maize (zea mays l.) responses to...

MAIZE (Zea mays L.) RESPONSES TO SUPPLEMENTAL

FOLIAR APPLIED PHOSPHORUS UNDER DROUGHT

STRESS

By

Zahoor Ahmad 2005-ag-1658

M.Sc. (Hons.) Agronomy

A thesis submitted in partial fulfillment of the requirement for the

degree of

DOCTOR OF PHILOSOPHY

in

Crop Physiology

Department of Agronomy,

Faculty of Agriculture,

University of Agriculture, Faisalabad, Pakistan.

2015

Declaration

I hereby declare that the contents of the thesis, “Maize (Zea mays L.) responses to

supplemental foliar applied phosphorus under drought stress” are product of my own

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

standard mathematical or genetic models/equation/formulate/protocols etc). I further

declare that this work has not been submitted for award of any other diploma /degree. The

university may take action if the information provided is found in accurate at any stage.

(In case of any default the scholar will be proceeded as against as per HEC plagiarism

policy).

Zahoor Ahmad

Regd. No. 2005-ag-1658

To,

The Controller of Examination,

University of Agriculture,

Faisalabad.

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

submitted by Zahoor Ahmad, Regd. No. 2005-ag-1658, have been found satisfactory

and recommend that it be processed for evaluation, by External Examiner(s) for the

award of the degree.

SUPERVISORY COMMITTEE:

Chairman: -------------------------------------------

Dr. EJAZ AHMAD WARAICH

Member: ------------------------------------------

DR. RASHID AHMAD

Member: -------------------------------------------

Dr. MUHAMMAD SHAHBAZ

Acknowledgements

I have only pearls of my eyes to admire the blessing of the compassionate and

omnipotent because words are bound, knowledge is limited and time is short to express

His dignity. ALMIGHTY ALLAH, the Propitious, the Benevolent and Sovereign Whose

Blessing and Glory flourished my thoughts and thrived my ambitions, giving me

affectionate parents, talented teachers, sweet brothers and unique friends.

Trembling lips and wet eyes praise for HOLY PROPHET HAZRAT MUHAMMAD

(P.B.U.H.) for enlightening our conscience with the essence of faith in ALLAH,

converging all His kindness and mercy upon him.

The work presented in this manuscript was accomplished under the sympathetic

attitude, scholarly comments and criticism, cheering perspective and enlightened

supervision of Dr. Ejaz Ahmad Waraich, Assistant Professor, Department of

Agronomy/Crop Physiology, University of Agriculture, Faisalabad.

I thank with deep motion of gratitude and great appreciation to Professor Dr. Rashid

Ahmad, Department of Agronomy/Crop Physiology and Dr. Muhammad Shahbaz,

Assistant Professor, Department of Botany, University of Agriculture, Faisalabad, for his

beneficial criticism, great support and sincere cooperation during the present study.

No acknowledgements could ever adequately express my obligations to my affectionate

and adoring parents, sisters and brothers (Particularly Mehboob Ahmad Malik), who

always raised their hands in prayers for me and without whose moral and financial

support; the present distinction would have merely been a dream.

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

and acknowledged for providing financial support for this study. I wish to express my

earnest thanks and deep appreciation to Dr. Muhammad Zia Ur Rehman, Assistant

Professor Institute of Soil and Environmental Science UAF, Dr. Masood Iqbal Awan,

Assistant Professor Department of Agronomy UAF, Dr. Tanveer Ahmad, Assistant

Professor Department of Horticulture, Ghazi University DG Khan, and Dr. Majid

Hussain, Assistant Professor Department of Food Science and Technology, BZU, for the

skilful guidance, constructive suggestions for the improvement of writing skills and above

all your patience. I wish to express my sincere thanks to my wife for indispensable

support. I also want to thanks my dear friends Rana Muhammad Sabir Tariq, Syed

Muhammad Zia-ul-Hassan Shah, Muhammad Sajjad Akhtar, Wasim Sajjad Hashmi,

Muhammad Azam, Muhammad Jaffir, Muhammad Aamir Iqbal, Rao Sajjad Sharif,

Muhammad Faisal, Usman Afzal, Fahad Munir, Ghulam Farid and Riaz Ahmad

whom moral support as well as guiding hands gave me a boost to accomplish my studies

and complete this endeavour in time.

Zahoor Ahmad [email protected]

(+92322-6008685)

mailto:[email protected]

C O N T E N T S

Chapter No. Title Page

1 Introduction 1

2 Review of literature 5

3 Materials and methods 32

4 Results 48

5 Discussion 122

6 Summary 137

- Literature cited 140

DETAILED CONTENTS

CHAPTER TITLE PAGE

Acknowledgements

Contents

Abstract

Chapter-I Introduction 1

Chapter-II Review of Literature 5

2.1 Overview of fertilizer use and needs in Pakistan 5

2.2 Drought effect on plants 6

2.2.1 Managing water stress in maize 9

2.2.2 Effect of water stress on germination and seedling growth 11

2.2.3 Physiological characteristics 13

2.2.4 Biochemical characteristics 15

2.2.5 Effect on yield and yield components 20

2.2.6 Breeding and genetics 22

2.3 Nutrient effect 23

2.3.1 Role of phosphorus in plants growth and development 23

2.3.2 Phosphorus use efficiency (PUE) and interaction with

other nutrients 25

2.3.3 Foliar nutrient uptake in plants 26

2.3.4 Foliar applied phosphorus 27

Chapter-III Materials and Methods 32

3.1 Experimental site and conditions 32

3.2 Maize germplasm collection 33

3.3 Laboratory experiments 34

3.3.1

Experiment # 1: Screening of different maize hybrids for

drought tolerance subjected to PEG-6000 induced water

stress

34

3.3.2 Experiment # 2: Screening of maize hybrids for drought

tolerance subjected to water stress 35

3.4 Experiment # 3: Optimization of phosphorus sources

in maize 35

3.5 Experiment # 4: Optimization of supplemental foliar

doses of phosphorus in maize 36

3.6 Experiment # 5: Optimization of stage of maize for

foliar applied phosphorus 37

3.7

Experiment # 6: The combined effect of optimum

source of P, dose of P and growth stage on

physiological and biochemical attributes of maize

hybrids under water stress

38

3.7.1 Seed sowing 38

3.7.2 Development and Maintenance of Water Stress Levels 38

3.7.3 Physiological parameters 39

3.7.3.1 Leaf water potential (Ψw) 39

3.7.3.2 Leaf osmotic potential (Ψs) 39

3.7.3.3 Turgor potential (Ψp) 39

CHAPTER TITLE PAGE

3.7.3.4 Relative water contents (RWC) 39

3.7.4 Gas exchange characteristics 40

3.7.5 Biochemical parameters 40

3.7.5.1 Chlorophyll contents 40

3.7.5.2 Total soluble proteins (TSP) 41

3.7.5.3 Total free amino acids (TFA) 42

3.7.5.4 Total soluble sugars (TSS) 43

3.7.5.5 Proline determination 43

3.7.6 Antioxidant enzymes 44

3.7.6.1 Catalase (CAT) 44

3.7.6.2 Peroxidase (POX) 44

3.7.6.3 Ascorbate Peroxidase (APX) 44

3.7.7 P determination 45

3.7.7.1 P concentration (%) 45

3.7.7.2 Phosphorus use efficiency (%) 45

3.8


source of P, dose of P and growth stage on yield and

yield components of maize hybrids under water stress

45

3.8.1 Yield and yield components 46

3.8.1.1 Cob length 46

3.8.1.2 Number of cobs per plant 46

3.8.1.3 Number of grains per cob 46

3.8.1.4 Cob weight without sheath 46

3.8.1.5 1000-grain weight 46

3.8.1.6 Grain yield (GY) 46

3.8.1.7 Biological yield (BY) 46

3.8.1.8 Harvest index (%) 46

3.9 Statistical Analysis 47

Chapter-IV Results 48

4.1

Experiment # 1: Screening of different maize hybrids

for drought tolerance subjected to PEG-6000 induced

water stress

48

4.1.1 Germination 48

4.2 Experiment # 2: Screening of maize hybrids for

drought tolerance subjected to water stress 51

4.2.1 Seedling growth 51

4.3 Experiment # 3: Optimization of phosphorus sources

in maize 54

4.3.1 Shoot length 54

4.3.2 Root length 54

4.3.3 Shoot fresh weight 55

4.3.4 Root fresh weight 55

4.3.5 Shoot dry weight 56

4.3.6 Root dry weight 56

4.3.7 Root-shoot ratio 57

CHAPTER TITLE PAGE

4.4 Experiment # 4: Optimization of supplemental foliar

doses of phosphorus in maize 62







4.4.7 Root-shoot ratio 65

4.5 Experiment # 5: Optimization of stage of maize for

foliar applied phosphorus 70







4.6


source of P, dose of P and growth stage on

physiological and biochemical attributes of maize


78







4.6.8 Photosynthetic rate (A) 85

4.6.9 Transpiration rate (E) 85

4.6.10 Stomatal conductance (gs) 86

4.6.11 Sub-stomatal CO2 rate (Ci) 86

4.6.12 Leaf water potential (-MPa) 90

4.6.13 Leaf osmotic potential (-MPa) 90

4.6.14 Leaf turgor pressure (MPa) 91

4.6.15 Relative water contents (%) 91

4.6.16 Chlorophyll a contents 94

4.6.17 Chlorophyll b contents 95

4.6.18 Total chlorophyll contents (a + b) 95

4.6.19 Total carotenoids 95

4.6.20 Total soluble proteins 99

4.6.21 Total free amino acid 99

4.6.22 Total soluble sugars 100

4.6.23 Proline contents 100

4.6.24 Catalase activity 103

4.6.25 Peroxidase activity 103

4.6.26 Ascorbate peroxidase activity 104

CHAPTER TITLE PAGE

4.6.27 P concentration in leaf 107

4.6.28 P concentration in stem 107

4.6.29 P concentration in root 108

4.6.30 P concentration in grain 108

4.6.31 Phosphorus use efficiency (PUE) 109

4.7 Experiment # 7: The combined effect of optimum

source of P, dose of P and growth stage on yield and

yield components of maize hybrids under water stress

113

4.7.1 Cob length (cm) 113

4.7.2 Number of cobs plant-1 113

4.7.3 Number of grains cob-1 114

4.7.4 Cob weight without sheath (g) 114

4.7.5 Thousand grain weight (g) 117

4.7.6 Grain yield (t ha-1) 117

4.7.7 Biological yield (t ha-1) 118

4.7.8 Harvest index (%) 118

Chapter-V Discussion 122

5.1 Screening of maize hybrids 122

5.2 Optimization of different sources of P in maize under

drought stress 124

5.3 Optimization of different rates of P in maize under

drought stress 125

5.4 Optimization of stage of maize for foliar P application

under drought stress 126

5.5 Physiological parameters 127

5.5.1 Gas exchange parameters 127

5.5.2 Water relations parameters 128

5.6 Biochemical parameters 129

5.6.1 Pigments 129

5.6.2 Total soluble sugars (TSS) 130

5.6.3 Total soluble proteins (TSP) 130

5.6.4 Total free amino acids (TFA) 131

5.6.5 Proline accumulation 131

5.6.6 Antioxidant enzymes 132

5.7 P concentration and PUE 133

5.8 Yield and yield components 134

5.9 Conclusion 136

Chapter-VI Summary 137

Literature Cited 140

Appendices 176

LIST OF TABLES

TABLE TITLE PAGE

3.1 Physiochemical characteristics of soil used for field

experiments 33

4.1 Analysis of variance (ANOVA) and mean parameter values of

germination percentage and promptness index for main effects

of hybrids under PEG (6000) induced water stress

50

4.2 Analysis of variance (ANOVA) and mean parameter values of

physiological indices for main effects of hybrids 53

4.3 Mean parameter values of growth parameters for main effects

of hybrids, Treatments, water levels and their interactions 61


of hybrids, Treatments, water levels and their interactions 69


of hybrids, stages, treatments and their interactions 77


of hybrids, water levels, treatments and their interactions 84

4.7

Analysis of variance table for photosynthetic rate, transpiration

rate, stomatal conductance and sub-stomatal CO2 rate of four

maize hybrids in well-watered and water stress conditions with

foliar applied phosphorus

87

4.8

Analysis of variance table for water potential, osmotic

potential, turgor pressure and relative water contents of four


supplemental foliar applied phosphorus

92

4.9

Analysis of variance table for chlorophyll a, chlorophyll b,

total chlorophyll contents and total carotenoids of four maize

hybrids in well-watered and water stress conditions with foliar

applied phosphorus

96

4.10

Analysis of variance table for total soluble proteins, total free

amino acids, total soluble sugars and proline contents of four


foliar applied phosphorus

101

4.11

Analysis of variance table for catalase activity, peroxidase

activity and ascorbate peroxidase activity of four maize

hybrids in well-watered and water stress conditions with foliar

applied phosphorus

105

4.12

Analysis of variance table for P concentration in grain, P

concentration in leaf, P concentration in stem, P concentration

in root and PUE of four maize hybrids in well-watered and

water stress conditions with foliar applied phosphorus

109

4.13a & b

Analysis of variance table for cob length, number of cobs

plant-1, number of grains cob-1, cob weight without sheath,

thousand grain weight (g) , grain yield (t ha-1) and biological

yield (t ha-1) of four maize hybrids in well-watered and water

stress conditions with supplemental foliar applied phosphorus

121

LIST OF FIGURES

FIGURE TITLE PAGE

2.1 Vegetative and reproductive stages of maize 11

3.1 Meteorological data of the experimental site for the growing

season 2013-14. 33

4.1

Germination percentage of eight maize (Zea mays L.) hybrids

under PEG (6000) induced water stress regimes (mean values ±

S.E).

48

4.2

Promptness index (PI) of eight maize (Zea mays L.) hybrids

under PEG (6000) induced water stress regimes (mean values ±

S.E).

49

4.3

Germination stress index (GSI) of eight maize (Zea mays L.)

hybrids under PEG (6000) induced water stress regimes (mean

values ± S.E).

49

4.4 Plant height stress tolerance index (PHSI) of eight maize (Zea

mays L.) hybrids under water stress regimes (mean values ± S.E) 51

4.5 Root length stress tolerance index (RLSI) of eight maize (Zea

mays L.) hybrids under water stress regimes (mean values ± S.E). 52

4.6 Dry matter stress tolerance index (DMSI) of eight maize (Zea

mays L.) hybrids under water stress regimes (mean values ± S.E). 52

4.7

Effect of different sources of phosphorus on shoot length (cm) of

four maize hybrids in well-watered (100% FC) and water stress

(60% FC) conditions (mean values ± S.E).

57

4.8

Effect of different sources of phosphorus on root length (cm) of


(60% FC) conditions (mean values ± S.E).

58

4.9

Effect of different sources of phosphorus on shoot fresh weight

(g) of four maize hybrids in well-watered (100% FC) and water

stress (60% FC) conditions (mean values ± S.E).

58

4.10

Effect of different sources of phosphorus on root fresh weight (g)

of four maize hybrids in well-watered (100% FC) and water

stress (60% FC) conditions (mean values ± S.E)

59

4.11

Effect of different sources of phosphorus on shoot dry weight (g)



59

4.12

Effect of different sources of phosphorus on root dry weight (g)



60

4.13

Effect of different sources of phosphorus on root-shoot ratio of


(60% FC) conditions (mean values ± S.E)

60

4.14




65

4.15 Effect of different sources of phosphorus on root length (cm) of 66

FIGURE TITLE PAGE



4.16




66

4.17




67

4.18




67

4.19




68

4.20

Effect of different sources of phosphorus on root-shoot ratio (%)



68

4.21




73

4.22

Effect of different sources of phosphorus on root length (cm) of



74

4.23




74

4.24




75

4.25




75

4.26




76

4.27

Effect of supplemental foliar phosphorus application on shoot

length (cm) of four maize hybrids grown under normal and water

stress conditions (mean values ± S.E).

81

4.28

Effect of supplemental foliar phosphorus application on root

length (cm) of four maize hybrids grown under normal and water


81

4.29 Effect of supplemental foliar phosphorus application on shoot 82

FIGURE TITLE PAGE

fresh weight (g) of four maize hybrids grown under normal and

water stress conditions (mean values ± S.E).

4.30

Effect of supplemental foliar phosphorus application on root

fresh weight (g) of four maize hybrids grown under normal and


82

4.31

Effect of supplemental foliar phosphorus application on shoot dry

weight (g) of four maize hybrids grown under normal and water


83

4.32

Effect of supplemental foliar phosphorus application on root dry

weight (g) of four maize hybrids grown under normal and water


83

4.33

Effect of supplemental foliar phosphorus application on net

photosynthesis rate (A) of four maize hybrids grown under

normal and water stress conditions (mean values ± S.E).

88

4.34

Effect of supplemental foliar phosphorus application on

transpiration rate (E) of four maize hybrids grown under normal

and water stress conditions (mean values ± S.E).

88

4.35

Effect of supplemental foliar phosphorus application on stomatal

conductance (gs) of four maize hybrids grown under normal and


89

4.36

Effect of supplemental foliar phosphorus application on sub-

stomatal CO2 rate (Ci) of four maize hybrids grown under normal


89

4.37

Effect of supplemental foliar phosphorus application on water

potential (-MPa) of four maize hybrids grown under normal and


92

4.38

Effect of supplemental foliar phosphorus application on osmotic

potential (-MPa) of four maize hybrids grown under normal and


93

4.39

Effect of supplemental foliar phosphorus application on turgor

pressure (MPa) of four maize hybrids grown under normal and


93

4.40

Effect of supplemental foliar phosphorus application on relative

water contents (%) of four maize hybrids grown under normal


94

4.41


chlorophyll a (mg/g fresh weight) of four maize hybrids grown

under normal and water stress conditions (mean values ± S.E).

97

4.42


chlorophyll b (mg/g fresh weight of four maize hybrids grown


97

4.43 Effect of supplemental foliar phosphorus application on total 98

FIGURE TITLE PAGE

chlorophyll contents (mg/g fresh weight of four maize hybrids

grown under normal and water stress conditions (mean values ±

S.E).

4.44

Effect of supplemental foliar phosphorus application on total

carotenoids (mg/g) of four maize hybrids grown under normal


98

4.45


soluble protein (mg/g fresh weight of four maize hybrids grown


101

4.46

Effect of supplemental foliar phosphorus application on total free

amino acid (mg/g fresh weight of four maize hybrids grown


102

4.47


soluble sugar (mg/g fresh weight of four maize hybrids grown


102

4.48

Effect of supplemental foliar phosphorus application on proline

contents (µg/g fresh weight of four maize hybrids grown under

normal and water stress conditions (mean values ± S.E).

103

4.49

Effect of supplemental foliar phosphorus application on catalase

activity (Unit min-1 g-1 fresh weight of four maize hybrids grown


105

4.50


peroxidase activity (Unit min-1 g-1 fresh weight of four maize

hybrids grown under normal and water stress conditions (mean

values ± S.E).

106

4.51

Effect of supplemental foliar phosphorus application on ascorbate

peroxidase activity (Unit min-1 g-1 fresh weight of four maize

hybrids grown under normal and water stress conditions (mean

values ± S.E).

106

4.52

Effect of supplemental foliar phosphorus application on P

concentration in leaf of four maize hybrids grown under normal


110

4.53


concentration in stem of four maize hybrids grown under normal


110

4.54


concentration in root of four maize hybrids grown under normal


111

4.55


concentration in grain of four maize hybrids grown under normal


111

4.56

Effect of supplemental foliar phosphorus application on PUE of

four maize hybrids grown under normal and water stress

conditions (mean values ± S.E).

112

FIGURE TITLE PAGE

4.57

Effect of supplemental foliar P application on cob length (cm) of

four maize hybrids under different water levels (mean values ±

S.E).

115

4.58

Effect of supplemental foliar P application on number of cobs per

plant of four maize hybrids under different water levels (mean

values ± S.E).

115

4.59

Effect of supplemental foliar P application on number of grains

per cob of four maize hybrids under different water levels (mean

values ± S.E).

116

4.60

Effect of supplemental foliar P application on cob weight without

sheath (g) of four maize hybrids under different water levels

(mean values ± S.E).

116

4.61

Effect of supplemental foliar P application on 1000-grain weight

(g) of four maize hybrids under different water levels (mean

values ± S.E).

119

4.62

Effect of supplemental foliar P application on grain yield (t ha-1)

of four maize hybrids under different water levels (mean values ±

S.E).

119

4.63

Effect of supplemental foliar P application on biological yield (t

ha-1) of four maize hybrids under different water levels (mean

values ± S.E).

120

4.64

Effect of supplemental foliar P application on harvest index (%)

of four maize hybrids under different water levels (mean values ±

S.E).

120

14

ABSTRACT

Water-limited conditions in early growth stages negatively affect germination and seedling

growth, often leading to suboptimal plant population and poor stand establishment. Germination

and seedling growth of eight hybrids of maize (Zea mays L.) genotypes in response to induced

water stress conditions and supplemental foliar fertilisation with P was investigated. In two

laboratory experiments, the observed germination parameters and calculated stress indices were

used as screening criteria for drought tolerance. Germination parameters viz. germination

percentage, promptness index and germination stress tolerance index declined in response to the

increasing polyethylene glycol induced stress levels. Water stress conditions imposed by with-

holding irrigation at seedling stage reduced plant height stress tolerance index and dry matter

stress tolerance index but increased root length stress tolerance index. Based on the results of

germination attributes and stress indices, 6525 and 32B33 were the most drought tolerant

genotype and Hycorn and 31P41 was the most drought sensitive genotype among all tested

genotypes. In wire house experiments, the source, dose and time for P application were

optimised. In 3rd experiment the KH2PO4 was the best source to improve all the growth

parameters under normal and stress conditions. Both the maize hybrids 6525 and 32B33

performed better than Hycorn and 31P41 under and normal and stress conditions. In 4 th

experiment the growth of maize was improved in 6525 and 32B33 than Hycorn and 31P41 where

P was applied @ 8 kg ha-1 as compared to all others doses under well watered and stress

conditions. In 5th experiment, the P applied @ 8 kg ha-1 when stress was applied at 8th leaf stage

of maize. Both maize hybrids 6525 and 32B33 improved growth of maize than Hycorn and

31P41. Subsequently, the best combination of P source, dose and time for P application was

tested in wire house / rain out shelter and field conditions. In 6th experiment the supplemental

folia applied P spray improved the water relations, gas exchange characteristics (i.e. through

accumulation of soluble sugars), total free amino acid and proline. The antioxidant activity was

also improved with foliar P spray at 8th leaf stage. In field experiments (Exp. # 7) supplemental

foliar application of P improved the number of grains per cob and 1000-grain weight, which

ultimately increased the grain yield at 8th leaf stage in normally irrigated plants as well as under

water stress conditions in all maize genotypes. The drought tolerant 6525 and 32B33 performed

better under well watered under water stress.

15

Maize is the third important cereal crop in the world after wheat and rice on the basis of area

and production (Tollenaar and Dwyer, 1999). In addition to use as food and feed for livestock

and poultry, maize grains are also utilized in many other commercial and industrial products. For

human consumption it is processed into a lot of products such as corn flour, pop corns, gruels,

porridges, bread, beverages, snacks and pastes (Ortiz-Monasterio et al., 2007; Menkir, 2008).

Increasing importance of maize in human food and nutrition could be estimated by its global

average production of 780 million tonnes during the year 2007-08. Maize added 22.2% in

Agriculture and 0.5% to GDP in Pakistan. In last year 0.2% maize crop was grown in less area

but its production 6.85 was increased. The yield per hectare in 2012-13 stood at 4268 (Kg ha-1)

posted a positive growth of 6.9% as compared to 4.9% growth last year. The production of maize

is enhanced due to using hybrid seeds and favourable environmental condition (Govt. of

Pakistan, 2014).

Maize being a C4 plant is more efficient user of CO2 and water in photosynthesis than C3

plants. Under water stress conditions, maize usually begins to feel stress when air temperature

exceeds 32oC during the tasseling, silking and grain filing stages. High temperature affects plant

growth and reduces grain yield by inhibiting photosynthesis and reducing pollination due to

pollen abortion and desiccation of silks. However, unlike photosynthesis, pollination generally

occurs in a relatively narrow span of plant’s life. Once the plant is pollinated, the developing

fruit is totally dependent on the supply of photosynthates. The activation state of Rubisco,

however, decreases progressively at temperatures above 30°C (Steven et al., 2002). This

inactivation of Rubisco effectively prevents photosynthesis with higher leaf temperature. Hence

decreases grain filling in maize. Maize is typically a monoaecious plant with staminate and

pistilate late flowers, separated much higher from the ground and prone to desiccating

temperature. Temperature affects the rate of photosynthesis and respiration. Maize needed

optimum temperature range is 22 to 32oC for day and 16.7 to 23.3oC for night for their growth

and development. The growth of maize was affected when the temperature fall below 5oC and

increase in temperature from 32oC because at high temperature the enzymes are inactive. Some

enzyme are completely inactivate when the temperature increase from 45°C (Steven et al., 2002).

16

Drought is the most important abiotic stress factor that limiting the all aspects of crop growth

and production. In many developing countries the water stress is the major constraint to

agricultural production and also reduced the quality, growth and production of crops (Golbashy

et al., 2010; Waraich et al., 2010, 2011; Ahmad et al., 2015). Worldwide, drought is considered

to be the leading cause of crops yield losses which reduces yield up to 50% depending upon its

severity (Wang et al., 2003). It is a recognized fact that a variety of metabolic and physiological

processes in plants are impaired by drought (Levitt, 1980). Significant reduction in growth,

chlorophyll and water contents along with different fluorescence parameters are changed with

drought (Ekmekçi et al., 2005; Mohsenzadeh et al., 2006; Yang et al., 2006). Impaired active

transport, reduced transpiration and membrane permeability caused by drought result in

decreased nutrients uptake due to decreasing power of roots to absorb water (Tanguilig et al.,

1987). Photosynthetic process in plants is affected to a great extent by drought. Reduced

photosynthetic activity caused by drought is due to the fact that there are several stomata related

limitations. (Shangguan et al., 1999; Yordanov et al., 2003; Zlatev and Yordanov, 2004) as the

first response offered by plants to drought is closure of stomata at once. this closure of stomata

though help in reducing water loss but it also causes significant reduction in CO2 absorption as

most field crops can absorb it through stomata only (Nayyar and Gupta, 2006; Yang et al., 2006).

In arid and semi- arid regions of the world the water stress seriously cause problems by limits

the agriculture production. The life cycle of different plants usually affected by water stress and

plants face water deficit from atmosphere and soils during its life cycle (Chaves et al., 2002).

Many growth variables (root length and shoot length etc.) of the plants are affected by water

stress. Different functions and fitness of the plants also reduced under water stress condition

(Tian and Lei, 2006 and Xu et al., 2007). Under water stress conditions the reactive oxygen

species (ROS) are produced and these ROS cause the serious problems inside the plants

(Waraich et al., 2011). The intake of CO2 is stopped due to close stomata and inside stomata the

more oxygen is produced that cause the production of ROS under water stress condition. After

ROS production the membranes are raptured and become leaky and which affect the rate of

respiration, photo-synthesis and growth of plant. The ROS also seriously damage the production

of many cellular constitutes such as lipids, proteins, nucleic acid and carbohydrates under

drought stress (Waraich et al., 2011).

17

Phosphorus (P) is the 2nd major nutrient after nitrogen (N) and it is deficit in Pakistan soils.

The growth and yield of many field crops are enhanced by the application of P and also

increased the root growth in many crops under water stress condition (Yaseen and Malhi, 2009).

For instance, 90% soils are deficient in nitrogen (N) and phosphorus (P) and 40% in potassium in

Pakistan (Ahmad and Rashid, 2004). Among all the nutrient elements required by a plant, P has

prime importance for crop production and emphasis is being given on the efficient use of P

fertilizers for sustainable crop production (Ryan, 2002). In Pakistan, over 90% soils are low in

available P, suffering from moderate to severe P deficiency due to alkaline calcareous nature of

soil (Iqbal et al., 2003).

It is investigated by the different scientists that the additional dose of P is used to increase

the over-all production of crops in Pakistan soil (Shah et al., 2006; Jabran et al., 2011). The

phosphorus application decreases the effect of drought in crops (Sing et al., 1981). Phosphorus is

an essential element for all living organisms and involved in nucleic acid and phospholipids

synthesis. It also activates many enzymes (Lambers et al., 2006). Phosphorus also plays a key

role in energy transfer and is thus essential for photosynthesis under drought condition. The

deficiency of P causes net photosynthesis reduction and decreased shoot and root biomass

production in maize crop (Wissuwa et al., 2005). Leaf growth depression under P deficiency is

well documented (Kavanova et al., 2006). Phosphorus deficiency affects the rate of emergence

and number of maize adventitious nodal roots (Pellerin et al., 2000; Kavanova et al., 2006). Its

deficiency also caused severely reduced LAI in maize that results low PAR absorption by canopy

and reduced crop growth (Pellerin et al., 2000).

Soils in Pakistan are naturally low in phosphorus and to maintain soil phosphorus at optimum

levels, it is inevitable to supplement maize crop with additional source of phosphorus, especially

under drought stress when nutrient absorption and uptake at root level is limited due to water

shortage in soil. Foliar application of phosphorus could improve phosphorus use efficiency

(PUE) by minimizing soil application in maize, when supply of nutrients either become deficient

or needed the most under water stress condition (Girma et al., 2007). This practice has gained

popularity in recent years. A reasonable research work on improving use efficiency of soil

applied phosphorus under drought stress has been reported in the literature, however very little

information is available on the interactive effect of foliar applied and soil applied phosphorus in

18

alleviating the adverse effect of drought stress especially in maize. The present study was carried

out with the following objectives:

To study the effect of water stress on various physiological and biochemical attributes of

hybrid maize

To assess the effect of supplemental foliar applied phosphorus on growth, yield,

physiological and biochemical traits of hybrid maize grown under water stress conditions

To find out optimum dose, time and source of phosphorus application for improving

drought tolerance in maize

19

Enhancing the plant growth as well as yield of different crops under water stress

condition has become an important method in different region of the world (Zhang et al., 2008).

The role of phosphorus (P) in plants under water stress conditions is an important. Many

researchers have adopted different mechanisms to evaluate the role of P in plants under drought

stress. Some of the relevant work available about the role of P in alleviating adverse effect of

drought stress is discussed in this chapter.

2.1: Overview of fertilizer use and needs in Pakistan

The total cropped area in Pakistan is about 22.2 million ha. The share of food grain crops

is 54%, followed by cotton and sugar cane 20%; pulses 6%; oilseed crops 3%; fruit/vegetables

4% and other crops about 13%. The wheat is the main food crop; it occupies about 36.3% of the

total cropped area, followed by cotton (14%), paddy (9.5%), sugar cane (4.5%), maize (4.5%)

and other crops (20.8%) (Govt. of Pakistan, 2014).

Fertilizers constitute a key component of the modern farm technology for achieving

increased production through improving soil fertility. The introduction of the high yielding

cereal varieties in 1966-67, having higher nutrient requirements, ushered in the ‘fertilizer era’ in

Pakistan and set the stage for ‘green revolution’. Prior to this, the use of fertilizer was nominal

(NFDC, 2000). Application of commercial fertilizers in Pakistan began in 1952-53, and the off-

take was only 1,000 nutrient tonnes of nitrogen. Phosphorus was introduced to farmers in 1959-

60 with an initial usage of 100 nutrient tonnes. Potash fertilizer off-take started in 1966-67 with a

volume of 120 nutrient tonnes. These trends in fertilizer usage emphasized the importance and

role of fertilizer in the economy of Pakistan. There has been a continuous rise in the consumption

of fertilizers. During the year 2000-01 total fertilizer sales were 2966,000 nutrients tonnes,

augmented by 851 percent from 312,000 nutrient tonnes sold in 1969-70. The major increase was

for nitrogen, which increased by 822 percent, i.e. from 274,000 nutrients tonnes to 2526 (000)

nutrient tonnes. Nitrogenous fertilizers now account for 78 percent of commercial fertilizer off-

take in Pakistan with phosphorous and potash accounting for 21 and about 0.7 percent,

respectively (NFDC, 2000).

20

Dholakia and Jagdip (1995) derived the fertilizer demand function in India and they

estimated short-run and long-run price elasticity. They found that fertilizer demand in India is

price inelastic both in the short-run and in the long-run. Hansen (2004) estimates nitrogen

fertilizer demand elasticity’s for Danish crop farms using the dual profit function approach on

micro panel data. The model includes several farm specific parameters, allowing estimating the

mean demand elasticity and testing for homogeneity of elasticity’s across panel farms. The mean

own price elasticity for nitrogen is –0.45, and there is a significant standard deviation from this

mean for individual farms of 0.24.

2.2: Drought effects on plants

The availability of water is one of the major abiotic factors for optimum plant growth, as

it affects many physiological and biochemical processes (Ashraf et al., 1994; Ahmad et al.,

2015). The severity, timing and period of stress as well as genetic variation and the growth stage

of the plant define its response to the imposed stress (Jefferies, 1995). The loss of turgor at low

relative water content improves the plants tolerance to the internal water deficit which also helps

to maintain the chloroplast function under water stress conditions (Gupta and Berkowitz, 1987;

Ranney et al., 1991). The decrease in relative water contents and water potential of leaves reduce

metabolism and carbon assimilation during photosynthesis in higher plants (Lawlor and Cornic,

2002). Ludlow et al. (1985) reported leaf turgor maintenance as an important adaptation trait for

stomatal regulation under drought stress. The water stress restricts cell expansion as plants lose

their turgor leading to reduced growth (Turner, 1986). Osmotic adjustment is an important

phenomenon that plays a vital role in improving drought tolerance in plants (Blum and Sullivan,

1986; Ludlow and Muchow, 1990). Ashraf and O'Leary (1996) observed that water stress

reduces osmotic potential of leaves and suggested that plants maintaining their water potential

under drought are more tolerant.

Water balance parameters in drought resistant line 1304 and susceptible line 389 in maize

exposed to 4 to 7 days of drought at the 5-leaf stage was studied. When influence of drought on

water regime was checked, total osmotic potential values were lower in both lines, (Jovnovic et

al., 1985). There was variation in effects of moisture stress on seed germination in sorghum.

Germination decreased with increase in level of water stress. In general, mean germination was

higher at moderate moisture than at low soil moisture conditions (Bijagare et al., 1994; Maiti et

al., 1996). When plants were re-watered after a short period of water stress, leaf elongation rate

21

was very rapid for a short time, but rate of growth after re-watering did not return to normal in

severely stressed plants (Hsiao and Jing, 1987). Westgate and Grant (1989) reported that the

response of productive tissues to plant water deficits varies with stage of grain development. The

high sensitivity to plant water deficits is higher in early reproductive development while it

decreases as reproduction progresses. Various mechanisms involved in drought resistance like

escape, avoidance, and tolerance etc were pointed out by many scientists (Arnon, 1972;

Osmanzai et al., 1987; Acevedo et al., 1991 and Acevedo et al., 1993).

Physiological characters were proposed for the assessment of drought tolerance (Martin

et al., 1989 and Castonguay and Markhart, 1992). Fang et al. (2011) were conducted a pot

experiment to study the physiological and morphological characteristics of maize at seedling

stage, growing under water stress condition. Three drought sensitive (DP-15, DP-27 and DP-35)

and two drought tolerant (DP-68 and DP-65) maize hybrids were used in this experiment.

Results showed that the drought tolerant hybrid accumulated more biomass and hence increased

the growth of plant. The production of antioxidant enzymes such as POD, SOD etc inside the

plant body under stress condition; enhanced the growth, physiological and morphological

characters of maize. Campos et al. (2004) studied that kernels per plant under drought conditions

can be increased by exploiting native genetic variation among elite breeding lines but

improvements in root distribution and function may need additional genetic variation from other

species. While Moreno et al. (2005) studied several strategies to develop transgenic maize lines

with improved drought tolerance.

Song and Dai (2000) studied the effect of drought stress on corn growth and development

of corn female flowers. Also height, diameter, dry and fresh weight reduce by water deficit.

Cakir (2004) reported that water stress during different corn growth stages decreases yield in

different degrees of intensity. The decrease of yield depends on both stress severity and plant

growth stage. Rezaverdinejad et al. (2006) by applying low irrigation treatments at different

growth stages of forage corn in Karaj (Iran) reported that drought stress at vegetative and

flowering stages compared to normal irrigation reduced yield by 28% and 29%, respectively.

Pandey et al. (2000) reported that deficit irrigation at early vegetative growth, slightly decrease

leaf area index, plant height, plant growth rate and dry matter in maize.

Ouattar et al. (1987) reported that water deficits in maize plants decreased photosynthesis

as judged by total plant dry weight, and concluded that grain growth was more sensitive to water

22

deficits during endosperm cell division than during the period of starch deposition. Rahman and

Hassaneinn (1988) reported that fresh and dry weight was decreased in maize with decreasing

soil moisture. Alam (1985) pointed out that water-limited conditions cause shoot elongate to a

great extent and particularly water stress causes noticeable damage at vegetative growth stages.

A variety of field trials showed that seed germination as well as seedling vigour is reduced to a

great extent in maize as water stress reduced seedling dry matter indices as well as germination

percentage (Lemcoff et al., 1998). Nagy et al. (1995) studied the response of maize and sorghum

seedlings under water stress and reported that leaf water content declined more in maize than in

sorghum.

Matsuura et al. (1996) also studied the drought tolerance in four crops (barnyard millet,

maize, pearl millet and sorghum). The irrigation was stopped in some plots 16 days after sowing

and was continued in others. Pearl millet and sorghum, which were identified as drought tolerant,

displayed the lowest reductions in relative growth rate. The stomatal conductance correlated with

leaf xylem water potential significantly. Pearl millet and sorghum showed the highest leaf water

status. Under water stress, total root length was significantly reduced in maize, was not affected

in barnyard millet, and was significantly increased in sorghum and pearl millet. Drought

tolerance in sorghum and pearl millet was associated with sustained water uptake ability by

increasing total root length and maintenance of high leaf water status under soil drying

conditions at the vegetative growth stage. Grzesiak (2001) conducted an experiment in

glasshouse and reported that different genotypes of maize are best tested when exposed to severe

water-limited conditions as compared to mild water stress conditions. Therefore global food

security depends on the development of crop plant with increased resistance to abiotic stresses

such as drought and salinity. Development of drought tolerant varieties is the cheapest method to

overcome the problem. Campos et al. (2004) studied that kernels per plant under drought

conditions can be increased by exploiting native genetic variation among elite breeding lines but

improvements in root distribution and function may need additional genetic variation from other

species. While Moreno et al. (2005) studied several strategies to develop transgenic maize lines

with improved drought tolerance.

Maize is considered to be more prone to drought at stages when plant switches to

separate its male and female parts (Grant et al., 1989). Drought is attributed to cause a significant

delay in silking while the timing of pollen shed are not effected to noticeable level. Different

23

studies have shown that female part of maize crop is more prone and susceptible to water stress

conditions in comparison with male parts (Moss and Downey, 1971; Herrero and Johnson,

1980). Westgate and Boyer (1986) reported that there was a significant change in water potential

of silk when the plants were exposed to water stress conditions while the change in the water

potential of pollen was negligible. They reported that stigmatic tissues in male parts were in

contact with vegetative tissues and this hydraulic contact prevented change in water potential of

pollen. Boyle (1990) by utilizing stem infusions of sucrose solution showed that the drastic

effects caused by drought at flowering stages of maize may alleviated, though partially only. It

was also reported by them that silk delay caused by water limited conditions may a symptom

showing less assimilates partitioning rather than the leading reason of bareness. They also

described that silking delay caused by water stress resulted in much increase in anthesis to

silking interval. Earlier researches have reported that there can be as much as 82% reduction in

grain yield when there is an increase in anthesis to silking interval from 0 to 28 days (DuPlessis

and Dijkhuis, 1967, as reported in Edmeades et al., 1993). Subsequent researches showed that

the increased grain yield of maize in temperate climate was due to better utilization of resources

and better resource capturing (Tollenaar and Lee, 2006). Mostafavi et al. (2011) in a study of

four maize hybrids in drought stress condition reported that hybrid KSC704 was tolerant, while

KSC500 was sensitive to drought. Khayatnezhad et al. (2010) and Mostafavi et al. (2011) in a

study of four corn hybrids in drought stress conditions also reported that hybrid golden west and

KSC-704 had the highest root, shoot and seedling length, respectively.

2.2.1: Managing water stress in maize

Maize being a C4 plant is more efficient user of CO2 and water in photosynthesis than C3

plants. Maize is typically a monoaecious plant with staminate and pistilate late flowers, separated

much higher from the ground and prone to desiccating temperature. Temperature affects the rate

of photosynthesis and respiration. The optimal day temperature ranges from 22 to 32oC and night

temperature ranges from 16.7 to 23.3oC for maize plant. At this temperature the photosynthetic

rate is rapid than respiration resulting in enhanced plant growth. Growth is affected adversely

when the temperature decreases to 5oC or increases from 32oC (Steven et al., 2002).

It is a matter of fact that very little can be done by farmers to avoid the drastic effects

caused by drought; however chances to minimize the damages caused by water limited

conditions can be increased if one has good knowledge of critical stages as these stages are most

24

important to be managed with. Moreover, appropriate management and by storing water in soil

before the drought sets in have the potential to increase the chances of a successful crop

husbandry (Heinigre, 2000).

The following strategies may go a long way in reducing the drastic effects inflicted by

drought:

• Matching the cropping season with periods of rains or drought while keeping in mind the

critical growth stages of crop

• Use no-till technique for maize plantation in order to conserve plentiful moisture in the

soil

• Reduce the tillage practices to the maximum level in order to reduce the evaporation

which gears up with soil opening causing hefty losses of water

• Herbicides must be applied judiciously and wisely keeping in mind that at the time of

herbicide application, crop should not be in stress conditions.

• Earlier application of nutrients with side dressing has the potential to supply nutrients

well advance of deficiencies appearance.

• In-row sub-soiling has the potential to increase the root penetration so much so that

drought effects are alleviated to a great extent.

• Several drought tolerant and resistant genotypes are present which should be cultivated.

• Sun-optimal plant population may also help to reduce water limited conditions drastic

effects (i.e. populations of 18,000 to 20,000 plants per acre)

• Low cost irrigation systems like tube well or moving pipe drip irrigation system must be

developed to reduce the effects of drought (Heinigre, 2000).

25

Vegetative Stages Reproductive Stages

VE= emergence (0 day) R1= silking (55-65 days)

V1= first leaf R2= blister (77 days)

V2= second leaf (7 days) R3= milk (85 days)

V3= third leaf R4= dough (91 days)

V4= fourth leaf R5= dent (101 days)

V5= fifth leaf (14 days) R6= physiological maturity (115-120 days)

V8= eighth leaf (28 days)

V12= twelfth leaf (42 days)

V16= sixteenth leaf (55 days)

VT= tasseling (55-65 days)

Vegetative and reproductive stages of corn

(Lauer, 2003; Omafra, 2002; Weedsoft, 2006; Wyffels, 2006; North Dakota State

University Extension Service, 1999 and Coffman, 1998)

2.2.2: Effect of water stress on germination and seedling growth

Water stress is a major environmental restriction in crop productivity. It was the reagent

of the great famines of the past. Because the demand for the food is increasing rapidly with

increasing population as compared to world’s water supply which is limiting (Somerville and

26

Briscoe, 2001). The drought harshness is unchangeable as it depends on many factors such as

evaporative demands, occurrence and distribution of rainfall and moisture storing capacity of

soils (Wery et al., 1994). Plants are faced many types of stresses while growing in nature such as

drought, low temperature, heat, salt and heavy metal toxicity. However one of major factor

limiting plant growth and yield worldwide is drought (water stress). Water deficit is a term that is

usually described as shortage of water that is necessary for a plant to complete its lifecycle and

grow normally (Manivannan et al., 2007). Drought is usually well defined as a period without

significant rainfall and it is meteorological term in which atmospheric conditions cause

continuous loss of water by evaporation or transpiration due to which available water contents

may be reduced. All plants show drought stress tolerance but it extents varies from species to

species and even within species. The major hurdle in sustainable food production and agriculture

crops is the water deficit (Jaleel et al., 2007).

The first critical and most sensitive stage considered in the life cycle of plant is seed

germination. High temperature, low atmospheric humidity and water deficit are major factors

that lead to drought and have negative effects on seed germination, plant growth and

development and in purpose of obtaining higher yield. Therefore the plants which are more

efficient in water use efficiency can produce better yield under limited environmental conditions

(Ahmad et al., 2009). Seedling establishment can be inhibited by the contrary environmental

conditions like water stress. Water stress has negative effects on germination and seedling

growth of many crops such as sorghum, maize, wheat and sunflower as reported by Oomah et al.

(2000). Like other oil seed crops sunflower is also particular sensitive to water shortage at

germination stage. Under water stress conditions seed germination may be decreased due to

some metabolic disorders because at imbibition’s and seed turgescence stages water absorption

may be reduced due to low water availability that results in low germination percentage and

germination rate (Hadas, 1977).

There are many hindrances faced by seed during germination among that water stress is

prominent one. Sunflower germination is susceptible to water shortage. As osmotic stress

increases a reduction in present germination and biomass accumulation has been observed

(Sajjan et al., 1999). An increment in mean germination time is more noticeable in response to

water deficit. In germination studies polyethylene glycol (PEG-6000) can be used to control

water potential in seeds. It has been observed that polyethylene glycol (PEG-6000) effects on

27

seeds germination by reducing the water availability to seeds, excessive absorption of nutrients

and metabolic disorder in protein synthesis (Bouaziz et al., 1990).

Dhanda et al. (2004) observed considerable variation for germination percentage, seed

vigor index, shoot length, root length, coleoptile length, root-to-shoot length ratio, and osmotic

membrane stability in thirty diverse bread wheat genotypes. They reported seed vigor index as

the most sensitive trait, followed by shoot length, germination percentage and root length. An

increase in root-to-shoot length ratio was recorded under osmotic stress. In a similar study, the

drought tolerance of twenty promising durum wheat genotypes was quantified by Moayedi et al.

(2009) using physiological indices. The germination stress tolerance index (GSTI) of all

genotypes was reduced under osmotic stress.

Shiralipour and West (1984) studied the response of maize seedlings under drought

condition. Drought reduced fresh and dry root and shoot weight by 58 and 40 percent,

respectively. Stress decreased the length and fresh weight of shoot and shorts in maize (Thakur

and Rai, 1984). Shoot elongation was reduced by water stress during vegetative period in maize.

In the green-house experiment to study the effect of water stress on the vegetative and root

growth of maize plants, found water stress reduced the shoot and root growth (Ramadan et al.,

1985) while Hoogenboom et al. (1987) observed that root weight increases under moisture stress

indicating greater density and greater depth of root penetration, both are important

morphological adaptations to moisture deficit and result in greater extraction of soil water.

Drought tolerant cultivars produced more dry and fresh weights of shoots compared to

susceptible one (Ashraf, 1989). Wu and Cosgrove (2000) found the root/shoot ratio of plants

increased when water availability is limiting. This ratio increases because roots are less sensitive

than shoots to growth inhibition by low water potentials.

2.2.3: Physiological characteristics

Water shortages and losses of soil water due to environmental change are challenges to

maize production. Hura et al. (2007) and Efeoglu et al. (2009) studied physiological response of

maize cultivars under drought stress condition in south of Poland and Turkey respectively.

Growth of cultivars was retarded under drought conditions. Relative water content decreased in

maize cultivars by drought and also fresh and dry biomass of the cultivars significantly decreased

while drought affected the minimum fluorescence. On the other hand chlorophyll a, b, total a+b

and carotenoid contents of maize cultivars were significantly reduced under drought. According

28

to Earl and Davis (2003) drought stress reduced maize yield by reducing canopy absorption of

photosynthetically active radiation (PAR), radiation use efficiency, and harvest index. Mild and

severe drought stress reduced grain yield up to 63% and 85%, respectively. On the other hand

crop dry matter accumulation was not linearly related to reduction of radiation use efficiency

under water stress. Drought can influence photosynthesis either through pathway regulation by

stomatal closure and decreasing flow of CO2 into mesophyll tissue (Chaves et al., 2004; Flexas

et al., 2004) or by directly impairing metabolic activities (Farquhar et al., 1989). While Medici et

al. (2003) reported drought stress in both vegetative and reproductive phases significantly

decreased the relative leaf contents and leaf osmotic potential. On the other hand, the evaluation

of relationship between relative leaf contents and leaf osmotic potential allowed a clear

difference in response to the water stress and normal condition in maize plant.

The leaf water potential has been reported to affect many other physiological processes

such as stomatal conductance, transpiration rate, photosynthesis and nutrient uptake in plants.

The non-availability of water from early or mid-grain filling until maturity reduced water

potential of maize leaves that ultimately decreased the rate of photosynthesis (Ouattar et al.,

1987). Liang et al. (2002) studied the relationships among stomatal conductance, water

consumption and growth rate to leaf water potential in spring wheat (Triticum aestivum L.). It

was observed that stomatal conductance and transpiration rate decreases with decrease in leaf

water potential. The water deficiency decreased osmotic regulation of wheat plants and osmotic

regulation induced by drying and re-watering alternation increased water use efficiency of plants

under drought stress conditions. Wang and Huang (2003) reported similar results in Kentucky

bluegrass. They reported that stomatal conductance, net photosynthesis rate, relative water

contents and leaf water potential declined under drought stress and found that leaf water potential

and ABA synthesis were negatively correlated to each other. Nawaz et al. (2012) concluded from

their studies that late drought stress imposed after six weeks of emergence has more deleterious

effect on the water relations, growth, yield and nutrient uptake of wheat than early drought stress

imposed after three weeks of seedling emergence. The decrease in leaf water potential

significantly reduced the uptake of nitrogen, phosphorous and potassium in plants under drought

stress. The decrease in mineral nutrition and growth of wheat (Triticum aestivum L.), chickpea

(Cicer arietinum L.) and lentil (Lens culinaris M.) under drought stress (Gunes et al., 2007) are

reported to be positively correlated with the low leaf water potential as water plays a significant

29

role in the transfer of nitrogen from other organs to seeds (Li et al., 2006). The leaf relative water

contents (RWC) are better indicators of leaf water status than water potential (Sinclair and

Ludlow, 1985) and can be used for the selection of drought tolerant cultivars in wheat breeding

programme (Bayoumi et al., 2008). Drought tolerant genotypes maintained high RWC and

moisture stress had little effect on their protoplasmic structure as compared to sensitive

genotypes (Song et al., 1995). There exists a highly positive correlation between RWC and

photosynthetic rate (Siddique et al., 2000). The ability of cultivars to maintain their turgor

potential and RWC during later stages of growth play a significant role in maintenance of

photosynthetic apparatus under limited soil water conditions (Xu and Ihii, 1996). The

maintenance of leaf water potential, RWC and stomatal conductance under severe moisture

deficit conditions can be helpful for plants to maintain photosynthesis under prolonged drought

(Wang and Huang, 2003). Moaveni (2011) investigated the changes in membrane stability,

chlorophyll contents and RWC of four spring wheat cultivars at flowering stage. He reported that

drought stress significantly reduced all these parameters and was of the view that photosynthesis

and RWC are positively correlated with each other.

2.2.4: Biochemical characteristics

Water stress in reproductive organs of maize changes the transcription that occurs during

meiosis in tassels and formation of floret in the ears. In the tassels, by water stress 1,513

transcripts were differentially expressed with 63% of these being up regulated. On the other hand

in same condition, 202 transcripts in ear were differentially expressed with 95% being up

regulated. The water stress-regulated transcripts are involved in biochemical activities.

Collectively, it was suggested that the transcripts differentially expressed during reproductive

organic stage under water-deficit stress (Zhuang et al., 2007). On the other hand, Muhammad et

al. (2006) evaluated drought tolerant maize hybrid by using different field capacity levels on the

basis of some agronomic observations, relative cell membrane injury (RCI%) and stomatal

conductance. It was found that RCI% could be used as main selection criterion for drought

tolerance in maize. Moreover, Vamerali et al. (2003) evaluated responses of maize to limited

water availability under greenhouse condition.

Among plant defense mechanisms, Osmotic adjustment is considered to be one of the

most important one which is supported and maintained by production of various salutes which in

turn protect different sensitive enzymes as well as membrane structures. This mechanism is also

30

believed to be involved in oxygen reactive species acting as scavengers (Bohnert and Shen,

1999). Various salutes produced in reaction to water stress help in preventing dehydration of

cells and help to regain water again (Bohnert et al., 1995), along with preventing deterioration of

macromolecules (Pugnaire et al., 1994). These kinds of molecules are produced in large

quantities during water limited conditions and play the role of osmoprotectant and prevent the

disintegration of biologically important protein (Yancey, 1994). These are classified as sugars,

glycerol, amino acid (proline and glycine betaine), sugar alcohols (manitol) and other low

molecular weight metabolites (Morgan, 1984). Various researches have demonstrated the

importance of osmotic adjustment in preventing dehydration as a result of water stress

(Hasegawa et al., 2000; Shao et al., 2005). It was also reported that different molecules and their

quantities may vary depending upon the severity of stress and many other factors (Rhodes and

Samaras, 1994).

Drought stress perturbs the balance between antioxidant defense and the amount of

reactive oxygen species (Gill and Tuteja, 2010). Reactive oxygen species (ROS) play an

important role in inter- and intracellular signaling to control plant growth and development

(Breusegem et al., 2001) and are involved in the regulation of photosynthesis and programmed

cell death (Turkan et al., 2005, Foyer and Shigeoka, 2011). They serve as substrates and signals

at low concentrations (Wang et al., 2012) and trigger defense responses in plant cells during

drought stress (Sofo et al., 2005) but high concentrations of ROS such as singlet oxygen (1O2),

superoxide radicals (O2), hydrogen peroxide (H2O2) and hydroxyl radical (OH) leads to damage

at various levels of the organization (Asada, 1999). The accumulation of ROS must be kept in

check through the action of scavenging enzymes and antioxidant molecules (Foyer and Shigeoka

et al., 2011; Wang et al., 2012). Reactive oxygen species are produced from the action of

photosynthetic apparatus, photorespiration pathways and mitochondrial respiration during

drought periods (Mittler, 2002). They can directly attack membrane proteins and lipids,

inactivate metabolic enzyme, damage nucleic acids and destroy cellular structures associated

with photosynthesis (Apel and Hirt, 2004; Miller et al., 2010).

In changing environments, a variety of antioxidants like catalase and peroxidase paly

important role in ensuring the survival of plant by their stimulating actions. These antioxidants

have been found to act as scavengers for H2O2 and convert their toxic levels to oxygen and

hydrogen (Apel and Hirt, 2004). H2O2 is reduced by peroxidase by using different reductants in

31

cells of plants under water limited conditions (Mittler, 2002). Wang et al. (2012) reported that

there was a significant increase the quantity of POD in drought stressed apple leaves. Similar

results were also demonstrated by Turkan et al. (2005) in field beans. Stoilova et al. (2010)

examined CAT activity in wheat leaves under severe soil drought and were of the view that

drought stress increases CAT activity especially in sensitive cultivars. Nazarli et al. (2011)

reported that CAT activity was not changed with drought stress in wheat leaves. In contrast,

Sharma and Dubey (2005) reported that decrease in CAT activity was witnessed in rice seedlings

when these were exposed to water stress conditions. Yang et al., 2008 reported that CAT activity

was increased to a great extent with stress conditions. Pan et al. (2006) evaluated the combined

effect of drought and salt stress and resulted showed that CAT activity in Glycyrrhiza uralensis

seedlings was decreased.

It is an established fact that production antioxidant is one of the most important

mechanisms that plants have developed in order to cope with the drought stress conditions with

the production of enzymes such as peroxidase, ascorbate, catalase, ascorbate peroxidase (APX),

superoxide dismutase (SOD) and glutathione peroxidase (GPx) (Li et al., 2009). Under the stress

condition the both types of antioxidant such enzymatic and non-enzymatic inhibit the effect of

reactive oxygen species (ROS) (Chaves and Oliveria, 2004). APX play an important role for the

protection of cells of higher plant to inhibit the effect of ROS (Gill and Tuteja, 2010). It is the

constituent of water-water and ascorbate-glutathione (ASH-GSH) cycles that scavenge hydrogen

peroxide in the chloroplasts of plant cells under stress (Asada, 1999; Gill and Tuteja, 2010). The

antioxidant defense system is activated/modulated by plant water relations (Srivalli et al., 2003;

Selote and Khanna-Chopra, 2004) that results in the synthesis of APX under drought stress.

Under stress condition the activity of APX was increased in P. asperata (Yang et al., 2008) and

P. vulgaris (Zlatev et al., 2006). The APX activity also enhanced in the chloroplast of rice

seedlings when rice plants grown under minimum water stress condition but higher levels of

drought decreased its activity in stressed plants (Sharma and Dubey, 2005).

Proline is a low molecular mass compound that accumulates in cytosol of plant cells

(Voetberg and Sharp, 1991) to improve their tolerance against drastic effects of drought (Gzik,

1996; Bajji et al., 2001). It not only function as an osmolyte for osmotic adjustment but also

stabilizes sub-cellular structures such as membranes and proteins (Rhodes et al., 1999; Ozturk

and Demir, 2002), act as a protein compatible hydrotrope (Srinivas and Balasubramanian, 1995),

32

regulates and activates multiple responses such as scavenging free radicals, and buffering

cellular redox potential that help plants to combat abiotic stresses (Maggio et al., 2002) and is

considered as a reliable indicator for the environmental stress in plants (Claussen, 2005). It is

well documented that proline accumulation under salinity or drought improves tolerance to these

stresses (Aspinall and Paleg, 1982 and Hsu et al., 2003) and this characteristic could be used for

the selection of stress tolerant cultivars (Ashraf and Haris, 2004). However, some investigators

have raised questions on physiological significance of stress-induced proline accumulation and

obtained contrasting results regarding its role in stress tolerance of plants (Brock, 1981; Dix and

Pearce, 1981; Lutts et al., 2000).

Proline accumulation under stress conditions is well reported for different crops

(Almansouri et al., 1999). Drought stress significantly increased proline contents in both young

and old leaves of sunflower (Cechin et al., 2006). Similar results were reported by Mostajeran

and Rahimi-Eichi (2009) in rice. They observed substantial increase in proline content of both

young and old leaves under drought stress and stated that young leaves accumulated more

proline than old leaves and proline accumulation was more in leaf sheath than blade under un-

submerged conditions. The accumulation of free proline under water stress has also been

reported in Vigna radiate (Hooda et al., 1999) and alfalfa plants (Irigoyen et al., 1992). Proline

contents were higher in wheat leaves during grain filling period than pre-anthesis stage and can

be used as a reliable parameter for the selection of drought tolerant genotypes (Farshadfar et al.,

2008). In a similar study, Qayyum et al. (2011) observed an increase in proline content of wheat

leaves from 0.33 mg g-1 in control to 2.65 mg g-1 under osmotic stress. They were of the view

that proline accumulation in stressed plants is an important adaptation for survival under field

conditions (Tatar and Gevrek, 2008). Nazarli et al. (2011) reported that proline accumulation is

related to irrigation regimes and recorded highest proline content (12 μmol g-1 DW) in irrigation

regime of 25% field capacity (FC) while lowest value (2.4 μmol g-1 DW) was recorded in

irrigation regime of 100% FC. Proline accumulation plays a predominant role in improving

drought tolerance of wheat seedlings (Simova-Stoilova et al., 2008). The degradation of proteins

with instantaneous decline in their synthesis result in intensive proline accumulation in all

stressed organs especially leaves and conversion of some of amino acids as arginine, glutamic

and ornithine to proline also facilitate its accumulation in plants (Chaitante et al., 2000).

33

Like proline, sugars are also important compatible solutes that play a significant role in

osmoregulation under drought stress (Fallon and Phillips, 1989). The accumulation of total

sugars and other compatible solutes such as polyols is characteristic feature of most plants under

stress (Delauney and Verma, 1993; Mohammadkhani and Heidari, 2008). They are involved in

the activation and regulation of nitrate reductase (NR) activity (Kaiser and Huber, 2001; Iglesias-

Bartolome et al., 2004; Lillo et al., 2004), enhance the transcription of NR genes (Sivasanker et

al., 1997; Klein et al., 2000; Larios et al., 2001) and regulate the activity of enzymes at post

translational level (Carpenter and Gowe, 1988 and Wolkers et al., 1998). Clifford et al. (1998)

observed significant role of sugars in osmotic adjustment of Ziziphus mauritiana under limited

water conditions while Patakas et al. (2002) reported contradictory results in grapevine plants

and stated non-significant differences in sugar contents of water stressed and unstressed plants.

The production of soluble sugars under drought stress may be the result of amylase

activity that decomposes starch and therefore increases soluble sugar contents (Ghasempour et

al., 1998; Vaezi, 2005). Farshadfar et al. (2008) evaluated the molecular indices of drought

tolerance in twenty bread wheat (Triticum aestivum L.) genotypes and recorded more soluble

sugar content during grain filling period than pre-anthesis stage under drought stress. They were

of the view that post-anthesis stage and grain filling period are the best stage for the screening of

drought tolerant cultivars (Hien et al., 2003). Mostajeran and Rahimi-Eichi (2009) studied the

accumulation of soluble sugars in sheath and blade of different age leaves of rice and found that

young leaves accumulated more sugars than old ones under non-submerged conditions. The

average amount of total soluble sugars in leaf sheath and blade was 219 and 212 mg g-1

respectively. The response of soluble sugars content in wheat leaves to different irrigation

regimes of 100%, 75%, 50% and 25% field capacity (FC) at two growth stages was evaluated by

Nazarli et al. (2011). They found that limited water supply increased production of total soluble

sugars in leaves and irrigation at 25% FC resulted in maximum total soluble sugars content (49

mg g-1of dry weight) in leaves. Non-significant difference was observed between different

irrigation regimes and growth stages. Qayyum et al. (2011) stated that increase in osmotic stress

progressively increases the production of total soluble sugars in wheat leaves. They observed that

sugars contents increased from 1.49 mg g-1 in control to 2.65 mg g-1 under osmotic stress of -8

bars.

34

2.2.5: Effect on yield and yield components

Water stress occurs when the demand for water exceeds the available amount during a

certain period. As deficit irrigation results in crop water stress and reduced crop yields, so water

must be applied adequately to avoid crop water stress and to recharge the active plant root zone

adequately. Understanding crop water needs under limited water supply it is necessary to follow

irrigation scheduling and water saving procedures in arid region (Chuanyan and Zhongren,

2007). While Lu et al. (2011) evaluated maize hybrids for drought resistance under both well-

watered and water stressed conditions. The evaluation was based on the multiple measurements

of biomass taken before and after the drought along with other selection criteria including leaf

senescence, anthesis-silking interval, chlorophyll contents, root capacitance, yield components

and grain yield. Kernel weight was a most stable trait under drought stress. Correlations between

grain yield and the secondary traits, except root capacitance and anthesis-silking interval under

drought condition, were all significant; since root capacitance had relatively less heritability and

low genetic correlation with the other drought resistance criteria. On the other hand, Haghighi et

al. (2010) reported under water stress condition of Iran the application of biological fertilizer

showed significant interaction. The maize physiological characteristics, yield and its components

significantly responded to the interaction of biological fertilizer and water stress condition. In

limited water supply yield of maize hybrid was enhanced with the application of biological

fertilizer due to increase of water holding capacity of soil while where chemical fertilizer was

applied, the crop growth rate was significantly affected with decrease of water. Maize yield is

closely associated with the physiological condition of the crop during the critical period. The

association of water and nitrogen availability is positive while growth as well as yield of crop is

influenced with the limited supply of water and nitrogen (Andrade et al., 2002; Rimski-Korsakov

et al., 2009).

Sasidharan (2005) conducted genetic analysis for yield and quality attributes in castor

under irrigated (E1) and un-irrigated (E2) conditions and concluded yield and yield components

excelled under normal condition, while quality traits such as oil and fatty acids displayed

marginal increase under drought conditions. Water stress, one week prior to silking and two

week after silking reduced grain yield (53% of the non-stressed) (Denmead and Shaw, 1960,

Claassen and Shaw, 1970). The adaptations of morphological, structural, anatomical, and

ecological characteristics of plants have been proposed for the genetic improvement of crop plant

35

for drought resistance in maize (Cultere et al., 1977). Benett and Hammond (1983) demonstrated

that whenever there is a mild water shortage, plants tend to save minimum water at vegetative

growth stages in comparison with severe drought conditions. Betran et al. (2003) reported that

hybrid and inbred lines of maize produce different quantities of grain under water stress

conditions which provide the basis for hetrosis (Tollenaar et al., 2004).

Khan et al. (2001) investigated the effects of water stress on growth and grain yield of

maize cultivar YHS-202. The study was comprised on six treatments one control (six

irrigations), five, four, three, two and one irrigation only. The data revealed that stem

height, stem diameter, leaf area and days to complete flowering decrease significantly with

increased water stress. Yield components i.e. number of grains per cob, 1000-grain weight and

yield also decreased by increased water stress. Maximum grain yield obtained from control (six

irrigations) was 3.5 tons ha-1 and minimum yield was 0.4 tons ha-1 obtained from one irrigation.

Ti-da et al. (2006) was conducted a field experiment to check the effect of drought stress

on yield and yield components of maize cop. He concluded that the yield as well as cobs was

decreased under stress condition. The reduction of yield of maize depends upon the number of

grains per cob and is weight. Xu et al. (2007) investigated the drought resistance from

regenerative plants of hairy root cultures, which included plant height stress index, dry matter

stress index, number, length, volume, dry weight, total absorbing area, active absorbing area, and

root/shoot ratio and showed that there is close relationships among dry weight, dry matter stress

index, leaf area of single plant, root/shoot ratio, volume and drought resistant index. Because of

high lines of the regenerative plants from hairy root cultures in maize, it has close

relationship with drought resistance and keeps strong hydraulic root signal under water stress.

Asefa et al. (2008) conducted an experiment to determine the combining ability of

highland maize inbred lines for ear length, 1000-kernel weight, ear height, shelling

percentage and grain yield. The crosses were made from five lines and three testers using line by

tester. The analysis of variance showed that the mean squares due to genotypes were significant

for all traits, except for 1000-kernel weight and shelling percentage. Generally, the magnitude of

mean squares due to general combining ability of lines was higher than that of the specific

combining ability in most of the cases, indicating that additive gene actions were more important

than non-additive with regard to inheritance of the traits studied. Worku and Zelleke (2008)

evaluated the twenty maize cultivars at nine locations in Ethiopia in randomized complete block

36

design with three replications for two years in variable environmental conditions. Most of the

cultivars had significant deviation mean square, implying that these cultivars had unstable

performance across the testing environments. However, additive main effect and multiplicative

interaction analysis showed Gibe-1 (mean yield, 7.40 t ha-1) had relatively stable performance

across the environments. BH-660 (mean grain yield, 8.14 t ha-1) had a relatively good

performance in the mid to high altitude.

2.2.6: Breeding and genetics

The drought stress effects the plant growth and development as well as the yield of the crop

plants. The grain number per plant of the maize also decreased under drought stress conditions

(Hall et al., 1981). The 53% reduction was done when drought come at least two week after

siliking as compared to the normal (Claassen and Shaw, 1970). Dass et al. (2001) evaluated 166

genetically diverse lines of maize under different artificially given stress conditions; viz. Control,

mild stress, intermediate stress and severe stress and confirmed that plant height was affected

severely when irrigation was restricted at knee height stage and majority of the genotypes

screened. Anthesis-silking hybrids were more tolerant as compared to inbred lines. While Arif

(1990) studied 6×6 diallel cross in maize and reported that 100-grain weight and grain yield per

plant were controlled by over-dominance type of gene action. Flower et al. (1990) reported

genotypic variability in sorghum is closely linked with drought tolerance and have a potential to

improve crop yield. Damborsky et al. (1994) studied 7 inbred lines and their hybrids from a

complete set of crosses and reported that grain yield was mainly conditioned by additive

type of gene action.

Yadav et al. (2003) carried out the genetic analysis according to variety cross diallel

model involving eight diverse maize composites possessing various levels of tolerance to

moisture stress under optimum moisture and rainfed conditions. The variance due to dominance

effect was significant for days to 50% silking and tasseling, leaf area, plant height, cob width

under both moisture situations. Variance due to additive effect, dominance effect, general and

specific combining abilities were significant for grain yield under both irrigated and rainfed

conditions. Sayar et al. (2007) studied the general combining ability and specific combining

ability effects in wheat and found to be significant for both traits (deeper root length and grain

yield) however, additive gene effects were predominant over non-additive effects. Broad- sense

and narrow-sense heritability were high for deeper root length, confirming the importance of

37

additive gene effects, whereas strict-sense heritability for grain yield was average, indicating the

importance of interaction effects as compared with the additive effects. While Srdic et al. (2007)

found the inheritance of maize grain yield and grain yield components, such as number of kernel

rows, 1000-kernel weight and number of kernels per row. General and specific combining

abilities were highly significant for all observed parameters. Dominant gene effects were more

significant in maize grain yield and number of kernels per rows, while additive gene effects were

more important for number of kernel row and 1000-kernel weight.

2.3: Nutrient effect

Sixteen elements are essential for the growth of a great majority of plants and these are

derived from the surrounding air and soil. The growth as well as yield of the different crops

enhanced by the application of nutrients because with-out application of nutrients the crop

growth as well as yield are decreased. Many researchers have previously reported that nutrients

like N, P, and K are readily taken up via plant leaves with much higher efficiency than nutrient

root uptake (Fisher and Walker, 1955). Ling and Silberbush (2002) compared the efficiency of

foliar fertilization to that of the soil-applied fertilizer. They evaluated the effect of application of

various forms of nitrogen–phosphorus–potassium (NPK) fertilizers and concluded that foliar

fertilization may be used as a supplement to compensate for the inadequate uptake of nutrients

by the roots from the soil applied fertilizer. The authors also note that it is important to

investigate how the nutrients would interact if more than one nutrient is applied as a foliar spray.

For instance one nutrient may enhance or inhibit the absorption of another nutrient when applied

together (Ling and Silberbush, 2002). Boote et al. (1978) stated that foliar application of

minerals such as N, P, and K help to maintain proper leaf nutrition, enhances leaf N, P, and K as

well as carbon balance, and promotes photosynthesis, which may lead to higher grain yields.

2.3.1: Role of phosphorus in plants growth and development

Phosphorus (P) is fundamentally important to plants and is an essential element for all

living organisms. Phosphorus can persist as inorganic phosphate or it can be incorporated into

the carbon chain through a hydroxyl group (phosphate ester) or an energy-rich pyrophosphate,

which is formed by the bond of phosphate to another phosphate (Marschner, 1995). Phosphorus

is a constituent of molecular structures such as nucleic acids that compose DNA and RNA

molecules, and is fundamental to transport and translation of genetic information in the plant

(Schönknecht, 2009). Furthermore, phosphate esters and energy-rich phosphates are responsible

38

for the formation of adenosine triphosphate and adenosine diphosphate (ATP and ADP

respectively), which are essential for starch synthesis. In addition, inorganic phosphates have a

regulatory role controlling some enzymatic reactions (Marschner, 1995).

Phosphorus (P) is considered to be one of the most important and major element required

for plant vegetative and reproductive growth as well as metabolism. A variety of plant processes

such as metabolism, nucleic acids and membranes synthesis, photosynthesis, respiration,

nitrogen fixation and enzyme regulation are affected by its concentration (Raghothama, 1999).

Plant development including flowering, fruiting and root growth has been found to be

accelerated with appropriate P nutrition. It has been reported that 40% soils are lacking P in

optimum quantities worldwide (Vance, 2001).

The leading causes of low P availability in soil and ultimately its low absorption by

plants are its low solubility in soil solution, formation of complexes with cations and its

integration with organic matter and ultimately becoming unavailable for plants. In cropping

systems, the harvested portions of crops are removed from the field and ultimately there is a

dearth of P in soil until and unless it is supplemented in the form of organic or inorganic P. Every

year, huge quantities of rock phosphate find its use in the fields to provide essential quantities of

P all over the world. The salts of rock phosphate are unstable chemically and more than 60% gets

get converted to mineral phosphate which is poorly soluble salt (Barrow, 1980).

Phosphorus (P) is an essential nutrient that, in a balance with other mineral nutrients such

as nitrogen (N) and potassium (K), is required in considerable amounts in plant tissues and is

necessary for plant growth and development. The major role of P in plants is storage and transfer

of energy in the form of ATP (adenosine triphosphate) and ADP (adenosine diphosphate).

Phosphorus is a key structural constituent of nucleic acids, coenzymes, phospholipids, proteins

and nucleotides; it also strongly affects plant photosynthetic activity (Guinn, 1984; Taiz and

Zeiger, 1991). Russell (1973) reported that P is a constituent of cell nuclei and thus it is essential

for cell division and development of meristematic tissue.

Many studies reported that basal P fertilization is important to obtain N response, while

some authors have reported that P addition resulted in improved plant N status and plant growth

(Israel, 1987). Large amounts of P are required in the symbiotic system in legumes (Robson,

1983; Graham and Vance, 2000) thus a strong interaction between response to N and P exists in

legumes (Sanginga et al., 2000). According to Grant et al. (2001), it is vital to supply P early in

39

the season of crops; moderate amount of P applied at sowing can help sustain early plant growth

vigor. In many crop production systems, P can be the most deficient, and therefore, limiting

nutrient after N. Because P has a very low diffusion coefficient in soil, plants can quickly

exhaust P within the root zone during the active growing period and may develop P deficiency as

available P is depleted (Tyree et al., 1990).

The amount of available P depends on many soil characteristics such as: pH, the amount

and make-up of organic matter in the soil, soil temperature, and the type of soil minerals present.

The degree of interaction between precipitated P and the soil solution, the rate of dissolution and

diffusion of solid phase phosphorus are other factors affecting P solubility and, therefore, plant

availability.

2.3.2: Phosphorus use efficiency (PUE) and interaction with other nutrients

Excessive P fertilization is often associated with the concept of sustaining a particular

sufficiency level of nutrients in soil. The sufficiency concept is viable in some instances for soil-

immobile mineral nutrients such as P. According to Bray’s mobility concept (Bray, 1954) the

plant response to immobile mineral nutrients, such as P, depends on the concentration of the

nutrient within the Root Surface Sorption Zone, not on the total amount of nutrient in soil. The

amount of P taken up by the plant is directly dependent on the root surface and the concentration

of plant available P within the roots reach. This is because the larger the root surface, the larger

the volume of soil it intercepts, and the higher the concentration, the larger amount of P is

potentially available to be taken up by the roots. Thus, the uptake of the soil immobile nutrients

is mainly due to diffusion and root interception. To increase the amount of P in the soil, adequate

P fertilizer should be applied since more than 80% of the amount applied may be strongly

adsorbed or precipitated in the soil (Sample et al., 1980; Sanyal and De Datta, 1991).

Mosali et al. (2006) reported the highest PUE of approximately 16% in wheat was

achieved when fertilizer is banded with the seed or knifed to the soil. As corn plants develop,

available P supplied by the inorganic fertilizer is being depleted, and plants begin to utilize the

slowly available organic forms of P present in soil. Karlen et al. (1988) observed a peak in P

uptake in corn during latter vegetative growth stages, a drop in P uptake during pollination, and a

continuous linear increase during the grain fill period. Several studies have shown that

maintenance of relatively high moisture and high frequency irrigation resulted in increased P

mobility and availability (Bacon and Davey, 1982; Mbagwu and Osuigwe, 1985; Bar-Yosef et

40

al., 1989; Kargbo et al., 1991). Also researchers have demonstrated that P fertilizer only moves 3

to 4 cm from the point of application (Khasawneh et al., 1974; Eghball and Sander, 1989).

Phosphate ion (PO4), a form of P absorbed by plants, is present in both dissolved (soil solution)

and particulate forms (Haygarth and Sharpley, 2000), and are readily absorbed by plant via

diffusion. Nye and Tinker (1977) reported that the diffusion rate of phosphate ions is influenced

by P concentration in the soil solution (intensity factor) and the P sorption capacity of the soil.

They further stated that since the radii of water-filled pores decreases when soil water content

decreases, P mobility also decreases As a result, lower soil moisture content can reduce P

availability and its absorption by the plant.

2.3.3: Foliar nutrient uptake in plants

During the past few decades, plant physiologists have attempted to identify the possible

mechanisms that foliar uptake of nutrients by plants. It has been determined that both leaf

stomata (Below et al., 1984) as well as hydrophilic pores of the leaf cuticle (Barel and Black,

1979) facilitate the mineral nutrient uptake. Tyree et al. (1990) noted that even though little is

understood about the mechanisms of infiltration of ions through leaf cuticles, the permeation

studies identified the size of the cuticle pores to be about 0.9 nm in diameter (Schonherr and

Bukovac, 1979). Since the diameter of many ions is less than 0.8 nm in hydrated state, the ion

permeation through cuticle pores is very probable. The efficiency of foliar applied fertilizer

compared to soil fertilization has not been established with certainty and has been found to be

dependent on the cropping system characteristics such as soil conditions and the type of crops

grown (Below et al., 1984 and Ro¨mheld and El-Fouly, 1999).

Many factors should be considered when dealing with foliar fertilization. For example, as

Ling and Silberbush (2002) noted that, the plant size as well as the leaf area should be adequate

in order for the foliar uptake to be sufficient. However, the plant and leaf size are generally not a

concern, since foliar application is usually carried out midseason, when the crop is well

established. Girma (2007) stated that the phosphorus use efficiency (PUE) was enhanced by

foliar applied phosphorus. The nine experiments were conducted at different places (Efaw,

Goodwell, Guymon, Lake Carl Blackwell, Perkins and Stillwater) in 2002 and 2003. These

experiments were conducted to observed foliar application of phosphorus at different growth

stages [V4 (Collar of fourth leaf visible), V8 (Collar of eighth leaf visible) and VT (Last branch of

the tassel completely visible but silks not yet emerged)] of maize. Results showed that the foliar

41

P @ 8 kg ha-1 applied at V8 (Collar of eighth leaf visible) growth stages and later, was performed

best than all others growth stages of maize.

2.3.4: Foliar applied phosphorus

Foliar fertilization (FF) is an important practice in agricultural that has been used more

than 40 years. The foliar application of major nutrients such as NPK enhanced the yield as well

quality of different crops (Romheld and El-Fouly, 1999). The foliar application of P and Zn

significantly enhanced the cob index and starch contents of maize but the dry matter production

of maize was not effected (Leach and Hameleers, 2001). Foliar P nutrition arose as an alternative

to minimize the impact related to soil fertilization, because it avoids contact between soil and

fertilizer. Consequently this reduces other drawbacks related to P management such as

eutrophication, reducing costs, and increasing P use efficiency, increasing crop productivity, and

quality. Recommendations regarding foliar P fertilization are directed to dry top soils in semi-

arid regions to slightly P deficient soils and as a supplemental source of P, and not as a substitute

of soil applications. Fritz (1978) stated that foliar P fertilization should be used as a supplement

to adequate soil applied P fertilizers because; by itself, foliar fertilization is not enough to fulfill

the nutrient demand of the crop. Boynton (1954) compared soil applied versus foliar P

fertilization and concluded that the absorption and metabolism of foliar applied P was superior to

the soil applied P but over the plant cycle, foliar fertilization was not enough to meet the plant’s

requirements for P.

Barel and Black (1979) conducted an experiment in pot and reported that within 10 DAS

phosphorus (P) such as ammonium triple-phosphate was absorbed 66% in young mature leaf of

maize by foliar application of P. In that time the translocation of P was 87% from the absorbed.

However the contradicting results of Harder et al. (1982) showed that the foliar applied P after

two weeks of silking, decreased the grain yield of maize significantly. The foliar application of

NPK enhanced the growth and yield of grain production in maize as compared to the soil applied

NPK. The reason is that the P demand is low as compared to N, so the foliar applied P is

beneficial and shows the positive results in maize (Ling and Silberush, 2002). The foliar

application of P at early stage of wheat and corn increased the growth. Benbella and Paulsen

(1998) reported that the grain yield of wheat was increased when foliar applied P @ 5-10 kg ha-1

KH2PO4. The P concentration of 0.19 to 0.23% enhanced the wheat grain yield up to 90% when

P applied at the critical stages of wheat (Elliott et al., 1997). Earlier, Bolland and Paynter (1994)

42

stated that during the growing season of wheat the P concentration reduced up to 0.91% to 0.23%

in shoot of plant and 0.27% in wheat grain. Haloi (1980) reported that after 25 DAS of sowing of

wheat the deficiency symptoms of P is appeared. As a result the foliar applied P improved the

deficiency symptoms and also enhanced the yield of wheat. The efficiency of both foliar applied

P and soil or basal applied P were same (Singh et al., 1981).

Mosali et al. (2006) applied 1, 2, 4, 8, 12, 16 and 20 kg P ha-1 with and without preplant

rates of P (30 kg P ha-1) and found that foliar P applications usually increased yield and P uptake

at Feekes 7 when compared to no foliar applied P, however the higher P use efficiency was

obtained at Feekes 10.5. Girma et al. (2007) reported a small yield increase and a large

improvement on forage and grain concentration in corn with foliar rates of 8 kg of P ha -1.

Another experiment conducted in Morocco, revealed up to 1 Mg ha-1 increase in wheat grain

yields with foliar application of KH2PO4 at rates of 1.1 to 2.2 kg P ha-1 after anthesis. The

increase in yield was attributed to the delayed senescence promoted by the fertilizer under water

and heat stress (Benbella and Paulsen, 1998). Leach and Hameleers (2001) stated that the foliar

applied P and Zn enhanced the cob index and starch contents of maize. During the dry season the

grain yield of wheat was increased by foliar applied P and it also delay the senescence of leaf

(Sherchand and Paulsen, 1985; Batten et al., 1986). The yield of barley was enhanced by the

foliar applied (Qaseem et al., 1978).

Early work by Wittwer and Teubner (1959) stated that all plants are known to obtain

water, gases and a wide spectrum of solutes from the environment through the foliage.

Considerable amount of research has been done to identify the factors affecting foliar nutrient

uptake (Swanson and Whitney, 1953; Fisher and Walker, 1955; Koontz and Biddulph, 1957). It

has been noticed that foliar-applied phosphate solution generally is taken up much faster at lower

pH levels (pH 2 to 3) (Wittwer and Teubner, 1959). Mono-ammonium phosphates are absorbed

at much high rate at lower pH values (Wittwer et al., 1957). According to Wittwer et al. (1957)

the total amount of P fertilizer taken up by the leaves is greater, because larger leaf area occurs

on plants grown on the P-rich media. Thus, the authors theorize that increased levels of

phosphates within the plant tissues, and in their vascular system especially, often inhibit P

transport from the leaves to a higher degree than decreased P absorption of P by the leaves. As

proposed by Mosali et al. (2006), use efficiency of the foliar fertilizer should be much higher,

since the many possible pathways for P loss associated with the application of nutrient to the soil

43

are eliminated. Instead, the nutrient is directly “fed” to the plant, and the available P is readily

taken up, translocated and utilized. Therefore, much smaller amounts of fertilizer would be

sufficient to satisfy crop nutritional requirements and to effectively correct P deficiency mid-

season. As stated by Mosali et al. (2006), for many decades the potential of foliar P application

has been underestimated due to generally lower levels of P in soils. Today, however, much

higher P concentrations are present in many cropping systems as a result of application of P

fertilizer in excess of crops needs.

According to Bundy et al. (2001), the amount of plant-available P in some soils has

increased significantly over the past 25 years due to P fertilizer and manure application in excess

of crops needs. The fact that solution P fertilizers were not as easily accessible to crop producers

in the past, also contributed to the traditional application of P to the soil. Application of foliar P

fertilizer to corn would allow for P deficiency correction if it occurs mid-season. This would

supply the crop with the P supplement needed to achieve higher grain yield as well as increase

phosphorus use efficiency (PUE). The efficiency of P fertilizer could be higher if P is applied

foliar compared to soil applied P fertilizer. Foliar mineral nutrient uptake is much more efficient,

because the nutrients taken up mid-season are translocated to the reproductive organs improving

grain formation. Application of nutrients like nitrogen (N), P, and potassium (K) as foliar sprays

were found to not only increase yield of various crops but also improve their quality (Römheld

and El-Fouly, 1999).

In a pot culture corn trial, Barel and Black (1979) observed that 66% of P applied to the

mature leaves as a foliar spray in a form of ammonium triple-phosphate was absorbed within 10

days and 87% of the absorbed amount was translocated, showing that corn plants were

successful and efficient in uptake and utilization of foliar P applied. Harder et al. (1982)

observed a significant reduction in corn grain yields when foliar P was applied 2 weeks after

silking. Sawyer and Barker (1999) evaluated the impact of foliar mono-potassium phosphate and

urea fertilizer on corn grain yield and grain constituents. They found that foliar fertilization had

no significant effect either on corn grain yield, nor grain characteristics. The achieved results can

be explained by the following: grain yield levels were quite high at the evaluated sites; soils did

not receive any P fertilizer pre-plant due to very high soil P levels. Therefore, the crop, most

likely, did not experience any P deficiencies and thus did not show any response to P fertilizer.

The utilization of foliar-applied phosphorus fertilizer has been found to be dependent on nutrient

44

availability of P in the soil for both peanuts (Halevy et al. (1987) and cotton (Halevy and

Markovitz (1988). Benbella and Paulsen (1998) reported that foliar P application after flowering

resulted in decreased grain yields due to a significant delay in senescence in winter wheat during

the grain fill stage. As proposed by Benbella and Paulsen (1998), foliar P should be applied to

the crop later in the growing season to effectively delay leaf senescence. According to the

findings by Mosali et al. (2006), foliar P fertilization can be delayed until Feekes 10 and may

result in increase in PUE by more than 10%. The authors note, however, that for the maximum

efficiency, it is preferable to combine N and P fertilization using the same approach earlier in the

growing season (Feekes 7). These findings suggest that timing is extremely important in foliar P

application, as well as suggest that mid-season foliar application of P fertilizer has the potential

to extend the grain fill stage and, thus, increase yield potential.

Investigating potential benefits of foliar fertilization application to cereal crops, Gooding

and Davies (1992) reported that foliar applications at or 2 weeks following anthesis can be of

greater benefit compared to soil applied fertilization. Multiple beneficial effects of foliar P

fertilizer application in corn (Pongsakul and Ratanarat, 1999; Leach and Hameleers, 2001 and

Thavaprakaash et al., 2006), wheat (Haloi, 1980; Sherchand and Paulsen, 1985 and Batten et al.,

1986) and barley (Qaseem et al., 1978) have been documented. Leach and Hameleers (2001),

observed a significant increase in both cob index and starch content when P was applied at four-

leaf growth stage. Sherchand and Paulsen (1985) and Batten et al. (1986) reported that foliar

application of KH2PO4 resulted in higher grain yield in winter wheat coupled with the delay in

leaf senescence in hot and dry growing conditions. Qaseem et al. (1978) achieved higher yields

when P fertilizer was applied to barley as a foliar spray solution. Mosali et al. (2006) found

wheat grain yield to be poorly correlated with P concentration. They noted that delayed maturity

is one of the main benefits of foliar P application in wheat production systems. The best results

were achieved when preplant P was coupled with mid-season foliar P fertilization.

Pongsakul and Ratanarat (1999) reported that foliar application of NPK fertilizers

increased grain yield of both field and sweet corn. Thavaprakaash et al. (2006) found that foliar

P applied 25 and 45 days after planting boosted growth parameters and resulted in significantly

higher corn yields. Haloi (1980) however, reported that higher rates of ammonium phosphate

applied as a foliar spray to wheat not only resulted in reduced P deficiency but also led to higher

grain yields. Mosali et al. (2006) noted that much larger increases in wheat grain yield are

45

expected with foliar P fertilization on low P soils compared to higher P fertility soils. They

achieved increases in wheat grain yield when the yield levels were generally lower, possibly -

due to water stress, which impaired the P uptake via contact exchange. Therefore, one would

expect the maximum response to foliar P fertilization when moisture stress is more severe. Foliar

application of urea in winter as well as NPK foliar sprays in spring are regularly used to intensify

flowering and increase yields in citrus production. Albrigo (2002) evaluated the effect of foliar

sprays on citrus orange trees. They observed a 9% increase in leaf N concentrations; leaf P and

K. However, these increased only when the initial P and K leaf concentrations were low prior to

spray application. Grain yields were not significantly different when the foliar sprays had been

used for one year, compared to trees that were not sprayed. Girma et al. (2007) was conducted an

experiment to study the different doses of foliar applied P (0, 2, 4 and 8 kg ha-1) on different

growth stages (4th, 8th and tasseling stage) of maize plants. He concluded that the P @ 8 kg ha-1

at tasseling stage of maize was enhanced the forage and grain yield of maize. He also concluded

that the foliar applied P at optimum rate and stage improved the growth and yield of maize.

46

The present study was intended to assess the response of water-stressed maize (Zea mays

L.) to supplemental foliar application of phosphorous to improve the growth and yield of maize

under drought stress conditions. The study was conducted in March (spring maize growing

season in Pakistan) during the year 2013-and 2014.

3.1. Experimental site and conditions

The study was conducted in the Stress physiology Laboratory, wire house/rain out shelter

and field conditions at Department of Agronomy, University of Agriculture Faisalabad, Pakistan.

A series of laboratory, wire house/rain out shelter and field experiment were conducted for this

study. The laboratory experiments were conducted in petri plates and plastic cups (containing

430 g of sterilized sand) whereas the wire house/rain out shelter experiments were conducted in

plastic pots (15 cm × 10 cm) containing 2 kg of sterilized sand. Laboratory and wire house

experiments were laid out in completely randomized design (CRD). The wire house experiment

was conducted at Department of Agronomy laid out in a completely randomized design (CRD)

in factorial arrangement with three repeats. The field experiment was laid out in randomized

complete block design (RCBD) in factorial arrangement with three repeats in sandy clay loam

soil at research area, Department of Agronomy, University of Agriculture, Faisalabad. The soil

texture was determined with the hygrometer method (Dewis and Freitas, 1970). The

physiochemical characteristics (Electrical conductivity, pH and ion contents) of the soil used for

this study were determined according to methods described by Jackson, (1962) and are present in

Table 3.1. The weather in respect of minimum and maximum (°C), relative humidity (%) and

rainfall (mm) of the experimental site (both wire house and field experiment) for the year 2013-

2014 is given in the Figure 3.1.

47

Fig. 3.1: Meteorological data of the experimental site for the growing seasons 2013 and

2014.

Table.3.1: Physiochemical characteristics of the soil used for field experiment.

Soil Characteristics Values

Physical

Soil texture Sandy clay loam

Chemical

Saturation percentage (%) 37.6

ECe (dSm-1) 0.72-0.92

Soil pHs 7.5-7.8

Organic matter (%) 0.4-0.6

Ca+Mg (meq L-1) 3.75-5.76

CO3 (meq L-1) Nil

HCO3 (meq L-1) 3.5-4.0

Available P (mg kg-1) 8.2-10.8

3.2. Maize germplasm collection

The seeds of eight maize genotypes viz. 7386, 6525, Hycorn, 9696, 32B33, 3672, MMRI

and 31P41 were used for the screening of these genotypes for drought tolerance. The seeds were

0

20

40

60

80

100

120

140

March April May June March April May June

2013 2014

Temperature (oc) Relative humidity (%) Rain fall (mm)

48

obtained from Maize and Millet Research Institute (MMRI) Yousaf Wala Sahiwal and local

market of Sahiwal, Pakistan.

3.3. Laboratory experiments

3.3.1. Experiment # 1: Screening of different maize hybrids for drought tolerance subjected

to PEG-6000 induced water stress

The experiments were carried out at Stress Physiology Laboratory, Department of

Agronomy, University of Agriculture, Faisalabad, Pakistan. During the first experiment,

polyethylene glycol (PEG) with the molecular weight of 6000 was used as a drought stimulator

and five stress levels of zero (control), -0.2, -0.4, -0.6 and -0.8 MPa were developed by

dissolving 6.65, 13.30, 20 and 26.6 g of PEG separately in 100 ml of distilled water. The

concentrations of the solution were confirmed by Viesper Osmometer (Model 5520) at 25°C

according to the method of Michel and Kaufmann (1973).

Maize genotypes viz. 7386, 6525, Hycorn, 9696, 32B33, 3672, MMRI and 31P41 were

evaluated for drought tolerance. Randomly selected 10 seeds of each cultivar were sterilized for

five seconds with 5% sodium hypochlorite solution, washed them with distilled water and then

air dried. Seeds were placed in covered sterilized petri dishes having 9 cm diameter and

containing filter paper moistened with 10 ml of PEG-6000.

The data for germination percentage (AOSA, 1983), promptness index (Ashraf et al.,

2006) and germination stress index (Fernandez, 1992) were recorded till ten days. Germination

data were recorded on daily basis. Seeds were considered to be germinated when gained

approximately 2 mm of root length and used for germination percentage calculations (Afzal et

al., 2004). Complete germination of seeds was considered when no further germination occurred

in two successive days.

Recorded data was used for calculation of promptness index (PI) and germination stress

tolerance index (G.S.I) according to the formulae given by Ashraf et al. (2006).

P.I = nd2 (1.00) + nd4 (0.75) + nd6 (0.5) + nd8 (0.25) + nd10 (0.0)

Where n is the number of seeds germinated on day d

G.S.I. = (P.I of stressed seeds / P.I control seeds) × 100

49

3.3.2. Experiment # 2: Screening of maize hybrids for drought tolerance subjected to water

stress

The second experiment was conducted under laboratory conditions in pots. The seeds of

eight maize genotypes (7386, 6525, Hycorn, 9696, 32B33, 3672, MMRI and 31P41) were grown

for 25 days in plastic pots having sand as a growth medium. All the pots were watered daily at

100% field capacity for 10 days. After the completion of seed germination the pots were divided

into two sets. One set of pots was watered daily at 100% field capacity while in other set the

irrigation was stopped to impose water stress. The data on root length stress index (RLSI), dry

matter stress index (DMSI) and plant height stress index (PHSI) were recorded after four weeks

of seedling growth by using the following formulae given by Ashraf et al. (2006).

PHSI = [Plant height of stressed plant/Plant height of control plant] × 100

DMSI = [Dry matter of stressed plant/Dry matter of control plant] × 100

RLSI = [Root length of stressed plant/Root length of control plant] × 100

Significant differences among cultivar means were determined by analysis of variance

according to Duncan's Multiple Range Test (P < 0.05) in an experiment with factorial structure

of treatments evaluated in a completely randomized design with three repeats for each variable.

The data so collected was analyzed statistically using analysis of variance technique and

the STATISTICA (Version, 8.1) Computer Program was used for this purpose.

3.4. Experiment # 3: Optimization of phosphorus sources in maize

A pot experiment was conducted in wire house/rain out shelter for optimization of

appropriate source helpful in improving drought tolerance in maize plants subjected to water

stress at seedling stage. Two drought tolerant (6525 and 32B33) and two drought sensitive

genotypes (Hycorn and 31P41) selected from laboratory experiments were grown under normal

(100% field capacity) and water stress (60% field capacity) levels. Randomly selected 10 seeds

of each cultivar and were sterilized for five minutes with 5% sodium hypochlorite solution and

washed three times with distilled water. Initially, each replication consisted of 5 seeds of each

cultivar sown in plastic pots (15 cm × 10 cm) containing 2 kg of sterilized sand as growth

medium but only five plants were kept after thinning in each replication.

50

The treatments were control, Diammonium phosphate (DAP), Potassium phosphate

(KH2PO4), Single super phosphate (SSP) and NitroPhos (NP). In this experiment, all the pots

were watered daily at 100% field capacity for 8 days. After the completion of seed germination

the pots were divided into two sets. One set of pots was watered daily at 100% field capacity

while in other set at 60% field capacity. The plants were harvested after 20 days. The experiment

was repeated three times to record data regarding growth characteristic of plants. Completely

randomized design (CRD) with three replications was used for this experiment. The data on root-

shoot length, root-shoot fresh and dry weight and root-shoot ratio were recorded after 20 days of

seedling growth.

3.5. Experiment # 4: Optimization of supplemental foliar doses of phosphorus

in maize

Experiment was conducted in pots in wire house/rain out shelter for optimization of

different rates of phosphorus helpful in improving drought tolerance in maize plants subjected to

water stress at seedling stage. The best source of phosphorus (KH2PO4) selected from experiment

# 3 was used in this experiment. Two drought tolerant (6525 and 32B33) and two drought

sensitive genotypes (Hycorn and 31P41) selected from laboratory experiments were grown under

normal (100% field capacity) and water stress (60% field capacity) levels. Randomly selected 10

seeds of each cultivar and were sterilized for five minutes with 5% sodium hypochlorite solution

and washed three times with distilled water. Initially, each replication consisted of 5 seeds of

each cultivar sown in plastic pots (15 cm × 10 cm) containing 2 kg of sterilized sand as growth

medium but only five plants were kept after thinning in each replication.

The treatments were control, water spray, KH2PO4 @ 4 kg ha-1, KH2PO4 @ 6 kg ha-1,

KH2PO4 @ 8 kg ha-1 and KH2PO4 @ 10 kg ha-1. In this experiment, all the pots were watered

daily at 100 % field capacity for 10 days. After the completion of seed germination the pots were

divided into two sets. One set of pots was watered daily at 100% field capacity while in other set

at 60% field capacity. The plants were harvested after 20 days. The experiment was repeated

three times to record data regarding growth characteristic of plants. Completely randomized

design (CRD) with three replications was used for these experiments. The data on root-shoot

length, root-shoot fresh and dry weight and root-shoot ratio were recorded after 20 days of

seedling growth.

51

3.6. Experiment # 5: Optimization of stage of maize for foliar applied

phosphorus

A pot experiment was conducted in wire house/rain out shelter for optimization of

different timing for foliar applied phosphorus helpful in improving drought tolerance in maize

plants subjected to water stress. The best source and best rates of foliar applied phosphorus

(KH2PO4 @ 8 kg ha-1) selected from experiment # 3 & 4 were used in this experiment. Two

drought tolerant (6525 and 32B33) and two drought sensitive genotype (Hycorn and 31P41)

selected from laboratory experiments were grown under normal (100% field capacity) and stress

conditions. Stress was applied by withholding irrigation for one week at 4 th leaf stage, 8th leaf

stage and tasseling stage. Randomly selected 10 seeds of each cultivar and were sterilized for

five minutes with 5% sodium hypochlorite solution and washed three times with distilled water.

Initially, each replication consisted of 5 seeds of each cultivar sown in plastic pots containing 4

kg of sterilized sand as growth medium but only five plants were kept after thinning in each

replication.

The treatments were control, stress applied at 4th leaf stage, stress applied at 8th leaf stage

and stress applied at tasseling stage. In this experiment, all the pots were watered daily at 100 %

field capacity for 10 days. The foliar application of phosphorus @ 8 kg ha-1 was applied at 4th

leaf stage, 8th leaf stage and tasseling stage. The plants were harvested after 70 days. Completely

randomized design (CRD) with three replications was used for these experiments. The data on

root-shoot length, root-shoot fresh and dry weight and root-shoot ratio were recorded after 70

days of growth.

52

3.7. Experiment # 6: The combined effect of optimum source of P, dose of P

and growth stage on physiological and biochemical attributes of maize


A pot experiment was conducted in rain-out shelter to check the best combination of four

maize genotypes, one source (KH2PO4), one dose (KH2PO4 @ 8 kg ha-1) and one timing (Stress

applied at 8th leaf stage) that is helpful in improving drought tolerance in water stressed maize

plants. In this experiment, four maize genotypes i.e. two drought tolerant (6525 & 32B33) and

two drought sensitive (Hycorn & 31P41) were grown under normal (100% field capacity) and

water stress (stress applied at 8th leaf stage) levels.

3.7.1. Seed sowing

For wire house experiment sand was initially sun dried, ground, sieved and mixed well in

order to avoid any plant residues and 4 kg sand was filled carefully in each pot. Five seeds were

sown in each pot and then watered with distilled water. In the beginning all pots were kept at

field capacity level for obtaining good germination and emergence. Later on the water was

applied according to the water stress level specified for that experiment. Before imposing water

stress the plants were thinned out and three healthy plants were kept in each pot. Recommended

doses of P, K and N were applied in solution form at the time of planting, but N was applied after

every 2 weeks.

3.7.2 Development and maintenance of water stress levels.

At 8th leaf stage the pots were divided into two groups i.e stress and non-stressed. The

above following condition was maintained for one week there after applying the supplemental

foliar P @ 8 kg ha-1 at this stage. For each treatment pots were weighed daily at about 9:00 am,

calculated the amount of water consumed in evapotranspiration and watered until the pot weight

reached to pre-determined weight.

Plants were grown up to 40 days and data regarding various growth and development

(root shoot length, root-shoot fresh and dry weight and root-shoot ratio), physiological,

biochemical parameters and P determination in leaf, stem, root and grain was recorded using

standard recommended methods.

53

3.7.3: Physiological parameters

3.7.3.1. Leaf water potential

The third leaf from top (fully expanded youngest leaf) of plants from each

treatment was used to determine the leaf water potential. The measurements were made from

8.00 to 10.00 a.m. with Scholander type pressure chamber.

3.7.3.2. Leaf osmotic potential

The same leaf used for water potential, was frozen at -20oC for osmotic potential

determinations. The frozen leaf material was thawed and cell sap was extracted while crushing

the leaves with a glass rod and then sap was sucked with a disposable syringe. The sap so

extracted was directly used for the determination of osmotic potential using an osmometer

(Wescor 5500).

3.7.3.3. Turgor potential

Turgor potential was calculated as the difference between osmotic potential (ψs) and

water potential (ψw) values.

(ψp) = (ψw) - (ψs)

3.7.3.4. Relative water contents (RWC)

Fully expand young leaf were taken from three plants of each treatment. Fresh weight

(FW) of each sample was taken on digital electrical balance and dipped in test tube containing

distilled water for 24 hours. Then it was wiped with the tissue paper and turgid weight (TW) was

taken. The samples were dried at 65°C for 72 hrs and dry weight (DW) of each sample was

taken. For each treatment, RWC was calculated by using the formula (Karrou and Maranville,

1995) given below:

RWC = [(FW-DW) / (TW-DW)] × 100

Where FW= fresh weight, DW= dry weight and TW= turgid weight

54

3.7.4: Gas exchange characteristics

A fully expanded youngest leaf of each plant after 8th leaf stage was used to measure the

instantaneous net CO2 assimilation rate (A), transpiration (E) and stomatal conductance (gs ) by

using an open system LCA-4 ADC portable infrared gas analyzer (Analytical Development

Company, Hoddesdon, England). These measurements were taken from 9.00 to 11.00 a.m. with

the following adjustments: molar flow of air per unit leaf area 403.3 mmol m -2S-1, atmospheric

pressure 99.9 kPa, water vapor pressure into chamber ranged from 6.0 to 8.9 m bar, PAR at leaf

surface was maximum up to 1711 mol m-2 s-1, temperature of leaf ranged from 28.4oC to 32.4oC,

ambient temperature ranged from 22.4 to 27.9oC and ambient CO2 concentration was 352 mol

mol-1.

3.7.5. Biochemical parameters

3.7.5.1: Chlorophyll contents

Chlorophyll contents were calculated by using the method of Arnon, (1949) and Davies,

(1976). Fresh leaves of (0.5 g) were chopped into segments of 0.5 cm and extracted with 5 mL

acetone (80%) at 10oC overnight. Centrifuge the material at 14000 rpm for 5 min. and measured

the absorbance of the supernatant at 645, 652 and 663 nm on spectrophotometer. Calculated a, b

and total chlorophyll.

Chl a = [12.7 (OD 663) -2.69 (OD 645) ] x V/1000 x W

Chl b = [22.9 (OD 645) -4.68 (OD 663)] x V/1000 x W

Total Chl = [20.2 (OD 645) + 8.02 (OD 663)] x V/100 x W

Carotenoids (g.mL-1) = Acar/Emx100

Where V is the volume of sample extract and W is the weight of the sample

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

55

3.7.5.2. Total soluble protein (TSP)

Total soluble proteins were determined using the method of Lowry et al. (1951).

Reagents

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

buffer.

1. One-molar solution of NaH2PO4.2H2O (156.01 g L-1) was prepared as the stock.

2. One-molar solution of Di-sodium hydrogen 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

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

One hundred g of sodium tungstate and 25 g of sodium molybdate were dissolved in 700

mL of distilled water. Fifty mL of 85% orthophosphoric acid and 100 mL of HCl were added and

the mixture was refluxed for 10 h. Then 150 g of lithium sulfate was added along with 50 mL of

distilled water. A few drops of Br2 were also added.

56

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 g for 5 min. The supernatant

was used for protein determination.

Procedure

One mL of the 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 (Hitachi, 220, Japan).

3.7.5.3. Total free amino acids (TFA)

Total free amino acids were determined according to Hamilton and Van Slyke (1973).

Fresh plant leaves (0.5 g) were chopped and extracted with phosphate buffer (0.2 M) having pH

7.0. Took 1 mL of the extract in 25 mL test tube, added 1 mL of pyridine (10%) and 1mL of

ninhydrin (2%) solution in each test tube. Ninhydrin solution was prepared by dissolving 2 g

ninhydrine in 100 mL distilled water. The test tubes with sample mixture, heated in boiling water

bath for about 30 min. Volume of each test tube was made up to 50 mL with distilled water.

Read the optical density of the coloured solution at 570 nm using spectrophotometer. Developed

a standard curve with Leucine and calculated free amino acids using the formulae given below:

Graph reading of sample x Volume of factor x Dilution factor

Total amino acids = ------------------------------------------------------------------------------

Weight of fresh tissue x 1000

57

3.7.5.4. Total soluble sugars (TSS)

Total soluble sugars were determined according to the method of Yemm and Willis,

(1954).

Extraction

Dried plant material was ground well in a micromill and the material was sieved through

1 mm sieve of micromill. Plant material (0.1 g) was extracted in 80% ethanol solution .The

extract was incubated 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 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 was read at 625 nm on a

spectrophotometer (Hitatchi, 220, Japan). The concentration of soluble sugars was calculated

from the standard curve developed by using the above method.

3.7.5.5. Proline determination

The proline was determined according to the Bates et al. (1973) method. Fresh leaf

material of 0.5 g was ground and dissolved in 10 mL of 3% sulfo-salicylic acid. The sample

material was filtered by using Whatman No. 2 filter paper. Two mL of the filterate was taken in a

test tube and reacted with 2 mL acid ninhydrin solution. Acid ninhydrin solution was prepared by

dissolving 1.25 g ninhydrine 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 kept for 1 h at 100oC. After

terminating the reaction in an ice bath, the reaction mixture was extracted with 10 mL toluene.

Continuous air stream was passed vigorously for 1-2 minutes in the reaction mixture. The

chromophore containing toluene was aspirated from the aqueous phase, warmed at room

58

temperature and the absorbance was noted at 520 nm on spectrophotometer. Toluene was used as

a blank. The proline concentration was calculated by using a standard curve and calculated on

fresh weight basis as follows:-

mmole proline g-1 fresh weight = (g proline mL-1 x mL of toluene/115.5) / (wt. of

sample/5)

3.7.6: Antioxidant enzymes

The activities of POX, CAT, and ascorbate peroxidase (APX) were determined

spectrophotometrically. Leaves were homogenized in a medium composed of 50 mM phosphate

buffer with 7.0 pH and 1 mM dithiothreitol (DTT) as described by Dixit et al. (2001).

3.7.6.1. Catalase (CAT)

Catalase activity was assayed by measuring the conversion rate of hydrogen peroxide to

water and oxygen molecules, following the method described by Chance and Maehly (1955).

The activity was assayed in 3 mL reaction solution comprising 50 mM phosphate buffer with 7.0

pH, 5.9 mM of H2O2 and 0.1 mL enzyme extract. The catalase activity was determined by

decline in absorbance at 240 nm after every 20 sec due to consumption of H2O2. Absorbance

change of 0.01 units min-1 was defined as one unit catalase activity.

3.7.6.2. Peroxidase (POX)

The activity of POD was determined by measuring peroxidation of hydrogen peroxide

with guaiacol as an electron donor (Chance and Maehly, 1955). The reaction solution for POD

consists of 50 mM phosphate buffer with pH 5, 20 mM of guaiacol, 40 mM of H2O2 and 0.1 mL

enzyme extract. The increase in the absorbance due to the formation of tetraguaiacol at 470 nm

was assayed after every 20 sec. One unit of the enzyme was considered as the amount of the

enzyme that was responsible for the increase in OD value of 0.01 in 1 min. The enzyme activity

was determined and expressed as units min-1 g-1 fresh weight basis.

3.7.6.3. Ascorbate peroxidase activity

Ascorbate peroxidase (APX) activity was measured by monitoring the decrease in

absorbance of ascorbic acid at 290 nm (extinction coefficient 2.8 mM cm-1) in a 1 ml reaction

59

mixture containing 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).

3.7.7. P determination

3.7.7.1: P concentration (%)

Five mL of the digested aliquot was taken in 50 mL volumetric flask, added 5 mL of

ammonium vanadate (0.25%) and ammonium molybdate (5 %), made volume and allowed to

stand for 15-30 minutes. Reading was recorded on spectrophotometer. Then from the standard

curve, P concentration (%) in grain, leaf, stem and root was calculated.

3.7.7.2 Phosphorus Use Efficiency (%)

The calculations were done on the basis of formulae as described by Fageria et al.

(1997a).

PUE % = Total P uptake (kg ha-1) in fertilized plot - Total P uptake (kg ha-1) in control × 100

P dose applied (kg ha-1)

3.8. Experiment # 7: The combined effect of optimum source, dose of P and

growth stage on yield and yield components of maize hybrids under water

stress

Field experiment was conducted over one year at research area of Department of

Agronomy, University of Agriculture, Faisalabad, Pakistan. The best combination of P source,

dose and stage for supplemental foliar P application using two drought tolerant and two drought

sensitive maize genotype. Best combination of source, dose and stage selected from wire house

experiments were applied at one water stress levels i.e at 8th leaf stage. The experiment was laid

out in RCBD factorial arrangement. Plants were allowed to grow up to maturity. Data regarding

yield and yield components were recorded. The following yield and yield components were

recorded:

60

3.8.1. Yield and yield components

3.8.1.1: Cob length (cm)

Five cobs were selected randomly from each plot and their length was measured with the

help of “a measuring tape” and then average length was calculated from five randomly selected

cobs.

3.8.1.2: Number of cobs per plant

Five plants were selected randomly from each plot and number of cobs per plant was

counted.

3.8.1.3: Number of grains per cob

Grain number per cob was counted from total number of grains of five cobs from each plot

selected randomly.

3.8.1.4: Cob weight without sheath (g)

Five cobs were selected randomly from each experimental unit, and with the help of

electrical balance their weight was measured and average weight was recorded.

3.8.1.5: 1000-grain weight (g)

A sample of 1000-grain was taken at random from the seed lot obtained from each plot and

weighed with an electric balance.

3.8.1.6: Grain yield (t ha-1)

All the cobs from plants of each plot were separated and then shelled with the help of

Sheller and grains were weighed to have grain yield, then grain yield is converted to tha -1.

3.8.1.7: Biological yields (t ha-1)

Biological yield contains Stover, pith and grain yield. Crop was harvested from each plot

manually, dried under sun and weighed to determine the biological yield in kg per plot and then

converted to this biological yield to t ha-1.

3.8.1.8: Harvest Index (%)

It was recorded for each pot by using the formula:

Economic yield (grain yield)

HI (%) = ----------------------------------------------- × 100

Biological yield (grain + straw)

61

3.9: Statistical Analysis

Data so collected in different experiments of this study were analyzed statistically using

analysis of variance technique and the STATISTIX (Version, 8.1) Computer Program was used

for this purpose. The Least Significant Difference test at 5% probability level was used to assess

the differences among significant means (Steel et al., 1997).

62

4.1. Experiment # 1: Screening of different maize hybrids for drought

tolerance subjected to PEG-6000 induced water stress

4.1.1. Germination

The analysis of variance (Table 4.1) showed a highly significant (P≤0.001) interaction

between hybrids and PEG-6000 induced stress levels for germination percentage (GP),

promptness index (PI) and germination stress index (GSI). In all tested hybrids, the non-stress

control treatment (0 -MPa) showed the maximum GP, PI and GSI which consistently decreased in

response to increasing osmotic stress (Figs.4.1-4.3). The highest value of germination and PI was

observed in both drought tolerant 6525 (72.0% & 2.38) and 32B33 (64.0% & 2.15) hybrid while

the minimum germination and PI observed in both drought sensitive Hycron (50.67% & 0.98)

and 31P41 (52.0% & 1.00) hybrid (Table 4.1). The treatment where water stress applied @ -

0.6MPa showed the maximum GP (13.33%) and PI (0.48) while the minimum GP (100%) and PI

(3.81) were observed in control treatment (Table 4.1).

Fig. 4.1: Germination percentage of eight maize (Zea mays L.) hybrids under PEG (6000)

induced water stress regimes (mean values ± S.E).

10

20

30

40

50

60

70

80

90

100

110

120

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

Germ

inati

on

%

HYBRIDS

Control 0.2 (-MPa) 0.4 (-Mpa) 0.6 (-MPa) 0.8 (-MPa)

63

Fig. 4.2: Promptness index (PI) of eight maize (Zea mays L.) hybrids under PEG (6000)

induced water stress regimes (mean values ± S.E).

Fig. 4.3: Germination stress index (GSI) of eight maize (Zea mays L.) hybrids under PEG

(6000) induced water stress regimes (mean values ± S.E).

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

Pro

mp

tness

In

dex

HYBRIDS

Control 0.2 (-MPa) 0.4 (-MPa) 0.6 (-MPa) 0.8 (-MPa)

10

20

30

40

50

60

70

80

90

100

110

120

130

140

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

GS

I (%

)

HYBRIDS

0.2 (-MPa) 0.4 (-MPa) 0.6 (-MPa) 0.8 (-MPa)

64

Table 4.1: Analysis of variance (ANOVA) and mean parameter values of germination percentage and promptness index for

main effects of hybrids under PEG (6000) induced water stress

a GP= Germination percentage, PI= Promptness index; b T = Treatment; H = Hybrids; T x H = Treatments x Hybrids;

*, **, *** = Significant at 0.05, 0.01 and 0.001, level respectively; NS = Non significant

Parametersa

Hybrids Osmotic stress ANOVAb

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41 Control

(0 MPa)

0.2

-MPa

0.4

-MPa

0.6

-MPa

0.8

-MPa

T H TXH

GP 58.67 bc 72.0 a 64.0 b 60.0 b 50.67 d 58.67 bc 57.33 bcd 52.0 cd 100 a 99.16 a 83.33 b 13.33 c 0.00 d *** *** ***

PI 1.41 c 2.38 a 2.15 ab 1.26 c 0.98 c 1.60 bc 1.28 c 1.00 c 3.81 a 1.89 b 1.35 c 0.48 d 0.00 d *** *** NS

65

4.2. Experiment # 2: Screening of maize hybrids for drought tolerance

subjected to water stress

4.2.1. Seedling growth

Analysis of variance showed highly significant difference (P≤0.001) among hybrids and

water stress treatments for root length stress index (RLSI), plant height stress index (PHSI) and

dry matter stress index (DMSI) (Table 4.2). In all tested genotypes maximum PHSI and RLSI

was calculated in 6525 and 32B33 (Fig. 4.4 & 4.5). The maximum value (108.64% & 102.67%)

of PHSI was recorded in 6525 and 32B33 while the minimum values of PHSI (77.64% &

81.21%) was recorded in Hycorn and 31P41, respectively (Table 4.2). Maximum value of RLSI

(120.67% & 112.86%) was recorded in 6525 and 32B33 while the minimum values of RLSI

(83.40% & 85.89%) was recorded in Hycorn and 31P41, respectively (Table 4.2).

Maximum DMSI (110.37% & 103.94) was recorded in 6525 and 32B33 while the

minimum values of DMSI (64.63% & 69.93%) was recorded in Hycorn and 31P41 (Table 4.2).

Fig. 4.4: Plant height stress tolerance index (PHSI) of eight maize (Zea mays L.) hybrids

under water stress regimes (mean values ± S.E)

0.00

20.00

40.00

60.00

80.00

100.00

120.00

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

Pla

nt

heig

ht

Str

ess

In

dex %

HYBRIDS

66

Fig. 4.5: Root length stress tolerance index (RLSI) of eight maize (Zea mays L.) hybrids


Fig. 4.6: Dry matter stress tolerance index (DMSI) of eight maize (Zea mays L.) hybrids


0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

Ro

ot

len

gth

str

ess

in

dex %

HYBRIDS

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

Dry m

att

er

stre

ss i

nd

ex %

HYBRIDS

67

Table 4.2: Analysis of variance (ANOVA) and mean parameter values of physiological indices for main effects of hybrids

a PHSI = Plant height stress index, RLSI= Root length stress index; DMSI = Dry matter stress index; *, **, *** = Significant at 0.05,

0.01 and 0.001, level respectively; NS = Non significant

Parametersa

Hybrids

ANOVA 7386 6525 32B33 9696 Hycorn 3672 MMRI 31P41

PHSI 98.31 bc 108.64 a 102.74 ab 90.98 cde 77.64 f 95.04 bcd 86.87 def 81.21 ef ***

RLSI 90.88 cd 120.67 a 112.86 a 93.87 bcd 83.40 d 101.90 b 96.59 bc 85.89 cd ***

DMSI 92.77 bc 110.37 a 103.94 ab 81.34 cde 64.63 f 76.90 def 86.81 cd 69.93 ef ***

68

4.3. Experiment # 3: Optimization of phosphorus sources in maize

The experiment were conducted in wire house/rain out shelter to optimized the different

source of phosphorus (DAP, KH2P04, SSP and NP) under normal and stress conditions.

4.3.1. Shoot length (cm)

Maize plants exhibited significantly lower (P≤0.001) shoot length under water stress

conditions as compared to well watered conditions (Fig. 4.7). The maximum value of shoot

length (17.63 cm) was recorded under well-watered conditions while minimum value of shoot

length (13.87 cm) was recorded under water stress conditions (Table 4.3).

The analysis of variance for the data regarding shoot length showed highly significant

difference among genotypes. The genotype 6525 and 32B33 maintained significantly higher

shoot length (16.72 cm & 16.01 cm) as compared to other genotypes (Table 4.3).

Among the foliar spray treatment, the foliar application of KH2PO4 significantly

(P≤0.001) improve the shoot length. The maximum value of shoot length (17.78 cm) was

recorded in treatment where foliar applied KH2PO4 as compared to all other treatments while

the minimum value of shoot length (14.42 cm) was recorded in control treatment (Table 4.3).

The interaction between H x W x T was non-significant (Table 4.3).

4.3.2. Root length (cm)


conditions as compared to well watered conditions (Fig. 4.8). The maximum value of root

length (11.44 cm) was recorded under well-watered conditions while minimum value of root


The analysis of variance for the data regarding root length showed highly significant


root length (10.81 cm & 9.87 cm) as compared to other genotypes (Table 4.3).


(P≤0.001) improve the root length. The maximum value of root length (12.06 cm) was


69

the minimum value of root length (7.50 cm) was recorded in control treatment (Table 4.3). The

interaction between H x W x T was non-significant (Table 4.3).

4.3.3. Shoot fresh weight (g)

Maize plants exhibited significantly lower (P≤0.001) shoot fresh weight under water

stress conditions as compared to well watered conditions (Fig. 4.9). The maximum value of

shoot fresh weight (2.54 g) was recorded under well-watered conditions while minimum value

of shoot fresh weight (1.79 g) was recorded under water stress conditions (Table 4.3).

The analysis of variance for the data regarding shoot fresh weight showed highly

significant difference among genotypes. The genotype 6525 and 32B33 maintained

significantly higher shoot fresh weight (2.33 g & 2.20 g) as compared to other genotypes

(Table 4.3).


(P≤0.001) improve the shoot fresh weight. The maximum value of shoot fresh weight (2.65 g)

was recorded in treatment where foliar applied KH2PO4 as compared to all other treatments

while the minimum value of shoot fresh weight (2.04 g) was recorded in control treatment

(Table 4.3). The interaction between H x W x T was non-significant (Table 4.3).

4.3.4. Root fresh weight (g)

Maize plants exhibited significantly lower (P≤0.001) root fresh weight under water


root fresh weight (2.29 g) was recorded under well-watered conditions while minimum value

of root fresh weight (1.54 g) was recorded under water stress conditions (Table 4.3).

The analysis of variance for the data regarding root fresh weight showed highly


significantly higher root fresh weight (2.11 g & 2.04 g) as compared to other genotypes (Table

4.3).


(P≤0.001) improve the root fresh weight. The maximum value of root fresh weight (2.27 g)

was recorded in treatment where foliar applied KH2PO4 as compared to all other treatments

70

while the minimum value of root fresh weight (1.48 g) was recorded in control treatment


4.3.5. Shoot dry weight (g)

Maize plants exhibited significantly lower (P≤0.001) shoot dry weight under water


shoot dry weight (0.30 g) was recorded under well-watered conditions while minimum value of

shoot dry weight (0.16 g) was recorded under water stress conditions (Table 4.3).

The analysis of variance for the data regarding shoot dry weight showed highly


significantly higher shoot dry weight (0.29 g & 0.24 g) as compared to other genotypes (Table

4.3).


(P≤0.001) improve the shoot dry weight. The maximum value of shoot dry weight (0.32 g) was


the minimum value of shoot dry weight (0.17 g) was recorded in control treatment (Table 4.3).


4.3.6. Root dry weight (g)

Maize plants exhibited significantly lower (P≤0.001) root dry weight under water stress

conditions as compared to well watered conditions (Fig. 4.12). The maximum value of root dry

weight (0.48 g) was recorded under well-watered conditions while minimum value of root dry

weight (0.28 g) was recorded under water stress conditions (Table 4.3).

The analysis of variance for the data regarding root dry weight showed highly


significantly higher root dry weight (0.44 g & 0.39 g) as compared to other genotypes (Table

4.3).


(P≤0.001) improve the root dry weight. The maximum value of root dry weight (0.49 g) was


71

the minimum value of root dry weight (0.27 g) was recorded in control treatment (Table 4.3).


4.3.7. Root-shoot ratio (%)

Maize plants exhibited significantly lower (P≤0.001) root-shoot ratio under water stress

conditions as compared to well watered conditions (Fig. 4.13). The maximum value of root-

shoot ratio (64.46%) was recorded under well-watered conditions while minimum value of

root-shoot ratio (56.01%) was recorded under water stress conditions (Table 4.3).

The analysis of variance for the data regarding root-shoot ratio showed highly


significantly higher root-shoot ratio (65.42% & 60.85%) as compared to other genotypes

(Table 4.3).


(P≤0.001) improve the root-shoot ratio. The maximum value of root-shoot ratio (67.39%) was


the minimum value of root-shoot ratio (51.67%) was recorded in control treatment (Table 4.3).


Fig. 4.7: Effect of different sources of phosphorus on shoot length (cm) of four maize

hybrids in well-watered (100% FC) and water stress (60% FC) conditions

(mean values ± S.E)

0.00

5.00

10.00

15.00

20.00

25.00

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Non stress (100% FC) Stress (60% FC)

Sh

oo

t le

ng

th (

cm

)

Hycorn 31P41 6525 32B33

72

Fig. 4.8: Effect of different sources of phosphorus on root length (cm) of four maize hybrids

in well-watered (100% FC) and water stress (60% FC) conditions (mean values

± S.E)

Fig. 4.9: Effect of different sources of phosphorus on shoot fresh weight (g) of four maize



0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP


Ro

ot

len

gth

(cm

)Hycorn 31P41 6525 32B33

0.000.501.001.502.002.503.003.504.00

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP


Sh

oo

t fr

esh

weig

ht

(g)

Hycorn 31P41 6525 32B33

73

Fig. 4.10: Effect of different sources of phosphorus on root fresh weight (g) of four maize



Fig. 4.11: Effect of different sources of phosphorus on shoot dry weight (g) of four maize



0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP


Ro

ot

fresh

weig

ht

(g)

Hycorn 31P41 6525 32B33

0.000.050.100.150.200.250.300.350.400.450.50

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP


Sh

oo

t d

ry w

eig

ht

(g)

Hycorn 31P41 6525 32B33

74

Fig. 4.12: Effect of different sources of phosphorus on root dry weight (g) of four maize



Fig. 4.13: Effect of different sources of phosphorus on root-shoot ratio of four maize



0.000.100.200.300.400.500.600.700.80

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP


Ro

ot

dry w

eig

ht

(g)

Hycorn 31P41 6525 32B33

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Co

ntr

ol

DA

P

KH

2P

O4

SS

P

NP

Non stress Stress

Ro

ot-

sho

ot

rati

o (

%)

Hycorn 31P41 6525 32B33

75

Table 4.3: Mean parameter values of growth parameters for main effects of hybrids, Treatments, water levels and their

interactions

a Shoot length (cm), Root length (cm), Shoot fresh weight (g), Root fresh weight (g), Shoot dry weight (g), Root dry weight (g) and

root-shoot ratio (%), b mean values across four hybrids; c DAP = Diammonium phosphate, KH2PO4 = Potassium phosphate, SSP =

Single super phosphate, NP = Nitrophos; d mean values across two water levels; WW= Well-watered, WS= Water stress; NS = Non

significant ;*’**’***significant at P≤ at 0.05, P≤ at 0.01, P≤ at 0.001, respectively. e H = Hybrid, W = water level, T = treatment

Parametersa

Hybridsb Phosphorus sourcec

Water leveld Interactionse

Hycorn 31P41 6525 32B33 Control DAP KH2PO4 SSP NP WW WS HxW HxT WxT HxWxT

Shoot length 15.02 d 15.24 c 16.72 a 16.01 b 14.42 e 15.64 c 17.78 a 15.86 b 15.05 d 17.63 a 13.87 b *** *** *** NS

Root length 8.78 d 9.06 c 10.81 a 9.87 b 7.50 e 8.83 d 12.06 a 10.47 b 9.29 c 11.44 a 7.83 b *** *** *** NS

Shoot fresh weight 2.02 d 2.10 c 2.33 a 2.20 b 1.74 d 2.04 c 2.65 a 2.37 b 2.03 c 2.54 a 1.79 b *** *** *** NS

Root fresh weight 1.75 c 1.76 c 2.11 a 2.04 b 1.48 e 1.80 d 2.27 a 2.13 b 1.90 c 2.29 a 1.54 b *** *** *** NS

Shoot dry weight 0.17 d 0.20 c 0.29 a 0.24 b 0.17 e 0.22 c 0.32 a 0.25 b 0.20 d 0.30 a 0.16 b *** *** *** NS

Root dry weight 0.33 d 0.35 c 0.44 a 0.39 b 027 e 0.36 c 0.49 a 0.43 b 0.33 d 0.48 a 0.28 b *** *** *** NS

Root-shoot ratio 78.82 d 58.85 c 63.42 a 60.85 b 51.67 e 56.45 d 67.39 a 65.21 b 60.44 c 64.46 a 56.01 b *** *** *** NS

76

4.4. Experiment # 4: Optimization of supplemental foliar doses of phosphorus

in maize

The experiment was conducted in wire house / rain out shelter to optimize the different

rates of phosphorus (KH2P04 @ 2 kg ha-1, KH2P04 @ 4 kg ha-1, KH2P04 @ 6 kg ha-1, KH2P04 @

8 kg ha-1 and KH2P04 @ 10 kg ha-1) under normal and stress conditions.









Among the foliar spray treatment, the foliar application of different rates of KH2PO4

significantly (P≤0.001) improve the shoot length. The maximum value of shoot length (20.19

cm) was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as compared to all

other treatments while the minimum value of shoot length (16.22 cm) was recorded in control

treatment (Table 4.4). The interaction between H x W x T was non-significant (Table 4.4).


Maize plants exhibited significantly lower (P≤0.001) root length under water stress








significantly (P≤0.001) improve the root length. The maximum value of root length (13.14 cm)

77

was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as compared to all other

treatments while the minimum value of root length (9.63 cm) was recorded in control treatment





shoot fresh weight (1.47 g) was recorded under well-watered conditions while minimum value

of shoot fresh weight (1.08 g) was recorded under water stress conditions (Table 4.4).




(Table 4.4).


significantly (P≤0.001) improve the shoot fresh weight. The maximum value of shoot fresh

weight (1.42 g) was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as

compared to all other treatments while the minimum value of shoot fresh weight (1.11 g) was

recorded in control treatment (Table 4.4). The interaction between H x W x T was non-

significant (Table 4.4).


Maize plants exhibited significantly lower (P≤0.001) root fresh weight under water






significantly higher root fresh weight (1.16 g & 1.14 g) as compared to other genotypes (Table

4.4).


significantly (P≤0.001) improve the root fresh weight. The maximum value of root fresh

78

weight (1.29 g) was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as

compared to all other treatments while the minimum value of root fresh weight (0.81 g) was

recorded in control treatment (Table 4.4). The interaction between H x W x T was non-





shoot dry weight (0.31 g) was recorded under well-watered conditions while minimum value of

shoot dry weight (0.15 g) was recorded under water stress conditions (Table 4.4).



significantly higher shoot dry weight (0.24 g & 0.23 g) as compared to other genotypes (Table

4.4).


significantly (P≤0.001) improve the shoot dry weight. The maximum value of shoot dry weight

(0.28 g) was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as compared to all

other treatments while the minimum value of shoot dry weight (0.15 g) was recorded in control



Maize plants exhibited significantly lower (P≤0.001) root dry weight under water stress

conditions as compared to well watered conditions (Fig. 4.19). The maximum value of root dry

weight (0.26 g) was recorded under well-watered conditions while minimum value of root dry

weight (0.14 g) was recorded under water stress conditions (Table 4.4).




4.4).


significantly (P≤0.001) improve the root dry weight. The maximum value of root dry weight

79

(0.24 g) was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as compared to all

other treatments while the minimum value of root dry weight (0.13 g) was recorded in control


4.4.7. Root-shoot ratio (%)

Maize plants exhibited significantly lower (P≤0.001) root-shoot ratio under water stress

conditions as compared to well watered conditions (Fig. 4.20). The maximum value of root-

shoot ratio (64.49%) was recorded under well-watered conditions while minimum value of

root-shoot ratio (62.60%) was recorded under water stress conditions (Table 4.4).

The analysis of variance for the data regarding root-shoot ratio showed highly


significantly higher root-shoot ratio (64.81% & 64.75%) as compared to other genotypes

(Table 4.4).


significantly (P≤0.001) improve the root-shoot ratio. The maximum value of root-shoot ratio

(65.61%) was recorded in treatment where foliar applied KH2PO4 @ 8 kg ha-1 as compared to

all other treatments while the minimum value of root-shoot ratio (59.64%) was recorded in

control treatment (Table 4.4). The interaction between H x W x T was non-significant (Table

4.4).




0.0

5.0

10.0

15.0

20.0

25.0

30.0

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Sh

oo

t le

ng

th (

cm

) Hycorn 31P41 6525 32B33

80

Fig. 4.15: Effect of different sources of phosphorus on root length (cm) of four maize



Fig. 4.16: Effect of different sources of phosphorus on shoot fresh weight (g) of four

maize hybrids in well-watered (100% FC) and water stress (60% FC)

conditions (mean values ± S.E)

0.00

5.00

10.00

15.00

20.00

25.00

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Ro

ot

len

gth

(cm

)Hycorn 31P41 6525 32B33

0.00

0.50

1.00

1.50

2.00

2.50

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Sh

oo

t fr

esh

weig

ht

(g)

Hycorn 31P41 6525 32B33

81







0.00

0.50

1.00

1.50

2.00

2.50

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Ro

ot

fresh

weig

ht

(g)

Hycorn 31P41 6525 32B33

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Sh

oo

t d

ry w

eig

ht

(g)

Hycorn 31P41 6525 32B33

82




Fig. 4.20: Effect of different sources of phosphorus on root-shoot ratio (%) of four maize

hybrids in well-watered (100% FC) and water stress (60% FC) conditions (mean

values ± S.E)

0.00

0.10

0.20

0.30

0.40

0.50

0.60

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Ro

ot

dry w

eig

ht

(g)

Hycorn 31P41 6525 32B33

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Co

ntr

ol

wa

ter

spra

y

P @

4 k

g/h

a

P @

6 k

g/h

a

P @

8 k

g/h

a

P @

10 k

g/h

a

Non stress Stress

Ro

ot-

sho

ot

rati

o (

%)

Hycorn 31P41 6525 32B33

83

Table 4.4: Mean parameter values of growth parameters for main effects of hybrids, Treatments, water levels and their

interactions

a Shoot length (cm), Root length (cm), Shoot fresh weight (g), Root fresh weight (g), Shoot dry weight (g), Root dry weight (g) and

root-shoot ratio (%), b mean values across four hybrids; c Con.= control, 1 = KH2PO4 @ 2 kg ha-1, 2 = KH2PO4 @ 4 kg ha-1, 3 =

KH2PO4 @ 6 kg ha-1, 4 = KH2PO4 @ 8 kg ha-1 and 5 = KH2PO4 @ 10 kg ha-1; d mean values across two water levels; WW= Well-

watered, WS= Water stress; NS = Non significant ;*’**’***significant at P≤ at 0.05, P≤ at 0.01, P≤ at 0.001, respectively. e H =

Hybrid, W = water level, T = treatment

Parametersa

Hybridsb Phosphorus dosec Water leveld Interactionse

Hycorn 31P41 6525 32B33 Con. 1 2 3 4 5 WW WS HxW HxT WxT HxWxT

Shoot length 18.21 d 18.48 c 18.62 a 18.52 b 16.22 f 17.24 e 18.80 d 18.93 c 20.19 a 19.35 b 20.63 a 16.28 b *** *** *** NS

Root length 11.67 d 11.74 c 12.01 a 11.87 b 9.63 f 10.87 e 12.26 d 12.39 c 13.14 a 12.64 b 13.43 a 10.21 b *** *** *** NS

Shoot fresh weight 1.26 b 1.27 b 1.30 a 1.29 a 1.11 e 1.18 d 1.28 c 1.29 c 1.42 a 1.37 b 1.47 a 1.08 b NS *** *** NS

Root fresh weight 1.01 c 1.02 c 1.16 a 1.14 b 0.81 f 1.01 e 1.06 d 1.13 c 1.29 a 1.19 b 1.44 a 0.72 b *** *** *** NS

Shoot dry weight 0.22 b 0.23 ab 0.24 a 0.23 ab 0.15 e 0.20 d 0.22 c 0.24 b 0.28 a 0.27 a 0.31 a 0.15 b NS ** NS NS

Root dry weight 0.19 b 0.20 ab 0.22 a 0.21 a 0.13 e 0.18 d 0.20 c 0.22 b 0.24 a 0.23 a 0.26 a 0.14 b NS ** ** NS

Root-shoot ratio 62.41 d 63.15 c 64.81 a 64.75 b 59.64 f 62.92 e 64.61 d 64.78 d 65.61 a 65.11 b 64.49 a 62.60 b *** *** *** NS

84

4.5. Experiment # 5: Optimization of stage of maize for foliar applied

phosphorus

The experiment was conducted in wire house/rain out shelter to optimize the sage of

maize (4th leaf stage, 8th leaf stage and tasseling stage) for foliar phosphorus application under

normal and stress conditions.


Maize plants exhibited significantly lower (P≤0.001) shoot length under control

conditions as compared to foliar applied P @ 8 kg ha -1 at different stages of maize plant (Fig.

4.21). The maximum value of shoot length (89.77 cm) was recorded under 8th leaf stage while

minimum value of shoot length (85.11 cm) was recorded under 4 th leaf stage of maize (Table

4.5).




Among the foliar spray treatment, the foliar application of KH2PO4 @ 8 kg ha-1

significantly (P≤0.001) improves the shoot length. The maximum value of shoot length

(100.72 cm) was recorded in treatment where foliar applied KH2PO4 while the minimum value

of shoot length (73.67 cm) was recorded in control treatment (Table 4.5). The interaction

between H x W x T was non-significant (Table 4.5).


Maize plants exhibited significantly lower (P≤0.001) root length under control


4.22). The maximum value of root length (61.38 cm) was recorded under 8th leaf stage while

minimum value of root length (57.69 cm) was recorded under 4th leaf stage of maize (Table

4.5).




85


significantly (P≤0.001) improves the root length. The maximum value of root length (72.21

cm) was recorded in treatment where foliar applied KH2PO4 while the minimum value of root

length (46.72 cm) was recorded in control treatment (Table 4.5). All others interactions were

non-significant (Table 4.5).


Maize plants exhibited significantly lower (P≤0.001) shoot fresh weight under control

conditions as compared to foliar applied P @ 8 kg ha-1 at different stages of maize plant (Fig.

4.23). The maximum value of shoot fresh weight (161.22 g) was recorded under 8 th leaf stage

while minimum value of shoot fresh weight (156.18 g) was recorded under 4 th leaf stage of

maize (Table 4.5).




(Table 4.5).


significantly (P≤0.001) improves the shoot fresh weight. The maximum value of shoot fresh

weight (175.72 g) was recorded in treatment where foliar applied KH2PO4 while the minimum

value of shoot fresh weight (142.12 g) was recorded in control treatment (Table 4.5). All others

interactions were non-significant (Table 4.5).


Maize plants exhibited significantly lower (P≤0.001) root fresh weight under control


4.24). The maximum value of root fresh weight (84.64 g) was recorded under 8th leaf stage

while minimum value of root fresh weight (80.29 g) was recorded under 4 th leaf stage of maize

(Table 4.5).



86

significantly higher root fresh weight (88.79 g & 85.45 g) as compared to other genotypes

(Table 4.5).


significantly (P≤0.001) improves the root fresh weight. The maximum value of root fresh


value of root fresh weight (70.64 g) was recorded in control treatment (Table 4.5). All others



Maize plants exhibited significantly lower (P≤0.001) shoot dry weight under control


4.25). The maximum value of shoot dry weight (116.34 g) was recorded under 8 th leaf stage

while minimum value of shoot dry weight (112.49 g) was recorded under 4th leaf stage of

maize (Table 4.5).



significantly higher shoot dry weight (120.00 g & 116.81 g) as compared to other genotypes

(Table 4.5).


significantly (P≤0.001) improves the shoot dry weight. The maximum value of shoot dry


value of shoot dry weight (102.21 g) was recorded in control treatment (Table 4.5). All others



Maize plants exhibited significantly lower (P≤0.001) root dry weight under control


4.26). The maximum value of root dry weight (24.96 g) was recorded under 8th leaf stage while

minimum value of root dry weight (22.31 g) was recorded under 4th leaf stage of maize (Table

4.5).

87




4.5).


significantly (P≤0.001) improves the root dry weight. The maximum value of root dry weight

(31.81 g) was recorded in treatment where foliar applied KH2PO4 while the minimum value of

root dry weight (15.50 g) was recorded in control treatment (Table 4.5). All others interactions

were non-significant (Table 4.5).




0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

Hycorn 31P41 6525 32B33 Hycorn 31P41 6525 32B33

Control Foliar applied P @ 8kg/ha

Sh

oo

t le

ng

th (

cm

)

4th leaf stage 8th leaf stage Tasseling stage

88

Fig. 4.22: Effect of different sources of phosphorus on root length (cm) of four maize



Fig. 4.23: Effect of different sources of phosphorus on shoot fresh weight (g) of four

maize hybrids in well-watered (100% FC) and water stress (60% FC)

conditions (mean values ± S.E)

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00



Ro

ot

len

gth

(cm

)4th leaf stage 8th leaf stage Tasseling stage

0.0020.0040.0060.0080.00

100.00120.00140.00160.00180.00200.00220.00



Sh

oo

t fr

esh

weig

ht

(g)


89







0.00

20.00

40.00

60.00

80.00

100.00

120.00



Ro

ot

fresh

weig

ht

(g)


0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00



Sh

oo

t d

ry w

eig

ht

(g)


90




0.005.00

10.0015.0020.0025.0030.0035.0040.0045.0050.00



Ro

ot

dry w

eig

ht

(g)


91

Table 4.5: Mean parameter values of growth parameters for main effects of hybrids, stages, treatments and their interactions

a Shoot length (cm), Root length (cm), Shoot fresh weight (g), Root fresh weight (g), Shoot dry weight (g) and Root dry weight (g), b

mean values across four hybrids; c S1 = Foliar P applied at 4th leaf stage, S2 = Foliar P applied at 8th leaf stage, S3 = Foliar P applied at

tasseling stage; d mean values across two levels; F0= No foliar applied P (Control), F1= Foliar applied P; NS = Non significant ;

*’**’***significant at P≤ at 0.05, P≤ at 0.01, P≤ at 0.001, respectively. . e H = Hybrid, S = stage, T = treatment

Parametersa

Hybridsb Stagesc

Foliar phosphorusd Interactionse

Hycorn 31P41 6525 32B33 S1 S2 S3 F0 F1 HxS SxT HxT HxSxT

Shoot length 76.59 c 87.70 b 95.14 a 89.33 b 85.11 c 89.77 a 86.70 b 73.67 b 100.72 a *** ** ** NS

Root length 51.51 c 59.97 b 64.90 a 61.31 b 57.69 c 61.38 a 59.19 b 46.72 b 72.12 a NS NS NS NS

Shoot fresh weight 146.50 d 160.17 c 166.34 a 162.67 b 156.18 c 161.22 a 159.36 b 142.12 b 175.72 a NS NS ** NS

Root fresh weight 72.49 c 83.97 b 88.79 a 85.45 b 80.29 c 84.64 a 83.10 b 70.64 b 94.71 a NS NS ** NS

Shoot dry weight 104.91 c 115.50 b 120.00 a 116.81 b 112.49 c 116.34 a 114.09 b 102.21 b 126.20 a NS NS NS NS

Root dry weight 18.02 c 24.32 b 27.19 a 25.09 b 22.31 b 24.96 a 23.69 ab 15.50 b 31.81 a NS NS NS NS

92

4.6. Experiment # 6: The combined effect of optimum source of P, dose of P

and growth stage on physiological and biochemical attributes of maize











(P≤0.001) improve the shoot length. The maximum value of shoot length (89.27 cm) was

recorded in treatment where foliar applied KH2PO4 while the minimum value of shoot length

(71.12 cm) was recorded in control treatment (Table 4.6). All the others interaction were non-



Drought significantly lower (P≤0.001) root length of maize plants under water stress








(P≤0.001) improve the root length. The maximum value of root length (61.33 cm) was

recorded in treatment where foliar applied KH2PO4 while the minimum value of root length

(47.70 cm) was recorded in control treatment (Table 4.6). All the others interaction were non-


93




shoot fresh weight (132.41 g) was recorded under well-watered conditions while minimum

value of shoot fresh weight (106.46 g) was recorded under water stress conditions (Table 4.6).




(Table 4.6).


(P≤0.001) improve the shoot fresh weight. The maximum value of shoot fresh weight (136.40

g) was recorded in treatment where foliar applied KH2PO4 while the minimum value of shoot

fresh weight (102.47 g) was recorded in control treatment (Table 4.6). The interaction between

H x S x T was non-significant (Table 4.6).


Drought significantly lower (P≤0.001) root fresh weight of maize plants under water






significantly higher root fresh weight (68.92 g & 60.96 g) as compared to other genotypes

(Table 4.6).


(P≤0.001) improve the root fresh weight. The maximum value of root fresh weight (70.92 g)

was recorded in treatment where foliar applied KH2PO4 while the minimum value of root fresh

weight (43.74 g) was recorded in control treatment (Table 4.6). The interaction between H x S

x T was non-significant (Table 4.6).

94




shoot dry weight (29.91 g) was recorded under well-watered conditions while minimum value

of shoot dry weight (17.93 g) was recorded under water stress conditions (Table 4.6).



significantly higher shoot dry weight (30.16 g & 25.51 g) as compared to other genotypes

(Table 4.6).


(P≤0.001) improve the shoot dry weight. The maximum value of shoot dry weight (32.06 g)

was recorded in treatment where foliar applied KH2PO4 while the minimum value of shoot dry

weight (15.78 g) was recorded in control treatment (Table 4.6). The interaction between H x S

x T was non-significant (Table 4.6).


Drought significantly lower (P≤0.001) root dry weight of maize plants under water


root dry weight (13.346 g) was recorded under well-watered conditions while minimum value

of root dry weight (12.30 g) was recorded under water stress conditions (Table 4.6).




4.6).


(P≤0.001) improve the root dry weight. The maximum value of root dry weight (17.53 g) was

recorded in treatment where foliar applied KH2PO4 while the minimum value of root dry

weight (8.11 g) was recorded in control treatment (Table 4.6). The interaction between H x S x

T was non-significant (Table 4.6).

95

Fig. 4.27: Effect of supplemental foliar phosphorus application on shoot length (cm) of four

maize hybrids grown under normal and water stress conditions (mean values ±

S.E).

Fig. 4.28: Effect of supplemental foliar phosphorus application on root length (cm) of four

maize hybrids grown under normal and water stress conditions (mean values ±

S.E).

0.00

20.00

40.00

60.00

80.00

100.00

120.00


Non stress Stress

Sh

oo

t le

ng

th (

cm

)Control Foliar applied P @ 8 kg/ha

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00


Non stress Stress

Ro

ot

len

gth

(cm

)

Control Foliar applied P @ 8 kg/ha

96

Fig. 4.29: Effect of supplemental foliar phosphorus application on shoot fresh weight (g) of

four maize hybrids grown under normal and water stress conditions (mean

values ± S.E).

Fig. 4.30: Effect of supplemental foliar phosphorus application on root fresh weight (g) of


values ± S.E).

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

200.00


Non stress Stress

Sh

oo

t fr

esh

weig

ht

(g)


0.00

20.00

40.00

60.00

80.00

100.00

120.00


Non stress Stress

Ro

ot

fresh

weig

ht

(g)


97

Fig. 4.31: Effect of supplemental foliar phosphorus application on shoot dry weight (g) of


values ± S.E).

Fig. 4.32: Effect of supplemental foliar phosphorus application on root dry weight (g) of


values ± S.E).

0.00

10.00

20.00

30.00

40.00

50.00

60.00


Non stress Stress

Sh

oo

t d

ry w

eig

ht

(g)


0.00

5.00

10.00

15.00

20.00

25.00

30.00


Non stress Stress

Ro

ot

dry w

eig

ht

(g)


98

Table 4.6: Mean parameter values of growth parameters for main effects of hybrids, water levels, treatments and their

interactions

a Shoot length (cm), Root length (cm), Shoot fresh weight (g), Root fresh weight (g), Shoot dry weight (g) and Root dry weight (g), b

mean values across four hybrids; c WW = Well watered, WS = Water stress; d mean values across two levels; F0= No foliar applied P

(Control), F1= Foliar applied P; NS = Non significant ; *’**’***significant at P≤ at 0.05, P≤ at 0.01, P≤ at 0.001, respectively. e H =

Hybrid, T = treatment and W = water level

Parametersa

Hybridsb Water levelsc

Foliar phosphorusd Interactionse

Hycorn 31P41 6525 32B33 WW WS F0 F1 HxW WxT HxT HxWxT

Shoot length 73.58 b 76.00 b 87.00 a 84.41 a 84.45 a 76.04 b 71.12 b 89.27 a NS NS NS NS

Root length 49.50 a 52.25 b 59.91 a 56.41 a 59.20 a 49.83 b 47.70 b 61.33 a NS NS NS NS

Shoot fresh weight 105.12 d 114.43 c 133.48 a 124.71 b 132.41 a 106.46 b 102.47 b 136.40 a NS NS ** NS

Root fresh weight 46.95 d 52.49 c 68.92 a 60.96 b 70.66 a 44.00 b 43.74 b 70.92 a *** NS *** NS

Shoot dry weight 18.39 d 21.62 c 30.16 a 25.51 b 29.91 a 17.93 b 15.78 b 32.06 a ** *** ** NS

Root dry weight 9.73 b 11.55 b 15.99 a 14.02 b 13.34 a 12.30 a 8.11 b 17.53 a NS ** NS NS

99

4.6.8: Photosynthetic rate (A)

Drought stress significantly reduced (P<0.001) the photosynthetic rate (Table 4.7). The

water deficit conditions at 8th leaf stage significantly decreased photosynthetic rate by 10.5% as

compared to normally irrigated (control) plants (Fig. 4.33). The maximum photosynthetic rate

(3.89 µ mol CO2 m-2 s-1) was observed under normal condition while minimum photosynthetic

rate (3.48 µ mol CO2 m-2 s-1) of maize was recorded under stress condition (Appendix 4.1). Maize

hybrids 6525 and 32B33 performed better than 31P41 and Hycorn. The maximum photosynthetic

rate (3.81 µmol CO2 m-2 s-1) was observed in hybrid 6525 while the minimum photosynthetic rate

(3.58 µmol CO2 m-2 s-1) was observed in Hycorn (Appendix 4.2).

The effect of supplemental foliar applied P spray treatment was also highly significant

(P<0.001) for photosynthetic rate. The foliar application of P spray at 8th leaf stage increased the

photosynthetic rate (3.83 µ mol CO2 m-2 s-1) as compare to no spray (3.55 µ mol CO2 m-2 s-1)

treatment (Appendix 4.1 & 4.2). The interaction between maize hybrids and treatments were

significantly affecting the photosynthetic rate under normal and stress conditions. The highest

photosynthetic rate was recorded in 6525 (3.98 µ mol CO2 m-2 s-1) where supplemental foliar P @

8 kg ha-1 was applied at 8th leaf stage while minimum photosynthetic rate was observed in Hycorn

(3.47 µ mol CO2 m-2 s-1) where no foliar spray under normal and stress conditions was applied

(Appendix 4.2).

All other interaction such as H x W x T was non-significant (Table 4.7).

4.6.9: Transpiration rate (E)

The transpiration rate was significantly (P<0.01) decreased by water stress in all four

maize hybrids (Table 4.7). The decrease in transpiration rate was more pronounced in water

stressed plants as compared to normal irrigated plants (Fig. 4.34). The water stress at 8th leaf stage

of maize reduced transpiration rate by 14.5% as compared to normally irrigated plants (Fig 4.36).

The maximum transpiration rate (2.56 mmol H2O m-2 s-1) was recorded under well watered

condition while minimum transpiration rate (2.19 mmol H2O m-2 s-1) of maize was recorded under

stress conditions (Appendix 4.3). Maize hybrids 6525 and 32B33 performed better than 31P41

and Hycorn. The maximum transpiration rate (2.45 mmol H2O m-2 s-1) was observed in hybrid

6525 while the minimum transpiration rate (2.30 mmol H2O m-2 s-1) was observed in Hycorn

(Appendix 4.4).

100


(P<0.001) for transpiration rate. The foliar application of P spray at 8th leaf stage increased the

transpiration rate (2.48 mmol H2O m-2 s-1) as compare to no spray (2.27 mmol H2O m-2 s-1)

treatment (Appendix 4.3 & 4.4).

All the interactions such as H x T and H x W x T were non-significant (Table 4.7).

4.6.10: Stomatal conductance (gs)

Drought stress significantly reduced (P<0.001) the stomatal conductance (Table 4.7). The

water deficit conditions at 8th leaf stage significantly decreased it by 11.3% as compared to

normally irrigated (control) plants (Fig. 4.35). The maximum stomatal conductance (4.07 mmol

H2O m-2 s-1) was observed under normal condition while minimum stomatal conductance (3.61

mmol H2O m-2 s-1) of maize was recorded under stress condition (Appendix 4.5). Maize hybrids

6525 and 32B33 performed better than 31P41 and Hycorn. The maximum stomatal conductance

(3.95 mmol H2O m-2 s-1) was observed in hybrid 6525 while the minimum stomatal conductance

(3.73 mmol H2O m-2 s-1) was observed in Hycorn (Appendix 4.6).


(P<0.001) for stomatal conductance. The foliar application of P spray at 8th leaf stage increased

the stomatal conductance (3.97 mmol H2O m-2 s-1) as compare to no spray (3.71 mmol H2O m-2 s-

1) treatment (Appendix 4.5 & 4.6). The interaction between maize hybrids and treatments were

significantly affecting the stomatal conductance under normal and stress conditions. The highest

stomatal conductance was recorded in 6525 (4.14 mmol H2O m-2 s-1) where supplemental foliar P

@ 8 kg ha-1 was applied at 8th leaf stage while minimum stomatal conductance observed in

Hycorn (3.64 mmol H2O m-2 s-1) where no P foliar spray was applied under normal and stress

conditions (Appendix 4.6).

All other interaction such as H x W x T was non-significant (Table 4.7).

4.6.11: Sub-stomatal CO2 rate (Ci)

The sub-stomatal CO2 rate was significantly (P<0.01) decreased by water stress in all four

maize hybrids (Table 4.7). The decrease in sub-stomatal CO2 rate was more pronounced in water


of maize reduced sub-stomatal CO2 rate by 21.5% as compared to normally irrigated plants (Fig

4.38). The maximum sub-stomatal CO2 rate (250.50 µ mol H2O m-2 s-1) was recorded under well

watered condition while minimum sub-stomatal CO2 rate (196.67 µ mol H2O m-2 s-1) of maize

101

was recorded under stress conditions (Appendix 4.7). Maize hybrids 6525 and 32B33 performed

better than 31P41 and Hycorn. The maximum sub-stomatal CO2 rate (235.42 µ mol H2O m-2 s-1)

was observed in hybrid 6525 while the minimum sub-stomatal CO2 rate (212.92 µ mol H2O m-2 s-

1) was observed in Hycorn (Appendix 4.8).


(P<0.001) for sub-stomatal CO2 rate. The foliar application of P spray at 8th leaf stage increased

the sub-stomatal CO2 rate (238.29 µ mol H2O m-2 s-1) as compare to no spray (208.88 µ mol H2O

m-2 s-1) treatment (Appendix 4.7 & 4.8).


Table 4.7: Analysis of variance table for photosynthetic rate, transpiration rate, stomatal

conductance and sub-stomatal CO2 rate of four maize hybrids in well-watered and water

stress conditions with foliar applied phosphorus

SOV Photosynthetic

rate

Transpiration

rate

Stomatal

conductance

Sub-stomatal

CO2 rate

Hybrids (H) *** *** *** ***

Water levels (W) *** *** *** ***

Treatments (T) *** *** *** ***

H x W * NS *** **

H x T ** NS ** NS

W x T NS * * *

H x W x T NS NS NS NS

*, **, *** = Significant at 0.05, 0.01 and 0.001 level respectively; NS = Non significant

102

Fig. 4.33: Effect of supplemental foliar phosphorus application on net photosynthesis rate

(A) of four maize hybrids grown under normal and water stress conditions (mean values ±

S.E).

Fig. 4.34: Effect of supplemental foliar phosphorus application on transpiration rate (E) of

four maize hybrids grown under normal and water stress conditions (mean values ± S.E).

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50


Non stress Stress

Ph

oto

syn

thesi

s rate

(µ

mo

lC

O2

mˉ²

sˉ¹

) Control Foliar applied P @ 8kg/ha

0.00

0.50

1.00

1.50

2.00

2.50

3.00


Non stress stress

Tra

nsp

irati

on

rate

(m

mol

H2O

mˉ²

sˉ¹

)


103

Fig. 4.35: Effect of supplemental foliar phosphorus application on stomatal conductance (gs)

of four maize hybrids grown under normal and water stress conditions (mean values ± S.E).

Fig. 4.36: Effect of supplemental foliar phosphorus application on sub-stomatal CO2 rate

(Ci) of four maize hybrids grown under normal and water stress conditions (mean values ±

S.E).

0.00

1.00

2.00

3.00

4.00

5.00


Non stress stress

Sto

mata

lco

nd

ucta

nce (

mm

ol

H2O

mˉ²

sˉ¹

)


0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00


Non stress stress

Su

bst

om

ata

lC

O2

Co

ncen

trati

on

(µm

ol

H2O

mˉ²

sˉ¹

)


104

4.6.12: Leaf water potential (-MPa)

Drought stress significantly reduced (P<0.001) the leaf water potential (Table 4.8). The

water deficit conditions at 8th leaf stage significantly decreased it by 24% as compared to

normally irrigated (control) plants (Fig. 4.37). The maximum leaf water potential (0.96 -MPa)

was observed under normal condition while minimum leaf water potential (1.19 -MPa) of maize

was recorded under stress condition (Appendix 4.9). Maize hybrids 6525 and 32B33 performed

better than 31P41 and Hycorn. The maximum leaf water potential (1.03 -MPa) was observed in

hybrid 6525 while the minimum leaf water potential (1.12 -MPa) was observed in Hycorn

(Appendix 4.10).


(P<0.001) for leaf water potential. The foliar application of P spray at 8th leaf stage increased the

leaf water potential (1.00 -MPa) as compare to no spray (1.15 -MPa) treatment (Appendix 4.9 &

4.10).


4.6.13: Leaf osmotic potential (-MPa)

The leaf osmotic potential was significantly (P<0.01) decreased by water stress in all four

maize hybrids (Table 4.8). The decrease in leaf osmotic potential was more pronounced in water


of maize reduced leaf osmotic potential by 65% as compared to normally irrigated plants (Fig

4.40). The maximum leaf osmotic potential (0.40 -MPa) was recorded under well watered

condition while minimum leaf osmotic potential (0.66 -MPa) of maize was recorded under stress

conditions (Appendix 4.11). Maize hybrids 6525 and 32B33 performed better than 31P41 and

Hycorn. The maximum leaf osmotic potential (0.45 -MPa) was observed in hybrid 6525 which is

statically at par with 32B33 (0.48 -MPa) while the minimum leaf osmotic potential (0.62 -MPa)

was observed in Hycorn (Appendix 4.12).


(P<0.001) for leaf osmotic potential. The foliar application of P spray at 8th leaf stage increased

the leaf osmotic potential (0.50 -MPa) as compare to no spray (0.56 -MPa) treatment (Appendix

4.11 & 4.12).


105

4.6.14: Leaf turgor pressure (MPa)

Drought stress non-significantly reduced (P<0.001) the leaf turgor pressure (Table 4.8).

The water deficit conditions at 8th leaf stage significantly decreased it by 5.4% as compared to

normally irrigated (control) plants (Fig. 4.39). The maximum leaf turgor pressure (0.56 MPa) was

observed under normal condition while minimum leaf turgor pressure (0.53 MPa) of maize was

recorded under stress condition (Appendix 4.13). Maize hybrids 6525 and 32B33 performed

better than 31P41 and Hycorn. The maximum leaf turgor pressure (0.58 MPa) was observed in

hybrid 6525 while the minimum leaf turgor pressure (0.50 MPa) was observed in Hycorn

(Appendix 4.14).


(P<0.001) for leaf turgor pressure. The foliar application of P spray at 8th leaf stage increased the

leaf turgor pressure (0.58 MPa) as compare to no spray (0.50 MPa) treatment (Appendix 4.13 &

4.14).


4.6.15: Relative water contents (%)

The relative water contents was significantly (P<0.01) decreased by water stress in all four

maize hybrids (Table 4.8). The decrease in relative water contents was more pronounced in water


of maize reduced leaf osmotic potential by 26.2% as compared to normally irrigated plants (Fig

4.42). The maximum relative water contents (266.32%) were recorded under well watered

condition while minimum relative water contents (196.49%) of maize were recorded under stress


Hycorn. The maximum relative water contents (270.61%) were observed in hybrid 6525 while the

minimum relative water contents (194.70%) were observed in Hycorn (Appendix 4.16).


(P<0.001) for relative water contents. The foliar application of P spray at 8th leaf stage increased

the relative water contents (263.67%) as compare to no spray (199.14%) treatment (Appendix

4.15 & 4.16).

All other interactions were non-significant (Table 4.8).

106

Table 4.8: Analysis of variance table for water potential, osmotic potential, turgor pressure

and relative water contents of four maize hybrids in well-watered and water stress

conditions with supplemental foliar applied phosphorus

SOV

Water

potential

(-MPa)

Osmotic

potential

(-MPa)

Turgor

pressures

(MPa)

Relative water

contents (%)

Hybrids (H) *** *** NS ***

Water levels (W) *** *** NS ***

Treatments (T) *** ** ** ***

H x W * NS NS NS

H x T NS NS NS NS

W x T ** * * NS



Fig. 4.37: Effect of supplemental foliar phosphorus application on water potential (-MPa) of


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40


Non stress Stress

Wate

r p

ote

nti

al

(-M

Pa)

Control Foliar apllied P @ 8 kg/ha

107

Fig. 4.38: Effect of supplemental foliar phosphorus application on osmotic potential (-MPa)


Fig. 4.39: Effect of supplemental foliar phosphorus application on turgor pressure (MPa) of


0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00


Non stress Stress

Osm

oti

c p

ote

nti

al

(-M

Pa) Control Foliar apllied P @ 8 kg/ha

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80


Non stress Stress

Tu

rg

or

Po

ten

tial

(MP

a)


108

Fig. 4.40: Effect of supplemental foliar phosphorus application on relative water contents

(%) of four maize hybrids grown under normal and water stress conditions (mean values ±

S.E).

6.16: Chlorophyll a contents

Drought stress significantly reduced (P<0.001) the chlorophyll a contents (Table 4.9). The


normally irrigated (control) plants (Fig. 4.41). The maximum chlorophyll a contents (0.93 mg g-1

FW) were observed under normal condition while minimum chlorophyll a contents (0.63 mg g-1

FW) of maize were recorded under stress condition (Appendix 4.17). Maize hybrids 6525 and

32B33 performed better than 31P41 and Hycorn. The maximum chlorophyll a contents (0.87 mg

g-1 FW) were observed in hybrid 6525 while the minimum chlorophyll a contents (0.70 mg g-1

FW) were observed in Hycorn (Appendix 4.18).


(P<0.001) for chlorophyll a contents. The foliar application of P spray at 8th leaf stage increased

the chlorophyll a contents (0.86 mg g-1 FW) as compare to no spray (0.70 mg g-1 FW) treatment

(Appendix 4.17 & 4.18).


0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00

400.00


Non stress Stress

RW

C %


109

4.6.17: Chlorophyll b contents

The chlorophyll b contents were significantly (P<0.01) decreased by water stress in all

four maize hybrids (Table 4.9). The decrease in chlorophyll b contents were more pronounced in

water stressed plants as compared to normal irrigated plants (Fig. 4.44). The water stress at 8th

leaf stage of maize reduced chlorophyll b contents by 41.6% as compared to normally irrigated

plants (Fig. 4.42). The maximum chlorophyll b contents (0.60 mg g-1 FW) were recorded under

well watered condition while minimum chlorophyll b contents (0.35 mg g-1 FW) of maize were

recorded under stress conditions (Appendix 4.19).


(P<0.001) for chlorophyll b contents. The foliar application of P spray at 8th leaf stage increased

the chlorophyll b contents (0.54 mg g-1 FW) as compare to no spray (0.41 mg g-1 FW) treatment

(Appendix 4.19 & 4.20).


4.6.18: Total chlorophyll contents (a + b)

Drought stress significantly reduced (P<0.001) the total chlorophyll contents (Table 4.9).

The water deficit conditions at 8th leaf stage significantly decreased total chlorophyll content by

36% as compared to normally irrigated (control) plants (Fig. 4.43). The maximum total

chlorophyll contents (3.94 mg g-1 FW) were observed under normal condition while minimum

total chlorophyll contents (2.52 mg g-1 FW) of maize were recorded under stress condition

(Appendix 4.21). Maize hybrids 6525 and 32B33 performed better than 31P41 and Hycorn. The

maximum total chlorophyll contents (4.58 mg g-1 FW) were observed in hybrid 6525 while the

minimum total chlorophyll contents (2.92 mg g-1 FW) were observed in Hycorn (Appendix 4.22).


(P<0.001) for total chlorophyll contents. The foliar application of P spray at 8th leaf stage

increased the total chlorophyll contents (3.62 mg g-1 FW) as compare to no spray (2.85 mg g-1

FW) treatment (Appendix 4.21 & 4.22).

All the interactions were non-significant (Table 4.9).

4.6.19: Total carotenoids

The total carotenoids were significantly (P<0.01) decreased by water stress in all four

maize hybrids (Table 4.9). The decrease in total carotenoids was more pronounced in water

110


of maize reduced total carotenoids by 34.7% as compared to normally irrigated plants (Fig 4.46).

The maximum total carotenoids (0.49 mg g-1 FW) were recorded under well watered condition

while minimum total carotenoids (0.32 mg g-1 FW) of maize were recorded under stress


Hycorn. The maximum total carotenoids (0.48 mg g-1 FW) were observed in hybrid 6525 while

the minimum total carotenoids (0.33 mg g-1 FW) were observed in Hycorn (Appendix 4.24).


(P<0.001) for total carotenoids. The foliar application of P spray @ 8 kg ha-1 increased the total

carotenoids (0.47 mg g-1 FW) as compare to no spray (0.34 mg g-1 FW) treatment (Appendix 4.23

& 4.24).


Table 4.9: Analysis of variance table for chlorophyll a, chlorophyll b, total chlorophyll

contents and total carotenoids of four maize hybrids in well-watered and water stress

conditions with foliar applied phosphorus

SOV Chlorophyll a Chlorophyll b

Total

chlorophyll

contents

Total

carotenoids

Hybrids (H) *** NS *** ***

Water levels (W) *** ** *** ***

Treatments (T) *** *** *** ***

H x W NS NS NS NS

H x T NS NS NS NS

W x T NS NS NS NS



111

Fig. 4.41: Effect of supplemental foliar phosphorus application on chlorophyll a (mg/g fresh

weight) of four maize hybrids grown under normal and water stress conditions (mean

values ± S.E).

Fig. 4.42: Effect of supplemental foliar phosphorus application on chlorophyll b (mg/g fresh

weight) of four maize hybrids grown under normal and water stress conditions (mean

values ± S.E).

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40


Non stress stress

Ch

loro

ph

yll

a(m

g/g

f.w

t.) Control Foliar applied P @ 8 kg/ha

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80


Non stress stress

Ch

loro

ph

yll

b(m

g/g

f.w

t.) Control Foliar applied P @ 8 kg/ha

112

Fig. 4.43: Effect of supplemental foliar phosphorus application on total chlorophyll contents

(mg/g fresh weight) of four maize hybrids grown under normal and water stress conditions


Fig. 4.44: Effect of supplemental foliar phosphorus application on total carotenoids (mg/g)


0.00

1.00

2.00

3.00

4.00

5.00

6.00


Non stress stress

To

tal

ch

loro

ph

yll

co

nte

nts

(m

g/g

f.w

t.)


0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80


Non stress stress

To

tal

caro

ten

oid

s (m

g/g

)


113

4.6.20: Total soluble proteins (TSP)

Drought stress significantly reduced (P<0.001) the total soluble proteins (Table 4.10). The


normally irrigated (control) plants (Fig. 4.45). The maximum total soluble proteins (6.73 mg g-1

FW) were observed under normal condition while minimum total soluble proteins (6.08 mg g-1

FW) of maize were recorded under stress condition (Appendix 4.25). Maize hybrids 6525 and

32B33 performed better than 31P41 and Hycorn. The maximum total soluble proteins (6.69 mg g-

1 FW) were observed in hybrid 6525 which is statically at par with 32B33 (6.56 mg g -1 FW) while

the minimum total soluble proteins (6.09 mg g-1 FW) were observed in Hycorn (Appendix 4.26).


(P<0.001) for total soluble proteins. The foliar application of P spray at 8th leaf stage increased

the total soluble proteins (6.85 mg g-1 FW) as compare to no spray (5.97 mg g-1 FW) treatment

(Appendix 4.25 & 4.26).


4.6.21: Total free amino acid (TFA)

The total free amino acid was significantly (P<0.01) increased by water stress in all four

maize hybrids (Table 4.10). The increase in total free amino acid was more pronounced in water


of maize enhanced total free amino acid by 25.6% as compared to normally irrigated plants (Fig

4.48). The maximum total free amino acid (27.82 mg g-1 FW) was recorded under stress condition

while minimum total free amino acid (20.69 mg g-1 FW) of maize was recorded under well

watered conditions (Appendix 4.27). Maize hybrids 6525 and 32B33 performed better than 31P41

and Hycorn. The maximum total free amino acid (25.69 mg g-1 FW) was observed in hybrid 6525

while the minimum total free amino acid (23.00 mg g-1 FW) was observed in Hycorn (Appendix

4.28).


(P<0.001) for total free amino acid. The foliar application of P spray at 8th leaf stage increased the

total free amino acid (26.15 mg g-1 FW) as compare to no spray (22.36 mg g-1 FW) treatment

(Appendix 4.27 & 4.28).


114

4.6.22: Total soluble sugars (TSS)

Drought stress significantly enhanced (P<0.001) the total soluble sugars (Table 4.10). The

water deficit conditions at 8th leaf stage significantly increased it by 37.9% as compared to

normally irrigated (control) plants (Fig. 4.47). The maximum total soluble sugars (2.66 mg g-1

FW) were observed under stress condition while minimum total soluble sugars (1.65 mg g-1 FW)

of maize were recorded under normal conditions (Appendix 4.29). Maize hybrids 6525 and

32B33 performed better than 31P41 and Hycorn. The maximum total soluble sugars (2.34 mg g-1

FW) were observed in hybrid 6525 while the minimum total soluble sugars (1.97 mg g-1 FW)

were observed in Hycorn (Appendix 4.30).


(P<0.001) for total soluble sugars. The foliar application of P spray at 8th leaf stage increased the

total soluble sugars (2.42 mg g-1 FW) as compare to no spray (1.90 mg g-1 FW) treatment

(Appendix 4.29 & 4.30).


4.6.23: Proline contents

The proline contents were significantly (P<0.01) increased by water stress in all four

maize hybrids (Table 4.10). The increase in proline contents was more pronounced in water


of maize enhanced proline contents by 38.2% as compared to normally irrigated plants (Fig 4.50).

The maximum proline contents (474.73 µ g g-1 FW) were recorded under stress condition while

minimum proline contents (293.51 µ g g-1 FW) of maize was recorded under well watered


Hycorn. The maximum proline contents (419.65 µ g g-1 FW) were observed in hybrid 6525 while

the minimum proline contents (250.55 µ g g-1 FW) were observed in Hycorn (Appendix 4.32).


(P<0.001) for proline contents. The foliar application of P spray at 8th leaf stage increased the

proline contents (433.20 µ g g-1 FW) as compare to no spray (335.04 µ g g-1 FW) treatment

(Appendix 4.31 & 4.32).


115

Table 4.10: Analysis of variance table for total soluble proteins, total free amino acids, total

soluble sugars and proline contents of four maize hybrids in well-watered and water stress


SOV Total soluble

proteins

Total free

amino acids

Total soluble

sugars

Proline

contents

Hybrids (H) *** *** *** **

Water levels (W) *** *** *** ***

Treatments (T) *** *** *** ***

H x W NS NS NS NS

H x T NS NS NS NS

W x T NS ** NS *



Fig. 4.45: Effect of supplemental foliar phosphorus application on total soluble protein



0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00


Non stress Stress

To

tal

solu

ble

pro

tein

(m

g/g

f.w

t.)


116

Fig. 4.46: Effect of supplemental foliar phosphorus application on total free amino acid



Fig. 4.47: Effect of supplemental foliar phosphorus application on total soluble sugar (mg/g

fresh weight) of four maize hybrids grown under normal and water stress conditions (mean

values ± S.E).

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00


Non stress Stress

To

tal

free a

min

o a

cid

(m

g/g

f.w

t.)


0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00


Non stress Stress

To

tal

solu

ble

su

gar

(mg

/g f

.wt.

)


117

Fig. 4.48: Effect of supplemental foliar phosphorus application on proline contents (µg/g

fresh weight) of four maize hybrids grown under normal and water stress conditions (mean

values ± S.E).

4.6.24: Catalase activity

Drought stress significantly enhanced (P<0.001) the catalase activity (Table 4.11). The

water deficit conditions at 8th leaf stage significantly increased catalase activity by 68% as

compared to normally irrigated (control) plants (Fig. 4.49). The maximum catalase activity

(166.07 units’ min-1 g-1 FW) was observed under stress condition while minimum catalase activity

(52.90 units min-1 g-1 FW) of maize was recorded under normal conditions (Appendix 4.33).

Maize hybrids 6525 and 32B33 performed better than 31P41 and Hycorn. The maximum catalase

activity (124.13 units min-1 g-1 FW) was observed in hybrid 6525 while the minimum catalase

activity (100.23 units min-1 g-1 FW) was observed in Hycorn (Appendix 4.34).


(P<0.001) for catalase activity. The foliar application of P spray at 8th leaf stage increased the

catalase activity (115.76 units min-1 g-1 FW) as compare to no spray (103.21 units min-1 g-1 FW)



4.6.25: Peroxidase activity

The peroxidase activity was significantly (P<0.01) increased by water stress in all four

maize hybrids (Table 4.11). The increase in peroxidase activity was more pronounced in water

0.00

100.00

200.00

300.00

400.00

500.00

600.00


Non stress Stress

Pro

lin

e c

on

ten

ts (

µg

/g f

.wt.


118


of maize enhanced peroxidase activity by 79.7% as compared to normally irrigated plants (Fig.

4.50). The maximum peroxidase activity (183.25 units min-1 g-1 FW) was recorded under stress

condition while minimum peroxidase activity (37.05 units min-1 g-1 FW) of maize was recorded

under well watered conditions (Appendix 4.35). Maize hybrids 6525 and 32B33 performed better

than 31P41 and Hycorn. The maximum peroxidase activity (124.25 units min-1 g-1 FW) was

observed in hybrid 6525 while the minimum peroxidase activity (99.19 units min-1 g-1 FW) was

observed in Hycorn (Appendix 4.36).


(P<0.001) for peroxidase activity. The foliar application of P spray at 8th leaf stage increased the

peroxidase activity (120.05 units min-1 g-1 FW) as compare to no spray (100.25 units min-1 g-1

FW) treatment (Appendix 4.35 & 4.36).


4.6.26: Ascorbate peroxidase activity

Drought stress significantly enhanced (P<0.001) the ascorbate peroxidase activity (Table

4.11). The water deficit conditions at 8th leaf stage significantly increased it by 57.3% as

compared to normally irrigated (control) plants (Fig. 4.51). The maximum ascorbate peroxidase

activity (3.84 units’ min-1 g-1 FW) was observed under stress condition while minimum ascorbate

peroxidase activity (1.64 units min-1 g-1 FW) of maize was recorded under normal conditions

(Appendix 4.37). Maize hybrids 6525 and 32B33 performed better than 31P41 and Hycorn. The

maximum ascorbate peroxidase activity (3.25 units min-1 g-1 FW) was observed in hybrid 6525

while the minimum ascorbate peroxidase activity (2.15 units min-1 g-1 FW) was observed in

Hycorn (Appendix 4.38).


(P<0.001) for ascorbate peroxidase activity. The foliar application of P spray at 8th leaf stage

increased the ascorbate peroxidase activity (3.26 units min-1 g-1 FW) as compare to no spray (2.23

units min-1 g-1 FW) treatment (Appendix 4.37 & 4.38).


119

Table 4.11: Analysis of variance table for catalase activity, peroxidase activity and

ascorbate peroxidase activity of four maize hybrids in well-watered and water stress


SOV Catalase activity Peroxidase activity Ascorbate

peroxidase activity

Hybrids (H) *** *** ***

Water levels (W) *** *** ***

Treatments (T) *** *** ***

H x W NS ** NS

H x T NS NS NS

W x T NS * NS

H x W x T NS NS NS


Fig. 4.49: Effect of supplemental foliar phosphorus application on catalase activity (Unit

min-1 g-1 fresh weight) of four maize hybrids grown under normal and water stress


0.00

50.00

100.00

150.00

200.00

250.00


Non stress Stress

Cata

lase

acti

vit

y (

Un

it m

in-1

g-1

F.w

t.)


120

Fig. 4.50: Effect of supplemental foliar phosphorus application on peroxidase activity (Unit

min-1 g-1 fresh weight) of four maize hybrids grown under normal and water stress


Fig. 4.51: Effect of supplemental foliar phosphorus application on ascorbate peroxidase

activity (Unit min-1 g-1 fresh weight) of four maize hybrids grown under normal and water


0.00

50.00

100.00

150.00

200.00

250.00

300.00


Non stress Stress

Pero

xid

ase

acti

vit

y (

Un

it m

in-1

g-1

F.w

t.)


0.00

1.00

2.00

3.00

4.00

5.00

6.00


Non stress Stress

Asc

orb

ate

pero

xid

ase

acti

vit

y

(Un

it m

in-1

g-1

F.w

t.)


121

4.6.27: P concentration in leaf

Drought stress significantly decreased (P<0.001) the P concentration in leaf (Table 4.12).


normally irrigated (control) plants (Fig. 4.52). The maximum P concentration in leaf (0.78 mg g-1

DW) was observed under normal condition while minimum P concentration in leaf (0.58 mg g-1

DW) of maize was recorded under stress conditions (Appendix 4.39). Maize hybrids 6525 and

32B33 performed better than 31P41 and Hycorn. The maximum P concentration in leaf (0.70 mg

g-1 DW) was observed in hybrid 6525 while the minimum P concentration in leaf (0.59 mg g-1

DW) was observed in Hycorn (Appendix 4.40).


(P<0.001) for P concentration in leaf. The foliar application of P spray at 8th leaf stage increased

the P concentration in leaf (0.72 mg g-1 DW) as compare to no spray (0.58 mg g-1 DW) treatment

(Appendix 4.39 & 4.40).


4.6.28: P concentration in stem

Drought stress significantly decreased (P<0.001) the P concentration in stem (Table 4.12).


normally irrigated (control) plants (Fig. 4.53). The maximum P concentration in stem (0.97 mg g-1

DW) was observed under normal condition while minimum P concentration in stem (0.67 mg g-1

DW) of maize was recorded under stress conditions (Appendix 4.41). Maize hybrids 6525 and

32B33 performed better than 31P41 and Hycorn. The maximum P concentration in stem (0.88 mg

g-1 DW) was observed in hybrid 6525 while the minimum P concentration in stem (0.76 mg g-1

DW) was observed in Hycorn (Appendix 4.42).


(P<0.001) for P concentration in stem. The foliar application of P spray at 8th leaf stage increased

the P concentration in stem (0.90 mg g-1 DW) as compare to no spray (0.74 mg g-1 DW) treatment

(Appendix 4.41 & 4.42).


122

4.6.29: P concentration in root

The P concentration in root was significantly (P<0.01) decreased by water stress in all

four maize hybrids (Table 4.12). The decrease in P concentration in root was more pronounced in


leaf stage of maize decreased P concentration in root by 36% as compared to normally irrigated

plants (Fig 4.56). The maximum P concentration in root (1.83 mg g-1 DW) was recorded under

well watered condition while minimum P concentration in root (1.17 mg g-1 DW) of maize was

recorded under stress conditions (Appendix 4.43). Maize hybrids 6525 and 32B33 performed

better than 31P41 and Hycorn. The maximum P concentration in root (1.62 mg g-1 DW) was

observed in hybrid 6525 while the minimum P concentration in root (1.38 mg g-1 DW) was



(P<0.001) for P concentration in root. The foliar application of P spray at 8th leaf stage increased

the P concentration in root (1.64 mg g-1 DW) as compare to no spray (1.35 mg g-1 DW) treatment

(Appendix 4.43 & 4.44).

4.6.30: P concentration in grain

The P concentration in grain was significantly (P<0.01) decreased by water stress in all

four maize hybrids (Table 4.12). The decrease in P concentration in grain was more pronounced

in water stressed plants as compared to normal irrigated plants (Fig. 4.55). The water stress at 8th

leaf stage of maize decreased P concentration in grain by 19.5% as compared to normally

irrigated plants (Fig. 4.55). The maximum P concentration in grain (2.62 mg g-1 DW) was

recorded under well watered condition while minimum P concentration in grain (2.11 mg g-1 DW)

of maize was recorded under stress conditions (Appendix 4.45). Maize hybrids 6525 and 32B33

performed better than 31P41 and Hycorn. The maximum P concentration in grain (2.46 mg g-1

DW) was observed in hybrid 6525 while the minimum P concentration in grain (2.28 mg g-1 DW)



(P<0.001) for P concentration in grain. The foliar application of P spray at 8th leaf stage increased

the P concentration in grain (2.50 mg g-1 DW) as compare to no spray (2.24 mg g-1 DW)



123

4.6.31: Phosphorus use efficiency (PUE)

The PUE was significantly (P<0.01) decreased by water stress in all four maize hybrids

(Table 4.12). The decrease in PUE was more pronounced in water stressed plants as compared to

normal irrigated plants (Fig. 4.58). The water stress at 8th leaf stage of maize decreased PUE by

34.5% as compared to normally irrigated plants (Fig 4.56). The maximum PUE (3.59%) was

recorded under well watered condition while minimum PUE (2.35%) of maize was recorded

under stress conditions (Appendix 4.47). Maize hybrids 6525 and 32B33 performed better than

31P41 and Hycorn. The maximum PUE (3.21%) was observed in hybrid 6525 while the

minimum PUE (2.74%) was observed in Hycorn (Appendix 4.48).

The effect of supplemental foliar applied P spray treatment was also highly

significant (P<0.001) for PUE. The foliar application of P spray at 8th leaf stage increased the

PUE (3.26%) as compare to no spray (2.68%) treatment (Appendix 4.47 & 4.48). All other


Table 4.12: Analysis of variance table for P concentration in grain, P concentration in leaf,

P concentration in stem, P concentration in root and PUE of four maize hybrids in well-

watered and water stress conditions with foliar applied phosphorus

SOV

P

concentration

in leaf

P

concentration

in stem

P

concentration

in root

P

concentration

in grain

PUE

Hybrids (H) *** *** *** *** ***

Water levels (W)

*** *** *** *** ***

Treatments (T) *** *** *** *** ***

H x W NS NS NS NS NS

H x T NS NS NS NS NS

W x T ** ** ** ** NS

H x W x T NS NS NS NS NS


124

Fig. 4.52: Effect of supplemental foliar phosphorus application on P concentration in leaf of


Fig. 4.53: Effect of supplemental foliar phosphorus application on P concentration in stem


0.00

0.20

0.40

0.60

0.80

1.00

1.20


Non stress stress

Leaf

P (

mg

/g d

.wt.


0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40


Non stress stress

Ste

m P

(m

g/g

d.w

t.)


125

Fig. 4.54: Effect of supplemental foliar phosphorus application on P concentration in root of


Fig. 4.55: Effect of supplemental foliar phosphorus application on P concentration in grain


0.00

0.50

1.00

1.50

2.00

2.50


Non stress stress

Ro

ot

p (

mg

/g d

.wt.


0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50


Non stress stress

Grain

P (

mg

/g d

.wt.

)


126

Fig. 4.56: Effect of supplemental foliar phosphorus application on PUE of four maize

hybrids grown under normal and water stress conditions (mean values ± S.E).

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

4.00

4.50


Non stress stress

PU

E (

%)


127

4.7. Experiment # 7: The combined effect of best source of P, dose of P and

growth stage on yield and yield components of maize hybrids under water

stress

4.7.1: Cob length (cm)

Drought stress significantly reduced (P<0.001) the cob length (Table 4.13a). The water

deficit conditions at 8th leaf stage significantly decreased it by 5.9% as compared to normally

irrigated (control) plants (Fig. 4.57). The maximum cob length (19.01 cm) was observed under

normal condition while minimum cob length (17.87 cm) of maize was recorded under stress

condition (Appendix 4.49). Maize hybrids 6525 and 32B33 maintained 6.7% and 5.9% more cob

length than 31P41 and Hycorn under normal and stress conditions (Fig. 4.57).


(P<0.001) for cob length. The foliar application of P spray at 8th leaf stage increased the cob

length (18.79 cm) as compare to no spray (18.10 cm) treatment (Appendix 4.49 & 4.50).

All the interactions such as H x T and H x W x T were non-significant (Table 4.11a).

4.7.2: Number of cobs per plant

The number of cobs plant-1 was significantly (P<0.01) decreased by water stress in all four

maize hybrids (Table 4.13a). The decrease in number of cobs per plant was more pronounced in


leaf stage of maize reduced number of cob per plant by 37.5% as compared to normally irrigated

plants (Fig. 4.58). The maximum number of cobs per plant (6.66) was recorded under well

watered condition while minimum number of cobs per plant (5.58) of maize was recorded under

stress conditions (Appendix 4.51). Similarly the maize hybrids 6525 and 32B33 performed better

than 31P41 and Hycorn. Data recorded maximum number of cobs per plant in hybrid 6525 (6.50)

statistically at par with the hybrid 32B33 (6.33) while the minimum number of cobs per plant was

observed in Hycorn (5.66) (Appendix 4.52).


(P<0.001) for number of cobs per plant. The foliar application of P spray at 8th leaf stage

increased the number of cobs plant-1 (6.54) as compare to no spray (5.70) treatment (Appendix

4.51 & 4.52).


128

4.7.3: Number of grains per cob

Drought stress significantly reduced (P<0.001) the number of grains per cob in all four

maize hybrids (Table 4.13a). The water deficit conditions at 8th leaf stage significantly decreased

number of grains per cob by 3.9% as compared to normally irrigated (control) plants (Fig. 4.59).

The more number of grains per cob (376.38) was observed under well watered condition while

minimum number of grains per cob (361.79) of maize was recorded under stress conditions

(Appendix 4.53). Maize hybrids 6525 and 32B33 maintained 3.8% and 3.7% more number of

grains per cob than 31P41 and Hycorn respectively (Appendix 4.53). The maximum number of

grains per cob in hybrid 6525 (372.17) was observed while the minimum number of grains per

cob (366.42) was observed in Hycorn (Appendix 4.54).


(P<0.001) for number of grains per cob. The foliar application of P spray at at 8th leaf stage

increased the number of grains per cob (372.54) as compare to no spray (365.63) treatment

(Appendix 4.53 & 4.54).


4.7.4: Cob weight without sheath (g)

The cob weight without sheath was significantly (P<0.01) decreased by water stress in all

four maize hybrids (Table 4.13a). The decrease in cob weight without sheath was more

pronounced in water stressed plants. The water stress at 8th leaf stage reduced cob weight without

sheath by 20.3% as compared to normally irrigated plants (Fig. 4.60). The maximum cob weight

without sheath (225.38 g) was observed under normal condition while minimum cob weight

without sheath (179.71 g) of maize was recorded under stress conditions (Appendix 4.55). Maize

hybrids 6525 and 32B33 performed better than 31P41 and Hycorn. The maximum cob weight

without sheath in hybrid 6525 (213.31 g) was observed while the minimum cob weight without

sheath (193.35g) was observed in Hycorn (Appendix 4.56).


(P<0.001) for cob weight without sheath. The foliar application of P spray at 8th leaf stage

increased the number of cob weight without sheath (214.53 g) as compare to no spray (190.56 g)



129

Fig. 4.57: Effect of supplemental foliar P application on cob length (cm) of four maize

hybrids under different water levels (mean values ± S.E).

Fig. 4.58: Effect of supplemental foliar P application on number of cobs per plant of four

maize hybrids under different water levels (mean values ± S.E).

0.00

5.00

10.00

15.00

20.00

25.00


Non stress Stress

Co

b l

en

gth

(cm


0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00


Non stress Stress

Nu

mb

er

of

co

bs/

pla

nt


130

Fig. 4.59: Effect of supplemental foliar P application on number of grains per cob of four


Fig. 4.60: Effect of supplemental foliar P application on cob weight without sheath (g) of

four maize hybrids under different water levels (mean values ± S.E).

330.00

340.00

350.00

360.00

370.00

380.00

390.00


Non stress Stress

Nu

mb

er

of

grain

s/co

b


0.00

50.00

100.00

150.00

200.00

250.00

300.00


Non stress Stress

Co

b w

eig

ht

wit

ho

ut

sheath

(g

) Control Foliar applied P @ 8 kg/ha

131

4.7.5: Thousand grain weight (g)

Drought stress significantly reduced (P<0.001) the thousand grain weight in all four maize

hybrids (Table 4.13b). The water deficit conditions at 8th leaf stage significantly decreased

thousand grain weights by 21.2% as compared to normally irrigated (control) plants (Fig. 4.61)

The maximum value of thousand grain weights (270.70 g) was observed under normal condition

while minimum thousand grain weight (213.47 g) of maize was recorded under stress conditions

(Appendix 4.57). Maize hybrids 6525 and 32B33 maintained 23.2% and 22.3% more thousand

grain weight than 31P41 and Hycorn respectively. The maximum thousand grain weight (253.76

g) was observed in hybrid 6525 while the minimum thousand grain weight (229.35 g) was



(P<0.001) for thousand grain weight. The foliar application of P spray at at 8th leaf stage

increased the thousand grain weight (258.28 g) as compare to no spray (225.88 g) treatment

(Appendix 4.57 & 4.58). The interaction between maize hybrids and treatments were significantly

affecting the thousand grain weight under normal and stress conditions. The highest thousand

grain weight was recorded in 6525 (272.60 g) where supplemental foliar applied P @ 8 kg ha-1 at

8th leaf stage while minimum thousand grain weight observed in Hycorn (216.23 g) where no

foliar spray under normal and stress conditions (Appendix 4.58).

All others interactions were non-significant (Table 4.11b).

4.7.6: Grain yield (t ha-1)

The grain yield was significantly (P<0.01) decreased by water stress in all four maize

hybrids (Table 4.13b). The decrease in grain yield was more pronounced in water stressed plants.

The water stress at 8th leaf stage reduced grain yield by 21.3% as compared to normally irrigated

plants (Fig. 4.62). The maximum grain yield (6.77 t ha-1) was recorded under normal condition

while minimum grain yield (4.62 t ha-1) of maize was recorded under stress conditions (Appendix

4.59). Maize hybrids 6525 and 32B33 performed better than 31P41 and Hycorn. The maximum

grain yield (6.15 t ha-1) was observed in hybrid 6525 while the minimum grain yield (5.30 t ha-1)



(P<0.001) for grain yield. The foliar application of P spray at 8th leaf stage increased the grain

yield (6.27 t ha-1) as compare to no spray (5.12 t ha-1) treatment (Appendix 4.59 & 4.60).

132


4.7.7: Biological yield (t ha-1)

Drought stress significantly reduced (P<0.001) the biological yield in all four maize

hybrids (Table 4.13b). The water deficit conditions at 8th leaf stage significantly decreased

biological yield by 22.4% as compared to normally irrigated (control) plants (Fig. 4.63). The

more biological yield (16.54 t ha-1) was observed under normal condition while minimum

biological yield (12.83 t ha-1) of maize was recorded under stress conditions (Appendix 4.61).

Maize hybrids 6525 and 32B33 maintained 23.1% and 22.9% more biological yield than 31P41

and Hycorn respectively.


(P<0.001) for biological yield. The foliar application of P spray at 8th leaf stage increased the

biological yield (15.68 t ha-1) as compare to no spray (13.69 t ha-1) treatment (Appendix 4.61 &

4.62). The maximum biological yield (15.52 t ha-1) was observed in hybrid 6525 while the

minimum biological yield (13.91 t ha-1) was observed in Hycorn (Appendix 4.62).

The interaction between maize hybrids and treatments were significantly affecting the

biological yield under normal and stress conditions. The highest biological yield was recorded in

6525 (16.63 t ha-1) where supplemental foliar applied P @ 8 kg ha-1 at 8th leaf stage while

minimum biological yield observed in Hycorn (12.87 t ha-1) where no foliar spray under normal

and stress conditions (Appendix 4.62).

All others interactions were non-significant (Table 4.11b).

4.7.8: Harvest index (%)

The harvest index was significantly (P<0.01) decreased by water stress in all four maize

hybrids (Table 4.13b). The decrease in harvest index was more pronounced in water stressed

plants. The water stress at 8th leaf stage reduced harvest index by 12.2% as compared to normally

irrigated plants (Fig. 4.64). The maximum harvest index (40.86%) was observed under normal

condition while minimum harvest index (35.90%) of maize was recorded under stress conditions

(Appendix 4.63). Maize hybrids 6525 and 32B33 performed better than 31P41 and Hycorn under

normal and stress conditions. Data recorded showed maximum harvest index (39.36%) was

observed in hybrid 6525 while the minimum harvest index (27.72%) was observed in Hycorn

(Appendix 4.64).

133


(P<0.001) for harvest index. The foliar application of P spray at 8th leaf stage increased the

harvest index (39.73%) as compare to no spray (37.04%) treatment (Appendix 4.63 & 4.64).


Fig. 4.61: Effect of supplemental foliar P application on 1000-grain weight (g) of four maize


Fig. 4.62: Effect of supplemental foliar P application on grain yield (t ha-1) of four maize


0.00

50.00

100.00

150.00

200.00

250.00

300.00

350.00


Non stress Stress

10

00

-grain

weig

ht

(g)


0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00


Non stress Stress

Grain

yie

ld (

t/h

a)


134

Fig. 4.63: Effect of supplemental foliar P application on biological yield (t ha-1) of four


Fig. 4.64: Effect of supplemental foliar P application on harvest index (%) of four maize


0.00

5.00

10.00

15.00

20.00

25.00


Non stress Stress

Bio

log

ical

yie

ld (

t/h

a)


0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

45.00

50.00


Non stress Stress

Harv

est

in

dex (

%)


135

Table 4.13a, b: Analysis of variance table for cob length, number of cobs plant-1, number of

grains cob-1, cob weight without sheath, thousand grain weight (g) , grain yield (t ha-1) and

biological yield (t ha-1) of four maize hybrids in well-watered and water stress conditions

with supplemental foliar applied phosphorus

a

SOV Cob length

(cm)

No. of cobs per

plant

No. of grains

per cob

Cob weight

without sheath

(g)

Hybrids (H) *** * *** **

Water levels (W) *** ** *** **

Treatments (T) *** ** *** **

H x W NS NS NS NS

H x T NS NS NS NS

W x T NS NS NS NS


*, **, *** = Significant at 0.05, 0.01 and 0.001 level respectively

NS = Non significant

b

SOV 1000-grain

weight (g)

Grain yield

(t ha-1)

Biological yield

(t ha-1)

Harvest index

(%)

Hybrids (H) *** *** ** **

Water levels (W) *** *** *** ***

Treatments (T) *** *** *** ***

H x W ** NS NS NS

H x T * NS * NS

W x T ** ** ** NS


*, **, *** = Significant at 0.05, 0.01 and 0.001 level respectively

NS = Non significant

136

5.1. Screening of maize hybrids

The screening of available maize hybrids before P application studies provided a useful

insight into the drought tolerance potential of selected hybrids. The hybrids were selected on the

basis of variation in their response to water stress. Lab screening was carried out in stress

physiology lab using PEG-6000 as an osmotic stress inducing agent (Experiment # 1). Several

methods are used for screening of genotypes for drought tolerance however, such approaches

should be easy, rapid and inexpensive (Austin, 1993). Polyethylene glycol (PEG) is one of the

most widely used drought simulator in laboratories (Mohammadi et al., 2003; Dhanda et al.,

2004). It is well established that PEG-6000 is a useful osmotic medium for the imposition of

drought stress to screen genotypes for drought tolerance (Blum et a1., 1986; Ashraf and Naqvi,

1995; Guttieri et al., 2001; Raza et al., 2012 and Ahmad et al., 2009). Polyethylene glycol (PEG)

is responsible for creating water stress in plants and acts as simulant (Ahsraf and O’leary, 1996;

Turhan, 1997). The current study the PEG-6000 was used for creating osmotic stress in plants.

PEG is commonly used it in lab experiment for creating artificial drought and to check the

response of plant (Hu and Jones, 2004).

The study regarding the effect of water stress created by PEG-6000 indicated that

germination percentage, promptness and germination stress indices (GSI) were limited by

enhancing the levels of PEG-6000. The germination stress tolerance index of different maize

hybrids was also affected by increasing concentration of PEG-6000. However 6525 and 3672

maize hybrid performed better than others. Many reports indicated that GSI can be utilized as

screening criteria for stress tolerance. However, many researchers are of the view that

germination criteria did not seem to reflect stress tolerance in different plants, but rather to

indicate seed quality differences, nor did this procedure reflect the yield stability of genotypes

(Ahsraf and O’leary, 1996). This agreed with the results of Khayatnezhad et al. (2010),

Khodarahmpour, (2011) and Mostafavi et al. (2011). According to Ayaz et al. 2001, the

germination of the seed crop was limited under water deficit condition. The seed germination was

also limited due to the imbibition of seed with water (Hadas, 1977). We calculated GSI values by

using promptness index (PI) values of stressed and non-stressed (control) seeds. Our results

137

showed that GSI decreased due to PEG-induced water stress irrespective of the genotype. Among

tested genotypes, 6525 and 32B33 showed the maximum values of GSI at all levels of water

stress. High values of GSI indicate the potential for drought tolerance (Zahra and Farshadfar,

2011; Ahmad et al., 2015).

Observations at the seedling stage in experiment 2 indicated that water stress induced after

germination caused an extension in root length in all tested genotypes. This extension in root

length happened at the cost of reduction in shoot length, thus shifting the relative root: shoot (R:

S) equilibrium in favour of the roots, also reported by Larson, (1992), Ashraf and Sarwar, (2002),

and Guoxiong et al. (2002). The increase in R: S length under water limitations may be attributed

to reduction in supply of water and nutrients to the shoot. The possible explanation for reduction

in shoot length under water-limited conditions might be the decrease in cell expansion, which

ultimately reduces the plant height (Bajji et al., 2000; Okçu et al., 2005; Shahbaz et al., 2011).

Moreover, plant hormones can also play their role in the extension of root length under water-

limited conditions (Sharp and Davis, 1985), which is particularly important to avoid drought

stress in dry soils (Dhanda et al., 2004). However, this may not be true for absolute root biomass

which may increase in plants grown under well-watered conditions (Sharp and Davis 1985;

Thornley, 1998).

Root length and seedling dry weight can be used as major selection criteria for screening

genotypes against drought stress (Leishman and Westoby, 1994; Al-Karaki, 1998). Results of the

study showed that the PHSI and DMSI are non-significantly affected but RLSI significantly

affected under drought stress. Ranking showed that the two maize hybrids such as 6525 and 3672

performed better than all others maize hybrids. Deep roots and ability to accumulate higher

biomass are considered typical characteristics of drought tolerant genotypes. Germination rate and

final germination percentage correlate with root length and how much biomass is accumulated per

unit area (Khan et al., 2002; Ghodsi, 2004; Okçu et al., 2005; Rauf et al., 2007; Yamur and

Kaydan, 2008). Water stress acts by decreasing the percent and rate of germination and seedling

growth (Farsiani and Ghobadi, 2009; Khayatnezhad et al. 2010). There are reports in the literature

of potential drought resistance traits like extensive viable radicle system that could explore deeper

soil layers for water (Mirza, 1956; Bocev, 1963). Maize plants with more radicles at seedling

stage subsequently developed stronger radicle system, produce more green matter and had higher

values for most characters determining seed yield (Bocev, 1963).

138

5.2. Optimization of different sources of P in maize under drought stress

Drought is the most important abiotic stress factor that limits all aspects of crop growth

and production. In many developing countries, water stress is the major constraint to agricultural

production and also reduces the quality, growth and production of crops (Hongbo et al., 2005;

Golbashy et al., 2010; Waraich et al., 2010, 2011). The life cycle of different plants usually

affected by water stress and plants face water deficit from atmosphere and soils during its life

cycle (Chaves et al., 2002). Many growth variables (root length and shoot length etc.) of the

plants are affected by water stress. Different functions and fitness of the plants also reduced under

water stress conditions (Tian and Lei, 2006 and Xu et al., 2007). Under water stress condition the

reactive oxygen species (ROS) are produced and these ROS cause the serious problems inside the

plants (Waraich et al., 2011). Water deficit affects shoot growth and P uptake in plants, Moreover

increasing moisture stress resulted in gradually less leaf area, plant height, crop growth rate, shoot

dry matter and harvest index. While biomass production responded to P and maximum yield was

produced under normal condition. But P uptake was more dependent on applied P than water

supply while P uptake decreased with water deficit (Pandey et al., 2000).

Phosphorus is a constituent of molecular structures such as nucleic acids that compose

DNA and RNA molecules, and is fundamental to transport and translation of genetic information

in the plant (Schönknecht, 2009). Furthermore, phosphate esters and energy-rich phosphates are

responsible for the formation of adenosine triphosphate and adenosine diphosphate (ATP and

ADP respectively), which are essential for starch synthesis. In addition, inorganic phosphates

have a regulatory role controlling some enzymatic reactions (Marschner, 1995). Bouma (1969)

found that glycerol did not improve the effectiveness of foliar-applied phosphoric acid (KH2PO4)

uptake. The present study showed that the different sources of P performed better under normal

and drought stress conditions. The maximum growth was observed in a treatment where KH2PO4

was foliarly applied as a source of P as compared to other sources. Under moderate stress

condition the P performed better for the production of dry matter. Prabhakar and Saraf (1991)

reported that the different source of P enhanced the growth and dry matter production under

moderate stress condition. Rahman and Hassaneinn (1988) reported that fresh and dry weight

decreased in maize with decreasing soil moisture. Alam (1985) pointed out that shoot elongation

was reduced by water stress during vegetative period in maize. Studies on seed germination and

139

seedling vigor for measuring drought tolerance of some maize genotypes indicated that

germination stress, germination rate stress and seedling dry matter stress indices were influenced

by both genotype and moisture stress level (Lemcoff et al., 1998).

5.3. Optimization of different rates of P in maize under drought stress

Phosphorus (P) is one of the most important elements for plant growth and metabolism. It

plays key role in many plant processes such as energy metabolism, the synthesis of nucleic acids

and membranes, photosynthesis, respiration, N fixation and enzyme regulation (Raghothama,

1999). Adequate P nutrition enhances many aspects of plant development including flowering,

fruiting and root growth. The mechanism of uptake and transport of foliar-applied nutrients

involves a complex plant tissue system including dermal, vascular, and ground systems

(Römheldl and El-Fouly, 1999 and Rathore, 2000). Previous research showed that a foliar-applied

nutrient passes through the cuticular wax, the cuticle, the cell wall, and the membrane in that

order (Middleton and Sanderson, 1965; Franke, 1967). Foliar fertilization with NPK can be

supplemented with soil applied fertilizers but cannot replace soil fertilization in the case of maize

(Ling and Silberush, 2002), because demand for P is 1/10 that of N, hence, a foliar application

might be beneficial. Therefore, correcting the plant’s deficiency by foliar application seems

plausible. Very little research has been conducted on the use of P as foliar spray at early stages of

wheat and corn.

In this study the growth parameters like shoot length, root length and fresh and dry

weights of shoot and root were favourably affected by the foliar application of P. The maximum

value of these growth parameters were observed in a treatment where KH2PO4 @ 8 kg ha-1 was

applied. The positive effects of P may be explained on the basis of the fact that P is a constituent

of cell membranes, a number of proteins, all nucleic acids and nucleotides (Waraich et al., 2011).

Phosphorus is a constituent of molecular structures such as nucleic acids that compose DNA and

RNA molecules, and is fundamental to transport and translation of genetic information in the

plant (Schönknecht, 2009). The foliar P @ 8 kg ha-1 increase the maize forage as well as yield in

some extent (Grima et al., 2007). Besides, P is involved in controlling key enzyme reactions and

in the regulation of metabolic pathways (Theodorou and Plaxton, 1993; Schachtman et al., 1998).

The observed advantageous effects of P application on the growth parameters are in conformity

with earlier workers (Shetty et al., 1990; Maitra et al., 1998; Kaushal et al., 2002; Aishwath,

140

2004; Pandey et al., 2006; Waraich et al, 2011). According to Grant et al. (2001), it is vital to

supply P early in the season of crops; moderate amount of P applied at sowing can help sustain

early plant growth vigor. The foliar P could be used as an efficient P management tool in corn

when applied at the appropriate rate (Grima et al., 2007). In a study by Barel and Black (1979) P

added in polyphosphate and orthophosphate form in maize. The maximum concentration of P

tolerated in solutions of tri- and polyphosphates as foliar sprays was 1.3% as compared to 0.5%

orthophosphate on maize leaves. Orthophosphate produced the lowest yield (even lower than the

control) which may have been due to leaf damage after the first application.

5.4. Optimization of stage of maize for foliar P application under drought

stress

Nutrients are the most important factors which are essential for the growth of plant and

ultimately enhanced the yield of crops. Water is basic requirement for all the growth stages of

plant and it also limited in some area such as arid and semi-arid region of the world. Water is

required for plant for its growth and development. Without water the plants goes under drought

condition and severely affect its growth stages and ultimately yield of crops is reduced. The most

important stage of crop is seed germination in the presence of water (Ashraf and Mehmood, 1990)

and the seeds exposed to unfavourable environmental conditions like water stress may have to

compromise the seedlings establishment (Albuquerque and Carvalho, 2003). Phosphorus

deficiency in many of the soils is largely due to low occurrence of P-containing minerals

(Nyandat, 1981; Bunemann, 2003) and P-fixation (Van der Eijk, 1997). Continuous cropping

without commensurate nutrient replenishment is reported to contribute to low P content of many

soils (Smaling et al., 1997; Sanchez, 2002; Bunemann, 2003; FAO, 2004). Maize varieties are

known to vary in P uptake and utilisation efficiencies, as well as in adaptability to different soil

types (Nielsen and Barber, 1978; Walker and Raines, 1988; Duncan and Baligar, 1990; Horst et

al., 1993; Machado et al., 1999).

In this study the growth parameters like shoot length, root length and fresh and dry

weights of shoot and root were enhanced by the foliar application of P @ 8 kg ha-1 at different

growth stages of maize such as 4th leaf stage, 8th leaf stage and tasseling stage under drought

stress. All the growth parameters performed better when KH2PO4 foliarly was applied @ 8 kg ha-1

at 8th leaf stage under normal and stress conditions. The P enhanced the growth of maize plant

positively due to the fact that P plays an important role in cell membranes, protein and all nucleic

141

acids (Waraich et al., 2011). The P also has positive role in balancing energy in plant under

drought conditions. Thavaprakaash et al. (2006) found that foliar applied P after 25 and 45 days

of planting improved growth parameters and resulted in significantly higher corn yields. Our

results are in line with the result of Grima et al. (2007). They reported that the forage growth and

grain concentration of maize increased with foliar applied KH2PO4 @ 8 kg ha-1 at 8th leaf stage. A

foliar P @ 8 kg ha-1 improved yield to some extent and increased forage and P concentrations

more than the lower rates. The foliar P could be used as an efficient P management tool in corn

when applied at the appropriate growth stage and rate (Grima et al., 2007).

5.5. Physiological parameters

5.5.1. Gas exchange parameters

Drought stress limits photosynthesis which determines the crop production (Tognetti et

al., 2005; Bacelar et al., 2006; Ben Ahmed et al., 2009), through metabolic impairment and

stomatal closure (Tezara et al., 1999; Lawson et al., 2003; Pieters and El Souki, 2005). Decrease

in CO2 assimilation rate (Pn), stomatal conductance (gs) and transpiration rate (E) under drought

stress has been reported in many crops such as wheat (Moud and Yamagishi, 2007), maize

(Ashraf et al., 2007), Brassica napus (Kauser et al., 2006) and mungbean genotypes (Ahmed et

al., 2002). In present study all the gas exchange parameters such as Pn, E, gs and substomata CO2

rate (Ci) improved with supplemental foliar application of P @ 8 kg ha-1 at 8th leaf stage of maize,

in both hybrids 6525 and 32B33 under normal and stress conditions. The increase in Pn, E, gs and

Ci rate was more pronounced with foliar P spray at 8th leaf stage under both well watered and

stress conditions. This also means that supplemental foliar fertilisation was also effective for

improving the plant growth of drought-sensitive genotype. The decline in Pn under water stress

may be associated with lower mesophyll capacity for net assimilation rate at the cellular level due

to lower P accessibility for investment into photosynthetic apparatus. The closure of stomata

decreases internal CO2 concentration (Ci) and inhibits ATP synthesis and activity of ribulose-1, 5-

bisphosphate carboxylase/oxygenase that may lead to a reduction in photosynthesis under drought

stress (Dulai et al., 2006). Literature indicated that a positive relation exists between RWC and

photosynthesis (Siddique et al., 2000; Moaveni, 2011).

Phosphorous under mild water deficit improved water use efficiency in P-treated wheat

plants (dos-Santos et al., 2004).The encouraging effects of P on plant growth under water stress

142

have been ascribed as to enhancing the efficiency of Pn, gs, and water use (Ackerson, 1985), to

effects on water relations, and to higher cell membrane stability (Sawwan et al., 2000). Several

studies confirmed decrease in E under limited water conditions (Egert and Tevini, 2002; Moud

and Yamagishi, 2005; Rahbarian et al., 2011; Bogale, 2011) that may be attributed to decrease in

Pn and gs in water deficit plants, as observed in present study. The major factor for enhanced

productivity is the net CO2 assimilation rate. The CO2 assimilation rate in plants is controlled by

stomatal conductance, specific metabolic processes in carbon uptake, photochemical capacity or a

combination of all these factors (Taiz and Zeiger, 2006; Waraich et al, 2011). In this study, the

net CO2 assimilation rate was more with foliar applied P than with-out application of P. The

possible reason of this variation in photosynthetic rate may be that P application increased the leaf

growth which in turn increases the photosynthetic rate. The increase in net CO2 assimilation rate

may be ascribed to increase in stomatal conductance and ribulose 1,5 bisphosphate (RuBP)

carboxylase regeneration capacity (Brooks, 1986). The reported accumulation of starch indicates

that photosynthates cannot be used for plant growth under P limited condition (Fredeen et al.,

1990).

5.5.2. Water relation parameters

Foliar application of P negatively affected the water relation parameters under water-

limited conditions as compared to normal water supply. The values of water potential, osmotic

potential, turgor potential and relative water contents were decreased where foliar P spray under

water-limited and normal conditions. The foliar application of P maintained the water potential

and osmotic potential and relative water contents at 8th leaf stages under water-limited conditions

in all maize hybrids, 6525, 32B33, Hycorn and 31P41. The turgor potential was significantly

improved by foliar P @ 8 kg ha-1 at 8th leaf stage. This shows that foliar spray of P helped in

maintaining the water status of plants possibly through osmotic adjustment, the accumulation of

organic and inorganic ions such as free amino acids and proline (Shabala and Lana, 2011).

Osmotic adjustment helps the plants in the maintenance of water uptake under drought

stress (Chen and Jiang, 2010; Abdelmalek and Khaled, 2011). It is reported that decrease in leaf

water potential and increase in bulk modulus elasticity (i.e a ratio of normal stress to a change in

volume) together with decrease in osmotic potential maintain the plant turgor potential (Saito and

Terashima, 2004). The maintenance of turgor by active lowering of osmotic potential (Ψs) is

143

generally considered as an adaptation of plants under water limited environment (Ludlow and

Muchow, 1990). The plants exposed to drought stress had more negative leaf water potential

(Ψw) than the normal plants. The plants tend to maintain favourable water relations that help to

develop resistance against drought stress (Passioura and Fry, 1992; Kaldenhoff et al., 2008).

Available literature indicated variation between drought tolerant and susceptible genotypes which

may be due to the maintenance of tissue turgor, physiological activities, water uptake from soil

and reduction in water loss through stomata (Song et al., 1995; Siddique et al., 2000; Terzi and

Kadioglu, 2006).

5.6. Biochemical parameters

5.6.1. Pigments

Pigments especially chlorophyll contents are necessary to maintain optimum

photosynthetic capacity in plant (Wright et al., 1994; Nageswara et al., 2001). Drought stress

inhibits photosynthesis by reducing chlorophyll contents (Chl) and affecting its synthesis, and

damaging the photosynthetic machinery of plants (Iturbe et al., 1998). The severity and duration

of drought stress determines the extent of damage to photosynthetic apparatus and Chl of plants

(Rensburg and Kruger, 1994; Kyparissis et al., 1995; Jagtap et al., 1998). The results of present

study showed that all the pigments showed maximum values with the supplemental foliar applied

P @ 8 kg ha-1 at 8th leaf stage in both hybrids 6525 and 32B33 under well watered and stress

conditions. Form the pervious study it is noted that the drought stress condition decreased the

pigments (Chl) in sunflower (Manivannan et al., 2007), wheat (Fotovat et al., 2007), chickpea

(Mafakheri et al., 2010) and corn (Khayatnezhad et al., 2011). Drought stress reduces leaf Chl

(Ommen et al., 1999) mainly due to damage to chloroplasts caused by reactive oxygen species

(ROS) (Smirnoff, 1995). The Chl contents were enhanced by the application of foliar applied P in

maize under normal and stress conditions. Contrasting results were reported by Schelmmer et al.

(2005) who reported that drought stress had no significant effect on Chl in maize. Recent

developments have shown that decrease in chlorophyll pigments increases the reflectance of the

incident radiation (Schelmmer et al., 2005) that can protect photosynthetic system against stress

(Arjenaki et al., 2012).

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5.6.2. Total soluble sugars (TSS)

Sugars are also important compatible solutes that play a significant role in osmoregulation

under drought stress (Fallon and Phillips, 1989). The accumulation of total sugars and other

compatible solutes such as polyols is characteristic feature of most plants under stress (Delauney

and Verma, 1993 and Mohammadkhani and Heidari, 2008). They are involved in the activation

and regulation of nitrate reductase (NR) activity (Kaiser and Huber, 2001; Iglesias-Bartolome et

al., 2004; Lillo et al., 2004), enhance the transcription of NR genes (Sivasanker et al., 1997; Klein

et al., 2000 and Larios et al., 2001) and regulate the activity of enzymes at post translational level

(Carpenter and Gowe, 1988 and Wolkers et al., 1998). Clifford et al. (1998) observed significant

role of sugars in osmotic adjustment under limited water conditions while Patakas et al. (2002)

reported contradictory results in grapevine plants and stated non-significant differences in sugar

contents of water stressed and non-stressed plants. The present study showed that under stress

condition, more total soluble sugar is produced as compared to the normal condition. The

supplemental foliar applied P @ 8 kg ha-1 significantly enhanced the total soluble sugar under

normal and stress conditions. Both maize hybrids 6525 and 32B33 performed better than Hycorn

and 31P41 hybrids under normal and stress condition. Qayyum et al. (2011) stated that increase in

osmotic stress progressively increases the production of total soluble sugars in leaves.

5.6.3. Total soluble proteins (TSP)

The decline in soluble proteins in the plants grown under drought stress condition was

observed in the present study which is associated with the decreased rate of protein biosynthesis

and proliferation in breakdown of proteins under water stress conditions (Rodriguez et al., 2002).

Total soluble proteins are essentially required in large amounts for osmotic adjustment or

osmoregulation (Nayyar and Walia, 2003). The present study revealed that the TSP increased by

the application of P @ 8 kg ha-1 at 8th leaf stage under normal condition as compared to the stress

conditions. Both hybrids 6525 and 32B33 produced more TSP as compared to the Hycorn and

31P41 maize hybrids. However, a decrease in TSP concentration of water stressed seedlings

complies with the findings of Kochaki (1997) and Sujin and Wu (2004) who reported that high

molecular weight soluble proteins concentration decreases while low molecular weight increases

in plants under moisture deficit conditions and concluded that biochemical attributes like TSS,

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TSP and TFA can be used as selection tool for screening of drought tolerant cultivars (Ashraf,

1998).

5.6.4. Total free amino acids (TFA)

In the present study TFA increased under stress condition. Amino acids contribute

significantly in plant metabolism as early products of photosynthesis and N assimilation.

Biosynthesis and accumulation of amino acids take place in response to environmental stresses

(Hsu and Kao, 2003) such as drought that results in breakdown of structural proteins into

component amino acids, which actively take part in osmotic adjustment under water deficit

environment (Good and Zaplachinski, 1994). An increase in total free amino acids under stressed

environment was also reported by Ashraf and Iram (2005). In the present study significant effect

of supplemental foliar applied P in combination on total free amino acids was observed under

both water-limited and normal water supply. But the accumulation of TFA helps the plants in

osmotic adjustment to stressed environment (Hsu and Kao, 2003) which is very true for present

study.

5.6.5. Proline accumulation

Proline is a low molecular mass compound that accumulates in cytosol of plant cells

(Voetberg and Sharp, 1991) to improve their tolerance against drastic effects of drought (Gzik,

1996; Bajji et al., 2001). The enhancement in leaf proline content was observed under water

stress conditions in the current study. Enhancement in leaf proline content in response to abiotic

stresses has been well documented (Ozturk and Demir, 2002; Hsu et al., 2003). In plants, a high

concentration of proline helps to endure and adapt the sub-optimal environment (Nayyar and

Walia, 2003). The proline content is commonly used as an indicator for the environmental stress

in plants (Claussen, 2005; Gunes et al., 2008). Proline accumulation under stress conditions is

well reported for different crops (Almansouri et al., 1999). Drought stress significantly increased

proline contents in both young and old leaves of sunflower (Cechin et al., 2008). Similar results

were reported by Mostajeran and Rahimi-Eichi (2009) in rice. The process of proline

accumulation in water stressed plants is possibly a result of decreased protein biosynthesis

(Cechin et al., 2006). The total soluble protein contents of plant grown in water-limited were

significantly lower than those of normal water supply. Therefore, high accumulations of proline

in the leaves of stressed plants were correlated with relatively low concentration of soluble

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proteins. The leaf proline contents improved with the foliar spray of P @ 8 kg ha-1 at 8th leaf

stage. Related results were also documented in other studies where application of P (Olgun et al.,

2006) enhanced the proline content in plant. Genetic make-up is accountable for differences in

the accumulation of proline in wheat plants in the present study, also reported previously by

Rhodes and Samaras, (1994). Therefore, production of proline in plants can be used as a criterion

for drought stress resistance assessment for varieties.

5.6.7. Antioxidant enzymes

In general, a well-known adaptive mechanism in plants in response to drought conditions

is the production of antioxidant enzymes such as peroxidase (POD), catalase (CAT), Ascorbate

peroxidase (APX), superoxide dismutase (SOD) and glutathione peroxidase (GPx) (Li et al.,

2009). Both enzymatic and non-enzymatic antioxidants such as glutathione (GSH), flavonoids,

ascorbic acid (AsA), α-tocopherol, β-carotene and hydroquinones collaboratively serve as

scavengers of ROS under stress (Chaves and Oliveria, 2004). Ascorbate peroxidase (APX) is one

of the most important antioxidant enzyme involved in the scavenging of ROS to protect cells of

higher plants (Gill and Tuteja, 2010). In present study, foliar application of P increased the

activity of antioxidants including catalase, peroxidase, and ascorbate peroxidase under stress

conditions. Increase in antioxidant enzymes is the most efficient mechanism against oxidative

stress (Farooq et al., 2008). Drought tolerant maize genotypes produced high amount of ascorbate

peroxidase and catalase under water stress condition as compared to drought sensitive ones

(Sairam et al., 1998). Nikolaeva et al., (2010) stated that APX activity varies in different wheat

cultivars and depends on duration of drought and stage of leaf development. They observed that

mild drought stress enhanced APX activity in leaves but prolonged drought reduced its activity

due to increase in malonic dialdehyde (MDA) content. The present study showed that the

supplemental foliar applied P @ 8 kg ha -1 at 8th leaf stage enhanced the activity of different

antioxidents under stress condition as compared to well watered condition in maize. Both maize

hybrids 6525 and 32B33 performed better than Hycorn and 31P41. CAT activity is enhanced in

high light conditions under drought stress (Yang et al., 2008). Pan et al. (2006) evaluated the

combined effect of drought and salt stress and reported a decrease in CAT activity in Glycyrrhiza

uralensis seedlings.

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5.7. Phosphorus concentration and PUE

Excessive P fertilization is often associated with the concept of sustaining a particular

sufficiency level of nutrients in soil. The sufficiency concept is viable in some instances for soil -

immobile mineral nutrients such as P. The present study showed that the P concentration in leaf,

root, stem, grain and PUE enhanced by the foliar application of P @ 8 kg ha -1 at 8th leaf stage.

According to Bray’s mobility concept (Bray, 1954) the plant response to immobile mineral

nutrients, such as P, depends on the concentration of the nutrient within the root surface sorption

zone, not on the total amount of nutrient in soil. The amount of P taken up by the plant is directly

dependent on the root surface and the concentration of plant available P within the roots

accessibility. Mosali et al. (2006) reported the highest PUE of approximately 16% in wheat which

achieved when fertilizer was banded with the seed to the soil. As corn plants develop, available P

supplied by the inorganic fertilizer is being depleted, and plants begin to utilize the slowly

available organic forms of P present in soil.

The results of present experiment revealed that foliar-applied P resulted in significantly

advantageous response for concentration of P in root, shoot, straw, grain and PUE in maize. PUE

measured by P physiological efficiency index (i.e. kg of grain or straw of P absorbed in the above

growth plant parts) identified in soil experiment was found associated with shoot and root growth

and shoot P uptake in the solution culture experiment. Increase in shoot and root growth was

associated with increased P uptake. The current study supported by Harder et al. (1982) who

reported that the foliar fertilization of P enhanced the P concentration in maize.

The PUE was generally higher for foliar applied with pre-plant than without applied P.

The results obtained here also revealed that foliar P rate applied at the 8th leaf resulted in higher

PUE than the earlier or later applied foliar P at the rate of 8 kg P ha-1. The lowest foliar P rate was

found more efficient than the higher rates. The decrease in efficiency with higher rates of foliar P

could be due to several reasons that influence the actual amount of applied P that comes in contact

with plant. Barel and Black (1979) found that ammonium salts of orthophosphate dried rapidly

and leave dry crystals on the surface of the leaf, which depending on moisture availability and

conditions such as temperature, humidity and moisture availability might be taken up later or

washed away. In moist conditions, KH2PO4 is rapidly absorbed by leaf. Since most of the foliar

ionic nutrients are absorbed through stomata, their opening and closure greatly affect the uptake

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of foliar P. Although, according to Linskens et al. (1965) leaf hairs have thinner cell walls near

their base which enhances entrance of ionic foliar nutrients at any time.

5.8. Yield and yield components

Water stress causes growth retardation (Baser et. al., 2004), consequently affecting the

yield and yield components. An obvious reduction in cob weight, number of grains per cob, 1000-

grain weight, biological yield and grain yield under water stress conditions for all maize hybrids

was observed. Foliar application of P at 8th leaf stage positively affected the yield parameters.

Foliar application of P (dos Santos et al., 2004) increased the grain yield of wheat. Under water

stress conditions, nutrient uptake by roots is limited and plant leaves were symptomatic of

nutrient deficiency, which in turn affects protein synthesis, cell structures, enzymatic activity, and

metabolism. At organ level, in such conditions, plants may have less and smaller leaves (Fricke

et al., 1997), which is the main site of photosynthesis. All these damaging effects of reduced

nutrient supply besides other foremost effects of drought stress on plant growth and development

could be responsible for decline in yield and yield components.

Application of foliar P to water stressed maize plants helped to mitigate the negative

effects of water limitations by improving several plants physiological and metabolic processes,

therefore ultimately improving the yield and yield components. Many researchers also describe

the mitigating water stress effects of nutrients in many crops. Application of combined P

fertilizers resulted in the greatest grain yield, largest grain number, and grain weight (Lu, 2011).

Benbella and Paulsen, (1998) showed that foliar applications P after anthesis @ 5 to 10 kg ha−1

(1.1 to 2.2 kg P ha-1) increased wheat grain yields by up to 1 Mg ha-1. Applying P as foliar spray,

in early growth stages, can increase the number of fertile tillers (Elliot et al., 1997; Grant et al.,

2001). Mosali et al. (2006) identified Zadoks 32 (i.e. when second node is detectable during stem

elongation) as the optimum time for foliar P spray as it increased both P-uptake and grain yield.

Applications of P to wheat after anthesis increased grain yield (Benbella and Paulsen, 1998).

Grain yields of corn positively responded to P at 2 kg ha-1 applied as foliar spray from eighth leaf

through to tasseling growth stages (Girma et al., 2007). Mixed nutrient solutions have been

widely tested (Garcia and Hanway, 1976; Alston, 1979; Ahmed et al., 2006; Arif et al., 2006,).

Arif et al. (2006) investigated the effect of applying numerous foliar applications of mixed

nutrient solutions (N, P and K) to wheat at tillering (Zadoks stage 26; main shoot and six tillers)

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and at booting (Zadoks stage 47; flag sheath opening) with one, two or three applications of the

nutrient mix. Gooding and Davies (1992) reported that foliar applications at or 2 weeks following

anthesis can be of greater benefit compared to soil applied fertilization. Multiple beneficial effects

of foliar P fertilizer application in maize (Leach and Hameleers, 2001; Pongsakul and Ratanarat,

1999 and Thavaprakaash et al., 2006), wheat (Haloi, 1980; Sherchand and Paulsen, 1985 and

Batten et al., 1986) and barley (Qaseem et al., 1978) have been documented. Leach and

Hameleers (2001), observed a significant increase in both cob index and starch content when P

was applied at four-leaf growth stage. Sherchand and Paulsen (1985) and Batten et al. (1986)

reported that foliar application of KH2PO4 resulted in higher grain yield in winter wheat coupled

with the delay in leaf senescence in hot and dry growing conditions. The supplemental foliar

applied P enhanced the number of grains per cob. They argued that less number of cobs plant-1 in

the control plots resulted in less number of grains plant-1 that finally resulted in minimum grain

yield. The results are in accordance with those of Sharma and Sharma (1991) who reported that P

fertilizer applications significantly affected the grains per cob. The results were similar with that

of Leon (1999) and Maqsood et al. (2001) who reported that number of grains cob-1 were

influenced significantly with NP application. Arain et al. (1989) reported that number of grains

per cob of maize increased with increase in P application. Fareed (1996) and Hussain et al. (2006)

observed an increase in 1000-grain weight with increase in NP application. Phosphorus being

responsible for good root growth directly affected the thousand grain weight because P at the rate

of 0 kg ha-1 (control plots) resulted in the least thousand grain weight (Hussain et al. 2006). Arain

et al. (1989) reported that number of cobs per plant of maize increased with increase in P

application. The soil application of P increased the grain yield in maize (Arain et al., 1989). The

supplemental foliar applied increased the grain yield of maize under drought condition. The

increase in grain yield due to P application was also reported by Khan et al. (1999), Maqsood et

al. (2001) and Sharma and Sharma (1996). Arain et al. (1989) reported that grain yield of maize

increased with increase in P application.

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5.9. Conclusion

Against the background of decreasing water availability and the need for hybrids, able to

withstand water-limited environments, we set up screening experiments testing eight modern

maize hybrids. Additionally, we investigated the possible role of supplemental foliar fertilisation

in ameliorating the water stress effects in early plant growth stages. Based on the germination

parameters (in experiment 1) and stress indices (in experiment 2), we screened out four hybrids:

6525 and 32B33 as drought tolerant and Hycorn and 31P41 as drought sensitive. Next, we

optimised the source, dose of P and stage of maize for P, both under well-watered and water-

stress conditions. After optimization the combined effect of source, dose of P and stage were

tested under wire house and field condition. We found that foliar spray P @ 8 kg ha-1 treatment at

8th leaf stage was the most effective in terms of improving plant growth, not only under well-

watered conditions but also under water-stress conditions though to a lesser extent. This means

that supplemental foliar fertilisation with foliar sprays can help correcting nutrient deficiencies in

water-limited environments where nutrient uptake is generally limited.

151

The present study comprised of different experiments conducted i) to screen out the maize

hybrids for drought tolerance under lab and wire house conditions ii) to find out optimum dose,

source and stage of phosphorus application for improving drought tolerance in maize iii) to assess

the effect of exogenously applied phosphorus on growth, yield, physiological and biochemical

traits of hybrid maize grown under water stress condition.

Water-limited conditions at early growth stages affect germination and seedling

development, often leading to poor stand establishment. In Experiment-1 germination and

seedling growth of eight maize hybrids viz. 7386 (Pak Hybrid), 6525 (Monsanto), Hycorn (H-

plus), 9696 (Swan Seed), 32B33 (Pioneer), 3672 (Swan Seed), MMRI hybrid and 31P41

(Pioneer) in response to PEG induced water stress conditions (PEG-6000) and by withholding

water supply were investigated. Two laboratory experiments were conducted to observe

germination parameters and to calculate stress indices as screening criteria for drought tolerance.

In first laboratory experiment, eight hybrids were tested against PEG induced water stress of -0.2,

-0.4, -0.6 and -0.8 MPa to screen out drought tolerant and drought sensitive genotypes. In tested

genotypes, germination parameters viz. germination percentage, promptness index, and

germination stress tolerance index declined in response to the increasing PEG-induced stress

levels. In second experiment, the same eight maize hybrids were grown in two sets of plastic pots;

one set was watered at 100% field capacity whereas water was withheld in the other set of plastic

pots at 8 to 25 days after sowing. Water stress conditions imposed by withholding irrigation at

seedling stage reduced plant height stress tolerance index and dry matter stress tolerance index

but increased and root length stress tolerance index. Based on results of germination parameters

and stress indices, 6525 and 32B33 were the most droughts tolerant and 31P41 and Hycorn was

the most droughts sensitive among tested eight hybrids.

In 3rd experiment, a pot experiment was conducted in wire house/ rain out shelter for

optimization of appropriate source helpful in improving drought tolerance in maize hybrids

subjected to water stress at seedling stage. Two drought tolerant, (6525 & 32B33) and two

drought sensitive, (Hycorn & 31P41) hybrids as screened out in experiment # 1& 2 were used in

this study, grown under normal (100% FC) and water stress (60% FC) conditions. All the sources

of phosphorus significantly improved the shoot and root length, shoot and root fresh weight, shoot

152

and root dry weight and root-shoot ratio as compared to control. Both drought tolerant (6525 and

32B33) and drought sensitive hybrids (Hycorn and 31P41) performed better where KH2PO4 was

applied followed by SSP. The phosphorus source of KH2PO4 was the most effective in improving

plant growth under both well-watered and water-stress conditions.

In 4th experiment, a pot experiment was conducted in wire house/ rain out shelter for

optimization of different rates of phosphorus helpful in improving drought tolerance in maize

hybrids subjected to water stress at seedling stage. Two drought tolerant, (6525 & 32B33) and

two drought sensitive, (Hycorn & 31P41) hybrids as screened out in experiment # 1& 2 were used

in this study, grown under normal (100% FC) and water stress (60% FC) conditions. All the four

hybrids perform better under the different rate of supplemental foliar applied phosphorus

(KH2PO4) as compared to control. Foliar applied phosphorus (KH2PO4) @ 8 kg ha-1 significantly

improved the shoot and root length, shoot and root fresh weight, shoot and root dry weight and

root-shoot ratio as compared to control.

In 5th experiment, a pot experiment was conducted in wire house/ rain out shelter for

optimization of different timing for foliar applied phosphorus helpful in improving drought

tolerance in maize plants under water stress condition. The best source and best rates of foliar

applied phosphorus (KH2PO4 @ 8 kg ha-1) selected from experiment # 3 & 4 were used in this

experiment. Two drought tolerant (6525 and 32B33) and two drought sensitive hybrids (Hycorn

and 31P41) selected from laboratory experiments were grown under normal (100% field capacity)

and stress conditions. Stress was applied by withholding irrigation for one week at 4 th leaf stage,

8th leaf stage and tasseling stage. Foliar applied phosphorus (KH2PO4) @ 8 kg/ha significantly

improved the shoot and root length, shoot and root fresh weight, shoot and root dry weight as

compared to control. Foliar application of phosphorus at 8th leaf stage of maize was significantly

better than all other stages.

In 6th experiment, a pot experiment was conducted in rain-out shelter to check the best

combination of four maize hybrids, one source (KH2PO4), one dose (KH2PO4 @ 8 kg ha-1) and

one timing (Stress applied at 8th leaf stage) that is helpful in improving drought tolerance in water

stressed maize plants. In this experiment, four maize hybrids i.e. two drought tolerant (6525 &

32B33) and two drought sensitive (Hycorn & 31P41) were grown under normal (100% field

capacity) and water stress (stress applied at 8th leaf stage) levels. In this experiment all the

physiological and biochemical attributes of maize were recorded. Drought stress significantly

153

reduced physiological and biochemical attributes of maize. The phosphorus application played a

significant role in improving drought tolerance in maize hybrids. The phosphorus application

through foliar at 8th leaf stage of maize enhanced drought tolerance potential of maize hybrids.

Maize hybrids 6525 and 32B33 responded to phosphorus application more positively than Hycorn

and 31P41 under non-stress and stress conditions.

In 7th experiment, field study was conducted over one year at research area of Department

of Agronomy, University of Agriculture, Faisalabad, Pakistan. The best combination of P source,

dose and stage for supplemental foliar P application using two droughts tolerant (6525 & 32B33)

and two drought sensitive (Hycorn & 31P41) maize genotype. Best combination of one source

(KH2PO4), one dose (KH2PO4 @ 8 kg ha-1) and one timing (Stress applied at 8th leaf stage) that is

helpful in improving drought tolerance in water stressed maize. In this experiment yield and yield

components of maize were recorded. Drought stress significantly decreased yield and yield

components of all maize hybrids. Foliar applied P at 8th leaf stage significantly increased grain

yield in maize plants exposed to limited water conditions. Maize hybrids 6525 and 32B33

responded to phosphorus application more positively than Hycorn and 31P41 under non-stress

and stress conditions.

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Appendix 4.1: Effect of supplemental foliar P application on photosynthetic rate of four maize hybrids under normal and

water stress conditions

Treatments Non stress Stress

Treatment

means


No spray

(Control) 3.74 g 3.77 f 3.83 e 3.79 f 3.26 n 3.31 m 3.43 k 3.37 l 3.55 B

P spray 3.88 b 3.98 c 4.19 a 4.05 b 3.49 j 3.54 i 3.71 gh 3.69 h 3.83 A

Level means 3.89 A 3.48 B

Appendix 4.2: Effect of supplemental foliar P application on photosynthetic rate of four maize hybrids

Treatments Hycorn 31P41 6525 32B33 Treatment

means

No spray

(Control)

3.47 h 3.52 g 3.63 e 3.58 f 3.55 B

Foliar P spray 3.69 d 3.76 c 3.98 a 3.88 b 3.83 A

Hybrid means 3.58 D 3.64 C 3.81 A 3.73 B

178

Appendix 4.3: Effect of supplemental foliar P application on transpiration rate of four maize hybrids under normal and



Treatment

means


No spray

(Control) 2.38 fg 2.41 f 2.53 d 2.47 e 2.01 n 2.07 m 2.17 kl 2.13 l 2.27 B

P spray 2.60 c 2.65 b 2.76 a 2.70 b 2.22 jk 2.27 ij 2.36 gh 2.31 hi 2.48 A


Appendix 4.4: Effect of supplemental foliar P application on transpiration rate of four maize hybrids


means

No spray

(Control)

2.19 h 2.24 g 2.35 e 2.30 f 2.27 B



179

Appendix 4.5: Effect of supplemental foliar P application on stomatal conductance of four maize hybrids under normal

and water stress conditions


Treatment

means


No spray

(Control) 3.82 g 3.86 f 3.94 e 3.92 e 3.46 n 3.52 m 3.59 l 3.54 m 3.71 B

P spray 3.99 d 4.21 c 4.50 a 4.30 b 3.62 k 3.66 j 3.78 h 3.74 i 3.97 A


Appendix 4.6: Effect of supplemental foliar P application on stomatal conductance of four maize hybrids


means

No spray

(Control)

3.64 h 3.69 g 3.77 e 3.73 f 3.71 B



180

Appendix 4.7: Effect of supplemental foliar P application on sub-stomatal CO2 rate of four maize hybrids under normal



Treatment

means


No spray

(Control) 219.67 gh 226.67 fg 242.00 e 235.33 ef 180.67 m 184.33 lm 193.33 jk 189.00 lm 208.88 B

P spray 262.33 c 252.00 d 292.00 a 274.00 b 199.33 jk 203.33 j 214.67 hi 209.00 ij 238.29 A


Appendix 4.8: Effect of supplemental foliar P application on sub-stomatal CO2 rate of four maize hybrids


means

No spray

(Control)

200.17 g 205.50 fg 217.67 e 212.17 ef 208.88 B

Foliar P spray 225.67 d 23283 c 253.17 a 241.50 b 238.29 A


181

Appendix 4.9: Effect of supplemental foliar P application on water potential (-MPa) of four maize hybrids under normal and



Treatment

means


No spray

(Control) 1.10 efg 1.08 def 1.01 cd 1.05 cde 1.27 l 1.25 l 1.20 j 1.23 k 1.15 B

P spray 0.94 bc 0.87 ab 0.79 a 0.83ab 1.18 j 1.16 hi 1.12 fg 1.14 gh 1.00 A


Appendix 4.10: Effect of supplemental foliar P application on water potential (-MPa) of four maize hybrids


means

No spray

(Control)

1.18 f 1.16 f 1.10 d 1.14 e 1.15 B

Foliar P spray 1.06 c 1.02 b 0.95 a 0.98 ab 1.00 A


182

Appendix 4.11: Effect of supplemental foliar P application on osmotic potential (-MPa) of four maize hybrids under normal



Treatment

means


No spray

(Control) 0.46 cdefg 0.42 cde 0.36 ab 0.37 bc 0.86 i 0.82 hi 0.60 fghi 0.64 hi 0.56 B

P spray 0.47 cdefgh 0.46 cdef 0.34 a 0.37 cd 0.69 hi 0.63 ghi 0.52 defghi 0.56 efghi 0.50 A


Appendix 4.12: Effect of supplemental foliar P application on osmotic potential (-MPa) of four maize hybrids


means

No spray

(Control)

0.66 d 0.62 cd 0.48 abc 0.50 bcd 0.56 B

Foliar P spray 0.58 cd 0.54 bcd 0.43 a 0.46 ab 0.50 A

Hybrid means 0.62 B 0.58 B 0.45 A 0.48 A

183

Appendix 4.13: Effect of supplemental foliar P application on turgor pressure (MPa) of four maize hybrids under normal



Treatment

means


No spray

(Control) 0.46 cde 0.41e 0.45 de 0.45 de 0.41 e 0.43 de 0.60 abcd 0.59 abcd 0.50 B

P spray 0.64 abc 0.66 ab 0.68 a 0.66 ab 0.49 bcde 0.54 abcde 0.59 abcd 0.58 abcde 0.58 A

Level means 0.56 A 0.53 A

Appendix 4.14: Effect of supplemental foliar P application on turgor pressure (MPa) of four maize hybrids


means

No spray

(Control)

0.47 b 0.48 b 0.52 ab 0.51 ab 0.50 B

Foliar P spray 0.52 ab 0.54 ab 0.64 a 0.63 a 0.58 A

Hybrid means 0.50 A 0.52 A 0.58 A 0.57 A

184

Appendix 4.15: Effect of supplemental foliar P application on relative water contents (%) of four maize hybrids under normal



Treatment

means


No spray

(Control) 188.69 cdef 215.20 bcdef 285.37 ab 229.92 bcde 139.65 f 155.63 ef 198.42 cdef 180.25 def 199.14 B

P spray 248.41 bcd 272.11 abc 355.40 a 335.47 a 202.05 bcdef 217.25 bcdef 243.24 bcd 235.40 bcde 263.67 A


Appendix 4.16: Effect of supplemental foliar P application on relative water contents (%) of four maize hybrids


means

No spray

(Control)

164.17 d 185.42 cd 241.90 abc 205.09 cd 199.14 B

Foliar P spray 225.23 bc 244.68 abc 299.32 a 285.44 ab 263.67 A

Hybrid means 194.70 C 215.05 BC 270.61 A 245.26 B

185

Appendix 4.17: Effect of supplemental foliar P application on chlorophyll a of four maize hybrids under normal and water

stress conditions


Treatment

means


No spray

(Control) 0.76 cdefg 0.85 bcde 0.90 abcd 0.89 abcd 0.50 h 0.53 gh 0.59 fgh 0.56 gh 0.70 B

P spray 0.87 bcde 0.99 abc 1.14 a 1.04 ab 0.65 defgh 0.63 efgh 0.84 dcdef 0.75 cdefgh 0.86 A


Appendix 4.18: Effect of supplemental foliar P application on chlorophyll a of four maize hybrids


means

No spray

(Control)

0.63 c 0.69 c 0.75 bc 0.73 bc 0.70 B


Hybrid means 0.70 B 0.75 AB 0.87 A 0.81 AB

186

Appendix 4.19: Effect of supplemental foliar P application on chlorophyll b of four maize hybrids under normal and water

stress conditions


Treatment

means


No spray

(Control) 0.50 abcde 0.52 abcde 0.59 abc 0.55 abcd 0.23 e 0.27 cde 0.32 cde 0.29 cde 0.41 B

P spray 0.63 ab 0.64 ab 0.71 a 0.65 ab 0.37 bcde 0.41 bcde 0.47 abcde 0.44 abcde 0.54 A


Appendix 4.20: Effect of supplemental foliar P application on chlorophyll b of four maize hybrids


means

No spray

(Control)

0.36 b 0.39 ab 0.46 ab 0.42 ab 0.41 B

Foliar P spray 0.50 ab 0.52 ab 0.59 a 0.55 ab 0.54 A

Hybrid means 0.43 A 0.46 A 0.52 A 0.48 A

187

Appendix 4.21: Effect of supplemental foliar P application on total chlorophyll contents of four maize hybrids under normal



Treatment

means


No spray

(Control) 3.16 cde 3.37 cd 3.79 bc 3.72 bc 1.99 g 2.09 g 2.39 fg 2.24 fg 2.85 B

P spray 3.89 bc 4.37 ab 4.80 a 4.43 ab 2.63 defg 2.63 efg 3.33 cde 2.87 def 3.62 A


Appendix 4.22: Effect of supplemental foliar P application on total chlorophyll contents of four maize hybrids


means

No spray

(Control)

2.78 d 2.73 d 3.09 cd 2.98 cd 2.85 B

Foliar P spray 3.26 bc 3.50 bc 4.06 a 3.65 ab 3.62 A

Hybrid means 2.92 C 3.11 BC 4.58 A 3.31 AB

188

Appendix 4.23: Effect of supplemental foliar P application on total carotenoids of four maize hybrids under normal and water

stress conditions


Treatment

means


No spray

(Control) 0.32 defgh 0.37 cdefgh 0.50 bcd 0.47 bcd 0.20 h 0.24 gh 0.29 efgh 0.27 fgh 0.34 B

P spray 0.46 bcde 0.52 abc 0.69 a 0.58 ab 0.35 cdefgh 0.38 cdefgh 0.44 bcdef 0.40 cdefg 0.47 A


Appendix 4.24: Effect of supplemental foliar P application on total carotenoids of four maize hybrids


means

No spray

(Control)

0.26 d 0.30 cd 0.39 bc 0.37 bcd 0.34 B

Foliar P spray 0.40 bc 0.45 ab 0.56 a 0.48 ab 0.47 A


189

Appendix 4.25: Effect of supplemental foliar P application on total soluble proteins of four maize hybrids under normal and



Treatment

means


No spray

(Control) 5.90 hi 6.11 gh 6.58 def 6.36 fg 6.41 j 5.63 ij 5.97 hi 5.80 hi 5.97 B

P spray 6.90 bcd 7.08 bc 7.68 a 7.27 b 6.14 fg 6.37 fg 6.82 cde 6.52 ef 6.85 A


Appendix 4.26: Effect of supplemental foliar P application on total soluble proteins of four maize hybrids


means

No spray

(Control)

5.65 f 5.87 ef 6.27 cd 6.08 de 5.97 B

Foliar P spray 6.52 bc 6.72 b 7.10 a 7.04 a 6.85 A

Hybrid means 6.09 C 6.30 B 6.69 A 6.56 A

190

Appendix 4.27: Effect of supplemental foliar P application on total free amino acids of four maize hybrids under normal and



Treatment

means


No spray

(Control) 15.93 j 16.25 j 20.24 hi 18.85 i 25.86 cde 26.73 bcd 27.94 abc 27.13 abcd 22.36 B

P spray 21.97 gh 22.96 fg 25.30 def 24.07 efg 28.24 abc 28.47 ab 29.27 a 28.92 ab 26.15 A

Level means 20.69 B 27.82 A

Appendix 4.28: Effect of supplemental foliar P application on total free amino acids of four maize hybrids


means

No spray

(Control)

20.89 f 21.49 ef 24.09 cd 22.99 de 22.36 B



191

Appendix 4.29: Effect of supplemental foliar P application on total soluble sugars of four maize hybrids under normal and



Treatment

means


No spray

(Control) 1.22 p 1.35 o 1.57 m 1.46 n 2.22 h 2.34 g 2.56 e 2.45 f 1.90 B

P spray 1.72 l 1.85 k 2.09 i 1.97 j 2.72 d 2.87 c 3.13 a 2.99 b 2.42 A


Appendix 4.30: Effect of supplemental foliar P application on total soluble sugars of four maize hybrids


means

No spray

(Control)

1.72 h 1.85 g 2.07 e 1.95 f 1.90 B



192

Appendix 4.31: Effect of supplemental foliar P application on proline contents of four maize hybrids under normal and water

stress conditions


Treatmen

t means


No spray

(Control) 186.25 k 219.16 jk 382.85 ij 242.90 jk 407.62 efg 424.88 def 473.52 bcd 443.12 cde 335.04 B

P spray 323.75 hi 346.17 ghi 382.56 efgh 364.40 fgh 484.58 abcd 503.16 abc 539.68 a 521.29 ab 433.20 A


Appendix 4.32: Effect of supplemental foliar P application on proline contents of four maize hybrids


means

No spray

(Control)

296.93 f 322.02 ef 378.19 cd 343.01 de 335.04 B

Foliar P spray 404.17 bc 424.67 ab 361.12 a 442.84 ab 433.20 A


193

Appendix 4.33: Effect of supplemental foliar P application on catalase activity of four maize hybrids under normal and water

stress conditions


Treatment

means


No spray

(Control) 41.57 f 45.97 ef 57.67 ef 47.33 ef 145.37 d 152.33 cd 178.80 ab 156.67 bcd 103.21 B

P spray 51.33 ef 53.83 ef 67.93 e 57.53 ef 162.67 bcd 168.00 abcd 192.13 a 172.63 abc 115.76 A


Appendix 4.34: Effect of supplemental foliar P application on catalase activity of four maize hybrids


means

No spray

(Control)

93.47 d 99.15 cd 118.23 ab 102.00 bcd 103.21 B

Foliar P spray 107.00 bcd 110.92 bcd 130.03 a 115.08 abc 115.76 A

Hybrid means 100.23 B 105.03 B 124.13 A 108.54 B

194

Appendix 4.35: Effect of supplemental foliar P application on peroxidase activity of four maize hybrids under normal and



Treatment

means


No spray

(Control) 28.86 f 31.04 f 37.71 ef 34.17 ef 157.44 d 162.38 cd 187.00 b 163.42 cd 100.25 B

P spray 37.16 ef 38.87 ef 47.53 e 41.05 ef 173.30 bc 183.80 b 224.78 a 213.91 a 120.05 A


Appendix 4.36: Effect of supplemental foliar P application on peroxidase activity of four maize hybrids


means

No spray

(Control)

93.15 d 96.71 cd 112.36 b 98.80 cd 100.25 B

Foliar P spray 105.23 bc 111.34 b 136.15 a 127.48 a 120.05 A

Hybrid means 99.19 C 104.02 C 124.25 A 113.14 B

195

Appendix 4.37: Effect of supplemental foliar P application on ascorbate peroxidase activity of four maize hybrids under

normal and water stress conditions


Treatment

means


No spray

(Control) 0.90 h 1.18 gh 1.38 gh 1.28 gh 2.91 def 2.95 def 3.79 bcd 3.44 cde 2.23 B

P spray 0.81 h 2.04 fg 2.86 def 2.71 ef 3.98 bc 4.23 abc 4.98 a 4.49 ab 3.26 A


Appendix 4.38: Effect of supplemental foliar P application on ascorbate peroxidase activity of four maize hybrids


means

No spray

(Control)

1.90 e 2.06 de 2.58 cd 2.36 de 2.23 B

Foliar P spray 2.39 de 3.13 bc 3.92 a 3.60 ab 3.26 A


196

Appendix 4.39: Effect of supplemental foliar P application on P concentration in leaf of four maize hybrids under normal



Treatment

means


No spray

(Control) 0.66 h 0.69 g 0.76 e 0.73 f 0.39 p 0.43 o 0.49 m 0.47 n 0.58 B

P spray 0.80 d 0.84 c 0.91 a 0.88 b 0.52 l 0.55 k 0.62 i 0.59 j 0.72 A


Appendix 4.40: Effect of supplemental foliar P application on P concentration in leaf of four maize hybrids


means

No spray

(Control)

0.52 h 0.56 g 0.63 e 0.60 f 0.58 B



197

Appendix 4.41: Effect of supplemental foliar P application on P concentration in stem of four maize hybrids under normal and



Treatment

means


No spray

(Control) 0.83 fg 0.85 f 0.95 d 0.89 e 0.56 m 0.59 lm 0.65 jk 0.62 kl 0.74 B

P spray 0.98 d 1.04 c 1.13 a 1.09 b 0.69 ij 0.72 i 0.80 gh 0.77 h 0.90 A


Appendix 4.42: Effect of supplemental foliar P application on P concentration in stem of four maize hybrids


means

No spray

(Control)

0.69 g 0.72 g 0.80 e 0.76 f 0.74 B



198

Appendix 4.43: Effect of supplemental foliar P application on P concentration in root of four maize hybrids under normal and



Treatment

means


No spray

(Control) 1.61 g 1.69 f 1.79 de 1.75 ef 0.86 n 0.96 m 1.12 kl 1.05 l 1.35 B

P spray 1.84 cd 1.89 c 2.10 a 1.97 b 1.19 k 1.29 j 1.49 h 1.38 i 1.64 A


Appendix 4.44: Effect of supplemental foliar P application on P concentration in root of four maize hybrids


means

No spray

(Control)

1.23 h 1.32 g 1.45 e 1.40 f 1.35 B



199

Appendix 4.45: Effect of supplemental foliar P application on P concentration in grain of four maize hybrids under



Treatment

means


No spray

(Control) 2.39 h 2.45 g 2.58 e 2.52 f 1.90 o 1.97 n 2.09 l 2.02 m 2.24 B

P spray 2.68 d 2.73 c 2.86 a 2.80 b 2.15 k 2.21 j 2.31 i 2.27 i 2.50 A


Appendix 4.46: Effect of supplemental foliar P application on P concentration in grain of four maize hybrids


means

No spray

(Control)

2.14 h 2.21 g 2.34 e 2.27 f 2.24 B



200

Appendix 4.47: Effect of supplemental foliar P application on PUE of four maize hybrids under normal and water stress

conditions


Treatment

means


No spray

(Control) 3.11 h 3.24 g 3.51 e 3.38 f 1.81 p 1.98 o 2.27 m 2.14 n 2.68 B

P spray 3.63 d 378 c 4.14 a 3.94 b 2.41 l 2.57 k 2.74 j 2.92 i 3.26 A


Appendix 4.48: Effect of supplemental foliar P application on PUE of four maize hybrids


means

No spray

(Control)

2.46 h 2.61 g 2.89 e 2.76 f 2.68 B



201

Appendix 4.49: Effect of supplemental foliar P application on cob length (cm) of four maize hybrids under normal and



Treatment

means


No spray

(Control) 18.42 ef 18.52 def 18.68 cde 18.62 de 17.48 i 17.56 i 17.85 ghi 17.71 hi 18.10 B

P spray 18.91 bcd 19.14 bc 20.56 a 19.28 b 17.92 ghi 18.05 fgh 18.76 efg 18.34 efg 18.79 A


Appendix 4.50: Effect of supplemental foliar P application on cob length (cm) of four maize hybrids


means

No spray (Control) 17.94 e 18.03 e 18.26 cde 18.16 de 18.10 B

Foliar P spray 18.41 bcd 18.59 bc 19.41 a 18.74 b 18.79A

Hybrid means 18.18 C 18.31 BC 18.84 A 18.45 B

202

Appendix 4.51: Effect of supplemental foliar P application on number of cobs per plant of four maize hybrids under



Treatment

means


No spray (Control) 5.67 def 6.33 cd 6.67 bc 6.33 cd 5.00 f 5.00 f 5.33 ef 5.33 ef 5.70 B

P spray 6.67 bc 6.67 bc 7.67 a 7.33 ab 5.33 ef 6.00 cde 6.33 cd 6.33 cd 6.54 A

Hybrid means 6.66 A 5.58 B

Appendix 4.52: Effect of supplemental foliar P application on number of cobs per plant of four maize hybrids


means

No spray (Control) 5.33 e 5.66 de 6.00 cd 5.83 cde 5.70 B

Foliar P spray 6.00 cd 6.33 bc 7.00 a 6.83 ab 6.54 A


203

Appendix 4.53: Effect of supplemental foliar P application on number of grains per cob of four maize hybrids under



Treatment

means


No spray

(Control) 371.33 f 372.00 ef 375.33 cd 373.67 de 355.67 m 257.00 lm 361.00 jk 359.00 kl 365.63 B

P spray 376.33 c 378.67 b 383.33 a 380.33 b 362.33 ij 364.00 i 369.00 g 366.33 h 372.54 A


Appendix 4.54: Effect of supplemental foliar P application on number of grains per cob of four maize hybrids


means

No spray (Control) 363.50 f 364.50 f 368.17 d 366.33 e 365.63 B



204

Appendix 4.55: Effect of supplemental foliar P application on cob weight without sheath (g) of four maize hybrids under



Treatment

means


No spray

(Control) 210.127 gh 207.17 fg 218.89 e 213.47 ef 163.78 n 169.24 mn 177.03 kl 173.63 lm 190.56 B

P spray 227.03 d 234.16 c 258.88 a 242.16 b 181.35 jk 184.21 ij 198.43 h 190.02 i 214.53 A


Appendix 4.56: Effect of supplemental foliar P application on cob weight without sheath (g) of four maize hybrids


means

No spray (Control) 182.52 g 188.21 f 197.96 e 193.55 e 190.56 B



205

Appendix 4.57: Effect of supplemental foliar P application on 1000-grain weight (g) of four maize hybrids under normal



Treatment

means


No spray

(Control) 234.07 g 243.77 f 259.57 e 252.40 e 198.40 m 201.80 lm 210.27 k 206.80 kl 225.88 B

Foliar P spray 269.80 d 286.13 c 314.37 a 305.47 b 215.13 jk 219.23 ij 230.83 gh 225.30 hi 258.28 A


Appendix 4.58: Effect of supplemental foliar P application on 1000-grain weight (g) of four maize hybrids


means

No spray (Control) 216.23 g 222.78 f 234.92 e 229.60 e 225.88 B

Foliar P spray 242.47 a 252.68 c 272.60 a 265.38 b 258.28 A


206

Appendix 4.59: Effect of supplemental foliar P application on grain yield (t ha-1) of four maize hybrids under normal and



Treatment

means


No spray

(Control) 5.70 h 5.90 g 6.57 e 6.23 f 3.80 n 4.00 m 4.57 k 4.20 l 5.12 B

P spray 7.00 d 7.23 c 7.93 a 7.60 b 4.73 jk 4.90 j 5.57 h 5.23 i 6.27 A


Appendix 4.60: Effect of supplemental foliar P application on grain yield (t ha-1) of four maize hybrids


means

No spray (Control) 4.75 h 4.95 g 5.56 e 5.21 f 5.12 B



207

Appendix 4.61: Effect of supplemental foliar P application on biological yield (t ha-1) of four maize hybrids under normal



Treatment

means


No spray

(Control) 14.33 f 14.98 e 16.02 d 15.71 d 11.41 l 11.98 k 12.83 ij 12.31 jk 13.69 B

Foliar P spray 16.86 c 17.31 c 19.11 a 18.02 b 13.06 i 13.28 hi 14.16 fg 13.67 gh 15.68 A


Appendix 4.62: Effect of supplemental foliar P application on biological yield (t ha-1) of four maize hybrids


means

No spray (Control) 12.87 g 13.48 f 14.42 d 14.02 e 13.69 B

Foliar P spray 14.95 c 15.29 c 16.63 a 15.84 b 15.68 A


208

Appendix 4.63: Effect of supplemental foliar P application on harvest index (%) of four maize hybrids under normal and



Treatment

means


No spray

(Control) 39.77 bcd 39.43 cd 41.00 abc 39.67 cd 33.32 h 33.40 h 35.59 fg 34.12 gh 37.04 B

P spray 41.53 ab 41.81 a 41.54 ab 42.19 a 36.26 f 36.90 ef 39.32 cd 38.30 de 39.73 A


Appendix 4.64: Effect of supplemental foliar P application on harvest index (%) of four maize hybrids


means

No spray (Control) 36.54 c 36.41 c 38.29 b 36.89 c 37.04 B

Foliar P spray 38.89 b 39.36 ab 40.42 a 40.24 a 39.73 A

Hybrid means 37.72 B 37.88 B 39.36 A 38.56 AB

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