Mechanisms of Drought Resistance

Abstract

There are different sources of genetic variation which introduce variability in the gene pool. Wide range of variability in germplasm provides multiple options for improvement against different stresses. For resistance against drought stress, there are different mechanisms that confer drought resistance in different biological ways. Drought escape induces earliness and prevents exposure of plants to terminal drought stress. Drought avoidance confers resistance by either increasing water uptake or reducing water losses. Drought tolerance maintains the physiological processes under drought stress and produces higher economic yield. Involvement of different drought resistance mechanisms in maize are described in this chapter.

Full text

123
SPRINGER BRIEFS IN AGRICULTURE
MuhammadAslam
MuhammadAmirMaqbool
RahimeCengiz
Drought Stress in
Maize (Zea mays L.)
Effects, Resistance
Mechanisms, Global
Achievements and
Biological Strategies
for Improvement

SpringerBriefs in Agriculture
aslampbg@uaf.edu.pk

More information about this series at http://www.springer.com/series/10183
aslampbg@uaf.edu.pk

Muhammad Aslam •Muhammad Amir Maqbool
Rahime Cengiz
Drought Stress in Maize
(Zea mays L.)
Effects, Resistance Mechanisms, Global
Achievements and Biological Strategies
for Improvement
123
aslampbg@uaf.edu.pk

Muhammad Aslam
Plant Breeding and Genetics
University of Agriculture
Faisalabad
Pakistan
Muhammad Amir Maqbool
Plant Breeding and Genetics
University of Agriculture
Faisalabad
Pakistan
Rahime Cengiz
Maize Research Station
Sakarya
Turkey
ISSN 2211-808X ISSN 2211-8098 (electronic)
SpringerBriefs in Agriculture
ISBN 978-3-319-25440-1 ISBN 978-3-319-25442-5 (eBook)
DOI 10.1007/978-3-319-25442-5
Library of Congress Control Number: 2015952763
Springer Cham Heidelberg New York Dordrecht London
©The Author(s) 2015
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Contents
1 Introduction ......................................... 1
2 Effects of Drought on Maize .............................. 5
2.1 Effects on Crop Stand Establishment . . . . . . . . . . . . . . . . . . . . . 6
2.2 Effects on Growth and Development . . . . . . . . . . . . . . . . . . . . . 8
2.3 Effects on Reproductive Growth Stages . . . . . . . . . . . . . . . . . . . 11
2.3.1 Pollen Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
2.3.2 Silk Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.3.3 Pollination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.4 Embryo Development . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3.5 Endosperm Development . . . . . . . . . . . . . . . . . . . . . . . . 15
2.3.6 Grain or Kernel Development . . . . . . . . . . . . . . . . . . . . . 16
3 Mechanisms of Drought Resistance ......................... 19
3.1 Drought Escape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
3.2 Drought Avoidance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3.3 Drought Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3.3.1 Osmotic Adjustment . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.3.2 Antioxidative Defense Mechanism . . . . . . . . . . . . . . . . . . 26
3.3.3 Plant Growth Regulators . . . . . . . . . . . . . . . . . . . . . . . . 28
3.3.4 Molecular Mechanisms of Drought Tolerance . . . . . . . . . . 30
4 Global Achievements in Drought Tolerance of Maize............ 37
4.1 Contribution of CIMMYT, IITA, and Other
Collaborative Partners . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.2 Contribution of Multinational Seed Companies . . . . . . . . . . . . . . 42
v
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5 Biological Practices for Improvement of Maize Performance ...... 45
5.1 Screening for Drought-Tolerant Maize Germplasm . . . . . . . . . . . . 45
5.2 Conventional Breeding Strategies. . . . . . . . . . . . . . . . . . . . . . . . 47
5.3 Marker-Assisted and Genomic-Assisted Breeding . . . . . . . . . . . . 50
5.4 Transgenic Maize Development . . . . . . . . . . . . . . . . . . . . . . . . . 54
6 Conclusions and Summary ............................... 57
References ............................................. 59
vi Contents
aslampbg@uaf.edu.pk

Abstract
Drought is one of the most detrimental abiotic stresses across the world which is
seriously hampering the productivity of agricultural crops. Maize is among the
leading cereal crops in world, but it is sensitive to drought. Maize is affected by
drought at different growth stages in different regions. Germination potential,
seedling growth, seedling stand establishment, overall growth and development,
pollen development, silk development, anthesis–silking interval, pollination,
embryo development, endosperm development, and kernel development are the
events in the life of maize crop which are seriously hampered by drought stress.
Plants have developed numerous strategies which enabled them to cope with
drought stress. Maize germplasms also have numerous features which enable some
accessions to cope with drought stress in better ways. One of the adaptive strategies
is the earliness which helps the plants to escape the drought stress. Some genotypes
have the ability to avoid the drought stress either by reducing water losses or by
increasing the water uptake. Drought tolerance is also an adaptive strategy which
enables the crop plants to maintain the normal physiological processes and harbors
higher economical yield under prevailing drought stress. Osmotic adjustment by
accumulation of osmolytes, plant self-defense by accumulation of antioxidants,
plant growth regulators, stress proteins, and water channel proteins, transcription
factors and signal transduction pathways are involved in conferring the drought
tolerance on maize. Maize genotypes have the differential capability to escape,
avoid, or tolerate the drought stress. Great efforts were made by CIMMYT, IITA,
and other multinational companies in the development of drought-tolerant maize
open-pollinated varieties (OPVs) and hybrids. Drought-tolerant hybrids and OPVs
of maize are being cultivated in numerous African countries. There is a need to
further improve the level of adaptability against drought stress for combating the
global issue of food security. Screening of available maize germplasms for drought
tolerance, conventional breeding strategies, marker-assisted and genomic-assisted
vii
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breeding, and development of transgenic maize are the prominent biological
strategies for the improvement of encounterability against drought stress. Complete
knowledge about the effects of drought stress, achievements, adaptive strategies,
and possible breeding tools are the prerequisites for any breeding plan. Hence, these
aspects were compiled in this brief.
Keywords Germination Seedlings Morphological traits Physiological traits 
Drought escape Drought avoidance Drought tolerance Reproductive growth
stage Molecular factors
viii Abstract
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Chapter 1
Introduction
Biotic and abiotic factors of environment had modified the primal living organisms
in such a way that modern plants were evolved. Any imbalance in these biotic and
abiotic factors act as stress and impose serious hampering effects on growth and
development of crop plants (Aslam et al. 2013a, b, c, 2014a, b; Naveed et al. 2014).
Survival of the fittest enabled the superiorly evolving plants to prevail in the
environment whereas, rest of the plants were eliminated during evolutionary events
due to their fitness issues. Still there are lots of factors which continually reshaping
the plant evolution; water availability is one of them. Water is among the basic
needs of living organisms on this globe. Without any exaggeration, water avail-
ability modulated the water responsive signaling which acted as critical factor for
shaping the flora on globe throughout the course of evolution. Different crops have
different delta of water (Box-1) required for their usual growth and development.
Maize required 368 kg, sorghum required 332 kg, barley required 434 kg and wheat
required 514 kg of water for 1 kg dry matter accumulation (Rana and Rao 2000).
Total 350–450 mm water is used by maize during its life cycle for completion of
growth and development. Every millimeter of water is responsible for production of
10–16 kg grains and single maize plant consumes 250 litres of water at maturity
(Du Plessis 2003).
Drought, salinity and low temperature stresses are known as main agricultural
problems which are seriously inhibiting the plants to show off their genetic potential
(Zhu 2002). Drought is sole factor which is affecting agricultural crops more than
any other stress and it is becoming even more severe in different regions of world
(Passioura 1996; Passioura 2007). Statistical data showed that globally area sub-
jected to drought stress has doubled from 1970 to 2000 (Isendahl and Schmidt
2006).
Maize is major cereal crop of the world which is currently grown in large
number of countries. It is a multidisciplinary crop and used in human food, animal
feed, fodder and bioenergy production. Area, yield and production of maize across
the world in comparison with rice and wheat have been shown in Figs. 1.1 and 1.2.
Comparison showed that wheat is cultivated on more area relative to other cereals
but yield and production of maize is more than wheat and rice (Figs. 1.1 and 1.2;
FAOSTAT 2013).
©The Author(s) 2015
M. Aslam et al., Drought Stress in Maize (Zea mays L.),
SpringerBriefs in Agriculture, DOI 10.1007/978-3-319-25442-5_1
1
aslampbg@uaf.edu.pk

Drought stress is seriously affecting the maize crop resultantly hindering the
productivity like other crops (Tai et al. 2011). Being drought sensitive crop, maize
is affected at each and every stage of growth and development by lesser moisture
availability. Prevalence of drought at seedling stage causes poor crop stand and
under extreme conditions can result in complete failure of seedling establishment
(Zeid and Nermin 2001). Shutting down of plant metabolism followed by plant
death due to stomatal closure and inhibited gaseous exchange occurs in response to
prolonged moderate drought stress (Jaleel et al. 2007). In case of maize repro-
ductive growth stage is comparatively more sensitive to drought stress and under
severe drought prevalence barren ear production might be the result (Yang et al.
2004). Global importance of maize and side effects of drought on maize triggered
the breeders to develop drought tolerance maize germplasm. Drought responsive
traits and adaptive mechanisms must be known for the development of drought
tolerant maize stock. Genetic diversity assessed on the basis of adaptive mechanism
like drought escape, drought avoidance and drought tolerance is present in maize
genotypes. So, in this book we have compiled the information regarding effects of
drought on maize plant from germination to harvest maturity. Different strategic
options for the improvement of maize performance under drought stress are also
included in this write up.
0
200
400
600
800
1000
1200
0
50
100
150
200
250
2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
Production (Mtonnes)
Area (mha)
Years
Wheat-Area Rice-Area Maize-Area Wheat-Production Rice-Production Maize-Production
Fig. 1.1 Area (Mha) and production (MTonnes) of wheat, rice and maize across the world for last
ten years (FAOSTAT 2013)
0
10000
20000
30000
40000
50000
60000
2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
Yield (Hg/Ha)
Years
Wheat-Yield Rice-Yield Maize-Yield
Fig. 1.2 Yield (Hg/Ha) of wheat, rice and maize across the world for last ten years (FAOSTAT
2013)
2 1 Introduction
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Box-1: Brief description of technical terms used in text
Delta of water is amount of water required for particular crop in complete
growing season.
Germination index is defined as cumulative daily total of germination over
specific days (Timson 1965). Speed of germination, totality and their inter-
action is also described as germination index (Brown and Mayer 1988).
Germination rate is defined as number of seeds of particular variety likely
to germinate over a specific period of time.
Germination velocity index is defined as daily germination counting for
estimation of seedling vigor (Throneberry and Smith 1955).
Seed vigor is cumulative properties of seed which determine the quick and
uniform emergence potential of seed and followed by potential seedling
development under diverse field conditions (AOSA 2002).
Seed priming is defined as maintenance of seed hydration level so, that
necessary metabolic activities needed for initiation of germination can start
but radicle emergence is avoided. Germination is improved by seed priming
treatment. Seed treatment with water before sowing to improve the germi-
nation is called hydro-priming.
Osmopriming: seed treatment with osmotic solutions before sowing to
improve the germination and stress tolerance.
1 Introduction 3
aslampbg@uaf.edu.pk

Chapter 2
Effects of Drought on Maize
Water is vitally needed for every organism in specified amount and any deficiency
in that particular amount imposes the stressful conditions. Water requirement is
variable across the tissues and across the growth stages of same species of crop
plant and maize crop has no exception so, far. Assessment of optimum plant water
requirements is prerequisite to determine the water deficiency in plants. Water
requirement of maize crop is low at early growth stages then reaches on peak at
reproductive growth stages and during terminal growth stages requirement of water
again lowers down. During reproductive growth stage, 8–9 mm water is needed per
day to single plant. Four weeks are most crucial regarding water requirement which
includes two weeks before and two weeks after pollination. Pollination is most
critical growth stage for water requirement and all leaves are kept unfolded and
grain yield is also decided at this stage. Grain filling and soft dough formation are
most sensitive to water deficiency, whereas, pre-tasseling and physiological
maturity are relatively insensitive to water deficiency. Drought stress during veg-
etative growth stages especially during V1–V5, reduces growth rate, prolong veg-
etative growth stage and conversely duration of reproductive growth stage is
reduced (Pannar 2012). Each millimeter of water produces 15.00 kg of kernels and
total 450–600 mm is needed across the whole season (Du Plessis 2003). Total 250 l
water is consumed by maize plant till maturity (Du Plessis 2003). Relative water
contents, stomatal resistance, water potential, leaf temperature and transpiration rate
maintain the plant water relation and any imbalance in these or any one of these
traits disturb the plant water relation (Anjum et al. 2011b). Relative water contents
determine the status of metabolic activities of the cell or tissue. During early leaf
development, relative water contents of the leaves were higher and tend to decline
towards maturity. Strong correlation is reported between relative water contents,
water uptake and transpiration rate. Under drought stress, relative water contents
and water potential is reduced, resultantly, leaf temperature is increased due to
reduced transpirational cooling (Siddique et al. 2001). It can be easily perceived
that plant water status is dependent on stomatal activity (Anjum et al. 2011b).
Transpiration ratio is described as number of water molecules lost in order to fix
one molecule of carbon. Soybean, wheat and maize have 704, 613 and 388 tran-
spiration ratio respectively which shows that maize is relatively efficient water user
crop (Jensen 1973). Despite of being efficient water user maize is badly affected by
©The Author(s) 2015
M. Aslam et al., Drought Stress in Maize (Zea mays L.),
SpringerBriefs in Agriculture, DOI 10.1007/978-3-319-25442-5_2
5
aslampbg@uaf.edu.pk

drought stress due to hypersensitivity against water deficiency. In maize, devel-
opmental stages starting from germination to harvest maturity including seedling
establishment, vegetative growth and development and reproductive growth stages
are very much prone to drought stress. Effects of drought on maize at different
growth stages and organizational levels have been presented in Fig. 2.1 and
described in subsequent sessions.
2.1 Effects on Crop Stand Establishment
Crop stand establishment comprised of germination, emergence and seedling
establishment. Concepts of germination and emergence prevailed under laboratory
conditions and field conditions respectively. Crop establishment accomplished up to
development of 7th or 8th leaf. These early growth stages are critical growth stages
regarding drought stress. Always there are prominent differences among different
levels of water treatments in maize regarding their effects at early growth stages
(Fig. 2.2). Proper seed germination is dependent on availability of appropriate
moisture contents for metabolic activation to breakdown the dormancy or to convert
stored food into consumable form. Crop density or number of emerged seeds, mean
time for emergence and synchronization of emergence are characteristic features
which determined the efficacy of seedling establishment (Finch-Savage 1995). Crop
survival, growth and development are determined by efficacy of seedling estab-
lishment (Hadas 2004). Drought stress reduces the germination potential of maize
Fig. 2.1 Effects of drought stress on vegetative and reproductive growth stages of maize
6 2 Effects of Drought on Maize
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seeds by reducing their viability. Poor maize seed germination is directly associated
with poor post germination performance (Radićet al. 2007). Severity of drought
stress is directly linked with poor imbibition, germination and seedling establish-
ment in maize (Achakzai 2009). Germination index (Box-1) is reduced by water
deficiency (Almansouri et al. 2001). Germination potential, germination rate (Box-1)
and seedling growth are the studied traits under drought stress because these traits
are direct representative of crop establishment and are badly affected by drought
stress (Delachiave and Pinho 2003). Germination velocity index (GVI) is corrobo-
rated with seed strength and always GVI (Box-1) was greater for maize hybrids than
landraces due to hybrid vigor (Mabhaudhi 2009).
Maize grain size is greater than other cereals like wheat, rice and barley there-
fore, water requirement is greater for maintenance of osmotic potential and con-
version of stored food into consumable form for proper germination (Gharoobi et al.
2012). Seed vigor (Box-1) is considered as important parameter in maize breeding
which is badly reduced by drought stress (Khodarahmpour 2011). Water absorp-
tion, imbibition and metabolic enzymatic activation are hindered under limited
water availability which reduces the maize grain germination. After germination,
water deficiency significantly reduced the plumule and radicle growth which
resulted in unusual seedling growth (Gharoobi et al. 2012). Hydropriming and
osmopriming (Box-1) of maize seed result in improved seed germination by reg-
ulation of enzymatic activity to break the dormancy which clearly highlights the
importance of water availability for exploitation of full germination potential
(Janmohammadi et al. 2008). Root and shoot elongations are parameter of seedling
growth and these are subjected to reduction by drought stress. At seedling stage in
maize, reduction in shoot elongation is more than root elongation under drought
stress (Khodarahmpour 2011). Seedling emergence rate of landraces is lower than
(a) (b) (c)
Fig. 2.2 Maize seedlings subjected to different treatments of water deficiency. a75 % of field
capacity, b50 % of field capacity, c25 % of field capacity. Visual observations show that same set
of maize genotypes is showing different pattern of growth and development due to differences in
water availability. Leaf area, seedling height, stem girth and leaf rolling are clearly showing the
significant differences among three different treatments. Leaf rolling seems highest in 25 % of field
capacity whereas, leaf area, seedling height, stem girth are lower in this treatment relative to others.
Under 75 % of field capacity, plant height, leaf area, stem girth seems to be greaterly higher than
other two treatments
2.1 Effects on Crop Stand Establishment 7
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hybrids whereas reduction in shoot elongation was less in landraces than hybrids
under drought stress (Mabhaudhi 2009). Rate and degree of seedling establishment
of maize are critical factors for determination of time of physiological maturity and
grain yield (Rauf et al. 2007).
So, it is evident from above discussion that seed vigor, imbibition, germination
potential, germination rate, plomule and radicle development and root and shoot
growth of maize are adversely affected by drought at early growth stages.
2.2 Effects on Growth and Development
Proper growth and development of crop plants is important for establishment of
normal plant structure that carry out all physiological and metabolic processes and
give potential yield. Drought stress seriously hindered the growth and development
of maize. Growth and development comprised of numerous component parameters
which are estimated by different traits like, plant height, leaf area, structural and
functional characters of root, plant biomass, plant fresh weight, plant dry weight
and stem diameter. Plant height, stem diameter, plant biomass and leaf area are
reduced under drought stress (Khan et al. 2001; Zhao et al. 2006).
Growth is described as increase in size of plant which is directly associated with
increase in number of cells and cell size. Meristematic tissues are involved in active
elongation of plant by active cell division. Cell division and cell size are reduced by
reduction in water potential of cells which causes the reduction in plant growth
(Nonami 1998).
Leaves in maize are ranged from 8 to 20 and these are present alternatively on
nodes. Leaf is comprised of structural and functional components. Leaf growth
consists of leaf size and number of leaves which are structural components.
Photosynthesis, transpiration and light interception are the functional traits of leaf.
Leaf size and number of leaves are reduced in maize by drought stress. Turgor
pressure, light interception and flux assimilation are determinant of leaf elongation
(Rucker et al. 1995). Wedge shaped motor cells are present on the upper leaf
surface and these keep the leaves unfold whereas, under drought stress turgor of
leaves is reduced and leaves are curled or folded (Du Plessis 2003). Leaf folding
reduces the leaf area and resultantly light interception is reduced which decreases
the photosynthetic activity. Leaf area and photosynthesis are directly proportional
to each other (Stoskopf 1981). Cell division and cell elongation are reduced under
drought stress which reduces the leaf area. Reduction in leaf area under drought
stress conditions is taken as adaptive strategy by maize plants. Leaf area index is
considered as an important parameter for maize breeding against drought stress
(Hajibabaee et al. 2012). Plant water requirement is reduced by reducing the leaf
area and probability of plant survival is increased under limited water availability
(Belaygue et al. 1996) but chlorophyll contents, chloroplast contents and photo-
synthetic activity are reduced which reduced the grain yield (Flagella et al. 2002;
Goksoy et al. 2004).
8 2 Effects of Drought on Maize
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Kinases protein family and cyclin-dependent kinases (CDKs) are involved in the
active progression of cell cycle. CDK activity is reduced under water deficit con-
ditions which increased the duration of cell division and decrease the number of cell
divisions per unit time that ultimately reduces the growth of leaves and plant
(Granier et al. 2000). Cell elongation is found to be reduced across all points on
leaf. Common regulatory pathway is involved in cell division and cell elongation
(Tardieu et al. 2000). Drought stress increases the leaf to stem ratio which is
indication of high level of growth retardation in stems than leaves (Hajibabaee et al.
2012). Reduced water potential in roots interrupts the optimal water supply to the
elongating cells and resultantly cell elongation is reduced. Water potential less than
−10.0 Bars causes the reduction in leaf growth (Tanguilig et al. 1987).
Light interception is reduced after reduction of leaf area. Less interception of
solar radiations causes the reduction in biomass production (Delfine et al. 2001).
Besides light interception, stomatal activity is also responsible for lower biomass
production (Delfine et al. 2001; Medrano et al. 2002). Rise in leaf temperature under
drought stress, inhibits the enzymatic activity and reduces photosynthesis (Chaves
et al. 2002). Photosynthetic machinery is inactivated by increase in leaf temperature
above threshold temperature which is 30 °C (Crafts-Brandner and Salvucci 2002).
Stomatal closure, reduced transpiration and its homeostatic effects are the cause of
rise in leaf temperature under limited water availability (Jones 1992).
Photosynthetic activity in maize plant is reduced by stomatal and non-stomatal
limiting factors. Reduced leaf turgor and root originated signals along with lower
plant water status trigger the stomatal closure. Reduction of water potential in the
roots transduces the signals for stomatal closure. CO
2
diffusion in the leaves is
reduced by stomatal closure and supply of CO
2
to the RUBISCO is hampered
(Flexas et al. 2007). Reduced CO
2
diffusion is considered as main reason for
decline of photosynthesis. Abscisic acid (ABA) accumulation is increased in the
leaves in response to drought induced signals which triggers the stomatal closure
(Wilkinson and Davies 2010). Cellular environment becomes alkaline under
drought stress. Rise in cellular pH increases ABA accumulation in the leaves (ABA
trapping) which induced the stomatal closure (Jia and Davies 2007).
Stomatal closure has protective role in saving the water loss and increasing water
use efficiency under mild drought stress but under severe drought stress stomatal
closure becomes inevitable evil (Chaves et al. 2009). Stomatal conductance and
transpiration rate modulate the CO
2
diffusion in leaves which are directly linked
with stomatal opening. CO
2
fixation rate, intercellular CO
2
concentration and net
photosynthetic rate are the parameters used for assessment of stomatal conductance
and photosynthetic activity under drought stress (Sage and Zhu 2011). Passive and
active stomata closures occur under normal conditions and stress prevalence
respectively (Fig. 2.3). Different genes are regulated to maintain the production and
consumption equilibrium by alteration of redox state in leaves under drought stress.
Reactive oxygen species (ROS), electron acceptors and electron carriers have
potential role in regulation of stomatal conductance (Chaves et al. 2009).
Leaf structural characters and biochemical parameters are components of
non-stomatal inhibition of photosynthesis. According to Von Caemmerer (2000)
2.2 Effects on Growth and Development 9
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and Ghannoum (2009) carboxylation is changed by RUBISCO (Ribulose
1,5-bisphosphate carboxylase/oxygenase), PEPC (phosphoenolpyruvate carboxy-
lase) and regeneration of PEP (phosphoenolpyruvate). Activity of the enzymes
involved in the photosynthesis are reduced in case of non-stomatal inhibition of
photosynthesis. Chlorophyll contents are reduced either by activation of cellular
protein degradation or by limited nitrate synthesis (Becker and Fock 1986;
Ghannoum 2009).
Maize is C4 plant and it is reported in C4 plants that intercellular spaces and
chloroplast positions are misplaced by drought stress resultantly CO
2
diffusion and
light penetration are disturbed followed by decreased photosynthetic activity
(Flexas et al. 2004). Photorespiration and Mahler’s reaction act as alternative
electron sinks under drought stress (Ghannoum 2009). Mahler’s reaction is
involved in generation of reactive oxygen species and develops oxidative stress
under drought stress. Oxygen molecule is converted into superoxide as a result of
direct reduction reaction in Photosystem-I (Haupt-Herting and Fock 2002).
Photosynthetic metabolism is reduced by reduction reaction of carbon substrate.
Carboxylation activity of RUBISCO, regeneration of RuBP and ATP are reduced
by inhibited CO
2
concentration in the leaves under drought stress (Tezara et al.
1999). CO
2
diffusion through mesophyll is reduced due to change in carbon
metabolism and leaf photochemistry under drought stress. Leaf biochemistry,
membrane permeability (aquaporin activity), leaf shrinkage, alterations in inter-
cellular spaces, intercellular structure, internal diffusion and internal conductance
are altered under drought stress which results in reduction of CO
2
diffusion through
mesophyll (Lawlor and Cornic 2002; Chaves et al. 2009).
Fig. 2.3 Passive and active
stomatal closure. Passive
stomatal closure occurs under
normal conditions and active
stomatal closure occurs under
drought stress (Arve et al.
2011). ©2011 Arve LE,
Torre S, Olsen JE,
Tanino KK. Originally
published in [short citation]
under CC BY-NC-SA 3.0
license. Available from http://
dx.doi.org/10.5772/24661
10 2 Effects of Drought on Maize
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Roots have the critical importance for plant because these are the primary
detectors or sensors of drought stress. Root length, root volume, root density and
number of roots are the characteristic structural traits which are disturbed under
drought stress and resultantly whole arial plant parts are disturbed. Spatial water
uptake and temporal water uptake are functional traits of roots. Root system of
maize comprised of axillary and lateral roots. Axillary roots are further comprised
of primary, seminal, nodal or crown roots (Cahn et al. 1989). Primary and seminal
roots are collectively known as embryonic roots. Seminal roots are permanent and
have functional role in growth and development of plant (Navara et al. 1994). Roots
of maize plant becomes elongated under mild drought stress to explore the more
soil foils for more water uptake whereas, under severe drought stress root length is
reduced. Root density, volume and number of roots are reduced under mild and
severe drought stress (Nejad et al. 2010).
Requirement of photosynthates and energy is reduced in leaves due to reduced
leaf area by leaf rolling or curling under mild drought stress. Photosynthetic
assimilates from leaves are directed toward roots for their elongation to increase the
water uptake (Taiz and Zeiger 2006). Roots act as primary sensor of water defi-
ciency in soil and transduce signals to the aerial parts to modulate the growth and
development. Signal from roots to the aerial parts are transduced through chemical
and hydraulic vectors (Davies et al. 1994). Decreased water and nutrient uptake
increase the pH of xylem (reduction of negative or positive ions) which transduces
ABA-mediated signals to the leaves for preventing water loss by stomatal closure
(Bahrun et al. 2002). Reduction in root growth under drought stress is also asso-
ciated with reduced cell division and cell elongation. Microtubules are critical for
cell division and cell elongation because these microtubules are involved in cellular
morphogenesis, embryo development, organogenesis, stomatal conductance and
organ twisting (Steinborn et al. 2002; Whittington et al. 2001; Marcus et al. 2001;
Thitamadee et al. 2002). Reduced root turgor under dehydrated conditions,
increases ABA accumulation and plasmolysis. Plasmolysis seriously damages the
microtubule skeleton and cellular geometry (Pollock and Pickett-Heaps 2005).
Disrupted microtubules in roots induce the ABA accumulation by increasing ABA
biosynthesis. Interactions between microtubules, cell wall, plasma membrane and
ABA biosynthesis are reported under osmotic stress (Lu et al. 2007).
2.3 Effects on Reproductive Growth Stages
Drought has adverse effects on maize life cycle; particularly reproductive growth
phase is most susceptible to drought stress. Translocation of photosynthetic
assimilates to the reproductive parts rather than roots for their extensive elongation
is most probable reason for more susceptibility of maize plant during reproductive
growth stage under drought stress (Setter et al. 2001; Taiz and Zeiger 2006).
Sequential effects of drought stress on reproductive growth stages of maize are
described in Fig. 2.1. Pollen and silk development, pollination, embryo
2.2 Effects on Growth and Development 11
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development, endosperm development and kernel development are the different
component phases of reproductive growth stage which are severely threatened by
drought stress.
2.3.1 Pollen Development
Pollens are produced in the tassel which is present on top of the plant. Almost 25
million pollens are produced by single tassel under normal conditions. Modern
hybrids produce 2–5 million pollens and landraces produce about 14–50 million
pollens on an average (Burris 2001). Breeding efforts are always focused to reduce
the tassel size and make tassel smart enough to ensure maximum photosynthetic
reserves supply to female inflorescence rather than male inflorescence development
because there is no problem of pollen availability in case of maize. Breeding efforts
had suppressed the male over-dominance which had reduced the pollen production
capability (Duvick and Cassman 1999). Maize pollens are produced in huge bulk
and crop yield is not affected even due to 40 % reduction in pollen production (Du
Plessis 2003). Timing for pollen shedding is effected negligibly by drought stress
so, pollen shedding occurs mostly at normal time even under drought stress but in
severe drought cases pollen shedding is adversely affected. Anthesis-silking interval
is increased by decrease in silk growth and development rate. Pollen shedding
depends on type of variety and environmental conditions; pollen shedding may
continue from 2 to 14 days under normal conditions. About 1 h after sunrise is the
time for initiation of pollen shedding which remain continue for 4–5 h and maxi-
mum pollen shedding occur during 5–8th day of pollen shedding (Burris 2001).
Pollens are affected by drought stress in different ways. Pollen mortality
occurred due to dehydration as moisture of pollen is lost due to drying conditions
(Aylor 2004). Settling speed, pollen viability, specific gravity, pollen shape and
dispersal are seriously affected in dehydrated pollens (Aylor 2002). Increased ABA
accumulation and reduced invertase activity are the main reasons for pollen sterility
under drought stress (Saini and Westgate 2000). Conversion of sucrose to hexoses
is impaired by reduced invertase activity (Sheoran and Saini 1996). Pollens of
maize were studied under drought and high temperature stresses which showed that
pollen weight, pollen viability, pollen size, pollen tube length and pollen moisture
contents were affected by these stresses (Fig. 2.4).
Maize pollens are of large size as compared to other angiosperms and have
relatively higher moisture contents. Pollen viability is reduced greatly if pollen
moisture contents are reduced below 0.4 g per gram of pollens (Buitink et al. 1996).
Pollens absorb moisture from hydrated silk to initiate proper germination so, pollen
germination is reduced in case of dehydrated silk under drought stress
(Heslop-Harrison 1979). Starch and certain osmolytes are present in the pollens
which protect them from losing viability. Drought stress reduced the accumulation
of starch in pollens during pollen development which rendered them nonfunctional
(Schoper et al. 1987). Upregulation of galactinol and vacuole invertase genes in
12 2 Effects of Drought on Maize
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pollen under drought stress showed that these protect the pollens through osmo-
protection and prevent the loss of viability (Taji et al. 2002). Gene expression is
changed in such a way that cell wall structure and synthesis is impaired which
results in loss of pollen viability under drought stress (Zhuang et al. 2007). Severe
drought stress at tasseling stage reduce the yield by affecting the number of kernels
per row, number of kernel rows, harvest index, number of kernels per cob and grain
yield per plant (Anjum et al. 2011b). Increase in ABA accumulation up to 0.5 µM
favor the pollen germination and pollen tube elongation but further increase in ABA
contents significantly reduces the pollen germination and pollen tube elongation
(Zhang et al. 2006).
2.3.2 Silk Development
Silk is female floral part of maize plant and should be receptive for proper polli-
nation and fertilization. Silks remain receptive for 21 days but receptivity tends to
reduce 10 days after silking (Du Plessis 2003). Silk elongation starts from the butt
of the ear and terminal portion of cob elongated at the end. Large ear size delayed
the silk appearance. However, it is reported that silking is delayed by 6–9 days by
prevalence of drought stress (Dass et al. 2001). Tassel emerges 2–4 days earlier
than silk emergence under normal conditions and this pattern is called protandry.
Delay in appearance of silk under drought stress conditions is responsible for
increased anthesis-silking interval (ASI) which is very critical index for efficient
completion of reproductive growth stage. Lower the value of ASI higher will be the
productivity and vice the versa. After silking, silk continue to elongate until it is
pollinated and lengthwise it may reach up to 15 cm (Bassetti and Westgat 1993).
After fertilization, elongation of silks stops and desiccation starts. Under drought
(a)
(d) (e)
(b) (c)
Fig. 2.4 Evaluation of different aspects of maize pollens under drought stress; apollens collected
from field and stored in zipper bags, bviable pollen grains; cmeasurement of pollen grain size;
dpollen tube length; epollen moisture contents measurement. These measurements showed that
pollen viability, pollen size, pollen tube length and pollen moisture contents were reduced under
drought stress
2.3 Effects on Reproductive Growth Stages 13
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stress, desiccation of silks starts earlier and pollen tube becomes unable to reach the
ovary resultantly no fertilization occurred. Fertilization failure occurs because of
earlier silks desiccation due to drought conditions and ear bareness becomes the fate
(Dass et al. 2001). So, assimilate partitioning towards the silk and hydration of silks
are of prime importance for higher grain yield.
2.3.3 Pollination
Release of pollens in bulk from tassel followed by proper landing on silks is
necessary for successful pollination process. Losses due to pollination failure can
never be recovered even after rehydration and yield losses may reach up to 100 %
(Nielsen 2002). Pollen grain productivity reduces from 3 to 8 % on daily basis
under drought stress (Rhoads and Bennett 1990). Pollen shedding is accelerated and
silking is delayed by drought prevalence for four consecutive days and this
increases the anthesis-silking interval followed by 40–50 % yield losses (Nielsen
2005a, b). Development of silk and ear is dependent on sufficient sugar supply
which results in potential seed setting (Zinselmeier et al. 1999). Invertase (carbo-
hydrate transporter) activity is reduced under drought stress which reduces the
carbohydrate supply to the developing reproductive plant parts. Glucose contents in
the pedicle of ovary are reduced due to IVR2 (soluble invertase) reduction during
pre and post pollination under drought stress (Qin et al. 2004). Starch contents of
the floral parts are reduced under drought stress due to impaired activity of the
enzymes involved in starch metabolism (Zinselmeier et al. 2002). Pollination
process is disturbed in following ways by drought stress; (a) silk becomes dried
under dehydrated conditions and no more supportive for pollen tube development
(Nielsen 2002), (b) pollen shedding occurs before silking which causes increase in
anthesis silking interval (Nielsen 2002), (c) silk elongation rate is reduced (Lauer
2012), (d) silk becomes non-receptive for pollen grains under dehydrated conditions
along with low humidity (Nielsen 2005a, b). So, the pollination process is badly
affected by drought stress in maize causing low productivity at the end.
2.3.4 Embryo Development
Embryonic development is very susceptible to drought stress. During early embryonic
development, embryo abortion occurs due to drought or heat stress (Setter et al. 2011).
Drought stress prior to fertilization can cause embryo abortion (Andersen et al. 2002).
Grain yield in maize is mainly dependent on the tolerance of female reproductive
part. Reactive oxygen species are accumulated in the ovary as a result of drought stress
and embryo is aborted in oxidative environment (Kakumanu et al. 2012). Embryo sac
14 2 Effects of Drought on Maize
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development is impaired due to imposition of drought stress during megaspore mother
cell formation and resultantly 80–90 % yield losses are reported (Moss and Downe
1971). Insufficient provision of photosynthetic assimilates and sugar substrates to the
developing embryo cause their abortion (Feng et al. 2011). Soluble invertases (Ivr2)
and cell wall associated invertases are responsible for the provision of hexose to the
developing embryos. These invertases are suppressed under drought stress causing
check to supply of sugars and assimilate to embryo resulting embryo abortion
(Andersen et al. 2002; Feng et al. 2011). Sucrose (substrate for invertase) to hexose
ratio is very important for normal embryo development which is impaired during
drought stress. Cell wall associated invertases and sugars are involved in signaling
pathways and theses signaling pathways are affected by disturbance in expression of
invertases and sugars (Kakumanu et al. 2012). Exogenous application of nutrients at
reproductive stages rescue the 80 % embryos which proves that assimilate translo-
cation is major reason for embryo abortion relative to lower water potential which
causes comparatively less damage (Boyle et al. 1991). Leaves upload sucrose in
phloem then it reach to pedicle where invertases hydrolyse sucrose into glucose and
sucrose. These hexoses are used for kernel development (Cheng et al. 1996) and starch
biosynthesis which participate in ovary development. ABA accumulation triggers the
embryo abortion under drought stress (Setter et al. 2001). So, embryo development is
very susceptible reproductive growth stage to drought stress which is affected by
different ways.
2.3.5 Endosperm Development
Endosperm is storage house of food for embryo in the seed and like other repro-
ductive stages; endosperm development is seriously affected by drought stress.
Storage capacity of the endosperm is determined by cell division during early
developmental stages of endosperm whereas; final volume of endosperm is deter-
mined by cellular elongation and multiplication of cellular organelles (Olsen et al.
1999). Cell division is reduced by imposition of drought stress during endosperm
development and resultantly storage capacity is reduced (Ober et al. 1991).
Prevalence of drought stress after fertilization, suppresses the cell elongation and
multiplication of organelles causing reduction in final endosperm volume.
Process of endoreduplication occurs in the endosperm after mitotic cell division.
Endoreduplication is repetition of S phase (synthesis phase) with mitotic cell
division. There is no cytokinesis but DNA ploidy becomes double after every
repetition of endoreduplication. Cell enlargement, cell differentiation, survival and
metabolic activities are the key functions of endoreduplication (Barow and Meister
2003). Comparative evaluation showed that endoreduplication is less affected by
drought relative to mitotic cell division (Artlip et al. 1995). Transition from mitotic
2.3 Effects on Reproductive Growth Stages 15
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cell division to endoreduplication is also affected by drought stress (Mambelli and
Setter 1998). Cell division is reduced during early stages (1–10 days after polli-
nation) of endosperm development in the apical kernels whereas; endoreduplication
is reduced during terminal stages (9–15 days after pollination) of endosperm
development (Setter and Flannigan 2001).
2.3.6 Grain or Kernel Development
Kernel development is very important phase as for as productivity is concerned and
comprised of following component stages; blister stage, soft dough stage, milking
stage, hard dough stage and dent stage. High moisture contents are needed during
blister stage for grain filling and drought stress at this stage results in poor quality
kernels. Moisture requirement during soft dough, milking and hard dough stages is
higher enough that drought stress at these stages can reduce the kernel quality and
yield. Drought stress during hard dough stage causes the premature hanging of the
cobs. Water requirement of dent stage is lower relative to pre-dent stages of kernel
development but drought stress at this stage still can cause potential loss in yield
and quality (Pannar 2012).
Kernel development in maize is comprised of three major stages; (a) lag phase;
sink capacity is developed, water contents increase and biomass accumulation
reduces (Saini and Westgate 2000), (b) effective grain filling stage or linear phase;
maximum biomass accumulation occurs in this stage and kernel size is determined
(Westgate et al. 2004), (c) physiological maturity; maximum dry weight is gained
and later on grain enters in quiescent phase (Saini and Westgate 2000).
Sink capacity and source strength interact with each other for grain filling.
Differences in grain weight are due to difference in source sink ratio. Source
strength is determined by photosynthesis and carbohydrate assimilation whereas,
sink capacity is determined by sink’s activity (Westgate et al. 2004; Yang et al.
2004). Drought stress reduces the photosynthesis and translocation of photosyn-
thetic assimilates followed by reduced grain filling. Source strength and sink
capacity are reduced by drought stress in maize. Grain size reduction is caused by
reduced remobilization of photosynthetic assimilates (Yadav et al. 2004). Grain
filling is also reduced due to decreased activity of sucrose and starch synthesizing
enzymes under drought stress (Anjum et al. 2011b). Numbers of kernels are
determined during pre-anthesis stages whereas; kernel weight is determined at
post-anthesis stages. Drought stress during post-anthesis stages is responsible for
kernel weight reduction (Oveysi et al. 2010). Interaction of water and biomass
during kernel development are the determinants of final kernel volume. Water
contents of the kernel are increased during early developmental stages of kernel and
later on water contents decrease followed by increase in biomass accumulation.
Biomass accumulation is dependent on source strength and sink’s capacity which
are seriously reduced by drought stress so final kernel volume is reduced by drought
stress (Gambín et al. 2006). Reduced water potential and kernel water uptake
16 2 Effects of Drought on Maize
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squeeze the duration of kernel filling resultantly kernel size is reduced (Brenda et al.
2007). It is reported that drought stress during, kernel development is responsible
for 20–30 % yield losses which are mainly due to under sized kernels (Heinigre
2000). Another report mentioned that drought prevalence during kernel develop-
ment can cause 2.5–5.8 % yield losses on daily basis (Lauer 2003).
2.3 Effects on Reproductive Growth Stages 17
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Chapter 3
Mechanisms of Drought Resistance
Effects of drought stress are very uncertain and unpredictable because they impair
the yield, yield potential and across the years performance. However, selection of
genotypes with better yield under drought prevailing conditions is effective tool for
combating against drought stress. Heterogeneity in nature of drought stress, variable
effects in space and time, degree and severity of stress are further increasing the
erratic and unpredictable behavior of stress. Nature has bestowed the plants to adapt
for survival and productivity under stressful conditions (Gill et al. 2003). Plants
harbor different morphological, physiological and biochemical traits which enable
them to adapt or resist under stressful conditions. Resistance can be described as
least reduction in yield under drought stress conditions relative to normal water
availability. Resistance can be in the form of escape, avoidance and tolerance
(Bohnert et al. 1995). In evolutionary reference, drought resistance is described as
ability of the varieties or species to survive and reproduce under limited water
availability. In agricultural context, drought resistance is described as ability of the
plants to produce economical yield under limited water availability (Qualset 1979).
Plant mechanisms which contribute to bring least losses in yield under drought
prevailing conditions compared to higher yield under normal water availability are
also described as drought resistance. Existence of genetic variability among dif-
ferent crop plants and varieties of same species for drought resistance was reported.
Yield stability in wheat, maize, rice, barley and sorghum was used to determine the
drought resistance (Singh 2010). Differences in yield, yield stability and level of
drought resistance showed that improvement can be made by proper exploitation of
this genetic variability. Different sources can be used for improvement of drought
resistance in crop plants and some of them are as following; cultivated varieties,
land races, wild relatives and development of transgenes. Landraces and wild rel-
ative are possible option to get the genes for drought resistance and to incorporate
them in modern cultivars to improve their status of drought resistance. Zea maxi-
cana or tripsacum floridanum, wild relative of maize, are tremendous source of
novel genes for improvement of tolerance against drought and other stresses (Singh
2010). Transgenes can be developed if genes for drought resistance are available in
non-crossable parents.
©The Author(s) 2015
M. Aslam et al., Drought Stress in Maize (Zea mays L.),
SpringerBriefs in Agriculture, DOI 10.1007/978-3-319-25442-5_3
19
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Existence of significant genetic variability among or between genetic popula-
tions is prerequisite for genetic improvement. Stress breeders suggested that elite
breeding populations have very low frequencies of stress resistant alleles so, first of
all these populations must be evaluated (Blum 1988). Significant genotypes ×en-
vironment interaction (GEI) in response of drought stress showed that substantial
genetic variation exists in breeding population. Breeders recommend that selection
of breeding population for improvement is perquisite (Hallauer and Miranda 1988).
Most importantly genetic variation for kernel yield under stress and stress free
conditions is conducive. Elite breeding genotypes or cultivars have significant
variability for drought resistance related traits which should be used for improve-
ment of drought resistance on priority basis. So yield is primary trait associated with
drought resistance whereas, appropriate secondary traits which confer drought
resistance are selected as selection criteria if following assumptions are full filled;
trait should be genetically correlated with yield, should have higher heritability,
should be of stable nature, easy to measure and traits should be correlated with yield
losses under normal prevailing conditions (Edmeades 2008; Barker et al. 2005).
Most of secondary traits do not full fill all of these prerequisite criteria however,
effective secondary maize traits associated with drought resistance are; leaf rolling,
stay green, shorter anthesis silking interval, cob barrenness (number of kernels per
ear), root system, increased leaf erectness, kernel weight and low canopy temper-
ature (Bolaños and Edmeades 1996; Edmeades et al. 2000). Genetic variability in
secondary traits of maize i.e. yield components and physiological traits can also be
exploited to accelerate the improvement in yield under drought stress.
Numerous factors contributed to the increase in resistance of maize germplasm
against drought stress i.e. high plant density in field during development of inbred
lines, prevalence of drought and heat stress in nurseries with insufficient water
availability, use of high yielding and stable progenitors for breeding program and
multi-location testing of material (Duvick et al. 2004; Tollenaar and Lee 2011).
Major breeding objectives are to cultivate maize hybrids with greater yield
potential, stable yield and improved grain traits for user whereas an additional on
demand objective is to produce the hybrids with enhanced resistance against
adversaries. New genotypes of maize developed through keeping in view the above
mentioned objectives, would overcome the water deficiency by lowering the yield
penalty. This implies that all maize hybrids should have significant level of drought
resistance (Kitchen et al. 1999). Heterosis acts as important mechanism for stress
tolerance, as maize hybrids give higher yield even under drought stress relative to
maize varieties (Blum 1988). In maize, kernel yield is critically determined during
flowering and early grain development (Claassen and Shaw 1970; Shaw 1976).
Drought resistance is not heritable plant trait but numerous mechanisms are in-
volved in conferring the resistance in different ways. These mechanisms are clas-
sified into three different types: drought escape, drought avoidance and drought
tolerance (Fig. 3.1).
20 3 Mechanisms of Drought Resistance
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3.1 Drought Escape
In low land tropics, 400–500 mm is lower limit of rainfall for optimum maize
cultivation, in mid tropics 350–450 mm and in highlands 300–400 mm. Water use
efficiency (WUE) is lower in low lands so more rainfall in needed for proper crop
growth than highlands. Synchronization of phenology with water availability is
selection objective in breeding for earliness (Edmeades 2013). Shortening of
growing season or life cycle of crop before the prevalence of drought stress is
described as drought escape. Drought escape is important against terminal drought
stress so, reproductive growth stage is to be involved for escaping drought (Araus
et al. 2002). Days to sowing, days to flowering and days to maturity are genetically
heritable traits and selection can re-modulated the phenology with water avail-
ability. Interaction between genotype and environment (G ×E) is determinant of
crop duration and induce the plants to complete the life cycle before the onset of
drought. Successful synchronization of phenological development with availability
of soil moisture and predominance of terminal drought stress are the factors which
necessitate the adoption of drought escape (Araus et al. 2002). Development of
short duration and early maturing cultivars is helpful for escaping the crop from
terminal drought stress (Kumar and Abbo 2003). Yield is directly linked with
duration of the crop and any reduction in crop duration ultimately reduces the yield
(Turner et al. 2001). Crop growing season can be partitioned in two parts; sowing to
Fig. 3.1 Mechanism of drought resistance in maize with background traits like, morphological,
physiological, biochemical and molecular traits
3.1 Drought Escape 21
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flowering and flowering to physiological maturity. Early, intermediate and late
maturing cultivars can be developed by making selection as these characters are
heritable. These stages are heritable in nature and selection can be made easily for
earliness as earliness is favorable for drought escape (Bänziger et al. 2000).
Earliness is characteristic feature of drought escape whereas, early maturing
genotypes have lower evapo-transpiration, lower leaf area index and lower yield
potential.
3.2 Drought Avoidance
Numerous physiological and metabolic processes are not exposed to water stress
and keep on performing normal functions in case of drought avoidance (Blum
1988). Drought avoidance is measured as estimate of tissue water status that is
expressed in the form of turgor water potential under drought stress. Plant water
status is maintained by either reducing transpiration rate or by increasing water
uptake. Leaf rolling, leaf firing, canopy temperature, stomata closing, leaf attributes,
and root traits are important selection criteria for drought avoidance in maize.
Stomata are involved in transpiration and gaseous exchange (photosynthesis and
respiration). Plant maintain water status by closing their stomata and avoiding the
water losses (Turner et al. 2001; Kavar et al. 2007). How stomatal closure prevents
the water losses under drought stress has been discussed in Sect. 2.2. However,
stomata closure confers yield penalty due to critical effects on photosynthesis and
respiration. Leaf rolling and leaf firing are important traits used for assessment of
drought avoidance in maize. Insufficient leaf transpiration cause the dryness of
leaves which results in leaf firing. There was report of negative correlation between
leaf senescence and yield in maize. Canopy temperature is also negatively corre-
lated with yield in maize under drought stress (Singh 2010; Edmeades 2013).
Maintenance of water uptake can be accomplished with the help of extensive
root system (Turner et al. 2001; Kavar et al. 2007). Structural/architectural or
phenotypic and functional or hydraulic root traits are very important for effective
drought avoidance in maize. Length of apical zone, length of basal zone, length of
branch inter-space, maximal number of branches, initial root elongation, insertion
angle, root radius, standard deviation of the root tip heading and tropism type
(geotropism for primary roots and exotropism for secondary roots) are used as
architectural or phenotypic traits in maize for estimation of water uptake pattern
with the help of mathematical models (Doussan et al. 1998; Leitner et al. 2014).
Hydraulic root traits e.g. axial conductance between crowns of maize nodal roots,
non-homogeneous soil water potential, transpiration during day time, water
potentials in the root and hydraulic conductance are examined for modeling of
water absorption and finding responses against water stress (Doussan et al. 1998;
Leitner et al. 2014). These structural and functional models showed that higher
water availability in root axis was responsible for higher transpiration. It is also
reported that root hydraulic properties were responsible for determination of
22 3 Mechanisms of Drought Resistance
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temporal dynamics of water deficit in root zone. Water saving and water spending
behavior of roots is attributed to lower equivalent root conductance and higher root
conductance respectively. Water uptake and root conductance is dependent on
hydraulic properties of roots, age of roots and order arrangements of roots. It is
found that radial conductivity of lateral roots is more influential than root structural
traits on total transpiration (Leitner et al. 2014). So, this structural and functional
root modeling in maize clarified that both architectural and hydraulic properties of
roots are important for avoiding drought stress.
Thick and deep root system is conducive for extraction of more water from soil.
Root characters like, root length, root density and root biomass are the main
determinants of drought avoidance (Turner et al. 2001; Kavar et al. 2007).
Thickness of individual root, maximum root depth (Ekanayake et al. 1985), root
length over weight ratio (Aina and Fapohunda 1986), weight of seminal roots
(Tuberosa et al. 2002a) and root length density (Aina and Fapohunda 1986) are the
traits of roots which ensure the excessive water uptake under prevailing drought
stress.
Pubescence or hairiness is very important character of xeromorphic plants.
Reduction of leaf temperature and transpiration are the characteristic benefits of leaf
hairs (Sandquist and Ehleringer 2003). Bicellular microhairs, prickle hairs and
macrohairs are the three hair types reported in maize leaves. Macrohairs are
prominently visible and act as morphological marker for identification of adult leaf
(Moose et al. 2004). Glaucousness or waxiness of leaves is involved in maintaining
the water potential of leaves (Ludlow and Muchow 1990). It is reported that
temperature of glaucous leaves remains lower than non-glaucous leaves. It is
claimed that 0.5 °C reduction in leaf temperature for six hours on daily basis can
increase the grain filling time for three days (Richards et al. 1986). Two pathways
are involved in the biosynthesis of wax in maize. One pathway biosynthesize the
wax till 5th or 6th juvenile leaf and wax of this pathway consists of mainly alcohols
and aldehydes. Second pathway biosynthesize the wax throughout the life and
consists of mainly esters (Bianchi et al. 1985). About twenty GLOSSY genes
(genes for cuticle wax production) are present in maize which determines the
quantitative and qualitative features of wax (Neuffer et al. 1997). There is signifi-
cant genetic variability observed in maize for glaucous versus non-glaucous and
hairy versus non-hairy. So, there is strong consensus that stomatal conductance,
hairiness and glaucousness/waxiness can reduce the water losses whereas; structural
and functional characteristics of roots can improve the water uptake to enable maize
plants to avoid drought stress.
3.3 Drought Tolerance
Potential of crop plants to maintain their growth and development under drought
stress is termed as drought tolerance. Yield stability is also associated with drought
tolerance under prevailing drought conditions. Tolerance is very complex
3.2 Drought Avoidance 23
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mechanism and plants have evolved numerous adaptations at physiological and
molecular levels to confer drought tolerance. Higher economic yield under drought
stress is the characteristic feature of drought tolerant accessions. Survival is
important at seedling stages whereas, later on just survival without economic yield
have no importance for breeders and farmers (Bänziger et al. 2000). Plant growth
and development, plant phenology, grain filling and translocation of photoassimi-
late reserves are important traits to be targeted for improvement of drought toler-
ance in maize (Edmeades 2013). Osmoprotection by osmotic adjustment and
antioxidant scavenging defense system, plant growth regulators, water channel
proteins, stress responsive proteins, transcription factors and signaling pathways
actively participate in conferring drought tolerance in crop plants.
3.3.1 Osmotic Adjustment
Osmotic adjustment is described as development of water gradient to increase the
water influx for maintaining turgor by lowering osmotic potential. Osmotic
adjustment help to maintain the tissue water status. Damaging effects of drought are
minimized by accumulation of solutes in cellular cytoplasm and vacuole. Protection
is provided by maintaining the turgor potential and physiological processes with the
help of osmotic adjustment (Taiz and Zeiger 2006). Plant water status is determined
by water potential, osmotic potential, turgor potential and relative water contents
(Kiani et al. 2007). Relative water contents act as integrative index for estimation of
drought tolerance. Stomata are closed followed by reduction in CO
2
accumulation
which is the result of reduction in relative water contents under drought stress
(Gindaba et al. 2004). Translocation of photosynthetic assimilates to the developing
kernels is also maintained by sustainable regulation of photosynthetic rate and
turgor potential (Subbarao et al. 2000). Osmoprotectants are categorized into two
main groups; first group is comprised of nitrogenous compounds like, proline,
polyols, polyamines and glycinebetaine whereas, second group consists of hydroxy
compounds like polyhydric alcohols, sucrose, and oligosaccharides (McCue and
Hanson 1990). Accumulation of organic solutes (proline, sugar alcohols, glycine-
betaine and soluble sugars) and inorganic ions (K
+
,Na
+
,Ca
2+
,Mg
2+
,Cl
−
,NO
3−
,
SO
4−
, and HPO
4
) are involved in osmotic adjustment (Morgan et al. 1986; Bajji
et al. 2001; Shao et al. 2006; Blum 2011). Osmolytes or osmoprotectants are
neutral, organic and non-toxic compounds for plants. These osmolytes protect the
cellular proteins and cellular membranes against the dehydrating effects of drought
stress (Yancey et al. 1994).
Glycinebetaine is organic, water soluble and non-toxic for plants and has very
important role in protection of plants against drought, salinity, cold and heat
stresses by acting as osmoprotectant (Ashraf and Foolad 2007; Chen and Murata
2008). Glycinebetaine protects photosynthetic apparatus, stabilize cellular proteins
(RuBisCo), reduces load of reactive oxygen species and acts as osmoprotectant
(Makela et al. 2000; Allakhverdiev et al. 2003). There are significant genetic
24 3 Mechanisms of Drought Resistance
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differences among maize genotypes for accumulation of glycinebetaine under
drought stress. Transgenic studies of maize using betA gene of Escherichia coli
which encodes for choline dehydrogenase (enzymes involved in the pathway of
glycinebetaine biosynthesis) showed improvement in drought tolerance. This
improvement in drought tolerance was reported right from germination to economic
yield in case of transgenic maize (Quan et al. 2004). Glycinebetaine confer drought
tolerance in transgenic maize by maintaining cell membrane integrity and cellular
enzyme activity. Exogenous application of glycinebetaine improves the kernel
morphology, kernel quality (Ali and Ashraf 2011), leaf area, 100 kernels weight,
biological yield, grain yield, harvest index, relative leaf water contents, proline
contents, total soluble proteins and antioxidative defense (Anjum et al. 2012) of
maize under water deficit conditions which proves that glycinebetaine is important
osmolyte for drought tolerance.
Proline an imino acid, acts as osmoprotectant under different abiotic stresses like,
drought salinity and extremes of temperature. Proline maintains water status by
maintaining osmotic potential, protects cellular membranes, prevents denaturation of
cellular proteins and maintains sub-cellular structures under osmotic stress (Ashraf
and Foolad 2007). Proline is well known as osmo-protector, osmo-regulator and
regulator of cellular redox potential (Mohammadkhani and Heidari 2008).
Accumulation of proline contents is directly associated with drought tolerance.
Proline being osmoprotectant, reported to be accumulated in maize under drought
stress and conferred drought tolerance (Bänziger et al. 2000). Proline oxidation
pathway is suppressed by down-regulation of proline dehydrogenase (PDH) enzyme
(first enzyme for proline oxidation) in maize under drought stress. Down-regulation
of PDH is ABA independent regulation which showed that ABA is not contributor
for its down-regulation under drought stress (Bruce et al. 2002). Hundred times
increase of proline contents in the primary roots of maize was reported under drought
stress. Accumulation of proline is dependent on the coordinated mechanism of
biosynthesis, catabolism and transportation within the plant under drought stress
(Mohammadkhani and Heidari 2008).
Accumulation of soluble sugars increases in roots and shoots of maize under
drought stress. This accumulation is dependent on degradation of starch. Ratio of
soluble sugars to starch increases due to increase in accumulation of soluble sugars
and decline in accumulation of starch because of starch degradation. Soluble sugars
being hydrolytic products are very important for plant metabolism and act as
substrate for biosynthesis processes, sugar sensing, signaling pathways, metabolic
regulation and energy production. Soluble sugars protect the plants under drought
stress either through substitution of water with hydroxyl group to maintain the
hydrophilic interaction with proteins and membranes or secondly through vitrifi-
cation. Vitrification is involved in the formation of biological glass in cytoplasm to
protect the cellular organelles under drought stress. Drought tolerance is reported to
be positively associated with accumulation of soluble sugars under drought stress
(Mohammadkhani and Heidari 2008).
3.3 Drought Tolerance 25
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Polyols/sugar alcohols well-known osmoprotectants, are reduced aldose and
ketose sugars. Cyclic polyols are ononitol and pinitol whereas; acyclic polyols are
sorbitol, glycerol and mannitol (Abebe et al. 2003). Hydroxyl group of polyols
make hydration sphere around the macromolecules and protect them from dehy-
dration (Williamson et al. 2002). Sorbitol increases the accumulation of proline,
nitrate reductase (involve in nitrate assimilation) and nitric oxide (NO) in maize
under osmotic stress (Jain et al. 2010).
However, it is reported that osmotic adjustment (OA) is governed by dominant
gene effects rather than additive gene effects and genetic variability for OA is low
among tropical maize populations which shows that small genetic gain can be
achieved by selection. Furthermore, genetic variability among tropical maize
germplasm for OA is narrow at vegetative stage relative to reproductive growth
stages which recommends that selection based on OA should be made during
reproductive growth stages. Weak correlation between yield and OA is obvious in
maize under drought stress which proves that improved OA brings very little
betterment in kernel yield. These results are specific to two tropical maize popu-
lations and furthermore genetic variability might be present across the regions and
across the seasons (Guei and Wassom 1993).
3.3.2 Antioxidative Defense Mechanism
Specific molecules which prevent the oxidation of other molecules by scavenging
reactive oxygen species are known as antioxidants. Antioxidants (AOX) act as
defense shield for plants against oxidative damage caused by reactive oxygen spe-
cies. Enzymatic and non-enzymatic compounds are part of antioxidant defense
system. Enzymatic components comprised of catalase (CAT), superoxide dismutase
(SOD), glutathione reductase (GR), ascorbate peroxidase (APX), peroxidase and
polyphenol oxidase while non-enzymatic antioxidant compounds are α-tocopherol,
ascorbic acid, β-carotene, glutathione and cysteine (Gong et al. 2005). High contents
of enzymatic and non-enzymatic antioxidants are critically important for conferring
drought tolerance in plants. Sequential generation of reactive oxygen species
(ROS) in response of osmotic stress has been depicted in Fig. 3.2. Hydrogen per-
oxide (H
2
O
2
) and superoxide radicals are scavenged by ascorbate-glutathione cycle
which consists of peroxidases, catalases, peroxiredoxins, ascorbate peroxidase,
monodehydroascorbate reductase, dehydroascorbate reductase and glutathione
reductase (Fazeli et al. 2007). Sequential scavenging of ROS by AOX has been
described in Fig. 3.3.
Enzymes of ascorbate-glutathione cycle are localized in stroma of chloroplast,
cytosol, mitochondria and peroxisomes. Superoxide molecules are dismutated into
O
2
and H
2
O
2
by superoxide dismutase in first step of ROS scavenging. Carotenoids
have potential capability of scavenging lipid peroxy-radicals and singlet oxygen.
Carotenoids prevent the generation of superoxides and lipid peroxidation under
drought deficit conditions (reviewed by Farooq et al. 2009). Hydrogen peroxide is
26 3 Mechanisms of Drought Resistance
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detoxified by peroxidase and catalase (Apel and Hirt 2004). Dismutation by
superoxide dismutase confers frontline defense shield against superoxide radicals
so, contents of superoxide dismutase are directly linked with oxidative stress tol-
erance (reviewed by Farooq et al. 2009). Comparison of well-known drought tol-
erant and susceptible maize genotypes showed that APX, GR, CAT and POX were
high in value in tolerant genotypes with increase of drought stress at reproductive
stages but these contents were decreased when drought prevailed for extended
period. In case of susceptible maize genotypes, these AOX components were
reduced with initiation of drought stress. H
2
O
2
and malondialdehyde
(MDA) contents of tolerant genotypes were lower even under drought stress which
showed that higher AOX contents have quenched the ROS and prevented the
cellular membrane damage by showing lower MDA contents (Chugh et al. 2013).
Another comparison among maize genotypes was conducted at seedling stage,
which showed that antioxidants were higher in tolerant genotypes and lower in
susceptible genotypes. Lipid peroxidation of cellular membranes and ROS load was
lower in drought tolerant genotypes due to strong AOX defense system (Chugh
et al. 2011).
Fig. 3.2 Sequential generation of reactive oxygen species in response of drought stress (Cristiana
et al. 2012). ©2012 C Filip, N Zamosteanu, E Albu. Originally published in [shortcitation] under
CC BY 3.0 license. Available from: http://dx.doi.org/10.5772/47795
Fig. 3.3 Sequential scavenging of reactive oxygen species by antioxidant defense system
(Ceron-Garcia et al. 2012). ©2012 A Ceron-Garcia, I Vargas-Arispuro, E Aispuro-Hernandez,
MA Martinez-Tellez. Originally published in [short citation] under CC BY 3.0 license. Available
from: http://dx.doi.org/10.5772/26057
3.3 Drought Tolerance 27
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3.3.3 Plant Growth Regulators
Plant hormones also called as plant growth regulators, phytohormones and growth
substances, are chemical substances governing the growth and development of
plants. Hormones act as signaling molecules, trigger cellular differentiation, act
locally at the site of origin or transported to distant targets (Gomez-Roldan et al.
2008; Wang et al. 2008). Plants respond to drought stress through numerous
adaptations and one of those is endogenous hormonal balance. Different plant
growth regulators confer drought tolerance in plants. Auxins, Cytokinins (CKs),
Abscisic acid (ABA), Indole acetic acid (IAA), Gibberellin3 (GA3), Salicylic acid
(SA), Brassinosteroids (BR), Methyl jasmonate (MeJA), Polyamines, Ethylene and
Zeatin (ZT) are the prominent plant growth regulators (Wang et al. 2008). Plant
hormones interact with each other to govern the plant responses and their inter-
action is dependent on specific stage of growth and development, specific tissue and
specific environmental conditions (Emam and Seghatoeslami 2005; Weiss and Ori
2007; Gomez-Roldan et al. 2008).
Auxins were involved in responses against drought stress. Ethylene, CK and
auxin are interactive and effect the biosynthesis of each other (Tsuchisaka and
Theologis 2004; Jones et al. 2010). Decline in concentration of IAA was observed
in maize leaves however, inconsistent response of this hormone was observed in
other crops (Wang et al. 2008; Bano and Yasmeen 2010). IAA accumulation
increased under moderate stress but reduced under severe drought stress. Decline in
IAA was due to increased degradation, reduced biosynthesis and higher ABA
accumulation (Pirasteh-Anosheh et al. 2013). IAA accumulation increased 13.4 %
under moderate stress whereas; decreased 63.2 % under severe drought stress in
maize.
GA
3
and ZT reduces in maize leaves when plants are subjected to drought stress
(Wang et al. 2008). Decline in GA
3
accumulation are either due to reduced
biosynthesis or increased degradation of this hormone by ROS (Pirasteh-Anosheh
et al. 2013). Increased ABA accumulation is one of the reasons for decline in GA
3
accumulation and 68 % reduction in GA
3
accumulation was observed (Wang et al.
2008). ZT is most sensitive phytohormone which is reduced up to 73.3 % in maize
leaves under drought stress (Pirasteh-Anosheh et al. 2013).
Salicylic acid (SA) is involved in plant growth, flower induction, thermogenesis,
nutrient uptake, stomatal regulation, ethylene biosynthesis, enzyme regulation and
photosynthesis (Hayat & Ahmed 2007). Photosynthesis is maintained by SA
through retaining the higher chlorophyll contents under drought stress and confers
drought tolerance (Rao et al. 2012).
Plant hormonal balance acts as regulator for different processes of growth and
development. Environmental factors and plant growth stages are determinants of
hormonal balance. ABA and ethylene govern stomatal conductance, number of
grains, grain filling rate and growth of plant apex (root and shoot) in antagonistic
way. Effects of ABA as stomata closing agent are suppressed by combined higher
accumulation of ethylene and CKs. CKs enhance the growth, development and
28 3 Mechanisms of Drought Resistance
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yield by improving the stay-green index, qualitative and quantitative characteristics
of grain. Effectiveness of these hormones depends on interaction between them
(Wilkinson et al. 2012).
ABA as a stress hormone is actively involved in modulation of growth, devel-
opment and responses against stresses. ABA dependent signaling pathway com-
prised of numerous components like light-harvesting chlorophyll a/b binding
proteins, chloroplast envelope-localized ABA receptor, chloroplastic antioxidant
defense enzymes and these components are highly responsive to ABA (Shen et al.
2006; Hu et al. 2007, 2008). Plastids in plants have thousands of proteins.
Chlorophyll biosynthesis and expression regulation of chloroplast proteins is
dependent on drought stress (Lawlor and Cornic 2002; Leister 2003). Maize
mutants were developed with deficiency in ABA biosynthesizing enzymes for
assessment of protein regulation in leaves. Study revealed that expression of
chloroplast proteins was dependent on ABA under drought and light prevalence as
ABA deficiency made the leaf devoid of photosynthesis and antioxidants.
Antioxidant defense mechanism and photosynthesis are found to be ABA depen-
dent in maize mutants which shows that maintenance of photosynthesis and
antioxidant defense are critical adaptation for drought tolerance (Hu et al. 2012).
Normal water availability does not induce even minute amount of ABA and
extremely severe drought reduces the ABA accumulation due to cessation of ABA
precursors like carotenoids and xanthophylls (Pirasteh-Anosheh et al. 2013).
Plants responses are modulated by polyamines against drought, oxidative stress,
heavy metal toxicity, salinity, osmotic stress, temperature extremes and flooding.
Polyamines act as signals and messengers to regulate the plant responses for
development of tolerance against stresses (Gill and Tuteja 2010). Polyamines are
organic compounds of low molecular weight with diverse functions due to having
diverse position and numbers of amino acids. These cationic ions have crucial role
in stabilization and destabilization of cellular contents through electrostatic linkage
with DNA, RNA and proteins. Polyamines involved in numerous developmental
processes, survival of plant embryos, membrane stabilization, cell signaling,
modulation in gene expression, cell proliferation, apoptosis, cell death, morpho-
genesis, cell differentiation, germination, seed dormancy, flower induction,
embryogenesis, grain ripening and aging (Rea et al. 2004). Polyamines are present
in the form of putrescine (Put), spermidine (Spd) and spermine (Spm). ABA
accumulation is responsible for increase in accumulation of polyamines in maize
(Liu et al. 2005). Photosynthetic pigments, leaf area and dry matter of maize
increase by exogenous application of polyamines. Accumulation, absorption and
compartmentalization of K
+
,Mg
++
and Ca
++
increase in tolerant maize genotypes
relative to susceptible ones under drought stress. Potassium contents increase in
roots and shoots of tolerant maize genotypes (Shaddad et al. 2011). Ca
++
and Mg
++
accumulation and absorption reduce in roots and shoots of maize under drought
stress but these improve by exogenous application of polyamine and phytohor-
mones. Potassium ions improve leaf water potential, relative water contents, turgor
potential, transpiration rate, photosynthetic rate, grain weight per cob, economical
3.3 Drought Tolerance 29
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yield and kernel yield (Aslam et al. 2014c) whereas, osmotic potential improved
significantly across the years.
Polyamines are positively associated with kernel yield only under mild water
deficiency whereas; under severe drought stress these have nothing to do with yield.
Polyamines are more effective to incorporate drought tolerance relative to other
phytohormones. Drought tolerant maize genotypes are more responsive to exoge-
nous application of polyamines and phytohormones whereas, susceptible genotypes
are least responsive (Shaddad et al. 2011). These small ionic molecules are reported
to be involved in osmotic adjustment under drought stress in different crops (Shao
et al. 2006; Liu and Bush 2006).
Brassinosteroid (BR) is steroid plant hormone which governs growth, devel-
opment, numerous physiological processes, and tolerance against biotic and abiotic
stresses (Bajguz 2007; Ren et al. 2009; Tanaka et al. 2009; Xia et al. 2009).
Improvement of drought tolerance in maize by exogenous application of BR shows
that this hormone is involved in important plant responses (Li et al. 1998). BR
alleviates the oxidative damage caused by reactive oxygen species in maize under
osmotic stress. Endogenous and exogenous BR availability is significantly and
effectively responsible for conferring tolerance against oxidative damage in maize.
BR-induced osmotic stress tolerance was dependent on ABA whereas,
ABA-induced osmotic stress tolerance is not dependent on BR and their molecular
background is elusive. BR upregulates the vp14 gene in maize leaves which is
involved in biosynthesis of ABA. BR increases the nitric acid in mesophyll cells of
maize leaf which regulates the ABA biosynthesis and confers the oxidative stress
tolerance (Zhang et al. 2011).
Methyl jasmonate (MeJA) is naturally occurring plant growth regulator and acts
as modulator of numerous morphological, physiological and biochemical plant
processes. MeJA actively participates in photosynthesis, plant growth, cell division
and stomatal closure (Ueda and Saniewski 2006; Norastehnia et al. 2007; Anjum
et al. 2011a) and its role in incorporation of drought tolerance across plant species is
very well known. Methyl-jasmonate confers drought tolerance in maize by
upregulating the osmoprotectants (proline, free amino acids and soluble sugars),
antioxidative defense and phytohormones (Abdelgawad et al. 2014).
In hormonal balance each hormone does not work independently but there is
strong interaction between them for regulation of growth and developmental pro-
cesses under drought stress. ABA has antagonistic effects on other hormones like
Auxin, IAA, GA
3
and ZT. ABA acts as growth suppressor and others (Auxin, IAA,
GA
3
and ZT) act as growth promoter so, their ratio provides the evidence for
growth responses (Weiss and Ori 2007).
3.3.4 Molecular Mechanisms of Drought Tolerance
Molecular events of the cell are affected in response of abiotic stresses. Water
channel proteins, stress responsive proteins, transcription factors and signaling
30 3 Mechanisms of Drought Resistance
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pathways are the molecular events which are responsive to drought stress. These
molecules are involved in conferring drought tolerance through protection of cel-
lular contents or by regulation of stress responsive genes.
3.3.4.1 Stress Proteins and Water Channel Proteins
Certain proteins have critical role in imparting tolerance against drought stress,
broadly these are known as stress proteins. Stress proteins are mostly water soluble
and ensure tolerance by hydration of cellular contents (Wahid et al. 2007). Late
embryogenesis abundant (LEA)/dehydrin, heat shock proteins (HSP), cold shock
proteins, aquaporins and Cyclophilins (CYP) are certain proteins which have crit-
ical role in enhancing drought tolerance. Aquaporins are water channel proteins and
confer drought tolerance by increasing water uptake through provision of opened
water channel.
LEA is a group of proteins which naturally accumulate in pollen grains, seeds
and vegetative tissues during prevalence of abiotic stresses. Accumulation of LEA
proteins is directly associated with desiccation tolerance in crop plants (Amara et al.
2012). LEA proteins are categorized into numerous classical groups based on
amino acid sequences, specific domains, distinct motifs and peptide composition
(Battaglia et al. 2008). Among these different groups, 1, 2 and 3 are main groups
which consist of large number of LEA proteins. Proteins belonging to Groups 1
undergo complex post-translational modifications (PTMs) including, acetylation,
phosphorylation, deamination and methylation and these PTMs depict their seed
specific role. Anti-aggregative properties are characteristic features of group-2 and
group-3 LEA proteins. Groups 2 LEA proteins provide protection to membranes by
preventing their denaturation. Groups 3 LEA proteins are linked in dehydration
tolerance and prevent the cell shrinkage which is the result of water loss. EMB564,
MLG3 and RAB17, are the representative LEA proteins of group1, 2, and 3
respectively for assessment of functional characteristics in maize (Amara et al.
2012).
Heat shock and cold shock proteins are chaperones which prevent the denatu-
ration and folding of cellular contents under stresses. Small heat shock proteins
(sHSP) constitute the larger HSP groups which are the indication of tolerance
against drought stress and heat stress. Cold shocks proteins (CSPs) accumulate
under stress and are the chaperones for protecting dysfunction of RNA. CSPA and
CSPB genes from bacteria were transgened in maize which increased the tolerance
against drought, heat and cold stresses by protecting photosynthesis, vegetative and
reproductive growth stages. Yield increased (11–21 %) in CSPA and CSPB
transgenic maize as evaluated through multi-year, multi-location and multi-hybrid
yield trials (Yang et al. 2010). Qualitative differences in the biosynthesis of heat
shock proteins were observed in maize lines. There are differences present among
maize lines for production of total proteins, low molecular and high molecular
weight heat shock proteins (Ristic et al. 1991). sHSP17.2, sHSP17.4 and sHSP26
are three identified small heat shock proteins by using MALDI-TOF mass
3.3 Drought Tolerance 31
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spectrometry in maize leaves in response to drought stress. Transcriptional analysis
showed that expression level of these sHSP is upregulated in response of heat and
drought stress in maize leaves. ABA was found to be responsible for
post-transcriptional expression regulation of these sHSPs (Hu et al. 2010).
Cyclophilin (CYP) proteins are involved in multiple functions like, cell division,
cell signaling, protein trafficking, transcriptional regulation, pre-mRNA splicing
and stress tolerance (Trivedi et al. 2012). Silico analysis revealed that stress
response was positively associated with cyclophilin proteins. CYP proteins are
more than 80 % similar in rice, maize, arabidopsis, sorghum and brachypodium
which show their high frequency of conserved sequences. CYP is frequently found
in chloroplast, cytosol, lumen and mitochondria (Trivedi et al. 2013). Comparative
evaluation showed that mRNA responsible for the synthesis of CYP was found in
high frequency in maize after 6–7 h of stress treatment whereas; in beans it took
about 48 h for that level. Gene family of 6–7 members encodes for CYP in maize
but only single gene is responsible in bean (Marivet et al. 1992).
Aquaporins act as facilitator for osmosis through water channels and increase the
membrane permeability. Expression of aquaporins is differentially effected in maize
under drought stress. Accumulation of large number of aquaporins in tolerant maize
genotypes shows that these have critical role in conferring the drought tolerance.
Tonoplast intrinsic proteins (TIP), nodulin-like intrinsic proteins (NIP), and plasma
membrane intrinsic protein (PIP), subfamilies of aquaporin proteins are upregulated
in maize (Hayano-Kanashiro et al. 2009).
Stress responsive proteins are very effectively involved in conferring drought
tolerance in maize genotypes. Selection of maize genotypes based on these stress
responsive proteins and their use in different hybridization program can further
improve drought tolerance potential. Aquaporins provide water channel and
increase rate of permeation so, genotypic selection based on higher contents of
aquaporins proteins under stress conditions can further increase water uptake and
improve drought tolerance.
3.3.4.2 Transcription Factors
Natural master regulators of cellular processes and modifier of traits in response of
stress are transcription factors. Single gene engineering which encodes for specific
protein is not sufficient to confer tolerance because of complex tolerance mecha-
nism (Bohnert et al. 1995). Transcription factors (TFs) have the potential to regulate
multistep complex pathways by modifying the metabolite fluxes in predictable
pattern. TF families in plants are extensively large compared to animals and
microorganisms. Stress responsive pathways are regulated at the level of tran-
scription factors. Studies of transcriptional factor regulation were mostly conducted
on model plant Arabidopsis thaliana. TFs of more than 50 different families
encoded by 1700 different genes were reported in Arabidopsis (Yang et al. 2010).
In present study we are going to discuss only those TFs which are responsive to
drought stress and confer drought tolerance. AP2/EREBP [DRE binding protein
32 3 Mechanisms of Drought Resistance
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(DREB)/CRT binding factor (CBF)], basic leucine zipper (bZIP) like ABA
responsive element binding protein (AREB)/ABRE binding factor (ABF),
zinc-finger e.g. C2H2 zinc finger protein (ZFP), NAM (no apical meristem), CUC2
(cupshaped cotyledon), ATAF1-2, CCAAT-binding e.g. nuclear factor Y (NF-Y)
and NAC e.g. stress-responsive NAC (SNAC) are known to have their crucial role
for drought tolerance (Shinozaki et al. 2003; reviewed by Yang et al. 2010).
ABA responsive element binding factors (AREB/ABF) are member of basic
leucine zipper (bZIP) TF family; they are involved in ABA signaling under drought
stress and seed maturation. AREB/ABF recognizes and binds ABA responsive
element (ABRE) and conserves cis-element to regulate the expression of down-
stream genes (Mundy et al. 1990). ABRE is located in the promoter region of ABA
responsive genes (Yamaguchi-Shinozaki and Shinozaki 2006). Stomatal closure
and reduced transpirational losses are the result of increased ABA contents due to
over expression of AREB1/ABF2, ABF3 or AREB2/ABF4 (Kang et al. 2002).
Over expression of ABF3 confer drought tolerance through increased chlorophyll
fluorescence (Fv/Fm) and reduced leaf wilting and rolling (Oh et al. 2005).
NAC (NAM, ATAF1-2, CUC2) is family of TFs which are specific to plants; it
consists of highly conserved DNA binding NAC domains (Guo et al. 2008).
NAC019, NAC055 and NAC072 are members of NAC TF family, they recognize
and bind to NACRS (NAC recognition sequence, CATGTG) element which is
located in promoter region of early responsive to dehydration (ERD1) and confer
tolerance against dehydration (Tran et al. 2004). About 40 NAC TFs out of 140 are
found to be responsible for drought and salinity tolerance in rice. Over expression
of SNAC1 reduces the stomatal aperture in response to drought stress. SNAC1 in
transgenic plants increases 17–22 % spikelet fertility and 22–34 % seed setting
under moderate and severe drought stress, however under well watered conditions
there were differences between transgenic and non-transgenic plants (Hu et al.
2006). TF is governed by maize ubiquitin (ZmUbi) promoter (Nakashima et al.
2007). OsNAC6 rice transgenic seedlings increases 42–57 % recovery after
removal from hydroponic growth medium.
Certain TFs are responsive to dehydration but not to ABA, these are called
ABA-independent dehydration-responsive TFs and these are different from
AREB/ABF and SNAC which are ABA responsive. Conserved Cis-element
localized in promoter region of concerned gene is potential binding site for these
TFs, which is known as dehydration responsive element (DRE). This typically
ABA-independent dehydration responsive TF family is known as DREB TF family.
These TFs act as response regulator under drought stress, cold stress and for the
developments of leaf, flower and seeds (reviewed by Yang et al. 2010). Two types
of DREB (DREB1 and DREB2) are found responsive for different stresses. DREB1
is responsive to cold stress whereas, DREB2 is responsive to drought, heat and salt
stresses. For improvement of stress tolerance in crops DREB1 signaling pathway is
extensively explored. Constitutive over-expression of ZmDREB1A (DREB1A of
Zea mays L.) and OsDREB1A (DREB1A of Oryza sativa L.) in arabidopsis
upregulated the downstream genes and conferred tolerance against drought, heat
and salinity stresses (Qin et al. 2004; reviewed by Yang et al. 2010). Transgenic
3.3 Drought Tolerance 33
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arabidopsis for DREB1A genes of maize and rice followed by improved stress
shows that DREB1 function and pathway is highly conserved in monocot and dicot
species. ZmUbi (Zea mays L. Ubiquitin) promoter is effectively regulat the
DREB1A, DREB1B and DREB1C genes in arabidopsis for improvement of tol-
erance against drought stress (reviewed by Yang et al. 2010).
HARDY is another member of DREB subfamily which is expressed in
inflorescence tissues. The constitutive over expression of this member has pleo-
tropic effects on vegetative growth and forms dense roots and thick green leaves.
HARDY improves water use efficiency through improving photosynthesis and
reducing transpiration (Karaba et al. 2007). DREB2A has little role for conferring
drought tolerance in arabidopsis. Over expression of DREB2 TFS like ZmDREB2A
(maize) and GmDREB2 (soybean) improved drought, heat and salinity tolerance.
Generally DREB2A needs certain modifications in Arabidopsis for improvement of
drought tolerance whereas, DREB2A of maize and soybean confer drought toler-
ance without any modification (Chen et al. 2007; Qin et al. 2007).
Zinc Finger Protein (ZFP) TFs, are strongly induced by salinity, drought, ABA
and cold treatments which depicts that these TFs are dynamically involved in stress
responses through functioning as transcriptional repressors or activators (Sakamoto
et al. 2000, 2004). Members of ZFP TF family like Drought and Salt Tolerance
(DST; Huang et al. 2009), OsZFP252 (Xu et al. 2008) and ZAT10 (Xiao et al.
2009) are actively involved in regulation of dehydration responses. ZFP252 is
found to be located upstream of DREB1A TF and upregulated the accumulation of
soluble sugars and proline. DST, a negative regulator, improves the salt and drought
stress tolerance without harboring yield losses through its knockdown expression
(Huang et al. 2009). C2H2 ZFP, EAR (ERF-associated amphiphilic repression),
cytoplasmic (non-TF) ZFP, and ZAT10 are the members of ZFP TF and improves
drought tolerance in rice (reviewed by Yang et al. 2010).
Nuclear Factor (NF-Y) consists of A, B and C subunits and belongs to
CCAAT-binding transcription factor family. AtNF-YB1 and AtNF-YA5 improves
drought tolerance in Arabidopsis. ABA and drought stress are responsible for
induction of AtNF-YA5 which resultantly close stomata by reducing aperture
(Li et al. 2008). Constitutive overexpression of ZmNF-YB2 in maize improves
drought tolerance which is accomplished through higher photosynthesis rate, low
leaf rolling, high stomatal conductance and low leaf temperature. Under drought
stress there was 50 % increase of yield whereas, under normal conditions there were
slightly compressed internodes and earliness of flowering in ZmNF-YB2 transgenic
relative to non-transgenic maize (Nelson et al. 2007).
ASR1 is a putative transcription factor which encodes for stress responsive
protein and ABA. Differential expression of ASR1 is responsible for determination
of leaf size and leaf senescence. Over-expression induces shorter leaves whereas
reduced expression triggers larger leaves in ASR1 transgene. ASR proteins protect
the DNA structure through maintaining the DNA topology in response of deficit
water. Re-channelization of assimilates from source to sink followed by senescence
of source is also one of the ASR1 functions (Jeanneau et al. 2002).
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There is huge variability reported among TFs across the plant species. But there
is still dire need to exploit their potential at best. Most of the literature shows that
role of TFs is assessed under screenhouse conditions and seedling stages. In
screenhouse conditions, drought stress is imposed for few hours whereas, in field
conditions duration of drought stress can prolong for many days. Assessment of
tolerance by TFs at seedling stage is also not true representative of yield. So, for
assessment of real time functional characterization of TFs, evaluation must be made
at reproductive growth stages under field conditions which will be true contribution
in economical yield through drought tolerance.
3.3.4.3 Signal Transduction Pathways
Adaptive responses in plants are broadly categorized into three categorical groups,
(a) osmotic adjustment or osmotic homeostasis; (b) stress damage control, detox-
ification and repair, (c) growth control (reviewed by Zhu 2002). Depending on plant
responses, drought stress signaling is again categorized into three functional
characteristics groups; (a) osmotic stress signaling for restoration of cellular
homeostasis; (b) detoxification stress signaling to prevent cell damage and to repair
cell damages; (c) signaling to maintain cell division and cell expansion to sustain
growth. Homeostasis and detoxification signaling confer drought tolerance and
regulate the stress responses to maintain growth (reviewed by Zhu 2002).
Osmotic stress signaling is accompanied by protein phosphorylation. Protein
kinases are activated in response of osmotic stress. Calcium signaling in response of
osmotic stress stimulates calcium-dependent protein kinases (CDPK) which further
regulates downstream responses. Constitutive overexpression of CDPK in maize
protoplast regulates the expression of certain genes which are responsive to ABA,
cold and osmotic stresses (Sheen 1996). These findings link the induction of gene
expression in response of osmotic stress with calcium signaling. Transcriptome for
protein kinases like, MAPK, MAPKK, MAPKKK and histidine kinase is increased
in response of osmotic stress (Mizoguchi et al. 2000). Understanding of signaling
pathways depends on clear insight of inputs and outputs of pathway. Osmotic stress,
changes in turgor and subsequent injury are inputs whereas, osmotic adjustment by
osmolytes, protection from damage and repair from damage (induction of LEA or
dehydrins) are outputs of osmotic signaling pathway (reviewed by Zhu 2002).
Membrane phospholipids (dynamic mechanism) produce numerous signaling
molecules (IP3, DAG, PA) and provide structural support to cells in response of
osmotic stress. At low level of osmotic stress, phospholipids act as messengers and
regulate downstream responsive genes whereas, under severe osmotic stress, high
level of phospholipids depict the cellular damage (Sang et al. 2001). Phospholipases
and other messengers are the basis for categorical grouping of phospholipid sig-
naling systems. Inositol 1, 4, 5-trisphosphate (IP3), a secondary messenger induces
stomatal closure by increasing Ca
+2
ions in cytoplasm. Calcium ion accumulation in
cytoplasm is responsible for regulation of osmotic stress responsive genes as
reported through pharmacological and microinjection experiments (Wu et al. 1997).
3.3 Drought Tolerance 35
aslampbg@uaf.edu.pk

IP3 is found to be osmotic stress responsive through numerous experiments which
shows that overexpression of ABA and stress responsive genes are directly asso-
ciated with higher IP3 accumulation (Sanchez and Chua 2001). Other inositol
phosphates, like, IP
6
,I
1
P
3
,I
3
P
3
and I
4
P
3
are involved in release of calcium ions
from internal cellular sources (reviewed by Zhu 2002). Phospholipase C (PLC) and
phospholipase D (PLD) are important components of osmotic stress responsive
signaling pathway. Phosphatidic acid (PA) is a secondary messenger and is pro-
duced by PLD through cleavage of membrane phospholipids. PLD enhances
lipolitic membrane disintegration and its activity is found to be higher in susceptible
genotypes. ABA treatment increases the PLD activity whereas; PA treatment
reduces the ABA effects (Jacob et al. 1999).
Osmotic stress tolerance and plant water balance regulation are among the main
characteristic functions of ABA. ABA mutants are developed in numerous crops
including maize and these mutants are unable to grow under drought and temper-
ature stresses (Koornneef et al. 1998). ABA mutants produce short stature plants
which show the involvement of ABA in regulation of cell cycle and other cellular
activities. Molecular mechanisms lying behind the higher accumulation of ABA in
response of osmotic stress became clear through recent advances in molecular
biology. Accumulation of ABA depends upon the equilibrium of ABA biosynthesis
and degradation. It is reported that numerous ABA biosynthesizing genes like ZEP,
9-cis-epoxycarotenoid dioxygenase (NCED), ABA aldehyde oxidase (AAO) and
LOS5/ABA3 are upregulated in response of drought stress (reviewed by Zhu 2002).
Genes involved in degradation of ABA like cytochrome P450 monooxygenase, are
downregulated in response of drought stress (reviewed by Zhu 2002).
ABA dependent gene upregulation is initiated after increase in ABA biosyn-
thesis in response of drought stress. Genes involved in osmotic homeostasis like,
aquaporins, osmolyte, LEA, dehydrins, chaperones, detoxification enzymes, ubiq-
uitination associated enzymes and proteases are upregulated (Zhu et al. 1996; Zhu
2002). Drought responsive genes are broadly classified into two groups; early
response and delay response genes. Induction of early response genes starts very
quickly even within minutes and encodes for transcription factors. Activated TFs
control further subsequent delay gene responses. Delay response genes are induced
after few hours of stress imposition and comprise of large number of stress
responsive genes. CBF/DREB gene family, AtMyb, ABF or ABI5 or AREB and
RD22BP are examples of early response genes for ABA regulation under drought
stress, salt stress, and cold stress (reviewed by Zhu 2002).
ABA intensive and ABA deficit mutants are developed to determine ABA
dependent responses. ABA intensives are not completely intensive and ABA def-
icits are not completely deficit; certain responses are independent of ABA.
Numerous studies reported that some genes are totally ABA dependent, some are
totally ABA independent and some are partially dependent (Shinozaki and
Yamaguchi-Shinozaki 1997). RD29A is an excellent example of ABA dependent
and ABA independent signaling. DRE (dehydration responsive element) sequence
is identified to be located in RD29A gene and activation of this element is inde-
pendent of ABA (Xiong et al. 2001).
36 3 Mechanisms of Drought Resistance
aslampbg@uaf.edu.pk

Chapter 4
Global Achievements in Drought
Tolerance of Maize
Contribution of public sector in improvement of drought tolerance is meaningful as
they developed methodologies and genetic resources. Comparison of public sector
with multinational private sector revealed that public sector is lacking in real
professional and technical staff, sustainable resources, discipline, and coordination
to develop the drought-tolerant material on consistent basis. Public sector projects
are dependent on continuous availability of funds. Private sector has done
tremendous work for improvement of maize especially against drought stress.
Pioneer and DeKalb hybrids (private companies) were credited for development of
drought-tolerant maize hybrid for temperate zone (Castleberry et al. 1984). From
1950 to 2001, 18 elite commercial hybrids were released for cultivation (Duvick
et al. 2004). Experiments showed that over the time selection has brought about
numerous changes in behavior and response of germplasm. As, leaf rolling scores
were higher in 1950s and reported to be lowered in 2000, whereas higher rolling
scores means the flatness or erectness of leaves and lower scores showed the
increased rolling. Tassel branch number and variation in stem volume were also
reduced from 1950 to 2000 due to extensive selections (Edmeades et al. 2006).
Improvement in drought tolerance is the output of extensive multi-environment
trials. Drought stress prevailed during flowering stage is purposed to be most critical in
effecting the kernel yield in maize. Different hybrids were compared which belongs
from different chronological time periods. Maize hybrids released in 1940s, when
subjected to drought stress at flowering, these hybrids yielded 2.20 t/ha, whereas
maize hybrids which were released in 1990s yielded 7.19 t/ha. These hybrids were
also subjected to drought stress at grain filling, and their yield was 4.97 and 8.69 t/ha,
respectively (Barker et al. 2005). These results showed that improvement in tolerance
and increased genetic gain under drought stress were due to increased kernel setting or
reduced barrenness. Whereas Hammer et al. (2009) attributed the genetic gains in
yield to increased water uptake in modern hybrids due to extensive root system. From
1940s to 1990s, irrigation water had increased the grain production up to threefolds
due to increased water uptake and increased yield potential and drought tolerance
(Butzen and Schussler 2009). Continuous evidences have supported that increase in
grain yield and drought tolerance in modern U.S. Corn Belt germplasm are associated
with root volume and root intensity (http://www.asgrowanddekalb.com/products/
corn/Pages/rootdig.aspx).
©The Author(s) 2015
M. Aslam et al., Drought Stress in Maize (Zea mays L.),
SpringerBriefs in Agriculture, DOI 10.1007/978-3-319-25442-5_4
37
aslampbg@uaf.edu.pk

4.1 Contribution of CIMMYT, IITA, and Other
Collaborative Partners
Breeding work is being carried out in different countries across the world by local
and international research institutes. Research experiments proved that betterment
in resistance against drought stress caused no yield penalty relative to normal water
availability. However, selection can be done for alteration in floral parts and
reproductive efficacy by changing the biomass partitioning within and to the maize
ear (Edmeades 2008). Pedigree maize breeding program was used for selection of
maize for reproductive traits under drought stress to improve the drought resistance
(Bänziger and Araus 2007). Along with grain yield, secondary traits were given due
importance for selection which have greater heritability under drought stress.
CIMMYT worked out at Zimbabwe and Kenya, which are key selection centers
and initial selection showed greater genetic gains under drought stress.
CIMMYT-selected hybrids were compared with commercial hybrids across 36–65
locations in Southern Africa, and results showed 13–20 % higher yield in 1–
5 ton/ha yield range and 3–6 % increase in the range of 5–10 ton/ha yield (Bänziger
et al. 2006). Preliminary improvement in CIMMYT trails, increased the interest of
donor agencies so, The Water Efficient Maize for Africa (WEMA) and The Drought
Tolerant Maize for Africa (DTMA) projects were funded for the duration of
10 years by Bill and Melinda Gates Foundation.
DTMA Project was initiated in 2007, with collaboration of CIMMYT,
International Institute of Tropical Agriculture (IITA) and 13 national institutes
from sub-Saharan Africa. DTMA focused on conventional selection methods and
marker-assisted selection (MAS) to genetically improve the maize germplasm
which was already adapted to drier sub-Saharan conditions. Conventional pedigree
hybrid breeding and biparental marker-assisted recurrent selection (MARS) were
focused techniques in DTMA. Regional location centers in Zimbabwe, Kenya,
Nigeria, and Zambia were selected for phenotyping of maize germplasm. In DTMA
project, association mapping was also accomplished to identify the genomic regions
associated with drought and heat tolerance (either drought and heat alone or their
combination) in 293 inbred lines (Cairns et al. 2013). Large-effect QTLs which
contribute more than 10 % of phenotypic variance were not identified in maize for
drought tolerance. So MARS was modified into genomic selection based on gen-
ome estimated breeding values (GEBVs). DTMA and WEMA projects also
developed a distinct database comprised 5000 lines which belongs to 27 interrelated
populations. This database is an excellent source for genetic studies on drought
tolerance of maize in tropical germplasm (DTMA 2012). It was also revealed in
DTMA project that some source lines of drought tolerance became susceptible with
increase in temperature which highlighted that selection for tolerance against
drought and heat stress should be carried out simultaneously (DTMA 2012).
Water Efficient Maize for Africa (WEMA) was initiated in 2009, with collab-
oration of CIMMYT, Monsanto and five other eastern and southern African
38 4 Global Achievements in Drought Tolerance of Maize
aslampbg@uaf.edu.pk

countries. Conventional selection, marker-assisted recurrent selection (MARS) and
transgenic maize development was key focus of WEMA. Maize researchers seems
to be confident that combination of doubled haploid (DH) inbred lines,
genome-wide association systems (GWAS) and precision field-based phenotyping
can bring the two times increase in genetic improvement of yield and drought
tolerance (Bernardo 2008; Lorenz et al. 2011; Yan et al. 2011).
Maize in Asia is also suffering from drought stress so, there were two projects for
drought tolerance of maize which were funded by GCP or the Syngenta Foundation
and led by CIMMYT for South East Asia. China, Indonesia, India, Philippines,
Thailand, and Vietnam are the component countries for Asian projects. Asian Maize
Drought Tolerance (AMDROUT) Project was governed by CIMMYT and IITA as
main partner. This Project was based on marker-assisted recurrent selection (MARS)
and genome-wide selection (GWS). Yellow drought-tolerant maize inbred lines
were developed in this project. This project was direly needed because about 80 % of
Asian maize is grown in rainfed conditions. Asia will further face more severe
drought stress in 2020s, and there is dire need to adapt the cropping pattern to
changing climate. Pakistan, China, and Indonesia can adapt to changing climate due
to their suitable geographical area, whereas other countries need priority breeding
preferences for future. Phenotyping, marker-assisted recurrent selection (MARS),
genome-wide selection (GWS) were the key techniques followed in this project. One
cycle of phenotypic selection followed by one cycle of only genotypic selection give
50–100 % more genetic gain relative to two cycles of only phenotypic selection. So
it is proved that GWS is beneficial technique for improvement of breeding popu-
lations. Biparental crosses were used between CIMMYT-Asia lines and African
drought-tolerant donors (Vivek 2013). Best donor lines for drought tolerance were
assessed through six location evaluation (China, Philippines, India, Thailand,
Indonesia, and Vietnam) and results showed that CML444 was the best donor line.
However, CML440, CML538, CZL0719, CML505, and CZL00009 donor lines
were also proved to be effective donor. VL1012767, CML470, VL108729,
VL108733, VL1012764, and CML472 were selected to be used as recipient parents
(CGIAR Generation Challenge Programme 2014).
Genetic resources for better traits are present in germplasm but their evaluation is
problematic due to linkage drag with non-desirable traits. However, to identify the
drought-tolerant genes in germplasm, a project was funded by Mexico Government
to CIMMYT and this project was named as Seeds of Discovery. International
Institute of Tropical Agriculture (IITA) and CIMMYT breeding programs have
established well-adopted pedigree breeding system for development of hybrids but
there is still demand for open-pollinated varieties (OPVs) among the farmers of
sub-Saharan Africa. OPV seed distributed from farmer to farmer without loss of
performance. For development of farmer participation in selection, adoption, and
production, mother–baby trail system was used in eastern and southern Africa.
Mother–baby trials resulted in development of drought-tolerant OPVs like, ZM523,
ZM521, and ZM409 (Edmeades 2013). Drought-tolerant maize hybrids and OPVs
developed by CIMMYT in collaboration of other partner organizations are listed in
Table 4.1.
4.1 Contribution of CIMMYT, IITA, and Other Collaborative Partners 39
aslampbg@uaf.edu.pk

Table 4.1 Drought-tolerant maize hybrids and OPVs
Country Drought-tolerant hybrids Drought-tolerant OPVs Reference
Angola CZH03030, CZH0819 ZM623, ZM423, ZM523, ZM725, ZM309 CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Benin –Ku Gnaayi, Mougnangui, Ya Kouro Goura Guinm,
Orou Kpinteke, Djéma- Bossi, DT SR W C2
CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Ethiopia MH130, MH138Q, MH140, BH546,
BH547
Melkasa5, Melkasa6Q, Melkasa7, Gibe-2,
Melkasa-1Q
CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Ghana Etubi, Enii-Pibi, Aseda, Opeaburoo,
Tintim
Abontem, Omankwa, Aburohemaa, Wang Taa,
Bihilifa, Sanzal-Sima, Ewul-Boyu
CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Kenya KDH3 KSCDT01 CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Malawi SC719, PAN53, CAP9001, MH27,
MH28, MH30, MH31, MH32, MH33,
MH34, MH35, MH36, MH37, MH38
ZM309, ZM523 CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Mali Tieba, Mata, Sanu, TZE-Y DT STR C4 x
TZEI 13
Jorobana, Brico, Diambal CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Mozambique SHluvukani, Olipa, Molocue, Pris 601,
SP-1
ZM309, ZM523, Dimba, Gema CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Nigeria Sammaz 22, Sammaz 23, Sammaz 24,
Sammaz 25, Oba Super 7, Oba Super 9,
Ifehybrid 5, Ifehybrid 6
Sammaz 15, Sammaz 18, Sammaz 19, Sammaz 20,
Sammaz 26, Sammaz 27, Sammaz 28, Sammaz 29,
Sammaz 32, Sammaz 33, Sammaz 34, Sammaz 35,
Sammaz 38
CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
(continued)
40 4 Global Achievements in Drought Tolerance of Maize
aslampbg@uaf.edu.pk

Table 4.1 (continued)
Country Drought-tolerant hybrids Drought-tolerant OPVs Reference
Tanzania WH403, WH502, WH505, VumiliaH1,
HB405, HB513, HB623, TZH 636, TZH
538, TZH 417, NATA H104, NATA
H105
ZM623, TZM 523 CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Uganda Longe 9H, Longe 10H, Longe 11H, UH
5051, UH 5052, UH 5053, UH5354,
UH5355
VPMAX CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Zambia KAM602, SC721, CAP5901, SC727,
ZMS606, ZMS623, GV 635, GV 638,
GV 628
ZM423, ZM523, ZM625, ZM721, Nelson’s Choice CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
Zimbabwe ZAP51, ZAP61, Pris 601, ZS263, ZS265,
PAN3 M-41, PGS53
ZM309, ZM401 CIMMYT, IITA, DTMA (http://dtma.
cimmyt.org/index.php/varieties/dt-
maize-varieties)
4.1 Contribution of CIMMYT, IITA, and Other Collaborative Partners 41
aslampbg@uaf.edu.pk

4.2 Contribution of Multinational Seed Companies
Multinational companies have done tremendous work for drought tolerance of
maize. Significant acceleration has been made by molecular markers in attaining the
higher genetic gains for yield and tolerance. MARS has doubled the rate of genetic
gains in maize population of Monsanto (Eathington et al. 2007; Edgerton 2009).
Regular pedigree breeding program was used by Pioneer under a program named as
“mapping as you go”which also doubled the rate of genetic gains (Podlich et al.
2004). Association mapping and genomic selection have been adopted and
exploited by large multinational companies more easily and quickly relative to
public sector due to their extensive capability of field testing of mapping popula-
tions, synchronization of phenotyping and genotyping, massive bioinformatics
resources, access to elite germplasm and capital (Eathington et al. 2007; Edgerton
2009; Schussler et al. 2011). Large multinational companies are producing about
500,000 double haploids in one calendar year which is also accelerating the speed
of inbred line development for them. Seed chipping methods are also used by
multinational companies for non-destructive assessment of DNA of double hap-
loids. Unwanted DNA combinations are discarded even before sowing of seeds.
However, combination of double haploids, genome-wide selection based on
genotyping and seed chipping are increasing the selection pressure and shortening
the number of generations needed for hybrid development which are achieving the
“speeding the breeding.”Multilocation testing, crop modeling, and combination of
above-mentioned techniques have doubled the genetic gains for drought tolerance
from 1930 to 2000 (Edgerton 2009). Drought-tolerant hybrids developed by using
combination of above discussed techniques are available in market of USA since
2011. “Leading competitor hybrids”are used for yield comparison of
drought-tolerant hybrids. Syngenta’s Agrisure Artesian™hybrids are better option
to be used for yield comparison of drought-tolerant hybrids because these hybrids
are based on series of 12 QTLs which are effective for broad range of genetic
backgrounds (http://www.freepatentsonline.com/y2011/0191892.html).
Pioneer Hi-Bred (a multinational company) launched AQUAmax™brand which
comprised a line of hybrids and this was launched in 2011. For development of
AQUAmax™, QTL-based approach was used which was commercially known as
Accelerated Yield Technology™. Molecular mapping-based genomic selection,
molecular markers as genetic covariates to highlight the genomic hotspots, and
multilocation testing were the key basis of AQUAmax™brand (Sebastian 2009).
Characteristics features of AQUAmax
TM
hybrids were having prolonged stay-green
property and vigorous silking (http://www.4-traders.com/news/Pioneer-Hi-Bred-
International-Inc). AQUAmax and Artesian hybrids were launched in market for
sale in 2013. Drought-tolerant maize hybrids developed through conventional
breeding by Pioneer Hybrid International, reported that 5 % yield was greater than
drought stress, whereas drought-tolerant maize hybrids by Syngenta claimed to give
15 % more yield under drought stress (Tollefson 2011).
42 4 Global Achievements in Drought Tolerance of Maize
aslampbg@uaf.edu.pk

Monsanto is also the leading body for development of drought-tolerant trans-
genic maize hybrid. Droughtgard™hybrids of Monsanto are transgenic and
launched in market for sale in 2013. MON87460, a drought-tolerant maize hybrid
of Monsanto has cold-shock protein gene (cspB); this gene was isolated from soil
bacteria “Bacillus subtilis”(http://www.biofortified.org/2012/08/monsantos-gm-
drought-tolerant-corn/). This gene encodes for a protein which act as chaperone
for other proteins and this gene remain active during whole plant life and also
increase the number of kernels per plant (Castiglioni et al. 2008). Recently
Monsanto collaborated with BASF (Badische Anilin-und Soda-Fabrik) for drought
tolerance gene discovery. Pioneer Hi-Bred and Syngenta are also working on
transgenic drought-tolerant maize development.
4.2 Contribution of Multinational Seed Companies 43
aslampbg@uaf.edu.pk

Chapter 5
Biological Practices for Improvement
of Maize Performance
Management of drought stress to reduce the yield losses in crop plants is practiced
in different forms across the world. Saving irrigation water with the help of different
water management practices, exploitation of agronomic practices to improve crop
performance under drought stress condition, and development of drought-tolerant
germplasm are the main tools which are exploited by agronomist and breeder.
Water saving and cultural practices are found to be inconvenient, expensive, and
requiring special skills. Development of drought-resistant germplasm is proved to
be effective, efficient, and feasible approach for improving yield in drought pre-
vailing territories (Athar and Ashraf 2009). Different strategic characters are
improved by numerous biological approaches which enable the plants to escape,
avoid, and tolerate the drought stress. Screening of germplasm for assessment of
tolerant variants, development of tolerant genotypes through conventional breeding,
mutation breeding, molecular breeding, and transgenic approaches are possible
options which are working and can further be employed for further improvement.
Schematic flowchart for biological or breeding strategies for improvement of
drought tolerance in maize is shown in Fig. 5.1.
5.1 Screening for Drought-Tolerant Maize Germplasm
Evolutionary pathway has diverged the biological diversity at different levels of
organization e.g., development of eukaryotes from prokaryotes followed by
diversion toward development of plants, animals, fungi, bacteria, viruses, and other
creations. Extensive prevailing diversity is categorized into different taxonomic
levels like, species, genus, order, class, phylum, and kingdom. Species are further
comprised of large number of varieties, strains, cultivars, and lines. Different groups
of populations even within species have genetic differences for numerous param-
eters. Drought resistance is one of the aspects for which lot of genetic differences
are present within species. These genetic differences could be assessed by screening
for drought stress resistance. Earliness is critical parameter which enables the plants
to escape the drought stress. Development of extensive root system and prevention
of water loss enabled the plants to avoid drought stress (reviewed by Athar and
©The Author(s) 2015
M. Aslam et al., Drought Stress in Maize (Zea mays L.),
SpringerBriefs in Agriculture, DOI 10.1007/978-3-319-25442-5_5
45
aslampbg@uaf.edu.pk

Ashraf 2009). Maintenance of normal physiological mechanisms with satisfactory
yield brings drought tolerance in plants. Detail description of drought escape,
avoidance, and tolerance has been discussed in Sects. 3.1,3.2 and 3.3, respectively.
Genetic variability among different genotypes of maize is present for drought
escape, avoidance, and tolerance which could be retrieved by suitable screening of
germplasm. Different characteristic parameters which are strongly linked with these
three mechanisms are focused in screening of germplasm. Screening of maize
germplasm could be done in growth room under controlled conditions and in field
under natural conditions. Parameters used for screening of germplasm must be
associated with grain yield because higher grain yield is ultimate objective of
screening. Traits which are affected severely by drought stress are targeted in
screening.
Biometrical tools assist in assessment of genetic variability among maize
germplasm. Analysis of variance, mean comparison tests, basic summary statistics,
metroglyph analysis, D2 statistics, principle component analysis and biplot
graphical analysis are extensively used for assessment of genetic diversity (Singh
and Chaudhary 1985). Drought tolerance indices are profusely used for efficient
screening of germplasm in different crop plants. Stress susceptibility index (Fischer
and Maurer 1978), geometric mean productivity (Fernández 1992), mean produc-
tivity (Rosielle and Hambling 1981), harmonic mean, tolerance index (Rosielle and
Hambling 1981), stress tolerance index (Fernández 1992), yield index (Gavuzzi
et al. 1997), yield stability index (Bouslama and Schapaugh 1984), ranking index,
integrated selection index, and integrated scoring are important drought tolerance
Fig. 5.1 Biological/breeding
approaches for improvement
of drought resistance in maize
46 5 Biological Practices for Improvement of Maize Performance
aslampbg@uaf.edu.pk

indices extensively used for effective germplasm screening. So, screening of pre-
viously available maize germplasm for drought resistance proved to be resource
efficient biological strategy.
5.2 Conventional Breeding Strategies
Creation of genetic variability and novel gene combination through intercrossing of
targeted parents is one of the practices used to develop tolerant genotypes.
Intercrossing followed by appropriate selection scheme enables to develop an
ideotype plant that is suitable for environment specific cultivation (Bänziger et al.
2000). Higher genetic variability, high heritability, and higher selection intensity
empower the breeder to make appropriate selection in the germplasm (Falconer
1989). Breeding strategies for development of drought-resistant germplasm are
economical and effective tool for combating the global issue of water deficiency
(Subbarao et al. 2005). Existence of genetic variability at generic, specific, and
varietal levels act as raw material for selection and breeding against drought stress
(Serraj et al. 2005a). Maize breeders have to focus on large number of traits for
improvement of drought resistance as it is well known that single trait could not
improve the resistance because plant responses interact with each other in complex
fashion. Gene pyramiding, efficient and systematic breeding method, can effectively
improve the drought tolerance by incorporation of large number of favorable traits
in one genotype. Morphological and physiological parameters which prevent water
loss, improve water use efficiency, and economic yield must be focused for pyra-
miding as recommended by Subbarao et al. (2005). Early vigor, rapid establish-
ment, structural and functional traits of roots, osmoprotection, stomatal
conductance, and leaf characteristics are suggested by Parry et al. (2005) as key
parameters for improvement of drought tolerance. Development of early maturing
varieties is important tool for escaping terminal drought stress by completing the
life cycle before the onset of drought stress. So, earliness could also be incorporated
by breeding to escape terminal drought (Athar and Ashraf 2009). Breeding for
development of genotypes which are efficient water user (collect more quantity of
water and loose less) could be effective to avoid the harmful effects of drought
stress. Structural and functional traits of roots and stomata should be focus of
breeders for development of drought avoiding genotypes. Breeding efforts for
improvement of drought tolerance concentrated on the traits which maintain normal
physiological mechanisms (osmolytes, antioxidants, plant growth regulators,
stress-responsive proteins, and transcription factors) and economic yield (yield and
yield components) of crop plant (Bänziger et al. 2000).
Breeders collect large number of germplasm with variable origin; initially,
selections (screening phase) are made on yield and yield component basis; after
reducing the number of genotypes by selection then selections are made for drought
resistance (testing phase). Quantitative inheritance, low heritability, and higher
genotype into environment interaction proved as barriers for quick improvement of
5.1 Screening for Drought-Tolerant Maize Germplasm 47
aslampbg@uaf.edu.pk

yield under drought stress (Babu et al. 2003). Assessment of yield limiting traits
with the help of morphological, physiological, biochemical, and molecular tech-
niques could supplement the conventional breeding methods for improvement of
yield (Cattivelli et al. 2008).
Conventional breeding methods rely on conducting multilocation, multiyear and
multiseason yield trials for evaluation of stability in the performance against
drought stress (Babu et al. 2003). Yield and yield components are primary target
traits to be focused for crop improvement against drought stress. Secondary traits
are equally important for breeding against drought stress. Secondary traits which
have strong correlation with grain yield, stable in nature, easy to measure, high
heritability, and improve yield are preferred even under normal environmental
conditions (Edmeades et al. 2001). Worth of secondary traits is realized through
selection indices, heritability in progenies, and genetic association. Development of
near isogenic lines and synthetics helped the breeder to know the association of
targeted secondary trait with economic yield (Bänziger et al. 2000). From practical
perception of breeder, secondary traits which are important for improvement of
drought tolerance and recommended by CIMMYT for use in breeding programs
have been enlisted in Table 5.1.
Interspecific and intervarietal differences are present for water use efficiency in
different crop plants and impairment of this trait is among early drought responses.
Water use efficiency is genetically governed trait and its higher value depicts
Table 5.1 Secondary traits targeted for drought tolerance improvement through conventional
breeding (Recommended by CIMMYT; Bänziger et al. 2000)
Trait Heritability Correlation
with yield
Selection
objective
Target growth
stage
Grain yield Medium to low under
flowering stress,
medium during grain
filling stress
High
positive
Increase
economic
yield
Flowering and
grain
development
Ears per plant High and increasing
with stress intensity
High More ears per
plant or low
barrenness
Flowering stage
Anthesis-silking
interval (ASI)
Medium under normal,
high level under severe
stress
High under
stress
Reduced or
negative ASI
Flowering stage
Tassel size Medium to high Medium Smaller tassel
with fewer
branches
Could be
measured under
normal and stress
conditions
Leaf senescence Medium Medium
under grain
filling stress
Delayed leaf
senescence or
stay-green
property
Grain filling
stage
Leaf rolling Medium to high Medium to
low
Unrolled
leaves
Flowering stress
48 5 Biological Practices for Improvement of Maize Performance
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drought tolerance. Water use efficiency is reduced under drought stress either due to
sustained biomass production or higher water losses. Higher ratio for biomass
production to transpired water, partitioning of biomass toward economical part,
reduced water loss, and increased water uptake are the components of water use
efficiency which could be focused for breeding against drought stress (Condon et al.
2004; Farooq et al. 2009).
Improvement in drought tolerance is complex due to polygenic nature and low
frequency of alleles for tolerance in maize. Open-pollinated varieties (OPV) and
hybrid products are targets in maize for improvement of drought tolerance.
Improving locally adapted germplasm, improving tolerant exotic germplasm for
adaptability, and development of new breeding population through introgression
are the recommended options for drought tolerance improvement in maize.
Development of source population and evaluation of that population are subcom-
ponents of introgression. Selection and development of source population must be
done on the basis of following characteristics; general adaptability, grain color,
grain texture, maturity, disease resistance, abiotic stress tolerance, heterotic pattern,
heterotic response, combining ability, and other value added traits. Evaluation of
developed population could be done through line evaluations, hybridization, diallels
of local or exotic populations or lines, population x local tester topcross, and line x
local tester topcross. Intrapopulation improvement for drought tolerance could be
done through individual plant selection, per se performance, test crosses using
individual plants, half-sib progenies and parental testers (Bänziger et al. 2000).
Mutation breeding is very important component of crop breeding program that is
involved in exploitation of mutations for improvement in agricultural and horti-
cultural crops. Chemical and physical mutagenic agents are used for induction of
mutations. Mutants are used either direct as new cultivars or as parent for devel-
opment of new cultivars (Waugh et al. 2006). Mainly improvement is brought by
mutation breeding through up-gradation of well-adapted genotypes which are
deficit for one or few traits (Wilde et al. 2012). Increase in genetic variability by
developing novel alleles, variety development in 2–3 generations, and generation of
chimera in somatic tissues are the characteristic benefits of mutation breeding
(Roychowdhury and Tah 2013). Seed, pollen, whole plant, tubers, cuttings, bulbs,
corms, stolons, tissues, and suspended cells could be used as plant material for
mutagenesis.
FAO/IAEA has developed a database called “Mutant Varieties Database”(http://
www-mvd.iaea.org). Details of all varieties developed through mutation breeding,
breeding methods, primary and secondary traits improved, year of registration, and
country of origin have been provided in this database. International Atomic Energy
Association (IAEA) has categorized the mutant database based on breeding
methods; (a) mutants directly used as commercial cultivars, (b) mutants used as one
parent in hybridization program, (c) commercial cultivars developed using both
mutant parents, (d) hybrid development using one mutant parent, and (e) commer-
cial cultivars developed through mutation of segregating populations. Aberrant
lateral root formation, unusual gravitropism behavior, lack of crown and brace
5.2 Conventional Breeding Strategies 49
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roots, and premature root degradation are observed by mutation of maize roots
(Feix et al. 1997).
Improvement in technology furnished mutation breeding with many
high-throughput techniques like, TILLING (Targeting Induced Limited Lesions IN
Genomes), EcoTILLING and high-resolution melt analysis (HRM). Efficiency and
efficacy of mutation breeding in crop breeding increased with the help of molecular
mutation techniques. TILLING is efficient reverse genetics tool which comprised
mutagenic treatment followed by detection of point mutations with the help of
sophisticated detection tools. Mutagenic treatment, development of segregating
population (M2), sample collection and their DNA extraction, pooling samples (8–
12), identification of induced point mutation, and validation followed by evaluation
of identified mutants are the key steps in the process of TILLING. Mutants
developed and identified through TILLING could be used in breeding programs and
gene-function assessment. TILLING was used for induction and identification of
point mutations in numerous crops. Maize pollen population was mutagenized
followed by detection of mutations through TILLING tools. DNA segment of 1 kb
was pooled from 11 different genes and 17 different-independent-induced point
mutations were obtained (Roychowdhury and Tah 2013).
Alternative TILLING approach, known as EcoTILLING, is effective tool for
identification of SNPs in natural populations which has been induced by sponta-
neous mutations. Point mutations, deletions, and insertion within target sequence
can be determined by EcoTILLING (Roychowdhury and Tah 2013).
High-resolution melt analysis (HRM) is an alternative screening approach which is
used for detection of mutations in genes that have multiple exons and introns.
Single-base mismatching is detected and effectively used for genotyping in medical,
single-nucleotide polymorphism (SNP) discovery and SNP genotyping in plants
(Zhou et al. 2004, 2005).
It is well admitted that conventional breeding approaches are effectively
involved in the improvement of drought tolerance in maize. These techniques are
effective because evaluation is made under field conditions and interaction with
environment is well considered. Mutation-assisted breeding alongwith modern
molecular techniques are effective in generation and identification of desired
mutations. Mutation breeding being nontransgenic approach is safe and secure
strategy for crop improvement. Ideotype for drought-tolerant maize genotype can
be developed through mutation-assisted breeding, so there is need to explore the
potential of mutations for maize improvement.
5.3 Marker-Assisted and Genomic-Assisted Breeding
Effectiveness of conventional breeding is reduced due to low heritability of traits in
field, high-field management cost, seasonal variability, time and space issues and
higher genotype into environment interaction. So marker-assisted selection
becomes effective tool because DNA present within cell is independent of
50 5 Biological Practices for Improvement of Maize Performance
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environmental and managerial effects. Pace of crop improvement increases by
marker-assisted breeding because these are based on cellular DNA.
Numerous DNA markers are available but plant breeder need ideal markers which
must have following characteristics features and must be strongly linked with trait
controlling genes, co-dominant inheritance, and PCR based; marker should depict
large variability for traits, polymorphic in nature, abundant in genome, and easy to
amplify (Varshney 2010). Marker-assisted selection is used for numerous tasks.
Genetic distance between parents, prediction of heterotic potential of hybrids, and
selection of inbred lines to be used as parent could be made effectively by fin-
gerprinting of inbred lines. Line conversion in maize can also be done through
marker-assisted backcrossing by transferring the one desired trait coding gene from
donor line to recipient line. Number of generations for backcrossing and probability
of linkage drag is reduced significantly in cases of marker-assisted backcrossing
comparative to conventional backcrossing (Bänziger et al. 2000).
Tolerance is complex feature governed by large number of traits, and these traits
are controlled by large number of chromosomal regions known as quantitative trait
loci (QTLs). Parents with contrasting phenotypic expression are crossed to develop
segregating progenies. Segregating populations are screened with the help of DNA
markers like, RAPD, RFLP, AFLP, SSR, and SNPs. Markers linked with specific
traits are then identified with bioinformatics tools. Exploitation of DNA-based
markers for identification of QTL mapping linked with morphological, physiological,
and biochemical traits could be targeted by breeder for drought resistance improve-
ment. After identification of QTLs linked with traits, drought tolerance can be
improved by introgression of these QTLs into modern promising cultivars.
Marker-assisted selection (MAS) based on trait-linked QTLs proved to be effective for
dissecting quantitative traits into unit genetic components and assisting plant breeder
to make appropriate-targeted selection (Chinnusamy et al. 2005; Hussain 2006).
Linkage mapping and association studies through association mapping and
candidate gene approach are effective for identification of QTLs. Most of QTL
studies in literature are based on segregation mapping but association mapping is
most vigorous tool than segregation mapping (Syvänen 2005). Monogenic traits like
plant height, osmotic adjustment, flowering time, and ear development are more
adaptive traits for drought tolerance. Genetic diversity for numerous morphological,
physiological, biochemical, and molecular drought responsive traits in maize is
reported. So, genetic variability could be exploited for improvement of drought
tolerance in maize through marker-assisted selection. MAS proved even more effi-
cient tool if markers are strongly linked with stress-responsive traits. Anthesis
silking interval (ASI) is very important trait in maize; lower ASI value is associated
with drought tolerance. CIMMYT identified six QTLs linked with ASI, which are
located on chromosome number 1, 2, 5, 6, 8, and 10 of maize genome. These QTLs
are contributing 50 % phenotypic variability of ASI and are stable across the years
and water regimes (Bänziger et al. 2000). Additive QTLs for ear length and kernel
weight, whereas epistatic QTLs for kernel number per row are observed in maize
recombinant inbred lines (RILs). Genetic background of QTLs is changed (additive
to apistatic and vice versa) under different water treatments for some maize traits.
5.3 Marker-Assisted and Genomic-Assisted Breeding 51
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Existence of additive and epistatic QTLs in maize shows that expression pattern of
traits is diverse and nature of drought tolerance is very complex (Lu et al. 2006).
Information obtained from MAS made the breeding programs more effective by
following ways; selection can be made at early generations and number of gener-
ations required for conventional breeding approach are reduced; accuracy of
selection is highly increased (Phelps et al. 1996). Depending on the targeted locus,
site of amplification, level of conservation, type of primers used, and breeding
objectives large number of DNA-based markers are being used, some of them are
enlisted here: random amplified polymorphic DNA (RAPD), selective amplification
of microsatellite polymorphic loci (SAMPL), restriction fragment length poly-
morphism (RFLP), sequence characterized amplified regions (SCAR), expressed
sequence tags (EST), simple sequence repeats (SSR), inter-simple sequence repeat
(ISSR), single nucleotide polymorphism (SNP), sequence specific amplification
polymorphisms (S-SAP), sequence tagged site (STS), sequence tagged
microsatellite site (STMS), single-primer amplification reactions (SPAR),
site-selected insertion PCR (SSI), single-stranded conformational polymorphism
(SSCP), short-tandem repeats (STR), diversity arrays technology (DART), and
variable number tandem repeat (VNTR) (Semagn et al. 2006).
Research efforts in maize were focused on the development of microsatellite
markers for germplasm analysis and genetic mapping. Gene mapping is helpful in
providing the information about specific locus of genes and number of genes gov-
erning the traits. Dubey et al. (2009) targeted the 24 accessions of tropical maize for
assessment of drought-linked SSR markers. They found that UMC1042,
DUPSSR12, UMC1056, BNLG1866, UMC1069, DUP13, BNLG1028, UMC1962,
and C1344 SSR markers were linked with drought responses. Tuberosa et al.
(2002a) and Sawkins et al. (2006) identified the QTLs in maize, which were linked
with drought. Introgression breeding in maize, introgression of transgenes, con-
version of simple or complex traits, and marker-assisted recurrent selection (MARS)
were breeding perspectives in maize for which markers were used with special focus
to drought stress (Ragot et al. 1995; Hospital et al. 1997; Sawkins et al. 2006).
Theoretically MAS is known to improve drought tolerance but practically
contribution of MAS in release of high yielding drought-tolerant genotype is
nonsignificant (Reynolds and Tuberosa 2008). So, focus should be targeted that
markers linked with drought tolerance should also be linked with higher yield
potential for getting two fold benefits. In literature, few cases had been reported
which showed the involvement of MAS in development of drought tolerance
cultivars with higher yield potential (Reviewed by Athar and Ashraf 2009). Ribaut
and Ragot (2007) mentioned that introgression of five QTLs in maize has increased
50 % yield comparative to standard hybrids under drought stress, and no yield
losses were observed under normal water availability. Introgression of yield-linked
QTLs in pearl millet improved the grain yield in drought sensitive genotypes (Serraj
et al. 2005b). Stay-green character of sorghum is improved by introgression of
QTLs (Harris et al. 2007).
QTL mapping enabled the breeders to identify the chromosomal regions linked
with different plant traits. Effect of genetic background, complex genetic basis,
52 5 Biological Practices for Improvement of Maize Performance
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stage of plant growth and development, environment ×QTL interaction (Tuberosa
et al. 2002b), gene by gene effects, inadequate phenotyping, cost, and skill issues
are limiting the effectiveness of QTL mapping (Campos et al. 2004; Xu, et al.
2009). QTL identification, validation in different populations, or under different
environments followed by their proper manipulation in breeding program could be
much more effective for real-sense improvement against drought stress. There is
still gap which must be filled for getting more benefits from marker-based selection.
Functional genomics and transcriptomics are recently used for extensive
understanding of plant responses against stresses. Identification of candidate gene,
followed by characterization, and determination of transcriptomic responses
through microarray or whole genome sequencing help to clearly highlight the
tolerance mechanisms. Drought responsive candidate genes are identified by
imposition of drought stress on stress-responsive genotypes followed by ESTs
generation from either normalized or nonnormalized cDNA library. Public data
bases are being exploited for retrieval of drought stress-responsive candidate genes
in major crops like wheat, maize, barley, and rice (Sreenivasulu et al. 2007;
Kathiresana et al. 2006).
Transcript profiling is used for identification of candidate genes through
assessment of differential gene expressions in a specific tissue at different times
(Hampton et al. 2010). Transcript profiling can be done through cDNA–amplified
fragment length polymorphism (cDNA–AFLP), PCR-based differential display
PCR (DDRT-PCR) analysis, digital expression analysis based on counts of ESTs,
cDNA and oligonucleotide microarrays, serial analysis of gene expression (SAGE)
technique, SuperSAGE, and next generation sequencing (reviewed by Mir et al.
2012). Among these techniques, next generation sequencing (NGS)-based tech-
niques are most preferred for routine transcript profiling of main crops for identi-
fication of drought-tolerant candidate gene followed by exploitation of that gene
through genomics and marker-assisted breeding. After identification of major
QTLs, contributing to drought tolerance, these are validated in target population.
Following validation, these QTLs could be exploited through their introgression
into high yielding and drought susceptible (recipient parent) from low yielding and
drought-tolerant parent (donor parent), this technique is known as marker-assisted
backcrossing (MABC). Birsa Vikas Dhan 111 (PY 84), a rice variety, was devel-
oped through marker-assisted backcrossing in India having improved drought tol-
erance (Steele et al. 2007). Complexity of mechanism of drought tolerance act as
barrier for substantial exploitation of MABC e.g., almost 10 % phenotypic vari-
ability was explained by identified QTLs in maize (Xu et al. 2009). These findings
conclude that extensively large population size is mandatory for achieving satis-
factory improvement through MABC.
MABC acts as effective tool when traits are governed by single or few genes but
in case of drought tolerance which is very complex feature and governed by large
number of genes, this technique becomes least effective. Marker-assisted recurrent
selection (MARS) has capability to deal with complex traits like drought tolerance
and involves the inter-mating of selected accessions in each recurrent cycle (Ribaut
and Ragot 2007). Population improvement is adequately accomplished by MARS
5.3 Marker-Assisted and Genomic-Assisted Breeding 53
aslampbg@uaf.edu.pk

because MAS is practiced in each selection cycle followed by interbreeding of
selected individuals, which validates and increases the frequency of desired genes
in target population (Eathington et al. 2007). MARS is being used for drought
tolerance improvement in different crops e.g., wheat, chickpea, cowpea, and sor-
ghum (reviewed by Mir et al. 2012). Plenty of work needed to be done for
improvement of drought tolerance in maize through MARS.
Genome selection (GS) and genome-wide selection (GWS) are important
molecular techniques for improvement of drought tolerance in crop plants by
developing superiorly drought-tolerant genotypes. Unlikely of MARS, genome
selection is done through genome-wide marker genotyping. Breeding methodology
for GS was described by Meuwissen et al. (2001) and Mir et al. (2012). GS has
numerous advantages over other techniques like, reduced selection time, increased
annual gain from selection, and reduced phenotyping frequency (Rutkoski et al.
2010). Initiative for exploration of GS potential in different crops has been taken but
its application for improvement of drought tolerance (Mir et al. 2012) especially in
maize is lacking. Exploration of GS in maize for the improvement of drought
tolerance is recommended.
5.4 Transgenic Maize Development
Genetically complex nature of drought tolerance makes the transgenic development
even more complex than for monogenic traits. Exploitation of signal transduction
cascades, transcription factors, or transformation with numerous genes regulates the
pivotal processes. But current research work is focused on single-gene transfor-
mation. Signal transduction pathways are activated by stress responses which
resultantly regulate the cascades of adaptations. These signaling pathways can be
modified or tailored using tool of genetic engineering. Development of
drought-tolerant transgenic crop basically involves the incorporation of one or more
genes from other donor source/sources in target crop to modify the signaling and
subsequent events (Vinocur and Altman 2005). Genes which can be manipulated in
genetic engineering are categorized into four main classes: (1) genes involved in
transcriptional and signal transduction pathways, (2) genes involved in protection of
cellular membranes and biosynthesis of stress-responsive proteins, (3) genes
involved in uptake of ions and water-like ion transporters and aquaporins (Wang
et al. 2003), and (4) genes involved in cellular metabolism e.g., free amino acids,
proline, soluble sugars, polyols, and glycinebetaine (Vinocur and Altman 2005).
Transgenic constitutive upregulation of transcription factors (TFs) improve
drought tolerance but these TFs also upregulate other genes which impair normal
plant growth and development resultantly reduced economic yield (Wang et al.
2003). Alternative to TFs, stress-induced promoters could be exploited for
improvement of drought tolerance because their side effects are far less than TFs
(Athar and Ashraf 2009). NADP-malic enzyme, key enzyme of C4 photosynthesis,
from maize was transgened in tobacco which reduced stomatal conductance and
54 5 Biological Practices for Improvement of Maize Performance
aslampbg@uaf.edu.pk

improved water use efficiency (Laporte et al. 2002). So functional sustainability of
this enzyme in maize under drought stress definitely will improve the tolerance by
improving water use efficiency. Transgenic plants for improved drought were
developed in arabidopsis, rice, wheat, tobacco, tomato, brassicas, and others (Athar
and Ashraf 2009). Monsanto developed drought-tolerant transgenic maize genotype
known as MON87460 which has permission for sale in United States Department
of Agriculture (Gilbert 2010). Monsanto and BASF developed another maize
transgene known as DroughtGuard Hybrid Corn which carried the cold shock
protein (CSPB) from Bacillus subtilis. Results of multilocation yield trails showed
that yield of transgenic maize was 5774.5 kg/ha and yield of nontransgenic standard
was 4770.25 kg/ha. mtID (bacterial mannitol-1-phosphate dehydrogenase) and
HVA1 (Hordeum vulgare) pyramiding in maize improved the drought tolerance
(Nguyen et al. 2013). Mitogen-activated protein kinase kinase kinase (MAPKKK)
is involved in conferring tolerance against different abiotic stresses. MAPKKK
from tobacco called NPK1 was transgened in maize which improved drought tol-
erance by improving photosynthesis rate and grain weight under drought stress
relative to nontransgenic maize (Shou et al. 2004).
Theoretically, it is possible to improve drought tolerance by developing trans-
genic crop plants but practically there are many limitations which reduce the
effectiveness of transgenic crops. Side effects of transgenes and complexity of
tolerance mechanism make improvement very difficult (Cattivelli et al. 2008).
Suitability of transgene, dosage effect, level of tolerance, side effects of transgene,
yield penalty and socio-scientific acceptance of transgenics in food crops like,
maize, are determinants for effectiveness of transgenic development.
5.4 Transgenic Maize Development 55
aslampbg@uaf.edu.pk

Chapter 6
Conclusions and Summary
Drought is major abiotic stress which hinders crop productivity across the world.
Drought affects numerous crop plants at different levels of growth and develop-
ment. Maize is important cereal crop and grown in large number of countries across
the world. Effects of drought stress on maize are prevalent from germination to
harvest maturity. Germination percent, germination potential, germination rate,
seedling establishment and seedling vigor are disturbed by drought at early growth
stages. Growth and development of vegetative parts of maize are seriously affected
by diminished cell division and cell proliferation which clarified that cell cycle is
critically dependent on water status of plants. Plant height, stem diameter, plant
biomass, leaf area and root development are disturbed in maize by drought stress.
Reproductive stage in maize is more critically impaired by drought stress.
Development of tassel and ear, pollination, fertilization, embryo development,
endosperm development and grain filling are seriously affected by drought stress in
maize.
All genotypes of maize are not equally affected by drought stress due to high
level of variability in genetic background of this crop. Different mechanisms have
been evolved in maize like other crops which enable them to effectively survive
under drought stress. Drought escape, drought avoidance and drought tolerance are
different mechanisms which work under the heading of drought resistance. These
mechanisms are evolved in different maize genotypes through course of evolution
and domestication. Drought escaper maize genotypes modulate their life to be
completed before the onset of drought and drought avoider maize genotypes avoid
themselves from drought either by reducing water losses or by increasing water
uptake. Drought tolerant maize genotypes maintain their growth and development
along with economical grain yield under drought stress. On the other hand, drought
susceptible maize genotypes may be lacking any one of these adaptive mechanisms
and facing severe damage in terms of growth, development and grain yield.
Drought tolerance is very complex mechanism which is collaboratively conferred
by osmotic adjustment, plant growth regulators, antioxidative defense, stress
responsive proteins, water channel proteins, transcription factors and signal trans-
duction pathways.
CIMMYT, IITA, Monsanto, Syngenta and Pioneer are the leading maize
research groups in the world. These groups have worked and working on numerous
©The Author(s) 2015
M. Aslam et al., Drought Stress in Maize (Zea mays L.),
SpringerBriefs in Agriculture, DOI 10.1007/978-3-319-25442-5_6
57
aslampbg@uaf.edu.pk

aspects of maize crop however, drought tolerance is also one of their key research
objectives. Numerous research projects have been completed in which different
breeding methods were practiced for improvement of drought tolerance in maize.
Drought tolerant maize hybrids and OPVs are practically cultivated in numerous
African countries whoever, Monsanto has developed drought tolerant transgenic
maize.
As demand of maize is increasing day by day and water scarcity is increasing so,
there is dire need to further improve the level of drought resistance in maize.
Different strategies are used for improvement of maize against drought stress e.g.
managerial strategies and biological strategies. Managerial strategies involve the
usage of water resources and adoption of water saving agronomic practices. On the
other hand biological approaches deal with manipulation of genetic background of
maize for improvement against drought stress. Biological strategies are preferred
over managerial practices due to long term and economical effectiveness. Biological
approach is practiced in different forms. Available germplasm has lot of genetic
variability which can be exploited for higher drought tolerance through effective
screening tools. Conventional breeding, mutation breeding, marker assisted and
genomic assisted breeding and development of drought tolerant transgenic maize
are numerous strategies which are enlisted under the heading of biological strate-
gies. There are few gaps in effectiveness of biological techniques which must be
filled under the umbrella of modern technology for the improvement of drought
tolerance of maize in such a way that it can fully combat with drought stress. Lots
of novel breeding and evaluation techniques have been developed in recent past and
practical application of other techniques will further help to cope the problem of
drought stress by development of more drought tolerant maize genotypes for res-
olution of food security.
58 6 Conclusions and Summary
aslampbg@uaf.edu.pk

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