very high amount in emerging

radicle and coleoptiles during germination cascades (Ozturk et al., 2006). Similarly, seed

coated with Zn improved the seedling dry weight due to better root and shoot growth.

Improvement in seedling growth, by Zn seed coating, may be attributed to provision of Zn for

growth cascades like protein synthesis, membrane functioning and cell elongation (Cakmak,

2000). Zn is a precursor of growth hormone auxin, and adequate Zn supply may help in auxin

regulated growth promotion (Alloway, 2003).

107

Zinc application, through seed coating, also improved the chlorophyll contents as its

deficiency is linked with reduced chlorophyll synthesis (Hisamitsu et al., 2001). Moreover,

improvement in chlorophyll contents during tillering and grain developing of wheat may be

attributed to catalytic and structural role of Zn in protein synthesis and enzyme activation,

which modulates the biosynthesis of photosynthetic pigments including chlorophyll

(Balashouri, 1995). This improvement in chlorophyll contents also contributed to better yield

formation.

108

Table 4.9: Analysis of variance for influence of seed coating with Zn on germination and seedling growth of bread wheat SOV DF Mean germination time Germination percentage Seedling shoot length Seedling root length Seedling dry weight Treatments (T) 16 0.449** 733.93** 5.779** 12.733** 216.455** Cultivar (C) 1 0.0002ns 1797.61** 2.599** 3.120* 65.201ns T × C 16 0.112 ns 73.08ns 0.789* 1.526** 11.642ns Error 102 0.074 53.18 0.367 0.678 38.914 Total 135

*= p< 0.05; **= P< 0.01; ns= Non-significant; SOV= Source of variation; DF= Degree of freedom

Table 4.10: Influence of seed coating with zinc on germination and seedling growth of bread wheat (Petri plate experiment)

Seed coating (MGT) (days) (g Zn kg-1 seed)

Control 4.77 4.82 4.80 ABC 87.50 86.11 86.81 D 9.45 h-m 9.10 k-o 9.28 EFG 9.79 g-j 9.95 f-i 9.87 EF 38.96 39.58 39.27 EF

ZnSO4 0.25 4.88 4.71 4.79 ABC 94.44 86.11 90.28 BCD 10.25 b-h 9.13 j-n 9.69 DE 11.35 bcd 10.15 e-h 10.75 CD 42.92 42.50 42.71 CDE

0.50 4.81 4.97 4.89 AB 91.67 88.89 90.28 BCD 10.28 b-h 10.05 d-i 10.16 BCD 11.45 bcd 11.83 bc 11.64 B 48.33 44.17 46.25 CD

0.75 4.69 5.06 4.87 AB 97.22 97.22 97.22 AB 10.00 d-i 10.80 a-e 10.40 BC 10.95 c-f 11.58 bcd 11.26 BC 46.67 46.67 46.67 BCD

1.00 4.44 4.73 4.59 C-F 100.00 97.22 98.61A 10.13 c-i 10.65 a-f 10.39 BC 11.18 cde 11.38 bcd 11.28 BC 47.50 46.67 47.08 BC

1.25 4.44 4.25 4.35 FGH 100.00 100.00 100.0 A 10.95 abc 11.30 a 11.13 A 11.75 bcd 13.98 a 12.86 A 53.33 53.33 53.33 A

1.50 4.47 4.53 4.50 D-G 100.00 94.44 97.22 AB 11.25 a 11.05 ab 11.15 A 12.34 b 13.70 a 13.02 A 50.00 55.00 52.50 AB

1.75 4.50 4.53 4.52 D-G 83.33 69.44 76.39 E 9.95 f-j 9.90 f-k 9.93 CD 10.65d-g 9.93 f-i 10.29 DE 41.67 40.00 40.83 DEF

2.00 4.73 5.26 5.00 A 86.11 63.89 75.00 E 9.35 i-m 8.48 no 8.91 GH 10.13 e-h 8.93 ij 9.53 EFG 43.33 41.67 42.50 CDE

ZnCl2 0.25 4.42 4.31 4.37 E-G 91.67 80.56 86.11 D 9.63 g-l 8.43 no 9.03 FGH 9.13 hij 9.53 g-j 9.33 FG 37.50 35.00 36.25 F 0.50 4.66 4.61 4.63 B-E 91.67 80.56 86.11 D 9.83 f-k 9.88 f-k 9.85 CDE 9.15 hij 9.55 g-j 9.35 FG 40.00 38.33 39.17 EF

0.75 4.53 4.44 4.48 D-G 94.44 83.33 88.89 CD 9.68 g-l 9.55 g-m 9.61 DEF 9.03 hij 9.83 f-i 9.43 FG 40.00 39.17 39.58 EF

1.00 4.42 4.31 4.36 FGH 100.00 88.89 94.44 ABC 10.25 b-h 9.98 e-i 10.11 BCD 9.48 hij 9.80 f-j 9.64 EFG 41.67 41.67 41.67 CDEF

Mean germination time Germination percentage (%) Shoot length (cm)

Root length (cm) Dry weight (mg)

LS - 2008

FSD - 2008

Mean (T)

LS - 2008 FSD -

2008

Mean (T) LS - 2008

FSD - 2008 Mean (T)

LS - 2008 FSD - 2008

Mean (T) LS - 2008

FSD - 2008

Mean (T)

109

Dry weight (mg)

LS - FSD - Mean (T) LS - FSD - Mean (T)

LS - FSD - Mean (T) LS - FSD - Mean (T)

LS - FSD - Mean (T)

1.25 4.53 4.06 4.30 GH 97.22 91.67 94.44 ABC 10.90 abc 10.23 b-h 10.56 AB 9.48 hij 9.55 g-j 9.51 EFG 48.33 45.83 47.08 BC

1.50 4.24 4.06 4.15 H 94.44 94.44 94.44 ABC 10.83 a-d 10.38 b-g 10.60 AB 9.70 g-j 9.25 hij 9.48 EFG 50.00 45.00 47.50 ABC

1.75 4.69 4.68 4.69 BCD 77.78 66.67 72.22 E 8.28 op 8.78 mno 8.53 HI 8.65 j 9.30 hij 8.98 G 38.33 35.83 37.08 EF

2.00 4.70 4.65 4.67 BCD 72.22 66.67 69.44 E 8.90 l-o 7.45 p 8.18 I 8.83 ij 9.95 f-i 9.39 FG 40.00 34.58 37.29 EF

Mean (C) 4.59 4.59

91.75 A 84.48 B 9.99 A 9.71 B

10.18 B 10.48 A 44.03 A 42.65

B

Means not sharing the same letter, for a parameter, don’t differ significantly at p≤ 0.05 LS-

2008 = Lasani-2008; FSD = Faisalabad-2008; C= Cultivar; T= Treatment

Table 4.11: Analysis of variance for influence of seed coating with Zn on germination and seedling growth of bread wheat

Mean sum of squares SOV DF Mean emergence time Emergence percentage Seedling shoot length Seedling root length Seedling dry weight

Treatment (T) 16 0.377** 712.259** 22.195** 21.839** 184.922** Cultivar (C) 1 1.980** 294.118* 3.679ns 2.051ns 247.791** T × C 16 0.079 56.849 1.043 2.080 17.312 Error 102 0.062 67.780 1.446 1.215 23.725 Total 135

*= p< 0.05; **= P< 0.01; SOV= Source of variation; DF= Degree of freedom

Table 4.12: Influence of seed coating with zinc on germination and seedling growth of wheat cultivar (Sand filled pot experiment)

Seed coating Mean emergence time (MET) Emergence percentage (%) Shoot length (cm) Root length (cm) (g Zn kg-1 seed) (days)

2008 2008 2008 2008 2008 2008 2008 2008 2008 2008

Control 4.62 4.79 4.70 B-E 86.11 83.33 84.72 CD 16.79 17.11 16.95 F 16.80 16.81 16.81 H 65.58 69.58 67.58 FGH

ZnSO4 0.25 4.31 4.62 4.46 EFG 88.89 86.11 87.50 BCD 17.00 17.18 17.09 EF 17.33 17.98 17.65 E-H 69.17 77.50 73.33 BCD

0.50 4.50 4.65 4.57 C-G 88.89 94.44 91.67 ABC 17.55 17.43 17.49 DEF 18.33 19.13 18.73 B-E 70.00 75.83 72.92 B-E

110

0.75 4.28 4.74 4.51 D-G 88.89 97.22 93.06 AB 18.93 17.73 18.33 CD 17.78 19.28 18.53 CDE 72.08 72.00 72.04 C-F

1.00 4.34 4.55 4.44 FG 97.22 91.67 94.44 AB 18.65 17.88 18.26 CDE 18.83 20.45 19.64 AB 71.67 74.59 73.13 B-E

1.25 4.26 4.44 4.35 G 94.44 94.44 94.44 AB 19.63 19.68 19.65 AB 19.83 20.45 20.14 A 77.50 77.50 77.50 AB

1.50 4.45 4.58 4.51 D-G 97.22 91.67 94.44 AB 19.88 20.23 20.05 A 19.30 19.58 19.44 ABC 79.17 77.50 78.33 A

1.75 4.66 4.86 4.76 BCD 80.56 80.56 80.56 DE 16.78 16.65 16.71 F 15.98 17.23 16.60 H 66.67 70.00 68.33 E-H

2.00 4.49 5.16 4.82 B 75.00 69.44 72.22 F 14.92 15.63 15.27 G 13.75 14.83 14.29 I 62.50 66.67 64.58 GHI

ZnCl2 0.25 4.79 4.69 4.74 BCD 94.44 83.33 88.89 ABC 18.73 17.90 18.31 CD 16.75 17.18 16.96 FGH 69.50 67.92 68.71 D-G 0.50 4.58 5.05 4.81 BC 91.67 86.11 88.89 ABC 17.95 17.65 17.80 DEF 18.48 17.50 17.99 DEF 70.00 71.67 70.83 DEF

0.75 4.77 4.67 4.72 BCD 88.89 88.89 88.89 ABC 18.53 18.83 18.68 BCD 18.20 17.63 17.91 D-G 69.79 73.33 71.56 C-F

1.00 4.51 4.76 4.63 B-F 97.22 91.67 94.44 AB 18.78 18.05 18.41 CD 18.85 17.10 17.98 DEF 74.17 72.71 73.44 BCD

1.25 4.50 4.66 4.58 B-G 94.44 97.22 95.83 A 19.08 19.00 19.04 ABC 19.45 18.95 19.20 ABC 75.21 76.67 75.94 ABC

1.50 4.49 4.63 4.56 D-G 97.22 94.44 95.83 A 19.20 18.80 19.00 ABC 19.03 18.75 18.89 BCD 74.17 78.33 76.25 ABC

1.75 4.90 5.32 5.11 A 77.78 69.44 73.61 EF 16.10 13.78 14.94 GH 17.53 16.18 16.85 GH 61.04 66.33 63.69 HI

2.00 4.95 5.33 5.14 A 69.44 58.33 63.89 G 14.33 13.70 14.01 H 13.78 15.14 14.46 I 58.92 64.89 61.90 I

Mean (C) 4.55 B 4.79 A

88.73 A 85.78 B 17.81 17.48

17.64 17.89 69.83 B 72.53 A

Means not sharing the same letter for a parameter don’t differ significantly at p ≤ 0.05; LS-2008 = Lasani-2008; FSD = Faisalabad-2008; C= Cultivar; T= Treatment

Table 4.13: Analysis of variance for influence of seed coating with Zn on stand establishment, productivity and grain biofortification of wheat cultivars

SOV DF Mean sum of squares

Mean emergence time Emergence percentage Plant height Grains per spike 100-grain weight Grain yield Harvest index Grain Zn

contents Treatment (T) 4 0.266* 207.969** 315.321** 49.756** 0.231* 12.117** 106.129* 73.795** Cultivar (C) 1 0.460* 18.906ns 90.963ns 11.556ns 0.148ns 0.388ns 22.320ns 0.052ns T × C 4 0.193ns 23.594ns 39.122ns 2.493ns 0.073ns 0.161ns 6.978ns 7.969ns

111

Error 30 0.076 43.698 36.182 5.115 0.075 0.223 15.285 5.418

Total 39


Table 4.14: Analysis of variance for influence of seed coating with Zn on chlorophyll a, b and straw Zn contents of wheat cultivars

SOV

DF

Before anthesis After anthesis

Chlorophyll a (mg g-1) Chlorophyll b (mg g-1) Straw Zn contents (µg g-1) Chlorophyll a (mg g-1) Chlorophyll b (mg g-1) Straw Zn contents (µg g-1) Treatment (T) 4 1.358** 1.849** 131.246** 0.531** 3.524** 107.378* Cultivar (C) 1 0.0002 0.040ns 0.924ns 0.021ns 0.818ns 0.077ns T × C 4 0.025 0.062ns 0.741ns 0.033ns 0.151ns 2.495ns Error 30 0.052 0.458 14.664 0.074 0.299 17.520 Total 39


Table 4.15: Influence of seed coating with Zn on stand establishment, productivity and grain biofortification of wheat cultivars (Glass house

experiment)

Treatments

ZnSO 6.21 6.16 BC 83.75 81.25 82.50 B 70.38 80.00 75.19 A 29.75 27.25 28.50 A

1.50 g Zn kg-1 seed 6.43 6.36 6.40 AB 87.50 82.50 85.00 AB 75.40 76.25 75.83 A 27.50 26.25 26.88 AB

ZnCl2 1.25 g Zn kg-1 seed 5.73 6.32 6.03 C 91.25 87.50 89.38 A 70.78 76.25 73.51 A 28.50 27.50 28.00 A

1.50 g Zn kg-1 seed 5.92 6.41 6.16 BC 82.50 83.75 83.13 AB 64.63 64.25 64.44 B 25.50 24.25 24.88 B

Mean(C) 6.13 B 6.35 A 83.75 82.38

68.81 71.83 26.68 25.60

ZnSO4 1.25 g Zn kg-1 seed 2.73 3.06 2.89 A 7.57 7.10 7.33 A 38.05 38.18 38.11 A 27.96 28.01 27.99 B 1.50 g Zn kg-1 seed 2.91 2.83 2.87 A 6.19 6.38 6.28 B 38.21 35.34 36.77 A 29.07 29.52 29.29 AB ZnCl2 1.25 g Zn kg-1 seed 2.74 2.92 2.83 A 6.85 6.50 6.67 B 35.60 36.39 36.00 A 30.50 28.14 29.32 AB 1.50 g Zn kg-1 seed 2.66 2.60 2.63 AB 4.70 4.34 4.52 C 32.50 29.04 30.77 B 31.56 30.72 31.14 A

100 - Grain weight (g) Grain yield (g pot - 1 )

Harvest Index (%) Grain Zn content (µg g - 1 )

Control

2.38 2.63

2.50 B 4.72

4.74 4.73 C

31.19 29.14

B 30.17 21.59

24.66 C 23.12

Mean emergence time (MET) (days) Emergence percentage (%)

Plant height (cm) Grains per spike

LS - 2008

FSD - 2008 Mean (T)

LS - 2008 FSD - 2008

Mean (T) LS - 2008

FSD - 2008 Mean (T)

LS - 2008 FSD - 2008

Mean (T)

Control 6.49

6.44 A 6.46

73.75 76.88

75.31 C 62.89

62.40 62.65 B

22.13 22.75

C 22.44

112

2.68 LS-2008 = Lasani-2008; FSD = Faisalabad-2008; C= Cultivar; T= Treatment

Table 4.16: Influence of seed coating with Zn on chlorophyll a, b and straw Zn contents of wheat cultivars (Glass house experiment)

Before anthesis Chlorophyll a (mg g-1) Chlorophyll b (mg g-1) Straw Zn contents (µg g-1)

Treatments LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) LS-2008 FSD-2008 Mean (T) Control 3.88 3.97 3.93 C 6.17 6.10 6.13 BC 32.15 31.74 31.94 C

ZnSO4 1.25 g Zn kg-1 seed 5.01 4.88 4.94 A 7.27 7.02 7.14 A 36.82 38.00 37.41 B

1.50 g Zn kg-1 seed 4.33 4.22 4.28 B 6.91 6.74 6.82 AB 42.23 42.31 42.27 A

ZnCl2 1.25 g Zn kg-1 seed 4.70 4.81 4.75 A 6.75 6.96 6.86 A 37.38 37.45 37.42 B

1.50 g Zn kg-1 seed 4.24 4.27 4.25 B 6.08 6.03 6.06 C 40.86 41.47 41.17 AB

Mean(C) 4.43 4.43 6.63 6.57

37.89 38.19

After anthesis

ZnSO4 1.25 g Zn kg-1 seed 3.73 3.46 3.59 AB 6.49 5.87 6.18 A 32.96 32.03 32.50 BC 1.50 g Zn kg-1 seed 3.11 3.13 3.12 C 5.56 5.13 5.34 B 34.07 35.77 34.92 AB

ZnCl2 1.25 g Zn kg-1 seed 3.59 3.66 3.63 A 5.36 5.39 5.38 B 35.50 34.76 35.13 AB 1.50 g Zn kg-1 seed 3.34 3.32 3.33 BC

4.63 4.60 4.62 C 38.91 38.13 38.52 A

LS-2008 = Lasani-2008; FSD = Faisalabad-2008; C= Cultivar; T= Treatment

Mean(C) 2.81

6.01 5.81

35.11 33.62

28.14 28.21

Means not sharing the same letter, for a parameter, don’t differ significantly at p≤ 0.05

Control 3.10

3.06 C 3.08

4.74 4.38

C 4.56 28.46

28.79 28.62 C

Mean(C) 3.37

3.33 5.36

5.07 33.98

33.89

Means not sharing the same letter, for a parameter, don’t differ significantly at p≤0.05

113

Zn application through seed coating improved the morphological, yield-related parameters

and grain yield of wheat as was visible from increase in plant height, grains per spike, 100-grain

weight and HI of wheat. Substantial increase in grains per spike, 100-grain weight, by Zn seed

coating, contributed for improvement in grain yield. Actually, adequate supply of Zn helps in grain

setting through synchronization of pollination and stigma secretions, which favors pollen

germination, pollen tube development, fertilization and, eventually, grain setting (Kaya and Higgs,

2002; Pandey et al., 2006). Zn also facilitates the assimilate translocation toward developing grains

(Rengel, 2001a; Ozkutlu et al., 2006; Peda Babu et al., 2007; Pooniya et al., 2012), which helped

in harvesting bold wheat grains and better HI from Zn seed coating.

Zn seed coating also improved the Zn contents in wheat grains and straw. This indicates

that Zn application in small amount as seed coating might have triggered the Zn uptake. Zinc is

highly mobile in phloem as it moves from leaves to roots; leaf to developing seed and within roots;

tissue Zn demand is the driving force for Zn phloem mobility (Rengel, 2001b). Furthermore, higher

mobility of Zn in phloem than xylem is due to presence of higher concentration of chelating

compounds in phloem sap (Gupta et al., 2016). However, Zn translocation into seed involves the

xylem loading with the help of Zn pumps HMA2 and HMA4 (Hussain et al., 2004); then it moves

to the leaf symplast via xylem parenchyma cells; it is transported then to leaf apoplast through

mesophyll cells which afterward load Zn into phloem (Olsen and Palmgren, 2014). After loading

into the phloem, Zn is translocated via long and short-distance pathways to developing sinks (seed)

and other plant parts (Gupta et al., 2016).

Zn concentration in wheat grains was significantly affected by rate of Zn application, as Zn

contents were the maximum from seed coated with 1.5 g Zn kg-1 seed possibly due to better

absorption and accumulation of Zn in vegetative parts (Lemes et al., 2015), which then moved to

developing grains.

Moreover, seeds coated with higher concentration of Zn, i.e., above 1.50 g kg-1 seed, using

either source, hampered the stand establishment possibly due to adverse effect of excessive Zn on

seed germination (Dirginčiutė-Volodkienė and Pečiulytė, 2011). Moreover, threshold level of Cl -

for wheat is 1.50 g kg-1 dry weight (Fixen et al., 1986). Zn application at higher rates also

suppressed the seedling growth (Tao et al., 2014). Actually, Zn is an essential micronutrient for

Table

114

plant growth but is required in small quantities. Zn accumulation at higher concentration may cause

Zn toxicity, which may affect the plant growth by disturbing the nutrient balance, causing leaf

necrosis and decrease in photosynthesis (Todeschini et al., 2011; Cambrolle´ et al., 2012).

In case of Zn sources, seed coating with Zn (ZnSO4) was better than ZnCl2 possibly due to

ills effect of additional Cl- ions, which might have influenced the photosynthesis (White and

Broadley, 2001; Stankovic et al., 2010) as high concentration of Cl- ions could be toxic for plants.

In crux, seed coating is a cost-effective, efficient and environment-friendly method of Zn

application as very little amount of nutrient is used in this technique. It also improved the stand

establishment, productivity, chlorophyll and grain Zn contents of wheat. More improvement in

wheat performance was observed from seed coated with 1.25 g Zn kg-1 seed using ZnSO4 as source

of Zn. Nevertheless, seed coating above 1.5 g Zn kg-1 seed using either source was not beneficial.


Diversity

4.3.1. Results

4.3.1.1 Yield related traits

Zinc application improved the yield and yield contributing traits of wheat. During both years

of study, the highest number of productive tillers were recorded in genotype Lasani-2008 with Zn,

and these were the lowest in Faisalabad-83 without Zn (Table 4.17). Highest number of grains per

spike were recorded in Sandal-73 and Sehar-2006 with Zn, while they were lowest in Pak-81 and

Blue silver without Zn during both years respectively (Table 4.17). The highest 1000-grain was

recorded in Faisalabad-85 with/without Zn while it was lowest in Iqbal-2000 during the first year.

During the second year, the highest 1000-grain weight was recorded in Faisalabad-2008 with Zn,

and the minimum of was recorded in Blue silver without Zn (Table 4.18). The highest harvest

index was recorded in Fsd-85 with Zn, and the lowest in Potohar without Zn during the first year

of experimentation. During the second year, the harvest index was the highest in Blue Silver and

lowest in Inqlab-91 without Zn (Table 4.18).

115

Zinc fertilization improved the grain yield of wheat as maximum grain yield (5.16 and 4.16

Mg ha-1) was recorded in Chakwal-50 with Zn, and the lowest (2.01 and 2.42 Mg ha-1) was

recorded in Sandal-73 without Zn during both years of experimentation (Table 4.19).

4.17: Mean comparison of productive tillers and grains per spike of wheat genotypes

under no and adequate Zn supply

Genotype

1

Productive tillers (m2) Grains per spike

2013-14 2014-15 2013-14 2014-15

-Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn

347±12 372±12 327±4.7 340±5 42.7±0.4 44.3±0.7 39.3±0.8 41.0±0.7

2 327±5 370±7 229±7.1 299±8 48.2±0.7 48.7±0.7 40.9±0.8 40.6±0.5

3 326±7 336±4 247±8.5 291±6 46.9±0.3 49.8±1.3 41.3±0.4 43.6±0.5

4 342±10 312±3 332±10 343±3 45.9±0.6 47.0±2.5 33.9±0.4 34.9±0.8

5 362±6 401±7 366±13 386±5 42.3±1.1 42.4±0.7 36.9±0.2 36.2±0.7

6 359±4 364±5 337±5.4 347±7 42.0±1.0 43.8±2.0 41.2±1.1 41.3±0.6

7 338±5 374±6 332±19 369±6 42.7±0.4 44.5±1.3 36.5±1.0 36.4±1.2

8 280±5 299±6 215±8.2 245±6 43.6±0.7 45.7±2.1 45.6±0.3 47.6±0.7

9 329±6 363±4 319±8.5 358±16 42.4±0.6 44.0±2.2 41.2±0.2 43.2±0.4

10 291±5 363±6 279±46 355±9 41.6±1.1 44.7±1.4 36.7±0.6 36.3±0.3

11 307±10 320±9 268±9.9 301±8 39.8±0.4 41.2±0.7 41.0±0.4 44.0±0.4

12 324±11 382±6 249±13 375±6 45.4±0.7 48.0±1.0 44.9±0.4 43.3±0.7

13 358±10 395±6 357±15 384±7 39.6±0.6 41.7±0.4 44.4±0.6 46.7±0.3

14 346±8 414±4 349±3.3 401±8 47.2±0.6 47.7±1.3 43.0±0.5 45.0±0.2

15 354±16 454±7 377±6 446±12 42.4±1.4 44.7±2.5 41.1±0.6 44.1±0.6

16 344±5 417±4 343±14 409±21 46.8±1.8 48.7±1.5 46.1±0.5 47.1±0.5

17 390±17 426±8 382±5.7 431±9 45.0±0.5 46.5±0.5 47.8±0.8 47.5±0.4

18 375±11 430±3 350±11 415±5 46.2±1.3 48.5±1.0 41.8±0.8 41.4±1.2

19 341±3 355±12 271±8.5 317±9 36.0±0.8 38.5±0.8 45.3±0.5 44.7±0.7

20 327±4 347±6 222±5 334±4 42.4±0.9 46.3±1.4 44.3±0.7 43.2±0.6

21 372±20 375±8 309±11 342±11 42.1±0.7 45.7±3.7 46.5±0.2 45.8±0.6

22 343±20 370±7 320±18 356±6 42.1±0.5 46.3±5.3 42.0±0.4 41.4±0.6

23 333±11 434±4 378±4.4 428±6 48.0±1.6 50.8±2.1 44.8±0.6 44.3±0.4

Table

116

24 348±5 410±18 384±5.3 400±17 45.8±0.4 53.2±1.4 47.2±1.0 48.8±1.0

25 389±5 416±9 370±5.4 377±7 44.4±0.3 45.7±1.2 47.9±0.3 49.9±0.3

26 345±7 407±5 383±5.7 405±5 45.1±0.7 45.7±1.2 42.1±0.3 43.7±0.3

27 366±32 394±10 341±19 379±8 39.4±1.2 39.8±1.3 40.6±0.8 42.8±0.2

28 323±16 331±4 309±5.3 321±5 48.6±0.7 49.7±0.9 47.1±0.3 49.4±0.3

Max 390 454 384 446 48.6 53.2 47.9 49.9

Min 280 299 215 245 36.0 38.5 33.9 34.9

Mean 342 380 320 363 43.7 45.8 42.5 43.4

-Zn= No Zn application; +Zn= soil application of Zn 10 kg ha-1; ± S.E.

Table

117

4.18: Mean comparison of 1000 grain weight and harvest index of wheat genotypes


Genotype

1

1000 grain weight (g) Harvest index (%)

2013-14 2013-14 2014-15


38.6±0.7 40.2±0.5 40.6±1.2 40.1±0.1 34.3±2.5 34.4±1.7 44.7±1.5 48.7±0.8

2 39.4±1.0 39.7±0.7 40.2±0.5 41.9±0.4 33.2±0.2 35.1±2.4 41.9±3.7 42.4±2.8

3 38.6±0.7 42.2±0.5 42.0±1.6 42.9±0.4 34.1±2 38.5±3.6 33.9±0.6 40.3±1.9

4 36.2±0.4 36.8±0.2 34.8±0.5 37.1±0.5 22.6±2.3 23.4±1.3 28.6±2.0 29.5±2.4

5 38.8±0.4 38.9±0.2 38.8±0.5 40.7±0.3 28.8±2.4 30.4±1.2 35.8±1.3 39.5±0.8

6 40.3±0.5 40.5±0.6 44.3±0.7 42.0±0.9 30.9±1 31.1±0.6 33.8±2.2 47.1±2.2

7 41.9±0.6 42.1±0.5 41.1±0.4 46.8±1.1 27.7±0.5 29.0±1.9 41.1±3.8 45.1±1.8

8 39.1±1.0 39.8±0.4 42.0±1.7 41.1±0.9 30.9±2.4 32.1±2.6 37.8±1.2 40.9±1.4

9 45.7±0.4 45.7±0.6 46.1±0.5 46.3±0.6 28.8±0.9 30.0±1.0 39.7±2.5 38.9±1.4

10 37.5±0.3 39.6±0.4 39.4±0.6 41.1±0.6 29.5±0.3 30.1±2.0 38.2±1.6 47.5±2.8

11 41.3±0.4 42.1±0.6 42.9±0.5 42.4±0.7 31.5±2.6 30.0±1.7 32.7±0.5 32.4±0.7

12 35.3±0.6 37.2±0.4 40.8±0.3 44.8±1.0 30.7±1.2 37.0±2.0 42.1±2.0 39.8±1.1

13 38.2±0.1 43.1±0.1 41.2±0.5 43.4±0.4 37.1±2.4 36.4±1.8 30.6±0.4 34.7±2.8

14 37.6±0.7 38.4±0.2 41.1±0.3 39.6±0.4 32.1±4 40.7±1.4 40.9±3.5 33.8±2.1

15 36.6±0.4 38.0±0.4 38.9±0.6 39.2±0.2 38.5±2.1 39.4±1.1 34.6±1.2 30.8±3.0

16 37.0±0.3 41.9±1.3 41.7±1.1 42.8±1.1 40.5±0.5 42.0±1.2 32.5±1.1 27.5±0.5

17 43.3±0.7 42.7±0.3 43.8±0.4 44.6±0.4 36.5±1.4 48.2±2.4 37.4±0.3 37.4±2.4

18 37.1±0.3 38.8±0.9 40.4±0.5 42.2±1.5 26.6±0.3 35.9±2.1 43.3±1.3 36.8±1.2

19 40.8±0.5 39.4±0.7 41.3±0.4 43.1±0.4 35.5±0.6 44.2±2.9 35.8±0.6 43.1±1.3

20 39.4±0.5 40.1±0.6 42.8±0.4 44.2±0.5 42.4±1.4 42.8±2.2 32.8±1.6 43.4±1.4

21 36.5±0.3 37.7±0.8 38.9±0.2 41.3±0.3 33.6±1.2 33.7±1.2 36.2±1.1 40.7±0.8

22 37.3±0.5 38.8±0.6 40.7±0.5 42.4±0.3 42.0±1.9 44.1±1.6 32.3±0.7 50.2±1.7

23 37.2±0.3 38.0±0.7 41.8±0.9 43.4±0.8 28.0±1.7 35.9±4.0 26.5±0.2 31.4±1.1

24 42.8±0.2 43.1±0.3 41.5±1.0 43.1±0.6 34.2±1.3 36.9±2.2 27.5±0.4 38.2±1.4

25 39.5±0.8 40.2±0.7 41.3±0.6 41.1±0.7 35.2±3.6 36.6±2.5 27.3±0.7 23.5±1.4

26 41.0±0.2 41.4±0.7 41.5±0.8 43.5±0.3 44.7±4.2 42.0±3.2 29.7±2.7 50.5±1.9

27 40.9±0.5 42.2±0.6 43.2±0.0 43.0±0.2 37.5±1.5 60.5±5.7 30.4±1.6 33.5±1.1

Table

118

28 42.1±0.6 42.2±0.6 44.1±0.5 46.4±0.3 39.7±3.5 49.6±3.2 33.3±2.2 39.8±1.1

Max 45.7 45.7 46.1 46.8 44.7 60.5 44.7 50.5

Min 35.3 36.8 34.8 37.1 22.6 23.4 26.5 23.5

Mean 39.3 40.4 41.3 42.5 33.8 37.5 35.1 38.8

-Zn= No Zn application; +Zn= soil application of Zn 10kg ha-1;± S.E

4.19: Mean comparison of grain yield and grain protein concentration in wheat


Genotypes

Grain yield (Mg ha-1) Grain protein (%)

2014-15 2014-15 2013-15 2014-15


1 3.52±0.04 3.84±0.08 3.14±0.15 3.41±0.06 13.3±0.5 14.0±0.1 14.0±0.2 15.6±0.9

2 3.56±0.06 3.95±0.04 3.06±0.05 3.52±0.10 13.0±0.1 13.9±0.1 13.1±0.2 14.4±0.1

3 3.37±0.07 3.64±0.11 3.16±0.06 3.64±0.11 13.4±0.2 13.8±0.2 13.7±0.1 14.0±0.3

4 2.36±0.18 2.60±0.05 2.48±0.10 2.64±0.07 13.1±0.3 13.9±0.3 13.3±0.3 14.5±0.5

5 3.60±0.1 3.79±0.11 2.87±0.07 3.22±0.06 13.2±0.2 13.5±0.1 13.2±0.2 13.4±0.1

6 3.54±0.04 3.69±0.06 2.64±0.06 3.58±0.03 13.4±0.3 13.9±0.3 13.4±0.3 13.8±0.2

7 3.39±0.15 3.45±0.1 2.75±0.15 3.45±0.10 12.3±0.2 15.2±0.0 12.3±0.2 15.8±0.3

8 3.29±0.07 3.30±0.03 3.24±0.07 3.27±0.07 12.7±0.1 13.8±0.5 12.7±0.1 13.8±0.5

9 3.45±0.16 3.63±0.16 2.93±0.10 3.13±0.19 12.7±0.3 15.0±0.3 12.7±0.3 15.0±0.3

10 3.45±0.13 3.50±0.04 2.54±0.18 3.14±0.09 12.4±0.0 14.2±0.2 12.4±0.0 14.2±0.2

11 3.40±0.07 3.60±0.09 2.91±0.09 3.04±0.05 12.7±0.2 13.7±0.2 12.7±0.2 13.7±0.2

12 3.56±0.25 3.79±0.05 3.00±0.07 3.69±0.02 11.8±0.0 14.3±0.2 11.8±0.0 14.3±0.2

13 3.87±0.11 3.97±0.05 3.26±0.09 3.87±0.07 13.0±0.2 14.4±0.6 13.0±0.2 14.4±0.6

14 3.20±0.2 3.99±0.11 2.89±0.09 3.89±0.10 12.1±0.3 14.0±0.2 12.1±0.3 14.0±0.2

15 3.83±0.09 4.33±0.05 3.25±0.05 4.14±0.06 11.9±0.2 13.8±0.2 11.9±0.2 13.8±0.2

16 3.92±0.02 4.08±0.0 3.10±0.11 3.68±0.06 12.1±0.2 13.9±0.2 12.1±0.2 13.9±0.2

17 4.81±0.1 5.09±0.04 3.65±0.03 4.04±0.09 13.5±0.4 13.6±0.1 13.5±0.4 13.6±0.1

18 2.99±0.05 4.06±0.07 3.35±0.05 3.89±0.07 12.6±0.3 13.7±0.4 12.6±0.3 13.7±0.4

19 3.51±0.1 4.05±0.06 3.53±0.06 3.78±0.04 14.0±0.2 14.0±0.1 14.0±0.2 14.0±0.1

20 4.00±0.11 4.31±0.08 3.13±0.04 4.08±0.10 13.7±0.3 14.3±0.3 13.7±0.3 14.3±0.3

21 3.90±0.08 4.27±0.11 3.61±0.11 4.14±0.03 12.5±0.2 13.6±0.2 12.5±0.2 13.6±0.2

22 4.05±0.03 5.16±0.07 3.69±0.03 4.16±0.07 12.2±0.1 14.1±0.2 12.2±0.1 14.1±0.2

23 2.01±0.06 3.03±0.06 2.42±0.06 2.86±0.08 12.7±0.0 13.0±0.2 12.7±0.0 12.7±0.4

24 3.47±0.01 3.75±0.08 2.52±0.05 3.51±0.11 13.1±0.3 13.2±0.3 13.7±0.5 12.9±0.4

25 2.94±0.11 3.42±0.05 3.25±0.11 3.25±0.14 13.8±0.3 14.3±0.3 14.3±0.3 13.9±0.2

26 2.88±0.06 3.57±0.07 2.80±0.15 3.57±0.07 12.2±0.6 14.6±0.3 12.2±0.6 13.0±0.3

119

27 2.61±0.03 4.23±0.04 2.92±0.18 3.76±0.08 14.4±1.1 12.8±0.2 14.4±1.1 12.8±0.2

28 3.67±0.05 3.88±0.02 3.21±0.04 3.44±0.02 12.0±0.7 12.6±0.4 12.0±0.7 12.0±0.2

Max 4.81 5.16 3.69 4.16 14.4 15.2 14.4 15.8

Min 2.01 2.60 2.42 2.64 11.8 12.6 11.8 12.0

Mean 3.43 3.86 3.05 3.56 12.9 13.9 12.9 13.9

Gen= Genotype; -Zn= No Zn application; +Zn= soil application of Zn 10 kg ha-1; ± S.E.

4.3.1.2 Grain mineral concentration

The highest grain protein was recorded in Ufaq-2002 (15.2 and 15.8%) with Zn, and the lowest

in GA-02 (11.8%) without Zn during both years (Table 4.19). The maximum grain Zn

concentration of 47.4 and 54.4 mg kg-1, during the first and second year respectively, was recorded

in Blue Silver with Zn. However, the grain Zn concentration was the lowest in AAS-2011 (21.2

mg kg-1), and Inqlaab-91 (25.1 mg kg-1) during the first and second year, respectively without Zn.

The lowest Fe concentration was recorded in Fsd-83 (29.9 mg kg-1) with Zn and the highest in

Millat-2011 (45 mg kg-1) without Zn during the first year. During the second year, Fe concentration

was the highest in Sehar-2006 (44.5 mg kg-1) and lowest in Sandal-73 (27 mg kg-1) without Zn.

The highest Ca concentration was recorded in Millat-2011 (481 and 511 mg kg-1) with Zn; while

that was the lowest with Sndal-73 (325 and 296 mg kg-1) during both years (Table 4.20).

The highest phytate concentration of 10.28 and 10.3 was recorded in Inqlab-91 without Zn,

during first and second year, respectively. The lowest phytate concentration of 6.92 and 6.72 mg

g-1 during first and second year, respectively was recorded in Kohinoor-83 with Zn. The highest

[phytate]:[Zn] molar ratio (44.1 and 40.6) during both years was recorded in Inqlab-91 without Zn;

and it was the lowest in Blue Silver (17 and 14.9) with Zn during both years. The highest

bioavailable Zn (3.08 and 3.28 mg day-1) during both years was recorded in Blue silver with Zn;

and it was lowest in Inqlab-91 (1.74 and 1.85 mg day-1) without Zn during both years (Table 4.21).

The highest embryo Zn was noted in Blue silver (156.7 mg kg-1) with Zn and it was the

lowest in Faisalabad-2008 (89.3 mg kg-1) without Zn. The highest embryo Fe was recorded in

Sehar-2006 (120.9 mg kg-1) and lowest in Chakwal-50 (82 mg kg-1) without Zn (Table 4.22). The

highest Ca concentration was noted in SH-02 (1264 mg kg-1) and the lowest in GA-02 (711 mg kg-

1) without Zn (Table 4.22). The highest aleurone Zn was noted in Blue Silver (94.7 mg kg-1) with

Zn and lowest in Shafaq-2006 (34 mg kg-1) without Zn. Aleurone Fe was the maximum (83.4) in

Table

120

Faisalabad-2008 and the minimum in Shafaq-2006 (32.2 mg kg-1) without Zn (Table 4.22). The

maximum aleurone Ca was noted in Fareed-2006 (1205 mg kg-1) with Zn and the minimum in

Miraj-2008 (306 mg kg-1) without Zn (Table 4.22).

The highest endosperm Zn of 20.0 mg kg-1 was recorded in Blue silver with Zn and that

was minimum (12.4 mg kg-1) in Ufaq-2002 with Zn (Table 4.23). The highest endosperm Fe was

recorded in Sehar-2006 (20.6 mg kg-1) and that was lowest in Chakwal-50 (9.95 mg kg-1) without

121

Zn. The maximum endosperm Ca (314 mg kg-1) was recorded in Iqbaal-2000 and the lowest in

GA-02 (189 mg kg-1) without Zn (Table 4.23).

4.3.1.3 AFLP primer combinations

The four primer combinations produced 507 alleles, with all being polymorphic. The primer

pairs E-AGT+M-CTG and E-AGA+M-CTC produced the highest number of polymorphic loci,

174 and 158, respectively. The level of gene diversity based on Nei (1973) gene diversity was

very low for all primer combinations (Table 4.24).

4.3.1.4 Genotypic and genetic diversity within wheat genotypes

The analysis of 87 wheat samples representing 28 wheat genotypes showed that the genotypes

had low genetic diversity based on Nei gene diversity estimates (H = 0.0194-0.0842) (Table 7).

The overall level of genetic diversity across all genotypes was also low (0.0558). The percent

polymorphic loci for each genotype ranged from 5 to 26% (Table 4.25). The samples produced 87

different AFLP genotypes (Fig. 4.1).

4.3.1.5 Genetic similarity and Analysis of molecular variance

The level of genetic similarity was high among the 28 wheat genotypes (98.51-99.98; avg.

99.43%) (Fig. 4.1). The highest level of genetic similarity was found between ‘Fareed-2006 and

GA-02’ genotype (99.98%), while the lowest level of genetic similarity was found between

‘Lasani-2008 and Mairaj-2008’ genotypes.

UPGMA cluster analysis of the 28 cultivars showed that 20 genotypes clustered together.

Genotypes AAS-2011, Sandal-73, Shafaaq-2006, Kohinoor-83 and Punjab-2011 formed one

cluster. However, genotypes Millat-2011, Lasani-2008 and AARI-2011 were the least similar to

the others (Fig. 4.1). No relationship was found between clustering of the genotypes and their place

of origin. Analysis of molecular variance (AMOVA) indicated the presence of very low level of

genetic variation among wheat genotypes (2.65%) (Table 4.26). Most of the genetic variation was

found to be within wheat genotypes. Pairwise analysis of genetic differentiation showed that low

levels of genetic differentiation exist among all genotypes, except between ‘Lasani-2008 and

Mairaj-2008’ (FST = 0.15094), ‘Lasani-2008 and Sehar-2006 (FST = 0.15714), and ‘Chakwall-50

and Ufaq-2002’ (FST = 0.15328).

122

Table 4.20: Mean comparison of grain Zn, Fe and Ca concentration of wheat genotypes under no and adequate Zn supply

Genotype

Grain Zn concentration (m

2013-14

g kg-1) 2014-15

Grain Fe concentration (mg kg-1) Grain Ca concentration (mg kg-

1)

2013-14 2014-15 2013-14 2014-15

-Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn 1 27.8±1.5 44.5±0.9 31.4±0.5 49.6±1.2 45.0±0.5 43.9±1.9 43.6±2.7 41.6±2.2 391±10 481±6 381±2 511±12 2 23.6±0.5 40.9±0.8 28.3±1.7 46.4±1.7 42.5±0.3 39.3±0.5 41.9±0.7 41.0±1.4 393±6 471±8 410±10 485±14 3 26.1±0.6 36.0±1.2 31.4±0.3 39.6±1.0 42.1±0.6 40.5±1.2 42.4±1.8 41.8±1.1 396±8 434±5 412±11 437±21 4 32.6±1.7 39.4±2.1 35.3±1.1 42.7±0.9 38.7±1.0 32.9±1.1 37.1±1.0 32.5±1.3 392±6 420±3 414±8 416±11 5 27.2±0.1 30.9±0.5 30.4±0.9 34.8±0.9 34.3±0.3 31.8±1.3 32.7±0.6 31.1±1.4 356±2 430±6 365±7 440±12 6 28.1±1.3 37.2±0.3 31.7±1.3 41.1±1.0 38.5±0.6 38.1±0.8 38.6±0.5 37.1±0.4 365±3 461±5 375±4 457±2 7 27.2±0.7 37.5±1.2 32.3±1.6 41.9±1.9 41.5±0.6 40.9±1.5 40.2±1.1 36.9±2.3 390±6 415±5 399±12 418±8 8 26.0±0.4 33.6±1.4 31.9±0.7 34.1±0.2 42.5±0.6 41.8±0.9 41.5±1.2 39.4±1.7 436±3 462±8 455±4 460±4 9 25.3±1.3 33.9±1.2 30.3±1.2 39.5±1.6 38.3±0.7 32.7±1.7 39.9±1.3 33.0±1.9 368±9 480±10 351±11 483±18 10 27.3±1.4 37.1±1.9 28.4±2.8 41.0±3.9 32.7±0.4 34.5±2.7 33.7±1.9 36.2±3.4 401±12 426±7 411±9 413±10 11 29.6±0.5 41.2±0.2 34.2±3.1 44.1±0.9 43.9±0.5 39.4±1.2 44.5±2.6 37.7±1.8 368±7 444±8 354±5 438±9 12 31.7±1.6 47.0±1.5 34.0±1.0 53.9±1.5 34.7±0.6 35.1±0.7 35.4±0.8 35.8±1.4 336±9 358±5 329±4 350±5 13 30.2±2.9 36.0±0.4 36.4±1.9 43.0±1.9 37.0±0.1 38.9±1.0 41.3±1.3 42.2±0.8 335±6 391±6 336±7 399±5 14 33.2±0.5 41.6±1.4 34.5±1.1 50.2±1.4 31.6±0.6 29.9±1.2 33.3±1.3 30.9±1.2 365±4 435±4 369±6 445±4 15 28.2±0.5 36.4±0.6 30.6±1.7 37.3±1.6 34.5±0.8 31.5±1.4 35.5±0.7 34.9±0.9 346±6 439±7 336±5 453±15 16 25.2±0.7 34.2±1.5 30.3±1.3 37.2±1.1 36.3±0.6 35.1±0.4 38.3±0.6 35.8±0.3 348±5 466±5 351±4 479±28 17 25.6±0.0 30.3±2.0 30.6±1.8 35.8±2.0 37.3±0.3 34.7±0.4 35.7±0.8 33.7±0.8 330±5 356±13 324±7 383±13 18 25.9±0.6 29.8±0.7 28.0±1.2 32.9±0.9 33.3±0.6 32.8±1.3 35.3±1.2 33.8±0.3 379±6 373±5 379±5 353±5 19 29.0±1.2 37.7±1.4 29.1±1.8 37.2±0.8 35.2±1 34.6±1.4 35.5±0.8 36.2±0.7 374±5 423±8 376±4 423±5 20 27.9±0.8 36.1±2.1 31.7±1.0 37.7±1.6 42.2±1.4 43.9±1.9 41.2±1.5 42.2±0.7 353±9 387±3 330±8 381±1 21 28.6±0.5 33.3±0.6 32.1±1.8 36.5±1.0 33.0±0.6 33.3±0.8 30.4±1.6 32.0±0.9 341±3 386±7 334±3 370±5 22 22.9±0.2 29.0±1.9 26.6±0.7 34.5±1.6 32.7±0.3 33.5±0.8 33.9±1.2 30.5±0.9 340±4 415±10 313±6 422±10 23 28.2±1.3 34.3±2.3 29.7±0.6 36.6±0.3 30.4±0.5 37.2±0.2 27.0±1.7 38.2±0.3 325±8 361±3 295±8 357±9 24 31.2±1.6 38.2±1.4 27.5±0.8 35.6±0.5 36.1±2.2 37.1±0.2 35.4±2 40.8±3.9 360±10 394±8 368±11 412±9 25 23.2±1.2 33.5±4.2 25.1±0.4 39.9±0.8 36.6±0.1 35.8±1.1 36.0±0.6 35.8±1.1 389±4 413±9 399±9 416±11 26 33.3±0.6 47.4±1.2 30.4±1.7 54.4±2.1 35.7±0.7 32.0±1.1 33.0±0.7 36.0±2.1 350±4 427±3 354±2 443±6 27 24.9±0.9 32.6±1.1 32.5±1.0 39.1±1.0 34.9±1.4 35.5±0.9 34.9±1.8 35.5±0.6 421±5 427±5 441±10 444±6 28 21.2±0.4 33.5±2.2 26.2±0.3 36.6±1.0 31.7±0.6 33.8±0.2 31.4±0.8 34.5±0.6 368±4 396±6 372±6 405±7 Max 33.3 47.4 36.4 54.4 45.0 43.9 44.5 42.2 436 481 455 511 Min 21.2 29.0 25.1 32.9 30.4 29.9 27.0 30.5 325 356 295 350 Mean 27.5 36.5 30.7 40.5 36.9 36.1 36.8 36.3 368 420 369 425


123

Table 4.21: Mean comparison of grain phytate concentration, [phytate]:[Zn] ratio and bioavailable Zn of wheat genotypes


Genotype

Phytate (mg g-1 ) Phytate/Zn ration Bioavailable Zn (mg day-1)

2013-14 2014-15 2013-14 2014-15 2013-14 2014-15

-Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn 1 9.28±0.03 8.61±0.09 9.1±0.03 8.58±0.13 33.3±1.8 19.2±0.2 28.7±0.4 17.2±0.7 2.11±0.07 2.90±0.02 2.31±0.02 3.08±0.06 2 9.51±0.03 8.91±0.07 9.2±0.05 8.64±0.12 40.0±0.9 21.6±0.2 32.5±1.8 18.5±0.8 1.86±0.03 2.73±0.02 2.14±0.08 2.96±0.07 3 9.10±0.04 8.46±0.17 9.0±0.05 8.33±0.14 34.6±0.8 23.3±1.2 28.5±0.4 20.9±0.6 2.04±0.03 2.61±0.08 2.32±0.02 2.77±0.04 4 8.78±0.05 8.31±0.04 8.7±0.05 8.27±0.06 26.9±1.5 21.0±1.1 24.5±0.8 19.2±0.4 2.40±0.08 2.77±0.09 2.54±0.05 2.90±0.03 5 8.71±0.04 8.28±0.02 8.6±0.04 8.28±0.02 31.7±0.2 26.6±0.5 28.1±0.7 23.6±0.6 2.16±0.01 2.40±0.02 2.33±0.04 2.58±0.04 6 9.30±0.04 9.11±0.04 9.3±0.10 9.01±0.04 32.9±1.6 24.2±0.2 29.0±1.4 21.7±0.4 2.12±0.07 2.56±0.01 2.30±0.07 2.72±0.03 7 8.98±0.05 8.55±0.13 8.9±0.07 8.45±0.13 32.7±0.8 22.6±0.9 27.4±1.3 20.0±0.7 2.12±0.03 2.65±0.06 2.38±0.07 2.84±0.06 8 8.15±0.05 7.72±0.14 8.1±0.05 7.62±0.14 31.0±0.5 22.9±1.4 25.2±0.7 22.1±0.3 2.18±0.03 2.62±0.09 2.48±0.04 2.66±0.02 9 9.61±0.03 9.18±0.06 9.5±0.06 9.01±0.09 37.8±2.0 26.9±0.9 31.3±1.4 22.7±1.1 1.94±0.07 2.41±0.05 2.19±0.06 2.66±0.07 10 7.15±0.09 6.92±0.06 7.1±0.06 6.72±0.06 26.1±1.2 18.6±0.9 25.3±2.5 16.6±1.8 2.40±0.07 2.92±0.07 2.46±0.15 3.10±0.16 11 9.49±0.08 8.39±0.28 9.5±0.05 8.26±0.10 31.7±0.8 20.2±0.7 28.0±2.7 18.6±0.2 2.17±0.03 2.82±0.04 2.37±0.14 2.95±0.02 12 10.28±0.09 9.62±0.15 10.2±0.04 9.42±0.12 32.2±1.5 20.3±0.9 29.8±1.0 17.3±0.3 2.17±0.07 2.84±0.07 2.28±0.05 3.08±0.02 13 9.09±0.04 8.19±0.12 9.1±0.08 8.13±0.11 30.4±3.1 22.5±0.3 24.9±1.2 18.8±0.7 2.24±0.14 2.65±0.02 2.52±0.08 2.93±0.05 14 9.30±0.05 9.08±0.03 9.3±0.06 8.91±0.06 27.8±0.5 21.7±0.8 26.8±0.8 17.6±0.6 2.36±0.03 2.73±0.06 2.41±0.04 3.04±0.05 15 9.32±0.07 8.96±0.08 9.2±0.11 8.83±0.07 32.7±0.4 24.4±0.3 30.0±1.3 23.5±1.2 2.12±0.02 2.54±0.02 2.25±0.07 2.60±0.07 16 9.19±0.03 8.72±0.06 9.3±0.07 8.55±0.04 36.2±0.9 25.4±1.3 30.4±1.2 22.8±0.5 1.98±0.03 2.49±0.07 2.23±0.06 2.64±0.04 17 9.88±0.04 9.17±0.07 9.8±0.04 9.01±0.08 38.2±0.1 30.3±2.1 31.9±2.0 25.1±1.5 1.92±0.00 2.24±0.10 2.18±0.09 2.51±0.09 18 8.15±0.05 7.79±0.03 8.1±0.08 7.55±0.05 31.2±0.6 25.9±0.7 29.0±1.5 22.8±0.7 2.17±0.03 2.43±0.04 2.28±0.08 2.62±0.05 19 8.70±0.06 8.26±0.06 8.5±0.11 8.11±0.05 29.8±1.3 21.8±0.8 29.1±2.2 21.6±0.6 2.25±0.06 2.71±0.06 2.28±0.10 2.71±0.04 20 8.96±0.05 8.33±0.03 8.9±0.05 8.16±0.06 31.9±0.8 23.0±1.3 28.0±1.0 21.5±0.9 2.15±0.04 2.63±0.09 2.34±0.05 2.72±0.06 21 8.71±0.06 8.2±0.05 8.7±0.07 8.06±0.03 30.1±0.6 24.4±0.6 27.1±1.8 21.9±0.7 2.23±0.03 2.53±0.04 2.39±0.09 2.69±0.05 22 8.25±0.05 7.69±0.03 8.2±0.03 7.45±0.05 35.7±0.5 26.5±1.7 30.7±0.8 21.5±1.1 1.98±0.02 2.40±0.10 2.19±0.03 2.71±0.08 23 8.73±0.04 8.16±0.05 8.7±0.10 8.09±0.03 30.8±1.6 23.8±1.6 29.0±0.9 21.9±0.2 2.20±0.07 2.58±0.10 2.29±0.04 2.69±0.01 24 8.77±0.03 8.24±0.08 8.9±0.04 8.1±0.05 28.0±1.5 21.4±1.0 32.0±1.0 22.6±0.3 2.34±0.08 2.73±0.07 2.15±0.04 2.65±0.02 25 10.27±0.04 9.57±0.1 10.3±0.08 9.31±0.08 44.1±2.2 29.1±3.3 40.6±0.8 23.1±0.6 1.74±0.06 2.32±0.17 1.85±0.03 2.63±0.04 26 8.58±0.05 8.15±0.06 8.6±0.09 8.18±0.10 25.5±0.4 17.0±0.4 28.1±1.9 14.9±0.5 2.47±0.02 3.08±0.03 2.34±0.10 3.28±0.05 27 9.39±0.04 8.82±0.04 9.4±0.05 8.69±0.05 37.4±1.4 26.9±0.9 28.7±1.0 22.1±0.6 1.94±0.05 2.40±0.05 2.32±0.05 2.69±0.04 28 9.19±0.03 8.62±0.05 9.2±0.08 8.54±0.03 42.9±0.6 25.7±1.8 34.7±0.6 23.1±0.7 1.76±0.02 2.47±0.10 2.04±0.02 2.62±0.04 Max. 10.28 9.62 10.3 9.42 44.1 30.3 40.6 25.1 2.47 3.08 2.54 3.28 Min. 7.15 6.92 7.12 6.72 25.5 17.0 24.5 14.9 1.74 2.24 1.85 2.51

124

Mean 9.03 8.50 8.98 8.37 33.0 23.5 29.3 20.8 2.13 2.61 2.29 2.79 -Zn= No Zn application; +Zn= soil application of Zn 10 kg ha-1 ; ± S.E.

Table 4.22: Mean comparison of Zn, Fe and Ca concentration (mg kg -1) of embryo and aleurone of wheat genotypes under no

and adequate Zn supply

Genotype Embryo Zn

2014-15 Embryo Fe

2014-15 Embryo Ca Aleurone Zn Aleurone Fe Aleurone Ca

2013-14 2014-15 2013-14 2014-15 -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn -Zn +Zn

1 108.8±4.9 147.6±4.6 106.5±3.5 105.1±2.71 880±51 1035±19 64.3±2.0 93.2±1.8 80.3±4.9 81.6±3.6 641±23 846.4±19 2 89.3±1.9 137.3±2.0 111.8±3.9 106.0±1.42 1066±35 1017±4 50.0±1.0 93.1±2.2 83.4±2.9 81.6±3.4 652±17 1140±11 3 111.2±1.8 128.7±1.6 114.4±3.0 115.9±1.06 1139±39 1058±35 34.0±1.3 90.6±1.2 32.2±3.6 44.7±1.9 432±43 1045±30 4 104.2±2.3 122.6±2.6 96.3±1.5 105.4±1.56 829±24 853±9 56.9±0.7 67.5±2.0 62.2±3.0 59.9±3.1 693±13 783.7±17 5 106.3±4.1 117.0±2.3 96.1±2.3 84.4±1.81 973±6 879±17 48.2±0.5 62.7±1.8 60.4±2.5 57.3±2.2 560±7.2 713.6±22 6 103.6±2.8 125.9±1.9 111.8±2.6 105.8±0.61 1264±21 865±18 48.7±1.0 60.2±1.5 61.5±1.9 56.1±2.9 769±27 622±7 7 114.1±2.4 125.7±1.7 102.8±0.6 112.5±1.29 907±16 1048±19 50.4±0.9 57.8±3.6 61.7±0.7 57.8±1.7 600±19 698.8±22 8 98.4±9.5 107.9±0.3 112.4±1.4 104.0±1.44 1110±46 1045±29 45.6±0.7 59.3±2.3 66.1±3.2 63.0±1.4 732±19 842.7±22 9 106.5±1.3 125.1±1.1 102.3±3.2 103.2±0.59 1076±12 774±14 51.3±1.0 48.0±3.1 56.4±2.2 54.7±1.9 582±32 627.8±23 10 105.3±3.8 133.6±2.1 94.4±3.2 106.5±2.72 913±21 971±15 41.5±0.8 86.0±4.2 45.1±1.7 68.0±3.2 648±43 846.2±37 11 114.0±2.9 130.3±1.6 120.9±3.4 110.1±3.00 876±16 1039±19 61.0±1.7 83.4±4.7 71.8±2.1 68.0±1.7 712±35 934±29 12 117.0±2.4 151.3±3.0 111.0±2.6 101.3±3.12 711±35 836±3 51.6±1.0 84.7±0.9 58.8±2.4 69.1±2.7 685±30 914.3±12 13 113.1±2.3 126.0±2.9 102.7±2.4 105.9±5.24 983±33 772±8 66.0±1.4 88.9±4.5 76.4±1.6 79.2±1.2 616±13 1028±45 14 105.1±1.9 153.6±2.2 101.4±0.9 103.6±6.11 1245±29 934±24 63.1±1.5 93.6±2.4 67.2±2.1 60.5±1.7 772±25 1010±14 15 108.0±4.3 116.3±1.0 96.4±2.3 91.5±0.25 746±8 1086±24 48.0±1.4 70.2±4.9 65.1±2.5 62.1±1.0 534±41 946.4±31 16 105.0±4.8 119.7±1.4 118.0±1.8 111.8±1.45 1250±33 748±23 47.0±0.8 80.4±1.4 72.3±2.9 67.6±4.3 555±33 1205±18 17 109.5±3.4 120.4±2.3 105.9±3.7 102.0±2.57 933±15 747±11 36.6±1.5 66.8±1.7 53.1±1.3 59.3±2.4 306±9.7 738.7±24 18 101.4±1.3 122.7±3.0 110.9±4.1 101.3±2.94 1041±35 884±8 57.5±4.7 82.2±3.5 66.0±3.5 69.7±2.6 684±41 920±37 19 108.9±4.4 128.6±2.6 114.5±2.4 109.2±3.11 942±24 1112±8 39.0±0.5 81.0±1.9 58.0±3.2 72.1±2.3 476±2.1 925±19 20 111.1±2.5 137.9±2.8 101.9±1.0 119.1±1.32 904±19 1151±14 51.7±1.0 62.4±1.9 51.2±0.5 55.3±2.3 516±3.9 689.3±16 21 108.8±2.0 120.9±1.5 95.5±0.9 97.0±0.99 904±11 1001±12 53.2±4.5 71.8±2.6 48.4±2.4 56.2±2.2 535±30 829.3±23 22 105.5±2.6 120.6±0.5 82.0±1.1 109.2±1.08 777±17 1053±15 59.9±1.6 73.1±2.9 64.0±1.6 63.3±1.4 647±9.4 912.1±35 23 109.8±4.1 120.2±3.5 94.8±3.6 104.2±4.55 719±6 1057±55 63.2±1.6 70.6±3.6 66.2±2.8 65.6±2.5 581±12 889.2±39 24 101.3±1.7 120.8±5.8 96.0±1.0 104.2±3.36 865±28 1034±32 53.9±0.7 60.4±1.4 62.0±5.1 65.0±2.3 687±14 715.8±64 25 102.0±5.3 119.0±3.0 99.6±3.7 97.2±3.76 876±47 1026±24 38.3±0.8 72.9±4.9 57.3±5.0 67.4±2.6 515±16 868.1±43 26 116.6±2.9 156.7±2.0 106.7±1.4 112.4±2.08 963±14 1150±17 63.6±0.9 94.7±2.5 70.6±0.9 75.7±2.2 740±6.4 1106±43 27 110.2±7.6 122.4±1.8 102.9±2.8 105.0±3.92 1045±33 1008±2 44.2±1.1 79.4±1.2 59.8±2.8 59.0±3.0 770±16 793.7±29

125

28 90.1±2.0 116.3±3.5 83.2±2.7 99.5±3.02 839±19 919±14 40.1±0.6 65.6±0.3 59.0±1.9 60.0±3.3 562±22 684.5±10 Max 117 157 121 119 1264 1151 66.0 94.7 83.4 81.6 772 1205 Min 89 108 82 84 711 747 34.0 48.0 32.2 44.7 306 622 Mean 107 128 103 105 956 968 51.0 75.0 62.0 64.3 614 867


126

Table 4.23: Mean comparison of Zn, Fe and Ca concentration (mg kg-1) of endosperm in

wheat genotypes under no and adequate Zn supply

Genotypes

Endosperm Zn

2014-15

Endosperm Fe

2014-15

Endosperm Ca

2014-15

-Zn +Zn -Zn +Zn -Zn +Zn

1 12.9±0.4 17.1±0.4 20.0±1.1 19.3±0.7 227±4.5 228±3.7 2 13.0±0.4 17.4±0.4 19.4±1.0 19.6±0.5 267±3.0 229±14

3 14.3±0.8 16.9±0.1 18.9±0.6 18.5±0.4 260±8.0 296±1.2

4 15.0±0.6 17.6±0.8 17.8±0.5 17.2±0.4 275±3.3 254±2.0

5 12.1±0.4 15.7±0.7 16.0±0.3 15.5±0.7 219±3.2 259±0.3

6 12.6±0.2 16.5±0.2 17.8±0.8 16.4±0.8 274±3.5 222±4.4

7 15.7±0.5 19.9±0.4 15.8±0.4 16.1±0.5 227±4.0 285±4.8

8 14.9±0.3 15.8±0.6 18.7±0.8 18.0±1.5 314±7.0 280±4.4

9 14.5±0.5 16.8±0.3 16.9±0.5 17.9±0.2 226±4.7 280±1.8

10 14.1±0.1 17.3±0.2 14.7±0.4 16.2±0.2 254±0.7 242±0.7

11 15.4±0.1 15.6±0.5 20.6±0.6 19.1±0.9 235±1.6 252±9.3

12 14.1±0.1 19.9±0.6 16.9±0.2 17.9±0.5 189±1.1 226±2.7

13 14.8±0.4 18.7±0.2 16.9±0.3 17.3±0.7 223±1.8 229±4.1

14 15.0±0.6 18.0±0.5 15.5±0.2 15.6±0.2 227±2.0 241±2.2

15 12.8±0.1 15.4±0.7 16.2±0.6 16.0±0.5 253±0.9 218±4.6

16 11.3±0.1 13.8±0.6 16.4±0.3 18.1±0.6 241±2.1 199±1.9

17 12.5±0.1 16.4±0.4 15.6±0.5 15.9±0.4 212±1.3 240±3.0

18 10.8±0.1 12.6±0.1 15.7±0.8 15.5±0.2 202±1.3 233±4.8

19 12.0±0.1 16.0±0.2 15.2±0.4 17.0±0.3 216±0.9 254±2.0

20 13.3±0.6 15.1±0.1 17.6±0.7 19.5±0.6 250±14 231±3.0

21 11.9±0.3 15.5±0.1 14.8±0.5 16.0±0.4 232±9.4 246±0.3

22 11.8±0.2 13.3±0.1 14.2±0.2 15.7±0.3 243±13 238±3.9

23 12.7±0.1 13.8±0.1 15.3±0.1 17.5±0.8 223±1.6 209±0.3

24 9.9±0.5 15.4±1.1 15.8±0.3 16.2±0.6 213±1.2 266±16

25 12.3±0.1 14.7±0.3 15.5±0.4 15.3±0.3 230±3.6 289±6.0

26 15.0±0.3 20.0±1.0 14.6±0.3 15.9±0.3 277±3.0 279±5.4

27 13.0±0.1 18.1±0.5 16.2±0.8 15.5±0.6 254±7.8 286±1.9

28 10.8±0.3 12.4±0.2 14.9±0.2 15.7±0.3 223±3.6 292±1.9

Max 15.7 20.0 20.6 19.6 314 296

Min 9.95 12.4 14.2 15.3 189 199

Mean 13.2 16.3 16.6 16.9 239 250

Gen= Genotype; -Zn= No Zn application; +Zn= soil application of Zn 10 kg ha-1; ± S.E.

127

4.3.1.6 Principal component analysis

The GGE-biplot indicated the interrelationship among different tested traits. Principal

components PC1 (16%) and PC2 (36%) indicated 52% of total variation observed among tested

genotypes with Zn application. Although, grain yield components were positively correlated

with each other, however, grain protein, grain Zn, grain Ca and bioavailable Zn were strongly

associated with each other. Whereas, grain Fe had negative relation with all other mineral traits

in this study. Grain minerals and yield components had positive but very weak association.

Furthermore, yield components were positively associated with phytate concentration and

[Phytate]:[Zn] ratio but had strong negative association with grain Zn, Ca, protein and

bioavailable Zn (Fig 4.2a).

Table 4.24: Evaluation of 4 primer pair combinations for use in studying genetic diversity

of wheat cultivars.

S. No. EcoRI MseI No. of alleles No. of polymorphic

alleles

Polymorphism

(%)

H

1 AGA CTC 158 158 100 0.0550

2 AGT CTG 174 174 100 0.0677

3 AGA CAT 117 117 100 0.0404

4 AGA CAA 58 58 100 0.0335

H = Nei's (1973) gene diversity

All genotypes responded positively to Zn application. Wheat genotypes Millat-11,

Kohinoor-83, SH-02, Fsd-85, and Sehar-2006 were better for grain protein, Zn, Ca

concentration and estimated bioavailable Zn under Zn application. Moreover, genotypes

AAS11, Mairaj-2008, Inqlaab-91, Fsd-85, Pak-81, and Chakwal-50 were high yielder under

Zn supply. The genotypes Mairaj-2008, Mairaj-2008, Inqlaab-91, Fareed-2006, Punjab-2011,

and Chakwal-50 have higher phytate and phytate/Zn ratio under no Zn supply (Fig. 4.2a).

Table 4.25: Population genetic analysis of 28 wheat genotypes using AFLP Fingerprinting

Genotypes N NPL PPL g % Ĝ/g H

AARI-2011 3 57 11 3 100 0.0367

AAS-2011 3 78 15 3 100 0.0494

128

Bakhar-2002 3 52 10 3 100 0.0344

Blue Silver 3 47 9 3 100 0.0315

Chakwal-50 3 71 14 3 100 0.0472

Chenab-2000 6 126 25 6 100 0.0524

Faisalabad-2008 3 76 15 3 100 0.0475

Faisalabad-83 3 55 11 3 100 0.0336

Faisalabad-85 3 50 10 3 100 0.0333

Fareed-2006 3 55 11 3 100 0.0351

GA-02 3 53 11 3 100 0.035

Iqbal-2000 3 47 9 3 100 0.0315

Inqlaab-91 3 100 20 3 100 0.0632

Kohinoor-83 3 76 15 3 100 0.0490

Lasani-2008 3 89 18 3 100 0.0582

MH-97 3 43 8 3 100 0.0273

Millat-2011 3 133 26 3 100 0.0842

Mairaj-2008 3 46 9 3 100 0.0305

Pak-81 3 48 9 3 100 0.031

Pasban-90 3 69 14 3 100 0.0426

Pothowar 3 52 10 3 100 0.0341

Punjab-2011 3 98 19 3 100 0.0628

Punjab-96 3 61 12 3 100 0.0405

Sandal-73 3 106 21 3 100 0.0675

Sehar-2006 3 29 5 3 100 0.0194

SH-02 3 66 13 3 100 0.0409

Shafaaq-2006 3 56 11 3 100 0.0372

Ufaq-2002 3 45 9 3 100 0.0314

Overall 87 507 100 87 100 0.0558

Where N is the sample size; NPL is the number of polymorphic loci, PPL is the percentage of

polymorphic loci (out of 507); g is the number of different genotypes recovered; Ĝ/g% is the percentage

of maximum diversity obtained in each population; and H is Nei (1973) gene diversity.

Table 4.26: Analysis of molecular variance (AMOVA) among wheat and within cultivars

Source of

Variation df

Sum of

squares

Variance

component

Percent

variation FST P

Among Populations 27 642.057 0.59719 Va 2.65 0.0265 <0.0001

Within Populations 59 1293.667 21.92655 Vb 97.35

Total 86 1935.724 22.52375 72.94

***= p<0.001

129

The vector view of the GGE-biplot (Fig. 4.2b) showed significant genetic variation

among wheat genotypes for Zn, Fe and Ca localization in different seed fractions with and

without Zn application. Principal components PC1 (19%) and PC2 (39%) explained 58% total

variation among genotypes based on mineral localization in seed fraction under no and soil Zn

application. Zinc concentration in embryo, aleurone, endosperm, aleurone Ca and aleurone Fe

were closely associated with each other, while embryo and endosperm Fe and Ca have strong

correlation with each other. Moreover, embryonic and aleurone Zn and aleurone Ca and Fe

concentration were higher in genotypes GA-02, Pasban-90, Fsd-83, Kohinoor-83, Fareed2006;

while, regarding endospermic Zn, genotypes Blue silver, Fsd-2008, Millat-11, and Fsd85 were

better. Furthermore, genotype Punjab-96 and Ufaq-2002 were better for embryo and

endosperm Ca and Fe concentration (Fig. 4.2b).

4.3.2. Discussion

This study examined genetic diversity of 28 wheat genotypes of Pakistani origin. The study

showed low level of genetic diversity and high gene flow among wheat genotypes. However,

these genotypes have great variation for yield and yield contributing traits due to morphological

differences. The level of genetic diversity of wheat genotypes was generally low. The level of

diversity is consistent with reports in China (Wang et al., 2013), but low compared to the

reported levels in Iran and Egypt (Mousavifard et al., 2015; Salem et al., 2015). This indicates

a high level of uniformity among seeds of each genotype, which could be a result of inbreeding

or selfing. However, the low level of genetic diversity of wheat genotypes observed in this

study may make them vulnerable for future disease outbreaks (AlSadi et al., 2012b).

130

Fig. 4.1: UPGMA dendrogram illustrating Nei's (1978) genetic distances of 28 wheat cultivars

based on AFLP fingerprinting analysis.

131

Fig. 4.2: Polygon view of the GGE biplot show the relation of wheat genotypes for (a) grain mineral,

yield components and Zn bioavaiblity (b) Zn, Fe and Ca localization in embryo, aleurone and

endosperm of wheat genotype under –Zn and +Zn conditions.

PT= productive tiller, GPS= grain per spike, TGW= 1000 grain weight, GY= grain yield; HI= harvest

index, GZn= grain Zn, GFe= grain Fe, Gca= grain Ca, Gpro= grain protien; Em. Embryo, Alu=

aleurone; Ed= endosperm

( a )

( b )

132

Table 4.27: Correlation coefficients of grain mineral concentrations with embryonic, aleurone and

endosperm Zn concentration and bioavailable Zn under no and Zn application treatments

Grain Zn Grain Fe Grain Ca Embryo Aleurone Endosperm Bioavailable

Zn Zn Zn Zn

Grain Zn

Grain Fe 0.172ns 1 0.390* 0.066ns -0.012ns 0.425* -0.013ns

Grain Ca 0.209ns 0.149ns 1 -0.365* -0.368* 0.237ns 0.022ns

Embryo Zn 0.874** 0.095ns 0.081ns 1 0.265ns 0.459* 0.437*

Aleurone Zn 0.562** 0.099ns 0.080ns 0.634** 1 0.235ns 0.475*

Endosperm Zn 0.718** 0.141ns 0.147ns 0.595** 0.285ns 1 0.472* Bioavailable Zn 0.876**

0.237ns 0.174ns 0.798** 0.6083** 0.631** 1

*= significant ≤ 0.05; **≤ 0.01

Most wheat genotypes clustered together in two groups. This was supported by AMOVA

analysis, which indicated the existence of the low levels of genetic differentiation among wheat

genotypes. These findings indicate that many wheat genotypes in Pakistan share a common

gene pool and there is high gene flow among wheat cultivars which could have been due to

breeding programs. The moderate levels of gene flow among three wheat genotypes

(‘Lasani2008 and Mairaj-2008’, ‘Lasani-2008 and Sehar-2006’ and ‘Chakwal-50 and Ufaq-

2002’) may be due to more genetic differentiation between their parent compared to other

genotypes used in this study. Many of the currently used genotypes have one similar parent as

these were developed for high yield which is possible reason for high gene flow among these

genotypes.

Application of Zn increased the yield and yield contributing traits of bread wheat

genotypes as was visible from an average increase in productive tillers, grains per spike,

1000grain weight, grain yield and harvest index from control treatment (Tables 4.17, 4.18,

4.19). The improvement in grain yield was due to close association of grain yield and yield

components with +Zn treatment (Fig. 4.2). The grain yield among wheat genotypes followed

the order Chakwal-50< Mairaj-2008< AARI-11< MH-97< Punjab-96< FSD-85< Bakhar2002<

FSD-83< Pasban-90< Chenab-2000< Fareed-06< GA-02< FSD-2008< AAS-11< Shafaq-

2006< SH-02< PAK-81< Millat-11< Blue Silver< LS-2008<Ufaq-2002< Punjab-11< Inqlaab-

91< Kohinoor-83 < Sehar-2006< Iqbal-2000< Sandal-73< Potohar (Table 4.19). Moreover,

1 0.143 ns - 0.129 ns 0.598** 0.510** 0.599** 0.797**

133

soil application of Zn enhanced the grain yield of wheat due to higher number of productive

tillers, grains per spike and 1000 grain weight which resulted in higher grain yield.

Improvement in grain yield due to Zn fertilization was owing to enhanced the seed setting and

nutrient uptake (Khalifa et al., 2011) which improved the yield and yield contributing traits.

In the present study, grain Zn concentration in wheat genotypes ranged from 23.7-33.9

mg kg-1, however, this range improved from 31.4-50.9 mg kg-1 with Zn application (Table

4.20) as soil Zn fertilization increases DTPA extractable Zn concentration (Poblaciones and

Rengel, 2016) which possibly enhanced the Zn uptake by wheat and ultimately increased the

grain Zn accumulation. Moreover, soil Zn fertilization has positive influence on grain Zn

concentration (Liu et al., 2017). The grain Zn concentration in wheat genotypes was highest in

Blue Silver< GA-02< Millat-11< FSD-83< FSD-2008< Sehar-2006< Potohar< Ufaq-2002<

Pasban-90< SH-02< Kohinoor-83< Shafaq-2006< Chenab-2000< Pak-81< Punjab-96<

AARI11< Inqlaab-91< Punjab-11< FSD-85< Fareed-06< Sandal-73<AAS-11< MH-97<

Iqbal2000< Mairaj-2008< LS-2008< Chakwal-50< Bakhar-2002.

Calcium is also very vital for human nutrition even if plants are receiving abundant Ca.

There was wide variation of Ca concentration in grain and grain fractions of wheat genotypes.

The Ca concentration in the present study ranged from 295-455 mg kg-1. This range was

relatively similar with Hussain et al. (2012a) and was broaden to 350-511 by application of Zn

as Zinc application increased the grain Ca concentration as soil application of Zn is associated

with higher Ca bioavailability (Hussain et al., 2012b). The grain Fe concentration of bread

wheat genotypes ranged 30.4-45 mg kg-1 which is relatively close to the previous reports of

Graham et al. (1999) and Ficco et al. (2009). In the present study, Zn fertilization did not

improve the Fe accumulation in grain and grain fractions of wheat as grain Zn concentration

has non-significant correlation with grain Fe concentration (Table 4.27, Fig 4.2). Moreover,

protein concentration in the wheat genotypes used in this study was 11.8-14.4% which was

increased by application of Zn (13.9-15.5%) as Zn fertilization increases the content and

quality of protein (Peck et al., 2008; Liu et al., 2015). Moreover, in the present study, grain

protein concentration is closely correlated with grain Zn concentration (Fig 4.2a) which support

the idea that grain Zn and protein concentration can be improved concurrently.

134

The wheat genotypes used in this study varied for grain Fe, Zn and Ca concentration.

The bread wheat genotypes ranged from 89-116, 33-66, 10-16 mg kg-1 for embryo, aleurone

and endosperm Zn concentration respectively for –Zn treatment; while soil Zn application

broaden this range from 108-157, 48-95 and 12-20 mg kg-1 in germ, aleurone and endosperm

respectively (Tables 4.22, 4.23) as indicated by strong positive correlation between grain Zn

concentration with embyo, aleurone and endospermic Zn concentration (Table 4.27). Zinc

concentration is high in embryonic and aleuronic regions of wheat grain (Oztruk et al., 2006);

while less Zn is present in starchy endosperm and is highly bioavailable due to presence of less

phytate in this seed fraction. Improvement in Zn deposition in endosperm of wheat genotypes

due to Zn fertilization (Table 4.23) was possibly due to high uptake of Zn and its translocation

to seed. Moreover, Fe concentration was ranged from 82-121, 32-83, 14-20 mg kg-1 in germ,

aleurone and endosperm of wheat genotypes irrespective of Zn application. Zinc application

substantially increased the Ca concentration in aleurone of wheat genotypes (Table 4.22) as

soil application of Zn has been found to increase Ca concentration in grain (Hussain et al.,

2013).

Zinc application increased the bioavailability of Zn as wheat genotypes fertilized

through soil application of Zn have higher bioavailable Zn with reduced phytate content and

lower phytate/Zn ratio compared to no Zn application (Table 4.21; Fig 4.2a). Wheat genotypes

have phytate range from 7.13-10.3 mg g-1 and phytate/Zn ratio 25.7-42.3 without Zn

fertilization while Zn application narrowed the range of phytate concentration from 6.8 to 9.5

mg g-1 with phytate/Zn ratio of 15.9-27.7, thus improving the range of estimated bioavailability

from 2.38 to 3.18 mg day-1 compared to 1.8-2.47 mg day-1 in -Zn treatment. The Zn

requirement for an adult is from 3 to 4 mg Zn/day (Hotz and Brown, 2004). In the present

study, Zn application enhanced the estimated bioavailable Zn > 3 mg in 300 g wheat flour

which is near to required level for a healthy human food. Reduced bioavailability under no Zn

treatment is due to high accumulation of phytate and higher phytate/Zn ratio which is key

indicator of Zn bioavailability of food, and for higher bioavailable Zn. Phytate: Zn should be

less than 15 for increased Zn absorption by human intestine (Brown et al., 2001).

The bioavailability of wheat genotypes used in this study is low as evident from high

phytate content, [phytate]:[Zn] ratio and low estimated bioavailable Zn. However, Zn

135

fertilization improved the yield, grain protein content, grain Zn concentration and its

localization in all the seed fraction, especially endospermic Zn fraction which is highly

bioavailable due to presence of low phytate. Moreover, increased Zn fertilization reduced the

phytate concentration and narrowed the [phytate]:[Zn] thus enhancing the bioavailable Zn.

The present study indicated that bread wheat genotypes of Punjab Pakistan have very

less genetic diversity which may make them vulnerable to disease. However, these respond

positively to Zn fertilization. Present wheat genotypes have high grain yield possibly due to

increase in endosperm size of wheat grain which reduces the nutrient rich seed parts. Therefore,

genetically diverse wheat genotypes with high endospermic Zn concentration with better crop

yield should be used in breeding programs together with agronomic approaches, aiming at

improving the Zn bioavailability.


Biofortification of Wheat

4.4.1. Results (Glass house experiment)

4.4.1.1 Zn solubilization activity

Data of Zn solubilization assay revealed that Pseudomonas sp. MN12 solubilized (halo zone

diameter mm) Zn from all ores but with variable efficacy. Maximum halo zone formation (35

mm) was observed in ZnO plate followed by 31 mm in Zn(CO3)2. The strain MN12 produced

halos diameter 13 and 8 mm in [Zn3(PO3)2] and ZnS plates, respectively.

4.4.1.2 Photosynthetic traits

Analysis of variance indicated that Zn application significantly affected the photosynthetic rate,

transpiration rate, intercellular CO2 and stomatal conductance during both years of

experimentation. Likewise, wheat cultivars also differed for photosynthetic rate, transpiration

rate, intercellular CO2 and stomatal conductance during both years of experimentation; results

being non-significant for the photosynthetic rate during the second year of experimentation.

The interaction of wheat cultivars with Zn application was significant for transpiration rate and

intercellular CO2 during both years and stomatal conductance during the first year of

experimentation (Table 4.28). During the first year, the highest photosynthetic rate was

recorded in Lasani-2008 than Faisalabad-2008. Among Zn application methods, the highest

136

photosynthetic rate during both years and stomatal conductance during second year was

recorded with soil application of Zn with Pseudomonas sp. MN12, which was statistically

similar with seed Zn priming and foliar Zn application + MN12, and soil application of Zn

without MN12 during first year for photosynthetic rate, and with Zn seed priming + MN12 for

photosynthetic rate and stomatal conductance during the second year of experimentation (Table

4.29). The interaction showed that the highest stomatal conductance during both years and

lowest intercellular CO2 and highest transpiration rate, during first year was recorded with soil

Zn application and seed Zn priming + MN12 in Faisalabad-2008 (Table 4.30). Moreover,

during second year of study, highest transpiration rate is with Faisalabad in 2014-15 with soil

application of Zn+MN12 followed by Lasani in 2014-15 with same treatment.

4.4.1.3 Water relation traits

Zinc application significantly affected the relative water content, water potential, osmotic

potential and pressure potential of wheat at anthesis during both years (Table 4.31). Moreover,

wheat cultivars differed significantly for water potential during the first year; and osmotic and

pressure potential during the both years of experimentation (Table 4.31).

The interaction of wheat cultivars and Zn application methods was only significant for

pressure potential at anthesis during first year (Table 4.31).Among wheat cultivars, the highest

water potential during first year, osmotic potential during both years and pressure potential

during the second year was recorded in Lasani-2008. The highest pressure potential was

observed in Faisalabad-2008 during the first year (Table 4.32).

Zinc application by either method, with and without MN12, was equally effective for

improvement in the RWCs at anthesis stage during both years. The highest water and osmotic

potential at anthesis during both years was recorded with seed Zn priming and soil application

of Zn + MN12 strain. During the first year, the lowest pressure potential was recorded with

seed Zn priming with/without Pseudomonas sp. MN12. During the second year, the pressure

potential was the lowest with soil application of Zn + MN12 (Table 4.32). The interaction

showed that the lowest pressure potential was recorded with seed Zn priming without MN12

in Faisalabad-2008 (Table 4.30).

137


Analysis of variance showed that Zn application significantly affected the grains per spike and

grain yield during the both years; 100-grain weight and harvest index first year; results being

non-significant for 100-grain weight and harvest index during the second year. Likewise, wheat

cultivars significantly differed for grains per spike and grain yield during both years; 100-grain

weight, and harvest index during the first year of experimentation. However, the interaction of

wheat cultivars with Zn application methods was significant for only grain yield during both

years (Table 4.33).

Among wheat cultivars, the highest 100-grain weight was recorded in Lasani-2008

during first year. Highest grains per spike and grain yield during both years; and harvest index

during first year was recorded in Faisalabad-2008 (Table 4.34).

Zn application improved the grain yield and yield contributes traits of wheat as

maximum number of grains per spike were recorded for seed priming and soil application of

Zn with and without Pseudomonas sp. MN12 during both experimental years. Foliar applied

Zn + MN12 resulted in maximum 100-grain weight during the first year of study. During the

first year, Zn application through either method (with/without MN12) was effective for

improvement in harvest index of wheat (Table 4.34). Grain yield was highest with soil applied

Zn + MN12 and seed Zn priming + MN12 during both years in Faisalabad-2008 (Table 4.35).

4.4.1.5 Grain quality

Zinc application significantly affected the bioavailable Zn, phytate concentration, phytate/Zn

ratio, during both years; and grain protein contents during the second year. Wheat cultivars

also differed significantly for phytate concentration during the second year of study; results

being non-significant for other traits during both years. The interaction of wheat cultivars with

Zn application was also significant for bioavailable Zn, and phytate/Zn ratio during both years;

and phytate concentration during the second year (Table 4.36).

The grain protein was the highest in Lasani-2008, while phytate concentration was the

highest in Faisalabad-2008 during the second year of experimentation. Among Zn application

methods, the highest grain protein contents during the second year were recorded with soil and

foliar application of Zn (with/without) + MN12. During first year, the lowest phytate

138

concentration was recorded with soil and foliar Zn application with Pseudomonas sp. MN12

(Table 4.34).

The interaction showed that the highest bioavailable Zn was noted in foliar Zn

application+ MN12 in Faisalabad-2008 during both years (Table 6). Phytate/Zn ratio was the

lowest with foliar and soil Zn applications with/without MN12 during the first year. During the

second year, it was lowest with foliar and soil Zn application + MN12 in Faisalabad-2008, and

in foliar Zn application without bacteria in Lasani-2008. During the second year, the lowest

phytate concentration was recorded with soil and foliar Zn application + MN12 in both

cultivars, and that was statistically similar with foliar Zn application without MN12 in

Lasani2008 (Table 4.37).

139

Table 4.28: Analysis of variance for effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on

photosynthetic traits of wheat cultivars

Cultivar (C) 1 33.54** 2.32ns 0.935** 0.095** 690.31* 1401.7** 0.014** 0.009** T × C 9 1.11ns 0.421ns 0.041** 0.012* 390.62** 246.3* 0.0004** 0.0001ns Error 60 0.81 0.689 0.010 0.006 110.45 105.2 0.0001 0.0001 Total 79

SOV= Sources of variation; DF= Degree of freedom; ns= non-significant; *= p ≤ 0.05; **= p ≤ 0.01

Table 4.29: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on photosynthetic traits of wheat

cultivars Treatments Photosynthesis (μmol CO2 m-2 s-1) Photosynthesis (μmol CO2 m-2 s-1) Stomatal conductance 2013-14 2014-15 mmol H2O m-2s-1) (2014-15)

Wheat cultivars Lasani-2008 14.8 A 15.1 0.209 Faisalabad-2008 13.5 B 15.5 0.233

No microbe Hydropriming 11.8 C 13.3 E 0.17 F

SP (0.5 M Zn) 14.3 AB 14.8 CDE 0.22 CD

SC (1.25 g kg-1 seed) 13.2 BC 14.0 DE 0.19 EF

SA (5 mg Zn kg-1 soil) 15.1 A 15.3 BCD 0.23 C

FA (0.025 M Zn) 14.5 AB 14.7 CDE 0.20 DE

MN12 Hydropriming 12.8 C 14.1 DE 0.19 F

SP (0.5 M Zn) 15.5 A 16.9 A 0.28 A

SC (1.25 g kg-1 seed) 14.3 AB 16.0 ABC 0.22 CD

SA (5 mg Zn kg-1 soil) 15.4 A 17.0 A 0.28 A

SOV DF Photosynthesis Transpiration Intercellular CO 2 Stomatal conductance 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15

Treatment (T) 9 11.57** 10.12** 0.439** 0.536** 9150.5** 8324.7** 0.009** 0.009**

HSD (p ≤ 0.05) 0.40 NS 0.004 Treatments

140

FA (0.025 M Zn) 14.9 A 16.5 AB 0.24 B

HSD

(p ≤ 0.05)

Table 4.30: Interactive effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on photosynthetic traits of

wheat cultivars

Treatments (2014-15) Transpiration (mmol H2O m-2s-1) Intercellular CO2Ci (μmol mol-1) Pressure Potential (-MPA)- (2013-14)

No microbe Hydropriming 1.61 i 1.71 hi 342 a 320ab 0.21 d 0.24 cd

SP (0.5 M Zn) 1.96 fg 1.99 efg 271 c-f 267 c-g 0.31 a-d 0.46 a

SC (1.25 g kg-1 seed) 1.76 ghi 1.91 fgh 285 cd 289 b-c 0.27 bcd 0.34 a-d

SA (5 mg Zn kg-1 soil) 1.96 fg 2.15 def 250 e-i 261 c-h 0.27 bcd 0.37 a-d

FA (0.025 M Zn) 1.87 gh 1.87 gh 256 d-i 256 d-i 0.25 bcd 0.25 bcd

MN12 Hydropriming 1.93 fgh 1.80 ghi 284 cd 272 cde 0.23 cd 0.27 bcd

SP (0.5 M Zn) 2.43 abc 2.56 ab 228 ijk 199 k 0.41 ab 0.37 a-d

SC (1.25 g kg-1 seed) 2.24 cd 2.25 cd 240 f-j 231 h-k 0.29 bcd 0.31 a-d

SA (5 mg Zn kg-1 soil) 2.46 abc 2.64 a 217 jk 203 k 0.32 a-d 0.39 abc

1.48 1.60 0.018 Figure of main effects sharing same letter for a parameter don’t differ significantly at p ≤ 0.05 SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application

141

SP (0.5 M Zn) 1.71 hij 2.17 a-d 251 c-g 257 b-f 0.183 ghi 0.215 def

SC (1.25 g kg-1 seed) 1.60 jk 1.72 g-j 274 bc 270 bcd 0.168 ij 0.193 f-i

SA (5 mg Zn kg-1 soil) 1.90 e-h 2.28 abc 226 ghi 240 efg 0.198 e-h 0.223 cde

FA (0.025 M Zn) 1.79 f-j 2.01 def 243 d-g 251 c-g 0.190 f-i 0.210 d-g

MN12 Hydropriming 1.78 f-j 1.73 g-j 281 b 261 b-f 0.173 hij 0.180 hi

SP (0.5 M Zn) 2.08 b-e 2.39 a 213 hij 201 ij 0.250 bc 0.293 a

SC (1.25 g kg-1 seed) 1.86 e-i 2.05 cde 265 b-e 236 fgh 0.180 hi 0.223 cde

SA (5 mg Zn kg-1 soil) 2.10 b-e 2.31 ab 198 j 198 j 0.233 cd 0.273 ab

FA (0.025 M Zn) 1.98 d-g 2.09 b-e 244 d-g 224 g-j 0.215 def 0.218 def

HSD (p ≤0.05) 0.26 27.51 0.028

Table 4.31: Analysis of variance for effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on water

relation of wheat cultivars at anthesis SOV DF

Treatment (T)

FA (0.025 M Zn) 2.23 cde 2.36 bcd 237 g-j 214 jk 0.33 a-d 0.24 cd

Treatments (2013 - 14) Transpiration l H o ( mm 2 O m - 2 s - 1

) Intercellular CO 2 Ci ( μmol mol - 1 ) Stomatal conductance gs mmol H ( 2 O m - 2 s - 1

)

Lasani - 2008 Faisalabad - 2008



No microbe

Hydropriming k 1.38

ijk 1.62 317 a

315 a 0.150 j

0.175 hij

0.244 31.69

0.17

Figure of interaction sharing same letter for a parameter don’t differ significantly at p ≤ 0.05

142

FA (0.025 M Zn) HSD (p ≤ 0.05) 6.43 6.99 0.11 0.057 0.057 0.107 0.088

Cultivar (C) 1 0.48ns 12.1ns 0.110** 0.016ns 0.009** 0.035** 0.023* 0.035* T × C 9 12.9ns 14.3ns 0.008ns 0.001ns 0.001ns 0.001ns 0.010* 0.002ns Error 18.2 11.2 0.005 0.002 0.001 0.001 0.002

Total SOV= Sources of variation; DF= Degree of freedom; ns= non-significant; *= p ≤ 0.05; **= p ≤ 0.01; RWC= relative water content; WP= Water potential; OP= Osmotic potential; PP= pressure potential

Table 4.32: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on water relation traits of wheat

cultivars

Treatments RWC (%) WP (-MPA) OP (-MPA) PP (-MPA) 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15

Wheat cultivars Lasani-2008 79.5 78.5 1.52 A 1.35 1.81 A 1.794 A 0.29 B 0.44 A Faisalabad-2008 79.7 77.5 1.45 B 1.35 1.77 B 1.778 B 0.32 A 0.42 B

No

microbe Hydropriming

SP (0.5 M Zn) 74.8 B 80.8 AB

72.7 C 80.4 AB

1.71 A 1.37 EFG

1.58 A 1.30 DEF

1.93 A 1.76 CD

1.89 A 1.75 DEF

0.23 C 0.39 A

0.31 D 0.46 AB

SC (1.25 g kg-1 seed) 78.9 AB 77.9 ABC 1.51 BCD 1.40 C 1.81 BC 1.81 BC 0.30 ABC 0.40 BC

SA (5 mg Zn kg-1 soil) 80.5 AB 75.7 ABC 1.42 D-G 1.27 EF 1.74 D 1.74 DEF 0.32 ABC 0.48 AB

FA (0.025 M Zn) 78.8 AB 78.8 ABC 1.55 BC 1.36 CD 1.80 BC 1.79 BCD 0.25 BC 0.43 AB

MN12 Hydropriming 77.4 AB 74.3 BC 1.60 AB 1.49 B 1.84 B 1.83 B 0.25 C 0.34 CD

SP (0.5 M Zn) 81.6 A 81.1 A 1.35 G 1.23 F 1.74 D 1.72 F 0.39 A 0.48 AB

SC (1.25 g kg-1 seed) 80.0 AB 77.6 ABC 1.48 C-F 1.34 CDE 1.78 CD 1.77 CDE 0.30 ABC 0.43 AB

SA (5 mg Zn kg-1 soil) 82.4 AB 81.2 A 1.37 FG 1.23 F 1.72 D 1.74 EF 0.36 AB 0.51 A

81.0 AB 80.2 AB 1.48 CDE 1.31 DEF 1.77 CD 1.76 C-F 0.29 ABC 0.45 AB 0.11

Figure of main effects sharing same letter for a parameter don’t differ significantly at p ≤ 0.05 SP=

Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application

HSD (p ≤ 0.05) NS NS 0.03 0.03 0.016 0.013 0.029 0.02 Treatments

143

ns= non - significant; p ≤ 0.05; **= p ≤ *= 0.01

Table 4.33: Analysis of variance for the effect of different Zn application methods with or without Pseudomonas sp. MN12 addition

on yield components of wheat cultivars

Grains per spike 100-grain weight Grain yield Harvest index

SOV DF 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 Treatment (T) 9 65.9** 64.3** 0.027** 0.035ns 1.28** 0.33** 492.9** 69.3ns Cultivar (C) 1 40.5** 26.4* 0.049* 0.001ns 1.28** 0.27** 1359.6** 6.80ns T × C 9 5.2ns 5.63ns 0.004ns 0.004ns 0.12** 0.013* 85.2ns 68.9ns Error 60 8.9 4.11 0.012 0.016 0.02 0.005 53.2 40.5 Total 79

SOV= Sources of variation; DF= Degree of freedom;

Table 4.34: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on yield components, grain protein and

phytate concentration of wheat cultivars

No microbe Hydropriming 35.9 B 35.0 D 3.80 B 3.81 34.6 D 35.9 12.3 12.3 B 9.38 A

SP (0.5 M Zn) 39.7 AB 41.0 AB 3.88 AB 3.85 51.1 AB 47.2 12.8 13.0 AB 8.57 C

SC (1.25 g kg-1 seed) 35.7 B 38.4 BCD 3.92 AB 3.93 44.2 BCD 35.7 12.4 12.8 AB 8.67 C

SA (5 mg Zn kg-1 soil) 42.3 A 39.7 BC 3.89 AB 3.83 51.9 AB 43.3 12.8 13.4 A 8.07 D

FA (0.025 M Zn) 36.3 B 36.8 CD 3.96 AB 3.91 38.2 CD 38.6 12.5 13.4 A 7.92 DE

MN12 Hydropriming 35.2 B 35.8 CD 3.83 AB 3.81 44.0 BCD 38.4 12.4 12.3 B 9.12 B

SP (0.5 M Zn) 41.6 A 43.8 A 3.90 AB 3.91 59.6 A 40.8 12.6 12.9 AB 8.44 C

SC (1.25 g kg-1 seed) 38.7 AB 37.7 BCD 3.95 AB 4.05 53.0 AB 40.9 12.4 12.8 AB 8.50 C

SA (5 mg Zn kg-1 soil) 42.0 A 44.6 A 3.89 AB 3.91 55.8 AB 41.0 13.0 13.7 A 7.76 E

FA (0.025 M Zn) 36.5B 36.9 CD 4.00 A 3.98 46.7 BC 40.7 12.3 13.5 A 7.81 E

144

Treatments Grains per spike 100-grain weight (g) Harvest index (%) Grain protein (%) Phytate (mg g-1)

2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14

Wheat cultivars Lasani-2008 37.7 B 38.3B 3.93 A 3.90 43.8 A 39.9 12.7 13.0 8.43 Faisalabad-2008 39.1 A 39.6A 3.88B 3.89 52.0 B 40.6 12.5 13.0 8.42

Figure of main effects sharing same letter for a parameter don’t differ significantly at p ≤ 0.05

SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application

Table 4.35: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on grain yield (g plant-1) of wheat cultivars

No microbe Hydropriming 2.11i 2.28ghi 2.08 j 2.28hij

SP (0.5 M Zn) 2.66ef 3.24bc 2.56 c-g 2.56 c-g

SC (1.25 g kg-1 seed) 2.27ghi 2.51fgh 2.24hij 2.38 e-h

SA (5 mg Zn kg-1 soil) 2.75def 3.06bcd 2.59cde 2.54 d-g

FA (0.025 M Zn) 2.18 hi 2.75def 2.16hij 2.28hij

MN12 Hydropriming 2.29ghi 2.48fgh 2.14ij 2.36 f-i

SP (0.5 M Zn) 3.28abc 3.64 a 2.68bcd 2.84 ab

SC (1.25 g kg-1 seed) 2.96cde 2.81def 2.34ghi 2.58 c-f

SA (5 mg Zn kg-1 soil) 3.02 b-e 3.35 ab 2.77abc 2.94 a

FA (0.025 M Zn) 2.66ef 2.58fg 2.26hij 2.39 e-h

HSD (p ≤ 0.05) 4.91 3.92 0.17 NS 11.98 NS NS 4.38 0.23

Treatments 2013 - 14 2014 - 15 Lasani - 2008 Faisalabad - 2008 Lasani - 2008 Faisalabad - 2008

HSD (p ≤0.05) 0.364 0.227

HSD (p ≤ 0.05) 1.33 1.05 0.04 NS 3.26 NS 3.12 2.86 NS Treatments

145

Table 4.36: Analysis of variance for the effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on grain

minerals composition of wheat cultivars

Cultivar (C) 1 0.851ns 0.033ns 0.0003ns 0.27** 0.016ns 0.002ns 14.5ns 5.58ns T × C 9 0.216ns 0.136ns 0.0175ns 0.08** 0.022** 0.109** 12.5** 21.4** Error 60 0.315 0.214 0.0205 0.01 0.009 0.010 4.30 4.02 Total 79


Table 4.37: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on phytate/Zn molar ratio,

Fe, P and Ca concentration in grains of wheat cultivars

2013-14 Treatments

Bioavailable Zn(mg/300 g flour) Phytate/Zn molar ratio Lasani-2008 Faisalabad-2008 Lasani-2008 Faisalabad2008

No microbe Hydropriming 1.63g 1.81fg 47.7a 41.4b

SP (0.5 M Zn) 2.26cde 2.24cde 29.5cde 29.9cde

SC (1.25 g kg-1 seed) 2.02ef 2.15cde 34.7c 31.8cde

SA (5 mg Zn kg-1 soil) 2.70ab 2.61b 21.8g 23.1fg

FA (0.025 M Zn) 2.76ab 2.65ab 20.9g 22.4g

MN12 Hydropriming 1.81fg 1.83fg 41.3b 40.5b

SP (0.5 M Zn) 2.30cd 2.33c 28.7de 33.5cd

SC (1.25 g kg-1 seed) 2.16cde 2.08de 31.4cde 27.9ef

SA (5 mg Zn kg-1 soil) 2.73ab 2.81ab 21.1g 20.2g

FA (0.025 M Zn) 2.72ab 2.87a 21.6 g 19.4g

SOV DF Protein Phytate Bioavailable Zn Phytate/Zn ratio 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15

Treatment (T) 9 0.499 ns 1.265** 2.3687** 3.12** 1.238** 1.545** 579.6** 437.4**

146

HSD (p ≤0.05) 0.24 5.42

2014-15 Bioavailable Zn (mg/300 g flour) Phytate/Zn Molar ratio Phytate (mg g-1)

No microbe Hydropriming 1.82 j 1.92 ij 40.9 a 38.1 ab 9.22 a 9.30 a

SP (0.5 M Zn) 2.33 fgh 2.29 fgh 28.0 cde 28.8 cde 8.25 ef 8.50 cde

SC (1.25 g kg-1 seed) 2.12 hij 2.24 fgh 32.7 bc 30.0 cde 8.50 cde 8.66 cd

SA (5 mg Zn kg-1 soil) 3.00 cd 2.70 de 17.8 gh 21.6 fg 7.71 h 7.77 h

FA (0.025 M Zn) 3.31 ab 2.73 de 14.5 h 21.3 fg 7.20 i 7.84 gh

MN12 Hydropriming 1.93 ij 2.17 ghi 37.9 ab 31.7 cd 8.79 bc 9.02 ab

SP (0.5 M Zn) 2.46 efg 2.54 ef 25.6 cde 24.2 cde 8.12 fg 8.21 ef

SC (1.25 g kg-1 seed) 2.27 fgh 2.25 fgh 29.2 def 29.6 ef 8.38 def 8.50 cde

SA (5 mg Zn kg-1 soil) 3.20 abc 3.34 ab 15.5 gh 14.2 h 7.24 i 7.11 i

FA (0.025 M Zn) 3.08 bc 3.43 a 16.7 gh 13.3 h 7.21 i 7.05 i

HSD (p ≤0.05) 0.31 6.19 0.32 Figure of interaction sharing same letter for a parameter don’t differ significantly at p ≤ 0.05 SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application

Table 4.38: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on organic acid

concentration of bread wheat ± S.E.

Treatments Pyruvic acid (µg mL-1) Tartaric acid(µg mL-1) Citric acid (µg mL-1)

Lasani-2008 Faisalabad2008 Lasani-2008 Faisalabad2008 Lasani-2008 Faisalabad-2008

No microbe Hydropriming 24.0 ± 0.58 29.3 ± 1.53 156 ± 2.8 72 ± 1.5 25.8± 3.5 nd

SP (0.5 M Zn) 42.5 ± 1.20 33.7 ± 0.88 185 ± 4.5 126 ± 6.1 53.0± 3.8 32.0± 1.1

SC (1.25 g kg-1 seed) 8.4 ± 0.88 43.2 ± 0.33 164 ± 5.1 110 ± 8.8 34.9± 2.6 nd

SA (5 mg Zn kg-1 soil) 18.7 ± 0.33 40.9 ± 1.45 185 ± 2.8 214 ± 6.7 42.1± 5.5 12.6± 1.2

147

FA (0.025 M Zn) 61.5 ± 0.33 69.1 ± 1.86 171 ± 7.0 110 ± 3.0 35.1± 5.0 nd

MN12 Hydropriming 83.5 ± 0.97 57.6 ± 0.70 272 ± 4.8 238 ± 8.8 103.5± 4.0 42.0± 1.1

SP (0.5 M Zn) 109.2 ± 4.17 118.6 ± 4.81 636 ± 22.7 389 ± 14.5 211.1± 7.5 178.7± 4.0

SC (1.25 g kg-1 seed) 69.9 ± 1.77 76.5 ± 1.86 303 ± 14.5 283 ± 3.3 181.8± 7.6 86.8± 3.1

SA (5 mg Zn kg-1 soil) 109.9 ± 11.1 176.1 ± 8.83 914 ± 8.1 376 ± 15.3 266.4± 6.4 202.9± 8.0

FA (0.025 M Zn) 92.3 ± 4.10 94.9 ± 0.67 325 ± 12.7 421 ± 8.8 163.5± 6.3 85.4± 5.3

Malonic acid (µg mL-1) Mallic acid (µg mL-1) Succinic acid (µg mL-1)

No microbe Hydropriming nd Nd nd nd Nd nd

SP (0.5 M Zn) 0.4 ± 0.03 Nd 4.7± 0.25 5.2± 0.06 Nd nd

SC (1.25 g kg-1 seed) nd Nd 3.7± 0.26 nd Nd nd

SA (5 mg Zn kg-1 soil) 0.1 ± 0.01 0.2 ± 0.02 5.2± 0.12 4.9± 0.11 Nd nd

FA (0.025 M Zn) nd Nd Nd nd Nd nd

MN12 Hydropriming 0.1 0.01 Nd 5.7± 0.57 24.1± 0.64 Nd nd

SP (0.5 M Zn) 0.8 ± 0.05 0.3 ± 0.03 34.1± 0.84 57.4± 0.44 Nd 13.7± 0.04

SC (1.25 g kg-1 seed) nd Nd 15.2± 0.55 25.6± 0.94 Nd nd

SA (5 mg Zn kg-1 soil) 0.7 ± 0.05 0.4 ± 0.02 37.7± 0.83 50.8± 2.18 Nd 16.6± 0.03

FA (0.025 M Zn) nd nd 6.7± 0.57 46.7± 1.68 Nd Nd

Oxaloacetic acid (µg mL-1) Oxalic acid (µg mL-1) Methyl malonic acid (µg mL-1)

No microbe Hydropriming nd nd nd nd Nd Nd

SP (0.5 M Zn) 5.1± 0.15 nd nd nd Nd Nd

SC (1.25 g kg-1 seed) nd nd nd nd Nd Nd

SA (5 mg Zn kg-1 soil) 6.5± 0.20 nd nd nd Nd Nd

148

FA (0.025 M Zn) nd nd nd nd Nd Nd

MN12 Hydropriming nd nd nd nd Nd Nd

SP (0.5 M Zn) 19.6± 1.15 7.8± 0.40 0.34± 0.04 nd 0.10± 0.03 0.24± 0.03

SC (1.25 g kg-1 seed) 3.0± 0.06 nd nd nd Nd Nd

SA (5 mg Zn kg-1 soil) 8.1± 0.34 20.4± 0.52 0.11± 0.03 nd 0.15± 0.03 1.84± 0.08

FA (0.025 M Zn) nd nd nd nd Nd Nd

Figure of interaction sharing same letter for a parameter don’t differ significantly at p ≤ 0.05 SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application; nd= not detected

149

Fig. 4.3: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on

grain and seed fractions Zn concentration of wheat cultivars (a) Lasani-2008 (b)

Faisalaabad-2008 ±S.E. SP= Seed priming; SC= Seed coating; SA= Soil application; FA=

Foliar application; B= MN12

150

Fig. 4.4:

Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on

grain and seed fractions Fe concentration of wheat cultivars (a) Lasani-2008 (b) Faisalaabad2008

±S.E. SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application; B=

MN12

151

Fig. 4.5: Effect of different Zn application methods with or without Pseudomonas sp. MN12 addition on

grain and seed fractions Ca concentration of wheat cultivars (a) Lasani-2008 (b) Faisalaabad2008

±S.E. SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application; B= MN12

152

(b)

Fig. 4.6: Zinc localizaton in seed fractions of wheat using DTZ in wheat cultivar Lasani-2008 as influenced by Zn application

methods and MN12 inoculation (a) Control (b) Seed priming with Zn (c) Seed coating (d) Soil application (e) Foliar spray (f)

Control + MN12 (g) Seed priming with Zn + MN12 (h) Seed coating with Zn + MN12 (i) Soil application of Zn + MN12 (j)

Foliar application of Zn + MN12

(b)

Fig. 4.7: Zinc localizaton in seed fractions of wheat using DTZ in wheat cultivar Faisalabad-2008 as influenced by Zn

application methods and MN12 inoculation (a) Control (b) Seed priming with Zn (c) Seed coating (d) Soil application (e)

Foliar spray (f) Control + MN12 (g) Seed priming with Zn + MN12 (h) Seed coating with Zn + MN12 (i) Soil application of

Zn + MN12 (j) Foliar application of Zn + MN12

4.4.1.6 Organic acids

Zinc application in combination with Pseudomonas sp. MN12 increased the organic acid

production in the root exudates of wheat cultivars. Zinc application + MN12 increased the

production of pyruvic acid as maximum pyruvic acid concentration was recorded for soil

application of Zn + MN12 in Faisalabad-2008 followed by seed priming with Zn + MN12 in

the same cultivar (Table 4.38). Use of Pseudomonas sp. MN12 improved exudation of tartaric

acid in both wheat cultivars, however, the maximum tartaric acid concentration was recorded

from the root exudates of Lasani-2008 with the soil application of Zn + MN12 (Table 4.38).

Combined application of Zn and strain MN12 improved the synthesis of citric acid as

( a )

) ( f ( g )

( ) h ( i )

( j )

e ) (

( c ) ( d )

) a ( ( c ) ( e )

( f ) ( g ) ( h ) ( i ) ( j )

( ) d

153

maximum citric acid concentration was recorded for soil application of Zn + MN12 and

followed by seed priming with Zn + MN12 (Table 4.38). Zinc application improved the

synthesis of malonic acid. Maximum malonic acid concentration was recorded for root

exudates of Lasani-2008 where Zn was applied through seed priming + MN12 followed by

soil application of Zn + MN12 (Table 4.38). Application of Pseudomonas sp. MN12 improved

the mallic acid exudation as maximum malic acid concentration in root exudates of

Faisalabad2008 were recorded where Zn was applied through seed priming + MN12 followed

by soil and foliar application of Zn + MN12 in the same cultivar (Table 4.38). Succinic acid

production was not detected from the exudates of cultivar Lasani-2008. However, maximum

succinic acid production was observed for seed priming + MN12 followed by soil application

of Zn + MN12

(Table 4.38). Oxaloacetic acid production was highest for Lasani-2008 with Zn seed priming

+ MN12, while in Faisalabad-2008 soil application of Zn + MN12showed maximum

oxaloacetic acid exudation (Table 4.38). However, oxalic acid production was only observed

in wheat cultivarLasani-2008 and it was the highest for Zn seed priming + MN12 followed by

soil application of Zn + MN12 (Table 4.38). Production of methyl malonic acid was maximum

in Faisalabad-2008 where Zn was applied through soil application + MN12 (Table 4.38).

4.4.1.7 Concentrations of Zn, Fe and Ca in seed fractions

i. Lasani-2008

Zinc application significantly affected the Zn, Fe, and Ca concentration within grain, embryo,

aleurone and endosperm of Lasani-2008. The highest grain Zn was detected with soil and foliar

applied Zn with/without Pseudomonas sp. MN12. The embryo and aleurone Zn was the highest

in soil applied Zn + MN12. The endosperm Zn was the highest in soil applied Zn + MN12 and

that was statistically similar with foliar applied Zn with/without bacteria (Fig. 4.3a). The grain

Fe was the highest in Zn seed priming and Zn seed coating + MN12. The embryo Fe was the

highest in Zn seed coating + MN12 and Zn foliar application without MN12. The aleurone Fe

was the highest in Zn seed priming + MN12 followed by foliar applied Zn with/without

Pseudomonas sp. MN12. The endosperm Fe was the highest in Zn seed priming + MN12 (Fig.

4.4a). The grain Ca was the highest with foliar applied Zn with/without MN12, while embryo

Ca was the maximum with foliar applied Zn + MN12 or soil applied Zn without MN12. The

154

highest aleurone Ca was detected in foliar applied Zn + MN12 and Zn seed coating without

MN12 (Fig. 4.5a).

ii. Faisalabad-2008

Zinc application significantly affected the Zn, Fe, and Ca concentration within grain, embryo,

aleurone and endosperm of Faisalabad-2008. The highest grain, embryo, aleurone and

endosperm Zn was detected with soil and foliar applied Zn with Pseudomonas sp. MN12 (Fig.

4.3b).

The grain Fe was the highest in foliar applied Zn + MN12 and Zn seed priming without

MN12, and vice versa for embryo Fe. The aleurone Fe was the highest in soil and foliage

applied Zn withoutMN12. The endosperm Fe was the highest in Zn seed priming and Zn seed

coating + MN12 (Fig. 4.4b). Zn application through any method with or without Pseudomonas

sp. MN12 was quite effective for improvement in grain, embryo and endosperm Ca except Zn

seed coating without MN12. The aleurone Ca was the highest with Zn seed priming and Zn

soil application without MN12 strain (Fig. 4.5b).

4.4.2 Discussion

Zinc deficiency is widely prevalent in wheat growing areas of the world (Alloway, 2008a;

Cakmak, 2008). Wheat is the staple for masses as it provides 50% of the daily calorie intake

and 1/4 of the total Zn supplied by food is obtained from wheat and its products (Ma et al.,

2008; Cakmak, 2008). Zinc concentration in wheat grains can be increased through Zn

fertilizer application. Moreover, Zn deficiency is predominant in plants grown on calcareous

and salt-affected soils by formation of ZnCO3 which limits Zn availability for roots (Hafeez et

al., 2013). Plant growth-promoting bacteria (PGPB) improve plant growth through increased

synthesis of phytohormones, better nutrient uptake by N fixation, phosphate/zinc

solubilization, ACC-deaminase and phytosiderophore production, and micronutrient

accumulation (Martínez-Viveros et al., 2010).

In the present study, Zn application improved photosynthesis, water relation traits,

yield and yield components, bioavailable Zn, organic acid production and grain Zn localization

of bread wheat. Zinc application increased the transpiration, photosynthesis and stomatal

conductance with low intercellular CO2 (Ci) (Ahmed, 2009) as was observed in this study.

Moreover, low photosynthetic rate in Zn deficient plants might be attributed to the lowering of

the stomatal conductance and augmenting the Ci (Sharma et al., 1995; Wang and Jin, 2005).

155

Zinc fertilization increased the photosynthesis of wheat (Tables 4.29, 4.30) possibly by

affecting the carbonic anhydrase activity (Han et al., 2003). Moreover, Zn application

improved the stomatal conductance (gs) possibly by increasing the stomatal aperture due to its

involvement in stomatal regulation as it maintains the membrane integrity (Khan et al., 2004).

Inoculation of Pseudomonas sp. MN12 with Zn application further enhanced the gas exchange

traits as bacterial inoculated plants had higher gs and transpiration (E) with higher

photosynthetic rate compared to uninoculated plants (Ahmad et al., 2012, 2013).

Zinc application through any method improved the water relations of wheat. However,

further improvement in leaf water relations were observed with seed and soil application of Zn

using Pseudomonas sp. MN12 due to Zn involvement in root growth (Nable and Webb, 1993);

modification in root morphology resulting in greater surface area of root (Saravankumar et al.,

2008); which helped the plant to use soil moisture efficiently. Moreover, improvement in plant

water relations was due to better photosynthetic rate, E and gs as higher gs are linked with better

plant water relations (Khan et al., 2004).

Zinc application improved the grain yield and yield components of wheat possibly by

improvement in photosynthetic rate, water relations and higher Zn uptake, resulting in better

seed setting and increased grain yield. Further improvement in yield and yield components of

wheat was recorded with seed priming and soil application of Zn using Pseudomonas sp.

MN12 as was evident from increase in 100-grain weight, grains per spike and ultimately grain

yield as Zn plays key role in seed setting and pollination (Pandey et al., 2006); which increased

the seed weight with better harvest index. Zinc nutrition improved the grain yield of wheat as

it regulates the auxin, carbohydrate metabolism, RNA and ribosome synthesis which leads to

better yield with good quality (Khalifa et al., 2011).

Use of PGPR improved the grain yield by enhancing the uptake of nutrients in wheat

(Ramesh et al., 2014). Recently, Abaid-Ullah et al. (2015) explored that PGPR species like

Pseudomonas, Bacillus, and Serratia increased the grain Zn translocation from 7-12%

compared to chemical Zn application. For instance, Iqbal et al. (2010) found that inoculation

of Zn solubilizing bacteria in Vigna radiata improved the Zn uptake. PGPR increase the plant

growth through multiple mechanisms, most probably by modulation of phytohormone and

nutrient solubilization/uptake (Dobbelaere et al., 2003; Naveed et al., 2015). Bacterial

inoculation improved the growth and yield of wheat possibly by increasing the solubilization

156

and uptake of nutrients by plants (Lucas et al., 2004; Cakmakci et al., 2006). In the present

study, improvement in plant growth and grain yield by inoculation with Pseudomonas sp.

MN12 might be due to increased production of IAA, phosphate/zinc solubilization and

phytosiderophore production (Rana et al., 2011). For instance, Zn solubilizing PGPB improved

the plant growth as these bacteria enhance P and Zn solubilization through production of

variety of organic acids (Shaikh and Saraf, 2017). Moreover, these bacteria also increase IAA

synthesis, modulate ethylene concentration and control other biotic stresses. Furthermore,

grain yield was positively correlated with N, Zn and Fe concentration in wheat (Saraf et al.,

2013).

Zinc application increased the Zn concentration in whole seed and seed fractions of

wheat. Further improvement in Zn application was recorded with soil and foliar application of

Zn with Pseudomonas sp. MN12 (Fig. 4.6, 4.7). Application of Zn increased the Zn

concentration in wheat grain (Ranjbar and Bahmaniar, 2007; Cakmak, 2008; Waters et al.,

2009). Moreover, Zinc concentration was highest in germ >aleuron and endosperm (Oztruk et

al., 2006; Cakmak et al., 2010b) as was observed in the present study (Fig 4.6, 4.7). Higher

absorption of Zn produced higher grain yield (Han et al., 2006). Further use of PGPB increased

the grain Zn concentration more than chemical Zn sources (Abaid-Ullah et al., 2015) due to

increased production of organic acid which possibly helped in Zn solubilization and uptake by

wheat roots. Soil microbes increase the metal (micronutrient) solubilization by changing the

metal properties through organic ligands, excretion of phytosiderophores and organic acid

which form cationic-nutrient complex (Hallberg and Johnson, 2005). Furthermore, PGPB

inoculation enhanced Zn and Ca accumulation in wheat due to improved micronutrient

translocation (Rana et al., 2012a), as was observed in current study. Inoculation of

Pseudomonas sp. MN12 in combination of soil and foliar Zn fertilizer further enhanced the Zn

deposition in whole grain and seed fractions of wheat possibly due to the solubilization of

complexed Zn in the soil (Mäder et al., 2011; Shaikh and Saraf, 2017). In the present study,

application of Zn increased the Zn concentration in all seed fractions. However, maximum Zn

concentration was recorded in embryo followed by aleuron while least was observed in

endosperm as high concentration of Zn has been reported in embryo and aleuron earlier (Lombi

et al., 2011; Lu et al., 2013). Unfortunately, a significant portion of seed Zn is removed through

removal of the embryo and aleuroneic fractions of seed during polishing and milling.

157

Therefore, seeds with high Zn concentration in the endosperm will provide more Zn in a

wheatbased diet. In the present study, Zn applied with Pseudomonas sp. strain MN12 increased

the endosperm Zn concentration > 75% over the control; suggesting a significantly higher

bioavailable Zn. Inoculation with Pseudomonas sp. increased the accumulation of Zn in grains

(Sharma et al., 2015) as was observed in this study.

Phytate acts as a metal chelator and is the main P storage compound in cereals which

limit the Zn bioavailability to humans (Bohn et al., 2008). In the present study, Zn application

by any method increased the estimated bioavailable Zn in wheat grains by reducing the phytate

concentration, phytate/Zn ratio and phosphorus concentration as Zn fertilization reduced the

phytate concentration earlier (Imran et al., 2015). Application of Zn as soil and foliar with

Pseudomonas sp. MN12significantly enhanced the bioavailable Zn as Zn bioavailability is

linked with reduced phytate and phosphorus concentration in grain (Cakmak et al., 2010b).

Root exudation involves secretion and excretion of plant photosynthates. In excretion,

gradient dependent waste material with unknown function is released while release of material

with known function like defense and lubrication is known as secretion (Uren, 2000; Bais et

al., 2004). Root exudates are of two type; low and high molecular weight compounds. Amino

acids, sugars, phenols and secondary metabolites are low organic root exudates (Hawes et al.,

2000; Vicre et al., 2005). Root exudates help in precipitation of heavy metal ions by binding

and absorbing metal outside of the root (Lin et al., 1998). Moreover, PGPB also enhanced the

organic acid production in the root exudates (Zhang et al., 2013). In the present study,

application of Zn alone did not improve the organic acid concentration in root exudates of

wheat. However, use of Pseudomonas sp. MN12 in combination with Zn applied as soil and

seed priming enhanced the organic acid concentration in root exudates of wheat as was visible

from increased production of pyruvic acid, tartaric acid, citric acid, malonic acid, mallic acid,

succinic acid, oxaloacetic acid, oxalic acid and methylmalonic acid (Table 4.38). Among these

organic acids, succinic acid was only detected in root exudates of Faisalabad-2008 while

oxaloacetic acid and oxaleic acid were present in root exudates of Lasani-2008. Root activities

of plants influence the availability of soil nutrients by production of compounds like organic

acids which increases the solubility and thus improving the availability of nutrients (Miransari,

2013). These organic compounds change the rhizospheric pH thus affect the nutrient

availability and soil microbial activity (van der and Heijden, 2010; Hodge, 2010; Johnson et

158

al., 2010) as PGPB found to enhance the phytosiderophore production which enhance the Zn

and Fe acquisition by plants (Carrillo-Castañeda et al., 2005).

In the present study, Zn application improved gas exchange traits, water relations, yield

related traits and bioavailable Zn. Among the Zn application methods used in this study, soil

Zn fertilization and seed priming in combination with Pseudomonas sp. MN12 proved

beneficial in improving the photosynthetic traits, water relation and grain yield. Moreover, soil

and foliar fertilization with Pseudomonas sp. MN12 significantly enhanced the grain Zn

concentration and Zn accumulation in all seed fractions with higher bioavailable Zn and lowest

phytate concentration and [phytate]:[Zn]ratio.

In conclusion, Zn application in combination with Pseudomonas sp. MN12 by any

method (soil application; foliar spray and seed treatment) improved gas exchange, water

relations, grain yield, Zn bioavailability and organic acid exudation in wheat. Soil and seed

priming in combination with the Pseudomonas sp. strain MN12 were the most effective

treatments to improve the physiology, root organic acid exudation and grain yield of wheat,

while, soil and foliar application of Zn with and without MN12 increased the Zn concentration

in whole grains and seed fractions with improved bioavailability.



4.5.1. Results (Field experiment)

4.5.1.1 Water relations

Zinc application significantly affected the RWC, water potential, osmotic potential and

pressure potential of wheat during both of the experimental years (Tables 4.39). Likewise,

wheat cultivars differ significantly for water potential, osmotic and pressure potential during

the first year (Table 4.39). During second year, wheat cultivars differ significantly for RWC

and water potential (Table 4.39). The interaction of Zn application with wheat cultivars was

significant for water potential during the second year (Table 4.39) and for osmotic potential

during the first year of experimentation (Table 4.39).

Among wheat cultivars, the highest RWC during the second year were recorded in

Faisalabad-2008 (Tables 4.40). However, water potential during the second year was the

lowest in Lasani-2008 (Table 4.40).

159

The maximum RWC during the first year were recorded with Zn seed priming + endophytic

strain MN12. During second year, the highest RWC were recorded with soil application of Zn +

MN12 (Table 4.40). The water potential during first year was the lowest with Zn seed priming +

MN12 (Table 4.40). Moreover, the lowest osmotic potential during the second year was recorded

with the soil application of Zn + MN12 (Table 4.40). The highest pressure potential was noted with

the Zn seed priming with endophytic Pseudomonas sp. MN12 during both years which was

statistically similar with soil application of Zn with or without MN12 during the second year of

experimentation (Table 4.40). The interaction showed that the lowest osmotic potential was recorded

with Zn seed priming along with MN12 in FSD2008 during the first year of experimentation. The

water potential was the lowest in Zn seed priming with MN12 in FSD-2008 (Table 4.41).


Zinc application significantly affected the 1000-grain weight, productive tiller, grain

yield and harvest index during the both experimental years; grains per spike during the first

year (Table 4.42). Moreover, wheat cultivars differed significantly for grains per spike and

1000- grain weight for both year; for grain yield during first year and for productive tillers

during the second year of experimentation (Table 4.42). However, wheat cultivars did not

differ significantly for harvest index during both years; productive tiller during first year and

grain yield during second year of experimentation (Table 4.42). The interaction of wheat

cultivars with the Zn application was found non-significant for all the studied traits (Table

4.42).

Among wheat cultivars, maximum grains per spike during both years, productive tillers

during second year and grain yield during the first year of experimentation were recorded in

Faisalabad-2008. However, 1000-grain weight was the highest in Lasani-2008 during the both

years (Table 4.43). Maximum grains per spike during the first year were recorded with the soil

application of Zn without strain MN12 or Zn seed priming + MN12 (Table 4.43). 1000-grain

weight was the highest with Zn seed priming + MN12 during both years which was statistically

similar with foliage applied Zn with/without strain MN12 or seed priming with Zn without

MN12 (Table 4.43). The highest productive tillers were recorded with Zn seed priming +

MN12 during both years which were statistically similar with soil applied Zn + MN12 (Table

4.43). Grain yield during the first year and harvest index during the both years was the highest

with Zn seed priming and Pseudomonas sp. MN12 (Table 4.43).

Table

160

- significant; p ≤ 0.05; **= p ≤ *= 0.01

4.39: Analysis of variance for effect of Zn application and Pseudomonas sp. MN12 on water relation traits of wheat cultivars

Treatment (T) 9 80.7** 59.9** 0.039** 0.069** 0.016** 0.023** 0.007** 0.015** Cultivar (C) 1 8.3ns 60.9* 0.034** 0.012** 0.007** 0.002ns 0.009* 0.004ns T × C 9 34.3ns 9.58ns 0.001ns 0.003* 0.002* 0.001ns 0.002ns 0.002ns Error 57 22.0 14.2 0.001 0.001 0.001 0.001 0.002 0.002 Total 79

SOV= Sources of variation; DF= Degree of freedom; ns= non

Table 4.40: Influence of Zn application and Pseudomonas sp. MN12 on water relation traits of wheat cultivars

Treatments Relative water contents Water potential (-MPA) Osmotic potential (-MPA) Pressure potential (- MPA) Wheat cultivars 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15

Lasani-2008 68.6 69.8 B 1.28 A 1.45 A 1.78 A 1.78 0.50 0.33 Faisalabad-2008 69.3 71.6 A 1.23 B 1.42 B 1.76 B 1.77 0.52 0.35

No microbe Hydropriming 65.5 AB 66.8 C 1.40 A 1.64 A 1.86 A 1.89 A 0.46 C 0.25 D

SP (0.5 M Zn) 71.2 AB 73.4 AB 1.23 CDE 1.39 E 1.76 CDE 1.76 CD 0.53 ABC 0.37 AB

SC (1.25 g kg-1 seed) 69.7 AB 69.9 ABC 1.30 B 1.47 BC 1.80 BC 1.80 B 0.50 ABC 0.34 ABC

SA (10 kg Zn ha-1) 68.2 AB 70.6 ABC 1.21 DEF 1.38 EF 1.74 DEF 1.75 CDE 0.53 ABC 0.37 A

FA (0.025 M Zn) 65.1 B 68.4 BC 1.27 BCD 1.43 CDE 1.78 BCD 1.79 BC 0.51 ABC 0.35 ABC

Pseudomonas sp.

MN12 Hydropriming

SP (0.5 M Zn) 68.1 AB 73.2 A

68.2 BC 74.2 AB

1.31 B 1.16 F

1.51 B 1.33 F

1.81 B 1.72 EF

1.82 B 1.72 DE

0.50 ABC 0.56 A

0.30 BCD 0.39 A

SC (1.25 g kg-1 seed) 70.7 AB 71.3 ABC 1.28 BC 1.46 BCD 1.76 CDE 1.76 CD 0.48 BC 0.29 CD

SA (10 kg Zn ha-1) 70.8 AB 74.9 A 1.17 EF 1.33 F 1.71 F 1.70 E 0.54 AB 0.38 A

FA (0.025 M Zn) 68.6 AB 69.2 ABC 1.23 CDE 1.41 DE 1.74 DEF 1.75 CD 0.52 ABC 0.34 ABC

SOV DF Relative water content Water potential Osmotic potential Pressure potential 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15

Replication 3 80.8 16.5 0.001 0.004 0.001 0.003 0.001 0.011

HSD (p ≤ 0.05) ns 1.69 0.016 0.016 0.011 ns ns ns Treatments - -

Table

161

Figures of main effect sharing same case letter, for a parameter, don’t differ significantly at p ≤ 0.05; NS= non-significant SP=

Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application; M= Molar

4.41: Influence of Zn application and Pseudomonas sp. MN12 on osmotic potential (2013-14) and water potential of wheat (2014-15)

No microbe Hydropriming 1.87 a 1.85 ab 1.65 a 1.62 ab

SP (0.5 M Zn) 1.75 c-f 1.76 bcd 1.38 g-k 1.40 e-j

SC (1.25 g kg-1 seed) 1.80 a-d 1.79 cde 1.46 c-g 1.47 c-f

SA (10 kg Zn ha-1) 1.75 c-f 1.73 def 1.37 g-k 1.38 f-k

FA (0.025 M Zn) 1.79 bcd 1.77 cde 1.44 d-h 1.43 d-i

Pseudomonas sp.

MN12 Hydropriming

SP (0.5 M Zn) 1.82 abc 1.74 def

1.80 a-d 1.69 f

1.54 bc 1.35 h-k

1.48 cde 1.31 k

SC (1.25 g kg-1 seed) 1.80 bcd 1.72 ef 1.51 cd 1.42 d-i

SA (10 kg Zn ha-1) 1.70 ef 1.71 ef 1.33 ijk 1.32 jk

FA (0.025 M Zn) 1.74 def 1.74 def 1.42 d-i 1.39 e-k

Means of interaction sharing same case letter, for a parameter, don’t differ significantly at p ≤ 0.05 SP=


HSD (p ≤ 0.05) 7.96 6.21 0.060 0.059 0.042 0.042 0.075 0.067

Treatments Osmotic potential at anthesis ( - MPA) Water potential at anthesis ( - MPA) LS - 2008 FSD - 2008 LS - 2008 FSD - 2008

HSD (p ≤ 0.05) 0.068 0.095

Table

162

Table 4.42: Analysis of variance for effect of Zn application and Pseudomonas sp. MN12 on yield parameters of wheat cultivars

Treatment (T) 9 6099** 2827.9* 30.7* 7.05ns 2.65** 3.19** 0.83** 0.68** 78.8** 97.1** Cultivar (C) 1 92.5ns 1862.5* 224** 23.5* 5.57* 12.4** 1.37** 0.08ns 6.96ns 9.66ns T × C 9 484.5ns 370.2ns 2.80ns 0.98ns 0.34ns 0.61ns 0.07ns 0.01ns 7.57ns 7.85ns Error 57 858.7 419.6 12.3 4.59 0.90 0.89 0.08 0.06 17.2 14.9 Total 79


4.43: Effect of Zn application and Pseudomonas sp. MN12 on yield parameters of wheat cultivars Treatments GPS HGW (g) Productive Tiller (m-2) Grain yield (Mg ha-1) HI (%)

2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 2013-14 2014-15 Wheat cultivars Lasani-2008 44.7 B 46.3 B 43.9 A 44.1 A 392 364.8 B 3.73 B 3.31 37.8 36.9

Faisalabad-2008 48.1 A 47.4 A 43.4 B 43.4 B 395 374.5A 4.00 A 3.37 38.4 37.6

ns ns ns Treatments

No microbe Hydropriming 42.8 B 45.2 42.3 B 42.6 C 359 C 336 C 3.51 D 3.05 C 35.9 B 32.4 C

SP (0.5 M Zn) 48.5 AB 47.5 44.0 A 44.4 AB 408 B 379 AB 4.11 ABC 3.71 AB 39.9 AB 41.5 AB

SC (1.25 g kg-1 seed) 46.2 AB 47.0 43.9 AB 43.0 BC 378 BC 353 BC 3.59 D 3.36 BC 34.9 B 37.9 ABC

SA (10 kg Zn ha-1) 48.8 A 47.2 43.5 AB 43.9 ABC 412 AB 379 AB 3.85 BCD 3.42 BC 38.3 AB 37.7 ABC

SOV DF Productive tillers Grains per spike 1000 - grain weight Grain yield Harvest index 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15 2013 - 14 2014 - 15 Replication 3 514 209.8 3.30 0.98 1.75 1.13 0.23 0.09 2.16 12.3

HSD (p ≤ 0.05) 1.57 0.96 0.42 0.42 9.17 9.17 0.13

Table

163

FA (0.025 M Zn) 44.9 AB 46.8 44.1 A 43.8 ABC 386 BC 366 ABC 3.60 D 3.08 C 34.9 B 36.1 BC

Pseudomonas

sp. MN12 Hydropriming SP

(0.5 M Zn) 45.1 AB 48.7 A

45.5 47.9

43.0 AB 44.1 A

43.3 ABC 44.7 A

391 BC 457 A

358 BC 396 A

3.75 CD 4.46 A

3.08 C 3.92 A

37.5 B 44.5 A

34.4 C 43.9 A

SC (1.25 g kg-1 seed) 46.9 AB 46.8 43.6 AB 43.8 ABC 386 BC 372 AB 3.91 BCD 3.27 C 38.9 AB 37.5 BC

SA (10 kg Zn ha-1) 47.0 AB 48.1 43.7 AB 43.9 ABC 392 BC 396 A 4.26 AB 3.40 BC 41.2 AB 37.3 BC

FA (0.025 M Zn) 45.1 AB 47.0 44.2 A 44.1 ABC 368 BC 363 ABC 3.60 D 3.11 C 35.1 B 33.6 C

Figures of main effects sharing same letter, for a parameter, don’t differ significantly at p ≤ 0.05; PH= Plant height; GPS= Grains per spike; HGW= 1000- grain weight; HI= Harvest index; NS= non-significant; SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application; M= Molar

Table 4.44: Analysis of variance for the effect of Zn application and Pseudomonas sp. MN12 on grain minerals composition of wheat

cultivars

Replication Treatment (T)

Cultivar (C) 1 0.265ns 0.058ns 0.078ns 0.313** 0.187** 0.070** 84.1** 39.9** T × C 9 0.558** 0.609ns 0.010 0.041* 0.007ns 0.016* 3.74ns 9.17**


HSD (p ≤ 0.05) 5.76 Ns 1.56 1.55 48.2 33.7 0.463 0.39 6.83 6.35

SOV DF

Grain protein content Phytate Bioavailable Zn [ Phytate]:[Zn ]

2013 - 14 2014 - 15

2013 - 14 2014 - 15

2013 - 14 2014 - 15

2013 - 14 2014 - 15

3

0.604 0.227 0.062 0.025 0.004 0.001 1.40 1.53 9

1.241** 1.460** 1.864** 1.670** 0.541** 0.599** 281** 256.5**

Error 57

0.199 0.317 0.024 0.021 0.012 0.006 6.13 2.36 Total

79

Table

164

165

Table 4.45: Effect of Zn application and Pseudomonas sp. MN12 on grain mineral composition of wheat

Treatments

Phytate (mg g-1) Bioavailable Zn (mg day-1) [Phytate]:[Zn] Grain protein (%) 2013-14 2013-14 2013-14 2014-15

Wheat cultivars Lasani-2008 8.57 2.28 B 29.8 A 11.8 Faisalabad-2008 8.51 2.38 A 27.7 B 11.7

ns Treatments

No microbe Hydropriming 9.46 A 1.80 F 41.9 A 11.2 BC

SP (0.5 M Zn) 8.54 BC 2.28 CD 29.2 CD 11.7 ABC

SC (1.25 g kg-1 seed) 8.68 B 2.14 DE 32.2 BC 11.8 ABC

SA (10 kg Zn ha-1) 8.36 CDE 2.52 AB 24.7 EF 11.8 ABC

FA (0.025 M Zn) 8.24 DE 2.48 AB 25.4 DEF 12.3 A

Pseudomonas sp.

MN12 Hydropriming

SP (0.5 M Zn) 9.25 A 8.32 CDE

2.07 E 2.46 AB

34.1 B 25.6 DEF

11.6 ABC 12.1 AB

SC (1.25 g kg-1 seed) 8.47 BCD 2.35 BC 27.7 DE 11.0 C

SA (10 kg Zn ha-1) 8.18 E 2.59 A 23.6 F 12.2 A

FA (0.025 M Zn) 7.90 F 2.62 A 22.9 F 11.9 ABC

SP= Seed priming; SC= Seed coating; SA= Soil application; FA= Foliar application; M= Molar

Table 4.46: Influence of Zn application and Pseudomonas sp. MN12 on grain protein (2013-14), phytate Concentration, phytate/Zn ratio and

bioavailable Zn of wheat cultivars (2014-15)

No microbe Hydropriming 11.3 abc 10.8 bc 9.48 a 9.30 a 42.0 a 35.8 b 1.80 j 2.00 ij

HSD (p ≤ 0.05) ns 0.024 1.11

HSD (p ≤ 0.05) 0.255 0.179 4.07 0.93 Figures of main effects sharing same letter, for a parameter, don’t differ significantly at p ≤ 0.05

Treatments Grain Protein (%) Phytate (mg g - 1 ) Phytate]:[Zn [ ] Bioavailable Zn (mg day - 1 ) LS - 2008 FSD - 2008 LS - 2008 FSD - 2008 LS - 2008 FSD - 2008 LS - 2008 FSD - 2008

166

SP (0.5 M Zn) 11.9 ab 11.7 ab 8.51 cde 8.39 c-g 27.7 def 26.9 efg 2.35 efg 2.39 def

SC (1.25 g kg-1 seed) 11.4 ab 12.0 a 8.69 bc 8.49 cde 31.4 cd 28.6 de 2.17 ghi 2.30 e-h

SA (10 kg Zn ha-1) 11.7 ab 11.3 abc 8.21 efg 8.26 d-g 22.4 hij 23.1 g-j 2.66 ab 2.61 abc

FA (0.025 M Zn) 11.6 ab 11.9 ab 8.19 efg 8.11 fgh 22.7 hij 22.5 hij 2.64 ab 2.65 ab

Pseudomonas sp.

MN12 Hydropriming

SP (0.5 M Zn) 11.2 abc 11.3 ab

11.6 ab 11.2 abc

9.33 a 8.37 c-g

8.90 b 8.37 c-g

35.3 bc 25.1 e-i

33.1 bc 26.2 e-h

2.02 i 2.49 b-e

2.10 hi 2.43 c-f

SC (1.25 g kg-1 seed) 11.3 ab 10.2 c 8.59 bcd 8.49 c-f 29.0 de 28.5 de 2.29 fgh 2.31 e-h

SA (10 kg Zn ha-1) 12.2 a 11.9 ab 8.10 gh 8.13 e-h 22.4 hij 21.7 ij 2.66 ab 2.71 a

FA (0.025 M Zn) 11.3 abc 11.2 abc 8.02 gh 7.80 h 23.7 f-j 21.0 j 2.57 a-d 2.75 a

Figures of main effects sharing same letter, for a parameter don’t differ significantly at p ≤ 0.05; SP=


HSD (p ≤ 0.05) 1.17 0.378 4.03 0.205

167

4.5.1.3 Grain analysis

Zinc application significantly affected grain protein, phytate, phytate/Zn ratio and

bioavailable Zn during both years of study. However, wheat cultivars did not differ

significantly for phytate during first year and grain protein content during both years of

experimentation (Table 4.44). Moreover, interaction of Zn application methods with wheat

cultivars was significant for phytate, bioavailable Zn, phytate/Zn ratio, during second year and

grain protein contents during first year of experimentation (Table 4.44).

Among wheat cultivars during first year, the highest bioavailable Zn was detected in

FSD-2008; however phytate/Zn ratio was the highest in LS-2008 (Table 4.45). Among Zn

application methods, the highest bioavailable Zn, and the lowest phytate concentration and

phytate/Zn molar ratio during the first year of experimentation was recorded with the foliage

and soil applied Zn + MN12 (Table 4.45). During 2014-15, the highest grain protein was

recorded with foliage applied Zn without endophytic bacteria and soil applied Zn with MN12

(Table 4.45). The interaction showed that the highest grain protein during the first year was

recorded with soil applied Zn with MN12 in LS-2008 and seed coating with Zn without

bacteria MN12 in FSD-2008 (Table 4.46). Bioavailable Zn during the second year were the

highest with foliage and soil applied Zn + MN12 in Faisalabad-2008 which was followed by

soil applied Zn + MN12 in LS-2008 (Table 4.46). The lowest phytate concentration and

phytate/Zn molar ratio was found with foliage applied Zn + strain MN12 in FSD-2008 during

the second year (Table 4.46).

In LS-2008, maximum grain Zn concentration was recorded with soil and foliar

application of Zn with and without endophytic bacteria. Nevertheless, embryo Zn was the

highest with foliar application of Zn with/without MN12 and soil application of Zn + MN12.

The highest aleurone Zn was recorded with foliar applied Zn + MN12. Endosperm Zn was

highest with soil application of Zn without bacterial strain MN12 (Fig. 4.8a). In cultivar

FSD2008, the highest grain Zn was recorded where Zn was applied as soil and foliar in

combination with endophytic strain MN12. In embryo and endosperm, Zn concentration was

maximum with foliar applied Zn + MN12. Likewise, Zn applied as foliar and soil application

in combination with endophytic bacteria MN12 resulted maximum aleurone Zn concentration

(Fig. 4.8b).

168

In LS-2008, the highest grain, embryo, aleurone and endospermic Fe were recorded with Zn

seed priming + MN12 (Fig. 4.9a). In FSD-2008, the highest grain Fe concentration was recorded

with seed coating without bacteria, seed priming + MN12, soil application + MN12, foliar

application with and without endophytic bacteria MN12. However, the maximum concentration of

embryo and endosperm Zn was recorded with seed coating and seed priming in combination with

Pseudomonas sp. MN12, respectively (Fig. 4.9b).

In LS-2008, highest grain, embryo, aleurone and endosperm Ca was recorded with soil

application of Zn with strain MN12 (Fig. 4.10a). In cultivar FSD-2008, the maximum grain Ca

was recorded with foliar applied Zn; while embryo and aleurone Zn was the maximum with

soil and foliar application of Zn + MN12. However, highest endospermic Ca was recorded

with soil application of Zn and Pseudomonas sp. MN12 (Fig. 4.10b).

4.5.1.4 Economic and marginal analyses

Economic analysis of different Zn application methods and endophytic bacteria MN12

of wheat cultivars LS-2008 and FSD-2008 is given in Table 4.47. Seed priming with Zn +

MN12 resulted in maximum gross income, net benefit and benefit: cost ratio in both wheat

cultivars. Similarly, the maximum marginal rate of return was recorded in the cultivar

FSD2008 (Table 7). Among the Zn application methods, seed priming of Zn in combination

with Pseudomonas sp. MN12 resulted in maximum marginal rate of return in both cultivars

(Table

4.48).

4.5.2. Discussion

Wheat covers about 1/4 of total cultivated area of the world (Shewry, 2009). Zinc

deficiency is wide spread in wheat as the 50% of the wheat cultivated soils are poor in plant

available Zn (Alloway, 2008a; Zou et al., 2008), resulting in low wheat yield with poor grain

quality (Zhao et al., 2011). Zinc deficiency in wheat can be corrected by application of Zn

fertilizers (Cakmak, 2008). Moreover, use of Zn solubilizing endophyte also increase the grain

Zn accumulation though enhanced uptake of Zn by different mechanisms viz. nutrient cycling,

transformation, mineralization and decomposition (Rana et al., 2012a, b).

169

Fig. 4.8:

Influence of Zn application and Pseudomonas sp. MN12 on grain and seed fractions Zn

concentration of wheat cultivars (a) Lasani-2008 (b) Faisalaabad-2008 ±S.E. SP= Seed

priming; SC= Seed coating; SA= Soil application; FA= Foliar application; B=

Pseudomonas sp. MN12

170

B

A A AB AB

Fig. 4.9: Influence of Zn application and Pseudomonas sp. MN12 on grain and seed fractions Fe




171

Fig. 4.10:

Influence of Zn application and Pseudomonas sp. MN12 on grain and seed fractions Ca




Table 4.47: Influence of Zn application and Pseudomonas sp. MN12 on economics of wheat

cultivars

Treatments Grain yield

(t ha-1)

Straw yield

(t ha-1)

Total Cost

(USD)

Gross income

(USD)

Net income

(USD)

Benefit-cost

ratio

Lasani-2008

Hydropriming 2.97 6.71 788.5 1104.1 315.6 1.40

Osmopriming 3.74 5.62 850.0 1293.8 443.8 1.52

172

Seed coating 3.30 6.40 827.7 1190.3 362.6 1.44

Soil application 3.46 5.98 895.3 1223.1 327.8 1.37

Foliar spray

3.04

6.17

827.6

1106.2

278.7

1.34

Hydropriming+ MN12 3.05 6.61 789.9 1121.7 331.8 1.42

Osmopriming+MN12 3.98 5.45 851.9 1357.5 505.6 1.59

Seed coating+ MN12 3.23 5.95 829.6 1153.4 323.9 1.39

Soil application+ MN12 3.36 6.57 897.2 1214.4 317.2 1.35

Foliar spray+ MN12 3.08 6.31 829.5 1120.4 290.9 1.35

Faisalabad-2008

Hydropriming 3.13 6.40 788.5 1139.1 350.6 1.44

Osmopriming 3.75 6.13 850.0 1314.3 464.3 1.55

Seed coating 3.35 6.11 827.7 1194.4 366.7 1.44

Soil application 3.44 6.32 895.3 1228.3 333.0 1.37

Foliar spray 3.14 6.66 827.6 1151.2 323.7 1.39

Hydropriming+ MN12 3.16 6.20 789.9 1143.0 353.1 1.45

Osmopriming+MN12 3.84 5.81 851.9 1328.8 476.9 1.56

Seed coating+ MN12 3.33 6.49 829.6 1200.3 370.7 1.45

Soil application+ MN12 3.46 6.29 897.2 1232.7 335.5 1.37

Foliar spray+ MN12 3.10 7.16 829.5 1156.6 327.1 1.39

Total cost, gross income and net income was calculated according to Pakistan price and expressed in USD;

1USD= 104.82 PKR

Table 4.48: Influence of Zn application and Pseudomonas sp. MN12 on marginal analysis of

wheat cultivars

Treatments Cost that vary

(USD)

Marginal cost

(USD)

Net benefits

(USD)

Marginal net benefits

(USD)

Marginal rate

of return (%)

Lasani-2008

Hydropriming 109.3 315.6

Osmopriming 170.8 61.5 443.8 128.1 208.3

Seed coating 148.5 39.2 362.6 47.0 119.9

Soil application 216.1 106.8 327.8 12.2 11.4

Foliar spray 148.3 39.0 278.7 -37.0 -94.7

173

Hydropriming+ MN12 110.7 331.8

Osmopriming+MN12 172.7 62.0 505.6 173.8 280.4

Seed coating+ MN12 150.4 39.7 323.9 -7.9 -20.0

Soil application+ MN12 218.0 107.3 317.2 -14.6 -13.6

Foliar spray+ MN12 150.2 39.5 290.9 -40.9 -103.4

Faisalabad-2008

Hydropriming 109.3 350.6

Osmopriming 170.8 61.5 464.3 113.7 184.8

Seed coating 148.5 39.2 366.7 16.1 41.0

Soil application 216.1 106.8 333.0 -17.6 -16.5

Foliar spray 148.3 39.0 323.7 -26.9 -69.0

Hydropriming+ MN12 110.7 353.1

Osmopriming+MN12 172.7 62.0 476.9 123.8 199.8

Seed coating+ MN12 150.4 39.7 370.7 17.6 44.5

Soil application+ MN12 218.0 107.3 335.5 -17.6 -16.4

Foliar spray+ MN12 150.2 39.5 327.1 -26.0 -65.7

Cost that vary, Marginal cost; Net benefits and Marginal net benefits were calculated according to Pakistan price and expressed in USD; 1USD= 104.82 PKR

In the present study, Zn application improved the water relations of wheat cultivars as

Zn deficient leaves have poor water status than leaves receiving the adequate Zn (Khan et al.,

2003; 2004). Delivery of Zn through soil and seed priming with and without Pseudomonas

MN12 were the most effective in improving plant water relations as Zn is involved in root

growth (Khan et al., 1998); thus improving the ability of plant to exploit the soil moisture.

Furthermore, improvement in plant water relations by application of Zn was possibly due to

involvement of Zn in regulation of stomatal conductance and plant water relations (Sharma et

al., 1984; Khan, 1998; Khan et al., 2004). Inoculation of Pseudomonas strain MN12 with

chemical Zn sources further improved the plant water relations of wheat possibly due to better

root growth (Dodd et al., 2010) which helped in higher water uptake deep from the soil as

endophytic bacteria have higher activities of ACC deaminase which enhanced the root growth

(Naveed et al., 2014).

174

Zinc delivery enhanced the yield and yield components of wheat as was visible from

increase in productive tillers, 1000-grain weight, grains per spike, grain yield and harvest index

of wheat. Furthermore, use of endophytic bacteria further improved the grain yield of wheat

possibly due to better plant growth, Zn accumulation in grains, root morphology and grain

weight (Wang et al., 2014).

Seed priming and soil application of Zn resulted in improvement in grain yield due to

substantial increase in productive tillers, grain weight and grains per spike. Moreover, the use

of MN12 strain in combination with soil and seed priming of Zn further improved the yield

and yield contributing traits as beneficial soil microbes enhance the solubility and availability

of Zn present in soil (He et al., 2010). This improvement in grain yield of wheat was possibly

due to involvement of Zn in carbohydrate metabolism, IAA, RNA and ribosomal function.

Furthermore, increase in grain yield due to Zn application was possible outcome of improved

plant growth, seed setting, nutrient uptake and plant biochemical traits (Khalifa et al., 2011).

In the present study, increase in grain yield by seed priming with Zn + MN12 was 24.5-27.1%

during both years. Improvement in grain yield by inoculating pseudomonas sp. MN12 with Zn

soil application and Zn seed priming might be due to enhanced IAA production, phosphate

solubilization and siderophore production (Naveed et al., 2014) which helped in increased Zn

uptake. Moreover, endophytic bacterial inoculation improved the grain yield of wheat possibly

due to better root growth (Wang et al., 2014; Naveed et al., 2014; Dodd et al., 2010),

photosynthesis and nutrient uptake (Naveed et al., 2014) and enhanced production of

phytohormones like indole acetic acid (IAA), cytokinins and gibberellins and some specific

amino acids (Marschner, 2006; Matthijs et al., 2007).

In the present study, application of Zn increased the Zn concentration in the whole seed

and all seed fractions of wheat as Zn application in wheat augments the Zn accumulation in

seed and leaves (Ranjbar and Bahmaniar, 2007; Cakmak, 2008; Waters et al., 2009). Zinc

fertilization increased the grain Zn concentration in wheat cultivars LS-2008 (58.1%) and

FSD2008 (32.5%). Likewise, improvement in Zn concentration in seed fraction i.e. embryo,

aleurone and endosperm was 37.4, 37.2 and 35.05% for Ls-2008; and 14.2, 26.4 and 24.8%

for Fsd-2008, respectively. Further improvement in Zn concentration was observed by use of

endophytic bacterial strain MN12 in combination with Zn application methods. The increase

in Zn concentration by use of beneficial bacteria MN12 is possibly due to mineralization of Zn

175

present in soil pool. Use of bio inoculant reduces the soil pH (Yu et al., 2011; Ramesh et al.,

2014); possibly due to production of organic acids as was observed in this study. Moreover,

low rhizospheric pH due to organic acid production is linked with H+ extrusion which causes

substantial decrease in soil pH (Dinkelaker et al., 1989; Neumann and Römheld, 2002).

Furthermore, endophytes enhance the production of organic acid which increase the

solubilization and ease the mobilization of Zn by reducing the sorption and changing the

characteristics of soil colloids (Jones, 1998). Use of endophyte increased the Zn concentration

in whole seed and seed fractions due to enhanced uptake of Zn owing to better root growth

(Singh et al., 2017) due to higher activity of ACC deaminase activity (Dodd et al., 2010) and

IAA production (Vessey, 2003), which possibly enhanced the Zn uptake deep from the soil. In

the present study, use of endophytic bacteria enhanced the Zn concentration in grain and seed

fractions of wheat as endophytic bacteria trigger the Zn uptake by modulating root morphology

and increasing the sugar, organic acid production in the root exudates of wheat and expression

of TaZIP genes which escalated the Zn accumulation in wheat (Singh et al., 2017).

Cereals are Zn deficient, and among the cereals, staple grains like wheat have low

micronutrient concentrations such as Zn and Fe and most of these minerals are removed by

milling (Borrill et al., 2014) as Zn concentration is high in embryo and aleurone layer of cereal

grains (Lombi et al., 2011; Lu et al., 2013). The localization of Zn is high in embryo and

aleurone layer (Ozturk et al., 2006; Cakmak et al., 2010b) which are removed during polishing.

Therefore, grains with high Zn concentration in endosperm will have more bioavailable Zn

(Olsen and Palmgren, 2014). In the present study, Zn application increased the Zn

concentration in all seed fractions (embryo, aleurone and endosperm) with Zn concentration

following order embryo > aleurone > endosperm. More importantly, Zn fertilization through

soil and leaf without or in combination of Zn solubilizing Pseudomonas strain (MN12)

enhanced the endosperm Zn concentration over control; suggesting the higher bioavailability

of Zn to human gut. In addition to increased Zn bioavailability, seed with high Zn concentration

results in better seedling emergence with higher vigor and high yield under Zn deficient

conditions (Rengel and Graham, 1995; Yilmaz et al., 1998; Harris et al., 2007). Soil and foliar

application of Zn increased the Ca concentration in whole grain and all seed fractions as soil

Zn fertilization increased the Ca accumulation and its bioavailability in wheat grains (Hussain

et al., 2012). Zinc application also increased the protein content of wheat as Zn fertilization

176

increased the quantity and quality of grain protein in wheat by escalation in concentration of

glutenins, albumins, gliadins and globulins (Liu et al., 2015).

Application of Zn enhanced the Zn bioavailability by lowering the phytate

concentration as phytate reduce the Zn absorption by human intestine (Weaver and Kannan,

2002). Generally, wheat cultivars have phytate concentration around 10 mg g-1 (Lott et al.,

2000). The decrease in phytate concentration in our study was possibly due to change in uptake

of P from soil and its translocation within plants (Huang et al., 2000). Moreover, increase in

estimated bioavailable Zn in wheat grain was due to reduction in [phytate]: [Zn] ratio due to

the presence of low phytate and P concentration in the seeds of wheat supplied with high Zn

concentration. Furthermore, improvement in Zn biofortification and bioavailable Zn by Zn

application was due to lower concentration of antinutrient concentration in seed and lower

phytate to Zn molar ration which is predictor of bioavailable Zn in food (Cakmak et al., 2010a).

Increasing the Zn concentration in whole grain through Zn fertilization also increased the Zn

deposition in starchy endosperm of wheat (Cakmak et al., 2010b), which enhanced the Zn

bioavailability (Table 4.45, 4.46) as endosperm has very low phytate concentration.

The main purpose of farmer is to get economic return from the introduction of any

innovative technology. This study highlighted that Zn application + MN12 not only increased

the grain yield but it also resulted in high net benefit, benefit: cost ration (BCR) and high

marginal rate of return. Zinc fertilization increased the grain yield and BCR as reported earlier

(Singh and Shivay, 2013; Manzeke et al., 2014; Wang et al., 2015). Seed priming of Zn in

combination with Pseudomonas sp. MN12 was the best method for Zn application. This

increase in net benefit in the present study due to seed priming of Zn in combination with

Pseudomonas sp. MN12 was due to lower cost of seed priming than soil application and higher

grain yield.

In conclusion, Zn application improved the performance of bread wheat. Soil

application and seed priming with Zn in combination of endophytic bacteria proved effective

in improving the water relation, grain yield. Moreover, leaf and soil application of Zn increased

the accumulation and localization of Zn in whole seed and all seed fractions. Moreover,

maximum net income was recorded for seed primed with Zn + MN12 in both wheat cultivars.

177

4.6. Improving the Drought Resistance in Wheat through Zinc Nutrition

4.6.1. Resutls

4.6.1.1 Gas exchange, biomass and relative water content

Analysis of variance depicted that drought stress significantly affected all the studied

parameters expect water use efficiency (WUE). Application of Zn also affected all the gas

exchange traits significantly (Table 4.49). However, wheat cultivars differed significantly only

for chlorophyll intensity and inter cellular CO2 (Ci) (Table 4.49). Moreover, interaction of

wheat cultivars and drought stress was significant for chlorophyll intensity, rate of

photosynthesis (A), rate of transpiration (E) and Ci, while, interactive effect of wheat cultivars

and Zn application was significant only for Ci, and quantum yield (QY). Furthermore,

interaction of drought stress and Zn treatment was significant for WUE and Ci, while,

interaction of Zn application, wheat cultivars and drought stress was only significant for

stomatal conductance (gs) and QY. However, results were not significant for rest of the

parameters (Table 4.49).

Zinc application increased the chlorophyll intensity. In this regard, maximum SPAD

value was recorded for cultivar Faisalabad-2008 (FSD-2008) under drought stress with

adequate Zn supply (Table 4.49). However, the maximum values of A, E and gs were recorded

for FSD-2008 with adequate Zn supply under well-watered conditions. Zinc application

improved the WUE of wheat cultivars, and the maximum WUE was noted in drought stressed

plants of cultivar Lasani-2008 (LS-2008) with adequate Zn supply followed by the same

treatment combination in cultivar FSD-2008 (Table 4.49). Zinc application reduced the

intercellular CO2 concentration and the minimum Ci was recorded with adequate supply of Zn

under well-watered conditions in cultivar LS-2008 followed by the same treatment

combination in cultivar FSD-2008. Adequate Zn supply enhanced the QY of wheat with the

maximum value recorded for cultivar LS-2008 with adequate Zn supply under both well-

watered and drought stress conditions (Table 4.49). Zinc application increased the shoot

biomass and relative water content as the highest shoot biomass and RWC were recorded for

FSD-2008 under well-watered condition with adequate Zn supply (Table 4.49).

4.6.1.2 Biochemical traits

Drought stress significantly affected all the studied biochemical parameters except

melanodialdehyde (MDA) and total soluble phenolics (TSP) (Table 4.50). Application of Zn

178

also affected all the biochemical traits significantly (Table 4.50). Moreover, the interaction of

wheat cultivars and drought stress was only significant for glutathione reductase (GR); while,

interactive effect of wheat cultivars and Zn application was significant for leaf protein (LP)

and GR activity. Furthermore, interaction of drought stress and Zn application was significant

for LP activity, while, interactive effect of drought stress, Zn application and wheat cultivars

was significant for only GR activity (Table 4.50).

Zinc application improved the biochemical traits. In this regard, the maximum LP and

TSP was recorded for cultivar LS-2008 under drought stress with adequate Zn supply (Table

2). However, the maximum specific activity of super oxide dismutase (SOD) activity and

lowest MDA contents were recorded for FSD-2008 under well-watered condition with

adequate Zn supply. Moreover, APX and GR activity was highest in cultivar LS-2008 under

low Zn supply in well-watered conditions (Table 4.50).

4.6.1.3 Leaf mineral concentrations

Analysis of variance indicated that drought stress significantly affected all the studied

parameters expect leaf potassium (K) concentration. Application of Zn also significantly

affected leaf mineral concentration (Table 4.49). Likewise, wheat cultivars differed

significantly for all the traits except leaf Ca concentration (Table 4.51). Moreover, interaction

of wheat cultivars and drought stress was significant for Zn, N and Ca contents/concentration.

Moreover, interactive effect of wheat cultivars and Zn application, wheat cultivars and drought

stress, wheat cultivars, drought stress and Zn application was only significant for Zn

concentration/contents (Table 4.51).

Zinc application improved the plant mineral status. In this perspective, maximum

concentration of Zn and N was noted in FSD-2008 under drought stress with adequate Zn

supply, while Zn and N contents were highest for seeds of LS-2008 under well-watered

condition with adequate Zn application (Table 4.51). Moreover, highest leaf K concentration

was measured with adequate Zn supply in FSD-2008 under both well-watered and drought

stress conditions. However, leaf K contents were maximum in LS-2008 under drought stress

condition with adequate Zn supply (Table 4.51).

4.6.1.4 Yield and yield related parameters

Drought stress and Zn application significantly affected the yield and yield related traits

expect 100-grain weight (HGW) (Table 4.52). Likewise, wheat cultivars also differed

179

significantly for all studied traits except grain yield (Table 4.52). Moreover, interaction of

wheat cultivars and drought stress was only significant for harvest index, while, interactive

effect of wheat cultivars and Zn application was only significant for HGW. Furthermore,

interaction of drought stress and Zn treatment was significant for all the yield related

parameters. Moreover, interaction of wheat cultivar, drought stress and Zn application was

only significant for the biological yield (Table 4.52).

Zinc application improved the yield related parameters. Grains per spike and biological

yield was the maximum in LS-2008 under well-water condition with adequate Zn supply.

However, highest HGW, GY and HI were recorded in FSD-2008 with adequate Zn supply

under drought stress condition (Table 4.52).

4.6.1.5 Grain quality and grain mineral concentration

Analysis of variance highlighted that the drought stress significantly affected all the

studied parameters expect embryo Zn concentration. However, wheat cultivars differ

significantly for embryo, aleurone, endosperm Zn concentration, phytate concentration,

[phytate]:[Zn] and bioavailable Zn. Moreover, Zn application significantly affected all the

grain quality traits. Interaction of wheat cultivars and drought stress was significant for phytate

and endosperm Zn concentration; while, interactive effect of wheat cultivars and Zn

application remained significant for aleurone and endosperm Zn concentration.

180

Table 4.49: Effect of zinc (Zn) nutrition on chlorophyll density, photosynthesis (A), transpiration rate (E), water use efficiency (WUE),

intercellular carbon dioxide (Ci), stomatal conductance (gs) and quantum yield (QY), relative water content and biomass prouduction

of wheat cultivars under drought and well-watered conditions

Cultivar Water Stress Zn Chlorophyll A E WUE Ci gs QY RWC (%) Shoot DW supply

(SPAD) (µmol m-2 s-1) (mmol m-2 s-1) (µmol mol-1) (mmol m ²s ¹) (g plant-1)

Adequate 44±0.2 15.8±0.4 3.3±0.10 4.9±0.02 200±15 0.18±0.00AB 0.56±0.013 85.6±1.49 1.53±0.11

Drought Low 44±1.0 11.8±0.8 2.8±0.05 4.2±0.25 242±8 0.10±0.02D 0.49±0.006 72.0±2.40 0.75±0.04

Adequate 46±1.0 15.2±0.8 3.0±0.06 5.1±0.22 167±7 0.15±0.01C 0.55±0.006 75.8±0.62 0.82±0.07

FSD-2008

Well-watered Low

Adequate

44±1.3

45±0.8

13.7±0.3

16.3±0.5

3.1±0.17

3.3±0.11

4.5±0.17

4.9±0.04

241±7

205±9

0.15±0.01C

0.21±0.02A

0.51±0.009

0.54±0.008

85.2±1.55

88.1±1.33

1.52±0.07

1.62±0.07

Drought

Low

Adequate

49±2.1

51±0.3

11.4±0.9

14.2±0.8

2.5±0.12

2.8±0.09

4.6±0.17

5.0±0.12

202±11

159±11

0.11±0.01D

0.14±0.01C

0.51±0.009

0.54±0.011

75.0±1.98

77.2±1.87

0.81±0.07

0.92±0.06

P C 0.000***

P T 0.000*** 0.000*** 0.000*** 0.2442 0.000*** 0.000*** 0.0169* 0.000*** 0.000*** P Zn 0.0001*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.0004*** 0.0003***

P C x T 0.0001*** 0.0085** 0.0001*** 0.1753 0.0029** 0.1159 0.0649 0.487 0.663

P C x Zn 0.900 0.394 0.8498 0.1386 0.0034** 0.3670 0.0129* 0.805 0.961

P T x Zn 0.415 0.517 0.5925 0.0448* 0.0256* 0.6187 0.7548 0.293 0.595

P C x T x Zn 0.183 0.735 0.1387 0.0617 0.1522 0.0017** 0.4374 0.097 0.413

Means sharing same case letter, for a parameter, don’t differ signficiatnly at p ≤ 0.05 by Tukey.s honesty signficant difference test.; ±S.D. P= p value; *=p≤ 0.05; **

p≤ 0.01; *** p< 0.001; well watered= 70% field capacity; Drought= 35% field capacity; C= cultivar, T= Treatment; Zn= Zinc; DW= Dry weight; LS-2008= Lasani- 2008; FSD= Faisalabad-2008

LS - 2008 Well - watered Low 42±1.1 12.9±0.7 2.9±0.15 4.5±0.10 248±8 0.16±0.01 BC 0.51±0.007 85.0±1.11 1.39±0.05

0.090 0.1167 0.0957 0.0018** 0.7032 0.2049 0.005** 0.0009***

181

Table 4.50: Effect of Zn nutriton on buffer-extractable protein concentration and specific activities of antioxidative enzymes superoxide

dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), melanodialdehyde content (MDA) and total soluble

phenolics (TSP) in wheat cultivars under drought and well-watered conditions

Leaf Protein SOD (U mg AP (μmol H2O2 GR (μmol [NADPH] MDA (nmol TSP (mg g-1

Cultivar Water Stress Zn supply (mg g-1 FW) protein) mg−1 prt.min−1) mg−1 prt. min−1) mL-1) FW)

0.54

Adequate 20.2±1.74C 28.9±2.18A 1.59±0.14AB 0.48±0.04AB 2.53±0.06 2.71±0.08

Drought Low 27.2±2.55B 14.3±0.83C 1.70±0.15BC 0.41±0.02AB 4.28±0.08 2.94±0.19

Adequate 27.9±2.50B 24.3±0.50B 1.65±0.12C 0.42±0.05AB 3.9±0.16 3.57±0.40

FSD-2008

Well-watered Low

Adequate

18.4±0.96CD

19.7±1.54C

16.4±2.05B

31.4±2.17A

1.22±0.11A

1.36±0.15B

0.40±0.03AB

0.44±0.06AB

3.27±0.14

2.48±0.15

2.39±0.14

2.80±0.09

Drought

Low

Adequate

23.0±2.95BC

33.1±3.17A

16.0±1.85C

21.1±2.33B

1.48±0.22C

1.22±0.13BC

0.51±0.09AB

0.39±0.09B

4.62±0.39

3.81±0.10

3.00±0.21

3.86±0.57

P C 0.129 0.075 0.714 0.214

P T 0.000*** 0.000*** 0.000*** 0.133 0.000*** 0.000***

P Zn 0.000*** 0.000*** 0.016* 0.195 0.000*** 0.000***

P C x T 0.422 0.422 0.159 0.008** 0.572 0.787

LS - 2008 Well - watered Low 14.1±1.08 D 22.5±2.54 C 1.95±0.11 AB ±0.08 A 3.12±0.07 2.24±0.13

0.183 0.143

182

0.000** 0.000*** 0.135 0.000***

P C x Zn 0.141 0.179 0.767 0.826 0.018* 0.693

P T x Zn 0.294 0.033* 0.181 0.355 0.433 0.139

P C x T x Zn 0.0001*** 0.0001*** 0.002** 0.015* 0.326 0.473


p≤ 0.01; *** p< 0.001; well watered= 70% field capacity; Drought= 35% field capacity; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD=

Faisalabad-2008

Table 4.51: Effect of zinc (Zn) nutrition on concentration and contents of Zn, N, K and Ca in wheat cultivars under drought and well-

watered conditions

Zn conc. Zn content N Conc. N content K conc. K content Ca Conc. Ca content Cultivar Water Stress Zn supply (mg kg-1) (ug plant-1) (%) (mg plant-1) (%) (mg plant-1) (mg kg-1) (mg plant-1)

13.5±1.4D 2.40±0.09

Adequate 23.2±1.3B 35.6±2.8A 2.59±0.11 39.6±2.1 1.68±0.04B 25.8±2.3 3.92±0.13 6.01±0.6

Drought Low 9.3±0.5C 14.1±1.1CD 3.45±0.20 52.3±3.5 1.57±0.10D 23.7±1.7 3.99±0.41 6.03±0.5

Adequate 23.3±1.3B 37.6±1.7A 3.53±0.09 57.1±3.3 1.66±0.07C 26.8±1.6 4.44±0.26 7.18±0.6

FSD-2008

Well-watered Low

Adequate

10.7±0.4C

22.6±1.3B

7.9±0.5E

18.6±2.5C

2.43±0.13

2.59±0.10

18.1±1.5

21.2±1.9

1.95±0.07BC

2.03±0.09A

14.5±0.5

16.6±2.2

3.55±0.25

3.72±0.38

2.65±0.2

3.04±0.5

Drought

Low

Adequate

10.4±1.1C

30.1±2.0A

8.4±1.5E

27.8±2.6B

3.65±0.08

3.73±0.10

29.5±2.0

34.4±3.0

1.93±0.06D

2.06±0.09C

15.6±1.0

19.0±1.3

4.45±0.10

4.83±0.21

3.60±0.3

4.46±0.3

P C 0.0001*** 0.000*** 0.0142* 0.000***

LS - 2008 Well - watered Low 9.7±0.7 C 33.3±2.2 1.54±0.08 BC 21.4±1.7 3.56±0.43 4.94±0.7

183

P T 0.0005*** 0.0001*** 0.000*** 0.000*** 0.947 0.007** 0.000*** 0.000***

P Zn 0.000*** 0.000*** 0.0142* 0.000*** 0.0005*** 0.000*** 0.003** 0.000***

P C x T 0.0002*** 0.0166* 0.047* 0.003** 0.911 0.975 0.019* 0.877

P C x Zn 0.0223* 0.000*** 0.891 0.417 0.806 0.427 0.528 0.182

P T x Zn 0.0001*** 0.0009*** 0.223 0.896 0.911 0.975 0.462 0.437

P C x T x Zn 0.0003*** 0.0129* 0.891 0.372 0.342 0.252 0.772 0.594


p≤ 0.01; *** p< 0.001; well watered= 70% field capacity; Drought= 35% field capacity; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD=

Faisalabad-2008

However, interaction of drought stress and Zn application was significant for all grain

mineral traits except protein and endosperm Zn concentration. Interaction of wheat cultivars,

drought stress and Zn application was only significant for embryo and endosperm Zn

concentration (Table 4.53).

Zinc application substantially increased the grain mineral concentration and

bioavailable Zn in the wheat cultivars. However, highest concentration/content of protein and

Zn in grain were recorded for the both wheat cultivars with adequate supply of Zn under

drought stress conditions (Table 4.54). Likewise, highest embryo and endosperm Zn

concentration was recorded with the adequate Zn supply under drought stress condition in

LS2008 and FSD-2008 respectively; while aleurone Zn concentration was impressively

increased with the adequate Zn supply in FSD-2008 both under well-watered and drought stress

conditions. Zinc application substantially reduced the phytate concentration, phytate/Zn ratio

and enhanced the bioavailable Zn as lowest phytate and phytate/Zn ratio with high bioavailable

Zn was recorded for FSD-2008 with adequate Zn supply under drought stress conditions (Table

4.55).

4.6.2. Discussion

Drought stress adversely affects the gas exchange traits by reducing the leaf growth,

cell division, root growth, WUE and plant water relation (Farooq et al., 2009). The reduction

in gas exchange traits was more severe under Zn deficient conditions. However, adequate Zn

supply enhanced the chlorophyll intensity under drought stress condition. Likewise, A, E and

gs and QY were higher with adequate Zn supply, while Ci was higher in low Zn treatment

(Table 4.49) as presence of higher Zn reduced the Ci (Wang and Jin, 2005). Earlier, higher

rates of Zn application were found to enhance the plant growth, gs and QY (Wang et al., 2009)

as Zn deficiency causes reduction in QY (Balakrishnan et al., 2000). Moreover, poor

photosynthesis in low Zn treatment was due to low activity of CA (Cakmak and Engles, 1999).

Adequate Zn treatment increased the photosynthesis possibly by enhancing activity of CA and

better chlorophyll contents.

Water deficient condition affects the plant growth by disturbing the plant water

relations by reducing relative water content, transpiration and leaf water potential (Siddique et

al., 2001). However, adequate supply of Zn enhanced water use efficiency (WUE) and RWC

possibly by increasing the rate of photosynthesis and stomatal regulation under water deficient

137

condition (Table 4.51). Zinc enhanced the high level of K+ in the leaves of wheat (Table 4.51)

which possibly maintained the stomatal conductance (Sharma et al., 1994, 1995). Moreover,

drought stress reduced the biomass production irrespective of Zn supply, however, dry matter

production was substantially reduced under Zn deficient conditions. However, supply of Zn in

adequate amount increased the chlorophyll contents, photosynthesis, gs and augmenting the

water status of leaf by improving WUE and RWC which resulted in high biomass production

under well-watered and water deficit conditions (Table 4.52). Moreover, poor biomass

production in Zn deficient plants under drought stress is due to reduction in the efficiency by

which water was used for dry biomass production (Khan et al., 2004). Furthermore, TSP

concentration was higher under drought stress irrespective of Zn supply. However, application

of Zn further enhanced the TSP production which might have increased the RWC as TSP is

positively correlated with RWC (Table 4.54).

Reactive oxygen species (ROS) production is increased under drought stress and ROS

generation is further enhanced under Zn deficiency (Cakmak 2000) due to enhanced lipid

peroxidation under Zn deficient conditions (Hong and Jin, 2007) as MDA content have strongly

negative correlation with A, E, WUE and SOD. Moreover, higher MDA production is

correlated with enhanced APX and GR activity (Table 4.53). Furthermore, a reduction in the

activities of SOD has also been reported under Zn deficiency. However, adequate Zn supply

enhances the activities of antioxidants like SOD (Table 4.50). Moreover, high Zn treatment

also enhanced the buffer extractable protein accumulation and it was accelerated under water

deficient conditions (Table 4.50) possibly due to involvement of Zn in protein synthesis

(Marschner, 2012). Specific activities of SOD were higher under adequate Zn supply as

application of Zn enhanced the SOD activity (Pandey et al., 2002).

138

187

Table 4.52: Effect of zinc (Zn) nutrition on grains per spike (GPS), 100 grain weight, biological yield, grain yield and harvest index

of wheat cultivars under drought and well-watered

conditions

(g pot-1) (g pot-1) (%)

29.4±1.8 14.2±0.15 48.3±3.0

13.7±0.8 6.54±0.48 47.9±2.3

14.7±1.1 7.11±0.35 48.6±2.4

25.9±0.9 12.2±0.18 47.2±2.3

26.1±1.7 15.5±1.07 59.7±7.9

13.3±1.5 6.39±0.53 48.1±2.0

13.9±0.9 7.56±0.27 54.6±3.0

P C 0.000*** 0.005** 0.007**

P T 0.000*** 0.027* 0.000*** 0.000*** 0.792

P Zn 0.0004*** 0.000*** 0.017* 0.000*** 0.0002***

P C x T 0.016* 0.955 0.101 0.133 0.179

P C x Zn 0.081 0.865 0.114 0.017* 0.006**

P T x Zn 0.789 0.0002*** 0.413 0.000*** 0.114

P C x T x Zn 0.184 0.895 0.264 0.367 0.454

Means sharing same case letter, for a parameter, don’t differ signficiatnly at p ≤ 0.05 by Tukey.s honesty signficant difference test. ; ±S.D.; P= p value; *=p≤ 0.05;

** p≤ 0.01; *** p< 0.001; well watered= 70% field capacity; Drought= 35% field capacity; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD=

Faisalabad-2008

Cultivar Water regime

Zn supply GPS

100 grain wt.

(%)

LS-2008 Adequate

46.8±1.69

49.2±2.08

3.02±0.03

3.45±0.04

Drought Low 37.8±0.88 3.08±0.10

Adequate 38.3±1.52 3.24±0.07

FSD-2008

Well-watered Low

Adequate

44.7±0.47

47.6±1.50

3.11±0.07

3.56±0.06

Drought Low 30.9±2.42 3.18±0.07

Adequate 35.2±2.19 3.33±0.20

0.024* 0.0008***

Biological yield Grain Yield Harvest index

26.7±0.8 12.1±0.39 45.3±0.4

188

Table 4.53: Effect of zinc (Zn) nutrition on concentration and contents of protein and Zn in whole grain, embryo, aleurone and

endosperm Zn concentration, phytate concentration, phytate/Zn ratio and bioavailable Zn in wheat cultivars under drought and

well-watered conditions

Cultivar Water Zn supply Prot conc. Pro Cont. Grain Zn Zn Cont. Emb. Zn Aleu. Zn End Zn Phy [Phy:Zn] Bio Zn

regime (%) (mg seed-1) conc. (ug seed-1) conc. conc. conc. (mg g-1) (mg day-1)

(mg kg-1) (mg kg-1) (mg kg-1) (mg kg-1)

watered Adequate 12.6±0.10 4.33±0.07 18.4±1.62 0.64±0.05 56.1±1.5B 29.3±2.3 5.90±0.29C 4.47±0.49 24.0±1.8 2.41±0.09

Drought Low 15.9±0.24 4.91±0.13 8.23±0.30 0.25±0.01 28.1±1.7D 12.3±0.9 2.81±0.06E 3.15±0.10 38.0±2.5 1.64±0.07

Adequate 16.4±0.71 5.31±0.24 24.5±2.85 0.79±0.09 63.1±1.7A 22.0±4.3 5.04±0.45D 3.06±0.28 12.5±2.3 3.31±0.27

FSD-2008

Wellwatered Low

Adequate

11.4±0.21

12.4±0.62

3.55±0.03

4.43±0.29

8.59±0.24

18.6±0.81

0.27±0.00

0.66±0.03

29.7±1.1D

57.0±2.3B

11.5±1.0

32.0±1.7

3.20±0.14E

7.85±0.09B

4.58±0.28

3.58±0.59

52.8±2.6

19.1±3.5

1.37±0.04

2.70±0.23

Drought

Low

Adequate

15.7±0.37

16.6±0.42

5.00±0.21

5.55±0.34

8.33±0.72

25.6±2.89

0.27±0.03

0.85±0.10

29.0±1.7D

60.9±3.1AB

11.9±1.2

31.5±2.4

3.43±0.09E

9.06±0.67A

3.10±0.12

2.84±0.22

37.1±3.3

11.1±1.2

1.67±0.11

3.46±0.16

P C 0.2070

P T 0.000*** 0.000*** 0.000*** 0.0002*** 0.4150 0.007** 0.8470 0.000*** 0.000*** 0.000***

P Zn 0.0001*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.0007*** 0.000*** 0.000***

P C x T 0.180 0.2590 0.5220 0.5490 0.2190 0.0076 0.000*** 0.017* 0.1020 0.3640

P C x Zn 0.101 0.2660 0.4550 0.2622 0.2190 0.050* 0.000*** 0.9220 0.6850 0.1590

P T x Zn 0.872 0.049* 0.000*** 0.000*** 0.000*** 0.0001*** 0.0980 0.0101* 0.005** 0.0002***

P C x T x Zn 0.872 0.8220 0.9220 0.7890 0.003** 0.1510 0.018* 0.6420 0.5510 0.7610

LS - 2008 Well - Low 12.1±0.45 3.65±0.15 9.11±0.44 0.28±0.01 35.7±3.3 C 14.1±1.6 3.48±0.29 E 5.58±0.89 60.9±10 1.28±0.16

0.2950 0.7360 0.2910 0.053* 0.007** 0.000*** 0.002** 0.022* 0.021*

189

Means sharing same case letter, for a parameter, don’t differ signficiatnly at p ≤ 0.05 by Tukey.s honesty signficant difference test.; ±S.D.; P= p value; *=p≤ 0.05;

** p≤ 0.01; *** p< 0.001; well watered= 70% field capacity; Drought= 35% field capacity; C= cultivar, T= Treatment; Zn= Zinc; Prot.= Protein; Conc.= concentration;

Cont.=Content Emb.= Embryo; Aleu.= Aleuron; End.= Endosperm; Phy= Phytate; Bio Zn= Bioavailable Zn LS-2008= Lasani-2008; FSD= Faisalabad-2008

Table 4.54: Correlation coefficients of gas exchange traits, enzyme activities, biomass production, relative water content, leaf Zn

and K content of wheat cultivars under well watered and drought stressed conditions (n=4)

A 0.86** 1 0.85** 0.86** 0.96** -0.17ns -0.37* -0.91** 0.87** 0.65** 0.55** 0.99** 0.57**

E 0.94** 0.98** 1 0.46 0.70** 0.22ns -0.64** -0.86** 0.58** 0.16ns 0.14ns 0.89** 0.88**

WUE 0.74** 0.97** 0.93** 1 0.94** -0.51** -0.04ns -0.71** 0.90** 0.93** 0.80** 0.80** 0.10ns

SOD 0.47* 0.84** 0.73** 0.92** 1 -0.21ns -0.13ns -0.79** 0.84** 0.76** 0.58** 0.94** 0.43*

APX -0.81** -0.42* -0.58** -0.22ns 0.10ns 1 0.13ns 0.30* -0.62** -0.75** -0.91** -0.05ns 0.64**

GR -0.83** -0.45* -0.60** -0.26ns 0.04ns 0.99** 1 0.71** -0.43* 0.06ns -0.23ns -0.39* -0.45*

MDA -0.64** -0.94** -0.86** -0.99** -0.96** 0.08ns 0.12ns 1 -0.89** -0.54** -0.61** -0.89** -0.53** TSP 0.89** 0.99** 0.99** 0.96** 0.81** -0.46* -0.49** -0.92** 1 0.85** 0.88** 0.81** 0.16ns RWC 0.72** 0.78** 0.77** 0.74** 0.72** -0.42* -0.52** -0.71** 0.78** 1 0.91** 0.56** -0.24ns

Biomass 0.99** 0.88** 0.95** 0.77** 0.53** -0.78** -0.81** -0.67** 0.90** 0.81** 1 0.45* -0.32*

LZn 0.31ns 0.66** 0.57** 0.75** 0.70** 0.18ns 0.22ns -0.78** 0.63** 0.12ns 0.28ns 1 0.66**

*=p≤ 0.05; ** p≤ 0.01; well watered= 70% field capacity; Drought= 35% field capacity; Zn= Zinc;Chl.= Chlorophyll; Pn= Photosyntehsis; E= transpiration;

WUE= water use efficiency; SOD= Super oxide dismutase; APX= Ascorbate peroxidase; GR= Glutathione reductase; MDA= melanodialdehyde; TSP= Total

soluble Phenolics; RWC= relative water content; LZn= Leaf Zn content; LK= Leaf K content

Chl A E WUE SOD APX GR MDA TSP RWC Biomass LZn LK Chl 1 ns 0.19 - ns 0.27 0.59** ns 0.28 - 0.98** 0.04 ns - 0.24 ns 0.61** 0.82** 0.91** ns 0.07 - 0.67**

190

Table 4.55: Correlation coefficients of grain yield, protein content, Zn content, endosperm Zn concentration, phytate and

bioavailable Zn in wheat cultivars under well watered and drought stressed conditions (n=4)

Grain yield Protein content Grain Zn content Endosperm Zn Phytate Bioavailable Zn Grain yield 1 0.962** 0.95** 0.99** - 0.86** 0.98** Protein content 0.96** 1 0.99** 0.95** - 0.73** 0.98** Grain Zn content 0.95** 0.99** 1 0.94** - 0.76** 0.99** Endosperm Zn 0.99** 0.95** 0.94** 1 - 0.82** 0.97** Phytate - 0.86** - 0.73** - 0.76** - 0.82** 1 - 0.83** Bioavailable Zn 0.99** 0.98** 0.99** 0.97** - 0.83** 1

*= p≤ 0.05; ** p≤ 0.01; well watered= 70% field capacity; Drought= 35% field capacity; Zn= Zinc

191

Ascorbate peroxidase and GR are two key enzymes of Halliwell Asada pathway,

present in chloroplast, have the higher activities under Zn deficiency suggesting more ROS

generation in Zn deficient plants (Table 4.50) as Zn deficiency results in ROS generation

(Cakmak, 2000). Drought stress foster the lipid peroxidation (Zhang et al., 2014) and it was

further aggravated under Zn deficiency as was visible from increased melanodialdehyde

content (MDA) under low Zn supply and drought stress. However, adequate supply of Zn

reduced the leaf MDA content under drought stress as Zn supply reduces the electrolyte leakage

and MDA content (Tavallali et al., 2010), thus help in reducing the adverse effect of drought

stress. Zinc deficient roots of wheat have higher membrane permeability with low – SH group

(Rengel, 1995b) indicating the involvement of Zn in maintenance and regulation of ionic

movement through membrane as it prevents peroxidation of –SH group and protein containing

ionic channels of root cell plasma membrane (Kochian, 1993; Welch, 1995).

Under limited water supply nutrient uptake is reduced with low tissue concentrations.

Drought stress restrict the nutrient uptake by plant roots and their transportation towards leaf

(Farooq et al., 2009). However, supply of adequate Zn increased the nutrient acquisition as Zn

fertilization enhanced the tissue Zn concentration (Table 4.51). It is well documented that Zn

positively interact with K as Zn maintains membrane integrity and reduces the leakage of K

and amides (Cakmak and Marschner, 1988). Zinc fertilizer enhanced the leaf K concentration

under well-watered and drought stress (Table 4.51). Moreover, higher Zn supply also enhanced

the uptake of N and K concentration in leaf tissues as was visible from the higher K and N

concentration/contents possibly due to the positive interaction of Zn and N (Kutman et al.,

2010; Erenoglu et al., 2011). Moreover, in the present study, adequate Zn supply improved

the plant growth under drought stress by improving the chlorophyll intensity, A, WUE, RWC,

TSP, LK and SOD activities as evident from positive correlation of Zn with these traits (Table

4.54) while Zn concentration reduced the lipid peroxidation as it has a negative correlation with

MDA content.

Water shortage severely hampers the wheat productivity. The sensitivity of Zn dearth

become more severe under drought stress as was visible from reduction in yield and yield

contributing traits of wheat. Drought stress drastically reduced the grains per spike; poor

biomass production with low grain yield, and this reduction was severe in Zn deficient plants

192

than high Zn plants as drought as yield and yield contributing traits were severely affected by

drought stress (Farooq et al., 2009; Kanani et al., 2013). However, adequate Zn supply

improved the wheat yield under drought stress by minimizing the adverse effect of drought

stress by improving the chlorophyll content, water relations, antioxidant enzymes and better

mineral uptake. Zinc supply has been found to increase the grain weight of wheat under drought

stress (Monjezi et al., 2013) as was observed in present study (Table 4.52)which ultimately

resulted in higher grain yield. Moreover, in the present study, higher values of harvest index

showed the higher transfer of Zn from photo assimilates towards reproductive tissue.

Zinc application enhanced the grain protein, Zn concentration/contents and Zn

accumulation in different seed fractions (Table 4.53). For instance, adequate Zn substantially

increased the grain Zn concentration (198%) and content (215%) compared to low Zn treatment

due to higher Zn uptake from soil as soil Zn fertilization substantially increased the grain Zn

accumulation (Cakmak et al., 2010a; Yilmaz et al., 1997; Zhang et al., 2012). However,

drought stressed seeds have higher concentration/contents of protein than seeds produced from

well-watered plants possibly due to dilution effect. Moreover, Zn supply increased the protein

concentration (33.9%) and content (56.3%) due to strong positive correlation of grain Zn

content with protein content (Table 4.55). Furthermore, it is wellestablished that higher Zn is

positively correlated with high grain N (Kutman et al., 2010; Erenoglu et al., 2011).

Moreover, Zn also play key role in protein synthesis as Zn application increases the

quantity and quality of protein (Liu et al., 2015). Zinc concentration enhanced the seed Zn

concentration (198%). Drought stress also resulted in an increased deposition of Zn in embryo,

aleuron and endosperm of wheat. Zinc deposition follows the order embryo > aleuron ˃

endosperm (Oztruke et al., 2006; Cakmak et al., 2010b) and high Zn treated seeds have about

2 fold higher concentration of Zn in embryo and aleuron, while increase in endospermic Zn

concentration was 3 fold for Zn fertilized drought stressed plants as seed Zn content positively

influenced the endospermic Zn concentration.

Drought stress reduced the phytate accumulation in wheat seeds. However, plants with

adequate Zn supply have lowest phytate value (72%) with very high bioavailability (153%)

compared to low Zn seed as Zn application has been found to increase the seed Zn

concentration and reduce the phytate accumulation in seed with lower phytate/Zn ration (Bharti

193

et al., 2013; Hussain et al., 2013). Indeed, Zn fertilization in adequate amount reduces he

phytate/Zn ration below 15 (Wang et al., 2015) thus improving the bioavailability of Zn.

Moreover, in the present study grain Zn concentration have very strong postivie and negative

correlated with bioavailable Zn and phytate concentration respectively.

In conclusion, drought stress drastically affected the physiology, tissue mineral

composition, yield and quality of wheat. However, adequate supply of Zn reduced the

detrimental effect of drought stress by improving the activities of antioxidants, membrane

stability, gas exchange traits, yield and quality of wheat.


4.7.1. Results

4.7.1.1 Gas exchange, water relation and biomass production

Analysis of variance indicated that salt stress significantly affected all the studied

parameters expect quantum yield (QY). Application of Zn also affected all the gas exchange

traits significantly (Table 4.56). However, wheat cultivars differed significantly only for

chlorophyll intensity and inter cellular CO2 (Ci) (Table 4.56). Moreover, interaction of wheat

cultivars and salt stress was significant for chlorophyll intensity, Ci and relative water content

(RWC). Likewise, interactive effect of wheat cultivars and Zn application was significant only

for Ci and RWC. Furthermore, interaction of salt stress and Zn treatment, wheat cultivars, salt

stress and Zn treatment remained non-significant for all gas exchange traits except RWC (Table

4.56).


value was recorded for cultivar Faisalabad-2008 (FSD-2008) with adequate Zn supply without

salt stress (Table 4.56). However, the maximum rate of photosynthesis (A), transpiration (E)

and stomatal conductance (gs) were recorded for FSD-2008 with adequate Zn supply under

normal growth conditions. Zinc application improved the WUE of wheat cultivars and the

maximum WUE was noted in salt stressed plants of cultivar FSD-2008 with adequate Zn

supply followed by the same treatment combination in cultivar LS-2008 (Table 4.56). Zinc

application reduced the intercellular CO2 concentration and the minimum Ci was recorded with

adequate supply of Zn under well- watered conditions in cultivar FSD-2008 followed by the

194

same treatment combination in cultivar LS-2008. Adequate Zn supply enhanced the QY of

both wheat cultivars as maximum value recorded with adequate Zn supply under both normal

and salt stress conditions (Table 4.56). Zinc application increased the shoot biomass. In this

context, maximum shoot biomass was recorded for FSD-2008 with adequate Zn supply under

normal growth condition. Zinc application improved the RWC under salt stress. However,

RWC were the highest in both wheat cultivars under normal growth condition irrespective of

Zn supply (Table 4.56).


Drought stress significantly affected all the studied parameters expect glutathione

reductase (GR) activity. Application of Zn also affected all the biochemical traits significantly

except ascorbate peroxidase (AP) and GR activity (Table 4.57). Moreover, interaction of wheat

cultivars and drought stress was only significant for total soluble phenolics (TSP), while,

interactive effect of wheat cultivars and Zn application was significant for APX activity.

Furthermore, interaction of drought stress and Zn application was significant for APX and TSP.

However, interactive effect of drought stress, Zn application and wheat cultivars was

significant for superoxide dismutase (SOD), APX and GR activity, results remained

nonsignificant for rest of the traits (Table 4.57).

Zinc application increased the buffer extractable protein (LP) and TSP in leaves of

wheat. In this regard, maximum LP and TSP was recorded for cultivar FSD-2008 under salt

stress with adequate Zn supply (Table 4.57). However, the maximum SOD activity was

recorded in both wheat cultivars under normal conditions with adequate Zn treatment (Table

4.57). The highest specific activity of APX was recorded for LS-2008 with low Zn supply

under normal growth conditions. However, highest specific activity of GR was monitored in

LS-2008 with adequate Zn under both normal and salt stressed conditions. Moreover, lowest

MDA contents were recorded in both cultivars with adequate Zn supply under normal growth

conditions (Table 4.57).

4.7.1.3 Leaf mineral concentrations

Analysis of variance depicted that salt stress significantly affected all the studied

parameters. Application of Zn also affected leaf minerals except Na concentration (Table 4.58).

Likewise, wheat cultivars differed significantly for all the traits except leaf, Zn, K

195

concentration and N contents (Table 4.58). Moreover, interaction of wheat cultivars and

drought stress was significant for N concentration, and leaf Na content and concentration,

whereas, interactive effect of wheat cultivars and Zn application was significant for Zn and Na

content, while, interaction of wheat cultivars and drought stress was significant for Zn and Na

concentration/contents. However, interaction of wheat cultivars, drought stress and Zn

application was only significant for Na concentration and contents. Results remained

nonsignificant for rest of the traits (Table 4.58).

Zinc application increased the mineral accumulation in wheat leaves. Leaf Zn

concentration was highest in the cultivar LS-2008 with adequate Zn supply under drought

stress condition. However, maximum Zn contents were recorded for FSD-2008 with adequate

Zn supply under normal growth conditions. Maximum N and K concentration were recorded

for FSD-2008 under salt stress condition (Table 4.58). However, highest N and K contents in

cultivar FSD-2008 were recorded with adequate Zn treatment under normal growth conditions.

Zinc application reduced the leaf Na concentration under salt stress. However, maximum Na

concentration/contents were recorded under normal growth condition with adequate Zn supply

(Table 4.58).

4.7.1.4 Yield and yield related parameters

Analysis of variance showed that salt stress significantly affected all the studied

parameters. Application of Zn also affected all the yield related traits except 100 grain weight

(HGW) (Table 4.59). However, wheat cultivars did not differ significantly for yield related

traits (Table 4.59). Moreover, interaction of wheat cultivars and salt stress was only significant

for grain per spike, while, interactive effect of wheat cultivars and Zn application was not

significant for yield parameters. Furthermore, interaction of salt stress and Zn treatment was

significant for HGW and grain yield. Moreover, interaction of wheat cultivar, drought stress

and Zn application was only significant for the grain yield (Table 4.59).

Zinc application improved the yield related traits. In this regard, grains per spike and

biological yield was the maximum in cultivar LS-2008 under normal growth condition with

adequate Zn supply. However, highest HGW, GY and HI were recorded in FSD-2008 with

adequate Zn supply under normal growth condition (Table 4.59).

196

4.7.1.5 Grain quality and grain mineral concentration

Analysis of variance highlighted that salt stress significantly affected all the studied

parameters expect embryo Zn concentration. However, wheat cultivars differ significantly for

embryo, aleurone, endosperm Zn concentration, phytate concentration and [phytate]:[Zn]

(Table 4.60). Moreover, Zn application significantly affected all the studied traits. Interaction

of wheat cultivars and salt stress was significant for whole grain and embryo Zn concentration,

phytate concentration and phytate/Zn ratio.

Moreover, interaction of wheat cultivars and Zn treatment was significant for protein

content/concentration, embryo, aleurone and endosperm Zn concentration (Table 4.60).

Furthermore, interaction of salt stress and Zn treatment was significant for all the traits except

protein and Zn contents, and bioavailable Zn. Interaction of wheat cultivars, salt stress and Zn

application significantly affected the protein concentration, Zn contents and embryo Zn

concentration; results being non-significant for rest of the traits (Table 4.60).

Zinc application impressively enhanced the protein contents/concentration. In this

regard, maximum grain protein concentration/contents were recorded in FSD-2008 with

adequate Zn supply under salt stress. However, highest Zn content/concentration of Zn in

whole grain, embryo, alerurone and endosperm were recorded in LS-2008 with adequate Zn

treatment under salt stress (Table 4.60). Application of Zn substantially reduced the phytate

concentration, [phytate]:[Zn] with high bioavailable Zn. For instance, lowest phytate

concentration, and [phytate]:[Zn] were recorded for FSD-2008 with adequate Zn supply under

salt stress. Moreover, bioavailable Zn was the maximum in both wheat cultivars under salt

stress with adequate Zn supply (Table 4.60).

4.7.2. Discussion

Increase salinization is biggest concern to the modern agriculture. Among abiotic stress,

salinity is the principal factor that severely hampers the quantity and quality of the agriculture

produce (Zhu, 2001). Indeed, salinity stress reduces the photosynthetic rate and chlorophyll

content (Lee et al., 2004; Kao et al., 2006; Saeidnejad et al., 2016), as was observed in this

study (Table 1). Salt stress disturbed the gas exchange traits by limiting the CO2 assimilation,

E, gs, WUE (Kanwal et al., 2013). Moreover, salinity affect the process of photosynthesis by

the accumulation of salts in the chloroplast which dreadfully disturb the photosynthetic process

197

(Munns and Tester, 2008). The effects of salt stress become more severe under Zn deficiency

as Zn deficiency disrupts the A, gs and QY (Wang and Jin, 2005).

Table 4.56: Effect of zinc (Zn) nutrition on chlorophyll density, photosynthesis (A), transpiration rate (E), water use efficiency (WUE),

intercellular carbon dioxide (Ci), stomatal conductance (gs) and quantum yield (QY), relative water content and biomass proudcitoin of

wheat cultivars under normal and salt stressed conditions

Chlorophyll A E WUE Ci gs QY RWC Shoot

Cultivar Salt Stress Zn supply (SPAD) (µmol m-2 s-1) (mmol m-2 s-1) (µmol mol-1) (mmol m-² s-¹) (%) DW

(g plant-1)

Adequate 44.2±0.20 16.4±0.66 3.25±0.11 5.05±0.21 202±14 0.19±0.01AB 0.55±0.015 86.7±2.1A 1.52±0.11

Salinity Low 38.2±3.65 11.5±0.65 2.19±0.07 5.27±0.43 236±10 0.15±0.02D 0.51±0.006 86.1±5.6C 0.58±0.08

Adequate 40.9±1.05 13.6±0.48 2.56±0.17 5.34±0.36 177±9 0.16±0.01C 0.54±0.006 88.2±4.2B 0.67±0.10

FSD-2008

Control Low

Adequate

44.1±1.16

45.4±1.50

13.1±0.72

16.4±0.48

3.05±0.18

3.32±0.10

4.29±0.18

4.93±0.04

248±4

212±5

0.15±0.01C

0.22±0.01A

0.49±0.017

0.54±0.004

50.9±1.0A

71.1±1.3A

1.56±0.08

1.62±0.06

Salinity Low 43.7±1.04 11.9±0.78 2.29±0.06 5.18±0.42 187±8 0.14±0.01D 0.51±0.006 73.0±1.2B 0.64±0.07

Adequate 44.7±0.92 13.3±0.74 2.39±0.04 5.58±0.26 173±6 0.16±0.01C 0.54±0.017 77.0±0.9B 0.86±0.09

P C 0.000*** 0.001**

P T 0.0005*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.1080 0.000*** 0.000*** P Zn 0.0089** 0.000*** 0.000*** 0.0004*** 0.000*** 0.000*** 0.000*** 0.000*** 0.0003***

P C x T 0.007** 0.835 0.0630 0.2440 0.0001*** 0.0730 0.0860 0.000*** 0.831

P C x Zn 0.394 0.328 0.0190 0.2950 0.0001** 0.0970 0.4156 0.0003*** 0.444

P T x Zn 0.692 0.002 0.2320 0.0950 0.3240 0.0002 0.0370 0.000*** 0.221

P C x T x Zn 0.525 0.835 0.5040 0.0609 0.0280 0.2380 0.3670 0.0003*** 0.179

Means sharing same case letter, for a parameter, don’t differ signficiatnly at p ≤ 0.05 by Tukey.s honesty signficant difference test; ±S.D.; P= p value; *=p≤ 0.05; ** p≤ 0.01; *** p<

0.001; Control= No stress; Salinity= 2500ppm/kg soil; C= cultivar, T= Treatment; Zn= Zinc; DW= dry weight; LS-2008= Lasani-2008; FSD= Faisalabad-2008

LS - 2008 Control Low 42.7±0.44 12.8±0.69 2.83±0.15 4.51±0.11 252±10 0.15±0.01 BC 0.50±0.013 84.6±1 .A 1.42±0.04

0.716 0.2210 0.6440 0.4560 0.1080 0.000*** 0.0003***

148

Table 4.57: Effect of Zn nutriton on buffer-extractable protein concentration and specific activities of antioxidative enzymes

superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), melanodialdehyde content (MDA)and total

soluble phenolics (TSP) in wheat cultivars under normal and salt stressed conditions

Leaf Prt. SOD APX GR MDA TSP Cultivar Salt stress Zn supply (mg g-1FW) (U mg-1 protein) (μmol H2O2 mg-1prt. min−1) (μmol [NADPH] mg-1 prt. min-1) nmol ml-1 mg g-1 FW

Adequate 19.9±1.21 29.2±1.81A 1.57±0.17ABC 0.47±0.04A 2.58±0.08 2.65±0.05

Salt stress Low 30.1±4.54 12.5±1.04D 0.93±0.06E 0.35±0.04A 3.91±0.22 3.02±0.32

Adequate 31.5±8.18 22.4±4.91B 1.43±0.33BCD 0.49±0.08B 3.00±0.16 3.71±0.19

FSD-2008

Control Low

Adequate

17.6±1.86

20.5±1.79

17.2±3.15BCD

30.5±2.08A

1.82±0.21AB

1.63±0.16ABC

0.43±0.08AB

0.45±0.05AB

3.32±0.16

2.56±0.13

2.43±0.20

2.68±0.12

Salt stress

Low

Adequate

27.3±7.04

35.6±3.29

15.8±3.73CD

20.7±1.34BC

1.34±0.22CDE

1.02±0.12DE

0.44±0.10AB

0.41±0.01AB

4.65±0.59

3.71±0.15

3.57±0.25

4.19±0.15

P C 0.476

P T 0.000*** 0.000*** 0.000** 0.081 0.000*** 0.000*** P Zn 0.0097** 0.000*** 0.166 0.187 0.000*** 0.000*

P C x T 0.749 0.3688 0.803 0.296 0.0007 0.005**

P C x Zn 0.457 0.9361 0.028* 0.113 0.682 0.511

P T x Zn 0.785 0.097 0.011* 0.154 0.226 0.019*

P C x T x Zn 0.164 0.021* 0.001** 0.022* 0.84 0.835

LS - 2008 Control Low 14.9±0.94 20.6±2.37 BC 1.94±0.09 A 0.49±0.03 AB 3.23±0.10 2.29±0.15

0.907 0.861 0.379 0.0003*** 0.0002

Means sharing same case letter, for a parameter, don’t differ signficiatnly at p ≤ 0.05 by Tukey.s honesty signficant difference test.; ±S.D.; P= p value; *=p≤ 0.05; **

p≤ 0.01; *** p< 0.001; Control= No stress; Salinity= 2500ppm/kg soil; C= cultivar, T= Treatment; Zn= Zinc; Prt= Prtoein; LS-2008= Lasani-2008; FSD=

Faisalabad2008

149

201

Under salt stress, high MDA content negatively correlated with the A, WUE due to high

Na+ (Table 4.62) which limit the A possibly due to injury of photosynthetic machinery.

However, adequate Zn supply increased the chlorophyll contents, A, E, gs and QY under both

normal and salt stressed conditions compared to low Zn plants (Table 4.56) as earlier

application of Zn to salt stressed plants has been found to enhance the chlorophyll contents and

gas exchange traits (Tavallali et al., 2009; Amiri et al., 2016). Moreover, lower photosynthetic

rate in salt stressed Zn-deficient wheat plants is possibly due to lower activity of carbonic

anhydrase (CA) and lower CO2 assimilation (Rengel, 1999).

Salt stress affect the plant growth by limiting water uptake (Qados, 2011) as relative

water content were reduced under salt stress irrespective of Zn supply possibly due to loss of

the cell turgor which affect the water availability during cell elongation (Katerji et al., 1997).

The reduction in RWC were less for plants receiving adequate Zn supply. The plants also have

the high WUE under salt stress with adequate Zn status possibly due to lower stomatal

conductance (Table 4.56) as Zn deficiency reduces the gs (Hu and Sparks, 1991).

Accumulation of TSP helped in better RWC under salt stress as TSP is positively correlated

with RWC (Table 4.61) which might have help wheat plants in osmotic adjustment under saline

condition. Moreover, improved gs in Zn sufficient plants is possibly due to increased K+ uptake

as leaf Zn concentration has positive correlation with leaf K concentration (Table 4.61).

Moreover, higher K uptake helped plant in maintaining the water relations as K regulates the

stomatal closure (Sharma et al., 1995). Salt stress reduced the wheat shoot biomass production

and this reduction was severe under limited Zn supply (Khoshgoftar et al., 2004). However,

reduction in dry mass was less with adequate Zn under salt stress than control plants due to

better gas exchange traits and water relation (Table 4.56) as was evident from positive

correlated of leaf Zn concentration with chlorophyll, A, WUE, RWC, TSP and RWC (Table

4.61)

Salinity enhanced the ROS production by enhancing the lipid peroxidation (Table

4.57). Moreover, Salt stress enhanced the buffered extractable protein accumulation and the

extent of protein accumulation was higher in the Zn sufficient plants. Specific activities of SOD

were highest with adequate Zn treatment under control conditions; while APX activities were

highest with low Zn supply under control conditions. However, GR activity was higher in Zn

202

sufficient plants of FSD-2008. Activity of SOD is influenced by the Zn availability as

expression of Cu/Zn SOD is higher in Zn efficient genotypes which might have helped the

plants to reduce the oxidative damage caused by salt stress (Gill and Tuteja, 2010). Similarly,

APX and GR activity are also enhanced by enhancing Zn supply (Table 4.57; Dai et al., 2015).

Salt stress also enhanced the lipid peroxidation (Tavallali et al., 2009) as was evident from

negative correlation of MDA content with SOD activity, RWC, leaf Zn and K concentration.

Higher MDA concentration under salt stress is possibly due to enhanced Na uptake

which resulted in membrane injury (Khoshgoftar et al., 2004) as MDA positively correlated

with leaf Na concentration under salt stress. Salt stress enhanced the leaf phenolic and TSP

was highest for adequate Zn treatment (Table 4.57) as application of Zn has found to increase

the phenolics (Tavallali et al., 2009; Song et al., 2015) possibly due enhanced expression of

genes involved in biosynthesis of phenolic (Song et al., 2015).

Salt stress causes ionic imbalance. However, adequate Zn application enhanced the Zn

concentration/contents in wheat leaves possibly due to high Zn uptake (Table 4.58) as Zn

fertilization increased the shoot Zn concentration (Liu et al., 2017). Moreover, Zn application

enhanced the K accumulation in wheat leaves (Table 4.58) as leaf Zn concentration is

positively correlated with leaf K concentration (Table 4.61) (Cakmak and Marschner, 1988).

The genotype LS-2008 have low Na concentration; while FSD-2008 have high Na

concentration (Table 4.58) due to genetic differences. However, leaf Zn and K concentration

are not correlated with the leaf Na concentration (Table 4.61). Moreover, Zn application

enhanced the leaf N concentration/contents as Zn concentration is positively correlated with N

(Erenoglu et al., 2011).

Salt stress severely reduced the wheat yield (Table 4.59; Khoshgoftar et al., 2004) and

this decline was more severe under Zn deficiency. However, adequate Zn supply counteracted

the adverse effect of salt stress (Table 4.59; Khoshgoftar et al., 2004). Salinity reduces the

wheat yield as reduction in spike weight, grains per spike and grain weight was observed in

wheat plants under salt stress (Table 4.59). However, addition of adequate Zn improved the

yield and yield contributing traits under control condition and yield loss was reduced under salt

stress due to Zn application (Table 4.59) as was affirmed by strong positive correlation of grain

Zn concentration with grain yield (Table 4.62). The improvement in yield and yield

203

contributing traits with adequate Zn application was due to increase in the photosynthesis

(Table 4.54), antioxidant activities (Table 4.57) and nutrient uptake (Table 4.58) which

ultimately boosted the wheat yield under optimal and salt stress conditions. Higher

concentration/contents of protein in wheat seeds were recorded under salt stress possibly due

to dilution effect. Moreover, addition of Zn further increased the protein concentration/contents

(Table 4.60) as seed Zn content are positively correlated with seed protein content (Table 4.61).

Higher protein concentration/content with high Zn treatment was possibly due to positive

interaction of Zn with N which ultimately enhanced the quantity of grain protein (Table 4.60;

Kutman et al., 2010; Erenoglu et al., 2011).

Moreover, increased Zn supply is associated with better wheat quality (Bharti et al.,

2013) as Zn application improve the protein composition and quantity of wheat (Table 4.60;

Liu et al., 2015). Adequate Zn application enhanced the Zn localization in wheat germ,

aleurone and endosperm of wheat. Moreover, increase in the Zn accumulation was aggravated

under salt stress. Among wheat fractions, higher Zn concentration was recorded in germ

(70.6)> aleuron (34.2) and endosperm (7.85) (Table 4.60; Oztruke et al., 2006; Cakmak et al.,

2010b). Furthermore, adequate Zn supply enhanced the Zn bioavailability by reducing the

phytate concentration (96.4%) and Phytate/Zn ratio (12) as [Phytate]:[Zn] < 15 is an indicator

of high Zn bioavailability (Wang et al., 2015). Moreover, grain Zn concentration was positively

correlated with endospermic Zn concentration and bioavailable Zn while it had strongly

negative correlation with phytate concentration (Table 4.62).

In conclusion, salt stress reduced the chlorophyll intensity, photosynthesis, RWC, plant

biomass production, grain yield and quality of wheat. However, adequate Zn supply helped

reducing the salt induced losses by improving the gas exchange traits, water relations,

membrane stability, enzyme activities, yield and quality of wheat.

204

LS - 2008 Control Low 9.74±0.7 BC 21.8±1.6 63±5 E 90±9 D

Table 4.58: Effect of zinc (Zn) nutrition on concentration and contents of Zn, N, K and Na in wheat cultivars under normal and

salt stressed conditions

Cultivar Salt Stress Zn supply Zn conc.

(mg kg-1)

Zn content

(ug plant-1)

N Conc.

(%)

N content

(mg plant-1)

K conc.

(%)

K content

(mg plant-1)

Na Conc.

(mg kg-1)

Na content

(ug plant-1) 13.8±1.4 2.42±0.11 34.3±2.0 1.54±0.08

Adequate 22.5±2.3B 34.4±5.6 2.60±0.11 39.6±3.5 1.68±0.04 25.6±1.4 78±6E 119±14D

Salinity Low 12.9±1.4D 7.5±1.8 4.07±0.07 23.5±3.5 2.27±0.07 13.1±1.9 74±9E 42±2D

Adequate 30.0±1.9C 20.1±2.6 4.27±0.07 28.6±3.9 2.39±0.06 16.1±2.6 73±5E 49±4D

FSD-2008

Control Low

Adequate

9.2±0.3BC

23.9±0.6A

14.3±1.1

38.7±1.9

2.39±0.18

2.60±0.08

37.2±3.5

42.3±2.6

1.54±0.08

1.67±0.06

24.0±2.4

27.1±1.1

1087±24B

1267±57A

1689.5±55B

2052.9±77A

Salinity Low 13.9±1.6D 8.9±0.9 4.47±0.20 28.7±3.0 2.25±0.14 14.6±2.4 985±39C 635.72±84C

Adequate 29.1±2.7C 25.0±2.4 4.55±0.16 39.2±3.6 2.49±0.12 21.5±2.8 857±53D 741.94±110C

P C 0.683

0.006** 0.0017** 0.0001 0.499 P T 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P Zn 0.000*** 0.000*** 0.0015*** 0.000*** 0.000*** 0.000*** 0.167 0.000***

P C x T 0.761 0.674 0.001** 0.033 0.451 0.278 0.000*** 0.000***

P C x Zn 0.958 0.0558* 0.663 0.283 0.384 0.264 0.409 0.000***

P T x Zn 0.047* 0.0002*** 0.536 0.265 0.551 0.315 0.000*** 0.003**

P C x T x Zn 0.132 0.946 0.454 0.231 0.305 0.135 0.000*** 0.011*

Means sharing same case letter, for a parameter, don’t differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test.; ±= S.D. P= p value; *=p≤

0.05; ** p≤ 0.01; *** p< 0.001; Control= No stress; Salinity= 2500ppm/kg soil; C= cultivar, T= Treatment; Zn= Zinc; Conc. Concentration; LS-2008=

Lasani-2008; FSD= Faisalabad-2008

0.001** 0.000*** 0.000***

205

Table 4.59: Effect of zinc (Zn) nutrition on grains per spike (GPS), 100 grain weight, biological yield, grain yield and harvest index of wheat

cultivars under normal and salt stressed conditions

100 grain weight

(%) (mg kg-1) (g plant-1)

3.03±0.03

Adequate 49.8±1.29 3.47±0.08 28.4±1.76 14.3±0.35B 50.7±3.8

Salinity Low 39.0±1.83 3.38±0.13 23.4±1.07 10.0±0.15F 42.7±2.6

Adequate 41.7±1.39 3.50±0.07 24.3±2.15 11.2±0.35DE 46.5±5.7

FSD2008

Control

Salinity

Low

Adequate

Low

44.6±0.50

47.2±0.79

42.1±1.87

3.14±0.04

3.59±0.04

3.30±0.04

25.5±0.88

27.8±2.06

24.7±1.15

12.2±0.15C

15.2±0.62A

10.7±0.22EF

47.9±2.1

54.8±3.9

43.3±2.0

Adequate 43.3±1.55 3.39±0.11 24.4±1.08 11.7±0.44CD 47.8±3.0

P C 0.754 0.677 0.886

P T 0.000*** 0.006 0.000*** 0.000*** 0.0006***

P Zn 0.0002*** 0.000*** 0.034* 0.000*** 0.0002***

P C x T 0.0001*** 0.0007 0.148 0.686 0.335

P C x Zn 0.471 0.817 0.792 0.265 0.576

P T x Zn 0.501 0.000*** 0.117 0.000*** 0.431

Cultivar Salt Stress Zn supply Grains per spike Biological yield Grain Yield Harvest index ( % )

LS - 2008 Control Low 47.3±1.73 26.7±0.81 12.2±0.41 C 45.6±0.8

0.001 0.087

206

P C x T x Zn 0.471 0.817 0.431 0.054* 0.792

Means sharing same case letter, for a parameter, don’t differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test.; ±S.D.; P= p value; *=p≤

0.05; ** p≤ 0.01; *** p< 0.001; Control= No stress; Salinity= 2500ppm/kg soil; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD=

Faisalabad-2008

Table 4.60: Effect of zinc (Zn) nutrition on concentration and contents of protein and Zn in whole grain, embryo, aleurone and endosperm

Zn concentration, phytate concentration, phytate/Zn ratio and bioavailable Zn in wheat cultivars under normal and salt stressed conditions

Stress (%) (mg seed-1) (mg kg-1) (ug seed-1) (mg kg-1) (mg kg-1) (mg kg-1) (mg g-1) (mg day-1)

Adequate 12.9±0.7DE 4.49±0.22 18.3±1.6 0.64±0.05B 58.3±4.7B 30.7±2.8 6.07±0.32 4.14±0.25 22.5±1.3 2.48±0.09

Salinity Low 16.0±0.6BC 5.37±0.29 9.07±1.1 0.30±0.03C 28.5±0.6D 11.7±1.2 3.62±0.16 3.05±0.20 33.8±5.9 1.79±0.19

Adequate 16.5±0.7B 5.77±0.25 24.0±2.8 0.84±0.09A 70.6±2.5A 34.2±1.2 7.85±0.20 2.87±0.07 12.0±1.5 3.34±0.19

FSD2008

Control

Salinity

Low

Adequate

Low

11.2±0.4E

12.6±0.6DE

14.2±0.9CD

3.52±0.10

4.51±0.26

4.69±0.34

8.63±0.3

19.4±1.8

8.53±1.1

0.27±0.01C

0.70±0.07B

0.28±0.03C

29.1±0.3D

57.5±2.8B

29.9±2.5D

12.0±0.5

21.8±1.8

9.69±0.6

2.87±0.05

5.04±0.38

2.68±0.11

4.85±0.33

3.41±0.14

2.93±0.26

55.6±2.8

17.5±2.1

34.5±6.1

1.33±0.04

2.81±0.18

1.75±0.21

Adequate 18.4±1.5A 6.19±0.67 20.6±1.5 0.69±0.06B 50.5±2.2C 28.3±2.1 6.11±0.53 2.47±0.19 11.8±0.7 3.29±0.08

P C 0.2450

P T 0.000*** 0.000*** 0.003** 0.003** 0.8760 0.057* 0.000*** 0.000*** 0.000*** 0.000***

Cultivar Salt Zn supply Prot. conc. Prot. cont. Zn conc. Zn cont. Emb. Zn Aleu. Zn End. Zn Phy tate [ ] Phytate:Zn Bio Zn

LS - 2008 Control Low 12.2±0.3 E 3.70±0.06 8.76±0.6 0.27±0.0 C 33.9±0.5 D 14.6±1.7 3.78±0.38 5.83±0.43 65.9±1.3 1.19±0.02

0.358 0.189 0.1840 0.000*** 0.000*** 0.000*** 0.000*** 0.005** 0.0870

207

P Zn 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P C x T 0.1950 0.858 0.035* 0.005 0.0009*** 0.1330 0.095 0.003** 0.003** 0.012

P C x Zn 0.0007*** 0.009** 0.473 0.3710 0.000*** 0.0003*** 0.0449* 0.940 0.3510 0.384

P T x Zn 0.031* 0.8159 0.005** 0.0710 0.009** 0.000*** 0.000*** 0.000*** 0.000*** 0.1420

P C x T x Zn 0.0119* 0.061 0.074 0.028* 0.000*** 0.3360 0.135 0.155 0.2070 0.335

Means sharing same case letter, for a parameter, don’t differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±S.D.; P= p value; *=p≤ 0.05; **

p≤ 0.01; *** p< 0.001; Control= No stress; Salinity= 2500ppm/kg soil; C= cultivar, T= Treatment; Conc. Concentration; Cont= Content; Emb. Embryo; Alu=

Aleurone; End= Endosperm; Zn= Zinc; LS-2008= Lasani-2008; FSD= Faisalabad-2008

Table

4.61:

Correlation coefficients of gas exchange traits, enzyme activities, biomass production, relative water content, leaf Zn and K

content of wheat cultivars under normal and salt stressed conditions (n=4)

A 0.75** 1 0.69** 0.98** 0.383* 0.70** -0.75** 0.79** 0.68** 0.67** 0.99** 0.85** -0.04ns WUE 0.50** 0.94** 1 0.60** -0.364* 0.01ns -0.48** 0.76** 0.89** 0.43* 0.76** 0.96** 0.14ns

SOD 0.63** 0.95** 0.97** 1 0.515** 0.80** -0.67** 0.81** 0.66** 0.76** 0.95** 0.79** 0.05ns

APX -0.76** -0.96** -0.85** -0.84** 1 0.92** -0.14ns 0.21ns -0.12ns 0.52** 0.26ns -0.10ns 0.07ns

TSP 0.87** 0.96** 0.82** 0.86** -0.980** -0.34* -0.92** 1 0.94** 0.91** 0.78** 0.85** 0.55** TDM 0.98** 0.61** 0.33* 0.46* -0.667** -0.81** -0.53** 0.77** 1 0.74** 0.72** 0.91** 0.53**

Chl A WUE SOD APX GR MDA TSP TDM RWC LZn LK LNa

Chl 1 0.40 * 0.42 * 0.48 ** ns 0.256 0.38 * 0.28 ns 0.85 ** 0.77 ** 0.92 ** 0.37 * 0.49 ** 0.89 **

208

Grain yield Protein content Zn content Endosperm Zn Phytate Bioavailable Zn

Bioavailable Zn 0.99 ** 0.96 ** 0.99 ** 0.83 ** - 0.93 ** 1 *= p≤ 0.05; ** p≤ 0.01; well watered= 70% field capacity; Drought= 35% field capacity; Zn= Zinc

GR

-

0.68** -0.09ns 0.24ns 0.12ns 0.248ns 1 -0.40* 0.50** 0.20ns 0.69** 0.61** 0.27ns 0.05ns

MDA -0.68** -0.98** -0.97** -0.99** 0.914** -0.02ns 1 -0.23ns -0.20ns -0.03ns -0.78** -0.58** 0.67**

*=p≤ 0.05; ** p≤ 0.01; well watered= 70% field capacity; Drought= 35% field capacity; Zn= Zinc;Chl.= Chlorophyll; Pn= Photosyntehsis; E= transpiration; WUE=

water use efficiency; SOD= Super oxide dismutase; APX= Ascorbate peroxidase; GR= Glutathione reductase; MDA= melanodialdehyde; TSP= Total soluble

Phenolics; RWC= relative water content; LZn= Leaf Zn content; LK= Leaf K content

Table 4.62: Correlation coefficients of grain yield, protein content, Zn content, endosperm Zn concentration, phytate and

bioavailable Zn in wheat cultivars under normal and salt stressed conditions (n=4)

Grain yield 1 0.66** 0.79** 0.68** -0.89** 0.87** Protein content 0.96** 1 0.79** 0.79** -0.71** 0.86** Zn content 0.99** 0.98** 1 0.98** -0.56** 0.98** Endosperm Zn 0.81** 0.94** 0.87** 1 -0.45* 0.94** Phytate -0.92** -0.81** -0.90** -0.61** 1 -0.70**

RWC 0.98** 0.83** 0.62** 0.74** -0.829** -0.56** -0.79** 0.92** 0.93** 1 0.62** 0.59** 0.67**

LZn 0.74** 0.99** 0.95** 0.98** -0.925** -0.04ns -0.99** 0.94** 0.59** 0.83** 1 0.90** -0.06ns

LK 0.71** 0.99** 0.95** 0.96** -0.955** -0.04ns -0.99** 0.95** 0.57** 0.81** 0.99** 1 0.13**

LNa 0.74** 0.12ns -0.18ns 0.00ns -0.152ns -0.88** -0.04ns 0.31* 0.83** 0.64** 0.12ns 0.07ns 1

209

4.8. Improving the Resistance against Cold Stress in Wheat through Zn Nutrition 4.8.1

Results

4.8.1.1 Gas exchange

Analysis of variance indicated that suboptimal temperature stress significantly affected

all the studied parameters expect stomatal conductance (gs). Application of Zn also affected

all the gas exchange traits significantly (Table 4.63). However, wheat cultivars differed

significantly only for chlorophyll intensity, rate of photosynthesis (A), transpiration (E) and

stomatal conductance (gs) (Table 4.63). Moreover, the interaction of wheat cultivars and cold

stress was significant for chlorophyll intensity, water use efficiency (WUE) and QY, while,

interactive effect of wheat cultivars and Zn application was significant only for E and gs.

Furthermore, interaction of cold stress and Zn treatment was significant for Ci and QY,

whereas, interaction of wheat cultivars, cold stress and Zn treatment was only significant for

gs. Results remained non-significant for all the other gas exchange traits (Table 4.63).


value was recorded for the cultivar Faisalabad-2008 (FSD-2008) with adequate Zn supply

under optimal temperature (Table 4.63). However, maximum A, E were recorded in both wheat

cultivars with adequate Zn treatment under normal temperature. Moreover, highest WUE was

recorded for Fsd-2008 with adequate Zn supply under suboptimal temperature stress. Zinc

application reduced the Ci and enhanced the gs as lowest Ci and maximum gs was recorded

for Ls-2008 with adequate Zn treatment under suboptimal temperature. Moreover, QY was

highest for Ls-2008 with adequate Zn supply under normal temperature.


Analysis of variance indicated that cold stress significantly affected all the studied

parameters expect glutathione reductase (GR) activity. Application of Zn also affected all the

biochemical traits significantly except GR activity (Table 4.64). However, the wheat cultivars

differed significantly only for total soluble phenolic (TSP). Moreover, interaction of wheat

cultivars and cold stress was only significant for total soluble phenolic (TSP); while, interactive

effect of wheat cultivars and Zn application was non-significant for all the biochemical

parameters. Furthermore, interaction of cold stress and Zn application was only significant for

ascorbate peroxidase (APX) and TSP. However, interactive effect of cold stress, Zn application

210

and wheat cultivars was significant for leaf protein (LP), super oxide dismutase (SOD) and

APX activity; results remained non-significant for rest of the traits.

Zinc application increased the LP. In this respect, maximum LP was recorded for

FSD2008 with adequate Zn supply under cold stress. However, highest specific activity of

SOD was recorded in cold stressed plants of FSD-2008 with adequate Zn treatment; while

specific activity of APX was highest in cold stressed plants of FSD-2008 with low Zn supply.

Whereas, higher specific GR activity was noted for low Zn plants of LS-2008 under normal

temperature. Cold stress plants possessed the highest MDA content. However, Zn application

reduced the MDA accumulation. In this regard, minimum MDA contents were recorded in the

cold stressed plants of FSD-2008 under normal growth condition. Zinc application enhanced

the synthesis of TSP. Highest value of TSP was recorded for cold stressed plants of Fsd-2008

with adequate Zn supply (Table 4.64).

4.8.1.3 Biomass and leaf mineral concentrations

Analysis of variance indicated that cold stress significantly affected all the studied

parameters. Application of Zn also affected the biomass and leaf mineral concentrations except

RWC. However, wheat cultivars differ significantly for shoot biomass, leaf Zn and N

concentration (Table 4.65). Furthermore, Interaction of wheat cultivars and cold stress was

significant for shoot biomass, leaf Zn concentration, N concentration/contents; whereas,

interaction of wheat cultivars and Zn treatment, cold stress and Zn treatment was only

significant for leaf Zn concentration. However, interaction of wheat cultivars, cold stress and

Zn treatment was only significant for K contents (Table 4.65).

Zinc application increased the biomass production and RWC. In this regard, maximum

shoot biomass and RWC were recorded in FSD-2008 with adequate Zn supply under normal

growth conditions. However, Zn concentration was maximum in cold stressed plants of

LS2008 with adequate Zn treatment; while, highest Zn contents were recorded in FSD-2008

under normal condition with adequate Zn application (Table 4.65). Zinc application increased

the N and K accumulation in wheat leaves as highest N, K concentration and N contents were

recorded in Fsd-2008 in cold stressed plants with adequate Zn supply. However, highest K

contents were recorded in both wheat cultivars receiving adequate Zn under optimal

temperature (Table 4.65).

211

4.8.1.4 Yield and yield parameters

Analysis of variance showed that suboptimal temperature significantly affected all the

yield parameters. Application of Zn also affected all the yield related traits (Table 4.66).

However, wheat cultivars did not differ significantly for yield related traits (Table 4.66).

Moreover, interaction of wheat cultivars and suboptimal temperature stress was only

significant for biological yield and harvest index, while, interactive effect of wheat cultivars

and Zn application was not significant for yield parameters. Furthermore, interaction of cold

stress and Zn treatment was significant for grains per spike, HGW and grain yield. Moreover,

interaction of wheat cultivar, drought stress and Zn application was not significant for the yield

parameters (Table 4.66).

Zinc application improved the yield related traits. In this regard, grains per spike and

biological yield was the maximum in cultivar LS-2008 under normal growth condition with

adequate Zn supply (Table 4.66). However, highest HGW, GY and HI were recorded in cultivar

FSD-2008 with adequate Zn supply under normal growth condition (Table 4.66).

4.8.1.5 Grain quality and mineral concentration

Analysis of variance enlightened that cold stress significantly affected the grain,

endosperm Zn concentration, phytate concentration, [phytate]:[Zn] and bioavailable Zn.

Moreover, Zn application significantly affected all the studied parameters. However, wheat

cultivars differ significantly for protein concentration/contents, phytate concentration,

[phytate]:[Zn] and bioavailable Zn (Table 4.67). Interaction of wheat cultivars and cold stress

was significant for contents/concentration of Zn in grain, aleuron and endosperm; whereas,

interactive effect of wheat cultivars and Zn treatment was significant for concentration and

contents of protein, embryo Zn concentration, phytate concentration and bioavailable Zn.

Further, cold stress and Zn treatment significantly affected the protein contents, Zn contents,

endosperm Zn and phytate concentration; while, interaction of wheat cultivars, cold stress and

Zn application significantly affected the aleuron Zn concentration. Results were nonsignificant

for rest of the traits (Table 4.67).

42.7± 0.45 12.9 ± 0.61

0.000*** 0.653 0.272 0.092***

Table 4.63: Effect of zinc (Zn) nutrition and cold stress on chlorophyll density, photosynthesis (A), transpiration rate (E), water use

efficiency (WUE), intercellular carbon dioxide (Ci), stomatal conductance (gs) and quantum yield (QY), relative water content (RWC)

and biomass proudcitoin of wheat cultivars

Cultivar Temperature Zn Chlorophyll A E WUE Ci gs QY RWC Shoot biomass

regime supply (SPAD) (µmol m-2 s-1) (mmol m-2 s- (xxxxxxx) (µmol mol-1) (mmol m ² s ¹) (%) (g plant-1) 1)

LS-2008 Optimal Low 2.88±0.11 4.47±0.14 249±10 0.15 ± 0.005B 0.50±0.016 84.4 ± 0.87 1.38 ± 0.06

Adequate 44.2 ±0.19 16.3 ± 0.72 3.23 ± 0.08 5.05 ± 0.22 206 ± 16 0.19 ± 0.013A 0.55±0.012 86.0 ± 2.88 1.52 ± 0.09

Suboptimal Low 36.4 ± 1.57 12.5 ± 0.54 2.06 ± 0.08 6.06 ± 0.23 200 ± 13 0.15 ± 0.007B 0.48±0.007 77.4 ± 0.63 0.73 ± 0.05

Adequate 39.0 ± 1.37 15.3 ± 0.62 2.24 ± 0.07 6.82 ± 0.34 184 ± 8 0.21 ± 0.013A 0.50±0.010 77.9 ± 4.03 0.79 ± 0.07

FSD-2008 Optimal Low 44.2 ± 1.13 13.1 ± 0.67 2.99 ± 0.10 4.36 ± 0.21 244 ± 8 0.14 ± 0.005B 0.49±0.022 86.1 ± 1.88 1.50 ± 0.07

Adequate 45.5 ± 0.89 16.5 ± 0.41 3.35 ± 0.09 4.91 ± 0.06 209 ± 12 0.15 ± 0.005B 0.54±0.008 87.3 ± 1.81 1.57 ± 0.04

Suboptimal Low 41.7 ± 1.55 13.3 ± 0.20 2.14 ± 0.11 6.21 ± 0.33 210 ± 12 0.14 ± 0.004B 0.50±0.006 77.1 ±1.89 0.69 ± 0.06

Adequate 44.6 ± 1.94 16.0 ± 0.54 2.21 ± 0.06 7.21 ± 0.36 198 ± 6 0.14 ± 0.007B 0.51±0.011 78.7 ± 2.19 0.81 ± 0.07

P C 0.000*** 0.031* 0.000*** 0.425 0.192

P T 0.000*** 0.046* 0.034* 0.000*** 0.000*** 0.913 0.000*** 0.000*** 0.000***

P Zn 0.0001*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.138 0.0001***

P C x T 0.0001*** 0.171 0.1730 0.043* 0.104 0.062 0.008** 0.444 0.033*

P C x Zn 0.956 0.926 0.002** 0.550 0.465 0.000*** 0.613 0.846 0.955

P T x Zn 0.152 0.076 0.460 0.093 0.005** 0.273 0.0002*** 0.834 0.867

P C x T x Zn 0.783 0.926 0.395 0.464 0.815 0.027* 0.467 0.647 0.187

p≤ 0.01; *** p< 0.001; optimal= 20/15ºC day/night; Cold stress= 10/7ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD= Faisalabad-2008

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant diffe rence test ; ± = S.D.; P= p value; *=p≤ 0.05; **

213

160

Table 4.64: Effect of Zn nutriton and cold stress on buffer-extractable protein concentration and specific activities of antioxidative

enzymes superoxide dismutase (SOD), ascorbate peroxidase (APX) and glutathione reductase (GR), melanodialdehyde content (MDA)

and total soluble phenolics (TSP) of wheat cultivars

Cultivar Temperature Zn supply Leaf Protein SOD APX GR MDA Phenolics regime (mg g-1 FW) (U mg-1 protein) (μmol H2O2 mg−1 prt. (μmol [NADPH] mg−1 (nmol ml-1) (mg g-1 FW)

min−1) prt. min−1)

Adequate 21.5 ± 1.12BCD 27.7 ± 1.72AB 1.49 ± 0.15 B 0.44 ± 0.04 2.59 ± 0.05 2.73 ± 0.12

Suboptimal Low 25.5 ± 4.15ABC 14.7 ±3.40D 1.35 ± 0.13 AB 0.50 ± 0.10 4.63 ± 0.48 2.92 ± 0.27

Adequate 27.8 ± 3.18ABC 23.4 ± 2.54BC 1.40 ± 0.26 B 0.45 ± 0.08 3.69 ± 0.26 4.14 ± 0.14

FSD-2008

Optimal Low Adequate

17.8 ± 1.90DE 20.3 ± 1.99CDE

18.1 ± 2.31CD 31.4 ± 2.96A

1.78 ± 0.23 B 1.64 ± 0.19 B

0.44 ± 0.07 0.43 ± 0.3

3.30 ± 0.11 2.48 ± 0.06

2.42 ± 0.16 2.79 ± 0.14

P T 0.000***

LS - 2008 Optimal Low 13.9 ± 1.36E 23.0 ± 3.46BC 2.14 ± 0.21 B 0.54 ± 0.08 3.26 ± 0.15 2.35 ± 0.22

Suboptimal Low 2.40A 24.2 ± - D 15.7 ± 2.74D 0.15 A 1.56 ± 0.47 ± 0.09 4.45 ± 0.25 3.59± 0.22 Adequate 29.2 ± 4.40A 23.4 ± 3.77BC 0.20 B 1.48 ± 0.03 0.41± 4.01 ± 0.22 4.43 ± 0.25

P C 0.502 0.948 0.787 0.092 0.871 0.0007*** 0.000*** 0.0001** 0.804 0.000*** 0.000***

0.000*** 0.006 0.041* 0.000*** 0.000* 0.621 0.087 0.587 0.517 0.006** 0.075 0.174 0.403 0.312 0.176 0.716 0.011* 0.960 0.767 0.0001***

P C x T x Zn 0.058* 0.031* 0.031** 0.303 0.064 0.204

P Zn 0.009** P C x

T 0.510

P C x Zn 0.526

P T x Zn 0.494

Means sharing same case letter, for a parameter, do not differ significantly at (p ≤ 0.05) by Tukey.s honesty significant difference test; ±S.D.; P= p value; *=p≤ 0.05; **

p≤ 0.01; *** p< 0.001; optimal= 20/15ºC day/night; Cold stress= 10/7ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD= Faisalabad2008

Zinc application enhanced the protein accumulation and mineral concentration in

wheat. In this context, highest protein concentration, grain, embryo, aleuron and endosperm

Zn concentration were recorded for FSD-2008 receiving adequate Zn (Table 4.67). Likewise,

lowest phytate concentration, [phytate]:[Zn] and highest bioavailable Zn were recorded for the

same treatment. However, protein content was the maximum in LS-2008 with adequate Zn

supply under optimal temperature. Moreover, grain Zn contents were the maximum in

FSD2008 with adequate Zn application under optimal temperature (Table 4.67).

4.8.2. Discussion

Cold stress can cause severe injuries eventually resulting in the limited plant growth,

yield, and sometimes the plant death (Shahandashti et al., 2013). Cold stress reduces the

photosynthesis, E and gs (Zhang et al., 2015), as was observed in this study. A further reduction

in gas exchange traits was recorded for Zn deficient plants as adequate Zn improves the gas

exchange traits under normal as well as suboptimal temperatures (Table 4.63).

Low photosynthesis under cold stress together with Zn deficiency might be due to the

ultrastructural changes in the chloroplast thus reducing the chlorophyll contents, and CO2

assimilation (Sharma et al., 1994). Furthermore, Zn deficiency limits the CA activity (Rengel,

1995b) which might have resulted in low photosynthetic rate for plants receiving low Zn.

Cold stress reduced the RWC (Turk and Erdal, 2015) irrespective of Zn supply which

was the possible outcome of the turgor loss which affects the water availability required for the

cell expansion (Katerji et al., 1997). However, supply of Zn to the cold stressed plants enhanced

the WUE efficiency due to the higher A with lower E. Moreover, adequate Zn treatment

increased the K and TSP accumulation in wheat which might have contributed in stomatal

regulation (Sharma et al., 1995).

Indeed, cold stress reduce the plant growth by inhibiting root development, shoot

growth due to low Zn status which can be counteracted by application of cold stress protectants

(Bradáčová et al., 2016) and Zn application (Table 4.65). Moreover, Cold stress reduced the

biomass accumulation of wheat, the extent of dry weight reduction aggravated in low Zn

treatment. High biomass production with Zn application treatment was due to increased

photosynthesis, RWC (Table 4.63) as leaf Zn concentration was positively correlated with

chlorophyll, A, E, RWC, WUE and SOD activities (Table 4.68).

162

217

LS - 2008 Optimal Low 9.6 ± 0.4 13.3 ± 0.47 2.39 ± 0.15 36.3 ± 3.8 1.52 ± 0.06 20.9 ± 1.4BC

Table 4.65: Effect of zinc (Zn) nutrition on concentration and contents of Zn, N, K and Na in wheat cultivars under optimal and

and cold stressed conditions

Cultivar Temperature Zn supply Zn concentration Zn content N Concentration N content K Concentration K content regime (mg kg-1) (ug plant-1) (%) (mg plant-1) (%) (mg plant-1)

Adequate 22.0 ± 1.8 33.5 ± 3.59 2.56 ± 0.06 44.4 ± 2.6 1.74 ± 0.07 26.4 ± 1.8A Suboptimal Low 13.8 ± 0.9 10.0 ± 0.28 4.15 ± 0.24 86.8 ± 7.2 2.09 ± 0.06 15.2 ± 1.2DE Adequate 35.6 ± 1.5 28.2 ± 2.65 4.52 ± 0.20 96.8 ± 5.4 2.14 ± 0.07 17.0 ± 1.4DE FSD- Optimal Low 9.4 ± 0.5 14.1 ± 1.20 2.26 ± 0.08 35.1 ± 3.2 1.56 ± 0.12 23.3 ± 1.6AB

2008 Adequate 22.3 ± 1.2 35.2 ± 2.39 2.52 ± 0.03 41.6 ± 1.8 1.65 ± 0.06 25.9 ± 1.0A Suboptimal Low 13.9 ± 1.1 9.5 ± 0.96 4.63 ± 0.14 95.6 ± 9.6 2.06 ± 0.15 14.1 ±

1.1E Adequate 39.0 ± 1.6 31.5 ± 1.46 4.86 ± 0.24 108.7 ± 10.4 2.23 ± 0.12 18.1 ± 1.6CD

P T 0.000*** P Zn 0.000***

P C x T 0.058*

P C x Zn 0.036*

P T x Zn 0.000***

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±S.D. P= p value; *=p≤ 0.05; **

p≤ 0.01; *** p< 0.001; optimal= 20/15ºC day/night; Cold stress= 10/7ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD=

Faisalabad2008

P C 0.048*** 0.061 0.008** 0.072 0.927 0.331 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.0002*** 0.0003*** 0.0005*** 0.000***

0.907 0.0002*** 0.01* 0.413 0.368 0.099 0.872 0.863 0.985 0.738

0.700 0.459 0.339 0.499 0.254 P C x T x Zn 0.118 0.316 0.282 0.602 0.069 0.0186*

218

Table 4.66: Effect of zinc (Zn) nutrition on grains per spike (GPS), 100 grain weight, biological yield, grain yield and harvest index of

wheat cultivars under normal and cold stressed conditions

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±S.D.; P= p value; *=p≤ 0.05;

** p≤ 0.01; *** p< 0.001; optimal= 20/15ºC day/night; Cold stress= 10/7ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD=

Faisalabad-2008

219

LS - 2008 Control Low 12.0±0.45 1.6C 14.1 ± 3.64 ± 0.26 5.06 ± 0.20 56.5 ± 5.2 1.32 ± 0.09

0.127 0.000*** 0.0009*** 0.000***

Table 4.67: Effect of zinc (Zn) nutrition on concentration and contents of protein and Zn in whole grain, embryo, aleurone and

endosperm Zn concentration, phytate concentration, phytate/Zn ratio and bioavailable Zn in wheat cultivars under optimal and cold

stressed conditions

Cultivar Temperature

regime Zn

supply Prot conc.

(mg kg-1)

Prot

content

(ug plant-1)

Zn Conc.

(mg kg-1)

Zn content

(ug plant-1)

Emb. Zn

(mg kg-1)

Alu. Zn

(ug plant-1)

End. Zn

(mg kg-1)

Phytate

(ug plant-1)

[Phy]:[Zn]

Bio Zn

(mg day-1)

3.66±0.11 8.91±0.6 0.27 ± 0.02 36.6 ± 3.5

Adequate 12.9±0.75 4.50±0.24 17.9±0.4 0.63 ± 0.02 56.2 ± 1. 27.7 ± 1.3A 5.89 ± 0.32 4.28 ± 0.15 23.6 ± 0.9 2.41 ± 0.05

Drought Low 11.3±0.42 3.63±0.15 8.52±1.3 0.27 ± 0.05 35.6 ± 2.9 10.1 ± 0.6C 2.67 ± 0.06 4.70 ± 0.22 55.4 ± 6.9 1.33 ± 0.15

Adequate 12.4±0.56 3.92±0.27 18.6±0.9 0.59 ± 0.03 58.5 ± 2.2 21.9 ± 0.8B 5.91 ± 0.08 4.01 ± 0.23 21.4 ± 0.9 2.55 ± 0.06

FSD- Control Low 10.5±0.69 3.32±0.18 8.58±0.3 0.27 ± 0.01 29.9 ± 1.1 12.6 ± 1.4C 2.86 ± 0.05 4.98 ± 0.52 57.6 ± 5.8 1.30 ± 0.08

2008 Adequate 11.9±0.50 4.22±0.23 19.5±1.6 0.69 ± 0.06 58.3 ± 2.7 21.3 ± 1.6B 5.01 ± 0.24 3.29 ± 0.31 16.8 ± 2.1 2.86 ± 0.17

Drought Low 12.9±0.71 4.20±0.35 8.74±0.7 0.29 ± 0.03 32.4 ± 1.7 13.0 ± 1.6C 3.19 ± 0.07 4.03 ± 0.21 45.9 ± 4.0 1.50 ± 0.10

Adequate 13.3±0.18 4.17±0.08 20.3±1.1 0.63 ± 0.03 60.4 ± 3.0 28.2 ± 3.7A 6.63 ± 0.18 3.10 ± 0.14 15.2 ± 0.5 2.99 ± 0.05

P C 0.032* 0.019* 0.960 0.5290 0.1070 0.6260

P T 0.3930 0.123 0.003** 0.4690 0.1070 0.3140 0.0008*** 0.0001*** 0.007** 0.003**

P Zn 0.000*** 0.000*** 0.0001*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P C x T 0.6200 0.843 0.000*** 0.0001*** 0.361 0.000*** 0.000*** 0.202 0.094 0.193

P C x Zn 0.023* 0.057* 0.871 0.3980 0.0005*** 0.5850 0.657 0.007** 0.4510 0.000***

P T x Zn 0.2390 0.030* 0.4010 0.0001*** 0.4140 0.0780 0.000*** 0.037* 0.1360 0.6760

P C x T x Zn 0.7660 0.623 0.151 0.2290 0.287 0.003** 0.229 0.093 0.0610 0.193

220

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±=S.D.; P= p value; *=p≤ 0.05;

** p≤ 0.01; *** p< 0.001; optimal= 20/15ºC day/night; Cold stress= 10/7ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD= Faisalabad-2008

Table 4.68: Correlation coefficients of gas exchange traits, enzyme activities, biomass production, relative water content, leaf Zn

and K content of wheat cultivars under optimal and cold stressed conditions (n=4)

A 0.52** 1 0.27ns 0.29ns 0.91** -0.46* -0.67** -0.47* 0.33* 0.19ns 0.17ns 0.98** 0.39* E 0.98** 0.49** 1 -0.84** 0.63** 0.71** -0.21ns -0.97** -0.82** 0.98** 0.98** 0.38* 0.96**

WUE -0.74** 0.15ns -0.79** 1 -0.12ns -0.96** -0.19ns 0.70** 0.99** -0.86** -0.88** 0.17ns -0.73** SOD 0.88** 0.81** 0.82** -0.35* 1 -0.09ns -0.57** -0.79** -0.09ns 0.56** 0.53** 0.93** 0.70** APX 0.53** -0.37* 0.47* -0.79** 0.24ns 1 0.14ns -0.55** -0.95** 0.78** 0.79** -0.32ns 0.63** GR -0.04ns -0.86** -0.04ns -0.55** -0.41* 0.78** 1 0.37* -0.25ns -0.28ns -0.26ns -0.77** -0.46* MDA -0.97** -0.70** -0.94** 0.57** -0.96** -0.36* 0.25ns 1 0.67** -0.95** -0.94** -0.58** -0.98**

TSP -0.46* 0.39* -0.57** 0.92** -0.00ns -0.62** -0.63** 0.26ns 1 -0.83** -0.85** 0.21ns -0.69** RWC 0.97** 0.35* 0.98** -0.87** 0.74** 0.57** 0.10ns -0.89** -0.67** 1 1.00bs 0.33* 0.97** Biomass 0.97** 0.34* 0.99** -0.86** 0.75** 0.58** 0.09ns -0.90** -0.66** 1.00ns 1 0.31ns 0.97** LZn 0.53** 0.99** 0.48** 0.15ns 0.83** -0.34* -0.85** -0.71** 0.41* 0.35* 0.36* 1 0.53** Lk 0.95** 0.62** 0.98** -0.67** 0.84** 0.29ns -0.23ns -0.95** -0.46* 0.94** 0.94** 0.61** 1

*=p≤ 0.05; ** p≤ 0.01; well watered= 70% field capacity; Drought= 35% field capacity; Zn= Zinc;Chl.= Chlorophyll; Pn= Photosyntehsis; E= transpiration; WUE= water

use efficiency; SOD= Super oxide dismutase; APX= Ascorbate peroxidase; GR= Glutathione reductase; MDA= melanodialdehyde; TSP= Total soluble Phenolics; RWC=

relative water content; LZn= Leaf Zn content; LK= Leaf K content

Chl A E WUE SOD APX GR MDA TSP RWC Biomass LZn LK Chl 1 0.74** 0.69** - 0.27 ns 0.85** 0.18 ns - 0.83** - 0.82** - 0.21 ns 0.71** 0.69** 0.86** 0.85**

221

Table 4.69: Correlation coefficients of grain yield, protein content, Zn content, endosperm Zn concentration, phytate and bioavailable

Zn in wheat cultivars under optimal and cold stressed conditions (n=4)

*=p≤ 0.05; ** p≤ 0.01; well watered= 70% field capacity; Drought= 35% field capacity; Zn= Zinc

Grain yield Protein content Grain Zn Content Endosperm Zn Phytate Bioavailable Zn GY 1 ns 0.11 0.656 ** ns 0.24 - ns 0.31 0.51 ** Protein content ns 0.11 1 0.59 ** 0.57 ** - 0.87 ** 0.64 ** Grain Zn Content 0.66 ** 0.59 ** 1 0.88 ** - 0.88 ** 0.98 ** End Zn 0.24 ns 0.57 ** 0.88 ** 1 - 0.88 ** 0.95 ** Phy - 0.31 ns - 0.87 ** - 0.88 ** - 0.88 ** 1 - 0.93 ** Bio Zn 0.51 * 0.64 ** 0.98 ** 0.95 ** - 0.93 ** 1

222

Cold stress enhanced the protein accumulation in wheat leaves irrespective of Zn

supply, whereas a further increase in buffer extractable leaf protein concentration was recorded

with adequate Zn supply. Cold stress enhanced the lipid peroxidation as was visible from

increase MDA content. Leaf MDA content was negatively correlated with SOD and APX

activity under cold stress while it had a positive correlation with leaf GR activity in cold

stressed plants. The total activities of SOD, APX and GR were higher under cold stress; while

specific activities of SOD, APX, and GR were lower under cold stress. Activity of SOD is

influenced by Zn availability as expression of Cu/Zn SOD is higher in Zn efficient genotypes

which might have helped plants to reduce the oxidative damage caused by cold stress (Gill and

Tuteja, 2010). Similarly, APX and GR activity are also enhanced by enhanced Zn supply (Dai

et al., 2015). Substantial decrease in temperature enhanced the lipid peroxidation (Table 4.64;

Turk and Erdal, 2015; Zhang et al., 2015). However, adequate Zn supply reduced the lipid

peroxidation as low level of Zn results in membrane leakage with low –SH level (Rengel,

1995b). Adequate Zn supply increased the TSP concentration in wheat under cold stress (Table

4.64) as Zn application enhance the phenolic concentration in leaves (Tavallali et al., 2009).

Moreover, high TSP concentration in wheat leaves was possibly due up regulation of genes

involved in phenolics biosynthesis (Song et al., 2015).

Adequate Zn supply enhanced the Zn concentration/contents in wheat leaves. Low

temperature restricts Zn uptake (Turk and Erdal, 2015; Bradáčová et al., 2016). However,

application of stress protectants enhanced the Zn concentration upto 3 fold than control plants

under low temperature (Bradáčová et al., 2016). In present study, adequate Zn supply enhanced

the tissue Zn, N and K concentration in wheat. The increase in tissue N and K concentration is

due to positive interaction of Zn with N (Kutman et al., 2010; Erenoglu et al., 2011) and K

(Cakmak and Marschner, 1988) as leaf Zn concentration have positive correlation with leaf K

(Table 4.65).

Cold temperature induces the flower abortion, pollen and ovule infertility, causes

breakdown of fertilization and affects seed filling, leading to low seed set and ultimately low

grain yield (Thakur et al., 2010). These yield losses increases under Zn deficiency (Yilmaz et

al., 1997). In the present study, cold stress reduced the grains per spike, 100 grain weight, grain

yield and harvest index. However, the losses due to suboptimal temperature were less in the

223

plants receiving an adequate Zn supply (Table 4.66). The yield increase with adequate Zn was

12% over control under suboptimal temperature, while it was 24.6% under optimal temperature

(Table 4.66) as grain Zn content have positive correlation with grain yield (Table 4.69).

Likewise, Zn application augmented the protein concentration/contents (Liu et al., 2015), as

grain protein contents have positive correlation with grain Zn content (Table 4.69). However,

grain yield has a negative correlation with grain protein content. Increase in grain yield reduced

the grain protein content due to dilution effect (Tables 4.69). The increase in protein

concentration in adequate Zn treatment was due to enhanced N uptake (Kutman et al., 2010;

Erenoglu et al., 2011) which might have resulted in increased protein quantity. Zinc

fertilization at adequate level enhanced the Zn concentration (137%) and content (156%) under

optimal and suboptimal temperature compared to low Zn treatment. Similarly, embryo,

aleurone and endosperm Zn concentration increased upto 2 folds with adequate Zn supply.

Higher endospermic Zn concentration is an indicator of the high bioavailability of Zn due to

the presence of low phytate in this fraction of grain. Zinc fertilization reduces the phytate

concentration and phytate/Zn ratio (Bharti et al., 2013). Moreover, Zn fertilization enhanced

the concentration of bioavailable Zn (130%) due to reduction in phytate concentration (60.6%)

and low phytate/Zn ratio i.e.15 which is an indicator of high bioavailable Zn (Wang et al.,

2015). In the present study, seed Zn content have strong positive correlation with endospermic

and bioavailable Zn concentration while the correlation was strongly negative for phytate

concentration (Table 4.69).

Cold stress affected the wheat growth by reducing the biomass production, disturbing

the water relations and gas exchange, nutrient imbalance and ultimately deteriorating the

quality and quantity of produce. However, application of Zn ameliorated the adverse effect of

suboptimal temperature by regulating the photosynthesis, ROS scavenging and improving the

yield and grain biofortification of wheat.

224

4.9. Improving the Resistance against Heat Stress in Wheat through Zinc Nutrition 4.9.1.

Results

4.9.1.1 Seedling growth traits

Analysis of variance showed that supra-optimal temperature significantly affected all

the seedling growth traits except shoot dry weight and root diameter. Similarly, Zn application

significantly affected all the studied traits; while, wheat cultivars differed only for the total

biomass, shoot dry weight and root dry weight. Interaction of wheat cultivars and supraoptimal

temperature was significant for root length and root diameter. However interaction of wheat

cultivars and Zn treatment was only significant for shoot dry weight (Table 4.70). Furthermore,

interactive effect of supra-optimal temperature and Zn application was significant for all the

seedling growth traits except root diameter and root volume. However, interactive effect of

wheat cultivars, supra-optimal temperature and Zn application was significant for only root dry

weight (Table 4.70).

Application of Zn enhanced the seedling growth traits. In this respect, highest total

biomass and root dry weight was recorded for FSD-2008 under optimal temperature with

adequate Zn. However, maximum shoot biomass was recorded for the plants of FSD-2008

growth under supra-optimal temperature with adequate Zn (Table 4.70). Likewise, highest

shoot: root ratio was recorded for the same treatment in both cultivars.

Moreover, plant of LS-2008 grown under optimal temperature with adequate Zn supply

produced the longest roots with highest surface area and root volume, while average root

diameter was maximum for roots of LS-2008 and FSD-2008 with adequate Zn supply under

supra-optimal and optimal temperature (Table 4.70).

4.9.1.2 Gas exchange

Analysis of variance indicated that the temperature regimes significantly affected the

gas exchange traits except chlorophyll intensity. Similarly, effect of Zn application was

significant for all studied traits. However, wheat cultivars differed significantly only for

quantum yield (QY). However, interaction of wheat cultivars and temperature regime was

significant for stomatal conductance (gs) and QY; while interaction of wheat cultivars and Zn

treatment was significant for all the traits except rate of photosynthesis (A), water use efficiency

(WUE) and intercellular CO2 (Ci) (Table 4.71). Furthermore, interaction of temperature

225

regimes and Zn treatment was significant for all the traits except the chlorophyll intensity and

WUE. Whereas, interactive effect of wheat cultivars, temperature regime and Zn treatment was

significant for only gs and QY.

Table 4.70: Effect of temperature regime and zinc (Zn) supply on total biomass, shoot and root dry matter production, shoot:root

ratio, root length, surface area, diameter and volume of two wheat cultivars LS-2008 and FSD-2008


regime Zn supply Total biomass

(mg plant-1)

Shoot

(mg plant-1)

Root

(mg plant-1)

[Shoot : Root] RL

(cm plant-1)

Root PA

(cm2)

Root SA

(cm2)

Root

diameter

( mm)

Root

volume

(cm3)

LS-2008 Optimal Low 242±4 143±5 99±3BC 1.4±0.1 1536±53 49.13±5 154.3±15 0.32±0.02 1.24±0.19

Adequate 338±8 231±7 107±3B 2.2±0.1 1819±71 59.4±8 186.6±11 0.33±0.02 1.53±0.16

Supraoptimal Low 184±3 121±9 64±11D 1.9±0.4 730±55 25.13±4 79.0±14 0.34±0.04 0.69±0.28

Adequate 337±10 246±7 91±4C 2.7±0.1 1227±61 44.21±5 138.9±15 0.36±0.01 1.25±0.16

FSD-2008 Optimal Low 254±7 153±7 101±7BC 1.5±0.2 1358±72 45.41±5 142.7±15 0.33±0.02 1.19±0.17

Adequate 351±5 229±7 122±2A 1.9±0.1 1602±73 57.06±1 179.3±5 0.36±0.04 1.63±0.19

Supraoptimal Low 207±6 134±\8 73±2D 1.8±0.2 759±69 24.64±4 77.4±16 0.32±0.01 0.63±0.21

Adequate 342±13 249±10 93±4C 2.7±0.1 1285±67 43.67±5 137.2±14 0.34±0.01 1.17±0.11

P C 0.0001*** 0.026* 0.0015** 0.241 0.0028** 0.3112 0.2643 0.7013 0.7619

P T 0.000*** 0.570 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.6147 0.000***

P Zn 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.0300* 0.000***

P C x T 0.858 0.481 0.418 0.717 0.000*** 0.4665 0.4274 0.0159* 0.4704

P C x Zn 0.136 0.051* 0.537 0.335 0.909 0.8356 0.8333 0.5939 0.6665

P T x Zn 0.000*** 0.000*** 0.0208* 0.0488* 0.000*** 0.0255* 0.0155* 0.9459 0.1793

P C x T x Zn 0.107 0.802 0.0115* 0.070 0.466 0.8242 0.8195 0.4675 0.516

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±= S.D. P= p value; *=p≤

0.05; ** p≤ 0.01; *** p< 0.001; optimal= 25/18ºC day/night; Supraoptimal = 36/28ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; RL= root length; PA=

projected area; SA= surface area; LS-2008= Lasani-2008; FSD-2008= Faisalabad-2008

170

Table 4.71: Effect of temperature regime and zinc (Zn) supply on chlorophyll density, photosynthesis (A), transpiration rate (E),

water use efficiency (WUE), intercellular carbon dioxide (Ci), stomatal conductance (gs) and quantum yield (QY) in two wheat


Cultivar Temperature Zn supply Chlorophyll A E WUE Ci gs QY

regime (SPAD) (µmol m-2 s-1) (mmol m-2 s-1) (µmol mol-1) (mmol m ²s ¹)

Adequate 44±1.5 20.3±0.5 5.2±0.8 4.0±0.6 264±18 0.29±0.02BC 0.57±0.01B

Supraoptimal Low 36±2.2 4.0±1.2 7.7±1.2 0.5±0.1 357±23 0.33±0.03B 0.16±0.03E

Adequate 45±2.3 22.7±4.5 15.0±2.4 1.5±0.1 278±7 0.27±0.01C 0.62±0.01A

FSD-2008 Optimal Low 24±3.9 17.0±1.5 5.8±0.9 3.0±0.8 295±25 0.17±0.01D 0.52±0.01C

Adequate 50±0.5 19.2±1.1 4.8±0.6 4.1±0.3 264±11 0.40±0.03A 0.57±0.01B

Supraoptimal Low 28±3.0 4.2±1.0 10.0±1.1 0.4±0.0 355±18 0.27±0.02C 0.27±0.03D

Adequate 51±3.8 20.7±1.7 13.9±1.6 1.5±0.0 285±3 0.39±0.02A 0.62±0.01A

P C 0.075

0.4603 P T 0.073 0.000*** 0.000*** 0.000*** 0.000*** 0.0045** 0.000***

P Zn 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P C x T 0.513 0.5494 0.3536 0.3775 0.6732 0.0011** 0.0037**

P C x Zn 0.000*** 0.1479 0.0465* 0.9229 0.5360 0.000*** 0.0001***

P T x Zn 0.324 0.000*** 0.000*** 0.7862 0.0012** 0.000*** 0.000***

P C x T x Zn 0.673 0.9147 0.1195 0.7059 0.8085 0.0063* 0.0035**

LS - 2008 Optimal Low 34±2.5 16.2±0.6 5.8±0.4 2.8±0.1 300±4 0.31±0.02 BC 0.49±0.02 C

0.6512 0.6952 0.9710 0.2466 0.0001***

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s hone sty significant difference test; ±= S.D. P= p value; *=p≤

171

0.05; ** p≤ 0.01; *** p< 0.001; optimal= 25/18ºC day/night; Supraoptimal = 36/28ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008;

FSD-2008= Faisalabad-2008

229

Zinc application increased the chlorophyll intensity under optimal and supra-optimal

temperature. For instance, maximum SPAD value was recorded for FSD-2008 with adequate

Zn supply under both optimal and supra-optimal temperature. However highest A, E were

recorded for the plants of LS-2008 grown in supra-optimal temperature with adequate Zn,

whereas, the maximum WUE and lowest Ci were recorded in plants of both wheat cultivars

grown under optimal temperature with adequate Zn (Table 4.69). Moreover, highest rate of gs

was noted for FSD-2008 with adequate Zn supply under both optimal and supra-optimal

temperature. However, highest QY was recorded for the both wheat cultivars with adequate Zn

under supra-optimal temperature (Table 4.71).

4.9.1.3 Enzyme activities

Supra-optimal temperature significantly affected the enzyme activities in seedling of

wheat except APX activity. Likewise, Zn treatment significantly affected all the enzyme

activities. However, wheat cultivars differed only for leaf and root protein concentration, leaf

SOD and GR activity. Interaction of wheat cultivars and supra-optimal temperature was only

significant for root SOD activity (Table 4.72). Furthermore, interactive effect of wheat

cultivars and the Zn treatments was significant for the leaf protein and SOD activity. Interaction

of supra-optimal temperature and Zn treatment was significant for leaf protein and specific

activity of root GR. Moreover, interaction of wheat cultivars, supra-optimal temperature and

Zn application was significant for only root SOD activity (Table 4.72). Adequate supply of Zn

enhanced the protein accumulation in wheat as maximum leaf and root protein concentration

was recorded for the plants of FSD-2008 receiving adequate Zn under supra-optimal

temperature. The highest SOD activities in leaf were recorded with similar treatment in both

cultivars. Highest specific activities of leaf APX was observed for LS-2008 with adequate Zn

treatment under optimal temperature. However, the maximum root APX activity was recorded

for LS-2008 with adequate Zn treatment under supra-optimal temperature (Table 4.72).

Adequate Zn enhanced the GR activities as the highest leaf GR activity was recorded for the

both wheat cultivars with adequate Zn treatment under optimal temperature. Whereas, root GR

activity was greatest with low Zn supply under the optimal temperature (Table 4.72).

4.9.1.4 Seedling mineral analysis

i. Seedling Zn analysis

230

Analysis of variance showed that supra-optimal temperature significantly affected the

Zn related traits except root Zn concentration. Likewise, Zn application significantly affected

all the studied traits; while, wheat cultivars differed significantly only for root Zn contents and

the total Zn uptake (Table 4.73). Moreover, interaction of wheat cultivars and temperature

regime did not differ significantly for studied traits; while interactive effect of wheat cultivars

and Zn treatment was only significant for root Zn contents. Moreover, interaction of wheat

cultivars, temperature regime and Zn supply was significant for all the studied traits; while,

interactive effect of wheat cultivars, temperature regime and Zn application was only

significant for root Zn contents (Table 4.73).

Zinc application increased the concentration/contents and total uptake of Zn under

optimal and supra-optimal temperature. In this regard, the highest Zn concentration/contents

in root and shoot of wheat and total Zn uptake was recorded in both wheat cultivars with

adequate Zn supply under optimal temperature (Table 4.73).

ii. Seedling potassium analysis

Supra-optimal temperature significantly affected the concentration/contents and total

uptake of wheat. Similarly Zn application significantly affected all the K parameters. Likewise,

wheat cultivars differed significantly for all studied traits except shoot K concentration.

Interaction of wheat cultivars and supra-optimal temperature was significant for shoot K

concentration/contents and total uptake, while, interactive effect of wheat cultivars and Zn

treatment was not significant for all studied traits. Moreover, interaction of wheat cultivars,

supra-optimal temperature, and Zn application was significant for only root K concentration

and shoot K contents (Table 4.74).

Zinc supply positively influenced the K uptake by wheat as the highest shoot K

concentration/ contents were recorded in the plants of LS-2008 with adequate Zn supply grown

under optimal condition. Similarly, maximum root K concentration was recorded with same

treatment in FSD-2008. Likewise, root K contents were the highest in both wheat cultivars with

adequate Zn supply under optimal temperature. Moreover, total Zn uptake was highest for

plants of FSD-2008 receiving the adequate Zn under optimal temperature (Table 4.74).

iii. Shoot nitrogen (N) analysis

231

Supra-optimal temperature and Zn treatment significantly affected N

concentration/contents. Likewise, wheat cultivars differed significantly for N

concentration/contents. However, the interaction of wheat cultivars and supra-optimal

temperature was non-significant for the N; while interactive effect of wheat cultivars and Zn

application, supra-optimal temperature and Zn treatment was significant for shoot N contents.

The interaction of wheat cultivars, supra-optimal temperature and Zn application was

significant for shoot N concentration (Table 4.74).

Zinc application increased the N accumulation in wheat as the maximum N

concentration and contents were recorded in shoot of LS-2008 with adequate Zn supply under

supra-optimal condition (Table 4.74).

iv. Seedling phosphorus (P) analysis

Supra-optimal temperature stress significantly affected the seedling P traits. However,

Zn application significantly affected only the shoot P concentration and root P contents.

Likewise, wheat cultivars differed significantly only for the root P contents.

Likewise, interaction of wheat cultivars and Zn treatments was not significant for any

P related traits. In the same line, interactive effect of wheat cultivars and Zn treatment was only

significant for total P uptake; while, interaction of supra-optimal temperature and Zn

application was significant for shoot P concentration and content. However, results for the

interaction of wheat cultivars, supra-optimal temperature and Zn treatment remained

nonsignificant for all P related traits (Table 4.75).

Zinc application reduced the P uptake by wheat as the lowest shoot and root P

concentration was recorded for LS-2008 with adequate Zn supply under supra-optimal

temperature. Moreover, shoot, root P contents and total P uptake were the minimum in LS2008

with low Zn supply under supra-optimal temperature (Table 4.75).

232

Table 4.72: Effect of temperature regime and zinc (Zn) supply on buffer-extractable protein concentration and specific

activities of antioxidative enzymes superoxide dismutase (SOD), ascorbate peroxidase (AP) and glutathione reductase (in

leaves and roots of two wheat cultivars LS-2008 and FSD-2008

Temperature Cultivar Zn supply Leaf Root Leaf Root Leaf Root Leaf Root

regime (mg g-1 FW) (U mg-1 protein) (μmol H2O2 mg−1 prt. min−1) (μmol [NADPH] mg−1 prt. min−1)

1.47±0.14

Adequate 15.3±1.5 6.8±0.7 27.1±2.5 30.5±2.9AB 1.84±0.33 1.88±0.25 0.87±0.07 1.01±0.09

Supraoptimal Low 20.3±1.8 10.0±1.7 19.7±1.5 26.1±2.9BC 1.45±0.16 1.78±0.22 0.71±0.06 0.80±0.14

Adequate 26.5±1.9 10.6±0.6 29.4±3.1 34.6±1.8A 1.68±0.19 2.02±0.26 0.62±0.06 0.77±0.04

FSD-2008 Optimal Low 14.8±2.5 5.2±0.7 16.9±4.5 22.9±4.5C 1.40±0.24 1.62±0.12 0.86±0.15 1.26±0.18

Adequate 18.5±1.2 8.3±0.8 20.7±0.5 29.9±5.5ABC 1.55±0.10 1.71±0.09 0.72±0.05 0.88±0.09

Supraoptimal Low 20.8±2.0 11.3±0.9 20.2±1.4 24.3±2.6BC 1.55±0.12 1.74±0.20 0.69±0.06 0.71±0.05

Adequate 30.8±1.3 12.1±1.1 25.3±1.3 34.4±1.1A 1.60±0.19 1.86±0.18 0.55±0.02 0.68±0.06

P C

0.0004*** 0.239 P T 0.000*** 0.000*** 0.0023** 0.000*** 0.9084 0.1051 0.000*** 0.000*** P Zn 0.000*** 0.0004*** 0.000*** 0.000*** 0.0081** 0.0356* 0.0007*** 0.001***

P C x T 0.8339 0.1975 0.174 0.0225* 0.1679 0.7392 0.1838 0.1942

P C x Zn 0.0559* 0.1011 0.0099** 0.0707 0.1578 0.5672 0.4816 0.0885

P T x Zn 0.0005*** 0.0723 0.3807 0.2377 0.3847 0.631 0.9516 0.0094**

P C x T x Zn 0.282 0.1952 0.9757 0.0147* 0.8665 0.7956 0.9516 0.083

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±= S.D. P= p value; *=p≤ 0.05; ** p≤ 0.01; *** p< 0.001; optimal= 25/18ºC day/night; Supraoptimal = 36/28ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008=

Lasani-2008; FSD-2008= Faisalabad-2008

Protein SOD AP GR

LS - 2008 Optimal Low 12.8±1.5 5.8±0.7 18.8±0.3 13.4±1.0 D 1.75±0.09 0.96±0.13 1.12±0.10

0.0098** 0.0014** 0.13 0.0709 0.01* 0.2417

233

LS - 2008 Optimal Low 1.39±0.04 D 2.75±0.14

Table 4.73: Effect of temperature regime and zinc (Zn) supply on concentration, content and total uptake of Zn in two wheat


Cultivar Temperature regime Zn supply

(mg kg-1) (μg plant-1) (μg plant-1)

9.52±0.7 14.0±0.8 1.36±0.14

Adequate 43.4±5.4 44.7±2.1 10.1±1.41 4.77±0.16B 14.83±1.52

Supraoptimal Low 10.1±1.3 16.5±0.7 1.21±0.11 1.05±0.14D 2.26±0.20

Adequate 23.8±0.7 37.8±3.3 5.85±0.27 3.44±0.18A 9.29±0.40

FSD-2008 Optimal Low 11.7±4.1 11.9±0.7 1.78±0.56 1.21±0.02D 2.99±0.55

Adequate 43.4±0.4 44.8±1.7 10.0±0.31 5.46±0.16C 15.42±0.38

Supraoptimal Low 11.1±0.5 17.1±0.6 1.48±0.04 1.25±0.06D 2.73±0.07

Adequate 27.2±2.3 40.4±3.4 6.78±0.57 3.74±0.42C 10.52±0.99

P C 0.0903

0.6498 0.0849 P T 0.000*** 0.2074 0.000*** 0.000*** 0.000***

P Zn 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P C x T 0.5619 0.0780 0.3096 0.9853 0.3882

P C x Zn 0.9626 0.1483 0.8695 0.0014** 0.2783

P T x Zn 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P C x T x Zn 0.2135 0.9171 0.1743 0.0074* 0.6904

Means sharing same case letter, for a parameter, do not differ significantly at p ≤ 0.05 by Tukey.s honesty significant difference test; ±= S.D. P= p value;

*=p≤ 0.05; ** p≤ 0.01; *** p< 0.001; optimal= 25/18ºC day/night; Supraoptimal = 36/28ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD-2008= Faisalabad-2008

Zn concentration Zn content Total Zn uptake

Shoot Root Shoot Root ( ) shoot+root

0.001** 0.0185*

234

Table 4.74: Effect of temperature regime and zinc (Zn) supply on concentration, content and total uptake of potassium (K) and

shoot N concentration and contents in two wheat cultivars LS-2008 and FSD-2008

K concentration K content Total K N concentration N content

Temperature uptake

Cultivar Zn supply regime Shoot

Root Shoot Root (shoot+root) Shoot Shoot

(%) (mg plant-1) (mg plant-1) (%) (mg plant-1)

6.10±0.21D

Adequate 4.57±0.24 4.92±0.07AB 10.6±0.29A 5.25±0.19 15.8±0.40 6.60±0.08B 15.3±0.54

Supraoptimal Low 3.06±0.07 4.28±0.12E 3.70±0.34E 2.72±0.46 6.42±0.16 6.25±0.28BC 7.56±0.83

Adequate 3.38±0.05 4.31±0.02E 8.30±0.27C 3.93±0.17 12.2±0.38 7.49±0.19A 18.4±0.86

FSD-2008 Optimal Low 4.15±0.08 4.93±0.15AB 6.35±0.34D 4.99±0.28 11.3±0.44 4.48±0.03E 6.87±0.35

Adequate 4.38±0.07 5.04±0.10A 10.0±0.27A 6.14±0.14 16.2±0.21 5.54±0.21D 12.7±0.77

Supraoptimal Low 3.22±0.07 4.34±0.13DE 4.31±0.26E 3.17±0.19 7.48±0.21 5.24±0.14D 7.00±0.42

Adequate 3.62±0.09 4.58±0.12CD 9.04±0.43B 4.24±0.28 13.3±0.65 6.08±0.11C 15.2±0.87

P C 0.6101 0.0002*** 0.0195* 0.000*** 0.000*** 0.000*** 0.000***

P T 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P Zn 0.000*** 0.0008*** 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P C x T 0.0001*** 0.8376 0.0011** 0.2219 0.0373* 0.6215 0.6667

P C x Zn 0.8561 0.5822 0.1546 0.2691 0.6797 0.8321 0.001**

P T x Zn 0.2116 0.6708 0.0126* 0.1692 0.0033** 0.1133 0.000***

P C x T x Zn 0.3684 0.0476* 0.0533* 0.0666 0.7217 0.0024** 0.0710

Means sharing same case letter for a parameter do not differ significantly at (p ≤ 0.05) by Tukey.s honesty significant difference test; ± S.D. P= p value;


LS - 2008 Optimal Low 4.28±0.04 4.69±0.13 BC 4.66±0.23 10.8±0.23 5.98±0.19 C 8.52±0.45

235

Table 4.75: Effect of temperature regime and zinc (Zn) supply on concentration, content and total uptake of phosphorus (P) in two

wheat cultivars LS-2008 and FSD-2008

Supraoptimal Low 0.99±0.07 0.55±0.02 1.19±0.16 0.35±0.04 1.54±0.13

Adequate 0.60±0.01 0.54±0.06 1.47±0.04 0.49±0.04 1.97±0.06

FSD-2008 Optimal Low 1.51±0.24 0.69±0.03 2.31±0.40 0.70±0.06 3.01±0.45

Adequate 0.76±0.01 0.66±0.04 1.75±0.07 0.81±0.06 2.56±0.08

Supraoptimal Low 1.05±0.02 0.57±0.00 1.40±0.09 0.41±0.02 1.81±0.07

Adequate 0.66±0.03 0.56±0.01 1.64±0.10 0.52±0.03 2.16±0.13

P C 0.9323 0.9593 0.1817

P T 0.000*** 0.000*** 0.000*** 0.000*** 0.000***

P Zn 0.000*** 0.2898 0.0181 0.000*** 0.4067

P C x T 0.109 0.2474 0.1183 0.9677 0.1549

P C x Zn 0.3547 0.9593 0.9117 0.4935 0.000***

P T x Zn 0.000*** 0.6469 0.000*** 0.1065 0.9421

P C x T x Zn 0.3547 0.7214 0.7856 0.0665 0.5383

Means sharing same case letter for a parameter do not differ significantly at (p ≤ 0.05) by Tukey.s honesty significant difference test; ±= S.D. P= p value;



regime Zn supply

P concentration P content Total P uptake

Shoot Root Shoot Root shoot+root ( )

( % ) mg plant ( - 1 ) mg plant ( - 1 )

LS - 2008 Optimal Low 1.64±0.14 0.69±0.01 2.34±0.17 0.69±0.02 3.03±0.17

Adequate 0.76±0.03 0.68±0.05 1.75±0.07 0.73±0.05 2.48±0.12

0.0094** 0.0733

236

Table 4.76: Effect of temperature regime and zinc (Zn) supply on concentration, content and total uptake of calcium (Ca) in

two wheat cultivars LS-2008 and FSD-2008

Adequate 0.46±0.02E 0.18±0.02 1.07±0.07 0.19±0.01BCD 1.26±0.07

Supraoptimal Low 0.65±0.01B 0.19±0.01 0.79±0.07 0.12±0.02F 0.91±0.05

Adequate 0.55±0.01CD 0.22±0.02 1.35±0.05 0.20±0.02ABC 1.55±0.04

FSD-2008 Optimal Low 0.54±0.01D 0.18±0.00 0.83±0.04 0.19±0.01CD 1.01±0.04

Adequate 0.44±0.02E 0.19±0.00 1.00±0.07 0.23±0.01A 1.23±0.06

P T 0.000*** P Zn 0.000***

P C x T 0.000***

P C x Zn 0.0487*

P T x Zn 0.0233*

Means sharing same case letter for a parameter do not differ significantly at (p ≤ 0.05) by Tukey.s honesty significant difference test; ±= S.D.; P= p value; *=p≤ 0.05; ** p≤ 0.01; *** p< 0.001; optimal= 25/18ºC day/night; Supraoptimal = 36/28ºC day/night; C= cultivar, T= Treatment; Zn= Zinc; LS-2008= Lasani-2008; FSD-2008= Faisalabad-2008


regime Zn supply

Ca concentration Ca content Total Ca uptake Shoot Root Shoot Root ( shoot+root )

) ( % ( mg plant - 1 ) ( mg plant - 1 ) LS - 2008 Optimal Low 0.57±0.01 CD 0.17±0.01 0.82±0.03 DE 0.17±0.01 0.98±0.04

Supraoptimal Low 0.76±0.02 A 0.21±0.01 1.02±0.05 E 0.16±0.01 1.17±0.05 Adequate CD 0.59±0.04 0.23±0.01 1.47±0.10 AB 0.21±0.01 1.68±0.11

P C 0.0038** 0.0055** 0.0026** 0.000*** 0.0001*** 0.000*** 0.000*** 0.0001*** 0.000*** 0.0015** 0.000*** 0.000*** 0.000*** 0.8164 0.0001*** 0.4804 0.0001***

0.3571 0.0511* 0.8682 0.0377* 0.0647 0.000*** 0.0002*** 0.000***

P C x T x Zn 0.0094** 1.000 0.6933 0.0345* 0.4481

237

Fig. 4.11 : (a) Influence of temperature regimes on Zn efficiency index of wheat cultivars (b) Effect of Zn supply on heat tolerance

index of wheat cultivars

A

B

A

B

0

20

40

60

80

100

Optimal Supraoptimal Optimal Supraoptimal

LS-2008 FSD-2008

B

A

B

A

0

20

40

60

80

100

120

Low Adequate Low Adequate

LS-2008 FSD-2008

( a ) ) b (

238

Table 4.77: Correlation coefficients of gas exchange traits, enzyme activities, biomass production, relative water content, Zn, K and Ca

uptake of wheat cultivars under optimal and heat stressed conditions (n=4)

Biomass

RL 0.68** 1 0.72** 0.54** 0.90** -0.49** 0.93** 0.87** 0.00ns 0.39* -0.18ns -0.16ns 0.16ns 0.42* 0.94**

Zn utpake 0.89** 0.66** 1 0.93** 0.66** -0.11ns 0.58** 0.69** 0.53** 0.77** 0.52** 0.39* -0.42* -0.30ns 0.91** 0.48*

Chl 0.90** 0.37* 0.90** 1 0.64** 0.27ns 0.29ns 0.69** 0.80** 0.95** 0.73** 0.70** -0.71** -0.54** 0.78** 0.77**

A 0.93** 0.74** 0.71** 0.72** 1 -0.07ns 0.67** 0.99** 0.31* 0.62** -0.01ns 0.16ns -0.14ns 0.24ns 0.87** 0.41*

E 0.34* -0.37* 0.05ns 0.48** 0.34* 1 -0.78** 0.00ns 0.78** 0.53** 0.64** 0.86** -0.84** -0.71** -0.30ns 0.81**

WUE 0.59** 0.98** 0.66** 0.31ns 0.62** -0.52** 1 0.62** -0.32* 0.06ns -0.39* -0.47* 0.46* 0.59** 0.81** -0.29ns

QY 0.91** 0.77** 0.68** 0.67** 0.99** 0.29ns 0.65** 1 0.39* 0.69** 0.07ns 0.25ns -0.22ns 0.16ns 0.87** 0.49**

LSOD 0.91** 0.33* 0.84** 0.99** 0.76** 0.59** 0.25ns 0.71** 1 0.9Z** 0.90** 0.99** -0.98** -0.82** 0.29ns 0.98**

RSOD 0.59** -0.10ns 0.66** 0.88** 0.32* 0.60** -0.13ns 0.25ns 0.86** 1 0.77** 0.85** -0.85** -0.60** 0.63** 0.93**

LAPX 0.90** 0.66** 1.00ns 0.91** 0.72** 0.08ns 0.65** 0.69** 0.85** 0.67** 1 0.91** -0.93** -0.97** 0.16ns 0.80**

RSOD 0.79** 0.09ns 0.63** 0.91** 0.68** 0.81** -0.02ns 0.62** 0.95** 0.86** 0.65** 1 -0.99** -0.87** 0.12ns 0.96**

LGR -0.13ns 0.63** -0.06ns -0.47* 0.03ns -0.85** 0.69** 0.10ns -0.52** -0.78** -0.07ns -0.70** 1 0.90** -0.13ns -0.95**

RGR 0.00ns 0.73** 0.01ns -0.37* 0.19ns -0.75** 0.76** 0.26ns -0.40* -0.74** 0.00ns -0.59** 0.98** 1 0.09ns -0.71**

LK 0.91** 0.91** 0.90** 0.73** 0.87** -0.07ns 0.87** 0.87** 0.69** 0.33* 0.90** 0.48* 0.26ns 0.37* 1 0.30ns

L Ca 0.89** 0.30ns 0.69** 0.91** 0.83** 0.73** 0.17ns 0.79** 0.96** 0.75** 0.71** 0.97** -0.53** -0.39* 0.63** 1

*=p≤ 0.05; ** p≤ 0.01 optimal= 25/18ºC day/night; Supraoptimal = 36/28ºC day/night; Zn= Zinc; Chl.= Chlorophyll; Pn= Photosynthesis; E= Transpiration;

WUE= Water use efficiency; QY= Quantum yield; L= leaf; R= root; RL= root length; SOD= super oxide dismutase; APX= ascorbate peroxidase;

biomass RL Zn uptake Chl A E WUE QY LSOD RSOD LAPX RSOD LGR RGR LK L Ca

1 0.81**

0.93** 0.92**

0.87** 0.02 ns

0.58** 0.90**

0.58** 0.85**

0.41* 0.44*

- 0.44* - 0.17 ns

0.94** 0.60

239

GR= glutathione reductase; Leaf Zn= Leaf Zn uptake content; LK= Leaf K content; LCa= Leaf Ca

240

v. Seedling calcium (Ca) analysis

Supra-optimal temperature significantly affected all the Ca related traits. Likewise, Zn

application and wheat cultivars differed significantly for all the Ca parameters. However,

interaction of wheat cultivars and supra-optimal temperature, wheat cultivars and Zn

application was significant for shoot Ca concentration/contents and the total Ca uptake (Table

4.76). Moreover, the interactive effect of supra-optimal temperature and Zn treatments was

significant for all the traits except root Ca concentration; while, the interaction of wheat

cultivars, supra-optimal temperature and Zn application were significant for shoot Ca

concentration and root Ca content; results being non-significant for rest of the traits (Table

4.76).

vi. Zinc efficiency index and heat tolerance index

Zinc application improved the Zn efficiency index and heat tolerance index as the

maximum Zn efficiency was recorded for both wheat cultivars under optimal temperature (Fig.

4.9a). The heat tolerance index was the highest in both wheat cultivars when supplied with

adequate Zn (Fig 4.9b).

4.9.2 Discussion

Wheat growth is reduced under Zn deficiency and supra-optimal temperature. This

study indicated that the adverse effect of the supra-optimal temperature can be overcome by

the adequate Zn supply at early growth stages of wheat. Indeed, heat stress reduces the

photosynthesis in wheat (Wahid et al., 2007), which results in poor growth and grain yield

(AlKhatib and Paulsen, 1990). In this study, Zn application increased the chlorophyll intensity

under supraoptimal temperature. The plants which received the low Zn supply had lowest

chlorophyll intensity under supraoptimal temperature due to less green color (Khalil et al.,

1998) as Zn dearth disrupts the chlorophyll synthesis (Hisamitsu et al., 2001), which ultimately

affects the chloroplast structure under low Zn availability (Peck and McDonald, 2010).

Moreover, enhanced accumulation of chlorophyll due application of Zn supply possibly due to

the role of Zn in the structure and catalytic activities of enzymes and protein, and the

biosynthesis of green plant pigments (Balashouri, 1995). Adequate Zn application improved

the plant biomass and root growth as Zn uptake is positively correlated with biomass

production, root length under optimal and supraoptimal temperature (Table 4.77).

241

Adequate Zn supply enhanced the gas exchange traits of wheat under normal and supra-optimal

temperature. Indeed, heat stress reduces the photosynthesis, transpiration, stomatal

conductance and increases the intercellular CO2 in wheat (Feng et al., 2014), as observed in

present study. On the other hand, Zn supply has been found to increase the photosynthetic rate,

E and gs with lower Ci (Table 4.71) as plant Zn uptake is positively correlated with chlorophyll,

A, E, WUE and QY (Table 4.77).

Biomass production (root and shoot, root: shoot) of wheat plants was reduced under

low Zn supply (Table 4.70; Impa et al., 2013). However, adequate Zn supply increased the

plant root growth, root morphology and shoot biomass (Table 1; Dong et al., 1995), which was

visible through finer and long roots, more surface area, and root diameter/volume (Table 4.70).

Zinc deficiency and heat stress induces the production of reactive oxygen species (ROS) which

cause oxidative cellular damage (Suzuki and Mittler, 2006). Indeed, elevated temperature

accelerates the photorespiration in mitochondria by disrupting electron transport chain (ETC)

which results in increased H2O2 synthesis due to high photorespiration (Wahid et al., 2007;

Farooq et al., 2011). Heat stress aggravates the specific SOD activities in shoot and root;

suggesting the increased requirement of superoxide radical dismutation in chloroplast and

other important cell organelles like cytosol and mitochondria (Scandalios, 1993). High activity

of SOD are linked with adequate Zn supply (Pandey et al., 2002). Moreover, Zinc application

enhanced the specific activity of APX and GR (Table 4.68: Li et al., 2013) as was observed

through enhanced specific activities of shoot and root APX activity with adequate Zn supply

(Table 4.71).

Improvement in plant biomass, photosynthesis and enzymatic activities was due to

higher Zn concentration in the leaf and roots of wheat receiving adequate Zn. The better growth

of plants supplied with Zn have higher concentration/content and total uptake of K and Ca and

N. In another study, adequate supply of Zn increased the K concentration/content and total K

uptake in wheat, thus suggesting positive correlation of plant Zn status with K uptake (Aktaş

et al., 2007) as in the present study, Zn uptake is positively correlated with K and Ca uptake

(Table 4.77). Improved K uptake helps the plants against heat stress owing to its role in osmotic

regulation and enzyme activation (Waraich et al, 2011). Potassium supply also maintains the

242

physiological processes by regulating the photosynthesis, assimilate translocation and cell

membrane turgidity and enzyme activation (Marschner, 2012; Mengel and Kirkby, 2001).

Likewise, we observed increased uptake of Ca with adequate Zn supply. Calcium application

has been found to increase the activities of antioxidant enzyme thus helping in heat tolerance

(Kolupaev et al., 2005). However, increased Zn level were linked with reduced P and higher

N uptake. Moreover, adequate Zn supply reduced the effect of supraoptimal temperature and

increased the biomass production by improving the chlorophyll density, photosynthesis,

enhanced antioxidant activities, root growth and mineral acquisition.

In crux, supraoptimal temperature reduced the growth of wheat by limiting

photosynthesis, enzyme activities and ionic imbalance, yield and grain quality. However,

adequate Zn supply improved the resistance against elevated temperature by regulating

photosynthesis, enzyme activities, leaf mineral concentration. Moreover, adequate Zn supply

enhanced the wheat yield and Zn bioavailability under optimal and suboptimal temperature

243

4.10. General Discussion

Zinc is one of the most important micronutrient and is vital for plants as well as for

humans (Stein, 2010). Although all crop plants are prone to Zn deficiency, wheat is more

relatively sensitive, which results in low yield of poor quality (Cakmak et al., 1999). Grain Zn

contents in the most of wheat genotypes ranges from 10 µg g-1 to 30 µg g-1. Growing wheat on

low Zn soils results in further decrease in the grain Zn concentration. Nonetheless supplemental

Zn nutrition can help meet the wheat Zn requirement. Zinc may be delivered through soil

application, foliar spray and/or seed treatments (viz. seed priming and seed coating).

In this comprehensive series of studies, the rate and concentration of Zn application

through seed priming and coating were optimized. For seed priming, seed priming hastened

the germination and improved the seedling growth of bread wheat possibly due to enhanced

breakdown of food reserves and supply of energy to growing embryos (Farooq et al., 2006a,

b). However, seed priming in Zn solution further improved the seedling emergence (Tables

4.1, 4.3) suggesting the role of Zn in early stage of seedling emergence as Zn concentration is

high during early stages of radicle and coleoptile development (Ozturk et al., 2006). Seed

priming with Zn also enahcned the the chlorphyll contents, grain yield and grain Zn

concentration. Improvement in grain yield of seed priming with 0.5 M solution was due to

more number of grains per spike, 100 grain weight (Table 4.8) owing to improved grain setting,

suggesting the role of Zn in pollination and fertilization possibly by improving the anther

(Sharma et al., 1987) and pollen development (Sharma et al., 1990); prompting the pollen tube

formation (Pandey et al., 2006).

Seed coating is another effective alternative to soil and foliar Zn fertilization. We

observed that seed coated with 1.25 g Zn kg-1 seed have better seedling root and shoot length

possibly due to increased uptake of Zn as in seed coating Zn is available in the vicinity of

germinating seedling. Fruher, improvement in seedling growth was due to participation of Zn

in gerimation metabolism as it is needed in high concentratin for emerging plumule and radicle

during (Ozturk et al., 2006). Similarly, seed coated with Zn improved the seedling dry weight

due to better root and shoot growth (Tables 4.10, 4.12). Seed coating with 1.25 g Zn kg-1 seed

Zn improved yield related traits of wheat (Table 4.15). Substantial increase in grains per spike,

100-grain weight, by Zn seed coating, contributed for improvement in grain yield as adequate

244

supply of Zn helps in grain setting through synchronization of pollination and stigma

secretions, which favors pollen germination, pollen tube development, fertilization and,

eventually, grain setting (Kaya and Higgs, 2002; Pandey et al., 2006). However, seed priming

beyond 0.5 M Zn solution (ZnSO4) and 0.1 M (ZnCl2) and seed coating > 1.25 g Zn kg-1 seed

suppressed the gerimination and wheat growth possible because higher Zn concentration

restrict the root growth and cell divison (Prasad et al., 1999).

Along with agronomic apporahces to increase the grain Zn concentration of wheat,

development of wheat genotypes with high mineral concentration is also integral component

of biofortification. In this situation, genetic diversity of wheat genotypes can be exploited to

select nutrient dense wheat genotypes. We screened 28 wheat genotypes of Punjab, Pakistan

for their genetic diversity, grain bioforticaltion and grain Zn localization. The wheat genotypes

used in this study had very less genetic diversity owing to selfing and inbreeding as the most

of the wheat cultivars were developed only for grain yield. The genetic diversity of most of the

wheat genotypes ranged from 0.0355-0.0677 with maximum genetic diversity of 0.0842

recorded Millat-2011. Moreover, green revolution has lead to development of high yielding

wheat cultivar which intensified the issue of Zn deficiency (Cakmak et al., 2010a; Stein, 2010).

However, Zn application substantially increased the grain yield, grain proteins, whole grain

Zn, embryo, aleurone and endosperm concentration with high bioavaille Zn.

Zinc availblity in soil is reduced due to high pH, CaCO3, salinity, waterlogging and

organic metter (Alloway, 2008a, 2009). However, Zn uptake and its availability to plants can

be increased by use of beneficial rhizospheric microbes, which help increasing plant ability to

assimilate Zn present in soil and thus offer a sustainable and low input option to improve plant

Zn status (He et al., 2010). Zinc solubilizing plant growth promoting rhizobacteria (PGPR) are

found on root surface in rhizosphere (Ahmad et al., 2008; Maheshwari et al., 2012). These

PGPRs enhance the concentration of soluble Zn by modulating rhizospheric pH through

enhaced organic acid production and mineralizing the CaCO3 and organic bound Zn (Ramesh

et al., 2014). In this study, application of Zn in combination of Zn solubilizing PGPR (MN12)

improved the water relations, photosynthesis, yield, grain Zn accumulation and bioaviblity of

Zn in wheat. The improvement in water relation traits (Tables 4.32, 4.40) may be attributed to

better root growth (Nable and Webb, 1993) as PGPRs improve the plant growth by root

245

colonization (Moulin et al., 2001). Moreover, higher photosynthetic rate, stomatal conductance

and transpiration rate were observed in this study as Zn application is linked with better gas

exchange traits (Khan et al., 2004).

Application of Zn increased the yield and yield contributing traits of wheat, Further

improvement in grain yield and yield components of wheat was recorded with seed priming

and soil application of Zn using PGPRs as was evident from increase in 100-grain weight,

grains per spike and ultimately grain yield (Tables 4.34, 4.43) as Zn play key role in seed

setting and pollination (Pandey et al., 2006); which increased the seed weight with better

harvest index. Zinc nutrition improved the grain yield of wheat as it regulate the auxin,

carbohydrate metabolism, RNA and ribosome synthesis which lead to better yield with good

quality (Khalifa et al., 2011).

Seed priming with Zn + PGPR impressively increased the grian yield of wheat followed

by soil Zn application + PGPR. This increased in grain yield by possibly due to enhanced

uptake of Zn by wheat as Zn solubilizing PGPR produce impressively higher soluble Zn by

reducing rhizospheric pH, increased organic acid production and depleting organic and CaCO3

bound Zn (Ramesh et al., 2014) thus improving phytoavailability of Zn. Plant growth

promoting rhizobacteria can also be used an alternative to chemical fertilization to increase the

density of micronutrients in wheat grains along with better crop yield (Blanchfield, 2004). We

noted that Zn application by either method proved effeictive in increasing grian Zn

concentration and Zn bioavailability. However, soil and foliage Zn applicaotn + bacterial strain

MN12 impressively increased the grain Zn concentration and bioavability.

Changing climate increases the incidence of abiotic stresses such as water shortage, salt

stress and thermal stresses. The adverse effect of these stresses is escalated under Zn

deficiency. We observed that these stresses affected the wheat growth by limiting the

chlorophyll intensisty, gas exhchange tratis, water use efficiency and relative water contents

of wheat (Tables 4.49, 4.56, 4.63, 4.70) enhaced lipid peroxidation (Tables 4.50, 4.57, 4.64),

reduced Zn, N and K uptake (Tables 4.51,4.58 4.65). However, adequate Zn supply increased

the chlorophyll contents, A, E, gs and QY under these stresses compared to low Zn plants

(Tables 4.49, 4.63,4.70) as earlier application of Zn to salt stressed plants has been found to

enhance the chlorophyll contents and gas exchange traits (Tavallali et al., 2009; Amiri et al.,

246

2016). Moreover, lower photosynthetic rate in abiotic stresses in Zn-deficient wheat plants is

possibly due to lower activity of carbonic anhydrase (CA) and lower CO2 assimilation (Rengel,

1999).

Abiotic stresses lead to reduction in plant water staus by causing loss in turgor (Katerji

et al., 1997). However, adequate Zn supply to drought, salt and heat stressed plants improved

the relative water contents of wheat possibly due to enahced accumulation of TSP (Tables 4.50,

4.57, 4.64) and K+ (4.51, 4.58, 4.65), which helped in maintainence of tissue water status.

Higher K uptake helped plant in maintaining the water relations as K regulates the stomatal

closure (Sharma et al. 1995). Moreover, increase tolerance in Zn treated plants against theses

abiotic stresses was due decrease in lipid peroxidation and higher activities of SOD an APX

(Tables 4.50, 4.57, 4.64, 4.72), which helped in ameliorating the adverse effect of these

stresses. Moreover, heat stressed reduced the root growth. However, adequate Zn application

improved the root morphology and growth under heat stress.

The ultimate effect of these stresses in on the quanity and quality of yield. We observed

that water shortage, salt stress and cold stress reduced the wheat yield and this extent of yield

reduction was higher under Zn deficient condition. However, we noted that adequate Zn supply

increased the grain yield of wheat as was evident from more number of grains per spike and

100-grain weight (Tables 4.52, 4.59, 4.66).

Moreover, abiotic stress also hampered the wheat productivity by reducing the yield

and yield contributing traits. Application of Zn enhanced the wheat yield by increasing the

grains per spike, 100 grain weight and ultimately grain yield. Moreover, the improvement in

yield and yield contributing traits with adequate Zn application was due to increase in the

photosynthesis (Table 4.49, 4.56, 4.63, 4.71), antioxidant activities (4.50, 4.57, 4.64, 4.72) and

nutrient uptake (Table Tables 4.51,4.58 4.65) which ultimately boosted the wheat yield under

optimal and suboptimal conditions. Moreover, adequate Zn applicaotn improved the grain

quality of wheat by increasing the protein, Zn concentration/content and Zn bioavailability as

Zn fertilization has been reported to increased the protein and Zn accumulation in grain (Liu

et al., 2017). High bioavailability was due to less phyate and phyate/Zn ration as phytate/Zn

ration <15 is critical for Zn bioavaiability in human intestine (Brown et al., 2001).

247

Zinc is vital for all living organisms and its deficiency causes serious health issues in

humans. Wheat is staple for masses in the world and is deficient in Zn. Biofortifiation offers a

cost effective and pragmatic option to increased Zn concentration in wheat grain. Wheat

genotyps with wide genetic diversity with high grain Zn concentration and yield potential

should be included in the breeding programmes. Moreove, concentration in grains can be

increased through Zn delivery via soil, leaf or seed treatment using PGPR. However, soil and

foliage leaf application was most effective for increasing the grain Zn concentration and Zn

bioavailability while seed priming produced more yield with high net benefit. In addition to

yield improvement, grain quality, adequate Zn application under abiotic stresses viz. drought,

salt, thermal stresses improved the resitance against theses stresses through increased

photosynthesis, antioxidant enzyme activities, Z, N, and K uptake and reduced lipid

peroxidation.

248

Chapter 5

SUMMARY

Zinc is an important microelement for all life forms. Its deficiency in plants can hamper

the wheat productivity. It is also very crucial for human body. Wheat is staple for masses in

the world and it is more prone to Zn deficiency than other cereals. Moreover, rapid changing

climate events are increasing the incidence of abiotic stress. Crop mineral nutrition can

improve the resistance against abiotic stress in plants. Zinc have certain roles in plant

metabolism as enzymatic regulator, membrane integrity and protein synthesis. Thus, Zn

nutrition can be helpful in alleviating the abiotic stresses. Experiments reported in this thesis

were conducted at Department of Agronomy, University of Agriculture, Faisalabad, Pakistan

Plant Physiology Lab, Sabanci Unviversity, Istanbul, Turkey to (i) optimize the source and

application of Zn through seed treatments, (ii) characterize the wheat genotypes for genetic

diversity, Zn bioavailability and localization in different seed fractions, (iii) improve the

productivity and grain biofortification of wheat by combined application of Zn and

pseudomonas and (iv) explore the potential of Zn nutrition in improving tolerance against

abiotic stresses (drought, salt, heat, chilling). The summary of finding of these experiments is

given below.



This study was comprised of three separate experiments. First two studies were

optimization experiment and were conducted in the Allelopathy Laboratory, University of

Agriculture, Faisalabad, Pakistan during 2012-13. Seeds of wheat cultivars Lasani-2008 and

Faisalabad-2008 were obtained from the Wheat Research Institute, Faisalabad, Pakistan. Seeds

of bread wheat genotypes were soaked in aerated solution of 0.01, 0.05, 0.1, 0.5 and 1 M Zn

(ZnSO4 and ZnCl2) or water (hydropriming) for 12 h at 25 ± 2°C and then dried back to their

original weight. Third experiment was conducted in glass house of Faculty of Agriculture,

University of Agriculture, Faisalabad, Pakistan during 2012-13. The seeds of both wheat

cultivars were primed with pre-optimized rate of 0.1, 0.5 M solution of Zn using ZnSO4 and

249

0.05 and 0.1 M solution of Zn using ZnCl2 and were sown in earthen pots (48 cm × 30 cm).

The day temperature of glass house during the whole crop season was 24±3ºC while it was

14±3ºC during night. The experimental design used in this study was completely randomized

design with factorial arrangement. The study indicated that seed priming is an effective method

of Zn application. Seed priming with 0.5 M Zn solution using ZnSO4 was the best treatment

to improve the seedling emergence and growth of bread wheat. Seed priming with Zn solution

beyond 0.5 M Zn using ZnSO4 and 0.1 M Zn solution using ZnCl2 proved toxic. Moreover,

seed priming with Zn improved the chlorophyll contents, yield related traits and Zn

concentration in grain of wheat. Moreover, seed priming with 0.5 M Zn using ZnSO4 was the

best treatment for improving the grain yield and grain biofortification of bread wheat.



This study was comprised of three independent experiments. The optimization studies

were conducted in Allelopathy Lab, Department of Agronomy, while the third experiment was

conducted in glass house, Faculty of Agriculture, University of Agriculture, Faisalabad,

Pakistan during 2012–2013 comprised of pre-optimized treatments from the first two

experiments. All experiments were laid out in completely randomized design in factorial

arrangement, with four replications. For the lab experiments, seeds of both wheat cultivars

were coated with 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75 and 2 g Zn kg-1 seed using ZnCl2 and

ZnSO4 as Zn source, while untreated seeds were taken as control. For glass house experiments,

seeds of both wheat cultivars were coated with pre-optimized rate at 1.25 and 1.5 g Zn kg-1

seed. Seed coating with 1.25 g Zn kg-1 seed using ZnSO4 were improved the stand

establishment, productivity, chlorophyll and grain Zn concentration of wheat. Seed coating

above 1.5 g Zn kg-1 seed using either source was not beneficial.

250


Diversity

This experiment was conducted for two growing seasons at Agronomic Research Area,

University of Agriculture, Faisalabad during 2012-13 and 2014-15. Seeds of twenty eight bread

wheat genotypes were obtained from Wheat Research Institute, Faisalabad, Pakistan.

Zinc was soil applied at 10 kg Zn ha-1 as basal dose; while no application of Zn was control.

Wheat genotypes of Punjab Pakistan have very less genetic diversity. Most of the genotyeps

have genetic diversity in the range of 0.0335-0.0677. However, among wheat genotypes

maximum genetic diversity was observed for Millat-2011 (0.0842) while it was lowest in

Sehar-2006 (0.0194). Most of the high yielding varities were low in Zn bioavailability due to

higher phytate concentration. However, soil applcation of Zn increased the yield and mineral

concentration of wheat genotypes. Among the tested genotypes, maximum grain yield was

recorded for Chakwal-50 (5.16 Mg ha-1), while, Faisalabad-2008 had higher protein

concentration. Moreover, grain Zn concentration (54.4 mg kg-1) and bioavailable Zn (3.08 mg

day-1) was the maximum in Blue silver. Highest values of grain Fe was recorded in Sehar-2006

(44.5 mg kg-1) without Zn while Ca was highest in grains of Millat-2011 (511 mg kg-1) with

Zn applciaiton. Zinc application increased the endosperminc Zn concentration and reduced the

phytate concentration. In this regard, highest Zn was present in the endosperm of Blue silver

(20 mg kg-1) while Kohinoor-83 have lowest phytate (6.72) in the tested genotypes.



This study was carried out for two growing seasons in glass house, Faculty of Agriculture,

and Agronomic Research Area, University of Agriculture, Faisalabad, Pakistan, and

Agronomic Research Area, University of Agriculture, Faisalabad, Pakistan during 2013-14 and

2014-15. Seeds of two bread wheat cultivars, Lasani-2008 and Faisalabad-2008 were very

kindly provided by Wheat Research Institute, Faisalabad, Pakistan. Zinc was applied as soil

application (10 kg Zn ha-1) and foliar application (0.025 M Zn solution); seed priming (0.5 M

ZnSO4 for 12) and seed coating with 1.25 g Zn kg-1 seed. Hydroprimed seeds were taken as

control. Zinc solubilizing PGPR strain Pseudomonas strain MN12 was also used in this study.

251

Use of Zn application method in combination with PGPR improved the productivity and grain

biofortification of wheat. Seed priming and soil application of Zn + PGPR increased the wheat

yield and organic acid production in root exudates of wheat. In the root exudates of wheat,

pyruvic acid, tartaric acid, citric acid, malonic acid, mallic acid, succinic acid, oxaloacetic

acid, oxalic acid and methylmalonic acids were noted. Organic acid production was higher for

soil application and seed priming of Zn with PGPRs, which helped in mobilization and uptake

of Zn from soil. However, soil and leaf Zn application with and without PGPR impressively

increased the Zn concentration in grain and all grain fractions. Moreover, it also improved the

bioavailable Zn. Seed priming with Zn using PGPR was produced more yield with highest net

ecomic return.

5.5 Improving the Resistance against abiotics Stresses in Wheat through Zinc Nutrition

This study was comprised of four experiments conducted in the growth chambers of Plant

Physiology Laboratory, Sabanci University, Istanbul, Turkey. Fifteen seeds of two bread wheat

genotypes Lasani-2008 and Faisalabad-2008 were grown in soil brought from central Anatolia

which was highly Zn deficient. The plants were thinned to ten after completion of germination.

Zinc was applied as low Zn (0.3 ppm kg-1 soil) while adequate Zn plants received 3 ppm Zn

kg/soil using ZnSO4.7H2O as source. Wheat cultivars, Zn levels and all other experimental

conditions were similar in all experiments. In the first experiment, plants were subjected to

drought stressed (35% field capacity), while well-watered plants were maintained at 70% field

capacity during the whole crop season. In second study, salt stress was applied as 2500 ppm

NaCl kg-1 soil while no salt application was taken as control. In third study, plants were grown

under optimal temperature 20/15ºC day/night (control). After 15 days of sowing half of the

pots were transferred to other growth chamber with low temperature i.e. 10/7ºC day/night

temperature. Cold stress was imposed till booting stage.

In the fourth experiment, wheat genotypes were grown in hydroponic solution culture

in growth rooms under controlled environmental conditions. All the pots were kept at 25/18°C

day/night temperature for 5 days; latter half of the pots were transferred to the heat stress

chamber viz. 36°C/28°C day night temperature. Moreover, high Zn pots were supplied with 1

μM ZnSO4.7H2O while the low Zn pot contained 0.1 μM ZnSO4.7H2O. Adequate Zn

252

application improved the productivity of wheat under optimal and suboptimal conditons. Zinc

supply increased the rate of photosynthesis, enzymatic activities (SOD, APX), relative water

contents, and reduced the lipid peroxidation. Adequate Zn supply resulted in maximum yield

under optimal condition while yield reduction was less in sufficient Zn treatment under

suboptimal conditions. Moreover, adequate Zn supply increased the grain quality of wheat by

increasing the concentration and contents of protein, Zn and bioavailable Zn. The increase in

grain Zn concentration/contents, embryo Zn, aleuron Zn and endosperm Zn was about 2 fold

compared to low Zn treatment.

Project Conclusions • Zinc application through seed priming with 0.5 M Zn solution and seed coating 1.25 g

Zn kg-1 using ZnSO4 improved the stand establishment, productivity and grain Zn

concentration of wheat.

• Seed prming with higher nutrient Zn concentration solution i.e. >0.5 M Zn solution and

seed coating beyon 1.25 g Zn kg-1 seed proved toxic.

• Bread wheat of Punjab Pakistan have less genetic diversity.

• Application of Zn increased the yield and Zn bioavaibiltiy of these genotypes.

• Application of Zn is effective in improving the yield and grain Zn bioavailability of

wheat.

• Application of microbial culture in combination with Zn application further enhanced

the effectiveness of Zn application.

• Use of PGPR improved the uptake and grain biofortification of Zn in wheat through

enhanced producton of organic acids.

• Seed priming with PGPR was more effective in enhancing yield net economic return.

• Application of Zn improved the resistance against drought, salt, cold and heat stress by

improving the water relation enzymatic activities and grain yield of wheat.

Future Research Thrusts

• Genetic basis for abiotic stress tolerance by Zn nutrition may be explored.

253

• Role of Zn nutrition under combination of different abiotic stresses should also be

evaluated.

• Proteomic of Zn nutrition under abiotic stress should be mapped.

• Metablomics of Zn application regarding grain biofortification should be investigated.

• Role of plant growth promoting rhizobacteria should also be investigated in enhancing

the nutrient uptake of other nutrient and also on growth improvement under stress

conditions.

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Statement Concerning Data

The original data have been lodged in the Department of Agronomy, University of

Agriculture, Faisalabad, Pakistan. Any person interested may approach Dr. Muhammad

Farooq, Associate Professor, Department of Agronomy, University of Agriculture,

Faisalabad, Pakistan.

Abdul Rehman

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