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In the Name of Allah, the Most Beneficent

And The Most Merciful!

Oh, Allah Almighty open our eyes,To see what is beautiful,Our minds to know what is true,

Our heart to love what is Allah.

Alleviation of Terminal Heat Stress in Wheat (Triticum aestivum L.) Through Potassium and Selenium Nutrition

By

MUHAMMAD SHAHIDM.Sc. (Hons.) Agriculture2008-ag-2268

By

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

DOCTOR OF PHILOSOPHY

in

AGRONOMY

DEPARTMENT OF AGRONOMY,FACULTY OF AGRICULTURE,UNIVERSITY OF AGRICULTURE, FAISALABAD-PAKISTAN2018

Dedicated To

The Sublime Love

Of

My Beloved and Kind Parents

Who taught me,

The first step to take,

The first word to speak,

The first alphabet to write,

Inspired me to higher ideas of life,

Whose hands always raise in prayer for me,

Who are with me to feel the bud of their wishes and prayers blooming into a flower

and

Under whose feet my heaven lies.

AND My Beloved BROTHER and SISTERS

Acknowledgements

Bounteous praise for ALMIGHTY ALLAH, the magnificent, the merciful, the propitious, the supreme, the omnipotent, the omnipresent, the omniscient and sovereign whose blessing and glories flourish my cogitation and all the eulogies for the HOLY PROPHET MUHAMMAD (SAW) for edifying our conscience of faith in ALLAH, converging all his kindness and mercy upon him.

I feel much honor to express my deepest sense of gratitude, philanthropy and magnanimity to my honorable supervisor, Dr. Muhammad Farrukh Saleem, Associate Professor, Department of Agronomy, University of Agriculture Faisalabad from the core of my heart for his dynamic supervision, marvelous guidance, keen interest and encouraging behavior. With humble, profound and deepest sense of devotion I wish to record my sincere appreciation to Dr. Shakeel Ahmad Anjum, Assistant Professor, Department of Agronomy, University of Agriculture Faisalabad and Dr. Irfan Afzal, Associate Professor, Department of Agronomy, University of Agriculture, Faisalabad for their sincere help, dynamic supervision and inspiring guidance throughout the course of this research work. I am genially thankful to Dr. Muhamad Shahid, Associate Professor, Department of Biochemistry, University of Agriculture Faisalabad for abetting in the conduct of biochemical analysis during the whole research work.

I feel inordinate appreciativeness for Higher Education Commission of Pakistan for economic backing to conduct this research work. I cordially applaud the facilities provided by Analytical Laboratory, Department of Agronomy, University of Agriculture Faisalabad and Medicinal Plants Biochemistry Laboratory, Department of Biochemistry, University of Agriculture Faisalabad for assistance in conduct of biochemical analysis.

I want to express my great appreciation and sincerest gratitude to my friends and juniors; Abdul Shakoor, Ubaid-Ur-Rehman and Siraj Ahmed for their dexterous, dynamic, untiring help, friendly behavior and moral support during my whole study.

Round out the picture, no acknowledgement could ever adequately express my obligation to my affectionate Parents whose endless efforts and best wishes sustained me at all stages of my life and encouraged me for achieving higher ideas of life. Just as importantly, I want to express my everlasting love for my loving brother Muhammad Faisal and caring Sisters they offered irreplaceable endorsement and my Nephews and Nieces whom countenances have bestowed me the blisses of life.

May ALLAH bless all these people with long, happy and peaceful lives (Aameen)!

Muhammad Shahid

LIST OF TABLES

Table

Title

Page

3.1

Physio-chemical analysis of experimental site during 2014-15, 2015-16 and 2016-17

15

3.2

Monthly averages of weather elements during growing season of crop in 2014-15, 2015-16 and 2016-17

16

3.3

Varying mean temperatures (C) 2014-15 for experiment 1

17

3.4

Varying mean temperatures (C) during heat imposition for experiment 2, 2015-16 and 2016-17

17

3.5

Varying mean temperatures (C) during heat imposition for experiment 3, 2015-16 and 2016-17

18

4.1.1

Effect of heat stress on fertile tillers of wheat varieties

31

4.1.2

Effect of heat stress on grains per spike and 1000-grain weight of wheat varieties

32

4.1.3

Effect of heat stress on grain yield of wheat varieties

34

4.1.4

Effect of heat stress on grain filling rate (GFR) and grain filling duration (GFD) of wheat varieties

38

4.1.5

Effect of heat stress on chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of wheat varieties

41

4.1.6

Effect of heat stress on superoxide dismutase (SOD) and peroxidase (POD) of wheat varieties

42

4.1.7

Effect of heat stress on catalase (CAT) and total phenolic contents (TPC) of wheat varieties

43

4.1.8

Effect of heat stress on leaf proline and glycine betaine of wheat varieties

47

4.1.9

Effect of heat stress on total soluble proteins of wheat varieties

48

4.1.10

Effect of heat stress on malondialdehyde of wheat varieties

50

4.1.11

Correlation analyses showing strength of association among recorded attributes of different wheat varieties under no heat stress (H0)

52

4.1.12

Correlation analyses showing strength of association among recorded attributes of different wheat varieties under heat from spike to grain filling (H1)

53

4.2.1

Effect of foliar applied potassium on fertile tillers and grains per spike of heat stressed wheat

57

4.2.2

Effect of foliar applied potassium on 1000-grain weight and grain yield of heat stressed wheat

59

4.2.3

Effect of foliar applied potassium on biological yield and harvest index of heat stressed wheat

64

4.2.4

Effect of foliar applied potassium on straw yield and plant height of heat stressed wheat

66

4.2.5

Effect of foliar applied potassium on spike length and spikelets per spike of heat stressed wheat

71

4.2.6

Effect of foliar applied potassium on grain filling rate (GFR) and grain filling duration (GFD) of heat stressed wheat

73

4.2.7

Effect of foliar applied potassium on chlorophyll a (Chl a) and chlorophyll b (Chl b) contents of heat stressed wheat

79

Table

Title

Page

4.2.8

Effect of foliar applied potassium on superoxide dismutase (SOD) and peroxidase (POD) of heat stressed wheat

81

4.2.9

Effect of foliar applied potassium on catalase (CAT) and total phenolic contents (TPC) of heat stressed wheat

83

4.2.10

Effect of foliar applied potassium on leaf proline and glycine betaine of heat stressed wheat

89

4.2.11

Effect of foliar applied potassium on total soluble proteins (TSP) and malondialdehyde (MDA) of heat stressed wheat

91

4.2.12

Effect of foliar applied potassium on osmotic (S) and water potential (W) of heat stressed wheat

96

4.2.13

Effect of foliar applied potassium on turgor potential (P) and shoot potassium (K) contents of heat stressed wheat

98

4.2.14

Effect of foliar applied potassium on grain crude proteins of heat stressed wheat

100

4.2.15 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied potassium during 2015-16

106

4.2.15 (b)


107

4.2.15 (c)


108

4.2.15 (d)


109

4.2.16 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied potassium during 2015-16

110

4.2.16 (b)


111

4.2.16 (c)


112

4.2.16 (d)


113

4.2.17 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied potassium during 2015-16

114

4.2.17 (b)


115

4.2.17 (c)


116

Table

Title

Page

4.2.17 (d)


117

4.3.1

Effect of foliar applied selenium on fertile tillers and grains per spike of heat stressed wheat

120

4.3.2

Effect of foliar applied selenium on 1000-grain weight and grain yield of heat stressed wheat

122

4.3.3

Effect of foliar applied selenium on biological yield and harvest index of heat stressed wheat

128

4.3.4

Effect of foliar applied selenium on straw yield and plant height of heat stressed wheat

130

4.3.5

Effect of foliar applied selenium on spike length and spikelets per spike of heat stressed wheat

135

4.3.6

Effect of foliar applied selenium on grain filling rate (GFR) and grain filling duration (GFD) of heat stressed wheat

137

4.3.7

Effect of foliar applied selenium on chlorophyll a (Chl a) and on chlorophyll b (Chl b) contents of heat stressed wheat

143

4.3.8

Effect of foliar applied selenium on superoxide dismutase (SOD) and peroxidase (POD) contents of heat stressed wheat

145

4.3.9

Effect of foliar applied selenium on catalase (CAT) and total phenolic contents (TPC) of heat stressed wheat

147

4.3.10

Effect of foliar applied selenium on leaf proline and glycine betaine contents of heat stressed wheat

154

4.3.11

Effect of foliar applied selenium on total soluble proteins (TSP) and malondialdehyde (MDA) contents of heat stressed wheat

156

4.3.12

Effect of foliar applied selenium on osmotic (S) and water potential (W) of heat stressed wheat

162

4.3.13

Effect of foliar applied selenium on turgor potential (P) and grain crude protein contents of heat stressed wheat

164

4.3.14 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under no heat stress (H0) and foliar applied selenium during 2015-16

168

4.3.14 (b)


169

4.3.14 (c)


170

4.3.14 (d)


171

4.3.15 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from spike to grain filling (H1) and foliar applied selenium during 2015-16

172

4.3.15 (b)


173

Table

Title

Page

4.3.15 (c)


174

4.3.15 (d)


175

4.3.16 (a)

Correlation analyses showing strength of association among recorded attributes of wheat under heat from flowering to grain filling (H2) and foliar applied selenium during 2015-16

176

4.3.16 (b)


177

4.3.16 (c)


178

4.3.16 (d)


179

LIST OF FIGURES

Figure

Title

Page

4.2.1

Regression analysis for effect of foliar applied potassium on grains per spike of heat stressed wheat

58

4.2.2

Regression analysis for effect of foliar applied potassium on 1000-grain weight and grain yield of heat stressed wheat

60

4.2.3

Regression analysis for effect of foliar applied potassium on biological yield and harvest index of heat stressed wheat

65

4.2.4

Regression analysis for effect of foliar applied potassium on straw yield and plant height of heat stressed wheat

67

4.2.5

Regression analysis for effect of foliar applied potassium on spike length and spikelets per spike of heat stressed wheat

72

4.2.6

Regression analysis for effect of foliar applied potassium on grain filling rate and grain filling duration of heat stressed wheat

74

4.2.7

Regression analysis for effect of foliar applied potassium on chlorophyll a and chlorophyll b contents of heat stressed wheat

80

4.2.8

Regression analysis for effect of foliar applied potassium on superoxide dismutase and peroxidase contents of heat stressed wheat

82

4.2.9

Regression analysis for effect of foliar applied potassium on catalase and total phenolic contents of heat stressed wheat

84

4.2.10

Regression analysis for effect of foliar applied potassium on leaf proline and glycine betaine of heat stressed wheat

90

4.2.11

Regression analysis for effect of foliar applied potassium on total soluble proteins and malondialdehyde of heat stressed wheat

92

4.2.12

Regression analysis for effect of foliar applied potassium on osmotic and water potential of heat stressed wheat

97

4.2.13

Regression analysis for effect of foliar applied potassium on turgor potential and shoot potassium contents of heat stressed wheat

99

4.2.14

Regression analysis for effect of foliar applied potassium on grain crude proteins of heat stressed wheat

101

4.3.1

Regression analysis for effect of foliar applied selenium on grains per spike of heat stressed wheat

121

4.3.2

Regression analysis for effect of foliar applied selenium on 1000-grain weight and grain yield of heat stressed wheat

123

4.3.3

Regression analysis for effect of foliar applied selenium on biological yield and harvest index of heat stressed wheat

129

4.3.4

Regression analysis for effect of foliar applied selenium on straw yield and plant height of heat stressed wheat

131

4.3.5

Regression analysis for effect of foliar applied selenium on spike length and spikelets per spike of heat stressed wheat

136

4.3.6

Regression analysis for effect of foliar applied selenium on grain filling rate and grain filling duration of heat stressed wheat

138

4.3.7

Regression analysis for effect of foliar applied selenium on chlorophyll a and on chlorophyll b contents of heat stressed wheat

144

4.3.8

Regression analysis for effect of foliar applied selenium on superoxide dismutase and peroxidase contents of heat stressed wheat

146

4.3.9

Regression analysis for effect of foliar applied selenium on catalase and total phenolic contents of heat stressed wheat

148

Figure

Title

Page

4.3.10

Regression analysis for effect of foliar applied selenium on leaf proline and glycine betaine contents of heat stressed wheat

155

4.3.11

Regression analysis for effect of foliar applied selenium on total soluble proteins and malondialdehyde contents of heat stressed wheat

157

4.3.12

Regression analysis for effect of foliar applied selenium on osmotic and water potential of heat stressed wheat

163

4.3.13

Regression analysis for effect of foliar applied selenium on turgor potential and grain crude protein contents of heat stressed wheat

165

XIII

ABSTRACT

Coincidence of high temperature at the terminal phenological stages of the wheat crop is a prime constraint to reach full yield potential in Pakistan. The present research work was conducted to determine the thermo-sensitivity of Pakistani wheat genotypes and alleviation of negative implications of heat through exogenous application of potassium and selenium. All research work was performed at the Agronomic Research Area, University of Agriculture Faisalabad, Pakistan from November 2014 to May 2017. In the first year, wheat genotypes were screened for terminal heat tolerance under field conditions. The experiment was laid out in a Randomized Complete Block Design (RCBD) in a split plot arrangement and was replicated 4 times. Treatments were comprised of heat stress in main plots viz. H0 = no heat imposition; H1 = Heat imposition from complete emergence of spike to grain filling initiation (Feekes Scale = 10.50 to 11.00) and wheat genotypes in sub plots viz. Punjab-2011, AARI-2011, Galaxy-2013, Millat-2011, Aas-2011, Fareed-2006, Chakwal-50, Mairaj-2008, Pakistan-2013, NIBGE-NIAB-1 and Kohistan-97. Imposition of heat stress deleteriously impacted the metabolism of all genotypes. The synthesis of antioxidants and osmo-protectants were enhanced in genotypes AAS-2011, Chakwal-50 and Mairaj-2008 under the high temperature environment compared to no heat stress. While, in all other genotypes biosynthesis of antioxidants and osmo-protectants was suppressed under heat compared to control. Likewise, adverse impacts of heat on spike growth, stay green trait, grain yield and yield components were relatively lesser in genotypes AAS-2011, Chakwal-50 and Mairaj-2008 than other genotypes. Statistically similar and relatively more grain yields compared to other genotypes were recorded for Aas-2011 (3.71 t ha-1), Chakwal-50 (3.36 t ha-1) and Mairaj-2008 (3.04 t ha-1) under heat stress. In the second year, two independent field experiments were conducted with the objective of mitigating heat stress using potassium and selenium as beneficial nutrients. The experimental design for both experiments was randomized complete block design (RCBD) with split plot arrangement having three replications. In both experiments, the main plot factor was comprised of three heat stress treatments viz. H0 = No heat imposition; H1 = Heat stress imposition from complete emergence of spike to grain filling initiation (Feekes scale = 10.50 to 11.0); H2 = Heat stress imposition from flowering initiation to grain filling initiation (Feekes scale = 10.5.1 to 11.0). In the second experiment, potassium was supplied via foliar application in sub plots at K0 = Control/ water spray; K15 = 15 g L-1; K30 = 30 g L-1; K45 = 45 g L-1 and K60 = 60 g L-1 to mitigate heat stress. In the third experiment, selenium was foliar applied at Se0 = Control/ water spray; Se25 = 25 mg L-1; Se50 = 50 mg L-1; Se75 = 75 mg L-1 and Se100 = 100 mg L-1 to alleviate heat stress. During the third year, heat stress mitigating experiments were repeated as described in the second year. Negative implications of heat were more pronounced under heat from spike to grain filling compared to heat from flowering to grain filling. Grain yield in second experiment was decreased by 42-45% under heat from spike to grain filling and 25-31% under heat from flowering to grain filling compared to no heat stress. While, in third experiment, heat from spike to grain filling and heat from flowering to grain filling caused decrease in grain yield compared to no heat stress by 43-44% and 33-36%, respectively. Whereas, varying concentrations of foliar potassium and selenium differed significantly from each other and remarkably improved response variables compared to control/water spray. Application of potassium at 45 and 60 g L-1 and selenium at 75 and 100 mg L-1 depicted statistically similar and relatively more grain yield, yield components, spike growth attributes, chlorophyll content and quality attributes compared to other concentrations under all treatments of heat stress. Likewise, statistically alike and comparatively more antioxidants, osmo-protectants and water relations attributes and statistically similar and relatively lesser malondialdehyde were observed with 45 and 60 g L-1 foliar potassium and 75 and 100 mg L-1 foliar selenium under no heat stress. However, application of 60 g L-1 potassium and 100 mg L-1 selenium showed significantly more antioxidants, osmo-protectants and water relations attributes and significantly lesser malondialdehyde under heat from spike to grain filling and heat from flowering to grain filling. Conclusively, genotypes AAS-2011, Chakwal-50 and Mairaj-2008 displayed terminal heat tolerance while genotypes Fareed-2006 and Punjab-2011 exhibited medium tolerance. In contrast, all other genotypes tested did not produce remarkable responses under heat and were characterized as terminal heat susceptible based on recorded parameters. Under no heat stress application of exogenous potassium at 45 g L-1 and selenium at 75 mg L-1 effectively alleviated the adverse impacts of heat. Whereas, application of potassium at 60 g L-1 and selenium at 100 mg L-1 provided more promising morphological and biochemical responses under heat from spike to grain filling and heat from flowering to grain filling. While, foliar applied potassium and selenium proved more important under heat treatments compared to ambient conditions. Moreover, biochemical attributes modulated regulations in growth, yield components and grain yield were significant under varying temperatures.

INTRODUCTION CHAPTER-1

Wheat is extensively grown all over the world and is an important source of starch and protein for humans (Ang and Fredriksson, 2017). Wheat is cultivated on more than 218 million hectares with 743 million tons annual production around the globe (FAO, 2017). The share of wheat in value addition in agriculture is 9.6% while it supplements 1.9% in gross domestic product of Pakistan. Its area of cultivation is 9. 052 million hectares and production is 25.75 million tons (Govt. of Pakistan, 2017). Each 100 g serving of wheat provides 247 calories, 58-62 g bioavailable carbohydrates, 12-13 g proteins, 1.8 g lipids, 13-14 g fiber and 1.7 g minerals (400 mg sodium, 248 mg potassium). Besides, 1 kg wheat of grains also provide 4.6-5.0 mg vitamin-B1 (thiamine), 0.9-1.2 mg vitamin-B2 (riboflavin), 51-55 mg nicotinamide, 12-13 mg pantothenic acid, 2.7-3.0 mg vitamin-B6 and 41 mg tocopherols (Koehler and Wieser, 2013).

Productivity of wheat in Pakistan is lagging far behind than the potential owing to numerous factors. Different factors that are responsible for low productivity of wheat in Pakistan are delayed soil preparation after rice and cotton, late sowing, low input use efficiency, unavailability of quality seed, fertilizers, irrigation water and terminal heat stress (Rehman et al., 2015).

Heat stress at reproductive stages (terminal heat stress) of wheat is one of the chief constraints hampering the full attainment of yield potential. The upper threshold temperature above which terminal stages of wheat are deleteriously impacted is 26C (Shahid et al., 2017). Terminal heat stress might be a consequence of rapid industrialization, deforestation, burning of fossil fuels, emission of chloro flouro carbons, rapid changes in land utilization and injudicious use of synthetic fertilizers in agriculture (Szymaska, 2017). In addition, the decline in rainfall over sub tropics including Pakistan has further intensified extreme temperature events (Rahut and Ali, 2017). Moreover, the rate of increase of temperature in the last decade (2000-2010) had been 2.2% higher than the rate of increase of temperature in previous 30 years (1970-2000) (IPCC, 2014). While, late sowing of wheat in rice-wheat, cotton-wheat and hybrid maize-wheat cropping systems leads to high temperature stress at reproductive stages of wheat (Mumtaz et al., 2015).

Wheat is a C3 and temperate plant and therefore is very susceptible to high temperature stress. High temperature stress at reproductive and grain filling stages of wheat is called terminal heat stress (Alghabari et al., 2016). It is usually 10-15C higher temperature than ambient temperature (Dwivedi et al., 2017). It is anticipated to increase in the near future due to global warming. Temperature will rise by 2.6-4.8C during the period of 2016-2035 (IPCC, 2014) while optimum temperature for reproductive stages of wheat is 12-22C (Dwivedi et al., 2017).

Damages due to high temperature stress depends on the duration of high temperature, the magnitude of rise of temperature and the rate of increase in temperature (Prasad et al., 2017). Temperature may rise slowly, rapidly or in cyclic pattern (increases during day while decreases during night). Cyclic increase is the most damaging while slow rise of temperature is the least damaging for wheat productivity (Rezaei et al., 2015).

The temperature optima for spikelet, anthesis and grain filling for wheat are 12, 23 and 21C, respectively (Innes et al., 2015). According to a previous assessment, an increase of 1C during the growing season declines grain yield by 3-17% in Pakistan and India (Mondal et al., 2013).

High temperature stress reduces wheat productivity by dehydration, pollen sterility, shortening of phenology, decreased CO2 assimilation, increased photorespiration and decreased growth rate (Altenbach, 2012). Under heat stress, photosynthesis is the most sensitive process. High temperature dissociates oxygen evolving complex of PS-II and initiates photorespiration (Mathur et al., 2014).

Chlorophyll enzymatic activity is also disturbed at higher temperature. Activity of adenosine diphosphate glucose pyro phosphatase (ADPG-PPase) is particularly reduced. It downregulates the synthesis of starch (Dwivedi et al., 2017). Diurnal fluctuations of temperature are more damaging to promote senescence (Laza et al., 2015).

Grain growth and development is also affected at higher temperature. Spike initiation stage is the most sensitive stage to high temperature as at this stage ridges development on spike rachis takes place. The number of ridges determines the number of spikelets in the spike (Iqbal et al., 2017).

Under heat stress excessive generation of reactive oxygen species (ROS) overcome scavenging mechanisms. Excessive ROS results in increased membrane damages, lipid peroxidation, protein carbonylation and damage to DNA by insertion, deletion, mutation and affecting nitrogen bases of DNA. High temperature stress increases superoxide radical (O2-) while hydrogen peroxide (H2O2) generation also rises above normal level. Other ROS that are excessively produced and aggravate lipid peroxidation at sub cellular level are singlet oxygen (1O2*) and hydroxyl radical (OH-) (Czgny et al., 2016).

To manage heat stress different strategies are available. These are breeding for heat stress tolerance and selection of tolerant genotypes (Mondal et al., 2016). While, agronomic management comprises of reduced tillage and stubble management, pre-sowing heat treatment, manipulation of sowing time and foliar sprays of various substances (Gouache et al., 2012).

Different types of compounds that can be foliar applied to mitigate heat stress include osmo-protectants, osmolytes, inorganic salts, compatible solutes, signaling molecules, plant growth regulators and oxidants (Farooq et al., 2011; Hu et al., 2016). Foliar application of mineral nutrients is one of solutions to the problem. Exogenous application of mineral nutrients augments tolerance against extreme temperature stresses (Waraich et al., 2012).

Potassium (K) is the most important osmoticum in the plant cell cytosol. The availability of K improves heat stress tolerance in plant. Potassium also helps the plant to make osmotic adjustments as it is the safest osmoticum (Zahoor et al., 2017a). It is an osmolyte and thus depresses the cellular water potential more than the apoplast. Net movement of water takes place into cell that helping them to maintain turgor and creating a favorable environment to maintain cellular enzymatic activities under heat stress (Jan et al., 2017; Xiaokang et al., 2017).

Potassium maintains the electrical charge balance at site of ATP synthesis and photophosphorylation remains continuous under stress conditions (Kanai et al., 2011). Potassium activates ATP for utilization by H+-ATPase pumps. Hydrogen pump ATPases exclude H+ out of the cell and create a favorable electrochemical gradient known as proton motive force (Ahmad and Maathuis, 2014). Most nutrient uptake utilizes proton motive force. Thus, K also helps in maintaining nutrient uptake under stressed conditions (Anschtz et al., 2014).

Potassium enhances dry matter accumulation by maintaining activities of different enzymes involved in starch and protein deposition. Exogenously applied potassium enhances activation of RuBisCO, sucrose phosphate synthase, sucrose synthase and soluble acid invertase under stressed environments. Increase in activities of these enzymes escalates the sucrose and starch accumulation in reproductive and vegetative organs under abiotic stress conditions. Moreover, easily available foliar applied potassium also increases stomatal conductance and gaseous exchange with the environment. Ultimately, dry matter accumulation of vegetative and reproductive parts, net assimilation rate, partitioning of starch and sucrose towards reproductive parts improves under stress conditions (Zahoor et al., 2017b). It also maintains activity of hydrolases (Pectinases, Cellulases) under stress condition. Activation of hydrolases loosens cell wall and concurrently K mediated depression of cell water potential causes influx of water into cell. Cell is able to expand and maintain growth under heat stress (Jin et al., 2011).

Foliar applied K enhanced net photosynthesis, stomatal conductance, yield and growth attributes of wheat under stress conditions (Zareian et al., 2013). Availability of K reduced photo oxidative damage, increased leaf potassium contents, water and osmotic potential, enhanced CO2 fixation, transpiration rate, maximum and actual quantum yield of photosystem-II (PS-II), non-photochemical quenching and increased utilization of light use efficiency under stressed conditions. Moreover, foliar application of potassium boosted the activities of superoxide dismutase, catalase, peroxidase and proline and consequence into decreased lipid peroxidation of bio-membranes (Zahoor et al., 2017c). Potassium availability under stressed conditions improved root hydraulic by increasing expression of aquaporin (Wang et al., 2013). Consequences of modulation in potassium balance and in physiochemical attributes are improved growth and yield under stress conditions. Availability of K reduced oxidative stress by reducing NADH oxidase activity, K deficiency augmented O2- generation and thus aggravated oxidative stress (Jimnez-Quesada et al., 2016).

Selenium (Se) down regulates ROS production under stress by upregulating the activity of antioxidants. It increases the activity of ascorbate peroxidase that is a key enzyme in detoxification of H2O2. It upregulates activity of catalase and glutathione peroxidase under heat stress (Cheng et al., 2016). Selenium compounds under heat stress quench 1O2* and OH-. It promotes stability of membranes as OH- is most damaging for lipid peroxidation and 1O2* causes mutation by reacting with nitrogen bases of DNA (Feng et al., 2013). It accelerates the non-enzymatic detoxification of O2- to H2O2 and protects cellular membranes. Selenium also acts as an activator of glutathione peroxidase, which detoxifies H2O2 (Huang et al., 2017).

In photosynthesis, elemental Se replaces sulfur from Fe-S cluster and reduces ROS synthesis through regulation of electron flow. Selenium enhances PS-I ability to produce reductants at the end of light reactions and promotes CO2 reduction under high temperature stress (Gupta and Gupta, 2017). Selenium reduces damage to PS-II light harvesting complex by excessive UV and high light intensity under heat stress (Feng et al., 2013).

Exogenous application of Se augmented synthesis of catalase, superoxide dismutase, peroxidase, glutathione and ascorbate reductase in wheat. Moreover, water retention capability of tissues was also enhanced with foliar applied selenium under stressed conditions over control in wheat (Nawaz et al., 2015). Improvement in accumulation of proline under exogenous selenium resulted in detoxification of reactive oxygen species and upregulated the biosynthesis of chlorophyll, total soluble sugars and phenyl ammonia lyase contents under stressed conditions (Manaf, 2016). Exogenous Se enhanced accumulation of ascorbate, carotenoids, anthocyanin, ascorbate peroxidase, chlorophyll a, b and reduced malondialdehyde (MDA) in wheat under high temperature stress (Iqbal et al., 2015). Selenium augmented antioxidant defense system under high temperature by increasing synthesis of glutathione reductase, dehydro ascorbate reductase and by maintaining high reducing power of NADH (Sieprawska et al., 2015). Selenium at low concentration acts as reductant for ROS. At higher concentration, it functions as pro antioxidant that improves signaling for upregulation of the antioxidant defense system (Ahmad et al., 2016). Moreover, application of selenium increased accumulation of anthocyanin, ascorbic acid, antioxidants and nutrients. Alleviation of stress under Se application can be attributed to selenium mediated improvements in redox buffering capacity of plant, phyto hormone regulations, antioxidant regeneration, ROS scavenging and enhanced cell division (Shekari et al., 2015).

Selenium reduced protochlorophyllide oxidoreductase contents, enhanced activities of starch biosynthesis enzymes and maintained normal function and shape of chloroplast (Kaur et al., 2014). Selenium improved the staygreen trait and maintained carbohydrates supply for longer duration of time (Haghighi et al., 2015). Selenium mediated synthesis of chlorophyll a and b, increased stomatal conductance, transpiration rate and exchange of gases with atmosphere under heat stress (Mora et al., 2015).

Different wheat cultivars depict assortment and heterogeneity in response to high temperature (Siebert and Ewert, 2014). Furthermore, numerous quantitative trait loci exist for a single targeted trait having complex inheritance pattern (Mwadzingeni et al., 2016). Therefore, selection of polygenic target traits can be accomplished indirectly employing biochemical markers closely related to heat tolerance (Sadat et al., 2013). Likewise, diversity among wheat cultivars combined with polyploidy and genes profusion makes it challenge to select a suitable genotype using morphological traits under high-temperature environment (Dube et al., 2016). Selection of wheat genotypes merely on the basis of morphological traits often leads to faulty inferences (Reynolds and Langridge, 2016). While, physiochemical markers assisted screening of genotypes depicts higher efficacy of selection than mere morphological markers-based selection for polygenic traits (Sadat et al., 2013).

Previous experiments were mainly comprised of heat imposition under controlled environments of glasshouse. Although, studies regarding manipulation of sowing dates are abundantly available to observe adverse effects of high temperature. Relatively little information is available regarding the imposition of heat stress under field conditions. Moreover, studying potassium and selenium mediated transformations in biochemical attributes in correlation with morphological traits might prove advantageous for agronomic management of heat stress. Information regarding the correlation of biochemical attributes with growth and yield parameters at terminal stages predisposed to heat are also scarce. Moreover, most of previous studies quantified biochemical attributes only at seedling stages without considering yield and other phenotypic traits at terminal stages.

In this context, a compendious understanding and boost of biochemical mechanisms using exogenous potassium and selenium is indispensable to induce heat tolerance. Moreover, distinctive biochemical response of varying heat stressed terminal pheno-stages leads us to a closer inspection of the problem and its management through exogenous potassium and selenium. Since, improvements in physiochemical traits might prove a potent tool to alleviate adversities on morphological attributes of wheat crop. Hence, elucidation of biochemical attributes in correlation with grain growth and yield will improve the efficacy of agronomic management of terminal heat.

It can be inferred that terminal heat stress in wheat badly impacts various growth, yield, biochemical and physiological attributes. As a consequence of negative implications of high temperature stress grain shriveling takes place under agro-climatological conditions of Pakistan. It reduces yield of wheat each year by sudden rises in temperature and increases the costs of wheat production. It is the hour of need to manage heat stress by devising strategies that are economical, everlasting and alleviate heat stress effectively. Foliar applied K and Se may have potential to regulate various physiological, biochemical, growth and yield related processes under high temperature stress.

Objectives

The study was conducted with the following objectives

1- Screening of Pakistani wheat genotypes for tolerance to terminal heat

2- Studying the comparative vulnerability of terminal phenological stages of wheat to high temperature

3- Exploring the morphological responses of wheat in relation to physiochemical perturbations under varying temperatures

4- Optimizing foliar potassium (K) and selenium (Se) to alleviate negative impacts of terminal heat in wheat

REVIEW OF LITERATURE CHAPTER-2

Wheat is among the widest grown cereals around the globe. Wheat chip in 21% to the worlds calorie intake and is grown on an area of 221 million-hectare worlds widely (Tao et al., 2015). Food security in Pakistan is affiliated with wheat production and consumption. Increasing prevalence of extreme temperatures is becoming a limiting factor for crop production specifically for cereals (Wang et al., 2015). Wheat production under changing climate has been an arduous task (Trnka et al., 2014).

The increasing accumulation of greenhouse gases will further intensify warm temperature together with the disturbance in water resources (Harris et al., 2015). Excessive emission of carbon dioxide from burning of fuels has increased the frequency of heat waves on wheat (Fernando et al., 2014). Carbon dioxide and other greenhouse gases are expected to increase by 50% of the current concentrations in atmosphere by 2050 due to incessant increasing demands for energy (OECD, 2012). Late sowing of wheat is one of the major reasons leading to grain shriveling in wheat by the abrupt rise of temperature during grain filling (Ihsan et al., 2016).

Heat stress negatively influences innumerable plant processes. High temperature increased catalytic activity of RuBisCO while its affinity for CO2 was decreased. Oxygen solubility into mesophyll cells of wheat was little affected while CO2 solubility decreased at higher temperature (Mathur et al., 2014). RuBisCO started to act as an oxygenase enzyme and photo respiration decreased yield. During photorespiration consumption of ATPs using assimilated carbohydrates promoted grain shriveling. RuBisCO sensitivity to higher temperature was more than any other enzyme in photosynthesis (Perez et al., 2011). RuBisCO activase (RCA) enzyme removes inhibitory sugar phosphates from active site of RuBisCO and makes it to react with CO2. At higher temperature, the activity of RCA was also reduced as well as photosynthesis (Carmo-Silva et al., 2012).

Photosystem-II (PS-II) is more labile to higher temperature than Photosystem-I (PS-I). Increase of temperature above 40C disrupted light harvesting complex of PS-II by separation of manganese (Mn) from the D1D2 complex (Ashraf and Harris, 2013). It inhibited the photolysis of water at start of photosynthesis, so electron flow was disturbed and generation of reductants at the end of light reaction for CO2 reduction were also reduced. Rise of temperature further disrupted the plastoquinone in electron pool in the transport chain of light reactions (Mathur et al., 2014).

High temperature stress reduced water potential and relative water content of leaves (Hasanuzzaman et al., 2013). Heat stress promoted respiration and water loss from leaves (Duan et al., 2017). Most species tend to close stomata and conserve water rather than regulation of temperature by transpiration. It impaired gaseous exchange with the atmosphere, thus photosynthesis was negatively affected (Marias et al., 2017).

The rise in temperature caused a rapid grain filling rate and reduced the duration of grain filling. The increased rate of grain filling could not compensate for the decreased duration of grain filling as assimilate partitioning towards the grain was less leading to the consequence of grain shriveling (Barlow et al., 2015).

Temperature above 30C caused completely infertile pollen grains and reduced the size of ovaries. Reduced size of ovaries was due to reduced activity of the acid invertase enzyme and partitioning of carbohydrates towards reproductive organs. Acid invertase governs the upper limit of sink size, so small sized grains were produced at high temperature stress (Dwivedi et al., 2017). Grain size was reduced due to shortening of phenology between anthesis and physiological maturity of grains (Hatfield and Prueger, 2015). Changes in the aleuron layer around the endosperm of wheat grains decreased starch deposition due to different enzymes involved in starch assimilation in endosperm (Iqbal et al., 2017).

Temperatures greater than 25C at grain filling stages reduced activity of starch synthase, granule bound starch synthase, sucrose fructosyltransferase, fructan fructosyltransferase and sucrose synthase. Reduced sucrose synthase activity dwindled phloem sucrose loading (Dwivedi et al., 2017). Diminished translocation of carbohydrates towards grain caused assimilate accumulation in the phloem that introduced a feedback mechanism to down regulate photosynthesis (Wang et al., 2012).

Different wheat cultivars display an assortment and heterogeneity in response towards high temperature (Siebert and Ewert, 2014). Diversity among wheat cultivars combined with polyploidy and genes profusion makes it challenging to select suitable genotypes under high temperature environment. Therefore, phenological and biochemical markers assisted screening of wheat cultivars increases cultivar selection efficacy (Sharma et al., 2014a).

Moreover, different management strategies are available to alleviate the adversity of heat stress in wheat. Soil application of minerals is an energy consuming process regarding plant metabolism. Most nutrients are taken up through secondary active transport that requires ATP. Plants under stress conditions with activated defense mechanisms are not able to extract nutrients from soil solution (Ma et al., 2017). Foliar application can resolve this problem under these hostile conditions of heat stress. Foliar applied nutrients are taken through diffusion that is driven by concentration gradient of nutrient across leaf epicuticular waxes (Wasaya et al., 2017).

Different agronomic strategies that can alleviate heat stress are water conservation, conservation tillage practices and timely sowing of crops (Farooq et al., 2011). Early sowing of wheat in different cropping systems may allow the wheat to escape from terminal heat stress (Suryavanshi and Buttar, 2016). Different foliar sprays i.e. compatible solutes, signaling molecules, plant growth substances and osmolytes enhance tolerance against heat stress. Application of mineral nutrients helps to mitigate high temperature stress in wheat. Nitrogen, phosphorous, potassium, zinc and boron are important in this regard (Hemantaranjan et al., 2014).

Foliar application of potassium (K) and selenium (Se) assists the plant to acclimatize under heat stress by regulation of various biochemical processes. Potassium regulates stomatal opening and closing under heat stress and aids the plant in gas exchange with the atmosphere. Thus, plants are able to uphold sufficient CO2 for RubisCO to act as carboxylase enzyme under heat stress (Wang et al., 2013; Nawaz et al., 2015).

Potassium mediated activation of ATP proved helpful for phloem sucrose loading and unloading. It sustained assimilate partitioning towards grain under heat stress (Marschner, 2012). Potassium diminished diffusible resistance of CO2 into leaf mesophyll by stomatal regulation that made RuBisCO to act as carboxylase enzyme and photorespiration was reduced (Jan et al., 2017).

Potassium enabled plants to make osmotic adjustments under heat stress by promoting accumulation of proline and glycine betaine. Proline acts as an osmoprotectant and alternate electron donor to PS-I and PS-II activity when photolysis of water was lessened at higher temperature (Hayat et al., 2012). Potassium declined malondialdehyde production under stressed conditions, which is an indication of membrane stability (Oosterhuis et al., 2013). Potassium enhanced activity of catalase that is involved in detoxification of excessive H2O2 produced under heat stress (Ahmad et al., 2016). Glycine betaine is a quaternary nitrogen compound, its accumulation was enhanced in presence of K as K is involved in activation of nitrate reductase and glutamine synthase. Glycine betaine also protects membranes from ROS damage under heat stress. Application of potassium improved glycine betaine accumulation, chlorophyll contents and yield related attributes of wheat under stress (Raza et al., 2014). Potassium improved growth and photosynthetic rate by regulating stomatal movement under stress conditions (Ahmad et al., 2014). Potassium application under stressed conditions enhanced dry matter content and relative leaf water content over control (Zahoor et al., 2017b).

Potassium enhanced grain quality by improving protein contents as well as protein quality (Zorb et al., 2014). Potassium is involved in each step of protein synthesis from nitrogen uptake by secondary active transport, activation of nitrate reductase, glutamine synthase, reading of genetic codes and binding of tRNA to ribosomes at ribosomal site of protein synthesis (Sharma et al., 2013).

Exogenous application of Se is more effective for improving plant selenium contents than soil application (Nawaz et al., 2014). Selenium is a beneficial element, but non-essential for growth. It improved relative water contents and water potential of cell under stress condition. Starch deposition in grain was increased under selenium application in high temperature environment (Malik et al., 2012). Selenium delayed the senescence and improved stay green trait under high UV light stress. Application of selenium improved - aminolevulinic acid dehydratase and porphobilinogin deaminase. These enzymes promoted chlorophyll biosynthesis under heat stress. Selenium application reduced protochlorophyllide oxidoreductase activity. Protochlorophyllide oxidoreductase converts protochlorophyllide (precursor of chlorophyll biosynthesis) to chlorophyllide (inactive chlorophyll), thus hindered chlorophyll deprivation in wheat (Yao et al., 2011). Selenium enhanced chlorophyll biosynthesis and reduced degradation. Maintenance of high chlorophyll content under high intensity of UV improved the staygreen trait. In addition, it maintained carbohydrate synthesis in high temperature environment (Yildiztugay et al., 2017).

Selenium assimilation boosted synthesis of glutathione reductase (GSH). It detoxified H2O2 and upgraded antioxidant defense mechanism of plant (Mehdi et al., 2013). Application of Se reduced oxidative stress by slowing down the synthesis of O2- and enhancing detoxification of H2O2 (Feng et al., 2013). Selenium improved superoxide dismutase activity in heat stressed wheat and alleviated oxidative stress significantly as compared to controls (Tedeschini et al., 2015). Selenium declined the reduction of tocopherol under stress conditions that improved glutathione peroxidase activity (Klusonova et al., 2015). Foliar application of Se improved uptake of Na, Fe, Ca and Zn. Increased antioxidant activity under heat stress might be due to enhanced uptake of micronutrients that act as cofactor for activation of enzymatic antioxidants (Nawaz et al., 2015).

Selenium enhanced non-enzymatic dismutation of O2- to H2O2. Selenium mediated synthesis of proteins act as reductants, which promoted non-enzymatic dismutation of O2- (Kaur et al., 2014). Together with non-enzymatic dismutation of O2-, Se also enhanced activity of superoxide dismutase. Different enzymes that are involved in detoxification of ROS are dehydro-ascorbate reductase, mono-dehydro-ascorbate reductase and glutathione reductase. For activation of these enzymes reductants are required. Selenium compounds-maintained reductants for activity of these enzymes (Nawaz et al., 2015).

Selenium improved PS-II stability of heat stressed wheat crop by regulating multiple processes. These processes include decreased excitation energy of PS-II, light absorption by antenna molecules, electron flux, energy quanta of PS-II and impairment of oxygen evolving complex (Labanowska et al., 2012). Selenium augmented cell membrane stability by increasing lipid to protein ratio and degree of unsaturation of lipids under stressed conditions (Feng et al., 2014). Selenium is useful to reduce lipid peroxidation of membranes as it reduces malondialdehyde production under stress conditions (Jiang et al., 2017). Selenium promoted lipid unsaturation and breaks ROS chain to reduce oxidative stress (Malik et al., 2012).

Application of Se improved starch accumulation and the stay green trait under UV light stress (Mostafa and Hassan, 2015). Selenium enhanced water uptake by roots under stressed conditions (Nawaz et al., 2014). Application of Se enhanced total soluble sugars, antioxidant activities, chlorophyll contents and yield in wheat under stressed conditions (Nawaz et al., 2015). Application of Se enhanced biosynthesis of chlorophyll, carotenoids and improved yield (Dong et al., 2013). Selenium alleviated oxidative stress by enhancing super oxide dismutase, catalase, glutathione peroxidase, ascorbate and tocopherol activities under stressed conditions (Lin et al., 2012). Selenium improved phenolic contents in stressed wheat by boosting phenylalanine ammonia lyase activity (Iqbal et al., 2015).

Furthermore, existence of numerous quantitative trait loci for a single targeted trait depicted complex inheritance pattern (Mwadzingeni et al., 2016). Hence, selection of wheat genotypes merely based on response of morphological traits often leads to faulty inferences (Reynolds and Langridge, 2016). While, biochemical markers assisted selection of genotypes exhibited more efficacy of selection than mere morphological markers-based selection for polygenic traits. Selection of genotypes using morphological attributes leads to poor selection efficacy studies (Jacoby et al., 2016). Selection of genotypes on basis of biochemical attributes in association to morphological attributes is lacking in previous experimentation.

The crux of the issues is that, high temperature negatively affects innumerable physiological, growth and yield attributes of wheat. Minor variations in ambient temperature affect physiochemical attributes of wheat crop. While, availability of potassium and selenium improves biochemical attributes that ultimately confer heat tolerance at morphological level. However, heat mediated changes and potassium and selenium triggered regulations in physiochemical attributes are not disclosed copiously so far. Moreover, data regarding potassium and selenium instigated regulations in physiochemical attributes of terminal heat stressed wheat are scarce. Hence, elucidation of thermo-tolerance at biochemical level is crucial for food security since improvements in biochemical attributes confer tolerance in growth and yield components. In addition, better understanding of the relation between biochemical attributes and yield components of heat stressed wheat provides sound basis for agronomic management of heat stress. Likewise, knowledge about heat caused deteriorations and potassium and selenium trigged improvements in in grain quality is also scarce.

It can be hypothesized that different genotypes and terminal growth stages will perform distinctly under high heat stress. While, varying concentrations of exogenous potassium and selenium might prove a potent tool to alleviate adversities of heat at biochemical and morphological level. Besides, foliar potassium and selenium instigated biochemical regulations will confer tolerance in growth and yield components of heat stressed wheat crop.

MATERIALS AND METHODS CHAPTER-3

The present research wok was carried out to alleviate deleterious impacts of terminal heat stress on wheat. Three years of field-based experiments were performed to accomplish this objective. For the 1st year (2014-15), wheat varieties were characterized for heat tolerance and a medium heat tolerant wheat genotype was selected for further experimentation. In the 2nd year (2015-16), two independent field experiments were performed whereby heat stress was alleviated through exogenous spray of potassium in one and selenium in other experiment. During the 3rd year (2016-17), the same experiments were repeated as in 2015-16. Variables such as grain yield, yield components, biomass accumulation, the stay green trait, antioxidants activities, osmo-protectants water relations and quality attributes were used as potential indicators of thermo-tolerance.

3.1. Experimental site

All research activities were carried out at Agronomic Research Area, University of Agriculture Faisalabad Pakistan during the period of November 2014 to May 2017. The site is located at latitude of 31-26N, longitude 73-06E and altitude of 184.4 m.

3.2. Physio-chemical analyses of soil

Soil samples were randomly taken from various points of the field at depths of 15 and 30 cm. Soil samples were mixed separately for the depths of 15 and 30 cm to record electrical conductivity (Rhoades, 1996), pH (Thomas, 1996), organic matter (Moodie et al., 1959), total nitrogen (Jackson, 1962), available phosphorous using 0.5 M sodium bicarbonate (NaHCO3) as extraction solution (Kuo, 1996) and available potassium using 1 N ammonium acetate (NH4OAc) as extraction solution (Helmke and Sparks, 1996). Textural class of experimental soil was loam (Table 3.1).

3.3. Weather elements

Data of different weather elements were collected from Meteorological Observatory, University of Agriculture Faisalabad Pakistan during the growing season of wheat. Data on average temperature, relative humidity, rainfall, pan evaporation, sunshine duration, evapotranspiration and wind speed were recorded on daily basis and averaged each month (Table 3.2).

3.4. Plant material

Numerous genotypes were collected from different institutes to determine thermo-tolerance and sensitivity for Experiment 1.

43

Table 3.1: Physio-chemical analyses of experimental site during 2014-15, 2015-16 and 2016-17

Soil characteristics

Depth of sample (cm)

Experiment I

Experiment II

Experiment III

2014-15

2015-16

2016-17

2015-16

2016-17

Sand (%)

0-15

45

45

44

43

46

15-30

43

44

43

45

44

Silt (%)

0-15

23

25

26

24

22

15-30

24

26

28

25

24

Clay (%)

0-15

29

27

29

31

33

15-30

28

26

28

29

31

Textural class

0-15

Loam

Loam

Loam

Loam

Loam

15-30

EC (dS m-1)

0-15

2.06

2.10

1.99

2.01

1.96

15-30

1.98

1.96

1.97

2.03

1.98

pH

0-15

7.7

7.5

7.6

7.8

7.9

15-30

7.6

7.8

7.9

7.7

7.8

Organic matter (g kg-1)

0-15

9.2

5.9

5.3

5.8

5.1

15-30

9.4

5.8

5.5

5.8

5.2

Total nitrogen (g kg-1)

0-15

0.44

0.46

0.45

0.46

0.44

15-30

0.41

0.45

0.43

0.45

0.42

Available phosphorous (mg kg-1)

0-15

7.7

8.02

7.7

7.8

7.3

15-30

8.04

7.9

7.4

7.7

7.1

Available potassium (mg kg-1)

0-15

177

179

162

177

159

15-30

165

176

159

177

155

Latitude = 31 - 26N; Longitude = 73- 06E; Altitude = 184.4 m

Table 3.2: Monthly averages of weather elements during growing season of crop in 2014-15, 2015-16 and 2016-17

Weather elements

Years

November

December

January

February

March

April

May

Average temperature (C)

2014-15

18.9

12.2

11.7

16.5

19.1

27.0

31.8

Relative humidity (%)

61.7

75.0

75.3

66.0

64.0

43.9

27.5

Rainfall (mm)

10.0

0.0

12.2

20.5

67.9

32.8

17.0

Pan evaporation (mm)

1.8

1.5

1.0

2.1

13.0

5.3

7.6

Sunshine duration (hours)

7.6

4.7

5.0

5.6

4.9

9.1

10.4

Evapotranspiration (mm)

1.5

1.3

0.7

1.8

2.8

3.7

5.3

Wind speed (km h-1)

3.1

2.0

3.6

5.3

5.6

6.2

5.7


2015-16

19.6

14.5

12.5

16.3

21.2

27.2

32.8


61.5

62.6

74.4

58.1

59.7

34.2

28.8

Rainfall (mm)

8.8

0.0

13.1

7.8

66.7

5.6

25.0


2.4

1.9

3.5

2.3

2.7

6.1

9.5


6.6

7.0

1.2

8.5

6.6

8.3

10.4


2.1

1.6

0.8

1.6

1.9

4.3

6.4

Wind speed (km h-1)

2.6

2.3

27.6

3.8

4.7

5.2

5.4


2016-17

20.1

16.4

12.9

16.8

23.7

29.3

33.5


60.1

68.7

72.0

53.0

49.5

30.6

29.8

Rainfall (mm)

0.0

0.0

11.5

4.1

16.2

28.3

10.1


2.4

2.1

3.6

2.7

3.9

7.5

9.2


6.4

6.7

1.3

6.6

7.2

9.2

10.4


1.8

1.7

0.9

1.9

2.7

5.2

5.7

Wind speed (km h-1)

2.6

2.8

3.5

4.0

3.9

5.8

5.4


Table 3.3: Varying mean temperatures (C) 2014-15 for experiment 1

Heat stress

Year

March 21

March 22

March 23

March 24

March 25

March 26

March 27

March 28

March 29

March 30

March 31

No heat stress (H0)

2014-15

30.02

30.20

31.90

32.60

32.15

31.55

31.40

33.40

31.30

30.50

32.40

Heat from spike to grain filling (H1)

39.37

39.77

38.76

38.82

38.23

40.40

41.60

40.80

41.20

39.30

39.70


Table 3.4: Varying mean temperatures (C) during heat imposition for experiment 2, 2015-16 and 2016-17

Heat stress

Year

March 1

March 2

March 3

March 4

March 5

March 6

March 7

March 8

March 9

March 10

March 11

March 12

March 13

March 14

No heat stress (H0)

2015-16

26.0

27.0

29.0

27.0

26.5

26.0

27.0

25.0

26.0

25.0

25.5

25.5

26.0

26.5


33.3

34.1

34.6

33.9

33.0

33.4

34.0

32.0

32.5

32.0

31.7

31.0

31.4

32.8

Heat from flowering to grain filling (H2)

-

-

-

-

-

-

-

32.2

32.4

32.3

31.5

31.0

31.5

32.5

No heat stress (H0)

29.0

28.0

30.5

29.0

28.5

28.0

29.0

28.0

28.5

28.0

28.5

27.5

28.0

27.5


2016-17

35.1

34.0

36.8

35.2

34.3

34.0

36.2

35.4

36.0

35.9

34.2

34.0

35.3

34.6


-

-

-

-

-

-

-

35.1

36.3

36.0

34.5

34.3

35.2

34.5


Table 3.5: Varying mean temperatures (C) during heat imposition for experiment 3, 2015-16 and 2016-17

Heat stress

Year

March 1

March 2

March 3

March 4

March 5

March 6

March 7

March 8

March 9

March 10

March 11

March 12

March 13

March 14

No heat stress (H0)

2015-16

26.0

27.0

29.0

27.0

26.5

26.0

27.0

25.0

26.0

25.0

25.5

25.5

26.0

26.5


32.9

34.3

34.2

33.3

32.8

33.1

34.6

32.6

32.1

32.3

31.3

31.4

31.8

33.0


-

-

-

-

-

-

-

32.6

32.7

32.1

31.8

31.3

31.4

32.7

No heat stress (H0)

29.0

28.0

30.5

29.0

28.5

28.0

29.0

28.0

28.5

28.0

28.5

27.5

28.0

27.5


2016-17

34.8

34.2

36.1

35.9

34.8

34.4

36.7

34.6

35.7

35.5

33.0

33.7

34.8

34.1


-

-

-

-

-

-

-

35.4

36.1

35.9

34.7

34.6

35.4

34.7


Plant material of genotypes Aas-2011 and Fareed-2006 was procured from Regional Agriculture Research Institute Bahawalpur, Pakistan. While, seeds of genotypes Mairaj-2008, AARI-2011, Punjab-2011, Millat-2011 and Galaxy-2013 were obtained from Ayub Agriculture Research Institute (AARI) Faisalabad, Pakistan. Whereas, seeds of genotype Pakistan-2013 were obtained from National Agriculture Research Center Islamabad, Pakistan. Seeds of genotypes Chakwal-50 and Kohistan-97 were procured from University of Agriculture Faisalabad (UAF), Pakistan. Plant material of genotype NIBGE-NIAB-1 was procured from Nuclear Institute for Agriculture and Biology (NIAB) Faisalabad, Pakistan.

3.5. Agronomic practices

Wheat was sown with the help of single row hand drill with R R of 22.5 cm. Seed was sown at the rate of 100 kg ha-1. During the 1st, 2nd and 3rd year, sowing was done on 17th November 2014-15, 25th November 2015-16 and 29th November 2016-17, respectively. Fertilizer was applied at the rate of 120:75:60 kg NPK ha-1 in Experiment 1. While, in Experiment 2 and Experiment 3 120:75 kg NP ha-1 was applied. Half of nitrogen fertilizer (urea) and all the phosphorus (SSP) and potash fertilizers (SOP) were applied as basal dose. While, remaining half nitrogen fertilizer was applied with first irrigation at crown root initiation. Fertilizers were band placed in inter row spaces with the help of single row hand drill. Irrigations were applied at four critical growth stages viz. crown root initiation, tillering, spike initiation and flowering. Two manual hoeings were performed in all treatments to maintain weeds population below economic threshold level; first after 40 days of sowing and second after 60 days of sowing.

3.6. 1st year (2014-15) trial

Experiment I: Biochemical markers assisted screening of wheat cultivars for terminal heat stress tolerance

Treatments:

Factor A: Heat stress (Main plot)

H0 = No heat stress imposition (Plots without polythene sheet)

H1 = Heat stress imposition from complete emergence of spike to grain filling initiation (early milk stage) (Feekes scale = 10.50 to 11.0)

Factor B: Varieties (Sub plot)

V1 = Punjab-2011

V2 = AARI-2011

V3 = Galaxy- 2013

V4 = Millat-2011

V5 = Aas-2011

V6 = Fareed-2006

V7 = Chakwal-50

V8 = Mairaj-2008

V9 = Pakistan-2013

V10 = NIBGE-NIAB-1

V11 = Kohistan-97

(a) Experimental design

Experiment was conducted using a Randomized Complete Block Design (RCBD) with split plot arrangement having 4 replications. Heat stress was imposed in main plots and genotypes were randomized in sub plots. Gross plot size of each experimental unit was 3.0 m 1.35 m.

(b) Imposition of heat stress

Five plants were randomly selected and tagged in each plot to notice for 50% complete emergence of spike and grain filling initiation. Heat stress was imposed when 50% of plants reached the complete emergence of spike and removed when 50% of plants had achieved grain filling initiation growth stage. The heat stressed main plot was covered with transparent polythene sheet from complete emergence of spike to grain filling initiation (Feekes Scale= 10.50 to 11.0) (Javed et al., 2014; Kamal et al., 2017; Shahid et al., 2017). Whereas, control (no heat stress) plots were left in ambient environment. Relative humidity under polythene sheet was maintained as in ambient conditions by making large number of small sized holes in the polythene sheet. Temperature of heat stress and control/no heat stress main plots was recorded three times a day (morning, noon and evening) and averaged. Temperature was recorded with the help of digital temperature and humidity probe (Digital Multimeter-50302). Comparative temperatures under no heat stress and heat from spike to grain filling are given as tabulated form (Table 3.3). Leaves were collected randomly from each experimental unit 1 day after removing stress, stored in liquid nitrogen and processed to record various biochemical response variables.

(c) Parameters recorded

Yield components and grain yield

1. Number of fertile tillers per m2

2. Number of grains per spike

3. 1000-grain weight (g)

4. Grain yield (t ha-1)

Growth of spike

1. Grain filling rate (g per day) (Hunt, 1978)

2. Grain filling duration (days) (Hunt, 1978)

Stay green and antioxidants

1. Chlorophyll a contents (mg g-1 FW) (Arnon, 1949)

2. Chlorophyll b contents (mg g-1 FW) (Arnon, 1949)

3. Superoxide dismutase (U mg-1 protein) (Giannopolitis and Ries, 1977)

4. Peroxidase (U mg-1 protein) (Liu et al., 2009)

5. Catalase (U mg-1 protein) (Liu et al., 2009)

6. Total phenolic contents (mg GAE g-1) (Ainsworth and Gillespie, 2007)

Osmo-protectants and lipid peroxidation

1. Proline (mol g-1) (Bate et al., 1973)

2. Glycine betaine (mol g-1) (Grieve and Grattan, 1983)

3. Total soluble proteins (mg g-1) (Bradford, 1976)

4. Malondialdehyde contents (mol g-1) (Cakmak and Horst, 1991)

(d) Statistical analysis

Data of recorded attributes were analyzed statistically (p 0.05) using the Fishers analysis of variance technique (Steel et al., 1997) and Tukeys Honestly Significant Difference (Tukeys HSD) test was employed to compare the means of different genotypes at 5% probability level. While type and strength of relationship among the recorded parameters was determined calculating correlation among these parameters using STATISTIX 8.1 software (Gomez and Gomez, 1984).

A medium heat tolerant genotype (Punjab-2011) was selected on the basis of recorded parameters and used in further experimentation (Van Esbroeck et al., 1998; Van Deynze et al., 2009; Conaty et al., 2012).

3.7. 2nd year (2015-16) trials

Experiment II: Exploring role of foliar applied potassium to induce terminal heat stress tolerance in wheat

Treatments:


H0 = No heat imposition (Plots without polythene sheet)


H2 = Heat stress imposition from flowering initiation to grain filling initiation (early milk stage) (Feekes scale = 10.5.1 to 11.0)

Heat stress was imposed by covering the plots with perforated, transparent polythene sheet (Javed et al., 2014; Kamal et al., 2014; Shahid et al., 2017).

Factor B: Potassium foliar application (subplot)

K0 = Control (0 kg K ha-1)

K15 = 15 g L-1 (4.5 kg K ha-1)

K30 = 30 g L-1 (9 kg K ha-1)

K45 = 45 g L-1 (13.5 kg K ha-1)

K60 = 60 g L-1 (18 kg K ha-1)

Experiment III: Alleviation of terminal heat stress in wheat through foliar application of selenium

Treatments:


H0 = No heat imposition (Plots without polythene sheet)


H2 = Heat stress imposition from flowering initiation to grain filling initiation (early milk stage) (Feekes scale = 10.5.1 to 11.0)

Heat stress was imposed by covering the plots with perforated, transparent polythene sheet (Javed et al., 2014; Kamal et al., 2017; Shahid et al., 2017).

Factor B: Selenium foliar application (subplot)

Se0 = Control (0 g Se ha-1)

Se25 = 25 mg L-1 (7.5 g Se ha-1)

Se50 = 50 mg L-1 (15 g Se ha-1)

Se75 = 75 mg L-1 (22.5 g Se ha-1)

Se100 = 100 mg L-1 (30 g Se ha-1)

3.8. 3rd year (2016-17) trials

Experiment II and experiment III were repeated as in 2015-16.

(a) Experimental design

Both the experiments were laid out in Randomized Complete Block Design (RCBD) with split plot treatments arrangement in 3 blocks. Heat was imposed in main plots whereas exogenous potassium was applied in split plots. Each experimental unit was comprised of 3.0 m 1.35 m gross area.

(b) Imposition of heat stress and foliar application of potassium and selenium

Five plants were randomly selected in each experimental unit and were observed for 50% complete emergence of spike, flowering initiation and grain filling initiation. When 50% plant reached the complete emergence of spike, heat stress was imposed by covering the plots with perforated polythene sheet (Javed et al., 2014; Kamal et al., 2017; Shahid et al., 2017). While, in the other main plot, heat was imposed in the same way on the 50% completion initiation of flowering. Polythene sheets (heat stress) in both main plots were removed at the same time i.e. on 50% initiation of grain filling. One main plot was also left in open environment as control/no heat stress. Recorded temperatures are given in tabulated form (Table 3.4 and Table 3.5).

Different concentrations of potassium and selenium as per treatments were applied after the imposition of heat stress on flowering initiation. Potassium and selenium were foliar applied with the help of a hand sprayer at the rate of 300 liter per hectare. Potassium was foliar applied using source potassium nitrate (KNO3) (K = 36.52%, K2O = 44%) and selenium was applied using sodium selenate (Na2SeO4) (Se= 41.79%). Leaf samples were collected 1 day after removing of heat stress, stored in liquid nitrogen and processed to record various attributes.

(c) Parameters recorded


1. Number of fertile tillers per m2

2. Number of grains per spike

3. 1000-grain weight (g)

4. Grain yield (t ha-1)

Biomass accumulation

1. Biological yield (t ha-1)

2. Harvest index (%)

3. Straw yield (t ha-1)

4. Plant height (cm)

Growth of spike

1. Spike length (cm)

2.Spikelets per spike

3. Grain filling rate (g per day) (Hunt, 1978)

4. Grain filling duration (days) (Hunt, 1978)


1. Chlorophyll a contents (mg g-1 FW) (Arnon, 1949)

2. Chlorophyll b contents (mg g-1 FW) (Arnon, 1949)

3. Superoxide dismutase (U mg-1 protein) (Giannopolitis and Ries, 1977)

4. Peroxidase (U mg-1 protein) (Liu et al., 2009)

5. Catalase (U mg-1 protein) (Liu et al., 2009)

6. Total phenolic contents (mg GAE g-1) (Ainsworth and Gillespie, 2007)


1. Proline (mol g-1) (Bate et al., 1973)

2. Glycine betaine (mol g-1) (Grieve and Grattan, 1983)

3. Total soluble proteins (mg g-1) (Bradford, 1976)

4. Malondialdehyde contents (mol g-1) (Cakmak and Horst, 1991)

Water relations and quality attributes

1. Osmotic potential (-MPa) (Scholander et al., 1964)

2. Water potential (-MPa)

3. Turgor potential (MPa)

4. Shoot potassium contents (g g-1) (Chapman and Pratt, 1961; Gupta, 1999) (Only for Experiment II)

5. Grain crude protein contents (%) (Bremner and Mulvaney, 1982; Ryan et al., 2001)

(d) Statistical analysis

Data of recorded attributes were analyzed statistically (p 0.05) using the Fishers analysis of variance technique (Steel et al., 1997) and Tukeys Honestly Significant Difference (Tukeys HSD) test was employed to compare the means of different genotypes at 5% probability level. While, type and strength of relationship among the recorded parameters was determined by calculating correlation among these parameters using STATISTIX 8.1 software (Gomez and Gomez, 1984). Moreover, regression analysis was performed to determine trends of response variables and improvements in different attributes towards different concentrations of foliar spray under varying treatments of heat stress. Years means were determined for each studied response variable without pooling of data for two years study period. Microsoft Excel-2016 was used for graphical work.

(e) Methodologies to record parameters


Number of fertile tillers was counted in 30 cm row length at five different places of each experimental unit and converted into fertile tillers for 1 m2 area through unitary method. Ten spikes were manually harvested, threshed and average number of grains per spike was calculated. Five samples of 1000 seeds were randomly taken from the seed lot of each experimental unit and averaged to calculate thousand seed weight. The crop in each experimental unit was harvested, threshed and grain yield was weighed and converted into tons per hectare.

Biomass accumulation

Ten plants in each experimental unit were randomly selected and plant height was measured from the base of plant to tip of spike with the help of meter rod at maturity. The biological yield of each experimental plot was weighed using a weighing balance and converted into tons per hectare. Harvest index was calculated by dividing the grain yield of each plot by respective biological yield (Gardner et al., 1985).

100

Straw yield of each treatment was computed by subtracting grain yield from the respective biological yield.

Growth of spike

Ten spikes were randomly selected in each plot, their length was measured and averaged. Similarly, spikelets per spike were counted and averaged for ten spikes. To determine grain filling rate, five spikes were randomly harvested from each plot on initiation of grain filling at interval of 5 days and their dry weight was recorded. Grain filling rate was calculated using formula described by Hunt (1978).

Whereas, W1 and W2 represent dry weight of spike at the time of first harvest (t1) and second harvest (t2). Grain filling duration was determined by tagging five plants in each plot and days taken from grain filling initiation to physiological maturity were counted (Hunt, 1978).


Green leaf samples were collected randomly from each experimental plot, 0.5 g sub sample was taken and soaked overnight in 80% acetone. Leaves extracts of 1.5 L were taken in ELISA plate and absorbance was recorded at 663 and 645 nm. Final readings of chlorophyll a and b were computed using formulae given by Arnon (1949)

Where A indicates absorbance, V volume of extract (mL) and W weight of fresh leaves tissue.

Superoxide dismutase (SOD) contents were quantified as enzyme units that inhibited photochemical reduction of nitro blue tetrazolium (NBT). Leaf tissues were extracted using potassium phosphate buffer (pH 4) prepared by dissolving KH2PO4 (7.45 g) + K2HPO4 (1.74 g) + KCl (7.45 g) + EDTA (0.58 g) in 1000 mL DI water. The reaction mixture was comprised of potassium phosphate buffer (pH 5) + 200 L methionine + 200 L triton X + 100 L NBT + 800 L distilled water. Enzyme extracts of 100 L volume was mixed with reaction mixture in Eppendorf tubes, placed under ultraviolet light for 15 minutes and added 100 L riboflavin and took 100 L in ELISA plate and recorded absorbance at 560 nm. Absorbance for blanks (standards) was also recorded using reaction mixture and riboflavin without adding enzyme extract (Giannopolitis and Ries, 1977). Regression equation was formed plotting blanks on x-axis and respective absorbance on y-axis and thus, finalized readings of SOD were computed from a calibration curve

Where, Y specifies absorbance of blanks solutions, X final concentration of SOD of unknown sample, a slope between blank and unknown (SOD) sample and b intercept.

Peroxidase (POD) contents were estimated as enzyme units that oxidize guaiacol. The same enzyme extracts as used for SOD contents was also used to quantify POD contents. Reaction mixture for determination of POD was comprised of 800 L potassium phosphate buffer (pH 5) + 100 L H2O2 (40 mM) + 100 L guaiacol (20 mM). Added 100 L enzyme extract + 100 L reaction mixture in Eppendorf tubes, took 150 L in ELISA plate and recorded absorbance at 470 nm (Liu et al., 2009).

Catalase (CAT) contents were measured as enzyme units that detoxified H2O2 to H2O and O2. Same enzyme extracts as prepared to quantify SOD were also used for determination of CAT contents. Enzyme extract of volume 100 L + 100 L H2O2 (5.9 mM) were mixed in cuvettes, took 150 L of mixture in ELISA plate and recorded absorbance at 240 nm (Liu et al., 2009).

Total phenolic contents were measured by extracting 0.5 g leaf tissues in 10 mL 80% acetone using Folin-Ciocalteu reagent method. Supernatant of 20 L volume was shifted in cuvettes. Then added 1.50 mL DI water + 100 L Folin-Ciocalteu (Rover and Brown, 2013) reagent, vortexed the cuvettes. Then added 300 L Na2CO3 (700 mM) solution in cuvettes and incubated for 2 hours at room temperature (25C), 150 L from cuvettes was shifted to ELISA plate and recorded the absorbance at 760 nm. Gallic acid (10-100 ppm) was used as standard to develop calibration curve for determination of TPC and results were reported as gallic acid equivalent (GAE) (Ainsworth and Gillespie, 2007).


Proline was determined by extracting 0.5 g leaf tissues with 3% 5 mL of sulfosalicylic acid. Obtained leaf extract was centrifuged for 15 minutes. Ninhydrin solution of concentration 3% was prepared in equal volumes of glacial acetic acid and 6 M orthophosphoric acid. Added 1 mL centrifuged leaf extract + 1 mL glacial acetic acid + 1mL 3% ninhydrin solution prepared in glacial acetic acid and orthophosphoric acid in cuvettes. The mixture was mixed and incubated at 100C for 1 hour. Afterwards, the mixture was cooled in an ice bath, added 1 mL toluene in mixture and vortexed it for 5 minutes. The upper aqueous layer was discarded after vortex and organic layer was retained. Volume of 150 L was placed in an ELISA plate and recorded absorbance at 520 nm using toluene as blank for standard curve (Bate et al., 1973).

Glycine betaine was measured homogenizing leaf tissues weighing 0.5 g with 5 mL distilled water and centrifuged the extracts for 5 minutes. Potassium tri-iodide solution was prepared dissolving 7.5 g iodine + 10 g potassium iodide in 10 mL, 1 M HCl solvent. Then 1 mL leaf tissue extract + 1 mL HCl (2 M) + 0.1 mL potassium tri-iodide solution were thoroughly mixed and incubated at 4C for 1 hour. After it, 5 mL chilled DI water + 10 mL 1,2- di-di-chloroethane was added and vortexed for 5 minutes. Upper aqueous layer was discarded, and absorbance was recorded at 365 nm using organic layer (Grieve and Grattan, 1983).

Total soluble proteins were analyzed by using same enzyme extract of leaves as was used for SOD determination. Enzyme extract of volume 40 L + 160 L Bradford Reagent was added in ELISA plate and recorded absorbance at 595 nm (Bradford, 1976).

To quantify malondialdehyde (MDA), leaf samples of weight 0.5 g were homogenized with 3 mL 0.1% (w/v) trichloroacetic acid (TCA). Then the samples were centrifuged for 15 minutes and the supernatant of 0.5 mL volume was transferred in a test tube. Then 3 mL 20% TCA solution containing 0.5% thiobarbituric acid was added to the supernatant. Afterwards, the mixture was incubated at a temperature of 70C for30 minutes and cooled with an ice water bath. Mixture containing leaf extracts and blanks were added in ELIZA plate taking volume of 150 L each and recorded absorbance at 532 nm and 600 nm (Cakmak and Horst, 1991).

Water relations and quality attributes

Leaves were collected early in the morning between 6-8 am randomly from each plot. Leaves were placed in Scholandar pressure gauge (ARIMAD-2, ELE, International) and pressure was applied until drop of sap appeared on midrib. Pressure applied from pressure gauge was considered equal to water potential (W) of leaf organs. Leaves used in water potential determination were frozen at -4C and ground to obtain cell sap which was taken to osmometer (Wescor 5520) and recorded osmotic potential (S). Turgor potential was determined by subtracting osmotic potential from water potential (Scholander et al., 1964).

Wheat shoots were collected at physiological maturity, sun dried, oven dried and ground to powder form. Ground powder weighing 0.5 was digested with nitric acid per-chloric acid (HNO3: HCLO4 in 2:1 ratio). The mixture was heated at 60C to complete reaction until synthesis of fumes from reaction mixture was stopped. Then the mixture was heated in a digestion chamber at temperature of 120C until clear aliquot was obtained. DI water was added to make volume of a 100 mL. Stock solution of concentration 1000 ppm was diluted to make concentrations of 0, 25, 50, 75 and 100 ppm. A regression (calibration) curve was developed plotting different concentrations on x-axis and respective absorbances on y-axis. Leaf samples were also loaded in a Flame photometer and recorded absorbances for different samples. Final readings of shoot potassium contents (g g-1) were computed from the regression equation (Chapman and Pratt, 1961; Gupta, 1999).

Grain crude proteins were quantified by using the method of Gunning and Hibbard. Sulphuric acid was used for digestion of wheat flour and it was followed by distillation of NH3 in boric acid with the help of Kjeldhal apparatus. Grains were milled to form flour and 1 g flour was taken in digestion tubes. Together with it, 25 mL concentrated H2SO4 + 5 g digestion mixture (K2SO4 + FeSO4 + CuSO4 in 85: 10: 5 ratio) were added. Digestion tubes were heated on digestion block at 400C until clear liquid was obtained. Then DI water added to make total volume 250 mL and 10 mL from digested and clear aliquot was taken to distillation unit. In the receiver flask of distillation unit, 10 mL 4% boric acid was taken and added a few drops of methyl red indicator. Upon distillation, the colour of boric acid in receiver flask was changed from purple to golden yellow. After it, boric acid was titrated against 0.1 N H2SO4 to get purple endpoint from golden yellow colour of boric acid and computed grain crude proteins (Jackson, 1962).

Nitrogen (%) =

Whereas DF represents dilution factor if there is any.

Whereas 5.83 is constant for wheat (Bremner and Mulvaney, 1982; Buresh et al., 1982; FAO, 2003).

RESULTS AND DISCUSSION CHAPTER-4

Experiment I: Biochemical markers assisted screening of wheat cultivars for terminal heat stress tolerance

Heat stress had an overall deleterious effect at reproductive stages of wheat. However, cultivars specific response was evident on different growth, yield, and biochemical attributes. However, heat stress, genotypes and their interaction unveiled distinctive response under controlled and stressed conditions and resulted in significant heat genotypes effect for various parameters.

4.1.1. Yield components and grain yield

(a) Results

Number of fertile tillers did not differ significantly in control and heat stress main plots. However, cultivars significantly varied from each other. All cultivars showed undistinguishable trend in both main plots to record non-significant interaction. The highest number of fertile tillers was observed for Punjab-2011 (377.13 m-2) and it was statistically alike to genotypes AARI-2011, Galaxy-2013, AAS-2011 and Pakistan-2013. Genotype Kohistan-97 produced minimum number of fertile tillers (278.88 m-2) (Table 4.1.1).

Heat stress and genotypes manifested significant distinction for number of grains per spike. The heat variety interaction was significant as varieties revealed unlike response in ambient and heat im

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