RESPONSE OF MAIZE (Zea mays L.) TO FOLIAR APPLICATION OF BORON AND CALCIUM UNDER DROUGHT STRESS

By

MUHAMMAD NAEEMM.Sc. (Hons.) Agriculture

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

of

DOCTOR OF PHILOSOPHY

IN

CROP PHYSIOLOGY

DEPARTMENT OF AGRONOMY, FACULTY OF AGRICULTURE,

UNIVERSITY OF AGRICULTURE,FAISALABAD - PAKISTAN

2016

The Controller of Examinations

University of Agriculture,

Faisalabad

We the supervisory committee, certify that the contents and form of thesis submitted

by Mr. Muhammad Naeem, Regd. No. 2005-ag-1735 have been found satisfactory and

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

degree.

SUPERVISORY COMMITTEE

1. CHAIRMAN: DR. MUHAMMAD SHAHBAZ NAEEM

2. MEMBER:

DR. RASHID AHMAD

3. MEMBER: DR. RIAZ AHMAD

DECLARATION

I hereby declare that contents of the thesis “Response of maize (Zea mays L.) to foliar

application of boron and calcium under drought stress” are the product of my own research

and no part has been copied from any published source (except the references, standard

mathematical or genetic modals/equation/formulate/ protocols etc.). I further declare that this work

has not been submitted for the awards of any other diploma/degree. The university may take action

if the information provided is found inaccurate at any stage (in case of any default, the scholar will

be proceeded against as per HEC plagiarism policy).

Muhammad Naeem

2005-ag-1735

ACKNOWLEDGEMENT

All praises and thanks for Almighty Allah, the most Merciful and Beneficent whose

copious blessings enabled me to complete this study and put it in this form. I offer my

humblest thanks to the Holy Prophet Muhammad (PBUH) who is forever a torch of

guidance and knowledge for the humanity as a whole.

With a proud sense of gratitude, I acknowledge that this manuscript has found this

shape under the kind supervision, inspiring guidance and sympathetic attitude of Dr.

Muhammad Shahbaz Naeem, Assistant Professor, Department of Agronomy, University of

Agriculture, Faisalabad, who benevolently extended all possible help for the smooth

execution of this humble presentation.

I offer my great sense of gratitude to Dr. Rashid Ahmad, Professor, Department of

Agronomy, University of Agriculture, Faisalabad, for providing valuable suggestions,

competent guidance and boosting up my morale during the conduct of this study.

I am highly indebted to Dr. Riaz Ahmad, Professor, Department of Agronomy,

University of Agriculture, Faisalabad, for his constructive criticism and valuable suggestions

to improve this manuscript.

I shall be missing something if I don’t extend my admiration and appreciation to my

sincere friends, who supported me morally with sentiment throughout my research. Last but

not the least gratitude is to be expressed to my family members, my affectionate father,

loving mother, brothers and beloved sister for their love, inspiration, good wishes and

unceasing prayers for me to achieve higher goals in life.

The role of Higher Education Commission (HEC) of Pakistan is highly appreciated

and acknowledged for not only providing financial support for this study but also giving me

an opportunity to work with Prof. Wayne Loescher, Department of Horticulture, Michigan

State University, MI East Lansing USA as a visiting scholar for six months under

International Research Support Research Initiative Program (IRSIP).

Muhammad Naeem

TABLE OF CONTENTS

CHAPTER # TITLE Page #

CHAPTER 1 INTRODUCTION 1

CHAPTER 2 REVIEW OF LITERATURE 5

2.1 Climate change and drought 52.1.1 Effects of drought stress on plant growth 52.1.2 Effects of drought stress on mineral nutrient availability 62.2 Foliar fertilization 72.3 Boron 82.3.1 Born uptake and mechanisms of absorption 82.3.2 Effect of drought stress on B uptake 82.3.3 Physiological roles of Boron 102.3.4 Role of B in alleviation of drought stress 112.4 Calcium 122.4.1 Calcium uptake and distribution under drought stress 122.4.2 Physiological roles of calcium 132.4.3 Role of calcium in alleviation of drought stress 15CHAPTER 3 MATERIALS AND METHODS 173.1 Experimental Site and Conditions 173.2 Maize Hybrids 213.3 Laboratory Experiments 21

3.3.1 Calculations of Germination attributes and Physiological Indices 22

3.4 Wire-house Experiments 233.5 Field Experiment 253.6 Data Collections 283.6.1 Estimation of Leaf Water Relations 283.6.1.1 Leaf Water Potential (‒MPa) 283.6.1.2 Leaf Osmotic Potential (‒MPa) 283.6.1.3 Turgor Potential (MPa) 283.6.1.4. Relative Water Contents (%) 283.6.2 Estimation of Pigment Contents 293.6.3 Gas Exchange Parameters 293.6.4 Biochemical parameters 293.6.4.1 Proline Determination 293.6.4.2 Total Soluble Proteins (mg g-1 DW) 303.6.4.3 Total Free Amino Acids (mg g-1 DW) 31

CHAPTER # TITLE Page #3.6.4.4 Total Soluble Sugars (mg g-1 DW) 323.6.5 Antioxidant Extraction and Lipid Peroxidation Assay 323.6.5.1 Superoxide Dismutase (Unit min-1 g-1 FW) 334.5.6.2 Catalase Activity (Units min-1 g-1 FW) 334.5.6.3 Peroxidase (Unit min-1 g-1 FW) 334.5.6.4 Ascorbate Peroxidase (ABA digested g-1 FW h-1) 334.5.6.5 Lipid Peroxidation (nmol g-1 DW) 343.6.6 Estimation of Leaf B Concentration (mg kg-1 DW) 343.6.7 Determination of Calcium (Ca+2) Concentration (mg g-1 DW) 363.6.8 Growth Parameters of Maize 373.6.8.1 Leaf Dry Weight (g plant-1) 373.6.8.2 Shoot Dry Weight (g plant-1) 383.6.8.3 Silk Threads (Number ear-1) 383.6.8.4 Length of Silk Threads (cm) 383.6.8.5 Tassel and Silk Dry Weight (g plant-1) 383.6.8.6 Plant Height at Harvest 383.6.9 Yield and Yield Components 383.6.9.1 Number of Grains per Ear 383.6.9.2 1000-Grain Weight (g) 383.6.9.3 Biological Yield (t ha-1) 393.6.9.4 Grain Yield (t ha-1) 393.6.9.5 Harvest Index (%) 393.6.10 Statistical Analyses 39CHAPTER 4 RESULTS 40

Experiment 1 40

4.1 Screening of maize hybrids for drought tolerance (Petri-plate experiment) 40

4.1.1 Germination Percentage 404.1.2 Mean Germination Time 404.1.3 Promptness Index 414.1.4 Germination Stress Tolerance Index 41

Experiment 2 45

4.2 Screening of maize hybrids for drought tolerance(Pot experiment) 45

4.2.1 Plant Height Stress Tolerance Index (PHSI) 454.2.2 Root Length Stress Tolerance Index (RLSI) 454.2.3 Dry Matter Stress Tolerance Index (DMSI) 45

EXPERIMENT 3 494.3 Optimization of boron concentration for spring maize 494.3.1 Plant Height Stress Tolerance Index (PHSI) 494.3.2 Root Length Stress Tolerance Index (RLSI) 494.3.3 Dry Matter Stress Tolerance Index (DMSI) 50

CHAPTER # TITLE Page #4.3.4 Total Fresh Biomass (g plant-1) 534.3.5 Shoot/Root Ratio 53

EXPERIMENT4 564.4 Optimization of calcium concentration for spring maize 564.4.1 Plant Height Stress Tolerance Index (PHSI) 564.4.2 Root Length Stress Tolerance Index (RLSI) 564.4.3 Dry Matter Stress Tolerance Index (DMSI) 564.4.4 Total Fresh Biomass (g plant-1) 594.4.5 4.3.5 Shoot/Root Ratio 59

EXPERIMENT 5 62

4.5 Foliar-applied boron and calcium ameliorates drought-induced physiological and biochemical changes in maize

62

4.5.1 Leaf Water Relations 624.5.1.1 Water Potential (-MPa) 624.5.1.2 Osmotic Potential (-MPa) 624.5.1.3 Turgor Potential (MPa) 634.5.1.4 Relative Water Contents (%) 634.5.2 Gas Exchange Characteristics 674.5.2.1 Net Photosynthetic Rate (µmol CO2 mˉ² sˉ¹) 674.5.2.2 Stomatal Conductance (µmol H2O mˉ² sˉ¹) 674.5.2.3 Transpiration Rate (mmol H2O m-2 sˉ¹) 674.5.3 Leaf Chlorophyll Contents (mg g-1 FW) 714.5.4 Total Carotenoids (mg g-1 FW) 714.5.5 Osmolyte Accumulation 754.5.5.1 Leaf Proline Contents (µmol g-1 DW) 754.5.5.2 Total Soluble Proteins (mg g-1 DW) 754.5.5.3 Total Free Amino Acids (mg g-1 DW) 774.5.5.4 Total Soluble Sugars (mg g-1 DW) 774.5.6 Antioxidant Activities 804.5.6.1 Superoxide Dismutase Activity (Units min-1 g-1 FW) 804.5.6.2 Catalase Activity (Units min-1 g-1 FW) 804.5.6.3 Peroxidase Activity (Units min-1 g-1 FW) 814.5.6.4 Ascorbate Peroxidase (ABA digested g-1 FW hr-1) 814.5.6.5 Lipid Peroxidation (nmol g-1 DW) 824.5.7 Leaf Nutrient Concentrations 864.5.7.1 Leaf Boron Concentration (mg kg-1 DW) 864.5.7.2 Leaf Calcium Concentrations (mg g-1 DW) 86

EXPERIMENT 6 89

4.6Foliar-applied boron and calcium improves growth, yield and yield components of maize under normal and water deficit conditions

89

4.6.1 Growth Parameters 894.6.1.1 Leaf Dry Weight (g plant-1) 894.6.1.2 Shoot Dry Weight (g plant-1) 89

CHAPTER # TITLE Page #4.6.1.3 Tassel dry weight (g plant-1) 894.6.1.4 Silk dry weight (g plant-1) 904.6.1.5 Silk length (cm) 904.6.1.6 Silk threads (Number ear-1) 904.6.1.7 Plant height (cm) 904.6.2 Yield and Yield Components 974.6.2.1 Number of grains per ear 974.6.2.2 Thousand-grain weight (g) 974.6.2.3 Biological yield (t ha-1) 994.6.2.4 Grain yield (t ha-1) 994.6.2.5 Harvest index (%) 99CHAPTER 5 DISCUSSION 1035.1 Laboratory Experiments 1035.2 Wire-house Experiments 1045.2.1 Seedling growth 1045.2.2 Leaf Water Relations 1065.2.3 Gas Exchange Characteristics 1085.2.4 Chlorophyll and Carotenoids 1095.2.5 Osmolytes Accumulation 1105.2.5.1 Proline 1115.2.5.2 Total Soluble Proteins 1115.2.5.3 Total Free Amino Acids 1125.2.5.4 Total Soluble Sugars 1135.2.6 Antioxidants 1145.2.7 Leaf Boron and Calcium Concentrations 1155.3 Field Experiment 1165.3.1 Growth parameters 1165.3.2 Yield and Yield Components 119CHAPTER 6 SUMMARY 120-121

CONCLUSION 122FUTURE PERSPECTIVES 122LITERATURE CITED 124

LIST OF FIGURES

Fig. # Title Page #

3.1 Metrological data of the experimental site during wire-house / rain-out shelter experiment for the growing season 2013 19

3.2 Metrological data of the experimental site during wire-house / rain-out and field experiment for the growing season 2014 19

3.3 Corn growth stages 21

3.4 Layout plan 26

4.1 Effect of PEG6000 induced osmotic stress on germination percentage (%) of different maize (Zea mays L.) hybrids 42

4.2 Effect of PEG6000 induced osmotic stress on mean germination time (Days) of different maize (Zea mays L.) hybrids. 43

4.3 Effect of PEG6000 induced osmotic stress on promptness index of different maize (Zea mays L.) hybrids 43

4.4 Effect of PEG6000 induced osmotic stress on germination stress index of different maize (Zea mays L.) hybrids 44

4.5 Effect of water stress on plant height stress tolerance index (PHSI) of different maize (Zea mays L.) hybrids 46

4.6 Effect of water stress on root length stress tolerance index (RLSI) of different maize (Zea mays L.) hybrids 46

4.7 Effect of water stress on dry matter stress tolerance index (DMSI) of different maize (Zea mays L.) hybrids 47

4.8Effect of boron foliar spray on plant height stress tolerance index (PHSI) of maize (Zea mays L.) seedlings under deficit moisture supply

50

4.9Effect of boron foliar spray on root length stress tolerance index (RLSI) of maize (Zea mays L.) seedlings under deficit moisture supply

51

4.10Effect of boron foliar spray on dry matter stress tolerance index (DMSI) of maize (Zea mays L.) seedlings under deficit moisture supply

51

4.11Effect of boron foliar spray on total fresh biomass of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions

54

Fig. # Title Page #4.12 Effect of boron foliar spray on total fresh biomass of maize 54

(Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions

4.13Effect of calcium foliar spray on plant height stress tolerance index (PHSI) of maize (Zea mays L.) seedlings under deficit moisture supply

57

4.14Effect of calcium foliar spray on root length stress tolerance index (RLSI) of maize (Zea mays L.) seedlings under deficit moisture supply

57

4.15Effect of calcium foliar spray on dry matter stress tolerance index (DMSI) of maize (Zea mays L.) seedlings under deficit moisture supply

58

4.16Effect of calcium foliar spray on total fresh biomass of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions

60

4.17Effect of calcium foliar spray on shoot/root ratio of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions

61

4.18

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf water potential (-MPa) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

64

4.19

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf osmotic potential (-MPa) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

64

4.20

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf turgor potential (MPa) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

65

4.21

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on relative water contents (%) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

65

4.22

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on net photosynthetic rate (μmol CO2 m-2 s-1) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

68

4.23

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on stomatal conductance (mol m-2 s-1) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

68

Fig. # Title Page #

4.24 Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on transpiration rate (mmol H2O mˉ² sˉ¹) of two maize (Zea mays L.) hybrids under

69

normal (100% WHC) and drought stress (30% WHC) conditions

4.25

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll a contents (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

72

4.26

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll b contents (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

72

4.27

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll a + b contents (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

73

4.28

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on total carotenoids (mg g-1

FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

73

4.29

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf proline contents (µmol g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

76

4.30

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on total soluble proteins (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

76

4.31

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on total free amino acids (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

78

4.32

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on total soluble sugars (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions.

78

Fig. # Title Page #

4.33

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on superoxide dismutase activity (Unit min-1 g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

82

4.34

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on catalase activity (Unit min-1 g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

83

4.35

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on peroxidase activity (Unit min-1 g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

83

4.36

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on ascorbate peroxidase activity (ABA digested g-1 FW hr-1) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

84

4.37

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on MDA content (nmol g-1

DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

84

4.38

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf boron concentrations (mg kg-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

87

4.39

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf calcium concentrations (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

87

4.40

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf dry weight (g plant-

1) of two maize (Zea mays L.) hybrids under normal and water stress conditions

91

4.41

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on shoot dry weight (g plant-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions

91

Fig. # Title Page #

4.42

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on tassel dry weight (g plant-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions

92

4.43 Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on silk dry weight (g plant-1)

92

of two maize (Zea mays L.) hybrids under normal and water stress conditions

4.44

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on silk length (cm) of two maize (Zea mays L.) hybrids under normal and water stress conditions

93

4.45

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on silk threads (number ear-

1) of two maize (Zea mays L.) hybrids under normal and water stress conditions

93

4.46

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on plant height (cm) of two maize (Zea mays L.) hybrids under normal and water stress conditions

94

4.47

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on numbers of grains per ear of two maize (Zea mays L.) hybrids under normal and water stress conditions

98

4.48

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on thousand-grain weight (g) of two maize (Zea mays L.) hybrids under normal and water stress conditions

98

4.49

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on biological yield (t ha-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions

99

4.50

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on grain yield (t ha-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions

100

4.51

Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on harvest index (%) of two maize (Zea mays L.) hybrids under normal and water stress conditions

101

List of Tables

No. Title Page #

3.1 Physiochemical characteristics of the soil for pot and field experiments 18

3.2 Corn vegetative and reproductive growth stages using the ISU and FCIC methods along with the estimated days 20

3.3 Crop husbandry operations for irrigation management 27

3.4 Boron standards prepared by 0.36 N H2SO4 35

4.1

Analysis of variance (ANOVA) of the data for PEG induced osmotic stress (‒MPa) on germination percentage (GP), mean germination time (MGT), promptness index (PI) and germination stress index (GSI) for eight different maize (Zea mays L.) hybrids

44

4.2

Analysis of variance (ANOVA) of the data for the effect of varying water stress levels on plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry matter stress tolerance index (DMSI) for eight different maize (Zea mays L.) hybrids

47

4.3Ranking order of eight maize (Zea mays L.) hybrids under water stress on the basis of their germination attributes and seedling stress tolerance indices

48

4.4

Analysis of variance (ANOVA) of plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry matter stress tolerance index (DMSI) in two maize (Zea mays L.) hybrids exposed to boron foliar spray under drought stress 52

No. Title Page #

4.5Analysis of variance (ANOVA) of total fresh biomass (g plant-1) and shoot/root ratio in two maize (Zea mays L.) hybrids exposed to boron foliar spray under drought stress

55

4.6

Analysis of variance (ANOVA) of plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry matter stress tolerance index (DMSI) in two maize (Zea mays L.) hybrids exposed to calcium foliar spray under drought stress

58

4.7

Analysis of variance (ANOVA) of total fresh biomass (g plant-1) and shoot/root ratio in two maize (Zea mays L.) hybrids exposed to calcium foliar spray under drought stress

61

4.8

Analysis of variance (ANOVA) of leaf water potential (ψw), osmotic potential (ψs), turgor potential (ψp) and relative water contents (%) in two maize (Zea mays L.) hybrids exposed to boron and calcium foliar spray under drought stress

66

4.9

Analysis of variance (ANOVA) for net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (E) of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

70

4.10

Analysis of variance (ANOVA) for leaf chlorophyll (Chl a), (Chl b) and (Chl a + b) and total carotenoids (CAR) of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

74

4.11

Analysis of variance (ANOVA) for leaf proline (PRO) contents, total soluble proteins (TSP), total free amino acids (TFA) and total soluble sugars (TSS) of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

79

4.12

Analysis of variance (ANOVA) for superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) activities and malondialdehyde (MDA) content in two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

85

4.13

Analysis of variance (ANOVA) for leaf boron and calcium concentrations of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

88

No. Title Page #

4.14

Analysis of variance (ANOVA) for leaf, shoot, tassel and silk dry weight of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under water deficit conditions

95

4.15

Analysis of variance (ANOVA) for silk length, number of silk threads per ear, and plant height of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under water deficit conditions

96

4.16

Analysis of variance (ANOVA) for number of grains per ear, thousand grain weight, biological yield, grain yield and harvest index of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under water deficit conditions

102

ABSTRACTPremise of the research- Drought stress perturbs the normal mineral-nutrient relations of plants that lead to reduced plant growth and results significant losses in crop yields. Various plant minerals have been known to be involved in stress amelioration, however reports regarding the effects of boron (B) and calcium (Ca2+) on maize (Zea mays L.) under drought are scant. Methodology- A series of laboratory, wire-house and field experiments were conducted. In laboratory experiments eight spring maize hybrids were germinated in petri plates under polyethylene glycol (PEG-6000) induced osmotic stress @ -0.2, -0.4, -0.6 and -0.8 MPa, and two water stress levels at 100% water-holding capacity (WHC) and 30% WHC. The wire-house / rain-out shelter study was done to optimize the foliar rate for B and Ca2+

supplies; one drought-tolerant (Dekalb-6525) and sensitive (Yousafwala Hybrid) maize cultivars selected from previous laboratory experiments were sprayed with distilled water (control) and B solutions at 2, 4 and 6 mg L-1 and Ca2+ solutions at 20, 40 and 60 mg L-1

under normal and water-deficit conditions. Furthermore, one drought tolerant and sensitive maize hybrids were sprayed with optimized rate of B (4 mg L-1), Ca2+ (40 mg L-1) and their combinations to evaluate their growth, physiological, biochemical and yield responses under both normal and water deficit conditions in a wire-house and field experiment. Pivotal results- The maize cultivars tested on the basis of germination and seedling growth revealed that cultivar, Dekalb-6525 performed better under water-deficit conditions, therefore categorized as drought-tolerant, whereas Yousafwala Hybrid was identified as drought-sensitive on the basis of its poor performance. Drought stress brings considerable growth inhibition and disturbance in gas exchange characteristics, leaf water relations, light harvesting pigments and osmolyte accumulation by increasing malondialdehyde (MDA) content and imbalancing of antioxidant system. Foliar treatment of B (4 mg L-1), Ca2+ (40 mg L-1) and their combinations considerably improved maize growth, photosynthesis, chlorophyll contents, water status as well as activities of antioxidants along with a decline in MDA accumulation in both cultivars under water-deficit conditions. However, optimum mineral nutrient supply markedly reduced accumulation of proline and total soluble proteins. In addition, foliar sprays of Ca2+ and B+Ca2+ substantially improved the content of total free amino acids and total soluble sugars, however B application noticeably reduced their accumulations as compared to control. Drought treatment considerably reduced the uptake of B and Ca2+, whereas foliar sprays of these nutrients alone and in combinations notably increased their concentrations in maize leaf tissues. Considerably improved growth rate, photosynthesis, water status, pigment contents, osmolyte accumulation as well as increased antioxidant activities along with decline in MDA accumulation was found to be key contributors to a stress-tolerant genotype to thrive under limited-water supply. Afterwards in a field study, foliar B and Ca2+ supplies significantly improved the vegetative and reproductive growth of both cultivars and caused improve grain weight and yield. Conclusion- From this study, it was likely to suggest that screening for drought tolerance at initial growth phases and their further improvement by foliar B and Ca supplies are effective approaches to make plants vigorous to thrive under limited water supply.

Keywords: Antioxidants, Boron, Calcium, Drought, Foliar spray, Maize, Osmolyte

CHAPTER 1 INTRODUCTION

Drought is one of key restrictions to sustainable agricultural production around globe,

especially arid and semi-arid regions of the world (Chaves et al., 2002; Nawaz et al., 2015b)

including Pakistan. Water stress disturbs a number of physiological processes within plant

body such as stomata opening, destroy chlorophyll contents and photosynthetic apparatus

which inhibits photosynthetic activity in plants. It interrupts the equilibrium between

production of reactive oxygen species (ROS) and antioxidant defense system within plants,

causing buildup of active oxides which brings oxidative tension to membrane lipids, proteins

and other cellular constituents (Waraich et al., 2011). Drought stress induce reduction in

plant growth and development causing less grain filling and flower production ultimately less

grains with smaller size are produced (Waraich et al., 2007). It generally prevents plant

growth through water absorption, nutrient uptake and nutrient mobility in soil which may

involve buildup of mineral elements in plant tissues and therefore, alter various physiological

and anti-oxidative plant responses (Basu et al., 2010; Luo et al., 2011). Worldwide, at least

60% of cultivated soils have growth limiting problems arising from plant mineral nutrient

deficiency and toxicity. Combination of such nutritional problems with other environmental

stress factors such as water deficit, salinity, chilling etc. are responsible for severe damages

in crop production (Cakmak, 2002). Many studies have verified the effects of various

abiotic stresses on crop growth and mineral nutrition (Broadley et al., 2007; Cakmak, 2000)

as well as role of various plant minerals in stress amelioration (Khan et al., 2004;

Upadhyaya et al., 2011). It has been reported that use of fertilizers can improve plants

resistance to various abiotic stresses such as drought and salinity (Movahed Dehnavi et al.,

2009; Cakmak, 2008). The positive effects of these nutrients like boron (B) has been mostly

reported in different plants under normal conditions (Brown et al., 2002; Goldbach and

Wimmer, 2007), whereas little attention has been paid to evaluate plant responses under

stress conditions. Among the mineral nutrients, foliar application of boron (B) and calcium

(Ca2+) can minimize drought-induced crop losses as they play several physiological and

biochemical roles.

Boron is required for biosynthesis of pectin polysaccharide in primary cell-wall of

plants and is associated with the rhamnogalacturonan-II (RG-II) fraction of pectic 1

polysaccharides, which has been described to be responsible for cross-linking of cell-wall

polymers and is therefore necessary for maintenance of cell wall stability (O'Neill et al,

2001). The stable membranes provide structural support and first line of defense against

environmental stresses (Hamann, 2012). Bulk of B in plants is related to RG-II polymer

however, under moisture deficit conditions there is a rise in the pectic chains RG I and RG II

in the roots of drought-tolerant wheat (Leucci et al, 2008). These authors suggest that, such

wall configurations might contribute to drought-resistance as gelling and anti-dehydrating

agents. B is participated in various processes of plant development such as cell expansion

and differentiation, maintenance of normal functioning and integrity of biological

membranes, phenolic metabolism and sugars translocation (Marschner and Marschner,

2012). Foliar spray of B under water-deficit conditions minimized the drought-induced

damages by improved root growth, paying to lower plant dehydration during dry periods in

Eucalyptus urophylla (Hodecker et al., 2014) and Picea abies (Mottonen et al., 2001;

Mottonen et al., 2005). It is essential for growth and development of pollen, for seed

formation and sugar translocation within plants (Christensen and Smart, 2005). It helps to

improve yield and yield components, water use efficiency, pollen grains viability, leaf area,

photosynthetic rate and stomatal conductance (Karim et al., 2012); hence, in turn improves

resistance of plants to drought stress (Azza et al., 2006). Literature evidence proposes that B

deficiency enhanced the accumulation of proline in brassica (Brassica juncea), maize (Zea

mays) and periwinkle (Catharanthus roseus) (Pandey, 2013; Pandey and Archana, 2009),

total soluble sugars in tobacco (Nicotiana tabacum) (Camacho-Cristobal et al., 2004) and

total free amino acids in citrus (Citrus sinensis) (Lu et al., 2014). However, B deficiency

disturbs secondary plant metabolism, caused oxidative stress (Camacho-Cristobal et al.,

2002; Kobayashi et al., 2004) and increased ascorbate per oxidase (APX) activity in

sunflower (Helianthus annus) (Dube et al., 2000) as well as increased (SOD EC 1.15.1.1),

catalase (CAT EC 1.11.1.6), peroxidase (POD EC 1.11.1.7), and ascorbate peroxidase (APX

EC 1.11.1.11) activity in Morus alba (Tewari et al., 2010), while decreased APX, SOD and

CAT activities in Glycine max (Peng and Yuai, 2000).

Calcium ion (Ca2+) has occurred as an important secondary messenger in plants

signaling networks (Cacho et al., 2013; Huda et al., 2013; Sarwat et al., 2013). It regulates

various essential processes as cytoplasmic streaming, gravitropism, thigmo-tropism, cell

division, elongation, differentiation, cell polarization, photomorphogensis and protect plants 2

under various stress conditions (Malho et al., 2006; Reddy, 2001). It is required for structural

roles in cell wall and membranes and affects membrane strength and respirational

frequency of a tissue and its resistance to fungal toxicities (Hepler, 2005; Marschner and

Marschner, 2012). Various environmental stimuli stimulated the increase of cytosolic Ca2+ to

activate the different biological and downstream responses (Zhu et al., 2013) that cause plant

adjustment to harmful environmental conditions of heat (Tan et al., 2011; Wang et al., 2009),

drought (Ma et al., 2005; Shao et al., 2008; Upadhyaya et al., 2011) and salinity (Amor et

al., 2010; Zehra et al., 2012). Further, calcium act as a regulator of plant cell metabolism

(Jaleel et al., 2007b), and is involved in signaling anti-drought responses (Shao et al., 2008).

It seems to perform an essential role in various resistance mechanisms that are brought by

drought and Ca2+ signaling are necessary for acquirement of drought resistance or tolerance

(Cousson, 2009).

Calcium uptake reduces in above-ground portions of plants (shoots and leaves) as

well as in roots under water-deficit conditions due to decline in transpiration rate (Brown et

al., 2006; Hu et al., 2008). Additionally, root tip area is a major site of calcium entry and this

zone of Ca2+ uptake is likely to be reduced under water deficit conditions (Raza et al., 2013).

Soil application of B and Ca2+ is less efficient in calcareous, high pH, low organic matter

soils (Ali et al., 2008; Rashid and Ryan, 2004) and this problem is further aggravated under

water deficit conditions due to their reduced mobility from soil to roots by mass flow (Brown

et al., 2006; Mattiello et al., 2009). Under mineral nutrient deficiency, activity of different

antioxidant enzymes declines, which in turn raises plant sensitivity to abiotic stresses

(Cakmak, 2000). Literature evidence proposes that foliar spray under such circumstances has

been described to be equal or more effective by different researchers (Rab and Haq, 2012;

Torun et al., 2001). A plant’s complete necessity for various nutrient elements may

frequently be provided by one or two foliar sprays as quantities required are small, rates of

uptake are sufficient and plants respond quickly to nutrient spray (Goatley and Hensler,

2011), consequently, foliar application of nutrients to growing crops will ensure improved

crop nutrition at reproductive stages (anthesis and seed filling), which in turn may result in

improved crop yield (Ali et al., 2009).

Maize is an imperative world food crops and is the third most important cereal with

the leading global production at 829 million tons annually. Maize grain yields in developing

tropical countries of Asia average 5.67 t/ha vs. temperate developed country of North-3

America 11.78 ton/ha (FAOSTAT, 2014). In both environments deficit moisture supply is

the most significant abiotic stress, limiting and threatening maize grain production. Average

annual yield losses by drought are supposed to around 15% of well-watered yield potential

on a worldwide basis, a number that compares to 120 million tons of grain (Edmeades 2013).

To best of our knowledge, there is inadequate information available in literature on

the effect of drought stress and foliar applied B and Ca on growth, water relations, gas

exchange characteristics, photosynthetic pigments, osmolyte content and antioxidant system

in maize cultivars differing in drought-resistance. We hypothesize that higher growth rate,

photosynthesis, water status, pigment contents, osmolyte accumulation and activation of

antioxidant system in maize leaf tissues are the principal constituents of differential tolerance

mechanism in two cultivars inconsistent in drought-resistance supplied with B and Ca under

water-deficit conditions. By this objective in notice, we planned series of experiments to

uncover the response of two maize cultivars in terms of plant growth, grain yield, gas

exchange characteristics, leaf water status, chlorophyll contents, osmolyte accumulation,

antioxidant activities and lipid peroxidation under drought stress and foliar sprays of boron

and calcium.

Drought stress severely affects crop production in north-western parts of Pakistan.

Keeping in view the problem, the proposed study is planned with the following objectives:

1. To screen-out the promising drought-tolerant and sensitive hybrids under drought

stress at germination and seedling growth stages of maize

2. To examine the effect of water stress on various physiological and biochemical

attributes of maize

3. To find out optimum rate and time of boron and calcium application for improving

drought tolerance potential in maize

4. To evaluate the effect of foliar-applied boron and calcium on growth, yield,

physiological and biochemical attributes of maize under water-deficit conditions

4

CHAPTER 2 REVIEW OF LITERATURE

2.1 Climate change and drought

Climate change has appeared as one of the most intricate tasks of the 21st century and

has established an area of attention in previous few decades. The intermittent droughts and

temperature fluctuation would modify the bio-physical relations of crops by changing their

growing periods, arrangement of cropping seasons, changing irrigation water supplies,

altering soil characteristics, and growing threat of drought stress, thus brutally limiting the

agricultural productivity (Nawaz et al., 2015a).

2.1.1 Effects of drought stress on plant growth

Drought as one of the key impacts of climate change and is reflected as one of the

primary limiting aspects responsible for reduced agricultural productivity (Ashraf, 2010;

Mahmood et al., 2009) and is the most communal abiotic stress that has restricted nearly

25% of the world's cultivated lands (Yousefi and Zandi, 2012). It can result in considerable

yield reduction of various crops containing maize (Gholipoor et al., 2013). Drought stress is

recognized to cause widespread changes in vital physiological and biochemical processes,

which is reflected as the primary effects of drought stress, while secondary effects include

oxidative impairment in various plants (Ashraf, 2010). Water stress causes stomatal closure,

which lessens the CO2/O2 ratio in plant leaves and hinders the photosynthesis of plants

(Griffin et al., 2004; Moussa 2006), triggering alterations of pigment contents, damages the

photosynthetic machinery and declines the activities of Calvin cycle enzymes (Monakhova

and Chernyadev, 2002). The reduction in relative water contents by drought is reflected as a

well indicator of plant tissue water contents (Zhang et al., 2011) and critical fluctuations in

water homoeostasis caused molecular impairment, growth inhibition and even complete

failure of plants. In addition of these reactions, free radicals and other dynamic byproducts of

oxygen i.e. hydroxyl radical (OH-) superoxide radical (O2-) and hydrogen peroxide (H2O2)

are increased predominantly in mitochondria and chloroplast (Mittler, 2002; Neill et al.,

2002) and these are unavoidable byproducts of biological redox reactions (Arora et al., 2002;

Ashraf, 2009, 2010). The production of active oxygen species (AOS) deactivates enzymes

5

and impairs key cellular constituents if the magnitude of their production surpasses the

antioxidant defense ability of plants cells uncovered to several environmental stresses

(Ashraf, 2009). The overproduction of ROS disturbs normal plant metabolism and damage

biological membranes through lipid peroxidation (Mafakheri, 2011; Zhang et al., 2011). The

dismutation product of superoxide radicals i.e. H2O2 can directly spasm the membrane

lipids and render inoperative SH enclosing enzymes (Sairam et al., 2000). The hydroxyl

radical causing damage to protein, lipids, DNA, chlorophyll and nearly all other organic

component of the living cell (Becana et al., 1998). To lessen these damaging effects of

drought stress on usual metabolism and warrant normal growth and development, plants have

developed different approaches to neutralize this problem (Ashraf, 2010). The synthesis and

accumulation of compatible osmolytes such as glycine-betaine and proline is one of the key

metabolic approaches to increase osmoregulation to hinder cell dehydration to maintain its

growth which is primarily a turgor-driven process (Ashraf and Foolad 2007; Bolen, 2004). In

addition, plants synthesize/accumulate a variety of antioxidants such as superoxide dismutase

(SOD), catalase (CAT) and peroxidase (POD) as well as other metabolites such as ascorbic

acid, glutathione, carotenoids and tocopherol that are commonly sufficient to defend plants

from oxidative impairment (Alscher et al., 2002).

The cascade of investigations on morpho-physiological alteration of crops elucidated

that variations in plant water status, osmolyte accumulation, antioxidant activities as well as

malondialdehyde (MDA) contents were linked with genotype and extent of drought stress

(Hamidou et al., 2007; Zhang et al., 2013; Zhang et al., 2011).

2.1.2 Effects of drought stress on mineral nutrient availability

Drought interrupts the nutritive status of plants by changing ion concentrations in

tissues as the supply of mineral nutrients via the roots is restricted under drought conditions

because of its negative effects on nutrient availability. Worldwide at least 60% of cultivated

soils have growth limiting problems arising from mineral nutrient deficiency and toxicity.

Combination of such nutritional problems with other environmental stress factors such as

drought, salinity, chilling etc. are responsible for the severe losses in crop production

(Cakmak, 2002). Drought stress is often the main factor for crop production under arid/semi-

arid conditions (Hussain et al., 2004) of Pakistan because it results in impaired plant growth,

6

variations in plant water status and reduced nutrient acquirement (Ali and Ashraf 2011;

Shahbaz et al., 2012). Among various metabolic processes disturbs in plants due to water

stress, diminishing in uptake and buildup of essential mineral nutrients is a communal

phenomenon (Akram et al., 2008; Shahbaz et al., 2012) which occurs mainly due to reduced

root growth and little nutrient accessibility in dry soil (Samarah et al., 2004). Nonetheless,

inter-specific and intraspecific difference exists in plant species in relation to nutrient uptake

under drought conditions (Garg, 2003). Soil moisture deficit declines the diffusion frequency

of nutrients in the soil to the absorbing root surface and ultimately declines nutrient uptakes

by the roots and transportation from the roots to the shoots (Alam, 1999; Hu et al., 2008).

Drought conditions results in reduced availability of nitrogen (Bloem et al., 1992),

phosphorus (Liebersbach et al., 2004), potassium (Hu et al., 2008), calcium, magnesium and

boron which may be attributed due to reduced transpiration rate (Hu et al., 2008; Mattiello et

al., 2009).

Limited water supply cause lower transpiration rate that results in lower calcium

content of plant tissues, consequently climate change which is expected to lessen

transpiration rate through the possessions of higher atmospheric CO2 and increased incidence

of drought and salt stress is also expected to decline plant Ca2+ content (Martinez-Ballesta et

al., 2010). Calcium uptake also reduces in above-ground portions of plants (shoots and

leaves) as well as in roots under drought conditions (Brown et al., 2006). Additionally, root

tip area is a major site of calcium entry and this zone of Ca2+ uptakes is likely to be reduced

under water deficit conditions (Johnson, 2010; Raza et al., 2013).

2.2 Foliar fertilization

Foliar application of micro-nutrients can enhance plants resistance to various abiotic

stresses such as drought and salinity (Movahed Dehnavi et al., 2009) and has been described

to be equally or more effective as soil application by different scientists (Rab and Haq 2012;

Torun et al., 2001). Foliar fertilization supplies the required nutrients directly to the site of

demand in the leaves and its comparatively rapid absorption and the independence of the soil

moisture availability and root activity (Romheld and El-Fouly, 1999). Moreover, foliar

fertilizers use small rates as well as efficiently absorbed by the plants under water deficit

conditions; hence, growing crops will ensure improved crop nutrition at reproductive stages

7

(anthesis and seed filling), which in turn may result in improved seed yield (Ali et al., 2009).

A plant’s complete necessity for various nutrient elements may frequently be provided by

one or two foliar sprays as quantities required are small, rates of uptake are sufficient and

plants respond quickly to nutrient spray (Goatley and Hensler, 2011).

2.3 Boron

2.3.1 Born uptake and mechanisms of absorption

Boron was suggested to absorbed by the plant roots predominately as undissociated

boric acid (H3BO3) and/or borate [B (OH)4]-1 (Jiao et al., 2005). Being the passive absorption

of B by the plant roots is well established, however its active absorption explained by the

clue that tissue B concentration could be higher than that of soil solution (Raven, 1980). This

estimation is supported also by the results that different plants did not show equal ability to

take up same B when delivered with the similar concentration in the growth media (Nable

and Paull, 1991). Likewise, active and passive absorption by the plant roots also elucidated

by the evidence that under sufficient B supply it is taken up in a continuous manner (passive)

and in case of B-deficient supply active mechanism is prevailed as supported by its higher

concentration in plant tissues than that of external concentration (Dannel et al., 1997; Dannel

et al., 2000; Pfeffer et al., 1999). In addition, H3BO3 uptake is assisted by membrane inherent

proteins in Cucurbita pepo (Squash) roots (Dordas and Brown, 2001; Dordas et al., 2000).

Membrane B carriers for xylem-loading have been recognized in Arabidopsis (Takano et al.,

2008; Takano et al., 2002). Similarly, B carrier was also identified by Miwa and Fujiwara

(2010) and revealed that plants sense both inside and outside B availability and regulate its

transport by modifying the manifestation and/or accumulation of these transporters but

families of transporters are probable to be diverse. The scope and peculiarity of B

transporters have yet to be recognized, but a plausible homologue for BOR1 has been

establish in silico in Eucalyptus (Domingues et al., 2005).

2.3.2 Effect of drought stress on B uptake

Boron availability reduces under water deficit conditions because of their reduced

mobility from soil solution by mass flow to plant roots (Barber, 1995; Hu and Brown, 1997).

The deficiency of water in soil decreases transpiration rate, thus decreasing B transport to

8

shoots (Lovatt, 1985). Edward and Moore, (2005) reported that alfalfa is sensitive to B levels

and drought reduces B availability. Assessments of the inconsistency of needle B

concentrations in Picea abies with the precipitation fall in the previous week (Sutinen et al.,

2006) or above a growing season in Pinus contorta, Pseudotsuga menziesii (Carter and

Brockley, 1990), Picea mariana (White and Krause, 2001) and Eucalyptus (Da Silva et al.,

2015; Mattiello, et al. 2009) provide support to this also in trees. Likewise, drought stress

reduces B content in Hordeum vulgare (Gupta, 1979) and reduces its uptake and transport in

leaves of Brassica napus (Huang et al., 1997) and Brassica rapa (Hajiboland and Farhanghi,

2011). In eucalypts, severe B deficiency symptoms were observed during dry season and

slightly affected trees may moderately improve during the subsequent wet season (Dell et al.,

2001). In seedlings of Picea abies, a comparatively slight drought treatment improved root B

absorptions in all B applied treatments, whereas needle B concentration remained unaffected

(Mottonen et al., 2001). After recurrent drought stress, concentrations of B reduced in

needles; proposing that stomatal closure caused more impairment of B translocation in

needles than that of roots in Picea abies seedlings (Mottonen et al., 2005). In contrast, Keren

et al. (1985) described that the distribution of B between the solid and liquid phases of soil

did not differ considerably with inconstant moisture. While, B mobility in soil is still

restricted by drought, this would mention that the causes for reduced B uptake during water-

deficit conditions could relatively be sought in declined transpiration rate through stomatal

closure than that of B mobility in soil. Nonetheless, in the findings on Picea abies mentioned

above, the trees most seemingly did not affect from drought stress, as water would still be

accessible for roots profounder in the mineral layers.

Various soil factors and situations reduces B availability i.e. little organic matter

content of soil, sandy/coarse textured soils, high pH, liming, water deficit, exhaustive

cultivation and additional mineral nutrients uptake by plants than their application, and the

usage of fertilizer meager in minor nutrients are thought to be the key aspects related with the

manifestation of B deficiency (Mengel et al., 2001; Niaz et al., 2007; Rashid and Ryan,

2004). According to National Fertilizer Development Centre, (NFDC), 40% maize crop

fields in Pakistan, surveyed for fertility status, have been described to be deficient in boron

(Government of Pakistan, 1998). Drought induced reduced availability of B has been

9

observed by many scientists (Hajiboland and Farhanghi, 2011; Hodecker et al., 2014; Hu and

Brown, 1997).

2.3.3 Physiological roles of Boron

Though the critical nature of boron was recognized 90 years ago, the molecular basis

of the B requisite of plants has been agreed for 15 years. The uncommon nature of B

chemistry proposes the prospect of diverse biological functions; however, the exact

metabolic roles are yet to be concluding. Boron is involved in several essential processes,

including, sugars transport, synthesis of proteins, respiration, carbohydrate and RNA

metabolism, and the plant hormonal metabolism (indole-acetic acid). Furthermore, it

involved in cell wall synthesis, lignification and cross-linking of cell-wall polysaccharides as

well as it maintains the structural integrity of biological membranes (Blevins and

Lukaszewski, 1998). Boric acid (H3BO3) forms di-esters bonds with alcohols in a pH

dependent mode (Power and Woods, 1997). The best steady borate esters are made with cis-

diols i.e. ribose and apiose. One of the important physiological roles of B is its association

with the rhamnogalacturonan (RG) II fraction of pectin polysaccharides, which has been

described to be responsible for cross-linking of cell-wall polymers and is therefore necessary

for maintenance of cell wall stability (O'Neill et al., 2001). This role of B was in accordance

with the explanations that B is largely (90%) found to the water insoluble portion of cells

under deficient B supply (Dannel et al., 1999; Hu and Brown, 1994; Marschner and

Marschner, 2012; Matoh et al., 1992; Noguchi et al., 2000). The existence of B dimers RG-II

complex was found in angiosperms, gymnosperms as well as in bryophytes and

pteridophytes (Matsunaga et al., 2004; O'Neill et al., 2004). Due to the great complication of

the glycosyl configurations and associations of RG II, complete RG-II synthesis progressions

are not entirely understood, while numerous enzymes for RG II synthesis have been

recognized (Bonin et al., 2003; Delmas et al., 2008; Egelund et al., 2006; Iwai et al., 2002).

Bassil et al. (2004) suggested that B executes a structural role in the cytoskeleton as

supported by the evidence that boronic acids contend with boric acid for binding to cis-diols,

disrupt cytoplasmic strands and cell to cell wall disconnection in cultured cells of tobacco.

The possible boron-binding proteins were also isolated from microsomes (Wimmer et al.,

2009). By the induction of plasmalemma ATPase it enhances the transport of phosphorus and

10

chlorine. Boron was established to support the structural integrity of biological membranes

and the development of lipid rafts (Marschner and Marschner, 2012). Meanwhile, all these

roles of B are necessary for growth of meristematic tissues and its deficient supply mainly

damage dynamically growing tissues such as root and shoot tips so that overall plant growth

may be arrested (rosetting). Flower retention, formation of pollen and its tube growth,

fixation of nitrogen and nitrate (NO3) assimilation are also varied by boron (Camacho‐Cristobal et al., 2008).

2.3.4 Role of B in alleviation of drought stress

Proper nutrition is the basic necessity of each living organism. There are now 17

elements which are considered crucial for plants to complete their life cycle. Some nutrients,

including boron (B), copper (Cu) zinc (Zn), manganese (Mn), iron (Fe), chlorine (Cl)

molybdenum (Mo) and nickel (Ni) are required at micro-amounts for normal plant growth

and development (Waraich et al., 2011). Under inorganic nutrient deficiencies the activity of

antioxidant enzymes reduces, which in turn raises plant sensitivity to various abiotic stresses

(Cakmak, 2000). Plant nutrients are not only essential for better plant growth and

development, but also useful to alleviate various kinds of environmental stresses like drought

stress. Marschner and Marschner, (2012) reported that inorganic nutrient status of plants

shows a critical role for plant resistance to moisture deficit conditions.

B is comparatively immobile in plants, and hence its availability is crucial at all

phases of growth, particularly during the period of reproductive development. B is

recognized to have various effects on biological processes in plants, predominantly courses

of cell wall development (Cakmak and Romheld, 1997; Dell and Huang, 1997). It is essential

for growth and development of pollen, for seed formation and for sugar translocation within

corn plants (Christensen and Smart, 2005). Boron may also defend cellular plasma

membranes against peroxidative impairment by reactive oxygen species and its deficiency

cause leakage of plasma membranes and lose their structural and functional integrity

(Cakmak and Romheld, 1997) which may consequently affect the resistance of plants to

drought stress. Boron mediated alleviation of negative effects of drought has been elucidated

in various plants like sunflower (Hassan et al., 2011), wheat (Karim et al., 2012), turnip

(Hajiboland and Farhanghi, 2011), tea (Hajiboland and Bastani, 2012), eucalyptus (Da Silva

11

et al., 2015; Hodecker et al., 2014), norway spruce (Mottonen et al., 2001; Mottonen et al.,

2005).

2.4 Calcium

2.4.1 Calcium uptake and distribution under drought stressPlants absorb calcium from the soil solution, where mass flow and root interception

are the primary mechanisms of Ca2+ transport to the root surfaces (Havlin et al., 1999). It

enters the root through Ca2+ permeable channels, some of them are selective for Ca2+ and

others are non-selective ion channels. The particular protein(s) that facilitates Ca2+ uptakes

still need to be identified (Demidchik and Maathuis 2007). The middle lamella of cell wall,

endoplasmic reticulum (ER) and vacuole contained high concentration of calcium. Most of

the water soluble Ca2+ ions tend to be impounded in the big vacuole of mature cells. Calcium

in the endoplasmic reticulum is linked with Ca2+ binding proteins. In contrast, Ca2+

concentration in cytosol is low (0.1–1.0 mM) (Marschner and Marschner, 2012) however,

stressed induced increased concentration of cytosolic Ca2+ has been elucidated by many

scientists (Boudsocq and Sheen, 2010; Medvedev, 2005; Reddy et al., 2011) that trigger

various physiological and downstream responses (Zhu et al., 2013). The stress induced

disturbance in cytosolic Ca2+ levels is distinguished by calcium-binding proteins called Ca2+

sensors. In plants, the Ca2+dependent protein kinases (CPKs or CDPKs) characterizes a

distinctive group of Ca2+ sensors (Harper et al., 2004). The increased level of cytosolic Ca2+

carried by abscisic acid was primarily triggered by calcium influx. The ABA induced

increase in cytosolic Ca2+ acts as secondary messenger that play role in stomatal closure. The

increased activity of inositol 1,4,5-triphosphate (IP3) induces increase cytosolic Ca2+

concentration, a vital step in stomatal closure (Takahashi et al., 2001). IP3 is recognized to

stimulate vacuolar Ca2+ channels (Allen and Sanders 1995). In addition, chloroplast-localized

Ca2+ sensing receptor (CAS) has been revealed to play an imperative part in the production of

extracellular Ca2+-prompted cytosolic Ca2+ transients and stomatal closure in Arabidopsis by

regulating IP3 concentrations, which cause in release of Ca2+ from internal stores (Han et al.,

2003; Nomura et al., 2008; Tang et al., 2007).

The key transporters catalyzing Ca2+ effluxes from cytosol to the apoplast and

endoplasmic reticulum are Ca2+-ATPases. At the membrane of vacuole (tonoplast), both Ca-

ATPases and Ca2+/H+ anti-porters catalyze Ca-efflux from the cytosol to the vacuole. The

12

latter is strengthened by the proton electrochemical gradient created by tonoplast H+-ATPase

and H+-PPiase activities (Marschner and Marschner, 2012; McAinsh and Pittman, 2009).

Chloroplasts can also enclose enormous amounts of calcium (6.5–15 mM total Ca2+,

generally bound to thylakoid membranes), but in the stroma of the chloroplast the free Ca2+

concentrations is only in the range of 2.4–6.3 μM (Kreimer et al., 1988). A plastid

Ca2+ATPase catalyzes Ca2+ uptake by plastids (Marschner and Marschner, 2012). The Ca2+

transporters for xylem loading still need to be identified and a fraction of xylem calcium may

reach through the apoplast (White, 2001), but Ca2+ mobility is also low in the plant vascular

structure (Maathuis, 2009). Calcium is relatively immobile in plants and their uptake reduces

in above-ground portions of plants (shoots and leaves) as well as in roots under drought

conditions due to decline in transpiration rate (Brown et al., 2006; Hu et al., 2008). The root

tip area is a major site of calcium entry and this zone of Ca2+ uptakes is likely to be reduced

under water deficit conditions (Raza et al., 2013). Therefore, continuous supply of calcium is

required by plants for vigorous leaves and roots development and overall canopy growth

(Del-Amor and Marcelis, 2003). Consequently, Ca2+ levels may drop below a dangerous

level in fast developing tissues triggering diseases such as ‘blossom end rot’ in tomatoes

‘black heart’ in celery, or ‘bitter-pit’ in apples.

2.4.2 Physiological roles of calcium

Calcium, a vital plant nutrient is necessary for structural parts in the cell wall and

biological membranes and act as secondary messenger (Hepler, 2005; Maathuis, 2009; White

and Broadley, 2003). It promptly complexes with organic compounds having negative groups

i.e. phosphates and carboxyl’s phospholipids, sugars and proteins. This is represented in cell

walls of plants where the cellulose micro-fibrils are cross linked by pectins and glycans.

Carboxyl groups from contrasting pectins can be electrostatically synchronized by Ca2+ that

consequently confers firmness to cell wall and plant tissues. Calcium plays an equivalent role

in cell membranes where Ca2+ organize with phosphate groups of phospholipids. This

complexion occurs largely at the outer face of the plasma membrane. The exclusion of

membrane Ca2+, or its restoration with other positively charged ions quickly compromises

membrane integrity. The second part elucidates the damaging effects of salinity stress and

13

why heavy metals such as copper (Cu) induce cellular leakage of electrolytes (Maathuis,

2009; Marschner and Marschner, 2012).

As Ca2+ readily form unsolvable salts with phosphates and sulfates, the free Ca2+

contents in the cytoplasm is reserved enormously low at about 100 nM. This makes Ca2+ an

ideal secondary messenger and a wide range of stimuli has been shown to evoke rapid

changes in cytosolic free Ca2+ in plants that include responses to biotic and abiotic stress,

stomatal regulation and physical damage (Mahouachi et al., 2006; McAinsh and Pittman,

2009). The root-shoot cell elongation needs acidification of the apoplasm and replacement of

calcium from the cross links of the pectic-chain, though this is only part of the process

(Carpita et al., 2001). The increased cytosolic concentration of free Ca2+ trigger the synthesis

of precursors for cell wall and their excretion into the apoplasm. The latter process is

repressed by eliminating apoplasmic calcium. The elongation of root-hairs and pollen tubes

also confide on the availability of apoplasmic calcium. The Ca2+ influx is restricted from the

apoplasm to the top of these cells and increases confined cytosolic Ca2+ concentration, which

acts as attention for the exocytosis of the cell wall material and creates a polarity for cell

elongation (Cole and Fowler, 2006; Krichevsky et al., 2007; White and Broadley, 2003). In

root caps, apoplasmic calcium is also required for the secretion of mucilage. The formation

of callose is one of another illustration of a Ca2+-induced secretory process. Under typical

conditions, cells produce cellulose (1.4 β-glucan units). Nonetheless, in reaction of injury or

the existence of toxic cations i.e. aluminium, a shift to callose (1.3 β-glucan units) production

can occur (Kartusch, 2003; Kauss, 1987; Rengel and Zhang, 2003). This switch is initiated

by a rise in cytosolic concentration of free calcium (Kauss, 1987). High concentration of

calcium (milimolar) stimulates α-amylase enzyme activity in germinating seeds and aleuron.

Calcium is an integral part of α-amylase, which is synthesized on rough endoplasmic

reticulum (ER). Transport of Ca2+ through the membranes of ER is improved by gibberellic

acid (GA) and repressed by abscisic acid (ABA), leading to the characteristic stimulation

(GA) and inhibition (ABA) of α-amylase activity in aleurone cells (Lovegrove and Hooley,

2000).

14

2.4.3 Role of calcium in alleviation of drought stress

Calcium is relatively immobile in plants and their uptake affected under drought

conditions due to limited water supply. Therefore, continuous supply of calcium is required

by plants for vigorous leaves and roots development and overall canopy growth (Del-Amor

and Marcelis, 2003). Calcium plays role as a regulator of plant cell metabolism and could

also contribute in the regulation mechanism of plants adjusting to adverse environmental

conditions such as water deficit (Bowler and Fluhr, 2000; Pietrobon et al., 1990). Application

of calcium can improve plant resistance to drought, prevent the synthesis of active oxides,

protect the structure of plasma membranes, and continue normal photosynthesis along with

control the metabolism of different plant hormones and other imperative chemicals.

Moreover, as a secondary messenger, cellular calcium also transfers drought signals,

consequently regulating the different physiological responses brought by water stress

(Guang_Min, 2001; Tuberosa et al., 2007; Zhang et al., 2001). Exogenous treatment of Ca2+

conferred improved tolerance to drought stress (Issam et al., 2012; Xu et al., 2013) and

modified stress induced active oxides metabolism, growth concert, photosynthetic

effectiveness, and nitrogen assimilation (Zhu et al., 2013). When the applied Ca2+ taken up

into plant body it participated in regulation of plant responses to numerous environmental

stresses by contributing directly or indirectly in plant defensive mechanisms. It alleviates the

adverse effects of heat, drought and salt by manipulation of antioxidant activities such as

glutathione reductase (GR) superoxide dismutase (SOD), catalase (CAT) and ascorbate

peroxidase (APX) reducing the membrane impairment (MDA), and aiding the plants to

survive in stress conditions (Fu and Huang, 2003; Jiang and Huang, 2001; Nayyar and

Kaushal, 2002). Calcium applied alleviation of drought-induced damages has been elucidated

in numerous plants such as Triticum aestivum (Nayyar, 2003; Nayyar and Kaushal, 2002),

Zea mays (Nayyar, 2003), Zoysia japonica (Xu et al., 2013), Camellia sinensis (Upadhyaya

et al., 2011), Lonicera japonica (Qiang et al., 2012), Phaseolus vulgaris (Abou El-Yazied,

2011) and Helianthus annuus (Hassan et al., 2011).

It is concluded that foliar application of boron and calcium at critical growth stages

helps to improve the resistance of plants against adverse conditions of drought which in turn

may result in improved economical yield. However, few studies have inspected drought

tolerance in plants in relation to boron-calcium availability. Efforts are needed to enhance

15

resistance of maize against water stress by managing the dose and time of boron and calcium

application that will ultimately help the farmers to improve the production of maize under

drought stress.

16

CHAPTER 3 MATERIALS AND METHODS

The present study was proposed to evaluate the response of water-stressed maize (Zea

mays L.) to foliar boron and calcium supply. The laboratory experiments were conducted

during 2012 to evaluate available (8) maize hybrids for drought tolerance using physiological

indices as screening tool. The further wire-house / rain-out shelter and field experiments were

conducted in February (spring maize growing season in Pakistan) during the years 2013 and

2014 to develop an appropriate rate of boron and calcium application to improve the growth

and yield of maize under water-deficit conditions.

3.1 Experimental Site and Conditions

The present studies were performed at the Department of Agronomy / Crop

Physiology, University of Agriculture, Faisalabad-38040 Punjab Pakistan. A series of

laboratory, wire-house / rain-out shelter and field experiments were conducted for this study.

Laboratory and wire-house experiments were laid out in completely randomized design

(CRD) with three replications. The field experiment was laid out in randomized complete

block design (RCBD) with split-split plot arrangement using three replications. Water stress

levels, hybrids and fertilizer doses were kept in main, sub-plot and sub-sub plot, respectively.

The field experiment was conducted at Agronomy / Crop Physiology Research Farm,

University of Agriculture, Faisalabad and experimental soil belonged to Lyallpur soil series

(Aridisol-fine-silty, mixed, hyperthermic Ustalfic, Haplargid in USDA classification and

Haplic Yermosols in FAO classification scheme). For pot (soil culture) and filed

experiments, soil samples were arbitrarily collected from field prior to crop sowing and

analyzed for soil textural class and physiochemical features by following the method of

Dewis and Freitas, (1970) and Jackson and Barak, (2005), respectively and present in Table

3.1. The weather data of the experimental site i.e. average monthly minimum-maximum

temperature (°C), relative humidity (%) and total rainfall (mm) for the year 2013 and 2014

are given in the Fig. 3.1 and 3.2 respectively. The corn growth stages according to the

calendar days as devised by the Iowa State University (Ritchie et al., 1993) are presented in

Table 3.2 and Fig. 3.3.

17

Table 3.1: Physiochemical characteristics of the soil for pot and field experiments

Soil CharacteristicsSoil Depth (cm)

0-15 15-30

Physical

Soil texture Sandy loam

Saturation percentage (%) 27 27

Chemical

ECe (dS m-1) 0.75 0.96

Soil pH 7.6 7.8

Organic matter (%) 1.19 1.12

Nitrogen (%) 0.061 0.051

Available Phosphorus (mg kg-1) 15.2 3.1

Available Potassium (mg kg-1) 80 60

Calcium (meq L-1) 3.08 3.47

Boron (mg kg-1) 0.58 0.55

18

Tem

pera

ture

CR

elat

ive

hum

idity

(%)

Rai

nfal

l (m

m)

MonthsFig 3.1: Metrological data of the experimental site during wire-house / rain-out shelter

experiment for the growing season 2013

Tem

pera

ture

CR

elat

ive

hum

idity

(%)

Rai

nfal

l (m

m)

MonthsFig 3.2: Metrological data of the experimental site during wire-house / rain-out and field

experiment for the growing season 2014

19

Table 3.2: Corn vegetative and reproductive growth stages using the ISU and FCIC methods along with the estimated days

Critical Points of Growth1,2,3

Vegetative growth stage Leaf Collar visible - *ISU -method1

Days after

seedingConcerns

V0 Germination, seeding to emergence 5 - 10 Low soil temperature will enhance

emergence timeVE Coleoptile opens, plumule emerges 7 - 14

V2 Collar of 2nd leaf 13 - 20

V3 Collar of 3rd leaf 16 - 23

V4 Collar of 4th leaf 19 - 26

V5 Collar of 5th leaf 22 - 29Cooler soil temperatures will slow

growth, potential for late harvest

V6 Collar of 6th leaf 25 - 32 Flooding up until this stage can kill a

plant in a few daysV8 Collar of 8th leaf 31 - 38

V10 Collar of 10th leaf 37 - 44 Drought conditions from V6 - V15 can

reduce yields up to 25%V12 Collar of 12th leaf 43 - 50V14 Collar of 14th leaf 49 - 56

Tasseling (VT) 65 - 72Water is still critical and hail can lead to

pollen damage*FCIC3 reproductive growth stage (R stage –*ISU method1) Silking (R1) – Silks emerged, tassel shedding pollen 69 - 76 Water is critical

Blister (R2) - Kernels are watery blister with clear fluid 78 - 89

Drought conditions from V15 - Blister

can reduce yields up to 50%

Milk (R3) - Full yellow kernels with milky fluid, no solids 91 - 98 Water is critical

Soft dough (R4) - Most kernels pasty with semi solids 100 - 107 Hails can reduce yields by 35 - 50%

Dent (R5) - Kernels dented, cut with fingernail 113 - 120

Drought conditions from Blister - Dent

can reduce yields up to 25%.

Maturity (R6) - Black layer kernel moisture from 25 – 30% 128 - 135  

1Ritchie, et al. (1993). 2Flowerday, (1995). FCIC3 - Federal Crop Insurance Corporation,*ISU - Iowa State University

20

VE V1 V3 V7V10

VTR1 R6

Fig. 3.3: Corn growth stages

3.2: Maize Hybrids

The seeds of eight available spring maize hybrids (Data-2368, Syngenta-8441, Data-

2468, Pioneer-31P41, Pioneer-32B33, FH-810, Dekalb-6525 and Yousafwala Hybrid) were

obtained from their respective independent dealers located in the local market of Sahiwal

(one of the core district for maize cultivation), Punjab-Pakistan.

3.3. Laboratory Experiments

The seeds of eight maize hybrids were germinated on filter paper placed in petri

plates (9 cm diameter) under artificial imposing drought stress by polyethylene glycol having

molecular weight of 6000 (PEG-6000). The solutions of different concentrations of PEG-6000

were prepared using Osmometer (Osmomat-030) by following the method of Michel and

Kaufmann, (1973). The seeds of maize hybrids were sterilized with sodium hypochlorite

(5%) for five minutes and 15 seeds of each hybrid were placed in petri plates containing filter

paper moistened with distilled water (control) and solutions having different osmotic

21

R2 R3 R4 R4 R5

potential (-0.2, -0.4, -0.6 and -0.8 MPa). The Petri-plates were kept under laboratory

conditions for eight days at 25±3°C (Ahmad et al., 2015). The data were recorded on daily

basis till for ten days. The seeds that grown approximately 2 mm of root were considered to

be germinated (Afzal et al., 2004). The numbers of seeds germinated were counted to

estimate germination percentage (GP), mean germination time (MGT), promptness index (PI)

and germination stress tolerance index (GSI).

In the same year, second experiment was conducted in laboratory for screening of

drought sensitive and tolerant maize hybrids under varying water stress levels. Ten healthy

seeds of same maize hybrids as used in previous experiment were grown in two sets of

plastic pots (11 × 9.5 cm) containing 500 g of fine, washed and sterilized river sand. The two

water stress levels i.e. 30% water-holding capacity (WHC) and 100% WHC maintained on

gravimetric basis (Nachabe, 1998). Drought stress was imposed by withholding water at V2

(Table 3.2). The water stress levels (30 and 100% WHC) were maintained by re-watering the

plants on daily basis by measuring the initially known pot weight by maintaining their

moisture content at original weight. As wilting symptoms appeared in stressed plants at V5

during four weeks of seeding (Table 3.2), five plants were randomly harvested from each pot

and the root-shoot length and seedlings biomass were recorded. The maize seedlings were

placed in an oven at 65°C for 72 hours and then their dry weights were recorded. From this

data the stress tolerance / physiological indices were calculated.

The laboratory experiments were repeated twice and the data obtained, presented as

mean values of the two experiments. From these experiments, germination attributes and

physiological indices were calculated using the formulae as described below.

3.3.1 Calculations of Germination attributes and Physiological Indices

The numbers of seeds germinated were counted for each treatment and replication.

The germination percentage (GP), mean germination time (MGT), promptness index (PI) and

germination stress tolerance index (GSI) were calculated as

GP = (Number of seeds germinated / Total number of seeds used) × 100

(Bouslama and Schapaugh, 1984)

MGT = (ΣDn/Σn) (Dezfuli et al., 2008)

n represents the number of seeds emerged on day D

22

D represents the number of days from the onset of seed germination

PI = nd2(1.00) + nd4(0.75) + nd6(0.50) +nd8(0.25) (Sapra et al., 1991)

Where nd2, nd4, nd6 and nd8 represent the number of seeds germinated on 2nd, 4th, 6thand

8th day respectively.

GSI = [PI of stressed seeds/ PI of control seeds] × 100

(Bouslama and Schapaugh, 1984)

The subsequent formulae as designated by Ashraf et al. (2006) were used to calculate

plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry

matter stress tolerance index (DMSI)

PHSI= (Plant height of stressed plants / Plant height of controlled plants) ×100

DMSI= (Dry matter of stressed plants / Dry matter of controlled plants) × 100

RLSI= (Root length of stressed plants / Root length of controlled plants) × 100

The maize hybrids were ranked as drought-tolerant and drought-sensitive on the basis

of their performance using germination attributes and physiological indices as screening tool.

3.4 Wire-house Experiments

Two experiments were conducted in pots (sand culture) to determine the suitable rates

of boron (B) and calcium (Ca2+) for foliar spray, effective in improving the seedling growth

of maize under water-deficit conditions. For this purpose seeds of maize hybrids, Dekalb-

6525 and Yousafwala Hybrid categorized as drought tolerant and sensitive in our previous

studies were used. Seeds of both hybrids were treated with recommended dose of fungicide

(Topsin-M 70 WP) and insecticide (Imidacloprid). Ten healthy seeds were grown in plastic

pots (8 cm diameter × 12 cm length) containing 2 kg of purified, washed, fine sand. After

emergence only five seedlings were retained in each pot. The plants were nourished with

essential nutrients by supplying Hoagland’s nutrient solution (Hoagland and Arnon, 1950)

initially applied to dry sand before sowing using auto-dispenser, thoroughly mixed with sand

and “wet up” to maximum water holding capacity. The fifteen days after emergence all pots

were flushed with water to avoid the buildup of salts in sand and fed with Hoagland’s

nutrient solution. The boron (B) and calcium (Ca2+) solutions of varied concentrations were

prepared using boric acid [B(OH)3] and calcium chloride di-hydrate [CaCl2.2H2O],

respectively (Sigma-Aldrich, USA).

23

Drought stress was imposed by withholding water at V2 during 14 days of seeding

(Table 3.2). The two water stress levels i.e. 100% water holding capacity (WHC) and 30%

WHC maintained on gravimetric basis (Nachabe, 1998). In first experiment, maize hybrids

(Dekalb-6525 and Yousafwala Hybrid) were sprayed with distilled water (control) or B

solutions of 2, 4 and 6 mg L-1 at V4 (Table 3.2). In second experiment, similar maize hybrids

were sprayed with distilled water (control) or Ca2+ solutions of 20, 40 and 60 mg L-1 under

normal and water deficit conditions at V4 during three weeks of seeding (Table 3.2). The

plant seedlings were harvested at V5 to record data regarding shoot length, root length and

plants fresh biomass. Plant samples were oven dried at 70±2 °C for calculating their dry

weights. The shoot/root ratios were recorded on dry weight basis. The best combinations of

foliar applied B (4 mg L-1) and Ca2+ (40 mg L-1) were selected on the basis of physiological

indices and seedling traits for further studies. The physiological indices were calculated by

the formulae as described in section 3.3.1

The third experiment was conducted in soil culture to evaluate the B and Ca2+

mediated physiological and biochemical changes in maize under drought stress. For this

purpose, soil collected from Crop Physiology / Agronomy Research Farm, University of

Agriculture, Faisalabad, Pakistan were sun dried, grounded, sifted and mixed well in order to

avoid any plant residue. Five healthy seeds of each hybrid, Dekalb-6525 and Yousafwala

hybrid were grown in 12 L plastic pots (30.5 × 24 cm) containing 10 kg soil. After

emergence only one plant per pot were retained. Plants were grown normally until V5, then

one set of pots were maintained at 100% WHC and other set at 30% WHC. In each pot,

recommended full dose of phosphorus (P), potassium (K) and 1/8 th nitrogen (N) in the form

of di-ammonium phosphate, sulphate of potash and urea, respectively were applied at

planting, while remaining N were applied in three equal splits at V5, V12 and V14 stages

(Table 3.2). Best dose of B (4 mg L-1) and Ca2+ (40 mg L-1) selected from previous

experiments, were foliarly applied alone and in combination, first at V10 and second at VT

(tasseling) stages (Table 3.2). Plants were protected from rain by physically operated shed

outfitted with adjustable plastic sheet. The plants were harvested on 14th day after the onset of

foliar B and Ca2+ supplies. The fully grown leaves from different experimental units were

used for determining gas exchange characteristics, leaf water relations, photosynthetic

24

pigment contents, osmolyte accumulation, activities of antioxidants and lipid peroxidation by

following the methods described in section 3.6.

3.5 Field Experiment

A field experiment was carried out at Crop Physiology / Agronomy Research Farm,

University of Agriculture, Faisalabad-38040 Punjab, Pakistan using same maize hybrids i.e.

Dekalb-6525 and Yousafwala Hybrid to determine the effects of foliar applied B (4 mg L -1),

Ca2+ (40 mg L-1) and their combinations on their yield under normal and moisture deficit

conditions. For this purpose, experimental plots of size 6 m × 3.5 m were made. The seeds

were hand-sown at ridges during March 01, 2014 in 70 cm spaced rows at 22.7 cm plant-

plant distance using three seeds per hill and then thinned at two leaf stage to maintain the

desired plant populations. Water deficit was imposed by withholding irrigations at V6 and

VT stages of maize (Table 3.2). By this way, normal plants received 793.2 mm water (675

mm irrigation + 118.2 mm rainfall) during the whole maize growing season, whereas water-

stressed received 643.2 mm water (525 mm irrigation + 118.2 mm rainfall). Foliar sprays of

B and Ca2+ alone and in combinations were applied at V6 and VT in drought stressed and

controlled plots. The fertilizer nitrogen (N), phosphorus (P) and potassium (K) were applied

in the form of urea (250 kg ha-1), diammonium phosphate (150 kg ha-1) and sulphate of

potash (150 kg ha-1), respectively. The full dose of P, K and 1/8th N was applied at sowing

and 1/5th N at V2, whereas, remaining N were applied in two equal splits at V12 and V14

stages of maize. Plants were fully grownup to maturity and data about growth, yield and

yield components were recorded.

25

Fig. 3.4: Layout PlanM

ain

Wat

er C

hann

el

Non

-Exp

erim

enta

l Are

aSub-Water Channel

Non

-Exp

erim

enta

l Are

a

Non-Stress Stress Stress Non-Stress Stress Non-Stress

Yousafwala Hybrid Dekalb-6525 Yousafwala Hybrid

T1 T0 T1 T3 T0 T1

T2 T3 T2 T1 T2 T3

T3 T2 T3 T2 T3 T2

T0 T1 T0 T0 T1 T0

Dekalb-6525 Yousafwala Hybrid Dekalb-6525

T1 T0 T2 T1 T0 T1

T3 T2 T1 T3 T2 T3

T2 T3 T3 T2 T1 T2

T0 T1 T0 T0 T3 T0

B1 B2 B3

Factor A: Maize hybrids-Drought tolerant (Dekalb-6525) and Drought sensitive (Yousafwala Hybrid); Factor B: Stress levels (Normal and Water deficit); Factor C: Foliar fertilization (T0= Control, T1 = Boron (4 mg L-1), T2 = Calcium (40 mg L-1), T3 = B+Ca2+ (4 + 40 mg L-1) B= Blocks

Table 3.3: Crop husbandry operations for irrigation management26

Operation DASCultivation 12.2.2014

22.2.201426.2.2014

Sample collection for Soil analysis 26.2.2014FertilizerN @ 32 Kg ha-1 (1/8th) 1.3.2014P2O5 @ 150 Kg ha-1 1.3.2014K2O5 @150 Kg ha-1 1.3.2014N @ 50 Kg ha-1 (1/5th) 18.3.2014N @ 84 Kg ha-1 (1/3rd) 17.4.2014N @ 84 Kg ha-1 (1/3rd) 28.4.2014Seedbed preparation 1.3.2014Sowing 1.3.2014Thinning (V2) 19.3.2014Weed Control Hand Hoeing 27.3.2014Crop establishment count 31.3.2014Irrigation (Normal)1. V2 18.3.2014 (75 mm)2. V6 2.4.2014 (75 mm)3. V12 17.4.2014 (75 mm)4. V14 28.4.2014 (75 mm)5. VT 11.5.2014 (75 mm)6. R2 21.5.2014 (75 mm)7. R3 31.5.2014 (75 mm)8. R3 6.6.2014 (75 mm)9. R4 12.6.2014 (75 mm)Total water (mm) 675 mmIrrigation (Water stress)1. V2 18.3.2014 (75 mm)2. V6 Skipped3. V12 17.4.2014 (75 mm)4. V14 28.4.2014 (75 mm)5. VT Skipped6. R2 21.5.2014 (75 mm)7. R3 31.5.2014 (75 mm)8. R3 6.6.2014 (75 mm)9. R4 12.6.2014 (75 mm)Total water (mm) 525 mmDate of Sampling1. 17.05.20142. 18.05.2014Harvesting 08.7.2014

3.6 Data Collections

27

3.6.1 Estimation of Leaf Water Relations

3.6.1.1 Leaf Water Potential (‒MPa)

The third fully expanded leaf from top of plants selected from each treatment at early

morning (06:00-8.00 a.m.) to estimate leaf water potential (Ψw) by “Scholander” type

pressure chamber Model 1000 (PMS, Oregon-USA).

3.6.1.2. Leaf Osmotic Potential (‒MPa)

The same leaves as used for water potential measurements were frozen at -20°C for 7

days and then thawed to extract cell sap for estimation of leaf osmotic potential (Ψs) using an

osmometer (Osmomat-030).

3.6.1.3. Turgor Potential (MPa)

The leaf turgor potential (Ψp) was determined by the following formula:

Ψp = Ψw-Ψs

3.6.1.4. Relative Water Contents (%)

The third leaf from top (fully expanded leaf) was collected for the measurement of

relative water content (RWR). Soon after cutting at base of the lamina, leaves were sealed in

plastic bags and quickly transferred to the laboratory. Fresh weight (FW) of each sample was

recorded using a digital electrical balance (Chyo, MK-500C) and leaves were dipped in test

tube containing distilled water for 24 hours. Then leaves were taken out, wiped with the

tissue paper and their turgid weight (TW) was recorded. The samples were dried at 65°C for

72 h and dry weight (DW) of each sample was recorded. Relative water contents were

calculated using the formula given by Cornic (1994).

RWC = [(FW-DW) / (TW-DW)] × 100

Where,

FW – sample fresh weight

DW – sample dry weight

TW – sample turgid weight

3.6.2. Estimation of Pigment Contents

28

Chlorophyll (Chl) and carotenoid (CAR) contents were quantified by the method of

Arnon, (1949) and Davies, (1986). Chl contents were extracted from 0.5 g leaf discs with 10

mL acetone (80%) at 100C over-night and then centrifuged the material at 14000 x g for 5

minutes. The absorbance of the supernatant was measured at 645, 652, 663 and 480 nm on

spectrophotometer (PG, T60U).

The Chl and carotenoid contents were calculated by following the formulae:

Chla (mg g-1 FW) = [12.7 (OD 663) -2.69 (OD 645)] × V/1000 × W

Chlb (mg g-1 FW) = [22.9 (OD 645) -4.68 (OD 663)] × V/1000 × W

Chla+b (mg g-1 FW) = [20.2 (OD 645) + 8.02 (OD 663)] × V/100 × W

Carotenoids (mg g-1 FW) = Acar/Emx100

Where

V = Volume of the sample extract and W = weight of the sample

Acar = (OD480) + 0.114 (OD663)-0.638 (OD645); Emax100 cm =2500

3.6.3. Gas Exchange Parameters

The net photosynthetic rate (Pn, μmol CO2 m-2 s-1), transpiration rate (E, mmol H2O

m-2 s-1) and stomatal conductance (gs, mol m-2 s-1) were measured using fully expanded middle

leaf between 9.00-11.00 a.m. by LCA-4 ADC portable infrared gas analyzer (Analytical

Development Company, Hoddesdon, England).

3.6.4. Biochemical parameters

3.6.4.1 Proline Determination

Leaf proline contents were determined according to the method given by Bates et al.

(1973). Fresh leaf tissues of 0.5 g was ground in 5 ml of 3% sulfosalysilic (SS) acid and the

homogenates were centrifuged at 3,000 × g for 20 min and filtered. Take 2 ml of the filtrate

in 19×150 mm test tubes and mixed with 4 ml of 2.5% acid ninhydrin reagent. The acid

ninhydrin solution was prepared by dissolving 1.25 g ninhydrin in 30 mL of glacial acetic

acid and 20 mL of 6 M orthophosphoric acid. Two mL of glacial acetic acid was added in the

test tube and heated for 1 h at 100 oC. After heating the mixture in a boiling water bath for

one hour, the reaction mixture was cooled in an ice bath for 5 min and extracted with 4 ml of

toluene which parted the chromosphere in the toluene fraction. Isolated colored phase was

allowed to stand for 2-3 min at room temperature and its absorbance read at 520 nm using 29

spectrophotometer (PG, T60U). A standard graph was prepared by using 5, 10, 15, 20, 25,

30, 35 and 40 µmol/ml of proline.

Toluene was used as a blank and proline contents of the unknown samples were

determined on FW basis by using the standard graph developed by Analar grade proline and

results expressed in micromole per gram on dry weight basis:

Proline (μmol g-1 DW) = [(µmol ml-1 proline × volume of toluene × volume of SS acid)] /

(sample FW x 115.5)

3.6.4.2. Total Soluble Proteins (mg g-1 DW)

Total soluble proteins (TSP) were estimated by following the method of Lowry et al.

(1951).

Reagents

Phosphate buffer (0.2 M): Following chemicals were used to formulate the phosphate

buffer.

1. One-molar solution of monobasic sodium phosphate (NaH2PO4.2H2O, 156.01 g L-1) was

prepared as the stock.

2. One-molar solution of Disodium phosphate (Na2HPO4.2H2O, 177.99 g L-1) was prepared

as the stock.

Copper Reagents

Solution A

Na2CO3 = 2.0 g

NaOH = 0.2 g

Sodium potassium tartarate = 1.0 g

All the three chemicals were dissolved in distilled water and the volume was made to 100

mL.

Solution B

CuSO4.5H2O solution: 0.5g CuSO4.5H2O was dissolved in 100 mL distilled water

Solution C

30

Fifty mL of solution A and 1.0 mL of solution B were mixed to prepare alkaline

solution. This solution was always prepared fresh.

Folin Phenol Reagent

Twenty five gram of sodium molybdate and 100 g of sodium tungstate were dissolved

in 700 mL of distilled water. Hundred mL of HCl and 50 mL of 85% orthophosphoric acid

were added and the mixture was refluxed for 10 h, followed by the addition of 150 g of

lithium sulfate and 50 mL of distilled water. A few drops of Br2 were also added. The

mixture was boiled without condenser for 15 min to remove extra Br2. The mixture was then

cooled and diluted to 1000 mL.

Standard Bovine Serum Albumin (BSA) solution (1 µg mL-1)

Ten mg of Bovine serum albumin (BSA) was dissolved in 10.0 mL of distilled water.

Extraction

Fresh leaf material (0.5 g) was chopped in 10 mL of phosphate buffer (0.2 M) of pH

7.0 and was ground. The ground leaf material was centrifuged at 5000 x g for 5 min. The

supernatant was used for protein determination.

Procedure

One mL of the supernatant (leaf extract) from each treatment was taken in a test tube.

The blank contained 1 mL of phosphate buffer (pH 7.0). One mL of solution C was added to

each test tube. The reagents in the test tube were thoroughly mixed and allowed to stand for

10 min at room temperature. Then 0.5 mL of Folin-Phenol reagent (1:1 diluted) was added,

mixed well and incubated for 30 min. at room temperature. The optical density (OD) was

read at 620 nm on a spectrophotometer (PG, T60U). The protein concentration was

calculated by using standard curve developed by different concentration of Bovine serum

albumin (BSA).

3.6.4.3. Total Free Amino Acids (mg g-1 DW)

Total free amino acids (TFA) were determined according to Hamilton and Van Slyke,

(1973). Half gram fresh plant leaves were chopped and extracted with 0.2 M phosphate

buffer (pH 7.0) then, took 1 mL of the extract in volumetric flask (50 mL), added 1 mL of

pyridine (10 %) and 1mL of ninhydrin (2%) solutions in flask. Ninhydrin solution was

freshly prepared by dissolving 2 g ninhydrin in 100 mL of distilled water. The flasks with

31

sample mixture, heated in boiling water bath for about 30 min. Volume of each flask was

made up to 50 mL with distilled water. Read the optical density (OD) of the colored solution

at 570 nm using spectrophotometer (PG, T60U). Developed a standard curve with leucine

and total free amino acids were calculated using the standard graph and the results were

denoted in milligram per gram dry weight (mg g-1 DW).

3.6.4.4. Total Soluble Sugars (mg g-1 DW)

Total soluble sugars (TSS) were determined according to the method of Yemm and

Willis (1954).

Extraction

Dried plant material was grounded well in a micro mill and the material was sieved

through 1 mm sieve of micro mill. Plant material (0.5 g) was extracted 5 mL of 80% ethanol

solution for 6 h at 60oC. This extract was used for the estimation of total soluble sugars.

Reagents

Anthrone reagent was prepared by dissolving 150 mg of anthrone in 72% H2SO4

solution. This reagent was freshly prepared whenever needed.

Procedure

Plant extract 0.1 mL was taken in 25 mL test tubes and 6 mL anthrone reagent was

added to each tube, heated in boiling water bath for 10 min. The test tubes were ice-cooled

for 10 min. and incubated for 20 min. at room temperature (25oC). Optical density (OD) was

read at 625 nm on a spectrophotometer (PG, T60U). The concentration of soluble sugars was

calculated from the standard curve developed by using different concentration of glucose

according to the above procedure.

3.6.5 Antioxidants Extraction and Lipid Peroxidation Assay

Fresh leaf samples (0.5 g) were homogenized in pestle and mortar on ice by a

medium composed of 5 ml extraction buffer (50 mM phosphate buffer pH 7.0 and 1 mM

dithiothreitol). The homogenate were then centrifuged at 12,000 × g for 20 min at 4 °C, and

the supernatant was used for analysis of superoxide dismutase (SOD EC 1.15.1.1), catalase

(CAT EC 1.11.1.6), peroxidase (POD EC 1.11.1.7), and ascorbate peroxidase (APX EC

1.11.1.11).

32

3.6.5.1 Superoxide Dismutase (Unit min-1 g-1 FW)

The SOD activity was determined by the method of Giannopolitis and Ries (1977).

The reaction mixture comprised of 0.95 ml of 50 mM phosphate buffer pH (7.8), 1ml of 1.3

μM Riboflavin, 1 ml of 50 μM NBT, 0.5 ml of 75 nM EDTA, 0.5 ml of 13 mM Methionine

and 0.5 ml of enzyme extract. The SOD activity was investigated by measuring the potential

of the enzyme to impede the photochemical reduction of nitroblue tetrazolium (NBT) by

photochemically-generated superoxide radicals. On unit of SOD was defined as the amount

of enzyme required to cause 50% inhibition of the rate of NBT reduction at 560 nm.

3.6.5.2 Catalase (Unit min-1 g-1 FW)

Activity of CAT was examined by measuring the conversion rate of hydrogen

peroxide (H2O2) to water and oxygen molecules, by using the method defined by Chance and

Maehly (1955).The activity was analyzed in 3 mL 50 mM phosphate buffer having 7.0 pH,

comprising 5.9 mM of H2O2 and 0.1 mL enzyme extract. The CAT activity was defined by

drop in absorbance at 240 nm after each 20 s due to depletion of H2O2. Absorbance change

by 0.01 unit min-1 was demarcated as one unit CAT activity.

3.6.5.3 Peroxidase (Unit min-1 g-1 FW)

POD activity was examined by computing peroxidation of H2O2 with guaiacol as an

electron donor (Chance and Maehly, 1955). The reaction mixture for POD contained 50 mM

phosphate buffer (pH 5), 20 mM guaiacol, 40 mM H2O2 and 0.1 mL enzyme extract. The

increase in the absorbance owing to the formation of tetraguaiacol at 470 nm was analyzed

after every 20 s. One unit of POD was reflected as the quantity of the enzyme that was

accountable for the increase in optical density (OD) value of 0.01 in 1 min. The POD activity

was determined and described as unit min-1g-1FW basis.

3.6.5.4 Ascorbate Peroxidase (ABA digested g-1 FW h-1)

The activity of APX was measured by observing the decline in absorbance due to the

formation of ascorbic acid at 290 nm (molar extinction coefficient 2.8 mM cm-1) in a 1mL

reaction mixture having 50 mM phosphate buffer (pH 7.6), 0.1 mM Na-EDTA, 12 mM H2O2,

0.25 mM ascorbic acid and the sample extract as described by Cakmak (1994).

3.6.5.5 Lipid Peroxidation

33

Lipid peroxidation was determined by following the method of Heath and Packer

(1968) using thiobarbituric acid reaction as index of malondialdehyde (MDA) content. The

absorbance of the TBA reactive substances (TBARS) was measured by the differences in

absorbance at 532 to 600 nm using the extinction coefficient of 155 mM-1 cm-1

3.6.6 Estimation of Leaf B Concentration (mg kg-1 DW)

At reproductive stage, leaf samples were collected for B analysis from below and

opposite ear leaf. The leaf samples were washed with distilled water and then dried in an air

forced oven at ±70 °C for 48 h and then crushed with plant grinding willey mill. B

concentrations in maize leaf tissues were determined by following the method of Mills and

Jones (1996) and Ho et al. (1986) as mentioned below:

Apparatus

1. Hot plate

2. Muffle Furnace

3. Spectrophotometer

Reagents

The subsequent reagents were used for the purpose of B estimation in maize leaf samples

Buffer- masking Reagent

We dissolved 250 g ammonium acetate (NH4OAC) and15 g di-Sodium salt of EDTA

(ethylene-diamine-tetra acetic acid) in 400 ml of distilled water, followed by gradual but

slow addition of 125 ml of glacial acetic acid. Throughout this whole process, samples were

carefully stirred and then heated to liquefied or dissolved the contents and sieved through

whatman No. 1 filter paper to eliminated any un-dissolved residues.

Azomethine-H Reagent

We dissolved 0.90 g Azomethine-H (Sigma-Aldrich, USA) and 3 g of AsA (ascorbic

acid) in distilled water with moderate heating and dilute up to 100 ml volume. The samples

were re-heated to eliminate turbidity of solution. This reagent was prepared freshly for each

analysis but under few situations this was stored for 7-10 days in brown flask in refrigerator

(Bingham, 1982).

Working Solution

34

Dissolved 80 ml of Azomethine-H reagent to 320 ml of buffer-masking reagent for

preparations of working solutions for 48 plant samples included standards.

Stock Solution of B

For preparation of 100 mg B kg-1 stock solution, we dissolved 0.57 g boric-acid in

water and diluted it to1 L.

Boron Standards

B standards were made in 100 ml volumetric flask.

Dilute Sulfuric Acid (0.36 N)

Diluted 10 ml concentrated sulfuric acid (H2SO4) to 1 L with deionized water to

prepare 0.36 N H2SO4 and stored in bottle for about 15 days.

Table 3.4 Boron standards prepared by 0.36 N H2SO4

B Concentration

(μg B ml-1)

Stock Solution

(ml)

0.36 N H2SO4

(ml)

Final Volume

(ml)

1.0 1.0 99 1002.0 2.0 98 1003.0 3.0 97 1004.0 4.0 96 1005.0 5.0 95 1006.0 6.0 94 100

Sample Preparation

Took 1 g leaf sample into 30 ml crucible and placed in muffle furnace for 1-3 hours at

450-600 oC. The ashed samples were cooled and wetted with 10 drops of deionized water.

Then, took 10 ml of 0.36 N H2SO4 using pipette into the crucible and samples were reacted at

room temperature for about 50 to 60 minutes. Periodically samples were agitated with the

help of plastic rod to break up ash exciting. Then these samples were sieved through

whatman # 1 filter paper. This filtrate was used for determination of B.

Color Development

The 4 ml of the sample filtrate pipetted in test tube, followed by the addition of 4 ml

of buffer-masking reagent and 1 ml of azomethine-H reagent that reacted to develop color for 35

1 hour and absorbance were measured at 420 nm using spectrophotometer. B concentrations

of the samples were determined using the standard curve developed by plotting the

absorbance against concentration of standards in μg B ml-1 as mentioned below:

10ml final volume

Sample B (μg ml-1) = μg B ml-1 ×

Sample weight (g)

3.6.7 Determination of Calcium (Ca2+) Concentration (mg g-1 DW)

Digestion

Dried ground material (0.5 g leaves) was taken in digestion tubes and 5 mL of

concentrated sulfuric acid (H2SO4) was added to each tube. All the tubes were incubated

overnight at room temperature. Then 0.5 mL of H2O2 (35%) was poured down the sides of

the digestion tubes. Then tubes were placed in a digestion block and heated at 350 oC until

fumes were produced. They were heated for another 30 minutes. The digestion tubes were

removed from the block and cooled. Then, 0.5 mL of H2O2 was slowly added and placed the

tubes back into the digestion block. The above step was repeated until the cooled digested

material became colorless. The volume of the extract was maintained up to 50 mL in

volumetric flasks. The extract was filtered and used for determination of calcium according

to the method proposed by Wolf (1982).

Reagents

Solution A: (Ammonium chloride–ammonium hydroxide buffer solution).

Ammonium chloride 67.5 g was dissolved in 570 mL of concentrated ammonium

hydroxide and made it to 1 L.

Solution B: (Approximately 4 N sodium hydroxide).

Sodium hydroxide 160 g was dissolved in 1 liter of water.

Solution C: (0.01 N calcium chloride solutions).

Pure calcium carbonate (calcite crystals) 0.5 g was dissolved in 10 mL of

approximately 3 N hydrochloric acid and diluted it to a volume of exactly 1 L.

36

Solution D: (Eriochrome black T (EBT) indicator).

EBT (F241) 0.5 mg and 4.5 g of hydroxylamine hydrochloride were dissolved in 100

mL of 95 % ethanol. This indicator is available under several different trade names.

Solution F: (Approximately 0.01 N ethylene diamine tetra acetate ‘versenate’ solution).

Disodium dihydrogen ethylene diamine tetra acetate 2 g and 0.05 g of magnesium

chloride hexahydrate were dissolved in distill water and diluted it to a volume of 1 L.

Solution E: (Ammonium pupurate indicator).

Standardized the solution against reagent C, using the titration procedure and each of

the indicators D and E, as the normality with E is 3 to 5 % higher than with D.

3.6.7a Estimation of Calcium

Aliquot/extract (5 to 25 ml) containing up-to 0.1 meq of calcium was poured into a 4

inch diameter porcelain casserole. It was diluted to a volume of approximately 25 mL and

0.25ml (5 drops) of reagent B and approximately 50 mg of E were added and titrated against

reagent F, using a 10 mL micro-burette. The color change was observed from orange red to

purple. When close to the end point, solution F was added at the rate of about a drop every 5

to 10 sec. as the color change is not instantaneous. A blank containing solutions B, E, and a

drop or two of F aided in distinguishing the end point.

Calculations

Milli equivalents per liter (mE L-1) of Ca = (ml of versenate solution used × normality of

versenate solution as determined by appropriate indicators × 1000) / (mL of aliquot). The

results were expressed in mg g-1 dry weight (DW).

3.6.8 Growth Parameters of MaizeTwo maize plants were harvested from each plot at reproductive stage and their

leaves, shoot, tassel and silks were separated.

3.6.8.1 Leaf Dry Weight (g plant-1)

Leaf samples were placed in an air forced oven at ±70 °C for 72 h and then their dry weights were

recorded.

37

3.6.8.2 Shoot Dry Weight (g plant-1)

The shoot samples were dried in an oven at ±70 °C to constant weight and their dry

weights were recorded.

3.6.8.3 Silk Threads (Number ear-1)

The silk threads of two ears were separated and their numbers were counted.

3.6.8.4 Length of Silk Threads (cm)

From the detached silk threads, 50 of them were selected randomly and measured for

their length.

3.6.8.5 Tassel and Silk Dry Weight (g plant-1)

The detached silk and tassel samples were kept individually within paper bag and

placed them in an air-forced oven at 70°C for 72 hours and then their dry weights were

recorded.

3.6.8.6 Plant Height at Harvest

Ten plants were selected randomly from each plot and their heights were measured

from ground surface to the top of the plant with the help of meter rod and their average

heights were calculated in cm.

3.6.9 Yield and Yield Components

3.6.9.1 Number of Grains per Ear

The number of grains from five randomly selected ear from each plot was counted

and then averaged to calculate number of grains per cob.

3.6.9.2 1000-Grain Weight (g)

Two random samples of one thousand grains were obtained from the total lot of each

experimental unit and weighed by means of electric balance.

3.6.9.3 Biological Yield (t ha-1)

38

The crop was harvested, tied into bundles and allowed to sun-dry in respective plots.

Biological yield of sun dried samples were recorded for each treatment with the help of hand-

held weighing balance.

3.3.9.4 Grain Yield (t ha-1)

Grain yield was recorded on plot basis in kg and then converted to ton ha-1.

3.6.9.5 Harvest Index (%)

Harvest index (H.I) was calculated by using the following formula of Beadle (1987).

Economic yieldH.I = -------------------- × 100

Biological yield

3.6.10 Statistical Analyses

Data collected in different experiments of this project were subjected to Fisher’s

analysis of variance and Statistix-8.1 program was used for analysis. Tukey's HSD (Honest

significant difference) test at 0.05 probability level was used to evaluate differences among

the treatment means (Steel et al., 1997).

39

CHAPTER 4 RESULTS

EXPERIMENT 1

4.1 Screening of maize hybrids for drought tolerance (Petri-plate experiment)

The experiment was conducted under laboratory conditions to screen-out available

maize hybrids for drought tolerance and water stress was imposed by using polyethylene

glycol (PEG6000) as an osmotic agent. Different germination attributes recorded was used as

screening tool for this experiment. The results obtained from this experiment are given

below:

4.1.1 Germination Percentage

The PEG-6000 induced osmotic stress significantly (P≤0.001) reduced the germination

percentage (GP) of all the tested maize hybrids (Data-2368, Syngenta-8441, Data-2468,

Pioneer-31P41, Pioneer-32B33, FH-810, Dekalb-6525 and Yousafwala Hybrid) (Table 4.1).

The gradual increase in osmotic stress reduced the GP of all maize hybrids (Fig 4.1). The

maximum GP (93%) was recorded in control treatment (0-PEG-6000), while it was minimal at

osmotic stress of -0.8 MPa. The significant (P≤0.001) difference was recorded among maize

hybrids for seed GP (Table 4.1). The maximum GP (93.33%) was recorded in maize hybrid

Dekalb-6525 which was statistically at par with hybrid Pioneer-31P41, Pioneer-32B33 and

Syngenta-8441. Maize hybrid FH-810 maintained minimum GP (42.67%), followed by

Yousafwala Hybrid (43.67%) (Fig. 4.1).

Significant (P< 0.001) interaction was recorded among PEG-6000 induced osmotic

stress levels (PEG-6000) × maize hybrids (H) for GP (Table 4.1). Maximum GP (100%) was

noted in hybrid Dekalb-6525 both in control (0- PEG-6000) and osmotic stress level of -0.4

MPa, however minimum (11.67%) in Yousafwala Hybrid at osmotic stress of -0.8 MPa (Fig.

4.1).

4.1.2 Mean Germination Time

Mean germination time (MGT) of maize seeds were influenced significantly

(P< 0.001) by different osmotic stress levels (Table 4.1). Data showed that germination was

40

delayed with the increase in osmotic stress. The significantly higher MGT (3.68 d) was noted

under osmotic stress of -0.8 MPa, however, minimum (2.51 d) was recorded under control

treatment (Fig. 4.2). All of the maize hybrids differed significantly (P< 0.001) with respect

to MGT (Table 4.1). Minimum MGT was observed in hybrid Pioneer-31P41 (2.45 d),

followed by the hybrid Dekalb-6525 (2.60 d). The hybrid FH-810 took maximum

germination time (4.07) followed by Yousafwala Hybrid (3.83 d) (Fig. 4.2)

Significant (P< 0.01) interaction (PEG-6000 × H) was recorded for this variable (Table

4.1). Under control treatment (0-PEG-6000), maize hybrid Pioneer-31P41 took minimum

MGT (1.80 d) which was statistically at par with hybrid Dekalb-6525. Hybrid Dekalb-

6525, even at highest osmotic stress (-0.8 MPa) took significantly lower germination time

(3 d). Maximum MGT (5.00 d) was recorded in Yousafwala Hybrid exposed to -0.8 MPa

PEG-6000 induced osmotic stress (Fig. 4.2)

4.1.3 Promptness Index

It refers to the number of seeds germinated at different days of observations. The

promptness index (PI) of maize seeds were differed significantly (P≤0.001) by PEG-6000

induced osmotic stress (Table 4.1). The seeds grown under controlled conditions (0- PEG -

6000) maintained significantly higher PI (3.74). The PI was gradually decreased by the

increase of osmotic stress and minimum was recorded at osmotic stress level of -0.8 MPa

(Fig. 4.3). Maize hybrids differed significantly (P< 0.001) for seeds PI. The significantly

higher PI value (3.07) was recorded in hybrid Dekalb-6525, followed by Syngenta-8441

(2.59), whereas minimum (1.07) was recorded in Yousafwala Hybrid (Fig. 4.3).

A significant (P< 0.01) interactions regarding PI were recorded among osmotic stress

levels (PEG-6000) and hybrids (H) (Table 4.1). The maximum seeds PI were calculated in

Syngenta-8441 (4.67) under control treatment (0- PEG-6000) and it was statistically at par with

Dekalb-6525 (4.42), Data-2368 (4.08), FH-810 (3.83) and Data-2468 (3.75) grown under

controlled conditions. The significantly lower seed PI was recorded in Yousafwala Hybrid

grown under osmotic stress level of -0.8 MPa (Fig. 4.3).

4.1.4 Germination Stress Tolerance Index

It was calculated by dividing the promptness index (PI) of stressed seeds to the PI of

controlled seeds. Data depicted in Table 4.1 indicated a highly significant (P< 0.001)

difference for GSI under varying osmotic stress levels. The GSI was gradually decreased by

41

the increase of osmotic stress and the maximum (58.13%) was recorded at osmotic stress

level of -0.2 MPa and minimum (36.39%) at -0.8 MPa (Fig. 4.4). The highly significant

(P< 0.001) variations for GSI were observed among maize hybrids (Table 4.1). The

significantly higher GSI (61.25%) was observed in hybrid Dekalb-6525 and minimal

(35.88%) in hybrid FH-810, which was statistically at par with Yousafwala Hybrid (Fig. 4.4).

A highly significant (P< 0.001) interaction was recorded among different osmotic

stress levels (PEG-6000) and hybrids (H) (Table 4.1). Maximum GSI (69.45%) was recorded in

Dekalb-6525 at osmotic stress level of -0.2 MPa and minimum (21.67%) in Yousafwala

Hybrid at -0.8 MPa (Fig. 4.4).

The results of the study revealed that germination attributes of all tested maize

hybrids significantly reduced by PEG-6000 induced water stress as compared to control (Fig.

4.1-4.4)

Ger

min

atio

n Pe

rcen

tage

(%)

Maize hybrids

Fig. 4.1: Effect of PEG6000 induced osmotic stress on germination percentage (%) of different maize (Zea mays L.) hybrids. Values are means + standard error

42

Mea

n G

erm

inat

ion

Tim

e (D

ays)

Maize hybrids

Fig. 4.2: Effect of PEG6000 induced osmotic stress on mean germination time (Days) of different maize (Zea mays L.) hybrids. Values are means + standard error

Prom

ptne

ss In

dex

Maize hybrids

Fig. 4.3: Effect of PEG6000 induced osmotic stress on promptness index of different maize (Zea mays L.) hybrids. Values are means + standard error

43

Ger

min

atio

n St

ress

Inde

x (%

)

Maize hybrids

Fig. 4.4: Effect of PEG6000 induced osmotic stress on germination stress index of different maize (Zea mays L.) hybrids. Values are means + standard error

Table 4.1: Analysis of variance (ANOVA) of the data for PEG induced osmotic stress (‒MPa) on germination percentage (GP), mean germination time (MGT), promptness index (PI) and germination stress index (GSI) for eight different maize (Zea mays L.) hybrids

SOVa DFc GP (%) MGT (day) PI GSI (%)

PEG6000 4 3365.62*** 6.38223*** 4.9147*** 1103.76***

Hybrids (H) 7 6750.92*** 4.79581*** 20.6379*** 2150.90***

PEG-6000 × H 28 244.67*** 0.54231*** 0.1890** 46.43***

Error 78 67.91 0.13459 0.0806 9.37

CVb 10.78 11.33 13.08 6.50

* = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

44

EXPERIMENT 2

4.2 Screening of maize hybrids for drought tolerance (Pot experiment)This experiment was conducted in wire-house / rainout shelter to screen out the maize

hybrids for drought tolerance and water was withheld to impose the drought stress. Different

physiological indices and seedling growth attributes were calculated and used as a screening

tool for drought tolerance in maize. The results of the study are given below:

4.2.1 Plant Height Stress Tolerance Index (PHSI)

Plant height stress tolerance index (PHSI) of different maize hybrids was influenced

significantly (P< 0.01) under drought conditions (Table 4.2). Data indicated that maximum

PHSI was recorded in hybrid Dekalb-6525 (90.15%), which was statistically at par with

Pioneer-31P41, Pioneer-32B33, FH-810, Data-2368 and Data-2468. Whereas, the minimum

PHSI was recorded in maize hybrid Syngenta-8441 (72.14%) which was statistically equal

with Yousafwala Hybrid (Fig. 4.5).

4.2.2 Root Length Stress Tolerance Index (RLSI)

Drought stress had significant (P< 0.01) effect on root length stress tolerance index

(RLSI) of different maize hybrids (Table 4.2). The significantly higher RLSI (130.69%) was

noted in Dekalb-6525 which was statistically equal with the hybrids Pioneer-31P41, Data-

2368, Pioneer-32B33 and Data-2468. The significantly lower value (73.96%) for this index

was noted in Yousafwala Hybrid (Fig. 4.6).

4.2.3 Dry Matter Stress Tolerance Index (DMSI)

Data concerning dry matter stress tolerance index (DMSI) showed significant

(P< 0.01) difference among maize hybrids (Table 4.2). The significantly higher value

(91.05%) for DMSI was recorded in Dekalb-6525, whereas it was lowest (72%) in

Yousafwala Hybrid. Maize hybrid Pioneer-31P41 showed the second highest value (86.36%)

for DMSI (Fig. 4.7).

Based on the results of these experiments conducted for screening of maize hybrids

for drought tolerance it was concluded that the hybrid Dekalb-6525 performed better on the

basis of improved germination and seedling growth under water limitations, therefore

categorized as drought tolerant. However, the Yousafwala Hybrid was categorized as drought

sensitive on the basis of it poor performance.

45

Plan

t Hei

ght S

tres

s Tol

eran

ce In

dex

(%)

Maize hybrids

Fig. 4.5: Effect of water stress on plant height stress tolerance index (PHSI) of different maize (Zea mays L.) hybrids. Values are means + standard error

Roo

t len

gth

Stre

ss T

oler

ance

Inde

x (%

)

Maize hybrids

Fig. 4.6: Effect of water stress on root length stress tolerance index (RLSI) of different maize (Zea mays L.) hybrids. Values are means + standard error

46

Dry

Mat

ter

Stre

ss T

oler

ance

Inde

x (%

)

Maize hybrids

Fig. 4.7: Effect of water stress on dry matter stress tolerance index (DMSI) of different maize (Zea mays L.) hybrids. Values are means + standard error

Table 4.2: Analysis of variance (ANOVA) of the data for the effect of varying water stress levels on plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry matter stress tolerance index (DMSI) for eight different maize (Zea mays L.) hybrids

SOV DFc PHSI (%) RLSI (%) DMSI (%)

Hybrid (H) 7 150.275** 830.630** 130.055**

Error 16 28.747 81.365 20.856

CV 6.55 8.35 5.67

* = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

47

Table 4.3: Ranking order of eight maize (Zea mays L.) hybrids under water stress on the basis of their germination attributes and seedling stress tolerance indices

GenotypesGP MGT PI GSI PHSI RLSI DMSI Total

Marks=8010 10 10 10 10 10 10

Dekalb-6525 9.33 7.4 10 10 10 10 9.11 65.84

P-31P41 9.33 7.55 7.14 9.27 9.91 9.17 8.64 61.01

P-32B33 9.06 6.93 7.71 9 9.82 8.62 8.55 59.69

Syng-8441 8.8 6.54 8.51 7.28 8 7.77 7.7 54.6

Data-2468 8.13 6.58 6.97 7.49 8.67 8.19 7.7 53.73

Data-2368 7.86 6.98 7.02 6.61 8.7 8.77 8.2 54.14

Yousafwala

Hybrid4.37 6.17 3.5 6 8.24 5.66 7.2 41.14

FH-810 4.27 5.93 6.12 5.86 9.29 7.95 7.4 46.82

48

EXPERIMENT 3

4.3 Optimization of boron concentration for spring maize This experiment was conducted in a wire-house to optimize the foliar rate for boron

(B) supply effective in mitigating the adverse effects of drought in maize; the two selected

maize hybrids i.e. Dekalb-6525 (drought tolerant) and Yousafwala Hybrid (drought-

sensitive) hybrids were sown under normal (100% Water-holding capacity) and water deficit

conditions (30% Water-holding capacity) conditions. Results of the experiment are given

below:

4.3.1 Plant Height Stress Tolerance Index (PHSI)

Plant height stress tolerance index (PHSI) was influenced significantly (P≤0.001) by

supplemental boron (B) supply to maize under drought conditions (Table 4.4). Foliar

treatment of B at 4 mg L-1 gave significantly higher PHSI (85.44%) as compared with plants

sprayed with distilled water (DW) and other B levels. The PHSI was also differed

significantly (P≤0.01) with respect to hybrids (Table 4.4). Drought tolerant maize hybrid

Dekalb-6525 maintained higher values for PHSI (77.55%) than that of drought sensitive

Yousafwala Hybrid (73.00%) (Fig. 4.8)

Significant (P< 0.01) interaction was recorded between hybrids (H) and boron (B)

levels (Table 4.4). Considerably higher value for PHSI (86.30%) was recorded in Dekalb-

6525 supplemented with B at 4 mg L-1, whereas minimum (57.69%) was recorded in

Yousafwala Hybrid sprayed with B at 6 mg L -1. Both cultivars sprayed with distilled water

and B at 2 mg L-1 gave statistically similar values for PHSI but significantly higher over

those plants applied with B at 6 mg L-1 (Fig.4.8).

4.3.2 Root Length Stress Tolerance Index (RLSI)

Foliar treatment of B significantly (P< 0.001) influenced the root length stress

tolerance index (RLSI) of maize under drought conditions (Table 1). Significantly higher

value for RLSI (122.46%) was recorded in maize plants foliarly treated with B at 4 mg L -1

and minimum (91.82%) was observed in plants treated with B at 6 mg L-1 (Fig. 4.9).

Considerable (P≤0.05) difference was also recorded between hybrids for this variable

(Table 4.4). Maize hybrid Dekalb-6525 maintained considerably higher value for RLSI

49

(111.58%) than that of Yousafwala Hybrid (Fig. 4.9). The non-significant interaction (H × B)

was recorded for RLSI (Table 4.4).

4.3.3 Dry Matter Stress Tolerance Index (DMSI)

Dry matter stress tolerance index (DMSI) of maize plants was significantly (P< 0.001)

affected by foliar application of B under limited moisture supply (Table 4.4). The highest

DMSI (95.67%) was noted in plants foliarly treated with B at 4 mg L-1, whereas minimum

(79.54%) in case of control treatment. Foliar treatment of B at 2 and 6 mg L -1 also caused

significantly higher dry matter accumulation as compared to untreated control (Fig. 4.10).

Significant (P≤0.01) difference was recorded between cultivars for DMSI and the drought-

tolerant cultivar Dekalb-6525 maintained higher value for DMSI (89.12%) than that of

drought-sensitive Yousafwala Hybrid (Fig. 4.10). However, non-significant interaction was

recorded for DMSI between hybrids (H) and boron (B) rates (Table 4.4).

Plan

t Hei

ght S

tres

s Tol

eran

ce In

dex

(%)

Boron concentration (mg L-1)

Fig. 4.8: Effect of boron foliar spray on plant height stress tolerance index (PHSI) of maize (Zea mays L.) seedlings under deficit moisture supply. Values are means + standard error

50

Roo

t len

gth

Stre

ss T

oler

ance

Inde

x (%

)

Boron concentration (mg L-1)

Fig. 4.9: Effect of boron foliar spray on root length stress tolerance index (RLSI) of maize (Zea mays L.) seedlings under deficit moisture supply. Values are means + standard error

Dry

mat

ter

Stre

ss T

oler

ance

Inde

x (%

)

Boron concentration (mg L-1)

Fig. 4.10: Effect of boron foliar spray on dry matter stress tolerance index (DMSI) of maize (Zea mays L.) seedlings under deficit moisture supply. Values are means + standard error

51

Table 4.4: Analysis of variance (ANOVA) of plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry matter stress tolerance index (DMSI) in two maize (Zea mays L.) hybrids exposed to boron foliar spray under drought stress

SOVa DFc PHSI (%) RLSI (%) DMSI (%)

Boron (B) 3 383.954*** 990.900*** 272.756***

Hybrids (H) 1 123.896** 176.855* 141.651**

B × H 3 95.203** 25.227NS 2.027NS

Error 14 5.531 23.513 7.628

CVb3.12 4.45 3.19

* = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

52

4.3.4 Total Fresh Biomass (g plant-1)

The significant (P≤0.001) difference was noted between water stress (W) levels for

this variable (Table 4.5). Drought treatment (30% WHC) considerably reduced the biomass

accumulations of both cultivars by 32% as compared to normal water supply (100% WHC).

Significant (P< 0.001) difference was recorded among boron rates (B) for biomass

accumulation in both maize cultivars. Foliar exposure of B (4 mg L -1) contributed

significantly higher biomass production (47%) in maize seedlings over water spray, but it

was statistically at par with 2 mg L-1 B. Plants treated with B at 2 and 6 mg L -1 maintained

36% and 29% higher biomass, respectively, compared with water spray. The maize hybrids

(H) differed significantly (P≤0.001) with respect to biomass accumulations. Maize hybrid

Dekalb-6525 maintained 22% more biomass than that of Yousafwala Hybrid (Table 4.5; Fig.

4.11).

Significant (P≤0.01) interaction (B × W) was recorded in seedling growth of maize in

terms of biomass accumulation (Table 4.5). The highest biomass accumulation (5.79 g plant-

1) was recorded in plants sprayed with B at 2 mg L-1 under normal water supply, whereas

minimum (2.63 g plant-1) in plants sprayed with water under drought stress. Two way

interaction (B × H) was significant (P≤0.05) for total biomass. The maximum biomass (5.17

g plant-1) accumulated by the cultivar Dekalb-6525 foliarly treated with B at 4 mg L -1,

whereas minimum (2.72 g plant_1) in Yousafwala Hybrid sprayed with water. The higher

order interaction (B × W × H) was also significant (P≤0.05) for this variable. Cultivar

Dekalb-6525 treated with B at 2 mg L-1 under normal water supply exhibited maximum

biomass (5.93 g plant-1), whereas minimum accumulation (2.24 g plant-1) was recorded in

Yousafwala Hybrid sprayed with distilled water under drought stress (Table 4.5; Fig. 4.11).

4.3.5 Shoot/Root Ratio

The shoot/root (S:R) ratio of maize plants did not influenced significantly (P< 0.001)

by different B levels and hybrids. The significant difference was recorded between water

stress levels for this seedling trait (Table 4.5). Maize plants under normal water supply

maintained considerably higher (42%) S:R ratio than that of drought stressed. Non-

significant interaction was recorded among all of the treatment combinations (Table 4.5; Fig.

4.12).

53

Tot

al F

resh

Bio

mas

s (g

plan

t-1)

Boron concentration (mg L-1)

Fig. 4.11: Effect of boron foliar spray on total fresh biomass of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Shoo

t/Roo

t Rat

io

Boron concentration (mg L-1)

Fig. 4.12: Effect of boron foliar spray on total fresh biomass of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

54

Table 4.5: Analysis of variance (ANOVA) of total fresh biomass (g plant -1) and shoot/root ratio in two maize (Zea mays L.) hybrids exposed to boron foliar spray under drought stress

SOVa DFcTotal biomass

(g plant-1)Shoot/root ratio

Water stress (W) 1 32.9064*** 2.83431***

Hybrids (H) 1 8.8468*** 0.01736NS

Boron (B) 3 5.3474*** 0.01771NS

W × H 1 0.0607NS 0.07008NS

W × B 3 1.0090** 0.00822NS

H × B 3 0.5165* 0.01436NS

W × H × B 3 0.5622* 0.01024NS

Error 30 0.1363 0.02269

CVb8.62 17.90

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

55

EXPERIMENT 4

4.4 Optimization of calcium concentration for spring maize The physiological indices and seedling traits of drought sensitive (Yousafwala

Hybrid) and drought-tolerant (Dekalb-6525) maize hybrids were studied in a wire-house

experiment to optimize the best rate of foliar calcium (Ca2+) under drought conditions. The

results of the experiment are given below:

4.4.1 Plant Height Stress Tolerance Index (PHSI)

Shoot length of normal and drought stressed seedlings was used to calculate plant

height stress tolerance index (PHSI). Different foliar applied Ca2+ rates exhibited significant

(P≤0.01) difference for PHSI (Table 4.6). Foliar treatment of Ca2+ at 40 mg L-1 gave

significantly higher PHSI value (82%), whereas the other Ca2+ rates did not show the

significant effect when compared with untreated control. A highly significant (P≤0.001)

difference was also noted for hybrids. The hybrid Dekalb-6525 maintained considerably

higher PHSI value (83%) as compared to Yousafwala Hybrid (Fig. 4.13). The non-significant

interaction was noted among Ca2+ rates (Ca2+) and hybrids (H) (Table 4.6).

4.4.2 Root Length Stress Tolerance Index (RLSI)

Analysis of variance showed highly significant (P≤0.001) variation between different

Ca2+ rates for RLSI (Table 4.6). Maximum RLSI (119%) was recorded in maize seedlings

treated with 40 mg L-1 Ca2+, while the 20 and 60 mg L-1 gave the statistically equal RLSI

values as in water spray. A significant (P≤0.05) variation was recorded for hybrids. The

significantly higher RLSI value (110%) was observed in Dekalb-6525 (Fig. 4.14), however

two way interactions (B × H) was found to be non-significant for RLSI (Table 4.6).

4.4.3 Dry Matter Stress Tolerance Index (DMSI)

Data regarding DMSI indicated highly significant (P≤0.001) difference among

different foliar applied Ca2+ rates (Table 4.6). Application of Ca2+ at 40 mg L-1 resulted

significantly higher DMSI value (114%), whereas minimum (89%) was recorded in plants

sprayed with water (control). Applications of Ca2+ at 20 and 60 mg L-1 significantly improved

DMSI as compared with no Ca2+ supply. A highly significant (P≤0.001) difference was also

recorded between hybrids for DMSI. The maize hybrid Dekalb-6525 maintained significantly

higher DMSI than that of Yousafwala Hybrid (Fig. 4.15).

56

Plan

t Hei

ght S

tres

s Tol

eran

ce In

dex

(%)

Calcium concentration (mg L-1)

Fig. 4.13: Effect of calcium foliar spray on plant height stress tolerance index (PHSI) of maize (Zea mays L.) seedlings under deficit moisture supply. Values are means + standard error

Roo

t len

gth

Stre

ss T

oler

ance

Inde

x

Calcium concentration (mg L-1)

Fig. 4.14: Effect of calcium foliar spray on root length stress tolerance index (RLSI) of maize (Zea mays L.) seedlings under deficit moisture supply. Values are means + standard error

57

Dry

Mat

ter

Stre

ss T

oler

ance

Inde

x (%

)

Calcium concentration (mg L-1)

Fig. 4.15: Effect of calcium foliar spray on dry matter stress tolerance index (DMSI) of maize (Zea mays L.) seedlings under deficit moisture supply. Values are means + standard error

Table 4.6: Analysis of variance (ANOVA) of plant height stress tolerance index (PHSI), root length stress tolerance index (RLSI) and dry matter stress tolerance index (DMSI) in two maize (Zea mays L.) hybrids exposed to calcium foliar spray under drought stress

SOVa DFc PHSI (%) RLSI (%) DMSI (%)

Calcium (Ca) 3 103.039** 405.016*** 644.96***

Hybrids (H) 1 661.802*** 161.915* 7764.84***

Ca × H 3 24.329NS 8.880NS 43.56NS

Error 14 17.600 18.668 31.13

CVb5.45 4.01 5.49

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

58

4.3.4 Total Fresh Biomass (g plant-1)

The biomass accumulation of plants was influenced significantly (P< 0.001) by

drought treatment (Table 4.7). The notable reduction in fresh biomass (31%) was recorded by

drought treatment (Fig. 4.16). Foliar supply of calcium (Ca2+) significantly (P< 0.001)

affected the seedlings growth of both genotypes. Plants exposed to Ca2+ at 40 mg L-1

significantly improved the biomass by 37% as compared to untreated control. Applications of

Ca2+ at 20 and 60 mg L-1 also significantly increased the accumulation of biomass by 20%

and 16% respectively, as compared with Ca2+ deficits. The two way interaction (W × Ca) was

also found to be significant (P< 0.05) for this variable. Plants exposed to Ca2+ at 40 mg L-1

under normal water supply maintained maximum biomass (12.45 g plant -1), while minimum

(6.25 g plant-1) was observed in untreated drought stressed plants. Foliar exposure of Ca2+ (40

mg L-1) considerably increased the seedlings growth (28%) of maize under water-deficit

conditions (Table 4.7; Fig. 4.16).

Non-significant interaction was recorded between hybrids in terms of biomass

accumulation, whereas significant (P< 0.01) interaction was observed between water stress

levels (W) and hybrids (H) (Table 4.7). Maximum biomass accumulation (10.92 g plant-1)

was recorded in Yousafwala Hybrid grown under normal water supply and minimum (6.79 g

plant-1) in similar hybrid under drought conditions (Fig. 4.16).

4.3.5 Shoot/Root Ratio

The shoot/root (S:R) ratio of normal and drought stressed plants was influenced

significantly (P< 0.001) and reduced by 18% in drought treatment (Table 4.7; Fig. 4.17).

Plants exposed to Ca2+ showed significant (P< 0.01) difference in S:R ratio. Application of

Ca2+ at 40 mg L-1 notably increased the S:R ratio (15%) as compared to untreated control.

Both cultivars differed significantly (P< 0.05) for S:R ratio and higher was observed in

Dekalb-6525 as compared to Yousafwala Hybrid. The two and three way-interactions were

found to be significant (P< 0.05) among all of the treatment combinations (Table 4.7).

Yousafwala Hybrid exposed to Ca2+ (20 mg L-1) under normal water supply maintained

considerably higher S:R ratio (1.05) which was statistically at par with both cultivars foliarly

treated with Ca2+ under normal water supply. Minimum S:R was recorded in Yousafwala

Hybrid exposed to Ca2+ (40 mg L-1) under water-limitations, whereas it was statistically at par

with control and other Ca2+ rates applied under drought stress (Fig. 4.17).

59

Tot

al F

resh

Bio

mas

s (g

plan

t-1)

Calcium concentration (mg L-1)

Fig. 4.16: Effect of calcium foliar spray on total fresh biomass of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

60

Shoo

t/Roo

t Rat

io

Calcium concentration (mg L-1)

Fig. 4.17: Effect of calcium foliar spray on shoot/root ratio of maize (Zea mays L.) seedlings under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Table 4.7: Analysis of variance (ANOVA) of total fresh biomass (g plant-1) and shoot/root ratio in two maize (Zea mays L.) hybrids exposed to calcium foliar spray under drought stress

SOVa DFc Total biomass (g plant-1) Shoot / root ratio

Water stress (W) 1 121.778*** 0.31183***

Hybrids (H) 1 0.040NS 0.03873*

Calcium (Ca) 3 15.185*** 0.03296**

W × H 1 10.721** 0.11883**

W × Ca 3 2.230* 0.02155*

H × Ca 3 1.301NS 0.01988*

W × H × Ca 3 0.544NS 0.01923*

Error 30 0.556 0.00635

CVb 8.45 9.50

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedomEXPERIMENT 5

61

4.5 Foliar-applied boron and calcium ameliorates drought-induced physiological and biochemical changes in maize

This experiment was conducted under wire-house (pot trial) using selected drought

tolerant (Dekalb-6525) and sensitive (Yousafwala) maize hybrids. For this purpose both

hybrids were grown under 30% and 100% water-holding capacity and the best rates of boron

and calcium selected from previous experiments, were foliarly sprayed individually and in

combination at ten leaf (V10) and tasselling (VT) stages.

4.5.1 Leaf Water Relations

4.5.1.1 Water Potential (-MPa)

Data regarding water potential (Ψw) indicated highly significant (P≤0.001) variation

between normal (100% WHC) and drought stressed (30% WHC) maize plants (Table 4.8).

Drought treatment considerably reduced the water potential (Ψw) by 26% as compared to

normal conditions (Fig. 4.18). A highly significant (P< 0.001) variation was also recorded by

foliar applied B and Ca2+ rates (F). Combined application of B (4 mg L-1) and Ca2+ (40 mg L-

1) significantly increased the Ψw by 27%, however, the individually applied B and Ca2+

increased the Ψw by 13% and 15%, respectively, compared with untreated control (Fig.

4.18). The substantial (P≤0.01) variation was recorded for Ψw in both hybrids, irrespective of

foliar treatment and watering-regimes. The drought-tolerant maize hybrid (Dekalb-6525)

conserved significantly higher Ψw (6%) than that of the drought-sensitive Yousafwala

Hybrid (Table 4.8; Fig. 4.18).

A highly significant (P≤0.001) interaction was recorded for water potential among

water stress levels (W) and foliar applied nutrients (F) (Table 4.8). Combined application of

B and Ca2+ under normal water supply maximally improved the Ψw (44%) as compared to

drought stressed plants sprayed with water. The lowest amount of Ψw (78%) was recorded in

leaves of drought-stressed untreated control plants (Fig. 4.18).

4.5.1.2 Osmotic Potential (-MPa)

The normal (100% WHC) and drought stressed (30% WHC) maize plants differed

significantly (P≤0.001) with respect to osmotic potential (Ψs) (Table 4.8). Considerably

lower Ψs (17%) was recorded under drought stress as compared to normal water supply.

Foliar treatment of B and Ca2+ significantly (P≤0.001) influenced the Ψs, irrespective of

watering-regimes. Application of B, Ca2+ and B+Ca2+ significantly reduced the Ψs by 9%,

62

13% and 14% respectively, as compared to untreated control (Fig. 4.19). Significant

(P≤0.01) difference was also recorded in hybrids for Ψs. Cultivar Dekalb-6525 maintained

significantly lower Ψs (5%) than that of Yousafwala Hybrid (Fig. 4.19).

Analysis of variance showed non-significant interaction among all of the treatment

combinations (Table 4.8).

4.5.1.3 Turgor Potential (MPa)

Analysis of variance showed non-significant (P≤0.001) difference in normal and

drought stressed plants (W) for turgor potential (Ψp), whereas a highly significant (P≤0.001)

difference was recorded in plants foliarly treated with B and Ca2+ (F) (Table 4.8; Fig. 4.20).

Plants treated with B, Ca2+ and their combination markedly improved the Ψp by 54%, 76%

and 106% respectively, as compared to untreated control. Maize cultivars differed

significantly (P≤0.01) with respect to turgor potential and significantly higher Ψp (19%) was

maintained by the cultivar Dekalb-6525 than that of Yousafwala Hybrid (Fig. 4.20).

A highly significant (P≤0.001) interaction (W × F) was recorded for this variable

(Table 4.8). Foliar treatment of B, Ca2+ and their combination under moisture deficit

conditions markedly improved Ψp by 220%, 175% and 141% respectively as compared to

drought-stressed plants sprayed with water (Fig. 4.20).

4.5.1.4 Relative Water Contents (%)

Data about relative water contents (RWC) indicated a highly significant (P≤0.001)

difference in normal and drought stressed plants (Table 4.8). Drought treatment considerably

reduced the leaf RWC of both cultivars by 14% as compared to normal water supply,

irrespective of foliar nutrient supply. A highly significant (P≤0.001) difference was also

observed for RWC under optimum B and Ca2+ supplies. B in conjunction with Ca2+

significantly improved the RWC by 20%, however, the individual application of B and Ca2+

substantially increased the RWC by 11% and 17%, respectively (Fig. 4.21).

Maize cultivars also differed significantly (P≤0.05) for this trait. Cultivar Dekalb-

6525 maintained slightly or significantly higher RWC (5%) than that of Yousafwala Hybrid

(Fig. 4.21). The non-significant interaction was observed among different treatment

combination (Table. 4.8).

63

Lea

f Wat

er P

oten

tial (

-MPa

)

Treatments

Fig. 4.18: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf water potential (-MPa) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Osm

otic

Pot

entia

l (-M

Pa)

Treatments

Fig. 4.19: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf osmotic potential (-MPa) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

64

Tur

gor

Pote

ntia

l (M

Pa)

Treatments

Fig. 4.20: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf turgor potential (MPa) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Rel

ativ

e W

ater

Con

tent

s (%

)

Treatments

Fig. 4.21: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on relative water contents (%) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

65

Table 4.8: Analysis of variance (ANOVA) of leaf water potential (ψw), osmotic potential (ψs), turgor potential (ψp) and relative water contents (%) in two maize (Zea mays L.) hybrids exposed to boron and calcium foliar spray under drought stress

SOVa DFc ψw

(‒MPa)ψs

(‒MPa)ψp

(MPa)RWC(%)

Foliar B and Ca2+

supply (F) 3 0.09228*** 0.06468*** 0.30122*** 503.63***

Hybrids (H) 1 0.02007** 0.04148** 0.11926*** 226.29*

Water stress (W) 1 0.29466*** 0.46276*** 0.01889NS 1843.63***

F × H 3 0.00038NS 0.00498NS 0.00283NS 0.07NS

F × W 3 0.03831*** 0.00893NS 0.08275*** 30.04NS

H × W 1 0.00115NS 0.00022NS 0.00236NS 1.31NS

F × H × W 3 0.00066NS 0.00186NS 0.00140NS 2.18NS

Error 30 0.00160 0.00475 0.00522 36.20

CVb5.92 5.56 12.85 7.33

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom4.5.2 Gas Exchange Characteristics

66

4.5.2.1 Net Photosynthetic Rate (µmol CO2 mˉ² sˉ¹)

Drought stress significantly (P≤0.001) influenced the net photosynthetic rate (Pn) of

both cultivars (Table 4.9). The plants exposed to drought treatment showed substantial

reduction in Pn (36%) as compared to normal conditions (Fig. 4.22). Foliar treatment of B

and Ca2+ significantly (P≤0.001) influenced the Pn. Foliar B, Ca2+ and their combination

substantially increased the Pn by 34%, 42%, and 49% respectively than that of control.

Maize cultivars also varied considerably (P≤0.05) with respect to Pn. The hybrid Dekalb-

6525 showed significantly higher Pn (6%) than that of Yousafwala Hybrid (Fig. 4.22).

The interaction was found to be non-significant among all of the treatment

combinations (Table 4.9).

4.5.2.2 Stomatal Conductance (µmol H2O mˉ² sˉ¹)

Stomatal conductance (gs) of both cultivars differed significantly (P≤0.001) under

normal and water-deficit conditions (Table 4.9). Drought stress considerably reduced the gs

by 38% as compared to normal water supply. A highly significant (P< 0.001) variation was

also observed for gs in both cultivars under foliar-applied B and Ca2+ rates regardless of

watering-regimes. Combined application of these nutrients considerably improved the gs by

54%, while the individual application of Ca2+ and B increased the gs by 42% and 26%,

respectively as compared to untreated control. Both cultivars did not differ significantly for

stomatal conductance (Fig. 4.23).

Non-significant interaction was recorded among all of the treatment combinations for

this variable (Table 4.9).

4.5.2.3 Transpiration Rate (mmol H2O m-2 sˉ¹)

Data concerning transpiration rate (E) indicated a highly significant (P< 0.001)

difference in normal and drought stressed plants (Table 4.9). Limited moisture supply

considerably reduced the E (32%) as compared to normal water supply. Foliar treatment of

B and Ca2+ significantly (P≤0.001) influenced the E of both cultivars. Plants supplemented

with B+Ca2+, Ca2+ and B markedly increased the E by 45%, 38% and 21% respectively as

compared to control (Fig. 4.24). The significant (P≤0.05) variation was also observed in

hybrids for transpiration rate. The drought-tolerant cultivar (Dekalb-6525) showed

noticeably higher E (8%) than that of drought sensitive Yousafwala Hybrid (Fig. 4.24). The

67

interactions were found to be non-significant among all of the treatment combinations for

transpiration rate (Table 4.9).N

et P

hoto

synt

hetic

Rat

e (P

n)

(μm

ol C

O2

m-2 s-1

)

Treatments

Fig. 4.22: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on net photosynthetic rate (μmol CO2 m-2 s-1) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Stom

atal

Con

duct

ance

(gs,)

(mol

m-2 s-1

)

Treatments

Fig. 4.23: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on stomatal conductance (µmol H2O mˉ² sˉ¹) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

68

Tra

nspi

ratio

n R

ate

(mm

ol H

2O m

-2 s-1

)

Treatments

Fig. 4.24: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on transpiration rate (mmol H2O m-2 s-1) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

69

Table 4.9: Analysis of variance (ANOVA) for net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (E) of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

SOVa DFc Pn(µ mol CO2 mˉ² sˉ¹)

gs

(µmol H2O mˉ² sˉ¹)E

(mmol H2O mˉ² sˉ¹)

Foliar B and Ca2+ supply (F)

3 125.003*** 0.03195*** 8.7608***

Hybrids (H) 1 14.092* 0.00399NS 2.2551*

Water stress (W) 1 867.796*** 0.22521*** 48.2623***

F × H 3 0.040NS 0.00002NS 0.0131NS

F × W 3 0.187NS 0.00244NS 0.1340NS

H × W 1 0.014NS 0.00039NS 0.4802NS

F × H × W 3 0.018NS 0.00002NS 0.0336NS

Error 30 2.068NS 0.00106NS 0.3247NS

CVb 7.35 11.24 10.67

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

70

4.5.3 Leaf Chlorophyll Contents (mg g-1 FW)

The normal (100% WHC) and drought-stressed (30% WHC) plants of both maize

cultivars differed significantly (P≤0.001) for leaf chlorophyll (Chl) contents (Table 4.10).

Chl contents viz. Chl a (Fig. 4.25), Chl b (Fig. 4.26) and Chl a+b (Fig. 4.27) contents

considerably decreased by 32%, 29% and 31% respectively by drought treatment.

Leaf Chl contents differed significantly (P< 0.01) by foliar B and Ca2+ supplies (Table

4.10). Combined foliar treatment of B and Ca2+ markedly improved Chl a (19%) Chl b (25%)

and Chl a+b (21%) contents as compared to untreated control. The individual treatment of

Ca2+ also considerably increased the leaf chlorophyll a, b and a+b contents by 13%, 20% and

15%, respectively as compared to control (Fig. 4.25-4.27).

Both maize cultivars also varied significantly for leaf chlorophyll contents (Table

4.10). The slightly but considerably higher Chl a (9%), Chl b (7%) and Chl a+b (8%)

contents were maintained by the cultivar Dekalb-6525 as compared to Yousafwala Hybrid

(Fig. 4.25-4.27).

Non-significant interactions were recorded for leaf chlorophyll contents (Table 4.10).

4.5.4 Total Carotenoids (mg g-1 FW)

Imposition of drought stress significantly (P< 0.001) influenced total carotenoids of

both maize cultivars differing in drought tolerance (Table 4.10). Plants exposed to drought

stress substantially reduced the total carotenoids (Car) by 18% as compared to normal water

supply (Fig. 4.28), irrespective of cultivars and foliar nutrient spray.

Foliar applications of B and Ca2+ significantly (P≤0.001) influenced the total

carotenoids (Table 4.10), regardless of watering regimes. Applications of B, Ca2+ and their

combination noticeably increased the contents of total carotenoids by 48%, 31% and 27%

respectively, as compared with untreated control (Fig. 4.28). Non-significant difference was

noted in hybrids for total carotenoids (Table 4.10).

The interactions W × F, W × H, F × H and W × F × H were found to be non-

significant among all of the treatment combinations for total carotenoids (Table 4.10).

71

Chl

orop

hyll

a C

onte

nts (

mg

g-1 F

W)

Treatments

Fig. 4.25: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll a contents (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Chl

orop

hyll

b C

onte

nts (

mg

g-1 F

W)

Treatments

Fig. 4.26: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll b contents (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

72

Chl

orop

hyll

a +

b C

onte

nts (

mg

g-1 F

W)

Treatments

Fig. 4.27: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll a + b contents (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Tot

al C

arot

enoi

ds (m

g g-1

FW

)

Treatments

Fig. 4.28: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on total carotenoids (mg g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

73

Table 4.10:Analysis of variance (ANOVA) for leaf chlorophyll (Chl a), (Chl b) and (Chl a + b) and total carotenoids (CAR) of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

SOVa DFc Chl a(mg g-1 FW)

Chl b(mg g-1 FW)

Chl a+b(mg g-1 FW)

CAR (mg g-1 FW)

Foliar B and Ca2+

supply (F) 3 0.13356** 0.03507** 0.26565*** 0.05716***

Hybrids (H) 1 0.08206* 0.01704* 0.17390** 0.00331NS

Water stress (W) 1 1.66381*** 0.44916*** 3.84192*** 0.09334***

F × H 3 0.0000034NS 0.00030NS 0.00032NS 0.00037NS

F × W 3 0.00907NS 0.00615NS 0.02366NS 0.00360NS

H × W 1 0.00000038NS 0.00653NS 0.00663NS 0.00124NS

F × H × W 3 0.00000020NS 0.00015NS 0.00015NS 0.00167NS

Error 30 0.01223 0.00381 0.01267 0.00196

CVb11.17 10.81 7.21 10.07

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom4.5.5 Osmolytes Accumulation

74

Accumulation of compatible osmolytes such as free proline, total soluble sugars and

free amino acids in leaves propose that osmotic adjustment may increase drought tolerance of

maize.

4.5.5.1 Leaf Proline Contents (µmol g-1 DW)

Leaf proline (PRO) contents of both genotypes significantly (P≤0.001) influenced by

drought stress (Table 4.11). The notably higher PRO (136%) contents were recorded in

plants under water-limitations (Fig. 4.29). A highly significant (P< 0.001) variation was also

recorded in plants treated with B and Ca2+ irrespective of watering-regimes. Considerably

higher PRO contents (23.27µmol g-1 DW) were recorded in control plants, whereas foliar

applied B, Ca2+ and their combinations markedly reduced its accumulation by 27%, 30% and

35% respectively (Fig. 4.29). Both cultivars did not differ significantly for PRO contents

(Table 4.11).

Two way interactions among water stress levels (W) and foliarly applied nutrients (F)

were found to be highly significant (P≤0.001) for leaf PRO contents (Table 4.11) and

maximum accumulation (35.9 µmol g-1 DW) was recorded in drought stressed plants sprayed

with water (Fig. 4.29).

4.5.5.2Total Soluble Proteins (mg g-1 DW)

Data regarding total soluble proteins (TSP) indicated a highly significant (P≤0.001)

effect of drought stress in both cultivars and reduced its accumulation by 34% as compared

to normal water supply (Fig. 4.30; Table 4.11). Foliar supplied B and Ca2+ significantly

(P≤0.05) influenced the TSP of both genotypes (Table 4.11), nonetheless of irrigation

regimes. Significantly higher accumulation of TSP (8.72 mg g-1 DW) was recorded in

untreated-control. Combined applications of B and Ca2+ noticeably degraded proteins by

13%, whereas their individual applications did not influence its concentration (Fig. 4.30.

Non-significant difference was recorded in both cultivars in terms of proteins synthesis

(Table 4.11). Significant (P≤0.05) interaction was between water stress levels (W) and foliar

applied B and Ca2+ (F) (Table 4.11). Maximum accumulation of TSP (9.96 mg g-1 DW) was

noted in untreated control plants grown under normal water supply and minimum (5.42 mg g-

1 DW) in plants supplied with B+Ca2+ under water-limitations (Fig. 4.30).

75

Lea

f Pro

line

Con

tent

s (µm

ol g

-1 D

W)

Treatments

Fig. 4.29: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf proline contents (µmol g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Tot

al S

olub

le P

rote

ins (

mg

g-1 D

W)

Treatments

Fig. 4.30: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on total soluble proteins (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

4.5.5.3 Total Free Amino Acids (mg g-1 DW)

76

Total free amino acids (TFA) significantly influenced by drought treatment (Table

4.11). Accumulations of TFA noticeably increased by 91% under drought stress compared to

normal water supply (Fig 4.31). A highly significant (P≤0.001) difference was also recorded

in plants foliarly treated with B and Ca2+, irrespective of cultivars and watering regimes

(Table 4.11). Applications of B+Ca2+ and Ca2+ notably increased the content of TFA by 25%

and 14% respectively, whereas B treatment reduced its accumulations by 13% as compared

to untreated control (Fig. 4.31). Both cultivars differed significantly (P≤0.01) for TFA and

more pronounced accumulation (8%) was noted in cultivar Dekalb-6525 than that of

Yousafwala Hybrid (Table 4.11; Fig. 4.31).

A highly significant (P≤0.001) interaction (W × F) was noted for this variable (Table

4.11). Maximum accumulation of TFA (1.76 mg g-1 DW) was recorded in plants treated with

B+Ca2+ under water-deficit conditions and minimum (0.71 mg g-1 DW) in normal plants

sprayed with water (Fig. 4.31).

4.5.5.4 Total Soluble Sugars (mg g-1 DW)

Accumulation of total soluble sugars (TSS) was influenced significantly (P≤0.001)

under limited-water supply and increased by 57% as compared to normal conditions (Table

4.11; Fig. 4.32). Foliar treatment of B and Ca2+ significantly (P≤0.001) affected the TSS

contents of both cultivars regardless of cultivars and watering-regimes (Table 4.11).

Application of Ca2+ and B+Ca2+ remarkably increased the accumulation of TSS by 31% and

37% respectively; as compared to untreated control. However, the control plants maintained

considerably higher TSS as compared to plants treated with boron (Fig. 4.32). A highly

significant (P≤0.001) difference was also recorded in both cultivars for this variable (Table

4.11). Accumulations of TSS was more distinct in cultivar Dekalb-6525 (50.0 mg g-1 DW)

than that of Yousafwala Hybrid (41.20 mg g-1 DW) (Fig. 4.32).

Two way interaction between water stress levels (W) and foliar applied nutrients (F)

was significant (P≤0.001) for this attribute (Table 4.11). Maximum accumulation of TSS

(71.96 mg g-1 DW) was recorded in plants treated with B+Ca2+ under water-deficit conditions

and minimum (33.12 mg g-1 DW) in well watered plants without nutrient spray (Fig. 4.32).

77

Tot

al F

ree

Am

ino

Aci

ds (m

g g-1

DW

)

Treatments

Fig. 4.31: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on total free amino acids (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Tot

al S

olub

le S

ugar

s (m

g g-1

DW

)

Treatments

Fig. 4.32: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on total soluble sugars (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

78

Table 4.11:Analysis of variance (ANOVA) for leaf proline (PRO) contents, total soluble proteins (TSP), total free amino acids (TFA) and total soluble sugars (TSS) of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

SOVa DFc PRO(µmol g-1 DW)

TSP(mg g-1 DW)

TFA(mg g-1 DW)

TSS(mg g-1 DW)

Foliar B and Ca2+

supply (F) 3 158.312*** 3.203* 0.34093*** 1200.12***

Hybrids (H) 1 2.06816NS 0.500NS 0.08030** 930.41***

Water stress (W) 1 2526.12*** 135.402*** 5.61679*** 4941.51***

F × H 3 4.90442NS 0.124NS 0.00013NS 6.41NS

F × W 3 167.681*** 2.294* 0.19629*** 666.50***

H × W 1 0.0000096NS 1.631NS 0.00023NS 1.36NS

F × H × W 3 3.08920NS 0.008NS 0.00008NS 2.91NS

Error 30 3.06658 0.770 0.00723 24.81

CVb9.76 10.85 7.76 10.92

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, c DF= Degree of freedomb CV-Coefficient of variation

79

4.5.6 Antioxidant Activities

The measurements of the activities of superoxide dismutase (SOD), catalase (CAT),

peroxidase (POD) and ascorbate peroxidase (APX) in leaf tissues are generally used to

evaluate antioxidant capability of plants under stress conditions.

4.5.6.1 Superoxide Dismutase Activity (Units min-1 g-1 FW)

Figure shows a highly significant (P≤0.001) variation in SOD activity under varying

water stress levels (Table 4.12). The substantially higher SOD activity (88%) was recorded in

plants exposed to drought stress than in normal water supply (Fig. 4.33). A highly significant

(P≤0.001) variation was also recorded in plants foliarly treated with B and Ca2+ (Table 4.12).

Activity of SOD was increased by foliar spray of B (10%), Ca2+ (25%) and their

combinations (35%) as compared to control. Both hybrids also varied significantly (P≤0.01)

for SOD activity (Table 4.12). The slightly higher SOD activity (8%) was observed in

cultivar Dekalb-6525 than in Yousafwala Hybrid. Significant (P≤0.01) interaction was

recorded among water stress levels (W) and hybrids (H) and considerably higher SOD

activity (11%) was observed in cultivar Dekalb-6525 as compared to Yousafwala Hybrid

under moisture-deficit conditions (Fig. 4.33).

A highly significant (P≤0.001) interaction was also recorded among water stress

levels (W) and foliar applied nutrients (F) (Table 4.12). Activity of SOD differed

significantly by foliar supplied nutrients under water stress but it remained unchanged in

normal water supply. The noticeably higher SOD activity was observed in plants supplied

with B (19%), Ca2+ (37%) and their combinations (53%) under water-deficit conditions (Fig.

4.33).

4.5.6.2 Catalase Activity (Units min-1 g-1 FW)

Activity of catalase (CAT) in both cultivars differed significantly (P≤0.001) by

drought treatment and its accumulations increased by 155% as compared to normal water

supply (Fig. 4.34; Table 4.12). Foliar sprays of mineral nutrients also significantly (P≤0.001)

influenced the CAT activity. Foliar treatment of B, Ca2+ and their combinations notably

increased the CAT activity by 11%, 19% and 28% respectively as compared to control (Fig.

4.34). Both cultivars also differed significantly (P≤0.001) with respect to CAT activity

80

(Table 4.12) and the drought-tolerant cultivar (Dekalb-6525) showed noticeable increase

(11%) in its activity than that of the drought-sensitive Yousafwala Hybrid (Fig. 4.34).

Two way interaction (W × H) was also found to be significant (P≤0.05) and the

cultivar Dekalb-6525 showed pronounced increase in CAT activity (11%) than that of

Yousafwala Hybrid under moisture deficit conditions, while both cultivars remained

unaffected under normal water supply (Table 4.12)

A highly significant (P≤0.001) interaction was recorded among foliar applied

nutrients (F) and water stress levels (W) (Table 4.12). Compared to control, foliar treatment

of B, Ca2+ and B+Ca2+ markedly improved CAT activity by 20%, 24% and 37%, respectively

under water limitations (Fig. 4.34).

4.5.6.3 Peroxidase Activity (Units min-1 g-1 FW)

The normal and drought-stressed plants varied significantly (P≤0.001) for peroxidase

(POD) activity (Table 4.12). Drought treatment markedly improved POD activity by 317%

than that of well watered supply. Activity of POD in both cultivars differed significantly

(P≤0.001) by foliar supplied B and Ca2+. Foliar treatment of B, Ca2+ and their combinations

distinctly increased POD activity by 24%, 24% and 36% respectively, as compared to

untreated-control. The POD activity also significantly (P≤0.01) changed in both cultivars

(Table 4.12) and more distinct increase (8%) was recorded in cultivar Dekalb-6525 than in

Yousafwala Hybrid (Fig. 4.35).

Two way interactions (W × F) were also found to be highly significant (P≤0.001)

(Table 4.12). The POD activity of drought-stressed plants sprayed with B, Ca2+ and their

combinations was substantially improved by 32%, 31% and 46% respectively as compared to

untreated control, whereas enzyme activity of normal plants did not change significantly by

foliar supplied nutrients (Fig. 4.35).

4.5.6.4 Ascorbate Peroxidase (ABA digested g-1 FW hr-1)

Imposition of drought stress significantly (P≤0.001) increased the ascorbate

peroxidase (APX) activity by 167% as compared to normal water supply (Table 4.12). A

highly significant (P≤0.001) difference was also noted in plants treated with mineral

nutrients. Foliar supply of B, Ca2+ and B+Ca2+ substantially increased the APX activity of

both cultivars by 32%, 33% and 42% respectively, with respect to control (Fig. 4.35).

Significant (P≤0.001) difference was also observed in both cultivars (Table 4.12) and the

81

hybrid Dekalb-6525 showed more pronounced increase in APX activity (38%) as compared

to Yousafwala Hybrid (Fig. 4.36). A highly significant (P≤0.001) interaction (W × F) was

also recorded for this antioxidant (Table 4.12). Application of B, Ca2+ and B+Ca2+ noticeably

increased the APX activity of both maize cultivars by 47%, 49% and 59% respectively, as

compared to control (Fig. 4.36).

4.5.6.4 Lipid peroxidation

Malondialdehyde (MDA) contents, an effective indicator of lipid peroxidation,

markedly (P≤0.001) increased under water-limitations (Fig. 4.37). Foliar supply of B, Ca2+

and their combinations noticeably (P≤0.001) reduced MDA accumulation by 19%, 21% and

23% in both cultivars under normal and water deficit conditions. The interactive effect of W

× F was significant (P≤0.001) and considerably lower MDA accumulation (28%) was

recorded in drought stressed plants supplemented with B+Ca2+ (Fig. 4.37; Table 4.12).

Yousafwala Hybrid showed more marked (P≤0.01) MDA accumulation (17%) than Dekalb-

6525. Significant (P≤0.01) interaction between hybrids (H) and water stress levels (W) was

recorded on MDA content and its accumulation was higher (22%) in Yousafwala Hybrid

than Dekalb-6525 under water-deficit conditions (Fig. 4.37; Table 4.12).

Supe

roxi

de D

ism

utas

e (S

OD

) Act

ivity

(Uni

ts m

in-1g-1

FW b

asis

)

Treatments

Fig. 4.33: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on superoxide dismutase activity (Unit min-1 g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

82

Cat

alas

e (C

AT

) Act

ivity

(U

nits

min

-1g-1

FW b

asis

)

Treatments

Fig. 4.34: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on catalase activity (Unit min-1 g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Pero

xida

se (P

OD

) Act

ivity

(U

nits

min

-1g-1

FW b

asis

)

Treatments

Fig. 4.35: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on peroxidase activity (Unit min-1 g-1 FW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

83

Asc

orba

te P

erox

idas

e (A

PX) A

ctiv

ity

(AB

A d

iges

ted

g-1 F

W h

r-1)

Treatments

Fig. 4.36: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on ascorbate peroxidase activity (ABA digested g-1 FW hr-1) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

MD

A C

onte

nt(n

mol

g-1 D

W)

Treatments

Fig. 4.37: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on malondialdehyde content (nmol g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

84

Table 4.12:Analysis of variance (ANOVA) for superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) activities and malondialdehyde (MDA) content in two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

SOVa DFcSOD CAT POD

APX(ABA

digested g-1 FW

hr-1)

MDAnmol g-1

DW(Units min-1 g-1 FW)

Foliar B and

Ca2+ supply

(F)3 8296*** 2379*** 4081*** 2.735*** 0.3540***

Hybrids (H) 1 2907** 2369*** 1395** 13.326*** 0.5275***

Water stress

(W) 1 174737*** 167563*** 389157*** 106.960*** 11.5583***

F × H 3 208NS 26NS 81NS 0.049NS 0.0013

F × W 3 5105*** 1503*** 3846*** 2.197*** 0.2309***

H × W 1 1453** 430* 208NS 11.879*** 0.3017**

F × H × W 3 435NS 18NS 20NS 0.037NS 0.0129

Error 30 159 57 161 0.114 0.0205

CVb6.36 5.57 8.63 10.31 10.42

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

85

4.5.7 Leaf Nutrient Concentrations

4.5.7.1 Leaf Boron Concentration (mg kg-1 DW)

Drought treatment significantly (P≤0.001) reduced the uptake of B (20%) with

respect to normal water supply. Leaf B concentration varied significantly (P≤0.001) by foliar

B and Ca2+ supplies. Foliar treatment of B+Ca2+ and B notably increased leaf B concentration

by 48% and 42% respectively, whereas the plants supplied with Ca2+ remained unchanged as

compared to control (Fig. 4.38; Table 4.13).

Both cultivars did not vary significantly for leaf B concentration. Two way

interactions (W × F) were found to be significant (P≤0.01) for this variable (Table 4.13).

Remarkably higher B uptake (16.19 mg kg-1 DW) was recorded in plants treated with B+Ca2+

under normal water supply, whereas minimal (8.62 mg kg-1 DW) in drought-stressed plants

sprayed with water (Fig. 4.38).

4.5.7.2 Leaf Calcium Concentrations (mg g-1 DW)

Data regarding leaf calcium (Ca2+) content indicated a highly significant (P≤0.001)

variation between normal (100% WHC) and water-deficit (30% WHC) plants (Table 4.13).

Drought stress markedly reduced the uptake of Ca2+ as compared to normal conditions. Foliar

applied B and Ca2+ significantly influenced the leaf Ca2+ concentration. Applications of

B+Ca2+ and Ca2+ significantly (P≤0.001) enhanced the leaf Ca2+ concentrations by 33% and

31%, respectively as compared to control. Both cultivars remained unchanged in terms of

leaf Ca2+ contents (Fig. 4.39; Table 4.13).

Significant (P≤0.05) interaction was recorded between water stress levels (W) and

foliar applied B and Ca2+ (F) for leaf Ca2+ concentrations (Table 4.13). Maximum Ca2+

contents (4.73 mg g-1 DW) was recorded in both cultivars treated with B+Ca2+ under water-

limitations and minimum (2.75 mg g-1 DW) in drought-stressed untreated control (Fig. 4.39).

86

Lea

f B

oron

Con

cent

ratio

ns (m

g kg

-1 D

W)

Treatments

Fig. 4.38: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf boron concentrations (mg kg-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

Lea

f Cal

cium

Con

cent

ratio

ns (m

g g-1

DW

)

Treatments

Fig. 4.39: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf calcium concentrations (mg g-1 DW) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions. Values are means + standard error

87

Table 4.13:Analysis of variance (ANOVA) for leaf boron and calcium concentrations of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under drought stress

SOVa DFcLeaf B

concentrations(mg g-1 DW)

Leaf Ca2+

concentrations(mg g-1 DW)

Foliar B and Ca2+ supply

(F) 3 88.646*** 4.20323***

Hybrids (H) 1 8.128NS 0.00819NS

Water stress (W) 1 105.403*** 9.98366***

F × H 3 0.263NS 0.01976NS

F × W 3 9.602** 0.27427*

H × W 1 0.114NS 0.02581NS

F × H × W 3 1.546NS 0.00557NS

Error 30 1.998 0.07700

CVb10.50 7.17

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

88

EXPERIMENT 6

4.6 Foliar-applied boron and calcium improves growth, yield and yield components of maize under normal and water deficit conditions 4.6.1 Growth Parameters

4.6.1.1 Leaf Dry Weight (g plant-1)

Imposition of drought stress significantly reduced the leaf dry weight (DW) by 22%

as compared to normal water supply. Significant (P≤0.001) difference was also observed in

both cultivars and the cultivar Dekalb-6525 showed more pronounced increase in leaf DW

(20%) as compared to Yousafwala Hybrid (Fig. 4.40; Table 4.14). Combined foliar treatment

of B and Ca2+ markedly improved the leaf DW by 40% as compared to untreated control;

however, their individual applications did not influence the leaf DW (Fig. 4.340). The two

and three way interactions were found to be non-significant (Table 4.14).

4.6.1.2 Shoot Dry Weight (g plant-1)

Data about shoot dry weight (DW) indicated a significant difference among water

stress levels and it was markedly reduced (22%) by limited-moisture supply. Both maize

cultivars also differed significantly (P≤0.01) with respect to shoot DW. The noticeably

higher shoot DW (19%) was maintained by the cultivar Dekalb-6525 than that of the

Yousafwala Hybrid. Applications of B, Ca2+ their combinations substantially improved the

shoot DW by 17%, 15% and 32% as compared to untreated-control. The interactions were

found to be non-significant among all of the treatment combinations for shoot dry weight

(Fig. 4.41; Table 4.14).

4.6.1.3 Tassel dry weight (g plant-1)

Data concerning tassel dry weight (DW) indicated a highly significant (P< 0.001)

difference in normal and drought stressed plants (Table 4.14). Limited moisture supply

considerably reduced the tassel DW (25%) as compared to normal water supply. Combined

applications of B and Ca2+ significantly increased the tassel DW by 28% as compared to

control; however it remains unaffected by their individual applications (Fig. 4.42). Both

hybrids did not change significantly for tassel dry weight. Two and three way interactions

were found to be non-significant for this variable (Table 4.14).

89

4.6.1.4 Silk dry weight (g plant-1)

Water limitations significantly reduced the silk dry weight of both cultivars by 38%

as compared to normal water supply (Table 4.14). Foliar sprays of B, Ca2+ and their

combinations notably increased the silk DW by 23%, 23% and 31% respectively, as

compared to untreated control. Both cultivars also varied significantly for silk DW (Fig.

4.43) and more pronounced increase (22%) was observed in cultivar Dekalb-6525 as

compared to Yousafwala Hybrid. Non-significant interactions were recorded among all of the

treatment combinations (Table 4.14).

4.6.1.5 Silk length (cm)

Data regarding silk length indicated a significant difference between normal and

drought stressed plants (Table 4.15). Drought treatment substantially reduced the silk length

by 29% as compared to normal water supply, irrespective of foliar nutrient supply and

cultivars. Silk length was influenced significantly by foliar nutrient supply, regardless of

cultivars and watering-regimes. Foliar supply of B and B+Ca2+ notably increased the silk

length by 21% and 34%, respectively as compared to control, while individual application of

Ca2+ did not influence it (Fig. 4.44).

4.6.1.6 Silk threads (Number ear-1)

Number of silk threads in both cultivars was significantly reduced (13%) by drought

treatment (Table 4.15). Foliar treatment of B, Ca2+ and their combinations markedly

improved the numbers of silk threads in an ear by 8%, 9% and 17% respectively, as

compared to untreated-control. Both cultivars also varied significantly in terms of silk

threads and noticeably higher (13%) was recorded in cultivar Dekalb-6525 as compared to

Yousafwala Hybrid (Fig. 4.45). Two way interaction was found to be significant (P≤0.05)

between water stress levels (W) and foliar treatment of mineral nutrient (F) (Table 4.15).

Maximum numbers of silk threads per ear (602) were recorded in plants supplied with

B+Ca2+ under normal water supply, whereas minimum (431) in untreated water-stressed

plants (Fig. 4.45).

4.6.1.7 Plant height (cm)

Plant height of both genotypes influenced significantly by drought stress and reduced

by 10% as compared to normal water supply (Fig. 4.46). Combined applications of B and

90

Ca2+ significantly improved the plant height (11%) of both genotypes; however it did not

change considerably by their individual applications as compared to control. Both genotypes

also remain unchanged with respect to plant height. The two and three way interactions were

found to be non-significant for this variable (Table 4.15).

Lea

f Dry

Wei

ght (

g pl

ant-1

)

Treatments

Fig. 4.40: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on leaf dry weight (g plant-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

Shoo

t Dry

Wei

ght (

g pl

ant-1

)

Treatments

Fig. 4.41: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on shoot dry weight (g plant-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

91

Tas

sel D

ry W

eigh

t (g

plan

t-1)

Treatments

Fig. 4.42: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on tassel dry weight (g plant-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

Silk

Dry

Wei

ght (

g pl

ant-1

)

Treatments

Fig. 4.43: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on silk dry weight (g plant-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

92

Silk

Len

gth

(cm

)

Treatments

Fig. 4.44: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on silk length (cm) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

Silk

Thr

eads

(num

ber

ear-1

)

Treatments

Fig. 4.45: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on silk threads (number ear-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

93

Plan

t hei

ght (

cm)

Treatments

Fig. 4.46: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on plant height (cm) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

94

Table 4.14:Analysis of variance (ANOVA) for leaf, shoot, tassel and silk dry weight of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under water-deficit conditions

SOVa DFc

Leaf DryWeight

Shoot Dry Weight

Tassel Dry Weight

Silk DryWeight

g plant-1

Water stress (W) 1 406.178** 25902.4** 128.758* 3.46903*

Error-Ι 2 1.903NS 9.21000NS 2.103 0.06040

Hybrids (H) 1 215.011*** 4446.75*** 11.043NS 0.63296**

W × H 1 0.024NS 1.370E-28NS 2.723NS 0.01092NS

Error-ΙΙ 4 0.238 5.653E-29NS 4.827 0.00845

Foliar B and Ca2+ supply (F) 3 134.624*** 5105.31*** 15.571** 0.20599***

W × F 3 4.744NS 90.7500NS 4.610NS 0.04063NS

H × F 3 0.223NS 3.748E-29NS 0.328NS 0.00229NS

W × H × F 3 0.248NS 6.019E-28NS 0.142NS 0.00256NS

Error- ΙΙΙ 24 10.548 291.630 2.996 0.01647

CVb 13.79 9.13 15.27 11.15

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variationb CV-Coefficient of variationc DF= Degree of freedom

95

Table 4.15:Analysis of variance (ANOVA) for silk length, number of silk threads per ear, and plant height of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under water-deficit conditions

SOVa DFc Silk Length(cm)

Silk Threads(Number ear-1)

Plant Height(cm)

Water stress (W) 1 198.697* 61261.2* 7467.28*

Error-Ι 2 2.750 2995.2 156.547

Hybrids (H) 1 0.099NS 46808.8** 329.077NS

W × H 1 0.350NS 130.5NS 1.271E-03NS

Error-ΙΙ 4 0.047 403.4 74.2035

Foliar B and Ca2+

supply (F) 3 28.679** 14034.0*** 1338.30*

W × F 3 4.406NS 1634.2* 0.04051NS

H × F 3 0.023NS 400.5NS 1.271E-03NS

W × H × F 3 0.035NS 1.8NS 1.271E-03NS

Error- ΙΙΙ 24 2.921 415.7 320.177

CVb14.14 3.89 7.84

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

96

4.6.2 Yield and Yield Components

Drought stress significantly reduced the yield and yield components (Table 4.16).

Foliar exposure of B, Ca2+ and their combinations under such circumstances improved the

maize yield and related traits (Fig. 4.47-4.51).

4.6.2.1 Number of grains per ear

Numbers of grains per cob (NGPC) were influenced significantly (P≤0.001) by

limited moisture supply and decreased by 14%, as compared to normal water supply (Fig.

4.47; Table 4.16). The NGPC in both cultivars were significantly (P≤0.001) changed by

foliar supplied nutrients. Foliar sprays of B, Ca2+ and their combinations markedly (P≤0.05)

increased the NGPC by 9%, 10% and 19% respectively, compared with untreated control.

The significant (P≤0.001) difference was recorded between both cultivars for this variable

(Table 4.16). Significantly higher NGPC (13%) was observed in cultivar Dekalb-6525 as

compared to Yousafwala Hybrid (Fig. 4.47).

Two way interactions were found to be significant (P≤0.01) between hybrids (H) and

foliar supplies of nutrients (F) (Table 4.16). Combined foliar treatment of B and Ca2+

maximally increased the NGPC (585) in cultivar Dekalb-6525, whereas minimum (441) was

recorded in untreated Yousafwala Hybrid (Fig. 4.47).

4.6.2.2 Thousand-grain weight (g)

Data regarding thousand-grain weight (1000-GW) of both cultivars indicated a

significant (P≤0.001) difference between watering-regimes (Table 4.16). Limited moisture

supply substantially reduced the 1000-GW by 17% as compared to normal water supply.

Thousand-grain weight differed significantly (P≤0.001) by foliar B and Ca2+ supplies (Table

4.16). Compared to control, foliar treatment of B, Ca2+ and their combinations markedly

improved the 1000-GW by 9%, 14% and 25% respectively, regardless of watering-regimes

(Fig. 4.48).

Significant (P≤0.05) interaction (W × F) was recorded for this variable and

considerably higher 1000-GW (36%) was recorded in plants exposed to B+Ca2+ under

moisture stress as compared to control. Both cultivars also varied significantly (P≤0.01) with

respect to 1000-GW and slight increase was recorded in drought-tolerant cultivar Dekalb-

6525 as compared to drought-sensitive Yousafwala Hybrid (Fig. 4.48; Table 4.16).

97

Num

ber

of G

rain

s Ear

-1

Treatments

Fig. 4.47: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on numbers of grains per ear of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

Tho

usan

d-G

rain

Wei

ght (

g)

Treatments

Fig. 4.48: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on thousand-grain weight (g) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

98

4.6.2.3 Biological yield (t ha-1)

Imposition of drought stress significantly (P≤0.001) influenced the biological yield

(Table 4.16). Limited water supply substantially reduced the biological yield of both cultivars

by 19% as compared to normal water supply, irrespective of foliar-applied nutrients.

Combined foliar treatment of B and Ca2+ significantly (P≤0.05) influenced the biological

yield and increased by 11% as compared to control. Both cultivars also varied significantly

(P≤0.05) with respect to biological yield and slight increase (6%) was observed in cultivar

Dekalb-6525 as compared to Yousafwala Hybrid (Fig. 4.49).

The interaction was found to be non-significant for this variable (Table 4.16).

4.6.2.4 Grain yield (t ha-1)

Data about grain yield (GY) indicated a highly significant (P≤0.001) difference in

normal and water stressed plants (Table 4.16). Water limitations significantly reduced the

GY of both cultivars by 22% as compared to normal water supply. A highly significant

(P≤0.001) difference was also observed in GY of both cultivars supplied with mineral

nutrients. Foliar sprays of B, Ca2+ and their combinations significantly improved the yield by

11%, 15% and 19% respectively, than untreated control. Both cultivars also changed

significantly (P≤0.001) for this trait (Table 4.16) and the cultivar Dekalb-6525 exhibited

considerably higher GY (20%) than Yousafwala Hybrid (Fig. 4.50).

The two way interactions was found to be significant (P≤0.01) between water stress

(W) and hybrids (H), and maximum increase in yield (51%) was recorded in Dekalb-6525

under normal water supply as compared to Yousafwala Hybrid grown under water-deficit

conditions (Fig. 4.50; Table 4.16).

4.6.2.5 Harvest index (%)

Harvest index of both cultivars did not differ significantly by water stress levels and

foliar-applied mineral nutrients (Table 4.16). Significant (P≤0.01) difference was observed

between hybrids for harvest index and more pronounced increase was noted in drought

tolerant genotype (Dekalb-6525) than drought sensitive one (Yousafwala Hybrid) (Fig. 4.51).

The two and three way interactions were found to be non-significant for this variable

(Table 4.16).

99

Bio

logi

cal Y

ield

(t h

a-1)

Treatments

Fig. 4.49: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on biological yield (t ha-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

Gra

in Y

ield

(t h

a-1)

Treatments

Fig. 4.50: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on grain yield (t ha-1) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

100

Har

vest

Inde

x (%

)

Treatments

Fig. 4.51: Effect of foliar sprays of B (4 mg L-1), Ca2+ (4 mg L-1) and their combinations (4+40 mg L-1) on harvest index (%) of two maize (Zea mays L.) hybrids under normal and water stress conditions. Values are means + standard error

101

Table 4.16:Analysis of variance (ANOVA) for number of grains per ear, thousand grain weight, biological yield, grain yield and harvest index of two maize (Zea mays L.) hybrids exposed to boron and calcium foliar sprays under water deficit conditions

SOVa DFcNumber of grains per ear

1000-grain Weight

(g)

Biological Yield

(t ha-1)

Grain Yield(t ha-1)

Harvest Index (%)

Water stress (W) 1 68359.2* 20716.8* 202.033* 19.2451** 10.009NS

Error-Ι 2 3477.6 355.7 5.499 0.0261 5.865

Hybrids (H) 1 45792.2** 2948.5** 16.000* 10.2962** 111.085**

W × H 1 153.6NS 400.2NS 0.059NS 1.0733* 13.676NS

Error-ΙΙ 4 452.5 129.3 1.319 0.0667 2.551

Foliar B and Ca2+ supply (F) 3 15752.0*** 5137.6*** 10.204** 1.8424*** 8.361NS

W × F 3 1652.9* 472.8NS 0.506NS 0.1729NS 2.676NS

H × F 3 534.4NS 433.8NS 0.080NS 0.1065NS 1.940NS

W × H × F 3 4.9NS 164.9NS 0.119NS 0.0410NS 2.162NS

Error- ΙΙΙ 24 407.7 178.8 2.047 0.1466 7.613

CVb 3.96 6.19 7.27 7.35 10.43

NS= non-significant, * = Significant at P≤0.05, ** = Significant at P≤0.01, *** = Significant at P≤0.001,a SOV- Source of variation, b CV-Coefficient of variation c DF= Degree of freedom

102

CHAPTER 5 DISCUSSION

5.1 Laboratory Experiments

The most effective prerequisite for drought tolerance concerning selection and

breeding depends on the degree of genetic variability occurring in gene pool of that crop

species. Therefore, in order to develop a selection approach for stress resistance in crop

species, considerate of genetic variation is of primary importance especially at initial growth

phases (Larsen and Bibby, 2004). The results of the study revealed that considerable genetic

variability exist among maize genotypes (Table 4.1 and 4.2). The adverse effects on seed

germination attributes of all tested maize cultivars were noted due to increasing polyethylene

glycol (PEG6000) induced osmotic stress (Fig. 4.1-4.4) that might be due to reduction in water

potential (Kulkarni and Deshpande, 2007). In sight of certain prior findings in maize (Ahmad

et al., 2015; Khodarahmpour, 2013; Mohammadkhani and Heidari, 2008) it has been strong

inference now that osmotic barrier reduced the steady water uptake during imbibition period

of seed germination due to stress brought by osmotica used externally that resulted in

impaired enzyme activities and decreased energy supply (Okcu et al., 2005; Taiz and Zeiger,

2010). In our study, PEG-6000 induced water stress (-0.8 MPa) markedly reduced germination

percentage (Fig. 4.1) and promptness index (Fig. 4.3) as well as increased mean germination

time in Yousafwala Hybrid, however the cultivar Dekalb-6525 even performed better for

these traits at this highest osmotic stress (Fig. 4.2). The variations in germination attributes of

tested maize cultivars (Fig. 4.1-4.4) are in accordance with the findings of Shamim (2013),

who reported that germination responses varies within the similar species or even within

dissimilar species as seeds of more tolerant species may germinate well under limited water

supply while sensitive species unable to germinate. Various reports indicate that germination

stress tolerance index (GSI) can be used as screening criteria for abiotic stress tolerance

(Ahmad, et al. 2015; Saensee et al., 2012). In our study, GSI of all tested maize cultivars

were gradually decreased by increase of water stress and cultivar Dekalb-6525 performed

better even at osmotic stress of -0.6 MPa, whereas Yousafwala Hybrid showed poor

performance (Fig. 4.4). The reduction in GSI of different maize cultivars under PEG-6000

103

induced water stress and considerably improved performance of cultivar Dekalb-6525 under

such stress has also been reported (Ahmad et al., 2015).

Drought stress significantly reduced the plant height stress tolerance index (PHSI),

root length stress tolerance index (RLSI), and dry matter stress tolerance index (DMSI) in

maize (Fig. 4.5-4.7) that might be due to reduction in plant growth rate and biomass

accumulation (Li et al., 2009). The decline in shoot-length under water-deficit conditions

(Fig. 4.5) might be due to reduction in cell expansion, which eventually reduced the plant

height (Okcu et al., 2005). The improved seedling growth in terms of greater root length and

dry matter production can be used as key selection criterion for screening genotypes against

drought stress (Qayyum et al., 2012). Among tested cultivars, drought-tolerant cultivar

Dekalb-6525 showed higher values for PHSI (Fig. 4.5), RLSI (4.6) and DMSI (4.7), whereas

drought-sensitive Yousafwala Hybrid showed poor performance in terms of these indices

(Fig. 4.5-4.7). The morphological damage and growth inhibition cause by water-limitation is

imparted by hormonal signals such as abscisic acid (ABA) and indole-3-acetic acid (IAA)

with rapid, quick and higher increase pragmatic in sensitive cultivars as compared to tolerant

ones (De-Micco and Aronne, 2012; Jiang et al., 2012, 2013). The variations in morphological

aspects are the eventual elements of stress effects on plants (Farooq et al., 2009; Jaleel et al.,

2009) and various plants of similar species can be categorized as drought tolerant on the

basis of improved root growth (Bruce et al., 2002), shoot length (Witt et al., 2012) and

biomass accumulations (Zhang et al., 2014). Final germination percentage and germination

time associate with root length and how greatly biomass is accumulated per unit area (Okcu

et al., 2005).

5.2 Wire-house Experiments

5.2.1 Seedling Growth

The results designate that maize seedling growth which is suppressed by exposure to

drought stress (Fig. 4.8-4.17) is due to hampering nutrient acquirement by roots from dry

medium (Karim and Rahman, 2015). Boron (B) and calcium (Ca2+) bioavailability hampered

under water-deficit conditions because of their reduced mobility from soil to roots by mass

flow (Brown et al., 2006; Mattiello et al., 2009). Few reports indicate that plants react

differently to water deficit conditions in presence of B as it being able to alleviate the adverse 104

effects of drought, deferring dehydration in plants (Mottonen et al., 2005; Waraich et al.,

2011), whereas Ca2+ has also been displayed to alleviate the harmful effects of drought on

plants (Jaleel et al., 2007a), as it is involved in signaling anti-drought responses (Shao et al.,

2008). Present study confirm the hypothesis that foliar fertilization of B and Ca2+ is effective

in improving maize growth through maintenance of water balance, photosynthesis, osmolyte

accumulation, antioxidant activities as well as reduction in MDA accumulation under water-

deficit conditions (Fig. 4.8-4.38).

Two selected maize hybrids i.e. Dekalb-6525 and Yousafwala Hybrid was used for

further study. Plant height (PHSI), root length (RLSI) and dry matter stress tolerance index

(DMSI) was equally susceptible to low B supply. However, application of B (4 mg L-1)

considerably improved these indices under water-deficit conditions. Root surface area is an

important measure for plants to grow under water-deficit conditions. A longer root length

(RLSI) of plants supplied with B leads to a greater ability of plants to water uptake with a

lesser transpiration area that could contribute in the maintenance of water status of drought-

stressed plants as has been noted in Eucalyptus urophylla (Hodecker et al., 2014) and

Brassica rapa (Hajiboland and Farhanghi, 2011). Drought treatment caused notable

reduction in B uptake that might be due to compact mass flow and diffusion rate and this

could be experienced not merely under short but also under sufficient B supply (Hajiboland

and Farhanghi, 2011). Consequently, B-deficiency produced substantial reduction in seedling

growth of maize that might be due to cessation of root growth and leaf expansion (Marschner

and Marschner, 2012). In addition, under B deficiency increase of hexoses activity suppress

the manifestation of genes that program for photosynthetic enzymes governing to reduced

photosynthetic rate. This could be a cause for reduced accumulation of biomass under B-

deficient conditions (Moore et al., 2003; Pandey et al., 2009). In current study, B deficiency

caused disturbance in gas exchange characteristics, water relations, photosynthetic pigments,

osmolyte content and antioxidant system through accelerating lipid peroxidation that might

also be involved in reduction of plant growth. Higher B concentration (6 mg L -1) showed the

adverse effects on seedling growth of corn that might be due to its toxic effect that caused

impaired root cell division and overall reduction in biomass accumulation (Nable et al.,

1997; Ben-Gal, 2007). Drought-tolerant cultivar, Dekalb-6525 showed the improved seedling

105

growth than drought-sensitive Yousafwala Hybrid (Table 1) that might be due to its greater

genotypic tolerance to drought stress (Farhad et al., 2011)

Dry matter production is thought to be important trait for estimation of tolerance in

plants under stress conditions. In current study, seedling dry weight (DMSI) was significantly

improved by foliar treatment of Ca2+ (40 mg L-1) as compared to control (Fig. 1c, 2a, 3). The

other plant stress tolerance indices namely PHSI and RLSI were also improved by

application of Ca2+ as compared to control (Fig. 4.13 and 4.14). However, water stress

substantially reduced the calcium uptake in above-ground portions of plants (shoots and

leaves) as well as in roots that could be due to decline in transpiration rate (Brown et al.,

2006; Hu et al., 2008). Additionally, root tip area is a major site of calcium entry and this

zone of Ca2+ uptake is likely to be reduced under water deficit conditions (Raza et al., 2013)

and brings considerable inhibition in seedling growth of maize. Therefore, continuous supply

of Ca2+ is required by plants for vigorous leaves and roots development and overall canopy

growth (Del-Amor and Marcelis, 2003). These results suggest that Ca2+ plays an imperative

role in mitigating damage to maize provoked by drought as variations in morphological

aspects are the eventual elements of stress effects on plants (Farooq et al., 2009; Jaleel et al.,

2009). Similar mitigation activity by employing Ca2+ has been described for different plant

species under varied stress conditions (Shoresh et al., 2011; Siddiqui et al., 2012; Xu, et al.

2013). Plants exposed to Ca2+ conferred improved tolerance to drought (Issam et al., 2012;

Xu, et al. 2013) by augmenting membranes integrity (Guimaraes et al., 2011; Ma et al.,

2005). Limited water supply significantly reduced the S:R ratio of both maize cultivars

compared with normal water supply (Fig. 4.12 and 4.17) and this ratio decreases because

shoots are more sensitive than roots to growth inhibition by low water potentials (Wu and

Cosgrove, 2000).

5.2.2 Leaf Water Relations

The maintenance of turgor by active lowering of Ψs is a principal resistance

mechanism in plants to cope with environmental stresses especially drought (Nawaz et al.

2012). Maize plants exposed to water deficit conditions showed significant reduction in Ψw

(Fig. 4.18) and Ψs (4.19) than normal water supply as the plants likely to conserve favorable

water balance that aid to improve resistance against water-deficit conditions (Kaldenhoff et

106

al., 2008; Passioura and Fry, 1992). In this study, deficient supply of B and Ca2+ considerably

reduced leaf Ψw in both normal and drought stressed plants (Fig. 4.18) as was reported in B-

deficient Brassica rapa (Hajiboland and Farhanghi 2011) and Camellia sinensis (Hajiboland

and Bastani, 2012). Nayyar and Kaushal (2002) described that deficient supply of Ca2+

markedly reduced the Ψw in sensitive genotype of Triticum aestivum under water stress.

However, Ψw in maize cultivars increased in response to foliar applied B and Ca2+ under

water stress (Fig. 4.18). It was described that phosphoenolpyruvate carboxylase (PEPC)

activity increased in plants provided with Ca2+ which contributes in generation of malate (an

osmoregulant) under stress conditions (Nayyar and Kaushal, 2002; Ortiz et al., 1994). The

higher Ψw in plants supplied with Ca2+ under water stress may be described to raise malate

production through higher content of PEPC. Indeed, water movement through the roots

influenced by numerous environmental aspects including drought (Aroca et al., 2012)

salinity (Carvajal et al., 2000) and nutrient stress (Clarkson et al., 2000). Boron scarcity

possibly decreases root hydraulic conductivity (Apostol and Zwiazek, 2004) due to

interruption in the development of functional xylem vessels as well as decline of fresh root

growth. Our data showed that foliar B and Ca2+ supplies under drought stress significantly

reduced Ψs that markedly improved Ψp (Fig. 4.19 and 4.20). The reduction in Ψs by B

supply might be attributed to its better water absorption as well as improved water use

efficiency (WUE) under water stress as elucidated by Hodecker et al. (2014) in Eucalyptus.

The less negative Ψs was recorded in Brassica rapa with respect to B supply under water

stress (Hajiboland and Farhanghi, 2011), however, Ca2+ in inorganic form give up-to 19% of

the pool in osmotic adjustment and thus plays an imperative role in maintenance of turgor

under water-limitations (Amede et al., 2004). It seems that application of CaCl2 might

impede damage from cellular dehydration by stabilizing osmotic vitality of the cytoplasm

(Arshi et al., 2006). Based on these result, it can be concluded that B and Ca2+ supply affects

the buildup of osmolytes during moisture stress that results in the reduction of ΨS (Fig. 4.19).

The fall in Ψp under water deficit conditions (Fig. 4.20) might be attributed to decline in Ψw

and lowering of net photosynthetic rate and stomatal conductance (Nawaz et al., 2012). The

interruption in osmoregulation causes reduction in turgor potential, subsequent reduction in

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cell elongation (Vassilev and Yordanov, 1997). It was reported that drought-prompted

decrease in water potential results in decreased RWC (Ali et al., 2011; Zhang et al., 2014)

which implies a fall of turgor that results in inadequate water availability for the cell

extension processes in plants (Zhang et al., 2011). Foliar applied B and Ca2+ significantly

improved RWC of both normal and drought stressed plants (Fig. 4.21). Reports have shown

that application of Ca2+ under drought stress enhanced RWC in Glycine max (Fioreze et al.,

2013) and Camellia sinensis (Upadhyaya et al., 2012; Upadhyaya et al., 2011) and ultimately

helped the plants to maintain cell growth which is primarily a turgor-driven process (Ashraf

et al., 2011; Ashraf and Foolad, 2007). In addition, Ca2+ modifies the extent of plasma

membrane hydration and increases the consistency of cell walls, which increases the

viscosity of the protoplasm and protects the cells from dehydration (Ma et al., 2009). The

positive role of B in increasing RWC has also been reported in Camellia sinensis

(Mukhopadhyay and Mondal, 2015; Upadhyaya et al., 2012). The present study revealed that

drought stress produced more marked decline in leaf water contents in the drought-sensitive

cultivar (Yousafwala Hybrid) than in drought resistant one (Dekalb-6525) that might be due

to their differential responses to drought resistance and is comparable to already observed in

maize (Farhad, et al., 2011; Zhang et al., 2009).

5.2.3 Gas Exchange Characteristics

The reduction in net photosynthetic rate (Pn) (Fig. 4.22) and stomatal conductance

(gs) (Fig. 4.23) in drought stressed plants was probably caused by degradation of chlorophyll

contents (Fig. 4.25-4.27), abolition of rubisco activity and stomatal closure that declines the

CO2/O2 ratio in maize leaves and eventually impedes photosynthesis (Moussa, 2006; Shen et

al., 2015). All these decrease the photosystem II (PSII) photochemical efficiency (Pieters

and El Souki, 2005) with the likely net deficit of photosystem-II (PSII) reaction centers

proteins (Liu et al., 2006; Zlatev, 2009) with the production of free radicals that caused

photo-inhibition and oxidative damage (Nishiyama et al., 2006). The reduction in Pn and gs

caused parallel reduction in transpiration rate (E) under water-deficit conditions (Rahbarian

et al., 2011; Shen et al., 2015). Drought induced reduction in photosynthetic efficiency has

been well established in numerous crop plants such as Zea mays (Shen et al., 2015), Triticum

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aestivum (Moud and Yamagishi, 2005), Cicer arietinum (Vineeth et al., 2015), Vigna radiata

(Sengupta et al., 2013) and Brassica juncea (Nazar et al., 2015).

Foliar treatment of B and Ca2+ considerably improved the Pn (Fig. 4.22), and gs (Fig.

4.23) under both normal and water-deficit conditions which may be linked to their

constructive role in conservation of photosynthetic apparatus. Our observations on improved

Pn and gs in plants supplied with B and Ca2+ under water stress correspond with those in

Brassica rapa (Hajiboland and Farhanghi, 2011) and Zoysia japonica (Xu et al., 2013),

respectively. In contrast, deficient supply of these nutrients markedly reduced the gas

exchange characteristics and our results are analogous with the previous investigations,

where B and Ca2+ scarcities reduce plant photosynthetic efficiency (Han et al., 2008; Xu et

al., 2013; Zhao and Oosterhuis, 2002). B deficiency increased starch content in plants and its

excessive accumulation may interrupt chloroplast structure, leading to lesser CO2

assimilation due to reduced activities of CO2 binding enzyme (RuBisCO) (Lu et al., 2014;

Han et al., 2008; Cave et al., 1981). However, Ca2+ scarcities reduced gs and net CO2

assimilation rates due to reduced CO2 mole fraction at carboxylation site in the chloroplast

(Ridolfi et al., 1996). It is notable that B and Ca2+ supplies considerably improved the E of

both cultivars under normal and water-deficit conditions (Fig. 4.24) and consistent with those

in Citrus sinensis (Lu, et al., 2014) and Vigna unguiculata (Murillo-Amador et al., 2006).

The markedly higher gas exchange characteristics (Fig. 4.22-4.24) were observed in drought-

tolerant cultivar Dekalb-6525 than in drought-sensitive Yousafwala Hybrid. The reduction in

Pn was caused by the oversensitivity early stomata closure, with more pronounced

manifestation in sensitive genotypes (Benesova et al., 2012). Recent study confirms that

drought tolerant cultivar of maize performed better in terms of photosynthesis due to higher

rates of CO2 assimilation (De-Souza et al., 2013; Sicher et al., 2015) that might be due to

stable photosynthetic pigments under drought conditions.

5.2.4 Chlorophyll and Carotenoids

Our data showed a reduction in chlorophyll (Chl a, b, a+b) and carotenoid contents

by drought treatment (Fig. 4.25-4.28) and this might be due to the damage of photosynthetic

pigments and the inconsistency of the pigment-protein complex (Efeoglu et al., 2009;

Huseynova et al., 2009). Drought induced reduction in Chl and carotenoid contents has been

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well documented in various plants such as Zoysia japonica (Xu et al., 2013), Zea mays

(Efeoglu et al., 2009), Triticum aestivum (Zlatev, 2009), Vigna unguiculata (Souza et al.,

2004) and Brassica rapa (Hajiboland and Farhanghi, 2011). However, foliar treatment of B

and Ca2+ notably increased the content of Chl and carotenoid (Fig. 4.25-4.28) and correspond

with those in Zoysia japonica where higher Chl contents were noted in plants treated with

CaCl2 under drought stress (Xu et al., 2013). It seems that application of Ca2+ might impede

damage from cellular dehydration by stabilizing the osmotic strength of the cytoplasm

(Arshi, et al. 2006). The positive role of B in improving the content of Chl and carotenoids

under drought stress has been reported in Brassica rapa (Hajiboland and Farhanghi, 2011)

and Camellia sinensis (Mukhopadhyay et al., 2013), however its deficiency increased the

accumulation of starch that may interrupt chloroplast structure, leading to lesser chlorophyll

contents (Cave et al., 1981; Han et al., 2008). Carotenoids, an accompanying light capturing

pigment, shielding the photosynthetic apparatus from photo-oxidative damage were also

reduced under B deficiency (Fig. 4.28) and in accordance with Pandey, (2013). In addition,

reduced carotenoid contents might be the reason of photo-oxidative impairment of

chloroplast and this injury could result in the reduction of chlorophyll contents (Siefermann‐Harms, 1987). The maintenance of considerably higher Chl contents by the drought-tolerant

cultivar (Fig. 4.25-4.27) might be due to stable photosynthetic pigments under water-deficit

conditions (De-Souza et al., 2013) and is analogous to already observed in Zea mays (Yang

et al., 2015), Glycine max (Hossain et al., 2014), Gossypium hirsutum (Parida et al., 2007)

and Triticum aestivum (Chandrasekar et al., 2000).

5.2.5 Osmolyte Accumulation

The tolerance efficiency in plants under water-deficit conditions linked with the

accumulation of compatible osmolytes such as free proline, amino acids and soluble sugars

which react actively to alleviates the adverse effects of drought stress on crop production

(Ashraf et al., 2011; Chernyadev and Monakhova, 2003; Javed et al., 2014). Accumulation

of osmoprotectants causes a reduction in cellular osmotic potential which helps in the

maintenance of tissue water balance and a rise in the turgor potential to enhance the

physiological performance of plants during drought stress (Anjum et al., 2011; Harb et al.,

2010).

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5.2.5.1 Proline

Accumulation of proline increases both by drought treatment and deficient supply of

B that could be due to down-regulation of proline dehydrogenase (Wei et al., 2009). Foliar

treatment of Ca2+ also reduced the leaf proline contents (Table 1) that might be due to

increasing the level of proline degrading enzymes and decreasing the proline synthesizing

enzymes (Jaleel et al., 2007a). Our findings are comply to the already explained in Brassica

juncea (Pandey 2013), Camellia sinensis (Hajiboland and Bastani, 2012), Zea mays (Pandey

and Archana, 2009) and Catharanthus roseus (Jaleel et al., 2007b). The noticeably higher

accumulation of proline under water and nutrient limitation may be a reaction to tissue

damage (Aghaz et al., 2013) that can be viewed as an approach for plants to lessen the

oxidative damage. B-starved accumulation of proline and activation of antioxidant system

can be viewed as an approach for tea plants to lessen the oxidative damage aggravated after

the deficient plants subjected to water stress (Hajiboland and Bastani, 2012).

5.2.5.2 Total Soluble Proteins

The reduction in total soluble proteins (TSP) in drought stressed plants (Fig. 4.30)

was probably caused by the reduced rate of protein biosynthesis and increased rate of protein

breakdown under drought stress (Krasensky and Jonak, 2012; Rodrıguez et al., 2005)

necessary for the production of compatible osmolytes for osmoregulation (Nayyar and Walia

2003). Drought-induced reduction in TSP has been well documented in various plants such

as Triticum aestivum (Nawaz et al., 2015a), Zea mays (Mohammadkhani and Heidari 2008;

Ti-Da et al., 2006), Phaseolus vulgaris (Ashraf and Iram, 2005) and Lycopersicon

esculentum (Rahman et al., 2004). Reduction of TSP in plants under water-deficit conditions

might be due to reduced rate of photosynthesis and materials for protein synthesis not

provided that results in dramatic reduction or even stopped protein synthesis (Ashraf and

Harris 2013). In addition, the amount of photosystem-II proteins (D1, D2 and LHC II) also

declined substantially under water-deficit conditions due to reduced rate of transcription,

translation as well as rapid degradation of proteins and mRNAs (Duan et al., 2006; Liu et al.,

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2009). The reduction in soluble protein contents of drought stressed plants might be due to

disintegration of proteins by proteolytic activities (Ashraf, 2004; Parida and Das, 2004),

subsequently low molecular weight amino acids increased in both maize cultivars required

for osmotic adjustment and in accordance with Javed et al., (2014). Likewise, Su and Wu

(2004) observed that high molecular weight soluble proteins accumulation reduces but low

molecular weight increases in crop plants under water limitations and established that

biochemical traits like total soluble sugars, proteins and free amino acids can be used as

selection tool for screening of drought-resistance cultivars. A considerable decrease in TSP

was recorded by supplemental B+Ca2+ supply (Fig. 4.30). In contrast, significantly lower

TSP and higher TFA were recorded in B-deficient leaves of Citrus sinensis that might be due

to reduced utilization of free amino acids for protein biosynthesis (Lu et al., 2014) and

Hajiboland et al. (2011) observed that TSP concentration did not change in Camellia sinensis

leaves under normal and deficient supply of boron. Likewise, Ca2+ applications considerably

increased the soluble proteins in water stressed seedling of Brassica napus (Alam, 2013).

5.2.5.3 Total Free Amino Acids

The marked accumulation of total free amino acids (TFA) was recorded by drought

treatment (Fig. 4.31) which is the most prevalent response to drought stress (Planchet et al.,

2011).This rise in amino acids could be due to their synthesis or inter-change of stress-

decreased protein biosynthesis and/or stress prompted protein biodegradation. Indeed,

drought stress caused plant proteins impairment and ultimately degradation by proteases

(Vierstra, 1996). Accumulations of free amino acids improve plant tolerance to drought by:

1. acting as compatible osmolytes, 2. regulating pH and detoxifying reactive oxygen species,

3. being a carbon and nitrogen reserves primarily for the biosynthesis of particular enzyme

and precursors of several secondary metabolites (such as lignin and flavonoids), 4. being an

accessible stock of free amino acids that would be beneficial during regaining from stress and

5. acting as regulatory and signaling molecules (Planchet et al., 2015).

Accumulations of TFA were considerably improved by sole spray of Ca2+ and in

combination with B (Fig. 4.31) and is in agreement with Alam (2013) who reported that

application of Ca2+ in Brassica napus improved the concentrations of TFA under both normal

and water-deficit conditions. Furthermore, aerobic pre-treatment of rice roots with CaCl2

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improved the accumulations of amino acids (Aurisano et al., 1995). Deficient supply of B

increased the accumulations of TFA in both cultivars (Fig. 4.31) that might be due to

disturbance in various plant physiological and biochemical mechanisms (Marschner and

Marschner, 2012) which resulted in enhanced leaves concentration of free amino acids in

Citrus sinensis (Lu et al., 2014). The authors suggest that B-scarcity enhanced leaf

concentration of TFA and activities of those enzymes involved in amino acid metabolism

indicating that synthesis of amino acids might be up-regulated in B-deficient leave (Lu, et al.

2014). In addition, B starvation impaired nitrate reductase (NR) activity and increased

concentration of nitrate in B-deficient leaves as have been noted in several plant species such

as Helianthus annuus (Kastori and Petrovic, 1989), Brassica napus (Shen et al., 1993), and

Lycopersicon esculentum (Ramón et al., 1989). The NR is crucial enzyme that catalyzes

principal reaction in nitrate (NO-3) assimilation pathway (Lee, 1999) and NO-3 must be

reduced to ammonia in order to synthesize structural component of the biological system

(Hamid et al., 2010; Heuer et al., 2005). Therefore, reduced NR activity resulted in

accumulation of free amino acids in B deficient leaves. In contrast, concentrations of TFA

did not alter in B-scarce tea and tobacco plants (Camacho-Cristobal and González-Fontes,

1999; Camacho-Cristóbal and González-Fontes, 2007; Hajiboland et al., 2011), whereas in

lupin, B-scarcity caused noticeable variations in accumulation of TFA in different organs

(Alves et al., 2011). Consequently, it seems that the effect of B-insufficiency on TFA

depends on plant species, organs, extent and degree of B-starvation (Lu et al., 2014).

5.2.5.4 Total Soluble Sugars

Plants respond to drought conditions by accumulating soluble sugars as these are

freely accessible organic osmolytes in the cell (Taiz and Zeiger 2006). Our results showed

enhanced accumulation of total soluble sugars (TSS) in maize leaves under drought treatment

as well (Fig. 4.32) which declines the cellular osmotic potential so as to stabilize membranes

and macromolecular configurations (Anjum et al., 2011; Harb et al., 2010). Like proline, a

decrease in TSS by B foliar spray is also observed in current study (Fig. 4.32). Our

elucidation are correspond with those in Gossypium hirsutum (Zhao and Oosterhuis, 2002),

Nicotiana tabacum (Camacho-Cristobal et al., 2004), Citrus grandis (Han et al., 2009) and

Phaselous vulgaris (Pandey et al., 2009). Accumulations of sugars under B deficient

conditions could result from the reduced demand for decreased carbon in developing sink 113

tissues due to growth inhibition (Han et al., 2009) and reduced transportation of sugars as B

plays part in translocation of sugars (Pandey et al., 2009). A considerable increase in TSS

was recorded by Ca2+ and B+Ca2+ foliar sprays (Fig. 4.32) that could help the plants to

sustain the intracellular osmotic balance indicating that application of nutrients effectively

improved drought tolerance of plants (Upadhyaya et al., 2011). Qiang et al., (2012) observed

increased accumulation of TSS in Honeysuckle (Lonicera japonica) grown in soil treated

with CaCl2 under water-deficit conditions. It seems that application of Ca2+ to the salt treated

plants of Gossypium hirsutum increased TSS (Amuthavalli et al., 2012) as it may increase

activity of α-amylase, required for conversion of starch into sugars, and Ca2+ is required for

its activity (Jaleel et al., 2007a).

The higher accumulation of osmotically active molecules like PRO (Fig. 4.29), TFA

(4.30) and TSS (4.31) in cultivar Dekalb-6525 proposed its greater genotypic tolerance to

drought stress than drought-sensitive Yousafwala Hybrid as these helps in maintaining plant

structures, and scavenging hydroxyl radicals under stress conditions (Guo et al., 2010; Kaul

et al., 2008; Rodriguez and Redman, 2005).

5.2.6 Antioxidants

Oxidative damage is a primary reaction of plant cells to environmental stresses and

optimum supply of B and Ca2+ has been establish to hamper the adverse effects of such

stresses through maintenance of structural and functional integrity of biological membranes

as revealed by reduced MDA accumulation (Fig. 4.37) and improved antioxidant defense

system (Fig. 4.33-4.36). It has been reported that plants antioxidant system activate in

response to B and Ca2+ supplies under stress conditions (Molassiotis et al., 2006; Zorrig et

al., 2012), while data about maize is limited. The up-regulation of antioxidant system is a

pervasive response, which results in decreasing lipid peroxidation and conserving

macromolecular configurations or functions (Zhang et al., 2014). In our study, foliar sprays

of B and Ca2+ substantially reduced the MDA content (Fig. 4.37) through up-regulation of

antioxidant activities in both cultivars and importantly synergistic effect were seen when

applied in combinations (Fig. 4.33-4.36). The increased SOD activity (Fig. 4.33) in plants

supplied with B and Ca2+ under water stress correlated with enhanced defense from oxidative

damage (Miller et al., 2010). The H2O2 is the product of SOD activity and still toxic until

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converted into H2O in consequent reactions (Amor et al., 2010). The activity of H2O2

catalyzing enzymes, CAT (Fig. 4.34), POD (4.35) and APX (4.36) also substantially

increased in water stressed plants supplied with B and Ca2+ and act simultaneously to

eliminate H2O2 at a highest rate (Siddiqui et al., 2011) that ultimately protect the plants from

oxidative damage. The reduction in lipid peroxidation and up-regulation of these enzymes in

B treated Glycine max (Liu and Yang, 2000; Peng and Yuai, 2000) and Ca2+ applied Zoysia

japonica and Festuca arundinacea (Xu et al., 2013; Jiang and Huang, 2001) have also been

reported under stress conditions. In contrast, increased antioxidant activity was recorded in

B-deficient Camellia sinensis (Hajiboland and Bastani 2012) and Morus alba (Tewari et al.,

2010). However, optimum supply of B in Citrus sinensis increased CAT and decreased APX

and SOD activity (Han et al., 2008). It appears that activation of plant antioxidant system in

response to B supply is species specific. It has been documented that stress tolerant cultivars

have greater ability to survive with abiotic stresses by prompting antioxidant defense systems

(Bor et al., 2003; Demiral and Turkan, 2005) and similar has been reported in our case as

well (Fig. 4.33-4.36). The drought-induced increased activities of antioxidant in tolerant

cultivars of numerous crops has been well established such as Zea mays (Zhang et al., 2014),

Triticum aestivum (Hassan et al., 2015), Gossypium hirsutum (Abdel-Kader et al., 2015),

Oryza sativa (Lum et al., 2014), Glycine max (Sakthivelu et al., 2008) and Cicer arietinum

(Mohammadi et al., 2011). The considerably higher antioxidant activities were recorded in

plants under water-deficit conditions (Fig. 4.33-4.36) which can be viewed as an approach

for plants to protect cellular membranes, proteins and metabolic apparatus, from adverse

effects of drought (Harb et al., 2010; Moussa and Abdel-Aziz, 2008).

5.2.7 Leaf Boron and Calcium Concentrations

Drought stress significantly reduced the uptake of B and Ca2+ which considerably

reduced their concentration in maize leaf tissues (Fig. 4.38, 4.39) as their bioavailability

hampered under water deficit conditions because of their reduced mobility from soil to roots

by mass flow (Brown et al., 2006; Mattiello et al., 2009). In addition, soil drying reduces B

availability due concentrated soil solution, compact mass flow and diffusion rate and this

could be experienced not merely under short but also under sufficient B supply (Hajiboland

and Farhanghi, 2011). Lisar et al., (2012) elucidated that drought stress disturbs plant mineral

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nutrition and ion equilibrium due to reduction in transpiration rate that declined half of the

Ca2+ contents in maize plants. However, foliar treatment of B considerably improved its

concentrations in both cultivars under normal and water deficit conditions (Fig. 4.38) and our

results are in agreement with Aref, (2011) who reported that application of B significantly

increased its concentration in plant leaves. B is associated with the rhamnogalacturonan II

fraction of pectin polysaccharides, which has been described to be responsible for cross-

linking of cell-wall polymers and is therefore necessary for maintenance of cell wall stability

(O'Neill et al., 2001), that ultimately provides the protection against drought stress.

Application of Ca2+ also improved its concentration in leaf tissues of both studied cultivars

(Fig. 4.38) which caused maintenance of structural and functional integrity of plant

membranes and other essential structures (Guimaraes et al., 2011; Ma et al., 2005; Nayyar

and Kaushal, 2002) as revealed by lesser accumulation of MDA content (Fig. 4.37). Foliar

supplied Ca2+ enhances plant ability to retain water (Shao et al., 2008) and maintain cell

growth which is primarily a turgor-driven process (Ashraf et al., 2011; Ashraf and Foolad

2007; Bolen, 2004). In addition, Ca2+ modifies the extent of plasma membrane hydration and

increases the consistency of cell walls, which increases the viscosity of the protoplasm and

protects the cells from dehydration (Ma et al., 2009).

5.3 Field Experiment

5.3.1 Growth parameters

Drought treatment considerably reduced the vegetative growth i.e. ‘leaf dry weight

(Fig. 4.40), shoot dry weight (Fig. 4.41) and plant height (Fig. 4.46)’ in both maize cultivars

that might be due to reduced rate of cell division, stem elongation, leaf size and root

proliferation, and disturbed stomata, plant-water-nutrient-relations with reduced crop

productivity and water-use efficiency (Farooq, et al. 2009; Li, et al. 2009). Drought induced

reduction in vegetative growth of maize has been documented earlier (Ashghizadeh and

Ehsanzadeh, 2008; Cakir 2004; Rahman et al., 2004; Yang et al., 2009; Zadehbagheri et al.,

2014). Our data showed that foliar sprays of B, Ca2+ and their combinations considerably

improved vegetative growth of maize and their deficient supply disturbed the normal growth

(Fig. 4.40, 4.41 and 4.46). Boron is a critical element for normal plant growth and

development and its proper supply is required for attaining high yield and quality crops

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(Brown et al., 2002; Goldbach and Wimmer, 2007). Reports have shown that B has role in

cell expansion and its deficiency caused cessation of root growth and leaf expansion that

ascribed its role in cell wall structure and function (Hajiboland and Farhanghi, 2011;

Marschner and Marschner, 2012). In addition, deficient supply of B caused damage to cell

membranes, enhanced accumulation of phenolics and induction of disproportion between

production and scavenging of free oxides that might be also involved in diminishing of plants

growth (Hajiboland and Farhanghi, 2010). B applied improvement in growth, and

physiological characteristics has been described in Zea mays (Lordkaew et al., 2011),

Triticum aestivum (Karim et al., 2012), Brassica rapa (Hajiboland and Farhanghi, 2011),

Eucalyptus urophylla (Hodecker et al., 2014), Camellia sinensis (Hajiboland and Bastani,

2012) and Citrus sinensis (Lu et al., 2014). In addition, calcium is also required by the plants

for vigorous leaves and roots development and overall canopy growth (Del-Amor and

Marcelis, 2003). It was described that exogenous application of Ca2+ improves the inhibition

of plant growth and development and maintains the structural and functional integrity of

plasma membranes under stress conditions (Guimaraes et al., 2011; Siddiqui et al., 2011;

Siddiqui, et al. 2012). Calcium applied improvement in growth under stress conditions has

been elucidated in numerous plants such as Triticum aestivum (Nayyar, 2003; Nayyar and

Kaushal, 2002), Zea mays (Nayyar, 2003), Zoysia japonica (Xu et al., 2013), Camellia

sinensis (Upadhyaya et al., 2011), 'Lonicera japonica (Qiang et al., 2012), Phaselous

vulgaris (Abou El-Yazied, 2011) and Helianthus annuus (Hassan et al., 2011).

Our data showed that reproductive growth of maize i.e. silk length (Fig. 4.44),

number of silk threads per ear (Fig. 4.45), tassel dry weight (DW) (Fig. 4.42) and silk DW

(Fig. 4.43) was markedly reduced by drought treatment. It was elucidated that drought stress

at flowering leads to reduction in ear growth and appearance of silk (Aslam et al., 2013).

Drought stress reduces the photosynthesis, disturbs the carbohydrate metabolism and level of

sucrose in leaves that falls over to a reduced export rate. This is probably due to drought-

induced improved activity of acid invertase (Kim et al., 2000). Inadequate photosynthesis

and sucrose buildup in the leaves may impede the rate of sucrose export to the growing sink

and eventually disturb the reproductive development (Farooq et al., 2009). In current study,

foliar treatment of B and Ca2+ notably improved the number and length of silk and dry matter

accumulations in tassel and silk as compared to control (Fig. 4.42-4.45). Our results are

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comply with the findings of Lordkaew et al. (2011) who reported that optimum supply of B

considerably improved the reproductive growth of Zea mays as compared to control. B

insufficiency has been described to cause reduced ovule and flower development in oilseed

rape (Xu et al., 2002), but under severe deficiency whole plant growth was affected. It has

occasionally been proposed that the anther and pollen may be more sensitive to B-scarcity

than the pistil (Brown et al., 2002; Dell and Huang, 1997).

The positive role of calcium in reproductive growth of flowering plants has been also

elucidated by Ge et al. (2007) who described that Ca2+ has an important physiological,

regulatory and signaling role during reproductive growth of flowering plants; elevation of

Ca2+ amounts is a precise interpreter of plant fertility. It accomplishes many crucial roles in

reproductive growth of plants. Connections between gametes of opposite sexes and

gametophytes contain different Ca2+ forms, happen at different levels of organization, and

need different forms of Ca2+ regulation at each level. Digonnet et al. (1997) described

varying spatial and sequential features of Ca2+ distribution in plant reproductive tissues and

cells that reveal the precarious role of Ca2+ during reproductive growth of flowering plants.

The results of current study revealed that drought tolerant maize hybrid perform

better in terms of improved vegetative and reproductive growth characteristics (Fig. 4.40-

4.46) as the stress tolerant plants have inherent capacity to overwhelm the damage instigated

by abiotic stresses at different growth stages (Nagarajan and Nagarajan, 2010). It was

reported that different maize plants performed differentially under drought conditions due to

differences in their metabolic activities (Farhad et al., 2011). A recent study confirms that

drought stress caused more marked reduction in dry matter production in drought sensitive

cultivars of maize than that of resistant one (Zhang et al., 2014). Various reports have shown

that drought tolerant genotypes of maize performed better in terms of improved root growth

(Bruce et al., 2002), shoot length (Witt et al., 2012) and biomass accumulations (Zhang et

al., 2014) as compared to sensitive cultivars.

5.3.2 Yield and Yield Components

Drought is reflected as one of the primary limiting aspects responsible for reduced

agricultural productivity worldwide and can consequence in significant yield reduction of

many crop plants including maize (Anjum et al., 2011; Ashraf, 2010; Gholipoor et al., 2013;

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Mahmood et al., 2009). We also observed a significant reduction in yield and yield

components of maize by drought treatment (Fig. 4.47-4.51) as its life cycle depends greatly

upon water accessibility and the drought at any phonological stage have varied response and

may result in severe grain yield reduction if it coincides with drought sensitive growth stage

of plant (Cakir, 2004; Earl and Davis, 2003; Edmeades et al., 1999; Zadehbagheri et al.,

2014). Our data showed that application B, Ca2+ and their combinations evidently improved

the maize yield and related traits (Fig. 4.47-4.51) as these are critical elements for normal

plant growth and development and its application especially under drought stress improves

leaf area, photosynthetic rate and stomatal conductance which in turn improves crop quality

and yield (Brown et al., 2002; Goldbach and Wimmer, 2007; Karim et al., 2012; Nayyar and

Kaushal, 2002). Boron applied increase in yield has been well documented in various crop

plants such as Zea mays (Heckman, 2002), Triticum aestivum (Karim et al., 2012),

Gossypium hirsutum (Dordas, 2006), Oryza sativa (Hussain et al., 2012), Glycine max

(Bellaloui et al., 2013), Cicer arietinum (Valenciano et al., 2011) and Medicago sativa

(Dordas, 2006). In addition, foliar exposure of chelated calcium improved the seed yield and

related attributes of Phaseolus vulgaris under water-deficit conditions (Abou El-Yazied,

2011). Naeem et al., (2013) reported that Ca2+ application enhanced crop productivity and

photosynthetic efficiency in a Senna occidentalis. Results of present study revealed that

drought tolerant cultivar Dekalb-6525 performed better in terms of improved yield and yield

components as compared to the drought-sensitive Yousafwala Hybrid (Fig. 4.47-4.51). It was

reported that the yield reduction was more pronounced in those cultivars which are most

sensitive to drought (Ashraf, 2010; Chandrasekar et al., 2000; Gholipoor et al., 2013; Wang

et al., 2002)

119

CHAPTER 6 SUMMARY

The present study included various experiments conducted in five phases to

investigate the 1) effect of polyethylene glycol (PEG6000) induced osmotic stress and varying

water stress levels on germination and seedlings attributes of eight different maize hybrids 2)

effect of foliar supply of different boron rates on seedling growth of maize cultivars (Dekalb-

6525 and Yousafwala) under drought stress 3) effect of foliar treatment of different calcium

rates on seedling attributes of water-stressed maize cultivars 4) effect of foliar B and Ca2+

supplies on physiological and biochemical traits of maize grown under water-deficit

conditions 5) effect of foliar treatment of B and Ca2+ on maize growth and yield under water

limitations.

In first phase, (laboratory experiments), eight available maize hybrids i.e. Data-2368,

Syngenta-8441, Data-2468, Pioneer-31P41, Pioneer-32B33, FH-810, Dekalb-6525 and

Yousafwala Hybrid were evaluated for their drought tolerance potential using PEG6000

induced osmotic stress (-0.2, -0.4, -0.6 and -0.8 MPa) and water stress (100% and 30%

water-holding capacity) at germination and seedling stages. Different germination attributes

and physiological indices were used as screening tool. The results of the study revealed

considerable genetic variation among maize genotypes and cultivar, Dekalb-6525 performed

better therefore categorized as drought tolerant, whereas the Yousafwala Hybrid was

identified as drought sensitive on the basis of its poor performance.

In second and third phase, two pot experiments were conducted in a wire-house /

rain-out shelter to optimize the foliar rate for B and Ca2+ supplies helpful in improving

drought tolerance in maize under drought stress at seedling stage. For this purpose, one

drought-tolerant (Dekalb-6525) and sensitive (Yousafwala) maize hybrids selected from

laboratory experiments were sprayed with distilled water (control) and B solutions at 2, 4 and

6 mg L-1 and Ca2+ at 20, 40 and 60 mg L-1 under normal and water-deficit conditions in two

separate experiments. It was noted that foliar treatment of B at 4 mg L-1 and Ca2+ at 40 mg L-1

considerably improved the plant height, root length and dry matter stress tolerance indices of

maize. In addition, results indicated that the maize cultivar Dekalb-6525 more responsive to

foliar B and Ca2+ supplies than Yousafwala Hybrid.

120

A wire-house / rain-out shelter experiment was carried out during fourth phase of this

study. The optimized rates of B (4 mg L-1), Ca2+ (40 mg L-1) and their combinations were

used to evaluate the physiological and biochemical changes in two maize cultivars differing

in drought tolerance under normal and water-deficit conditions. Drought stress brings

considerable growth inhibition through reduction in uptake of B and Ca2+, disturbance in gas

exchange characteristics, water relations, chlorophyll contents and osmolyte accumulation by

increasing MDA accumulation and disproportioning antioxidant system as compared to

normal conditions. Foliar treatment of B and Ca2+ alone and in conjunction at six leaf and

tasseling stages of maize substantially improved physiological and antioxidative responses of

both maize cultivars under water limited environment. Accumulation of proline and total

soluble proteins were notably reduced by foliar supply of these nutrients; however

application of Ca2+ and B+Ca2+ improved the accumulation of TFA and TSS under water-

deficit conditions. In addition, plants supplied with B maintained considerably lower TFA

and TSS than in control. Results of the study revealed that maize cultivar, Dekalb-6525

performed better in terms of improved water balance, chlorophyll contents and gas exchange

characteristics through enhanced accumulation of compatible osmolytes and increasing

antioxidant activities than Yousafwala Hybrid.

In fifth phase of this study, a field experiment was conducted to evaluate the effect of

supplemental B and Ca2+ supplies on growth and yield of maize (Dekalb-6525 and

Yousafwala Hybrid) under water-deficit conditions. Our data showed that drought treatment

considerably reduced the growth, yield and yield related attributes of maize, whereas the

optimum supply of B and Ca2+ significantly improved vegetative and reproductive growth of

maize that caused improves grain weight and yield under stress conditions. The results

indicated that drought sensitive cultivar (Dekalb-6525) produced significantly higher grain

yield under normal and deficit-moisture supply.

ConclusionBased on these results, we have established that screening of drought-tolerant

cultivars at germination and seedling stages of maize can be valuable in decreasing the risk

of poor stand-establishment under water deficit conditions. We have established the

differences in B and Ca2+ induced physiological and biochemical changes and modifying

ability in two maize cultivars differing in drought tolerance. The results of the present study

121

suggest that drought stress brings considerable growth inhibition through reduction in

nutrient uptake and disturbance in gas exchange characteristics, water relations and

photosynthetic pigments by accelerating MDA accumulation and disproportioning

antioxidant system as compared to normal water supply. Considerably improved growth rate,

water status, photosynthesis, pigment contents, osmolyte accumulation, antioxidant activities

as well as reduced MDA accumulation were found to be the key contributors to a stress-

tolerant genotype to thrive under limited-water supply. Moreover, plant growth,

photosynthesis, water relations, pigment content, osmoprotectant accumulation, antioxidant

defense system, lipid peroxidation and final economic yield could be adjusted by foliar B and

Ca2+ supplies under water-deficit conditions. Thus, we suggest synergistic effect between B

and Ca2+ and the hardening for drought tolerance by their optimum supplies should rather be

pragmatic to a drought-sensitive cultivar under drought stress to improve its potential to grow

vigorously under drought-prone situations.

Future Perspectives

Our considerate of B and Ca2+ acquisitions from dry soils by roots has greatly been

reduced; but, the processes of distribution of these nutrients within different vegetative and

reproductive organs of plants under water-deficit conditions are still poorly understood. In

certain, more prominence should be placed on the molecular learning of B and Ca2+ transport

to propagative organs during the reproductive development under water-limited conditions,

as B and Ca2+ inadequacy causes bareness, which is the main reason of yield reduction in

staple crops under drought conditions. Comprehensive study of soluble carbohydrate profile

is suggested to work out the synthesis of stress induced sugars. In current study, limited

osmolyte have been explored. Studies on accumulations of other osmolytes like trehalose and

various other organic acids are suggested to understand osmoregulation in maize under

water-deficit conditions.

122

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Conflict of Interest

All authors disclose there is no conflict of interest.

156

APPENDICES:Appendix 4.1: Effect of PEG6000 induced osmotic stress on germination percentage (%) of different maize hybrids

PEG6000 /hybrids

Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

Grand Means

Control 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 100.00 a 73.33 bcd 73.33 abcd 93.33 A

-0.2 MPa 93.33 ab 93.33 ab 100.00 a 93.33 ab 80.00 abc 86.67 abc 46.67 de 60.00 cd 81.67 B

-0.4 MPa 100.00 a 100.00 a 80.00 abc 86.67 abc 73.33 bcd 80.00 abc 46.67 de 46.67 de 76.67 B

-0.6 MPa 80.00 bc 86.67 abc 80.00 abc 86.67 abc 80.00 abc 66.67 bcd 20.00 ef 26.67 ef 65.83 C

-0.8 MPa 93.33 ab 86.67 abc 80.00 abc 86.67 abc 73.33 bcd 60.00 cd 26.67 ef 11.67 f 64.79 CGrand Means 93.33 A 93.33 A 88.00 A-C 90.67 AB 81.33 BC 78.67 C 42.67 D 43.67 D

Appendix 4.2: Effect of PEG6000 induced osmotic stress on mean germination time [(MGT) days)] of different maize hybridsPEG6000 /hybrids

Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

Grand Means

Control 2.07 no 1.80 o 2.07 no 2.77 k-o 3.00 g-o 2.40 l-o 3.09 f-n 2.86 i-o 2.51 D

-0.2 MPa 2.63 l-o 2.30 m-o 3.13 e-n 2.95 h-o 2.89 i-o 2.33 l-o 4.27 a-f 3.13 e-n 2.96 C

-0.4 MPa 2.37 l-o 2.80 j-o 4.02 a-i 2.93 h-o 2.93 h-o 2.98 g-o 4.33 a-e 4.14 a-h 3.31 B

-0.6 MPa 2.91 i-o 2.60 l-o 4.58 a-c 3.33 d-m 4.73 ab 3.53 b-l 4.17 a-g 4.00 a-j 3.73 A

-0.8 MPa 3.00 g-o 2.73 k-o 3.50 c-m 3.36 d-m 3.53 b-l 3.85 a-k 4.50 a-d 5.00 a 3.68 AGrand Means 2.60 E 2.45 E 3.46 BC 3.07 CD 3.42 B-D 3.02 D 4.07 A 3.83 AB

157

Appendix 4.3: Effect of PEG6000 induced osmotic stress on promptness index (PI) of different maize hybrids

PEG6000 /hybrids

Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

Grand Means

Control 4.42 ab 3.33 c-f 4.67 a 3.66 b-e 3.75 a-d 4.08 a-c 3.83 a-d 2.17 g-j 3.74 A

-0.2 MPa 3.07 d-g 2.05 h-k 2.63 f-i 2.13 g-j 2.25 g-j 2.25 g-j 1.91 h-k 1.17 k-o 2.18 B

-0.4 MPa 2.80 e-h 2.00 h-k 2.22 g-j 2.25 g-j 1.73 i-m 1.83 i-l 1.58 j-m 0.90 l-o 1.91 C

-0.6 MPa 2.63 f-i 1.87 h-k 1.92 h-k 1.92 h-k 1.47 j-n 1.42 j-n 1.15 k-o 0.63 no 1.62 D

-0.8 MPa 2.32 g-j 1.63 j-m 1.53 j-n 1.78 i-l 1.42 j-n 1.11 k-o 0.83 m-o 0.47 o 1.39 E

Grand Means 3.05 A 2.18 CD 2.59 B 2.35 BC 2.12 CD 2.14 CD 1.86 D 1.07 E

Appendix 4.4: Effect of PEG6000 induced osmotic stress on germination stress tolerance index [(GSI) %)] of maize hybrids

PEG6000 /hybrids

Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

Grand Means

-0.2 MPa 69.44 a 61.39 a-c 56.67 b-f 58.27 b-e 60.05 a-d 55.02 b-g 50.29 d-i 53.90 b-h 58.13 A

-0.4 MPa 63.46 ab 60.42 a-c 47.75 f-j 61.31 a-c 46.54 g-k 44.85 h-k 41.36 i-l 41.78 i-l 50.93 B

-0.6 MPa 59.69 a-d 56.25 b-g 41.25 i-l 52.38 c-h 39.14 j-m 34.68 l-n 30.11 m-o 29.56 m-o 42.88 C

-0.8 MPa 52.39 c-h 49.02 e-j 32.70 l-n 48.63 e-j 37.66 k-m 27.30 no 21.72 o 21.66 o 36.38 D

Grand Means 61.24 A 56.77 B 44.59 C 55.14 B 45.85 C 40.46 D 35.87 E 36.72 DE

158

Appendix 4.5: Effect of water deficit on plant height stress tolerance index [(PHSI) %)] of different maize hybrids

Hybrids Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

90.14 a 89.35 ab 72.13 c 88.55 ab 78.16 abc 78.39 abc 83.78 abc 74.30 bc

Appendix 4.6: Effect of water deficit on root length stress tolerance index [(RLSI) %)] of different maize hybrids

Hybrids Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

130.69 a 119.85 ab 101.60 b 112.69 ab 106.99 ab 114.57 ab 103.89 b 73.962 c

Appendix 4.7: Effect of water deficit on dry matter stress tolerance index [(DMSI) %)] of different maize hybrids

Hybrids Dekalb-6525

Pioneer-31P41

Syngenta-8441

Pioneer-32B33

Data-2468

Data-2368

FH-810

Yousafwala Hybrid

91.05 a 86.35 ab 76.98 bc 84.50 abc 77.04 bc 82.09 abc 73.96 bc 71.99 c

Appendix 4.8: Effects of boron on physiological indices of maize hybrids under drought

159

Treatments PHSI (%) Grand Means

RLSI (%)Grand Means

DMSI (%)Grand MeansB

doses/hybridsDekalb-

6525Yousafwala

HybridDekalb-

6525Yousafwala

HybridDekalb-

6525Yousafwala

Hybrid

Distill water 74.84 b 75.55 b 75.196 A 111.70 104.17 107.94 B 81.31 77.77 79.54 C2 mg L-1 74.97 b 74.20 b 74.584 B 113.63 112.85 113.24 B 86.82 82.27 84.55 B4 mg L-1 86.30 a 84.58 a 85.441 A 127.44 117.48 122.46 A 98.17 93.16 95.67 A6 mg L-1 74.09 b 57.69 c 65.886 C 93.54 90.09 91.82 C 90.17 83.83 87.00 B

Grand Means 77.55 A 73.00 B Grand Means 111.58 A 106.15 B Grand

Means 89.12 A 84.26 B

PHSI Plant height stress tolerance index; RLSI Root length stress tolerance index; DMSI Dry matter stress tolerance index

Appendix 4.9: Effects of boron on total fresh biomass (g plant-1) of maize hybrids under droughtTreatments 100% Water-holding capacity 30% Water-holding capacity

Hybrids/B doses

0 2 4 6 0 2 4 6mg L-1 mg L-1

Dekalb-6525

R1 5.2 5.665 6 5.63 3.35 4.025 4.66 3.91R2 5.02 5.69 6.04 5.54 2.89 3.402 4.48 5.13R3 4.6 6.43 5.17 5.07 2.8 3.5 4.68 4.21

Means 4.94 5.92 5.73 5.41 3.01 3.64 4.60 4.41

YousafwalaHybrid

R1 2.69 5.47 4.7 4.92 2.47 3.15 3.81 2.35R2 3.415 6.13 5.55 4.64 1.98 2.73 4.2 2.7R3 3.5 5.36 5.4 4.83 2.26 3.03 4.2 3

Means 3.20 5.65 5.21 4.79 2.23 2.97 4.07 2.68

Appendix 4.10: Effects of boron on shoot/root ratio of maize hybrids under drought

160

Treatments 100% Water-holding capacity 30% Water-holding capacityHybrids/B

doses0 2 4 6 0 2 4 6

mg L-1 mg L-1

Dekalb-6525

R1 0.66 0.53 0.54 0.54 0.46 0.37 0.33 0.47R2 0.52 0.71 0.49 0.55 0.31 0.25 0.32 0.46R3 0.43 0.51 0.61 0.70 0.31 0.38 0.42 0.32

Means 0.54 0.58 0.55 0.60 0.36 0.33 0.36 0.42

YousafwalaHybrid

R1 0.59 0.50 0.58 0.37 0.53 0.32 0.29 0.49R2 0.46 0.60 0.53 0.79 0.34 0.46 0.33 0.68R3 0.63 0.54 0.50 0.58 0.42 0.31 0.34 0.47

Means 0.56 0.55 0.54 0.58 0.43 0.36 0.32 0.55

Appendix 4.11: Effects of calcium on physiological indices of maize hybrids under drought

Treatments PHSI RLSI DMSI(%)

Calcium levels (Ca)Control 73.271 b 104.36 b 88.61 c20 (mg L-1) 78.729 ab 106.60 b 101.52 b40 (mg L-1) 82.497 a 119.49 a 113.98 a60 (mg L-1) 74.802 b 100.55 b 102.37 bHybrids (H)Monsanto-6525 82.576 a 110.35 a 119.61 aYousafwala Hybrid 72.074 b 105.15 b 83.63 bCa × H NS NS NS

PHSI Plant height stress tolerance index; RLSI Root length stress tolerance index; DMSI Dry matter stress tolerance index

Appendix 4.12: Effects of calcium on shoot/root ratio of maize hybrids under drought

161

Treatments 100% Water-holding capacity 30% Water-holding capacity

Hybrids/B doses

0 2 4 6 0 2 4 6mg L-1 mg L-1

Dekalb-6525

R1 0.70 0.78 0.89 1.10 0.81 0.78 0.87 0.80R2 0.81 0.91 0.70 1.06 0.89 0.68 0.64 0.97R3 1.00 0.84 0.64 0.82 0.78 0.68 0.85 0.93

Means 0.84 0.84 0.74 0.99 0.83 0.72 0.79 0.90

YousafwalaHybrid

R1 0.71 1.05 0.93 0.64 0.66 1.15 0.79 0.90R2 0.62 0.91 0.80 0.65 0.82 0.77 1.06 0.90R3 0.68 1.09 1.02 0.77 0.85 0.67 1.05 0.84

Means 0.67 1.01 0.92 0.69 0.77 0.87 0.97 0.88

Appendix 4.13: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf water relations of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H) W×F W×H F×H W×F×H CV

100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525Yousafwala

Hybrid NS NS NS NS

Water potential 0.60 b 0.75 a 0.78 a 0.69 b 0.66 b 0.57 c 0.66 b 0.70 a *** NS NS NS 5.92

Osmotic potential 1.14 b 1.33 a 1.14 b 1.24 a 1.28 a 1.29a 1.27 a 1.20 b NS NS NS NS 5.56

Turgor potential NS NS 0.35 c 0.55 b 0.62 b 0.73 a 0.61 a 0.51 b *** NS NS NS 12.85

Relative water

contents88.25 a 75.85

b 73.20 b 81.38 a 85.80 a 87.80 a 84.22 a 79.88 b NS NS NS NS 7.33

162

Appendix 4.14: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (E) of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525Yousafwala

Hybrid

Pn 23.82 a 15.32 b 14.93 c 19.96 b 21.20 ab 22.19 a 20.11 a 19.03 b NS NS NS NS 7.35

gs 0.36 a 0.22 b 0.22 c 0.28 b 0.31 ab 0.34 a NS NS NS NS NS NS 11.24

E 6.35 a 4.34 b 4.24 c 5.12 b 5.86 a 6.15 a 5.56 a 5.13 b NS NS NS NS 10.67

Appendix 4.15: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on chlorophyll (Chl a), (Chl b), (Chl a+b) and total carotenoid contents of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525Yousafwala

Hybrid

Chl a 1.77 a 0.80 b 0.93 bc 0.88 c 1.05 ab 1.11 a 1.03 a 0.95 b NS NS NS NS 11.17

Chl b 0.67 a 0.47 b 0.50 b 0.57 a 0.60 a 0.62 a 0.59 a 0.55 b NS NS NS NS 10.81

Chl a+b 1.84 a 1.28 b 1.42 b 1.45 b 1.64 a 1.73 a 1.62 a 1.50 b NS NS NS NS 7.21Total Carotenoids 0.48 a 0.40 b 0.35 c 0.44 b 0.46 b 0.51 a NS NS NS NS NS NS 6.55

163

Appendix 4.16: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on osmolytes accumulation of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525Yousafwala

Hybrid

Proline 10.68 b 25.19 a 23.27 a 17.03 b 16.24 b 15.20 b NS NS *** NS NS NS 9.76Total soluble proteins

9.77 a 6.41 b 8.72 a 8.28 ab 7.79 ab 7.56 b NS NS * NS NS NS 10.85

Total free amino acids 0.75 b 1.44 a 1.03 c 0.90 d 1.17 b 1.28 a 1.14 a 1.05 b *** NS NS NS 7.76

Total soluble sugars

35.45 b 55.75 a 40.24 b 34.27 c 52.64 a 55.26 a 50.00 a 41.20 b *** NS NS NS 10.92

Appendix 4.17: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on antioxidant activities of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525

Yousaf-wala

Hybrid

SOD 137.93 b 258.60 a 168.95 d 185.23 c 210.87 b 228.02 a 206.05 a 190.49 b *** ** NS NS 6.36

CAT 76.47 b 194.64 a 118.50 d 131.17 c 141.03 b 151.52 a 142.58 a 128.53 b *** * NS NS 5.57

POD 56.80 b 236.88 a 121.22 c 150.29 b 150.61 b 165.23 a 152.23 a 141.44 b *** NS NS NS 8.63

APX 1.78 b 4.77 a 2.58 b 3.42 a 3.43 a 3.67 a 3.80 a 2.75 b *** *** NS NS 10.31

164

Appendix 4.18: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on leaf B and Ca+2 concentrations of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525

Yousaf-wala

Hybrid

Leaf B Concentration

14.54 a 11.58 b 10.81 b 15.04 a 10.63 b 15.75 a NS NS ** NS NS NS 10.82

Leaf Ca+2 Concentration

4.32 a 3.41 b 3.32 b 3.39 b 4.34 a 4.41 a NS NS * NS NS NS 7.17

Appendix 4.19: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on vegetative and reproductive growth of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525

Yousaf-wala

HybridLeaf dry weight 26.45 a 20.64 b 20.21 b 23.51 b 22.32 b 28.13 a 25.66 a 21.43 b NS NS NS NS 13.79

Shoot dry weight 210.23 a 163.77 b 161.62 c 188.92 b 185.42 b 212.02 a 196.62 a 177.37 b NS NS NS NS 9.13

Tassel dry weight 12.97 a 9.70 b 9.71 b 11.67 a 11.59 ab 12.37 a NS NS NS NS NS NS 15.27

Silk dry weight 1.42 a 0.88 b 0.96 b 1.18 a 1.19 a 1.27 a 1.27 a 1.04 b NS NS NS NS 11.15

Silk threads per ear 560.34 a 488.89 b 482.91 c 520.93 b 528.26 b 566.36 a 555.84 a 493.39 b NS NS NS NS 3.89

Silk length 14.12 a 10.05 b 10.43 c 12.58 ab 11.34 bc 13.99 a NS NS NS NS NS NS 14.14Plant height 240.79 a 215.84 b 213.79 b 228.67 ab 232.02 ab 238.79 a NS NS NS NS NS NS 7.84

165

Appendix 4.20: Effect of foliar sprays of B (4 mg L-1), Ca+2 (4 mg L-1) and their combinations (4+40 mg L-1) on yield and yield components of two maize (Zea mays L.) hybrids under normal (100% WHC) and drought stress (30% WHC) conditions

Parameters/ Treatments

Water Stress Levels (W) Foliar B and Ca supplies (F) Hybrids (H)

W×F W×H F×H W×F×H CV100% WHC

30% WHC Control B Ca B+Ca Dekalb-

6525

Yousaf-wala

Hybrid

Number of grains ear-1 547.64 a 472.16 b 465.12 c 507.29 b 513.56 b 553.63 a 540.79 a 479.01 b * NS NS NS 3.96

1000-grain weight 236.72 a 195.17 b 192.72 c 208.80 b 220.33 b 241.92 a 223.78 a 208.10 b NS NS NS NS 6.19

Biological yield 21.73 a 17.62 b 18.40 b 19.81 ab 19.90 ab 20.60 a 20.25 a 19.10 b NS NS NS NS 7.27

Grain yield 5.85 a 4.58 b 4.68 b 5.20 a 5.39 a 5.59 a 5.68 a 4.75 b NS * NS NS 7.35Harvest index NS NS NS NS NS NS 27.98 a 24.94 b NS NS NS NS 10.43

166