Plant-Environment Interaction: Responses and Approaches to Mitigate Stress, First Edition. Edited by Mohamed Mahgoub Azooz and Parvaiz Ahmad.

© 2016 John Wiley & Sons, Ltd. Published 2016 by John Wiley & Sons, Ltd.

1

1.1 Introduction

For food, humans rely on approximately 275 crops

(Tilman et al., 2011). Out of these, three crops, wheat,

maize and rice, are significant cereal crops that con­

tribute to major dietary requirements as staple foods for

humans – a reason why they are collectively termed the

‘big three cereal crops’ (Shewry, 2009). Comparatively,

wheat is the most important cereal crop that contributes

a major portion of the daily diet for humans (Slade

et al., 2012). It is estimated that wheat is a source for

one‐fifth of total calories utilized by humans globally

(Waines & Ehdaie, 2007). Wheat grains contain vital

constituents such as carbohydrates, including 60–70%

starch (Slade et al., 2012) and 8–15% protein such as

glutenin (Shewry et al., 1995) and gliadin (D’Ovidio &

Masci, 2004). From the total wheat grain produced

globally, 65% is utilized as food by humans while the

remaining 35% is distributed among livestock feed

(21%), seed material (8%) and raw material (6%) in

industries such as the production of vitamins and anti­

biotics, manufacturing of paper; it is also used as a

fermentation substrate or as adhesives in various prod­

ucts (Shewry & Jones, 2005).

1.1.1 History of wheat: from domestication to revolutionsIn ancient times, wheat was a product of the activities

of  hunter‐gatherers but about 10,000 years ago, the

Neolithic Revolution laid the basis for domestication of

various crops (Waines & Ehdaie, 2007). This domestica­

tion process focused mainly upon cereal crops, and

wheat is considered the originator of domesticated crops

(Peleg et al., 2011). With the passing of time, problems

arising in the domestication process compelled scientists

to analyse and study various concerns such as local con­

ditions, yield maximization, development of improved

cultivars and storage techniques (Cavanagh et al., 2013).

Eventually, these findings resulted in major events such

as the Agricultural Revolution in the 19th century

(Godfray et al., 2010) and the Green Revolution in the

20th century (Waines & Ehdaie, 2007).

Wheat domestication followed by major revolutions

and scientific achievements contributed to speciation

and initiation of new varieties (Shewry, 2009). The

factors involved in such speciation primarily include

adaptations to the ecology of an area as soon as wild‐

type wheat cultivars were moved for domestication

purposes (Chaudhary, 2013). These adaptations under

the influence of epigenetics offered the opportunity to

select the desired traits in wheat such as yield, grain

quality, grain size and many other phenotypic attributes

(Burger et  al., 2008). Thus, wheat evolved into many

varieties in response to human cultivation practices,

selection procedures and the phenomena of epigenetics

(Fuller, 2007).

Since the Green Revolution, technologies have

been  incorporated into crop improvement practices,

specifically wheat, in various ways (Schmidhuber &

Tubiello, 2007). These include successful development

of hybrids with enhanced desired traits, development of

pathogen‐resistant plants, enhanced yield, improved

nutrient contents, affordable fertilizer requirements

and improved irrigation systems (Godfray et al., 2010).

Biotechnological applications to improve salinity stress in wheatSami ullah Jan1, Ghulam Kubra1, Mehreen Naz2, Ifrah Shafqat2, Muhammad Asif Shahzad1, Fakiha Afzal1 and Alvina Gul Kazi1

1 Atta‐ur‐Rahman School of Applied Biosciences (ASAB), National University of Sciences and Technology (NUST), Islamabad, Pakistan2 Department of Bioinformatics and Biotechnology, International Islamic University, Islamabad, Pakistan

CHapter 1

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COPYRIG

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ATERIAL

2 Chapter 1

The consequences of all aspects of the Green Revolution

increased yield to fulfil the world’s food requirements

(Tilman et al., 2011).

1.1.2 Wheat genomeModern wheat includes six sets of genomes, called hexa­

ploidy, and is a result of domestication and scientific

processes practised by man. Polyploid genomes of wheat

cultivars evolved after crossing or hybridization, selec­

tion procedures and cultivation practices in domestication.

The wild wheat ancestor Triticum turgidum sp. dicoccoides is

considered as the first domesticated wheat species in

the Near East region (Maier, 1996). This wheat species

was spread across Europe and gave rise to new varieties

like Triticum turgidum sp. dicoccum and Triticum turgidum

sp. durum (Buckler et  al., 2001). Durum wheat is still

widely grown in the Near East crescent around the

Mediterranean Sea (Thuillet et al., 2005).

In reference to common bread wheat, this is an

allopolyploid consisting of three genomes designated

as  A, B and D originating from wild wheat grasses of

the  genera Triticum and Aegilops (Zohary et  al., 1969).

Modern wheat is hexaploid, existing in three sets,

A‐Genome, B‐Genome and D‐Genome. The ancestor

of  A‐genome wheat Triticum urartu contained 14

chromosomes in two sets, and was crossed with Aegilops

speltoides (B‐genome) that resulted in a hybrid which

contained both genomes (AB) which after doubling

yielded a viable tetraploid containing 28 chromosomes

(AABB). This hybrid, known as wild emmer (Chen

et al., 2013), upon further crossing with Aegilops squar-

rosa (a diploid grass), produced a new hybrid with 21

chromosomes (42 chromosomes in diploid form). The

later hybrid produced is the hexaploid wheat utilized

today and contains genomes from three ancestors

(AABBDD) (Levy & Feldman, 2002).

1.1.3 Wheat production and concernsDuring the past 50 years, research and technological

applications in the cultivation of wheat have increased

its yield to a rate of 41 kg per hectare (Ewert et  al.,

2005). But the world’s population is increasing all the

time (Godfray et al., 2010). If this continues, by the mid‐

century, the world’s population is estimated to be 9–10

billion (DeLong et al., 2010). Simultaneously, demands

for more food and energy resources will also be raised

such that, by the middle of the century, necessary food

production will be double that of the present (Ray et al.,

2013). Numerically, the required rate of increase in food

production by the year 2050 is 100–110% compared to

the current rate of production (Tilman et  al., 2001).

About 600 million metric tons of wheat is produced per

year worldwide but with the increment in population,

by 2020 we would require an estimated yield of 1 billion

metric tons (Shewry, 2009). In 2005, calculated yield

per hectare of wheat was 2.5 tons which was forecasted

to reach a figure of 4.0 t/ha by 2020 (Rajaram, 2005).

Despite these important facts, only 3 billion hectares

of land out of 13.4 billion hectares is available for crop

cultivation (Smith et  al., 2010). One solution to over­

coming the world’s food requirements is to turn more

land over to arable in order to increase wheat global pro­

duction (Gregory et al., 2002). It has been estimated that

by utilizing only 20% of untilled land, we could increase

crop yields up to 67% (Bruinsma, 2003). In 2007, total

yield of cereal crop was 3.23 tons per hectare which

could be increased to 4.34 tons per hectare by increasing

land under cultivation to 25% (Bruinsma, 2009). The

actual figure for per capita arable land is continuously

decreasing due to industrialization, housing and defor­

estation as well as some environmental concerns

(Gregory & George, 2011). However, environmental

concerns are among the major problems that cause the

loss of yield such that only 50–80% yields are achieved

(Lobell et al., 2009). Various scientific communities con­

tribute to minimize the gap between actual and potential

yields (Jaggard et al., 2010) but the problems remain the

same and the environmental concerns are important,

such as abiotic (salinity, drought, temperature) and

biotic stresses (Atkinson & Urwin, 2012).

1.2 Salinity stress is a striking environmental threat to plants

Agricultural production all over the world is constrained

by salinity stress and it is becoming a growing universal

issue that affects almost 20% of cultivated land globally

(Flowers & Yeo, 1995). From the agricultural point of

view, salinity is the aggregation of dissolved salts within

soil or agricultural water to an extent which adversely

affects plant growth (Gorham, 1992). High salinity

influences the physiological mechanism that adversely

affects plant growth and development which necessi­

tates detailed investigation of tolerance mechanisms in

salinity (Abogadallah, 2010).

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Biotechnological applications to improve salinity stress in wheat 3

Salinity‐induced stress increases the accumulation of

salts in plant roots (Zhang et al., 2014). Such hyperac­

cumulation of salts in roots restricts water absorption

from the soil surface and thus also causes water stress, in

spite of available water at the root zone. Water absorption

from saline soils requires extra energy expenditure.

Thus, higher salinity will always lead to decreased levels

of water as well as inducing analogous stresses like

water and osmotic stress (Bauder & Brock, 1992).

1.2.1 Statistics of salinity stress‐affected landSaline soils are widespread in arid and semiarid regions,

especially in areas where heavy irrigation or overfertili­

zation is common (Reynolds et al., 2005). It is estimated

that 800–930 million hectares (7%) of the world’s total

arable land is influenced by salt stress (Shannon, 1997;

Szabolcs, 1994) while 230 million hectares of irrigated

land are affected by salts (Oldeman et  al., 1991).

Extensive salts in soil arise due to natural processes such

as rainfall containing salts as well as irrigation practices

such as the use of fertilizers, resulting in poor water

quality (Reynolds et al., 2005).

1.2.2 Causes of salinity stressSalinity is a primary abiotic stress that hinders plant

growth. Numerous causes are responsible for salinity.

Some prominent causes include extensively irrigated

lands, use of improper waters, inefficient drainage,

practising inappropriate irrigation, standing water for

prolonged time and water seepage from reservoirs.

Underground leakage or seepage from water reservoirs

tends to raise the water table which mobilizes salt and

thus causes salinity stress (Awad, 1984). Further,

increment of the saline water table to 2 meters speeds

up the evaporation process, leaving excessive salt in the

soil, resulting in waterlogged soil. Due to the limited

oxygen in water‐logged soils, the survival of vegetation

is endangered. Another important reason causing

salinity is heavy rainfall which drains salts away with it

and may saturate cultivated land.

Despite such diverse causes, the main reasons for

salinity are (1) natural (primary) salinity, and (2) human‐

induced (secondary) salinity.

Primary salinity or natural salinity is a result of

a  prolonged natural build‐up of salt in water or soil

which occurs by the natural process of breakdown of

rocks containing SO4

2–, Ca2+, CO3

2–, Cl–, Na+ and Mg2+.

Furthermore, evidence of ocean salt conveyed by wind

and downpour is likewise a reason, which changes with

the types of soil.

More specifically, the main reason for natural salinity

is the excess accumulation of salts at the soil surface.

Among all these causes, studies have revealed that those

salts which accumulate and cause salinity stress are com­

posed of a particular set of ions, including Na+, K+, Ca2+,

Mg2+ and Cl–. However, among these, Na+ is more domi­

nant and its excess makes soil sodic. Sodic soil is more

challenging because it has a poor architecture, thus

limiting or preventing infiltration of water as well as

drainage. Some soils harbour large amounts of salt which

are flushed away along with rain or irrigation water

drainage. Salt accumulation in the form of precipitation

or resulting from weather changes as well as mineral

degradations also leads to salinity. Salt accumulation in

dry lands is very common and rapidly causes salinity

conditions (Brinkman, 1980).

Secondary salinity or human‐induced salinity is

induced because of anthropogenic activities that disturb

the hydrological stability of the soil between water

connected (watering system or precipitation) and water

utilized by plants (transpiration) (Garg & Manchanda,

2008). The water table is increased because of the large

quantity of water supplied to the irrigated fields with

poor drainage systems. Globally all these watering sys­

tems may result in secondary salt stress and saturation

of soil with water (Garg & Manchanda, 2008).

1.2.3 types of salinity stressSalinity stress can be classified on the basis of its initia­

tion process, mobilization means and mode as well as

their impacts. Regardless of the sources or types of

salinity, the effects of salinity on plant development

remain constant but these effects are variable in terms

of salt concentration encountered by plants. A compre­

hensive classification of salinity is presented as follows.

1.2.3.1 Dry land salinity stressDry land salinity refers to a type of salinity that occurs in

unirrigated land. Such land is usually colonized by

shallow‐rooted plants instead of deep‐rooted crops

because the shallow‐rooted plants can withstand less

water and their growth cycle is short. Dry land, as com­

pared to normal or moist land, increases seepage rates to

ground water and induces mobilization of salts pre‐

stored in soil. In low areas or slopes, ground water along

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4 Chapter 1

with its salts is exposed to evaporation, leaving salts

at  the soil surface and thus increasing salinity–plant

interactions. Likewise, ground water may also finally

be  added to streams and rivers, in which case, salt

concentration in water resources is increased and when

these resources are utilized for irrigation purposes, they

will cause salinity stress to plants (Dias & Thomas, 1997).

Dry land salinity is dependent upon type and compo­

sition of soil when subjected to irrigation. Predominantly

sodic or naturally saline clays fall under the category of

dry land salinity which may also spread if such soil is

transported by any means and/or mixed with other

soils. However, when sodic soil becomes wet, it dis­

perses and causes blockage of pores within the soil

which are common routes of water seepage towards

ground water. In other cases, when sodic soil is dry, it

becomes dense and hard and forms a crust on the soil

surface. In the latter case, soil structure loses its capacity

for infiltration of water, leaving no or little salt at the

root. In some conditions, subsodic soils can produce a

perched water table which creates water logging at the

root zone. In view of such conditions, if large amounts

of water are applied in irrigation, it will increase the

ground water level which eventually causes irrigation

salinity. This problem will tend to increase if such soils

are accompanied with crops that utilize lower amounts

of water. The worst condition of irrigated salinity can

occur if water for irrigation purposes is obtained from

salty ground water or salty rivers (Dumsday et al., 1983).

1.2.3.2 Urban salinity stressUrban salinity includes the activities involved in urban­

ization and its development as well as excessive use of

water for gardens and other low‐level cultivation prac­

tices. Other phenomena include leakage from pipelines,

tanks and drainage systems or changing water flow

direction from its normal routes. All these factors

support the rise of the water column in the ground

water table. Sources of salt arising from these processes

include material used in buildings, waste from indus­

tries, use of chemicals and fertilizers, salt effluents as

well as naturally occurring salt.

Industrial waste and effluents are excreted from

industrial and some domestic areas; those which have

high salt concentrations have proved more dangerous

compared to other categories in urban salinity. Industries

using coal for fire also use huge amounts of water for

cooling purposes which results in evaporation of water

while salts are deposited and finally added to soil.

Similarly, mining activities also play a role in causing

urban salinity (Murray, 1999).

1.2.3.3 River salinity stressRivers are the major source of irrigation but this source

is very much contaminated with salts. All the drainage

from domestic and industrial systems, as well as affected

dry lands, finally end in a river. The same water is recy­

cled for irrigation purpose which creates a stress

environment for plants. With the passage of time, the

quality of river water becomes more saturated as water

level decreases in saline rivers which in turn irrigates

plants, resulting in impaired plant survival (Awang

et al., 1993).

1.2.3.4 Irrigation salinity stressWhen the source of water used for plants is a saline

water reservoir, a condition arises known as irrigation

salinity. It is different from river salinity because river

salinity refers to salinity caused by river water used for

irrigation purposes while irrigation salinity encompasses

the salinity caused by any water source for plants (Ayres &

Westcot, 1985). Sources of salts in such conditions are

mostly similar to those mentioned in rivers and dry land

salinity. However, in addition, climate and routes of irri­

gation system also determine the irrigation salinity

levels (Bauder & Brock, 2001).

1.3 effects of salinity stress on wheat

Wheat can tolerate salt to some extent but as the salinity

concentration increases, more serious risks of damage

are likely to occur. The vast dimensions of threats to

wheat and other plants by salinity stress range from its

physiological characteristics to vital biochemical path­

ways and its genetic make‐up. These effects include

hindrance of seed germination and seedling growth and

dry mass accumulation in wheat (Shirazi et  al., 2001;

Sourour et  al., 2014), disturbed biochemical pathways

(Radi et  al., 2013) and disorganized patterns of gene

expression. Many investigators have screened numerous

wheat cultivars, including salt‐tolerant varieties, to

check the effects and variations due to salinity on var­

ious growth levels (Munns & Termaat, 1986). Some of

the patterns of salt effects on numerous attributes of

wheat are discussed below.

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Biotechnological applications to improve salinity stress in wheat 5

1.3.1 effects of salinity stress on phenological attributes of wheatTo assess the impact of salinity on some physiological

(Yang et al., 2014) and phenological qualities in durum

wheat, a factorial investigation was directed at time of

stem lengthening, time to heading and development,

chlorophyll content and chlorophyll fluorescence of

leaf. Results demonstrated that the relation between

salinity and cultivars (61130 and PGS) causes expansion

in time to heading (Kahirizi et  al., 2014). Distinctive

attributes like grain number obtained on a meter square

area, grain yield, 1000 grains weight, leaf’s Na+ and

K+ ion contents and plant’s height had been measured

in field analyses for bread wheat. Numerous quantitative

trait loci (QTL) were determined by ecological contacts

which demonstrate several phenological characteristics

for salt resistance. Numerous QTL for biomass of seed­

lings and end of Na+ particles were seen in hydroponic

and field tests (Genc et al., 2013).

Excessive salinity has always diminished development,

yield and related yield characteristics, photosynthetic

traits (Mathur et  al., 2013), ionic substances and

biochemical activities in all wheat cultivars. Nonetheless,

application of potash composts in soil and also in foliar

application counteracts the unfavourable impacts of salt

stress on all wheat cultivars and the application of sul­

phate of potash (SOP) in soil and as a foliar splash was

more compelling in triggering salt stress tolerance. No

unfavourable impact of chloride on plant development

was noticed. Among the cultivars, ‘S‐24’ and ‘Sehar’

demonstrated remarkable development, yield and

biochemical matter and subsequently could be utilized

as parental material for achieving better yield under

saline conditions (Ashraf et al., 2013).

1.3.2 effects of salinity stress on morphological attributes of wheatMorphologically, salt stress causes reduction in overall

plant growth but shoots are seen to be more affected

than roots and the ratio between them is increased

(Allen, 1995). Salt stress also reduces the total dry

matter yield and number plus appearance of wheat tiller

(Mass & Poss, 1989). Salt stress also causes stunting of

shoots (Walker et al., 1981). Two reasons why increased

salt levels found in soil water cause reduction of plant

growth are (1) plants’ ability to take up water from soil

is decreased due to ionic imbalance which decelerates

natural growth processes, and (2) when salts get into a

plant through any possible route, such as transpiration

stream into the transpiring leaves, this eventually

injures cells which also restricts growth (Munns, 2005).

Initial contact with salinity causes water stress in

plants, resulting in reduction of leaf expansion ability.

Another associated problem, osmotic stress, also arises

in plants on initial exposure to salt stress and can lead to

inhibition of cell division, expansion and function of

stomata (Munns, 2002).

Soil salinity causes formation of Na+ and Cl– that

affects the ionic composition taken up by plants

(Rengasamy, 2006). Salt stress directly affects plant

growth through osmotic stress and ionic toxicity caused

by Na+ and Cl– ions which promote imbalance in plant

nutrient metabolism (Rowell, 1994). Adverse effects of

salt stress on cell morphology include accumulation of

toxic ions that disrupts intra‐ and extracellular compo­

nents like DNA, enzymes, membranes, mitochondria,

chloroplasts and many more by the development of

reactive oxygen species (ROS) (Allen, 1995; Saqib

et al., 2012).

1.3.3 effects of salinity stress on physiological attributes of wheatSalt stress has numerous consequences for germination

methods. Germination is delayed due to high salt con­

centrations in soil which create high osmotic pressure,

thus reducing water intake by seeds (Khan & Weber,

2008), that may cause the metabolism of nucleic acid

digestion (Gomes‐Filho et al., 2008), changes in metab­

olism of protein (Dantas et al., 2007) and aggravation of

hormonal offset (Khan & Rizvi, 1994), as well as less­

ening the ability to utilize seed stores (Othman et  al.,

2006). It might likewise influence the fine structure of

cell, tissues and organs (Al‐Maskri et al., 2014; Rasheed,

2009). However, there are different intramural (plant)

as well as external (natural) aspects that influence seed

germination under saline conditions which incorporate

nature of seed layer, seed torpidity, seedling power, seed

polymorphism, seed age, water, gases (Mguis et  al.,

2013), light and temperature (Wahid et al., 2011).

Death of the plant occurs at higher concentrations of

the salt as a result of hyperionic and hyperosmotic

stress. The result of these impacts may cause membrane

layer harm, nutrient unevenness, distorted levels of

enzymatic hindrance, developmental regulators and

metabolic abnormality, including photosynthesis which

at last prompts plant demise (Hasanuzzaman et al., 2012;

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6 Chapter 1

Mahajan & Tuteja, 2005). Similar consequences have

been observed in various varieties globally, including

Triticum aestivum (Akbarimoghaddam et  al., 2011),

Brassica spp. (Ulfat et al., 2007), Zea mays (Khodarahmpour

et  al., 2012), Oryza sativa (Xu et  al., 2011), Vigna spp.

(Jabeen et  al., 2003), Helianthus annuus (Mutlu &

Buzcuk, 2007) and Glycine max (Essa, 2002). It was

observed that germination of seeds has a negative rela­

tionship with salinity (Rehman et  al., 2000). Higher

salinity represses the germination of seeds while seed

dormancy is incited by lower levels of salt stress (Khan &

Weber, 2008).

The most harmful ions which cause several

physiological impacts on plants are Na+ and Cl–

(Tavakkoli et al., 2010). Many stresses, abiotic and biotic,

affect photosynthesis which is a complex process

involving many components such as photosynthetic

pigments, photosystems, the electron transport system,

CO2 reduction pathways, etc. Stress of any kind can

affect any of these components, reducing the photosyn­

thetic capacity of plants. To combat this harm, harvested

plants typically utilize protein kinases, for example,

MAPKs and transcription factors (Ashraf et  al., 2013;

Saad et al., 2013; Zhang L et al., 2012).

Research undertaken by Rajendran et  al. (2009)

showed the impact of ion exchange under salt stress in

advanced stages of plant development. They observed

that hazardous ions accumulated after 2–4 weeks of

exposure of salt stress. The stress caused by ions (Na+

and/or Cl–) overlaps with the osmotic impacts and dem­

onstrates more hereditary variety than osmotic impacts

(Munns et al., 2002).

1.3.4 effects of salinity stress on biochemical attributes of wheatFrom the biochemical point of view, life‐sustaining

pathways like respiration (Shekoofa et  al., 2013) and

photosynthesis as well as their associated enzymes are

affected by high salt levels while responses to these

salts by cellular machinery mostly use enzymes (Walker

et  al., 1981). In such conditions, salt stress triggers

further phenomena such as hyperosmotic shocks, cell

turgidity is lost, ROS are formed and stomatal size is

minimized (Price & Hendry, 1991). Eventually, these

conditions collectively or individually may restrict

plant growth.

As NaCl is an actual component of saline soil, plants

gather Na+ and Cl– particles up to levels that are

detrimental (Cuddy et al., 2013). Shoot Na+ poisonous

quality is connected with the decline of stomatal con­

ductance while high shoot Cl– levels immediately

influence chlorophyll and repress photosystem II

(Ashraf & Ashraf, 2012; Tavakkoli et al., 2011).

Higher Na+ and Cl– in plant cells are seen as the key

components of ionic damage (Cuin et al., 2009; Munns &

Tester, 2008; Rajendran et al., 2009). There are various

studies and evaluations that discuss the connection of

Na+, K+ and K+/Na+ homeostasis with salt stress toler­

ance in harvest plants (Horie et al., 2009). The effect of

Cl– homeostasis in salt tolerance is little understood

(Teakle & Tyerman, 2010). The elevated levels of Cl–

that congregate in the plant leaves developed under

saline conditions will affect the whole plant (White &

Broadley, 2001). Hence, it is striking that minimal

investigation has been undertaken into the impacts of

Cl– content in connection to salt tolerance; however,

there are no reports on the hereditary control of this

attribute. Just a few studies showed that treatment of

Cl– may be essential for salt tolerance in a few products

including grain (Tavakkoli et al., 2010a; Tavakkoli et al.,

2010b; Teakle & Tyerman, 2010).

Metabolically harmful quantities of Na+ to a greater

extent are a consequence of its capability to contend

with K+ for binding components vital for cell capacity.

High Na+/K+ proportions can disturb different enzymatic

courses of action in the cytoplasm (Tester & Davenport,

2003). Stress caused by ions is connected with a decline

in chlorophyll content and restrains photosynthesis,

impelling leaf senescence and early leaf fall. Ionic stress

consequently diminishes photosynthesis limit, biomass

and yield (Tester & Davenport, 2003).

In one study, the impact of salicylic acid or indole­

acetic acid (IAA) was tested by spraying them on

Triticum aestivum genotypes which were subjected to

different saline levels in order to check their impact on

growth of different plant organs. It was concluded

that  under such circumstances, cell enzymes having

antioxidant properties like reducing sugars, catalase,

peroxidase, ascorbate peroxidase, photosynthetic

shades, amino acid, proline in shoot and root were

enhanced (Nassar et al., 2013).

Salinity has been reported to induce oxidative stress

as the ROS are enhanced by producing superoxide (O2),

hydorxyl (OH) and hydrogen peroxide (H2O

2) radi­

cals. As the accumulation of ROS increases, scavengers

initiated protection mechanisms in plants from

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Biotechnological applications to improve salinity stress in wheat 7

salinity‐induced damage (Al‐Quraan et al., 2013; Hayat

et  al., 2014). Plants harbour an antioxidative defence

mechanism, activated in salinity to overcome oxidative

stress mediated by ROS, which is composed of ions

(Asgari et al., 2012), antioxidant enzymes and osmotic

homeostatic conditions. Ionic homeostasis under salinity

stress is to be maintained for physiological and

biochemical conditions of the plant because such ionic

balance regulates the essential ion concentration rather

than toxic ions (Gupta & Huang, 2014; Hajiboland,

2012). Certain plants possess a potential to maximize

the cellular level of osmotically compatible solute

concentration. Such osmotically compatible solutes can

mediate ionic homeostasis related to water concentration,

eventually leading to minimized impact of ionic concen­

trations upon cell proteome including enzymes, protein

complexes plus membrane proteins and other proteins

required in cell stability under stress (Ma et al., 2012).

The role of antioxidative enzymes, for instance APX

(ascorbate peroxidase), SOD (superoxide dismutase),

CAT (catalase) and POD (peroxidase), was important in

minimizing oxidative stress/damage induced during

salinity stress (Ahmad et al., 2010a).

Under saline conditions, the activities of the cell anti­

oxidant system, for example, SOD (superoxide dismutase)

and CAT (catalase), in susceptible cultivars were lower as

compared to controls. With respect to APX, there was no

significant difference between saline and control condi­

tions. Under salt stress, the MSI (membrane stability

index) of two tested cultivars was adversely affected.

Hydrogen peroxide (H2O

2) content of salinity susceptible

cultivars was higher than controls. Salt‐tolerant mixtures

had more K+ levels and Na+ ratio; relative water sub­

stance, yield and chlorophyll under saline condition and

susceptible cultivars accumulate higher Na+ content at

the tillering stage. The method of salt stress may be

accomplished because of low lipid peroxidation, presum­

ably, fewer changes in MSI, evasion of Na+ quantity and

release of antioxidant enzymes (Rao et al., 2013).

Salt tolerance in plants is dependent on their ability to

alter osmotically to decreased soil water potential as

well as keeping intracellular levels of ROS under con­

trol. Both of these techniques are accepted to depend on

accumulation of natural osmolytes. In one study,

outside NaCl complex was connected by the dynamic

congregation in leaf Na+. This aggregation was much

higher in old leaves and juvenile ones. In old leaves,

three inorganic ions (Cl–, K+ and Na+) contributed 70.4%

and 67.7% of leaf osmotic potential in wheat and grain

individually when presented to 200 mm NaCl treatment,

while in junior leaves their assertion was just

46.8%and  43.9% separately. It was suggested that

salinity prompted increase of natural osmolytes in

wheat grain and leaves corresponding with exaggerated

oxidative stress tolerance and provides the confirmation

of a system of cross‐tolerance between these two stresses

(Puniran‐Hartley et al., 2014).

So as to overcome perverse intercellular ROS, plants

have created cell antioxidant guard frameworks assist­

ing them in management of ROS levels. Plants utilize

different components to protect themselves from the

distant impacts of salinity (Zhang Y et al., 2012). Plants

react to excessive salinity by accretion of osmoprotec­

tants including proline and sugar (Gurmani et al., 2013).

Plant hormones have outstanding effects on the

framework of cell division and development of a whole

plant. Salt stress‐induced decline in general development

signifies endogenous irregular hormonal levels (Iqbal &

Ashraf, 2013).

1.4 Wheat natural tolerance and defence against salinity

Wheat also possesses particular mechanisms that help it

to combat harmful effects of primary as well as secondary

stress with the accretion of osmolytes and antioxidants

production (Ahmad et al., 2010a; Ahmad et al., 2010b;

Ashraf & Foolad, 2007; Devi & Prasad, 1998; Foyer et al.,

1994). The principal antioxidant enzymes that assist

plants to withstand numerous environmental stresses

include peroxidases, superoxide dismutases, catalases

and glutathione reductases. These enzymes co‐ordinate

in orchestra; for instance, superoxide dismutase con­

verts superoxide anions to H2O

2 (Alscher et al., 2002).

Similarly, catalase stands as a second line of defence

against varied stresses by converting H2O

2 to molecular

oxygen and water. With reference to their potential of

free radical quenching, quantization and other patterns

of analysing antioxidant enzymes and non‐enzymatic

antioxidants levels are utilized to determine the effec­

tiveness of oxidative defence mechanisms in plants

(Geebelen et al., 2002).

Tolerance to salinity stress is generally evaluated from

the biomass produced under saline compared to con­

trolled conditions grown over a convenient time period

0002580878.indd 7 10/3/2015 6:20:17 PM

8 Chapter 1

(Munns, 2002). Tolerance levels in plants also vary

depending upon genetic potential plus biochemical and

physiological characteristics. A halophytic salt‐tolerant

plant upon abrupt exposure to prevailing salinity or

salinity shock will develop diverse strategies that could

lead to gradual acclimation, depending on develop­

mental stages and sensitivity of plants. Conversely,

tolerance mechanism is determined by interaction bet­

ween environment and particular plant species. Some

plant species may demonstrate stage‐specific sensitivity,

exhibiting greater susceptibility to salt in the germina­

tion phase while others may be highly sensitive in the

reproduction stage (Mahmoodzadeh et  al., 2013).

Evolution is also important in this regard and numerous

mechanisms are evolved in plants to defend against

salinity stress.

1.4.1 Mechanisms of salt tolerance in wheatSalinity in plants is counteracted by diverse components

(Maas, 1986). Agricultural productivity of certain plants

appears to be more sensitive to high levels of salt con­

centrations, such as glycophytes. While halophytic

species are exceedingly salt tolerant and can survive and

maintain development even at saline levels much

higher than that of seawater, none of the radical exam­

ples can tolerate more than 25% salt levels without

yield loss and development impairment. The high salt

tolerance of halophytes is ascribed to rare anatomical

and morphological adjustments, or avoidance tools

(Greenway & Munns, 1980). However, halophytes are

exceptional among the 250,000 types of promising

plants (Flowers & Flowers, 2005). Past studies have

grouped plants into two classifications: salt includers

and salt excluders. Sodium ions (Na+) are transported to

shoots with the help of salt includers where it is used in

vacuole‐mediated osmoticum‐intended tolerance fol­

lowed by salt excluders which adjust these ions to saline

conditions by eluding uptake of Na+ ions (Mian et al.,

2011a). Overall, plant response to salinity is categorized

in three classes: (1) osmotic stress tolerance; (2)

excluding Na+ from leaves; and (3) tolerance at tissue

level (Munns & Tester, 2008).

Halophytic species form systems like proficient Na+

sequestration into the vacuole, which preserve low

cytosolic Na+, as well as allowing these plants to utilize

Na+ as an osmoticum to maintain cell turgor and

development. Accordingly higher Na+ uptake can lead

to rapid development of halophytes (Blumwald, 2000).

Change of salt tolerance in harvested glycophytes like

rice and durum wheat has been attained by the advance­

ment of cultivars with low Na+ in shoot or high K+/Na+

proportion (Munns et al., 2012; Thomson et al., 2010).

Plants use three normal instruments of salt tolerance

(Rajendran et al., 2009): osmotic change; suitable con­

firmation for Na+ uptake via roots and restricting its

entry into susceptible tissue; and tissue tolerance (Na+

incorporation, Na+ compartmentation). These systems

are controlled by integrated physiological, biochemical

pathways (Zhou et  al., 2012). Osmotic alteration

includes the combination and aggregation of perfect

solutes inside the cytoplasm. Compatible solutes are

smaller water‐solvent particles that contain nitrogen‐

holding blends, for example, betains, amino acids,

additionally natural sugars, polyols and acids (Chen

et al., 2007). The ability of the compatible solutes is not

constrained to maintaining osmotic balance. Compatible

solutes are usually hydrophilic as well as they may have

the capacity to displace water at the protein surface

advancing towards low subatomic weight chaperones

(Carillo et  al., 2011). Furthermore, these solutes have

the ability to maintain cell structure through ROS

scavenging (Hasegawa et al., 2000).

High quantities of Na+ and Cl– are lethal to all plant

cells. The ability of plants to keep up a high K+/Na+

proportion in the cytosol is a contributory element of

plant salt resistance. A few genes and transporters that

plants utilize to maintain high K+/Na+ proportion have

been described (Jamil et  al., 2011). These include the

following.

1 Na+/H+ antiporters in plasma layers that expel Na+

from the cytosol as a major aspect of the administrative

SOS pathway (Zhu, 2001). Three obviously subtle

(SOS) proteins (SOS 1, 2 and 3) suggest an organiza­

tional fraction in the communication of particle trans­

porters to maintain small cytoplasmic amounts of Na+

under salt stress (Lu et al., 2014). Zhu (2003) proposed

that a protein kinase complex composed of calcium‐

bound protein (SOS3) and serine/threonin protein

kinase (SOS2) is activated by stress induced in salinity‐

mediated calcium signals. Subsequently, transporter

particles such as Na+/H+ plasma layer and SOS1 anti­

porters are phosphorylated by protein kinases, as

shown in Figure 1.1.

2 Vacuolar Na+/H+ antiporters (NHXs) and energy

suppliers of these NHXs (like H+ pumps: HVA/68 and

0002580878.indd 8 10/3/2015 6:20:17 PM

Biotechnological applications to improve salinity stress in wheat 9

Hvp1) (Blumwald et  al., 2000; Ligaba & Katsuhara,

2010). NHX proteins sequester Na+ in the vacuoles

and provide an effective component to avoid the

harmful impacts of Na+ in the cytosol and sustain

osmotic equivalence (Glenn et al., 1999). Thus, Cl– is

likely transported into the vacuole by anion trans­

porters, for example, CLC proteins (Teakle & Tyerman,

2010; Zifarelli & Pusch, 2010).

3 High‐ and low‐partiality K+ transporters (HKT).

The HKT family comprises two classes which work

either as particular Na+ transporters or Na+ and K+

co‐transporters (Hauser & Horie, 2010; Shabala et al.,

2010). HKT21 was demonstrated to improve Na+

uptake and higher Na+ levels in xylem sap (salt

including conduct) which are associated with pro­

longed salt tolerance (Mian et al., 2011a). Numerous

researchers proposed that Na+ avoidance from the

shoot is connected with salt tolerance and that genes

from the HKT1 subfamily, for example, HKT1;4 and

HKT1;5, are included (James et al., 2011; Munns et al.,

2012). Shabala et  al. (2010) indicated that both salt

exclusion and deliberation are vital for grain salt toler­

ance. In fact, grain is an upright illustration of a harvest

that links halophytic and glycophytic properties, and

accordingly may be an outstanding model to study

both the glycophytic and halophytic components that

might be used to adapt to salt stress (Mian et al., 2011b).

1.4.2 Conventional strategies to enhance salinity stress toleranceVarious tools and techniques have been devised by

many researchers around the world to support plants

in acquiring tolerance against salinity and producing

higher yields. The most common strategy to enhance

induction of salinity tolerance in plants is seed priming

which requires less energy and is quite successful in

minimizing the harmful effects of salinity on plants

(Fercha et al., 2014; Hou et al., 2013). Speed and rate

of germination in equipped seeds under salt stress

have reportedly increased. This pre‐sowing priming

technique with different growth regulators, osmoprotec­

tants and halotolerant non‐pathogenic micro‐organisms

Na

Na

ADP

ATP

Vacuole

NaNa

Ca

CaCa

H+

H+

H+

H+H+

SOS1

SOS2

SOS1

AtHKT

AtNHX1

AtHKT

Plasma membrane

SOS3

Figure 1.1 SOS pathway indicating Na+/H+ antiporters in plasma layers that expel Na+ from the cytosol.

0002580878.indd 9 10/3/2015 6:20:17 PM

10 Chapter 1

(Ramadoss et  al., 2013) as well as water has also

proved  supportive in developing field‐level salinity

tolerance in  plants because it establishes an aid to

germination (Qiu et  al., 2014). Seeds initially primed

with CaCl2 followed by KCl and NaCl remained effec­

tive  in  minimizing adverse effects of salinity of wheat

plants  as  they change levels of numerous phytohor­

mones. Physiological attributes of plants have also shown

improve ment after priming seeds at both laboratory and

field level. This technique also illustrates the complete

representation of salinity tolerance mechanisms in plants

(Cantliffe, 2003).

Some other strategies utilized to minimize salinity‐

induced effects on plants, especially in wheat, include

removal of excessive salts from soil surface or harvesting

aerial parts containing accumulated salt in areas where

low rainfall occurs, and utilizing practices that aid in

saline soil amelioration (Bacilio et al., 2004).

1.5 Biotechnological applications to improve salinity stress in wheat

Advancements in agricultural biotechnology are

employed to overcome several problems related to

plants, including salinity. The primary goals of agricul­

tural biotechnology include minimizing production

cost  by derogating inputs like pesticides and nutrient

requirements grown in diverse climates. The main goal

is to enhance plant quality by producing strains which

have better yield with low inputs, can also yield good

crop rotation for conservation of natural resources and

also obtain more nutritious products that are suitable

for long‐term storage or travel and low purchase costs

for consumers.

After years of expensive and intensive studies, today

agricultural biotechnology has made it possible to pro­

duce transgenic plants commercially. In 1990, it was

speculated that 40 million hectares of land had been uti­

lized in the growth of 20 important species including

corn, rapeseed, soybean and cotton (ISAAA 1999). But

due to widespread criticism and opposition at the

beginning of the 21st century, transgenic wheat was not

accepted to be cultivated and commercialized. However,

in 2009 the NAWG (National Association of Wheat

Growers) conducted a survey which revealed that 76%

of the growers supported the cultivation of transgenic

wheat for large commercial‐scale purposes.

Over the previous decades, diverse methodologies

have been used to enhance salt resistance in plant har­

vests (Munns et  al., 2006). New salt‐tolerant hybrids,

including tobacco (Hu et al., 2012), rice and wheat, have

been utilized in nations far and wide like Pakistan, India

and the Philippines (Bennett & Khush, 2003). Screening

of a substantial accumulation (~5000 increases) of

common wheat in Australia and 400 Iranian wheat

mixed bags in California for salt tolerance has recog­

nized lines that delivered seeds under high salt focus

(half seawater) or provided exceptional profits on saline

soil. So far no new cultivar has been developed from

these tolerant lines (Munns et al., 2006).

Two broad types of approaches can be employed to

overcome the problems caused by salt stress. Soil

affected with salt can be managed and brought under

cultivation by reclamation, or alternatively, modern bio­

technological techniques have been utilized to exploit

the germplasm for the development of salinity tolerant

varieties (Farshadfar et al., 2008).

Among conventional germplasms, an Indian cultivar/

landrace named Kharchia 65 was tested against salt stress

and was found to be salt resistant up to a certain level

(Mujeeb‐Kazi et al., 2008), making plant breeders select

this variety over others for cultivation. Numerous

examples/landraces of wheat with the potential for salt

tolerance have been reported from several areas of

Pakistan, Iran and Nepal (Martin et  al., 1994). In

addition to these, several other wheat varieties, for in­

stance KRL1‐4 and KRL 19 (India), LU26 S and SARC‐1

(Pakistan) and Sakha 8 (Egypt), are also considered as

salt‐tolerant cultivars (Munns et al., 2006).

Numerous genes control the characteristics that may

be included in salt tolerance. These genes are communi­

cated diversely throughout the plant lifespan and are

affected by numerous ecological variables (Roy et  al.,

2011). Plant agriculturists are searching for more

authentic methodologies with the assistance of molec­

ular markers or transgenic methodologies (Arzani, 2008).

The vast majority of the genes that may help salt tol­

erance still remain a mystery, even in model plants like

Arabidopsis and rice. Likewise, salt tolerance is a multi­

genic attribute; consequently, extensive change focused

around alteration of a single gene is not likely to occur

(Colmer et al., 2005). Distinctive evidence of new attrib­

utes aiding in salt tolerance is possible through immediate

traditional selection in stress situations or focused

around mapping investigations of QTL (Holland, 2007).

0002580878.indd 10 10/3/2015 6:20:17 PM

Biotechnological applications to improve salinity stress in wheat 11

At present, association mapping seems an alluring and

effective methodology to distinguish extra genes serving

the regular occurrence of changes for salt tolerance in

varieties, landraces and wild relatives of yields.

Previously the molecular foundation of the characters

that aid in salt tolerance has been determined: marker‐

aided selection (Munns et  al., 2012). This might be

utilized to productively exploit the new qualities and

genes or to induce hereditary adjustment which could

produce genetically engineered crops with new genes

exhibiting enhanced levels of salt tolerance.

The relevant selection approach to screen expansive

mapping populations and produce precise data on

attributes is crucial for recognizing the characteristics

and genes for assisting salt tolerance (Ramezani et al.,

2013). This will provide understanding on the vicinity/

extent of the heritable variety for tolerance attributes,

their inheritance and the magnitude of genotype and

environmental co‐operation.

1.5.1 plant phenotypingTo reveal the hereditary premise of complex qualities

like salt tolerance, it is important to assist genotypic

marker data with the relating phenotypic information.

The precise phenotyping is a basis to ascertain and

present new genes for salt tolerance into productive

plants (Munns et al., 2006). Recently, advancement in

DNA marker and sequencing advances has allowed

high‐throughput genotyping of numerous individual

plants with moderately minimal effort. Rapid strategies

to assess huge amounts of genotypes are critical to

completely exploit the immediate improvement of bio­

technological systems and to encourage hereditary

analysis of complex qualities.

Traditional selection for execution and yield under

saline field conditions has different impediments identi­

fied by natural variables, for example, soil heterogeneity

and climate conditions (Chen et  al., 2005; Isla et  al.,

1998; Munns et al., 2006). The supportive physiological

characteristics serving salt tolerance and the genes

underlying these qualities could be distinguished more

proficiently under natural conditions (Cuin et al., 2008).

Effective screening systems that were used recently to

assess the response of grains to salinity were plasticized

on hydroponics (Chen et  al., 2005; Munns & James,

2003) or on sand as well as soil‐based substrates (Munns

et al., 2002; Tavakkoli et al., 2010b). The shoot Na+ (Cl–)

content and K+/Na+ degree have been recommended as

dependable characteristics for salt tolerance determina­

tion in products (Munns & Tester, 2008; Munns et al.,

2002; Tester & Davenport 2003). Hereditary investiga­

tions employing traits that influence particle homeostasis

have distinguished QTLs characterized by Na+ and K+

transporters which facilitate salt tolerance in rice

(Bonilla et  al., 2002; Ren et  al., 2005) and in wheat

(Munns et  al., 2012). Comparative studies in barley

have not yet unravelled genes for salt tolerance, despite

the fact that it is the most salt‐tolerant oat crop.

Shabala et al. (2010) and Mian et al. (2011a) demon­

strated that both particle exclusion and deliberation

assist grain salt tolerance. Further precise and appro­

priate screening strategies may be required that permit

numerous stage estimations of salt stress throughout

the life cycle of barley. Also, the processes ought to

allow examination of the synthesis and collaborative

impacts between distinctive qualities and incorporate

Cl– risk as Cl– is an ‘overlooked adversary’ for salt

tolerance research (Munns & Tester, 2008; Teakle &

Tyerman, 2010).

1.5.2 QtL mappingQTL mapping has been a key apparatus in studying

the genetic structure for engineering of complex

characteristics in plants (Kearsey, 1998). Most agro­

nomically significant characteristicss, for example

yield, grain quality and tolerance to biotic and abiotic

stresses, are indistinct (Azadi et  al., 2014). Genetic

assembly modelling alludes to various genome areas

with genes that influence the attributes, the magni­

tude of the impact, and the relative assurance,

prevailing and epistatic impacts (Holland, 2007). The

discovery of QTLs of agronomical significance and the

underlying genes has significantly extended our under­

standing of the intricacy of characteristicss (Salvi &

Tuberosa, 2005).

Advancement in distinguished QTLs that trigger the

characteristics will fundamentally assist breeding through

marker‐aided selection (Collard & Mackill, 2008) and

pyramiding of numerous suitable alleles (Yang et  al.,

2012). Biparental (customary) QTL mapping focused

around a single dividing population inferred from two

homozygous parental genotypes has been the basic

methodology for genetic investigation of salt tolerance in

rice (Lee et al., 2006), wheat (Genc et al., 2010) and grain

(Xue et al., 2009). A few loci were found to encode parts

of the HKT group of particle transporters which together

0002580878.indd 11 10/3/2015 6:20:17 PM

12 Chapter 1

enhance salt tolerance like the Kna1 locus in common

wheat (Dubcovsky et al., 1996) as well as Nax1 plus Nax2

in durum wheat (Munns et al., 2012). In the meantime,

biparental QTL mapping has constraints identified with

the reduced investigation of allelic variety exhibited in

the gene pool for each of the loci influencing the qual­

ities, absence of isolation for some characteristics, and

poor determination (Rock Garcia, 2003). Biparental QTL

mapping discovers genomic areas connected with char­

acteristics with precision successively in normal range

from 10 to 30 centimorgan (cm) (Bernardo, 2008). Such

chromosomal locales could harbour up to a few thou­

sand genes (Ingvarsson et al., 2010), thus proving the fact

that effective QTLs which are currently being cloned are

underlined by more than one gene (Mackay & Powell,

2007). Further research is required to determine the

mapping requisite in order to overcome the utilization

of the hereditary variety for salt tolerance in barley

germplasm.

1.5.3 association mappingAssociation mapping, also known as linkage disequi­

librium mapping, is a system utilized for mapping QTLs

which interprets outstanding disequilibrium linkage

with phenotype connection (perceptible characters) to

genotypes. Recently, association mapping has been

supported as the technique for distinguishing loci

included in the inheritance of complex characters in

genetics. This technique includes distinctive markers

connected with the phenotypes of interest found

among a group of irrelevant individuals (Pritchard

et  al., 2000). Association mapping has recently been

presented in plant genetics (Kloth et  al., 2012) and

demonstrated promise to utilize the exact capacity of

novel molecular markers and sequencing progress

(Zhu et al., 2008).

Association mapping depends on the local character­

istics related to linkage disequilibria in accrual of

normally different germplasms (Mackay & Powell,

2007). It provides dynamic utilization of all the recom­

bination incidents that have occurred throughout the

long evolutionary history of a crop species, distributing

meagre linkage obstructions than those found in bipa­

rental QTL mapping studies (Nordborg & Tavare, 2002).

Furthermore, association mapping deals with all actual

allelic variants of QTLs, influencing the attributes of

study when performed with an adequate association

mapping group dialogue to the majority of the crop

gene pool (Figure 1.2).

In association mapping, linkage disequilibrium (LD)

is a central element. LD is a population detail for

non‐arbitrary relationship between alleles of distinc­

tive polymorphic loci. The destruction in LD among

adjacent markers determines the marker thickness

and trial outline required to perform association map­

ping effectively. Linkage, choice, transformation and

assortment all influence the level of LD. Furthermore,

LD depends upon the mating framework and hence

fluctuates from species to species among populations

(Rostoks et al., 2006).

An association mapping group includes vast land

regions, areas of adjustment with an upright represen­

tation of its evolutionary history typically non‐arbitrary

because of familial relatedness and may indicate dis­

tinctive sorts of structure (Pritchard et al., 2000). This

may suggest counterfeit marker quality affiliations

(Zhao et  al., 2007). In this way it is essential to have

appropriate statistical procedures and methods to eluci­

date such complexities (Patterson et  al., 2006). The

most prevalent course is to assemble the constituents

affiliated with mapping section and consolidate the

data in measurable models in which markers are being

discovered within the familiar subpopulations (Balding

et al., 2006).

Other important approaches to control population

structure are the utilization of intermingled models

to  document contrast in genetic relatedness between

section representatives (kinship matrix) (Malosetti

et al., 2007). While evaluating, population structure is

an important computational demand; Patterson et  al.

(2006) presented a methodology utilizing principal

component analysis (PCA) to manage the issue of coun­

terfeit associations. This is quick and simple and works

well with extensive information sets.

1.5.4 proteomic approachProteomics is the most advanced approach to categori­

zation of diverse proteins that are included in unique

and/or distorted structures (Maleki et  al., 2014).

Particular genes or families of genes might regulate a

few protein types to control specific characteristics.

Advancement in proteomics has been used to focus

information on instruments that manage complex inherent

characteristics (Capriotti et  al., 2014). Peng et  al. (2009)

0002580878.indd 12 10/3/2015 6:20:17 PM

Biotechnological applications to improve salinity stress in wheat 13

assessed that cultivars of wheat Shanrong 3 and progenitor

cultivar Jinan 177 are utilized in two‐dimensional gel

electrophoresis and mass spectroscopy for protein pro­

filing. Consequences revealed that 6 and 34 protein

conserved differentially in leaves (Maleki et  al., 2014)

and roots, respectively.

A few uniquely conserved proteins could further be

described in terms of their capacities in metabolomics or

other indicators for transduction cross‐communication

in salt tolerance systems in plants. This was also the

case with seedlings of wheat when treated with sali­

cylic acid (0.5 mM) and sodium chloride NaCl (250 mM)

for 3 days. In both salicylic acid and salt analysis, 39

proteins are demonstrated by 2d PAGE and MALDI‐

TOF/TOF‐MS is used to control 38 protein (Kang

et  al., 2012). The research proposed that communi­

cated proteins are being included in diverse cells

along with metabolic methodologies, for example

metabolism, stress safeguard, signal transduction and

photosynthesis.

Selection of germplasm with genetic diversity

Phenotypic measurementin multiple replication

trials in differentconditions

Genotypingwith molecular markers

SNPs, SSRs,and AFLPs

Quanti�cationof LDusing

molecular markersdata

Measurement ofcharacteristics

ofpopulation

Marker traitcorrelation with suitable method

Marker tags are identi�ed that areassociated with trait of interest

Cloning and annotation of tag loci forpotential biological functions

Figure 1.2 Schematic representation of association mapping.

0002580878.indd 13 10/3/2015 6:20:17 PM

14 Chapter 1

1.5.5 Salt tolerance‐related genesEnhanced salt tolerance is controlled by genes (Table 1.1)

that can be classified into three categories (Munns, 2005)

as discussed below.

1.5.5.1 Genes for salt uptake and transportA considerable amount of research is dedicated globally

to investigating the processes and understanding the

interactions occurring among the genome and pro­

teome within a plant cell during salt stress (Hirayama &

Shinozaki, 2010). One of these processes is the primary

phase of gene expression, transcription, which is a vital

process because the production of a vigorous transcrip­

tome is a promising feature for better protein yield and

stabilized cellular activities (Chew & Halliday, 2011;

Christov et al., 2014). Salt stress influences the genome

and gene expression patterns, thus resulting in a diverse

proteome (Nakashima et al., 2009).

Researchers from the Commonwealth Scientific and

Industrial Research Organization (CSIRO) isolated two

salt‐tolerant wheat genes, Nax1 and Nax2, evolved

from an old relative of wheat, Triticum monococcum. Both

genes were responsible for limiting toxic sodium passage

from root to shoot, thus causing inhibition of toxic

sodium. In one study evaluating the Nax2 gene in field

trials, a variety with the Nax2 gene showed a 25%

greater yield than without Nax2 under saline environ­

ment (James et al., 2006).

Proteins implanted within the membrane lipid bilayer

are involved in controlling Na+ ion uptake from soil and

in transport across the whole plant body. The Na+ ion

may be regulated by the K+ ion transporter and chan­

nels directly since they are not completely selective for

K+ ions as it buffers the cell for uptake of Na+ ions by

rigorous K+ homeostasis (Munns, 2005). Ion selective

channels are involved in transportation of ions passively

under electrochemical gradient. Non‐selective channels

allow transport of Na+/K+ (Demidchik et  al., 2002).

Active particle transport occurs through symporters and

antiporters. Transport occurs under conditions of elec­

trochemical potential distinction of a conjugated solute,

generally H+. Numerous genes significantly maintain

Na+ or K+ homeostasis in higher plants while possibly

being promising for hereditary controls (Munns, 2005).

Different genes like AKTI, AKT2 and KATI encode for

K+ ion channels such as AKTI, AKT2, KATI (which encode

for shaker‐type inward channels having a single pore),

KCOI (two‐pore channel of KCO family): K+ antiporters

like KEA, SKOR (shaker‐type outward channel), CPA

(KVlf antiporter). K+ transporters like HAKI IO, KUPI

4 and SOS I, HKTI; Na+ antiporters like NHXI‐5 and

proton pumps like AVPI code for H+‐ATPase. AIIA2 plus

H+‐PPase (Mäser et  al., 2002) can be employed to

enhance salt tolerance.

1.5.5.2 Genes for osmotic functionSolutes that show a protective or osmotic effect are clas­

sified into four categories: (1) N‐containing compounds

like glycine betaine and proline; (2) sugars including

sucrose and raffinose; (3) straight‐chain polyhydric

alcohols like mannitol and sorbitol; and (4) cyclic poly­

hydric alcohols such as myoinositol, pinitol and ononitol.

Numerous genes have been recognized that code for

such osmotically important compounds, such as P5CS

gene coding for proline (Hong et  al., 2000), mtID for

mannitol, otsA and S6PDH for sorbitol (Gao et al., 2001),

codA for glycine betaine, ots8 for tetrahalose (Gong

et al., 2002) and imtl for myoinositol. These genes are

Table 1.1 Genes for salinity tolerance in wheat.

Serial no. Gene category Genes References

1 Genes encoding

for K+ channels

AKTI, AKT2, KATI (encoding for shaker‐type single‐pore inward channels), SKOR

(shaker‐type outward channel), KCOI (two‐pore channel of KCO family), K+ antiporters

like KEA, CPA (KVlf antiporter), K+ transporters like HAKI‐IO, KUPI‐4 and HKTI

Mäser et al. 2002

2 Na+ antiporters NHXI‐5 and SOS I and proton pumps like AIIA2 and AVPI encoding for H+‐ATPase and

H+‐PPase, genes for Na+ exclusion, named Nax1 and Nax2

Mäser et al. 2002

Munns and James

2003

3 HKT gene family HKT7 (HKT1;4), HKT8 (HKT1;5), TmHKT7 (TmHKT1;4‐A2), TmHKT8 (TmHKT1;5‐A)

(origin in T. monococcum)

Huang et al. 2006

Byrt et al. 2007

4 HKT gene family TaHKT8 (TaHKT1;5‐D), AtHKT1;1 (origin in T. aestivum) Byrt et al. 2007

0002580878.indd 14 10/3/2015 6:20:18 PM

Biotechnological applications to improve salinity stress in wheat 15

mainly present in model cash crops as well as other

plants required to be transformed followed by field trials

to produce and commercialize easy, cost‐effective and

high‐yield varieties (Yamaguchi & Blumwald, 2005).

1.5.5.3 Genes for cell growthCertain genes have also been identified that play vital

roles in plant growth such as development of new roots

or leaves, or may also be involved in life‐sustaining

biochemical pathways like photosynthesis. Once such

genes are well characterized and transformed efficiently,

this can lead to mediating cell division, growth rate

and  other measurements under varied environmental

conditions. Similarly rate of photosynthesis, stomatal

closure and opening or measuring mesophyll cells, sig­

nalling pathways (Choi et al., 2014; Schmidt et al., 2014)

and co‐workers like hormones, a variety of proteins and

enzymes (Kahrizi et al., 2012) like kinases and phospha­

tases are easily studied and regulated via detailed

assessment of such genes (Zhang et al., 2004). It is pro­

posed that such genes can simultaneously also be

instructive in water stress (Chaves et al., 2003). Some

other factors like CBFs (C‐repeat binding factors) and

ABFs (ABRE binding factors) have also proved enhance­

ment in tolerance against varied abiotic stresses (Oztur

et al., 2002).

1.5.5.4 Genes for reducing Na+ accumulationCereal crops like durum wheat (Cuin et al., 2010; Munns &

James, 2003), rice (Haq et  al., 2010), pearl millet

(Krishnamurthy et al., 2007), Medicago sativa (Castroluna

et al., 2014) and grain (Shavrukov et al., 2010) all pos­

sess the pattern of salinity tolerance with exclusion of

Na+ from plant leaves. On the other hand, wheat wild

progenitors like Triticum tauschii (Schachtman et  al.,

1991), durum (pasta) wheat (Triticum turgidum), tall

wheatgrass (Colmer et al., 2006) and hordeum species

are more susceptible to salinity stress as compared to

Triticum aestivum (bread wheat) due to its low Na+

exclusion potential (Munns & James, 2003).

In order to obtain salinity‐tolerant durum wheat,

durum or durum‐associated wheat, genotypes were

gathered across the world. Initial studies revealed ’Line

149 genotype’ among gathered genotypes which sur­

prisingly possessed the property to avoid Na+. Following

detailed investigations, it was found that two genes,

Nax1 and Nax2 from Line 149, are both responsible for

Na+ exclusion (James et al., 2011). QTL analysis revealed

that Nax1 is located on chromosome 2A, distinguished

by mapping as Na+ ion transporter of HKT7 (HKT1:4)

HKT gene family (Huang et al., 2006). Nax2 is located on

chromosome 5A with HKT8 (HKT1:5) (Byrt et al., 2007).

Currently wheat does not exhibit Nax genes but both

Nax1 and Nax2 genes were coincidentally shifted into

Line 149 after crossing Triticum monococcum (C68 101)

with durum wheat with the intention to exchange rust

resistance genes (James et  al., 2006). Following that,

these genes were named as TnHKT8 (TmHKT1: 5‐A)

and TmHKT7 (TmHKT1: 4‐A2) in Triticum monococcum,

respectively.

These genes in durum wheat play their role in

exclusion of Na+ from xylem so that leaves may receive

lesser amounts of Na+ ions (James et  al., 2006). More

specifically, Nax1 ejects Na+ ions from roots, lower parts

of leaves and xylem, while Nax2 plays the same role in

root xylems. Nax2 bears a phenotype for expulsion of

Na+ and enhances K+/Na+ selection in Triticum aestivum

(bread wheat) while Nax1 has a phenotype of high

sheath‐blade proportion of Na+ ion concentration

(Dvořak et  al., 2004). It was demonstrated that, in

Triticum aestivum, Nax2 is homologous to Kna1, espe­

cially Tahkt8 (Tahkt1:5‐D) (Byrt et al., 2007). The HKT

gene family also encodes transporters in plasma mem­

brane which mediate Na+ or K+ uptake from apoplast

(Hauser & Horie, 2010). These are crucial for cell

homeostasis in terms of Na+ and K+, and if carried to

stele, more specifically the parenchymatous lining in

xylem, they recover Na+ ions from the transpiration

path and thus protect leaves from Na+ ions (Hauser &

Horie, 2010; Munns & Tester, 2008). Transportation of

Na+ to leaves is decreased if stele‐specific supporting

genes are upregulated, thus increasing salt tolerance to

Arabidopsis (Moller et  al., 2009). These Nax genes are

associated with Triticum monococcum, a diploid wheat

progenitor, while they have vanished from advanced

wheat cultivars (Huang et al., 2008).

These genes are brought into bread wheat by the

usual combination of tetraploid wheat, i.e. durum being

crossed with hexaploid wheat and resultant F1 genera­

tion, i.e. pentaploid was again backcrossed to bread

wheat. Offspring of hexaploid wheat that contain one or

both NaX genes were selected as four Australian culti­

vars of wheat. Genes near Nax genes were examined for

their ability to promote Na+ exclusion and division of

Na+ ions between the sharpened and sheath stele as well

as their photosynthetic implementation in 150 mM

0002580878.indd 15 10/3/2015 6:20:18 PM

16 Chapter 1

NaCl. Saline soils were frequently wet throughout the

time in advance of planned vegetative development

(Colmer et al., 2005).

1.5.6 Molecular markersMolecular markers are principally related to evaluating

polymorphisms in DNA arrangements (i.e. base pair

cancellations, substitutions, augmentations or patterns).

Molecular markers are amongst the most effective

machinery for the assessment of genomes and allow the

relationship of heritable qualities with underlying

genomic diversity to be determined (Table 1.2).

The most widely used DNA marker systems for

assessment of genetic diversity in wheat are SNPs (single

nucleotide polymorphisms), SSRs (simple sequence

repeats), ISSRs (inter‐simple sequence repeats), AFLP

(amplified fragment length polymorphism), RFLP (restric­

tion fragment length polymorphism), RAPD (random

amplified polymorphic DNA), ESTs (expressed sequence

tags) and microarray technology. All molecular marker

methods can be utilized for diverse applications inclu­

ding germplasm characterization, hereditary diagnostics,

characterization of transformants, investigation of

genome association, marker‐assisted selection (MAS)

and phylogenic dissection (Mishra et al., 2014).

1.5.6.1 Single nucleotide polymorphismsSingle nucleotide polymorphisms (SNPs) are a single

base‐change or small insertions or terminations in

homologous sections of DNA. In human genome

sequencing, 10–30 million SNPs were discovered and

were the greatest source of polymorphisms (Collins

et  al., 1998), present both in coding and non‐coding

locales (Aerts et  al., 2002). As markers, SNPs are

favoured over other marker frameworks owing to their

more continuous, co‐dominant nature and occasional

connection with morphological progressions (Lindblad‐

Toh et  al., 2000). Genomes of higher plants such as

barley (Kanazin et al., 2002), soybean (Choi et al., 2007),

maize (Tenaillon et  al., 2001), sunflower (Lai et  al.,

2005), sugar beet (Schneider et al., 2001), rye (Varshney

et al., 2007) and cotton (Ahmad et al., 2007; Lu et al.,

2005; Shaheen et  al., 2006) have furthermore been

studied for SNP revelation and characterization. Since

SNPs are exceptionally polymorphic, each gene must

contain few SNPs even among strains (Cho et al., 1999).

Table 1.2 Advantages and disadvantages of some commonly used DNA markers.

Serial no. Molecular marker Advantages Disadvantages References

1 RFLP Robust

Reliable

Transferable across population

Time consuming

Laborious

Expensive

Large amount of DNA required

Limited polymorphism

Beckman and Soller

1986

Kockert 1991

2 RAPD Quick and simple

Inexpensive

Multiple loci from single primer

possible

Small amount of DNA required

Problems with reproducibility

Generally non‐transferable

Penner 1996

Williams et al. 1990

3 SSR Technically simple

Robust and reliable

Transferable between populations

Large amount of time and labour required

for production of primers

Usually require PAGE

McCouch et al. 1997

Rodcr et al. 1995

4 AFLP Multiple loci

High level of polymorphism generated

Large amount of DNA required

Complicated methodology

Vos et al. 1995

5 SNP High abundance

Cross‐study comparisons are easy

Low mutation rate

Easy to type

Expensive to isolate

Low information content of the single SNP

Substantial rate of heterogeneity among

sites

Christian 2004

AFLP, amplified fragment length polymorphism; RAPD, random amplified polymorphic DNA; RFLP, restriction fragment length polymorphism;

SNP, single nucleotide polymorphism; SSR, simple sequence repeat.

0002580878.indd 16 10/3/2015 6:20:18 PM

Biotechnological applications to improve salinity stress in wheat 17

The MT‐shsp gene is a vital gene which serves to protect

against high temperature shock. MT‐shsp ensures

NADH; ubiquinone oxidoreductase of the electron

transport chain throughout temperature stress in plants

(Herman et  al., 1994). SNP markers, joined with QTL

information for phenotypic character, can provide

another arrangement of breeding, i.e. gene‐interceded

background rather than marker‐aided determination

(Lange & Whittacker, 2001). Genetic advances in crops

and farming profits will be improved by the accessibility

of rapidly developing genetic assets and tools, including

high‐density genetic maps (Lacape et  al., 2005).

Polyploid genomes are more useful to investigate for

SNPs than diploids. The proportion of SNP alleles shifts

up in polyploid genomes (Adams et al., 2003).

1.5.6.2 Simple sequence repeatsSimple sequence repeats (SSRs), also called microsatel­

lites, are 1–6 nucleotide repeats of DNA which provide

characterization of genetic contents for marker‐assisted

selection to detect deletions, insertions, duplication of

genes and allelic variations (Sehgal et al., 2012). SSRs

are more significant compared to other molecular

markers used for detection of polymorphic levels

because SSRs exhibit more potential co‐dominance in

inheritance. Simple detection of SSRs shows another

advantageous attribute. In wheat, many studies have

been conducted using SSRs both for wheat germplasm

improvement (Borner et al., 2000; Huang et al., 2002)

and the assessment of genetic diversity (Hammer et al.,

2000). One such research report that determined the

level of genetic diversity among 12 wheat cultivars with

the aid of four SSRs was conducted by Islam & Begum

(2012). They reported 10 alleles while per locus aver­

aged 2.5 with a range from 2 to 4. Among these wheat

cultivars grouped in clusters, ‘Gourab’ and ‘Akbar’ plus

‘Gourab’ and ‘BAW‐1064’, the value of genetic distance

obtained was 0.4697 while the groups ‘Balaka’ and

‘Aghrani’ plus ‘Triticale’ and ‘BAW‐1036’ were the

lowest. The genetic diversity, allelic number, size and

type of repeats were all positively correlated, suggesting

that the maximum number of SSRs can be applied to

find more characterized genetic diversity (Islam &

Begum, 2012). Similar studies have also suggested the

same persuasive explanation and numerous SSR

markers have been applied (Song et al., 2005).

These SSR‐mediated genomic analyses show a prom­

ising future to combat natural problems like abiotic

stress, more specifically salt stress. In polymorphism

identification, the allelic variations can also be detected

which during salt stress can lead us to detect the effects

of salt stress on genetic level based on polymorphism.

Salinity‐induced genetic alterations and identification

of  salt‐tolerant genes are the likely outcomes for this

technique which can be further utilized in genetic engi­

neering to develop salt‐tolerant varieties.

1.5.6.3 Inter‐simple sequence repeatsInter‐simple sequence repeats (ISSRs) are a technique that

joins the SSR/microsatellite primers coupled with poly­

merase chain reaction which yields multi‐locus markers

which contain advantages of SSRs and AFLP as well as

RAPD (Reddy et  al., 2002). Compared to others, ISSR

markers are more polymorphic and are used in phylogenic

analysis, genetic diversity, genome mapping, gene tagging

as well as evolutionary biology. Like other molecular

markers, ISSRs have been utilized in evaluating genetic

diversity and gene mapping as well as crop breeding

studies (Gupta & Varshney, 2000; Staub et al., 1996).

ISSRs have minimized major limitations in various

plant improvement studies in terms of cost, reproduc­

ibility and expertise required as compared to RAPD and

AFLP (Godwin et al., 1997). They are applied in areas of

phylogenetic analysis, gene/genome mapping, gene tag­

ging and related evolutionary studies. ISSR lies within

the SSR or microsatellite repeats, variable number of

tandem repeats (VNTRs) or short tandem repeats (STRs)

(Tautz & Renz, 1984).

Although wheat possesses a huge genome, and usu­

ally such huge genomes pose problems in genome‐based

studies, yet these ISSRs could be a promising solution

for salt resistance studies in wheat. ISSRs serve the same

function as SSRs but with its advantages like gene tag­

ging, it provides a more developed tool for further

analysis. For instance, in order to obtain insight into a

particular gene under salinity stress, ISSR is a good tool

to use. Once the gene is mapped, followed by tagging

technique to analyse that gene during salt stress, it will

be more convenient to figure out and procure salt‐

tolerant genes or related genes that influence cellular

mechanisms during such harsh conditions.

1.5.6.4 Amplified fragment length polymorphismAmplified fragment length polymorphism (AFLP) serves

as a precise tool for identification and estimation of

genetic diversity in plant germplasms (Martos et al., 2005).

0002580878.indd 17 10/3/2015 6:20:18 PM

18 Chapter 1

Widely studied genetic diversity of common crops, sig­

nificantly wheat, reveals that with the passage of time,

practices like domestication and other breeding tech­

niques have caused a reduction in genetic composition

of germplasm, thus neglecting genome‐wide recombi­

nation (Donini et al., 2000). In a study by Martos et al.

(2005), phylogenetic relationship was investigated in

24 wheat (durum) cultivars of Italy and Spain. From the

AFLP‐based results, old Spanish cultivars were found to

have high level of genetic similarity with collected

Italian varieties. Furthermore, genetic variation was

also found to be constant from last century among

overall breeding practices. Another similar study used

seasonal variations in winter and spring wheat cultivars

(Tyrka, 2002). This AFLP analysis was accomplished

using enzyme PstI and capitulated in 111 fragments.

Results demonstrated higher variation in spring‐grown

wheat cultivars as compared to winter wheat.

In addition to identification and phylogeny, polymor­

phism among wheat genomes can be calculated based

on landraces, adaptation and environmental conditions

(Bozzini et al., 1998) as well as physiological and mor­

phological characterization. The genetic potential and

genetic erosion analysis can be achieved through molec­

ular characterization which constricted the genetic

background among the cultivars (Gupta et al., 1999).

AFLP thus supports genetic‐level investigations for

plants in stressed conditions. Genetic responses of plants

to stress (Makhloufi et al., 2014), specifically wheat in

salinity, can be better understood with the advantage of

gene mapping and its activities via PCR‐based high‐

throughput AFLP markers (Manifesto et al., 2001).

1.5.6.5 Restriction fragment length polymorphismRestriction fragment length polymorphism (RFLP) is a

powerful tool for developing a genetic map, commonly

in plants and animals. Genetic maps of many crops

including rice, maize, barley, rye and tomato have been

developed (Bonierbale et  al. 1988; Graner et  al. 1991;

Helentjaris et al. 1986; McCouch et al. 1988; Wang et al.,

1991). RFLP analysis yields genetic maps based on dis­

tance between two markers but it was observed that

distances in genetic maps are different from physical

maps. This is because two genes, for instance, located

closely on a genetic map may be far apart on a physical

map or vice versa. Such problems are encountered using

more advanced techniques with RFLP such as in situ

hybridization (Werner et al., 1992).

A study by Shah et al. (2000) was conducted to check

polymorphism in two wheat varieties, ‘Cheyenne’ and

‘Wichita’, based on their chromosome 3A. For this

purpose, the authors used 52 RFLP, 40 RAPD, 77 STS

and 10 SSR probes upon group 3 homologous

chromosomes of wheat. Detected polymorphisms

included 3.9% STS, 60% SSR and 20% RAPD while the

highest polymorphism was 78.8% detected via RFLP.

This suggested that RFLP is a more suitable tool in poly­

morphism studies. These observations indicate that

RFLP can also be informative and a promising tool in

stress assays.

1.5.6.6 Random amplified polymorphic DNARandom amplified polymorphic DNA (RAPD) uses arbi­

trary primers to determine genetic diversity. Various

scientific communities have investigated genetic diversity

of numerous wheat cultivars. Bibi et al. (2009) studied 12

hybrid wheat genotypes for their genetic diversity using

RAPD markers. In their investigations, 14 primers were

used to amplify 102 loci and the authors observed that

89.2% (91 loci) showed polymorphism while the remain­

ing 10.8% (11 loci) were monomorphic; the size of

fragments ranged between 142 bp and 5.3 kb.

The major difference between RAPD and RFLP is the

use of an arbitrary‐sequence primer in RAPD. However,

both RAPD and RFLP are promising in studying effects

of stress conditions upon plants. Wheat grown under

salinity stress can be assessed using RFLP and RAPD that

can give a better insight into genetic variations and

linkage analysis and thus can be very helpful in allevi­

ating salt stress in wheat.

1.5.6.7 Expressed sequence tagsExpressed sequence tags (ESTs) are small fragments of

cDNA which help in tagging those genes from which

mRNA was produced. Sometimes, an unknown EST is

sequenced which results in a 200–700 base pair long

sequence and is used to search for similar genes among

proteomic and genomic databases (Adams et al., 1991).

ESTs can be employed to search for gene‐specific

sequence or motifs or, simply, similar gene sequences in

various organisms. However, EST‐based analysis of

wheat has been limited because only nine ESTs were

available for wheat in May 2000, although this has

increased a great deal recently (Lazo et al., 2004).

In an experiment conducted by Lazo et al. (2004), a

large‐scale map of hexaploid wheat (Triticum aestivum L.)

0002580878.indd 18 10/3/2015 6:20:18 PM

Biotechnological applications to improve salinity stress in wheat 19

genome was exploited based on deletion bin. The EST‐

based chromosome bin map revealed seven homologous

chromosome groups from wheat. In their overall contri­

bution, EST sequencing, processing and nomenclature

as well as assembly plus unassembled ESTs were used

for selecting various motifs in genes. They further sub­

mitted data from southern hybridization into databases

which has proved supportive in wheat genome‐related

researches. The same findings can also be utilized in

gene identification and finding similarities among wheat

cultivars such as genes for salt tolerance and many other

abiotic as well as biotic stresses.

1.5.6.8 Microarray technologyRecent developments in biological sciences for solving

various problems have focused on more advanced tools

and technologies. One such is microarray technology

that reveals the dynamics of gene expression. Using this

technology, various attributes or vital agricultural traits

in plants can be controlled. However, the judicious use

of this technology can lead to better understanding of

genome (Coram et al., 2008). In wheat under salt stress,

such technology can be more helpful as the expression

pattern of genes under salinity stress can be studied in

detail. The expression patterns during salt stress as well

as other phenomena like co‐expression and related

networks leading to transcriptome plus proteome may

also help in discovering genes that can ensure salinity

tolerance in wheat. Due to its potential, microarray

technology is the most outstanding option for finding

biological solutions to biotic as well as abiotic stress

in plants.

1.6 Conclusion and future perspectives

Environmental stresses threaten plant growth,

development and yield and these threats to wheat are a

major concern because wheat is a daily staple of the

human diet worldwide. With growth in population,

increase in wheat production is mandatory to meet

world requirements but abiotic and biotic stresses con­

fine its yield. However, among all stresses, salinity is

most alarming because it is widespread in both arable

and non‐arable lands. The consequences of excessive

salts have been observed to have a disastrous effect

upon wheat. Global scientific communities have been

investigating various aspects to try to resolve this issue.

These range from conventional breeding practices to

modern biotechnological techniques based on molec­

ular studies. Conventional breeding practices such as

hybridization and application of supportive chemicals

are helpful to some extent but are limited due to lack of

detailed integrated knowledge about the molecular‐

level processes occurring in cells under such harsh

environmental circumstances.

Hence, it is crucial to understand the cellular mecha­

nisms that occur in wheat under saline conditions in the

hope of finding an integrated solution to combat such

problems. Biotechnology holds promise for both aspects:

(1) with the latest tools and techniques, detailed studies

can be carried out to understand the processes within

the cell in various conditions; and (2) the integrated

potential of biotechnology can be employed to combat

such problems by altering the genetic contents or the

expression patterns or by optimizing the biochemical as

well as molecular pathways in many ways.

Despite these advantages, however, some limitations

exist in biotechnological applications although the

future looks promising. Most biotechnological strat­

egies utilized for salt tolerance are based on avoidance

mechanisms. But, in accordance with targets, future

endeavours have the potential of optimizing or directing

plants in such a way that a plant will be able to posi­

tively utilize available salts with the advantage of

reverting sodic or saline soil to normal soil. Thus, bio­

technological applications applied in wheat may be the

solutions to improve salinity stress.

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