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Introduction and Review of literature
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1.1. Introduction: Wheat
1.1.1. Economic importance and taxonomy of the wheat
Wheat (Triticum aestivum L.) is the most important cereal crop for the
majority of world’s populations. It is the most important staple food of about two
billion people (36% of the world population). Worldwide, wheat provides nearly 55%
of the carbohydrates and 20% of the food calories consumed globally (Breiman and
Graur, 1995). Wheat is cultivated over a wide range of climatic conditions and
therefore understanding of genetics is of great value for genetics and plant breeding
purposes.
Wheat belongs to family Poaceae (Gramineae) which includes major crop
plants such as wheat (Triticum spp. L.), barley (Hordeum vulgare L.), oat (Avena
sativa L.), rye (Secale cereale L.), maize (Zea mays L.) and rice (Oryza sativa L.).
Triticeae is one of the tribes containing more than 15 genera and 300 species
including wheat and barley.
Linnaeus in 1753 first classified wheat. In 1918, Sakamura reported the
chromosome number sets (genomes) for each commonly recognized type. He
separated wheat into three groups viz. diploids (2n=14), tetraploids (2n=28) and
hexaploids (2n=42) chromosomes.
1.1.2. Wheat cultivation in India
Wheat cultivation in India started 5000 years ago (Feldman, 2001). Today, India
ranks second in wheat production with a harvest of 80.4 million ton during the season
08-09 (Fig 1.3).Its cultivation area is 28 M Ha.
Breeding programs are traditionally empirical, that is selection is generally based on
yield only which has limitations under stress environment. To meet the increasing
demand of wheat production without increasing area, there is need to incorporate new
physiological tools. These tools will help for the improvement of breeding programme
under abiotic stress environment. If specific physiological trait associated with yield
could be identified under stress environment, selection efficiency could be increased.
These traits will contribute to more objective screening of yield for selection in early
generations, when grain yield may not be properly assessed.
On the basis of agro climatic diversity wheat cultivation in India has been divided in
to six mega zones (fig 1.1). Maharashtra and Karnataka comes under Peninsular Zone.
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Wheat is grown in all the states in India except Southern and North Eastern states.
Uttar Pradesh, Haryana, Punjab, Rajasthan are the major wheat producing states and
accounts for almost 80% of total production in India. Only 13% area is rainfed. Major
Rainfed wheat areas are in Madhya Pradesh, Gujarat, Maharashtra, West Bengal and
Karnataka. Central and Peninsular Zone accounts for total 1/3rd of wheat area in India.
All India basis only 1/3 irrigated wheat receives desired irrigations and remaining is
limited irrigation only. Breeding programmes are generally aimed for rainfed and
irrigated environments and there is need to develop varieties which are responsive to
limited irrigation conditions.
Thus to increase the productivity of this region different physiological techniques
need to be adopted , for improving water use efficiency and breeding wheat genotypes
tolerant to water stress and heat.
1.2 Water stress Water stress is of common and wide occurrence in nature. It occurs whenever water
absorption by the crop is lower than the evaporative demand of the atmosphere. There
are two major processes involved in that i) water absorption by the crop which is
controlled by root characteristics and soil properties. ii) Crop evapotranspiration (ET)
which depends on atmospheric properties like net radiation, vapour pressure deficit
(VPD) and crop characteristics. Wheat may experience water stress in any
environment. CIMMYT has defined 12 mega environments (ME) as irrigated region,
high rainfall areas, acid soils, semi arid zones tropical areas and winter wheat zones
(Rajaram et al., 1995). ME definition is based on water availability, soil type,
temperature regime, production system and associated biotic and abiotic stress.
Taking into account all these factors India comes under ME4C.In this mega
environment wheat crop suffers from continuous or subcontinent type of drought
which is associated with stored moisture after monsoon rain that is rainfed condition.
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Fig 1.1: Different Wheat growing zones in India
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Fig 1.2: Monthly temperature variation during crop season
Fig 1.3: Area, production and productivity of wheat in India.
Source: Project Directors report DWR Karnal
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1.3. Drought tolerance 1.3.1. Drought
World food production is limited primarily by environmental stresses. It is
very difficult to find ‘stress free’ areas where crops may approach their potential
yield. Abiotic environmental factors are considered to be the main source (71%) of
yield reductions (Boyer, 1982). Drought is one of the most common environmental
stresses that affects growth and development of plants through alterations in
metabolism and gene expression (Leopold, 1990). It is a permanent constraint to
agricultural production in many developing countries (Ceccarelli and Grando, 1996).
Wheat production suffers from variability in yield from year to year and from
location to location. One of the main environmental abiotic stress responsible for
yield instability is drought stress, which may occur early in the season or terminally at
grain filling and grain development stages. Improvement of productivity of wheat
cultivars under drought conditions is one of the important breeding objectives in
wheat.
1.3.2. Mechanism of drought tolerance
Drought tolerance in wild plant species is often defined as survival, but in crop
species it is defined in terms of productivity (Passioura, 1983). Rosielle and Hamblin
(1981) defined drought tolerance as the difference in yield between stress and non-
stress environments, while productivity is the average yield in stress and non-stress. A
different definition regards drought tolerance as minimization of reduction in yield
caused by stress compared to yield under non-stress environments (Fischer and
Maurer, 1978;; Blum, 1983a; Blum, 1988). Also, it is defined as the relative yield of a
genotype compared to other genotypes subjected to the same drought stress (Hall,
1993). Drought tolerance comprises drought escape, dehydration avoidance and
dehydration tolerance mechanisms (Blum, 1988).
1.3.2.1. Drought escape
Drought escape through early flowering and/or short growth duration is advantageous
in environments with terminal drought stress and where physical or chemical barriers
inhibited root growth (Turner, 1986; Blum, 1988; Blum et al., 1989). On the other
hand, late flowering can be beneficial in escaping early-season drought, if drought is
followed by rains (Ludlow and Muchow, 1990). Under non-stress conditions, late-
Introduction and Review of literature
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flowering varieties tend to yield higher than early-flowering varieties (Turner, 1986;
Ludlow and Muchow, 1990). This is because the early-flowering varieties are likely
to leave the yield potential unutilized (Muchow and Sinclair, 1986).
1.3.2.2. Dehydration avoidance
Dehydration avoidance can be defined as the plant’s ability to retain a relatively
higher level of ‘hydration’ under conditions of soil or atmospheric water stress (Blum,
1988). Levitt (1980) identified two plant types with respect to dehydration avoidance
i.e. ‘water savers’ and ‘water spenders’. Important features of these plants are root
characteristics (increased water uptake), leaf and stomatal characteristics (reduced
water loss) and osmotic adjustment to lower the osmotic potential (Blum, 1988;
Acevedo and Fereres, 1993).
1.3.2.3. Dehydration tolerance
Dehydration tolerance describes the ability of plants to continue metabolizing at low
leaf water potential and to maintain growth despite of dehydration of the tissue.
According to Hsiao (1973) and Boyer (1976), translocation is one of the more
dehydration tolerant processes in plants. It would proceed at levels of water deficit
sufficient to inhibit photosynthesis. When water stress occurs and the current
photosynthetic source is inhibited, the role of stem reserves as a source for grain
filling increases. Stem reserves may therefore be considered as a powerful resource
for grain filling in stress-affected plants during the grain filling stage.
1.3.3. A conceptual model for drought tolerance
The conceptual model was described by Reynolds et al., (2000). This model includes
following traits:
Large seed size
o Helps in emergence, early ground cover and initial biomass.
Long coleoptiles
o Helps for emergence from deep sowing (Radford, 1987). This enables
to help seedlings to reach the receding moisture profile and to avoid
high soil surface temperatures which inhibit germination.
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Early ground cover
o Thinner, wider leaves (i.e. with a relatively low specific leaf weight)
and a more prostrate growth habit help to increase ground cover, thus
conserving soil moisture and potentially increasing radiation use
efficiency (Richards, 1996).
High pre-anthesis biomass
o Up to 40% of available water may be lost by evaporation directly from
the soil surface in Mediterranean types of environments (Loss and
Siddique, 1994), so early ground cover and biomass production may be
useful to permit a more efficient use of soil water.
Good capacity for stem reserves and remobilization
o Stored fructans can contribute substantially to grain filling especially
when canopy photosynthesis is inhibited by drought (Rawson and
Evans, 1971). Traits that may contribute include long and thick stem
internodes, with extra storage tissue perhaps in the form of solid stems.
High spike photosynthetic capacity
o Spikes have higher WUE than leaves and have been shown to
contribute up to 40% of total carbon fixation under moisture stress
(Evans et al., 1972). Awns contribute substantially to spike
photosynthesis and longer awns are a possible selection criterion.
High RLWC/CTD during grain filling to indicate ability to extract water
o A root system that can extract whatever water is available in the soil
profile is clearly drought adaptive (Hurd, 1968), but that is difficult to
measure. Traits affected by the water relations of the plant, such as
relative leaf water content (RLWC) , canopy temperature depression
(CTD) during the day and C13 discrimination or ash content of grain or
other tissues, can give indications of water extraction patterns.
Osmotic adjustment
o Osmotic adjustment will help to maintain leaf metabolism and root
growth at relatively low leaf water potentials by maintaining turgor
pressure in the cells (Morgan and Condon, 1986).
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o
Accumulation of abscisic acid (ABA)
o It appears that plants can be pre adapted to stress by reducing stomatal
conductance, rates of cell division, organ size and increasing
development rate. The benefit of ABA accumulation under drought has
been demonstrated (Innes et al., 1984).However, high ABA can also
result in sterility problems since high ABA levels may abort
developing florets.
Leaf anatomy: waxiness, pubescence, rolling and thickness
o These traits decrease radiation load to the leaf surface (Richards,
1996). Benefits include a lower evapotranspiration rate and reduced
risk of irreversible photo-inhibition.
High tiller survival
o Comparison of old and new varieties have shown that under drought
older varieties over-produce tillers many of which fail to set grain
while modern drought tolerant lines produce fewer tillers most of
which survive (Loss and Siddique, 1994).
Heat tolerance
o The contribution of heat tolerance to performance under moisture
stress needs to be quantified, but it is relatively easy to screen
(Reynolds et al., 1998).
Stay green
o The trait may indicate the presence of drought avoidance mechanisms,
but probably does not contribute to yield .If there is no water left in the
soil profile by the end of the cycle to support leaf gas exchange, it may
be detrimental if it indicates lack of ability to remobilize stem reserves
(Blum, 1998). However, research in sorghum has indicated that stay-
green is associated with higher leaf chlorophyll content at all stages of
development and both were associated with improved yield and
transpiration efficiency under drought (Borrell et al., 2000).
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1.4 Identification of the physiological traits for use as a selection criterion The success of a physiological approach to improve yield under drought will
depend on the effective identification of the limiting trait and then on its genetic
complexity. The identity proposed by Passioura (1977) provides the most valuable
framework. Passioura proposed that grain yield in water- limited environments
is the product of three factors, viz:
Grain yield = crop water use x water-use efficiency x harvest index.
Improving any one of these should improve grain yield provided that the
components are largely independent on each other. This identity should be
considered in relation to the target environment.
The traits to be considered as candidates for yield selection must be
genetically correlated with yield. The trait should have greater heritability
than yield and less subject to genotype x environment interaction. Different
approaches can be used to identify potential traits for selection .One of the
approach is to identify the main physiological process involved in yield
determination and the plant attributes influencing them.
Yield can be divided into several integrative components. Yield itself is the
most integrative trait, because it is influenced by all factors that determine
productivity. However, there are many limitations in empirical breeding
approach based only on yield. Therefore, any breeding strategy based on a
physiological approach should use screening tools to evaluate the integrative
physiological parameters that determine harvestable yield. Although harvest
index has been the most successful trait when modified to improve yield.
Genetic advances in grain yield under rainfed conditions have been achieved
by empirical breeding methods and selection is generally based on yield.
There is urgent need to develop varieties responsive to limited irrigation. To
meet future challenges of increasing productivity under these situations
breeding strategies should incorporate the new physiological traits that can
increase water use efficiency. If specific physiological trait related to yield
could be identified, selection efficiency could be increased and may contribute
to meaningful screening for yield in early generations where grain yield may not be
properly assessed.
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Several morpho-physiological traits are traditionally used as screening criteria for
drought tolerance such as Relative Water content (RWC), stomatal traits, water use
efficiency (WUE) etc. Crop WUE can be increased by optimizing crop transpiration.
Under field conditions variation of environmental factors makes it difficult to reveal
genetic variation. Hence several workers have proposed carbon isotope discrimination
(CID), ash content and canopy temperature depression (CTD) as criteria for
transpiration efficiency. These may be used as surrogate measures for grain yield. The
proposed work therefore aims to assess the efficacy of these traits in breeding wheat
under water stress conditions.
1.5 Carbon Isotope Discrimination Carbon isotope discrimination is the ratio of 13C/12C. In C3 cereals like wheat, ∆ is
positively correlated to CO2 levels in intercellular air spaces (fig 1.4) (Farquhar et
al., 1982; Farquhar and Richards, 1984; Ehdaie et al., 1991) and, negatively related to
water use efficiency, (Farquhar and Richards, 1984; Hubick and Farquhar, 1989).
Plants with high WUE would be less able to discriminate against 13C, and thus
would accumulate more of the heavy carbon isotope in their tissues than less efficient
water users.
Carbon isotope discrimination provides an indication of WUE throughout plant
growth when measured in plant dry matter (Farquhar et al., 1982, 1989). Carbon
isotope discrimination has been proposed as a possible screening tool for identifying
variations in WUE in wheat (Farquhar and Richards, 1984; Ehdaie et al., 1991;
Condon and Richards, 1993) and barley (Hubick and Farquhar, 1989). The
relationship between WUE and ∆ indicate that ∆ may be useful for modifying the
WUE and yield under water stress condition. (Condon et al., 1987; Condon and
Richards, 1992, 1993).
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Fig 1.4: Carbon isotope discrimination under irrigated and dry conditions
STOMATA OPEN (irrigated conditions): Discrimination in favour of 12 co2fixation at high internal co2 concentration
STOMATA closed (moisture stress): Discrimination less favorable to 12Co2fixation as internal low co2 concentration falls
Introduction and Review of literature
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1.5.1 Sample selection criterion
Considerable genotypic variations for carbon isotope discrimination have been
found in bread wheat (Condon and Richards, 1992), barley (Romagosa and Araus,
1991; Acevedo, 1993), and durum wheat (Araus et al., 1993a), but environmental
factors may cause even larger changes in the value of ∆, which could compromise
the effective
use of ∆ in breeding programs. Experiments carried out by Condon and Richards
(1992) concluded that assessing genotypic variation in ∆ would be most effective
under well-watered conditions. In this regard, Richards and Condon (1993) pointed
out that under WW conditions, ∆ is highly heritable and exhibits substantial
genetic variation and few genotypes x environment interactions.
In rainfed environments, Condon and Richards (1992) proposed sampling for ∆ at
early crop stages. However, the information available on rainfed environments does
not support the above hypothesis. When dry material from seedlings is analyzed
correlation between ∆ and yield is weak (Bort et al., 1998), although it increases
when upper plant parts are used in ∆ analysis. The best genetic correlations between
∆ and yield, together with the high broad sense heritability of ∆, have also been
reported for the upper parts of durum wheat (Araus et al., 1998b).
The effects of progressive stress after anthesis on yield revealed that the
correlation between yield and ∆ increases with plant age. On the contrary, ∆ usually
decreases from the oldest to the youngest plant parts, under well-watered
conditions (Hubick ‘and Farquhar, 1989; Acevedo, 1993). This decrease may lead to
stomatal closure in response to declining soil water or increasing vapor-pressure
deficit during the later stage of crop growth (Condon and Richards, 1992; Condon et
al., 1992; Araus et al., 1993b). Thus, mature kernels could be the most adequate plant
part to sample.
1.5.2 Carbon isotope discrimination (High or low)
In water-limited environments, genotypes with low ∆ should have greater
biomass and hence potential for higher yield, assuming that all genotypes use the
same amount of water for transpiration (Richards, 1996). Under same conditions,
selection for high WUE (Passioura, 1977) or low ∆ (Craufurd et al., 1991) has
been proposed as an important alternative in breeding strategies. However, under
Introduction and Review of literature
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well-irrigated or rainfed conditions ∆ values often correlate positively with grain
yield and/or total biomass in wheat (Condon et al., 1987; Kirda et al., 1992; Araus
et al., 1993c, 1997b; Morgan et al., 1993; Sayre et al., 1995) and barley
(Romagosa and Araus, 1991; Richards, 1996).
From an agronomic point of view, a positive relationship between ∆ and
yield may exist if plants are not using all available soil water. Assuming the same
phenology, a genotype with high ∆ will be able to sustain a high level of
transpiration. Therefore, ∆ can be considered as an indicator of WUE, but also
depends on the water transpired by the crop. The positive association between ∆
and yield suggests that variations in stomatal conductance are predominant than
intrinsic photosynthetic capacity in determining ∆ (Romagosa and Araus, 1991;
Condon et al., 1992). High ∆ is related to a high level of CO2 in the cellular air
spaces due to greater stomatal conductance (Farquhar and Richards, 1984). This
leads to higher photosynthetic rates and, higher yield even in the absence of stress.
In this situation, decrease in WUE decreases (and ∆ increases) reduces
transpiration more than photosynthesis, even when yield may be positively
affected by low stomatal limitation.
Alternatively, lower stomatal conductance, may limit yield potential because of
the intercellular levels of CO2, resulting decrease in photosynthesis. These
genotypes will consistently show low ∆ values (Morgan et al., 1993). Stomata
that close only in response to severe water stress may be more useful in terms of
yield than stomata that permanently show low conductance values (Jones, 1987).
Selection for low ∆ i.e., high WUE may favor low-yielding genotypes under
drought conditions. Therefore, low ∆ may not be a good selection criterion for
improving yield in dry environments. Grain yield under drought conditions
depends not only on WUE but largely on the genotype’s capacity to sustain
transpiration (Blum, 1993).
1.5.3 Optimal yield conditions
Carbon isotope discrimination has been proposed as a useful trait to select
for yield potential (Araus et al., 1993c; Sayre et al., 1995; Araus, 1996). A positive
relationship between ∆ and growth has also been reported for seedlings grown
under adequate water conditions (Febrero et al., 1992; Lopez Castaneda et al.,
1995). On the other hand, increased early growth and leaf area development may
Introduction and Review of literature
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be closely linked to decreased WUE (Turner, 1993) and thus resulted in higher ∆.
Blum (1996) reported that carbon isotope discrimination and yield support a
consistent positive relationship between crop yield and photosynthetic capacity for
wheat and other crops. He also reported that higher crop productivity brings an
increase in stomatal conductance, with related increase in ∆ (or a decrease in crop
WUE).
Carbon isotope discrimination (∆) has been proposed by several workers as a
predictive selection criterion for grain yield under drought (Araus et al., 2001;
Merah et al., 2001b; Condon et al., 2002; Monneveux et al., 2005;Misra et al.,
2006; Xu et al., 2007, Monneveux et al., 2007, Cabera –Bosquet et al.,2009). In C3
species, ∆ positively correlates to Ci/Ca, the ratio of internal CO2 leaf
concentration to ambient CO2 concentration (Farquhar et al., 1989).It also
provides an integrated measurement of long term Ci/Ca ratio and transpiration
efficiency (Condon et al., 1990). Carbon isotope discrimination in grain was found
to positively correlate to yield in the conditions of South Australia (Condon et al.,
1987), North West Mexico (Sayre et al., 1995; Monneveux et al., 2005), Spain
(Araus et al., 1998) and South of France (Merah et al., 2001c). All these conditions
correspond to Mediterranean-type environments, characterized by limited
irrigation water stress and moderate heat during grain filling (Rajaram et al., 1995).
Under limited irrigation significant correlation was found between grain ∆ and
yield in the conditions of North West Mexico (Monneveux et al., 2004). He also
reported significant correlation between grain ∆ and yield at pre-anthesis and
residual moisture water stress (Monneveux et al., 2005). However, the magnitude
of the correlation was lower than under post-anthesis water stress. Under severe
post-anthesis water stress, significant correlations were found between grain yield
and ∆ in flag leaf (Merah et al., 1999) and awns (Merah et al., 2001c). Under
residual moisture stress, negative correlation was found between grain yield and ∆
in young seedlings by Condon and Hall (1997).
Introduction and Review of literature
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1.5.4 Role of phenology in genotypic differences in carbon isotope discrimination
In the absence of stress, ∆ in wheat is independent of phenological
differences (Sayre et al., 1995). In Mediterranean environments phenology is the
most important factor that accounts for increasing wheat yield, as it affects
assimilate partitioning, the pattern of water use. (Slafer et al., 1993; Loss and
Siddique, 1994). In addition, some of the genotypic differences in ∆, as well as
their positive association with yield, can be due to phenology. Thus, early
flowering lines are more likely to have high ∆ than late-flowering lines due to the
lower transpirative demand, which maintains higher stomatal conductance
(Ehdaie et al., 1991; Acevedo, 1993). In, short early flowering in wheat and other
cereals is related to higher yield, which is in accordance with higher ∆ in the early
genotypes.
1.6 ∆ Surrogates The cost and technical skills involved in carbon isotope analysis is very high
therefore, different surrogates such as mineral accumulation in vegetative plant
parts, leaf structure, ash content and canopy temperature depression (CTD) have
been proposed. These selection criteria allow the evaluation of traits other than
WUE that determines yield. Regarding the mineral accumulation, if passive
transport driven by transpiration is the mechanism of mineral accumulation in
vegetative parts, then mineral content will also be an indicator of the total water
transpired. The second trait i.e. leaf structure corresponds to structural criteria that
indicate the amount of photosynthetic tissue per unit leaf area and is therefore
related to photosynthetic capacity. The third trait canopy temperature depression is
a good indicator of a genotype's physiological fitness, since a high value is
indicative of good expression of all of those traits under a given set of
environmental conditions. When water evaporates from the surface of a leaf it
becomes cooler, and the rate of evaporative cooling is directly affected by stomatal
conductance (besides VPD), which is itself affected by feedback mechanisms of
other processes such as photosynthetic metabolism and vascular transport.
Introduction and Review of literature
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1.7 Ash content Ash content accumulated in vegetative tissues have been proposed as surrogates
of ∆ in cereals, forage crops, and soybean (Walker and Lance, 1991; Masle et al.,
1992; Mayland et al., 1993; Mian et al., 1996). Masle et al., (1992) reported for
all the herbaceous C3 species they assayed a positive linear relationship between
total mineral content of vegetative tissues and the inverse relationship with WUE
or ∆. Therefore, the amount of minerals accumulated by plants in the field could
be a potentially useful indicator of ∆ and WUE (Walker and Lance, 1991; Masle
et al., 1992; Mayland et al., 1993). In theory, total mineral and ash content seem
to be better surrogates than the content of any single mineral, such as silicon or
potassium (Masle et al., 1992; Mayland et al., 1993). Therefore, estimating plant
mineral content, especially ash content, which requires a muffle furnace, might
be a good alternative to ∆ for preliminary screening of large, genetically diverse
populations (Masle et al., 1992; Araus et al., 1998b).
1.7. 1 Determination of ash content
Ash content was analyzed by using the Method 08-01 of the American Association
of Cereal Chemists, (1995). Ash content is expressed (on a concentration basis) as
a percentage of sample dry weight as follows:
[(Burnt crucible mass – empty crucible mass)
% Ash = ………………………………………………… x100
[(Filled crucible mass-empty crucible mass)]
1.7.2 Choice of environment and type of sample
The positive correlation between ash content and ∆ may indicate that plants
which are able to maintain higher stomatal conductance and transpiration will
accumulate more ash in a transpirative organ. Mineral accumulation seems to be
better related to ∆ and yield under well-watered conditions (Masle et al., 1992;
Mayland et al., 1993), as well as under drought conditions (Araus et al., 1998b).
Ash accumulated in the flag leaf may be positively related to the ∆ of kernels
(Araus et al., 1998b). Leaves must be mature to allow minerals accumulate, but
not senescent because minerals can remobilize to other plant parts. Ash content
measured at maturity in straw did not correlate with either ∆ of mature grains or
yield (Voltas et al., 1998). This may be explained by the fact that ash content on a
Introduction and Review of literature
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kernel mass basis may be an indirect indicator of total reproductive sink per culm
attained at maturity (Araus et al., 1998b). Under Mediterranean conditions kernel
ash has been proposed as complementary to kernel ∆ in assessing genotypic
differences in cereal yield (Febrero et al., 1994; Voltas et al., 1998). In theory the
pattern of ash accumulation in kernels is different from that in vegetative tissues
because, unlike mineral accumulation in vegetative tissues, grain filling does not
take place via the xylem (driven by transpiration) (Slafer et al., 1993). Such
differences in mineral accumulation could explain the interrelationship of ash
content and ∆ kernels as necessary traits predicting grain yield (Febrero et al.,
1994; Voltas et al., 1998).
In short, kernel ash combined with either kernel ∆ or leaf ash can be independent
trait when assessing differences in grain yield (Febrero et al., 1994; Araus et al.,
1998b; Voltas et al., 1998). Thus selecting for low ash content in kernels,
combined with high ∆ in kernels or, high ash content in the flag leaf, may be
useful in wheat breeding programme. This approach, if coupled with a new
analytical technique such as near infrared reflectance spectroscopy (NIRS) would
allow a fast, reliable estimation of ash content and ∆ in intact kernels (Araus,
1996).
Correlation was found between ∆, leaf ma and grain yield in different C3
species by Masle et al., (1992), Mayland (1993) and Merah et al., (1999, 2001a).
Negative correlation was reported between ma in grain and grain yield by
Febrero et al., (1994), Voltas et al., (1998) and Merah (1999, 2001a), Misra et al.,
(2006), Xu et al., 2007 and L.Cabera-Bosquet et al., (2009) in Zea Mays.
1.8 Canopy temperature depression (CTD) Canopy temperature depression data was recorded at different stages of crop
growth for unirrigated crop using a portable infrared thermometer (Model AG-42,
Telatemp Corporation, Fullerton, CA).Reynolds et al., 2001b showed a clear
association of CTD with yield in both warm and temperate environments. Work
has been carried out to evaluate its potential as an indirect selection criterion for
genetic gains in yield. While CTD is affected by many physiological factors
making it a powerful integrative trait, its use may be limited by its sensitivity to
environmental factors.
Introduction and Review of literature
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Table 1.1: Factors affecting canopy temperature depression (CTD) in plants
Sr.No. Biological factor Environormental factor
1 Partitioning of assimilates Radiation
2 Evapotranspiration Wind
3 Soil water availability Clouds
4 Carbon metabolism Temperature
5 Vascular transport Moisture
1.8.1 Factors affecting expression of CTD
Leaf temperatures are depressed below air temperature when water evaporates from
their surface. It is one of the factors determining evapotranspiration which is directly
related to stomatal conductance. This process is regulated by carbon fixation and
vascular transport of water. Canopy temperature depression is a good indicator of a
genotype's physiological fitness. Its high value will be indicative of good expression for
all of those traits under a given set of environmental conditions. CTD is also affected by
the ability of a genotype to partition assimilates to yield.CTD is a function of number
of environmental factors like soil water status, air temperature, relative humidity and
incident radiation. The trait is best expressed at high vapour pressure deficit (Amani et
al., 1996), low relative humidity and warm air temperature.CTD will not be useful
trait for cool and humid conditions and it is quite sensitive to environmental changes.
Therefore, it is important to measure the trait when it is best expressed that is on
warm and relatively cloudless day.
The trait can be measured in a few seconds with an infrared thermometer,
which measures the surface temperature of a field plot. Since the reading integrates the
temperatures of plant leaves and spikes over a small area of the canopy, error associated
with plant to plant variability is reduced. CTD measured on irrigated yield trials showed
a good association with plot performance, but in addition a good predictor of yield.
1.8.2 Combining selection for CTD and leaf conductance
Canopy temperature depression and leaf conductance show an association with each
other and with yield (Amani et al., 1996). Combining selection for both the traits is a
good criterion. At CIMMYT, work is in progress to evaluate the application of CTD
for yield potential. The objective is to evaluate families of F3 for higher expression of
Introduction and Review of literature
21
CTD. From these, individual plants were assessed for leaf conductance using a
viscous flow porometer (Thermoline & CSIRO, Australia). This instrument can give a
relative measure of stomatal conductance in a few seconds (Rebetzke et al., 1996),
permitting the possibility of identifying physiologically superior genotypes within
bulks that have already been selected for CTD and other important selection criteria.
Thus CTD can be used as an efficient tool for screening in early generation which can
be further used for evaluating advanced lines. Experiments carried out by Reynolds et
al., (1997, 1998a) suggest that CTD can also be used for selections in smaller plots.
Fig 1.5 Potential use of CTD in a breeding programme
1.9 Water Use Efficiency (WUE) Carbon isotope discrimination (∆) has also been proposed by several workers for
estimating water use efficiency (WUE). WUE of a crop has been defined, as the ratio
of total biomass, or above-ground biomass, or harvested yield, against total available
water, or evapotranspiration, or plant transpiration (Jones, 2004). According to
Gregory et al. (1997), the ratio of total biomass produced against total available water
may be expressed as:
WUEB = M / (Es + T + R + D)
Introduction and Review of literature
22
Where M, Es, T, R and D are the biomass produced, the evaporation from the soil
surface, the transpiration during the growing season, the runoff, and the drainage
below the root zone, respectively.
Under drought conditions, carbon isotope discrimination (∆) was shown to be
negatively correlated to transpiration efficiency (Farquhar et al., 1982).It is also
negatively related to M/T (the ratio of biomass produced to water consumed, at the
plant level) and WUE (biomass or grain) under water stress conditions. A significant
negative correlation has been obtained in wheat between ∆ and M/T (Condon et al.,
1990; Ehdaie et al., 1991). In rice, Impa et al., (2005) also found a negative
association between leaf ∆ and WUE. Kirda et al. (1992), by using water balance
model (Hatfield, 1990) to evaluate evapotranspiration and by measuring soil water
with a neutron probe, reported a highly significant negative association between ∆ and
WUE in durum wheat grown in Austria. However, for bread wheat no significant
correlation was observed between ∆ and WUE in Australia (Condon et al., 1993).
WUE and the other traits can be used as an indirect selection for yield particularly in
drought prone environments (Monneveux et al., 2005; Misra et al., 2006; Xu et
al., 2007).
1.10 Molecular mapping in plants Biotechnology brings new and powerful tools to plant breeders, to meet
the increasing demand of food production. One method receiving growing
attention is the mapping of Chromosomal regions affecting qualitative or
quantitative traits. Polygenic characters, which were very difficult to analyse using
traditional plant breeding methods, can now be tagged using DNA molecular
markers. Molecular markers allow geneticists and plant breeders to locate and
follow the numerous interacting genes that determine a complex trait. Genetic
linkage maps can provide a more direct method for selecting desirable genes via
their linkage to easily detectable molecular markers (Tanksley et al., 1989).
Combining marker-assisted selection methods with conventional breeding schemes
can increase the overall selection gain and, therefore, the efficiency of breeding
program. With the use of molecular techniques it is possible to hasten the transfer of
desirable genes between varieties and to introgress novel genes from wild species
into crop plants. The plant breeder would like to exercise indirect marker aided
selection (MAS) at the seedling stage in early generations, if possible.
Introduction and Review of literature
23
Availability of tightly linked molecular markers for a trait will facilitate such an
indirect selection and help plant breeding by saving time and expense.
1.10.1 Molecular marker technologies for genetic mapping
The development of molecular marker technologies during the last ten
years has revolutionized the genetic analysis of crop plants. A significant progress
has been made towards the use of molecular approaches in plant breeding. From
the time of Gregor Mendel until the mid-eighties, morphological characters had been
the major types of markers readily available for genetic mapping. Molecular marker
technology has changed dramatically during the past two decades. The first molecular
markers were isozyme markers, which based on the different mobility of differently
charged protein with the same enzymatic function on the gel. Enzyme markers have
limited genome coverage and numbers. The term molecular marker is taken here to
refer to markers identifying variation at the level of DNA, though biochemical
markers such as isozyme have made a valuable contribution to the development of
genetic maps in the late seventies and eighties for example, of tomato (Tanksley and
Rick, 1980) and maize (Edwards et al., 1987).
The molecular markers may be used for improving the efficiency of
traditional plant breeding by facilitating indirect selection through molecular
markers linked to genes for the traits of interest. These markers are not
influenced by the environment and can be scored at all stages of plant growth.
This saves time, resources and energy that are needed for raising large
segregating populations for several generations. In addition to these applications,
DNA markers can also be used for germplasm characterization, genetic
diagnostics, study of genetic diversity, study of genome organization, etc.
(Rafalaski et al., 1996). Molecular markers have already been used not only for the
preparation of molecular maps but also for tagging genes, controlling traits of
interest, for use in marker assisted selection (MAS). In plants, using markers,
several genetic maps were initially constructed in tomato (Bernatzky and
Tanksley, 1986). Subsequently, maps were constructed in different crops such as
rice (McCouch et al., 1988, Kishimoto et al., 1989); maize (Burr et al., 1988;
Beavis and Grant, 1991; Burr and Burr, 1991), barley (Heun et al., 1991; Graner et
al., 1991; Hinze et al. 1991) or wheat (Chao et al., 1989; Liu and Tsunewaki, 1991;
Liu et al., 1992; Devos et al., 1992; Devos and Gale, 1993; Röder et al., 1998).
Introduction and Review of literature
24
1.11 Progress in wheat molecular genetics
1.11.1 Use of molecular markers for mapping and gene identification
Progress in gene identification and marker development has been slow in wheat due
to its hexaploid nature and the large size of its genome. However, in the recent past,
a significant number of genes involved in various functions have been mapped to
specific wheat chromosomal regions. Characterizing genes that control flowering in
wheat has benefited from chromosome manipulations involving aneuploidy as well
as molecular markers.
Using intervarietal chromosome substitution lines and single-chromosome
recombinant line populations, genes controlling vernalization response Vrn1 and
Vrn3 have been located on the long arms of chromosomes 5A and 5D, respectively
(Law et al., 1976), and VrnB1 on chromosome 5B (Zhuang, 1989). Similar
procedures have been utilized to identify genes controlling photoperiod response
(Ppd genes) (Worland and Law, 1986). Plant height, important for determining
adaptation and yield in wheat, is genetically complex; so far about 21 genes have
been identified to be associated with this trait (McIntosh et al., 1995). A
microsatellite marker has recently been developed that is linked to Rht8 (Korzun et
al., 1998).
Efforts have been made to genetically dissect complex physiological traits
associated with drought tolerance such as accumulation of abscisic acid in rice and
to investigate possible relationship between rice and wheat homologous loci
controlling abscisic acid accumulation (Quarrie et al., 1997). In wheat, using single-
chromosome recombinant lines, Quarrie et al. (1994) located a genetic factor
controlling drought-induced abscisic acid production on the long arm of
chromosome 5A. Molecular genetic tools have also been used to study complex
traits such as carbohydrate metabolism and the association between abscisic acid
concentration and stomatal conductance (Prioul et al., 1997). Comparative RFLP
mapping in cultivated and wild wheat (Triticum dicoccoides) has led to the
identification of molecular markers associated with resistance to the herbicide
chlorotoluron which is a selective phenylurea herbicide (Krugman et al., 1997). A
list of wheat genes that control various physiological and agronomic parameters that
have been identified with the use of molecular markers is presented in table 1.2.
Introduction and Review of literature
25
The existence of numerous sets of wheat near-isogenic lines (NILs) differing in the
presence/absence of a resistance allele for various biotic stress factors (diseases and
pests) has facilitated the mapping of genes for which such lines exist. Large number
of genes conferring disease or pest resistance have been identified and associated
with molecular markers (reviewed in Hoisington et al., 1999). When the
chromosomal location of a particular gene is known from previous genetic studies
but no NILs are available, markers mapped to that chromosome (Anderson et al.,
1992) can still be used to score parental lines for polymorphisms, construct a single-
chromosome map, and determine which markers are close to the gene of interest.
This strategy was followed by Dubcovsky et al. (1996) to tag the Kna1 locus in
wheat, which is responsible for higher K+/Na+ accumulation in leaves, a trait
correlated with higher salt tolerance.
In wheat, bulked segregate analysis, initially used mostly with RAPDs, can now be
used with any type of marker including AFLPs (Goodwin et al., 1998; Hartl et al.,
1998), which have the advantage that a high number of DNA fragments can be
amplified with one primer combination. Also, with AFLPs the problem of highly
repetitive DNA is overcome by using methylation sensitive endonucleases such as
PstI and SseI.Many genes that have been tagged with molecular markers in wheat
have been introgressed from alien species (Hoisington et al., 1999). In the case of
translocations from wheat’s wild relatives known to carry genes for agronomically
important traits, markers can be successfully established due to the high level of
polymorphisms between the wheat and introgressed genome and the low level of
recombination between the translocated segment and the corresponding wheat
chromosomes.
1.12 Mapping populations Mapping is putting markers (and genes or QTL) in order, indicating the
relative distances among them and assigning them to their linkage groups on the
basis of their recombination values from all pair wise combinations. Knowledge
about the genetic concepts of segregation and recombination is essential to the
understanding of mapping. The construction of a linkage map is a process that
follows the segregation of molecular markers in a segregating population and put
them in linear order based on pair wise recombination frequencies. Thus, a mapping
population with high number of polymorphisms over the total genome is highly
Introduction and Review of literature
26
desirable. Towards this end, various ways have been used to create mapping
populations. Populations used for mapping are usually derived from F1 hybrids
between two lines (either homozygous or heterozygous), which show allelic
differences for selected probes. Genetic maps of plants are constructed based on
several different kinds of populations (Paterson, 2002), with each population structure
having unique strengths and weaknesses. Four types of population are commonly
used for map construction and mapping experiment. These are F2 population, back
cross population (BC), double haploid (DH) population, and recombinant inbred
lines (RILs). Most genetic mapping populations in plants have been derived from
crosses between largely homozygous parents.
1.13 Mapping QTLs in wheat Utilizing a base map and linkage data from a range of other segregating wheat, rye,
and barley populations, a consensus map with more than 1000 data points has been
developed (Gale et al., 1995). This detailed linkage map has confirmed that the order
of genetic loci across the A, B, and D genomes has been conserved (Gale et al., 1995).
A RIL mapping population developed utilizing ‘Opata 85’ and a synthetic hexaploid
from CIMMYT has been used extensively in mapping and genome relationship studies
(Van Deynze et al., 1995; Nelson et al., 1995a, b, c). The genetic map of this
population, developed by the International Triticeae Mapping Initiative (ITMI), contains
over 1000 RFLP loci. Two other published maps are available in wheat (Liu and
Tsunewaki, 1991; Cadalen et al., 1997). Linkage maps in wheat have confirmed
evolutionary chromosomal translocation rearrangements involving chromosomes 2B,
4A, 5A, 6B, and 7B, which were based on cytological evidence, and have established
synteny among closely related grass species such as rice, maize, oats, and wheat (Ahn
et al., 1993; Devos et al. 1994; Van Deynze et al., 1995; Borner et al., 1998).
The low number of quantitative traits dissected into their QTLs in wheat is a
reflection of the focus on simply inherited traits and the difficulty of building
comprehensive linkage maps. ITMI map is one of the densest and the population from
which it was developed is segregating for a number of traits. It has been used to map
important traits and several major genes. Known genes include vernalization (Vrn1 and
Vrn3), red coleoptile (Rc1), kernel hardness (Ha), and powdery mildew (Pm1 and Pm2)
genes (Nelson et al., 1995a), as well as genes conferring and suppressing leaf rust
resistance (Nelson et al., 1997). Quantitative trait loci have been identified for kernel
Introduction and Review of literature
27
hardness (Sourdille et al., 1996), Karnal bunt (Nelson et al., 1998), and tan spot (Faris
et al., 1997).
A large number of studies have reported QTL for yield and yield components in
wheat in different environments including water-limiting stress environments. QTL
for grain yield, anthesis, plant height, grain weight, grain number have been reported
(e.g. Huang et al., 2006;Marza et al., 2006; Snape et al., 2007; Rebetzke et al., 2008;).
Three significant QTLs have been identified for CID in Barley by This D (2005). ,
two of them being antagonist on chromosome 4, and one other located on
chromosome 5. Arvindkumar et al., (2008) has identified the presence of significant
QTLs for ∆ on chromosomes 1B, 3A and 5A.
Research on developing molecular markers for traits associated with drought
tolerance in wheat started recently at CIMMYT. A RIL population is being utilized to
identify genomic regions associated with a range of physiological parameters
controlling drought tolerance.
Introduction and Review of literature
28
Table 1.2 Genes identified and mapped with molecular markers for physiological and agronomic traits in wheat.
Traits Genes Species Chromosomes References
Physiological and agronomic
Preharvest sprouting QTL Triticum aestivum Anderson et al., 1993
Galiba et al., 1995; Korzun et al., 1997 Vrn1 5AS
Kato et al., 1998 Vernalization
Vrn3
5DS Nelson et al., 1995a
Photoperiod response Ppd1 T. aestivum 2DS Worland et al., 1997
Ppd2 T. aestivum 2BS Worland et al., 1997
Dwarfing Rht8 2DS Korzun et al.,1998
Rht12 5AL Korzun et al., 1997
Cadmium uptake Penner et al., 1995
Aluminum tolerance Alt2 4D 4DL
Luo and Dvorak, 1996; Riede and
Anderson, 1996
Drought induced ABA 5AL Quarrie et al., 1994
Na+/K+ discrimination Kna1 T. aestivum 4D 4DL Allen et al., 1995; Dubcovsky et al.,
1996
Water-soluble carbohydrate Wheat 1A, 1 D, 2D, 4A, 6B,
7B, 7D
Yang et al., 2007
Carbon isotope ratio, osmotic potential,
chlorophyll content, flag leaf rolling index
Wheat 2B, 4A, 5A, 7B Peleg et al., 2009
Introduction and Review of literature
29
1.14 Genesis and structure of the thesis: Very few efforts have been made to understand the correlation between yield and
physiological parameters under water stress conditions in India. Based on the agro
climatic diversity, wheat cultivation in India has been divided in to six mega zones.
Maharashtra comes under Peninsular Zone. Wheat is grown in all zones except Southern
and North Eastern states. Uttar Pradesh, Haryana, Punjab, Rajasthan are the major wheat
producing states and accounts for almost 80% of total production in India. Central zone
and Peninsular zone accounts for 1/3rd of total wheat area in India. On all India basis only
1/3 irrigated wheat receives desired irrigations and remaining 2-3 irrigations (limited
irrigation). Breeding programmes are generally aimed for rainfed and irrigated
environments and there is need to develop varieties which are responsive to limited
irrigation condition. Breeding strategies in this region should aim for limited irrigation
requirement with high yield. Thus, to improve the productivity of this region the
technologies need to be adopted, to improve water use efficiency and breed for wheat
genotypes having higher tolerance to water stress and heat.
Several morpho physiological traits are traditionally used as screening criteria for drought
tolerance such as relative water content (RWC), stomatal traits, water use efficiency
(WUE) etc. Under field conditions variation of environmental factors makes it difficult to
reveal genetic variation. Several workers have proposed carbon isotope discrimination
(CID), ash content and canopy temperature depression (CTD) as criteria for transpiration
efficiency, Misra et al., (2006, 2010), Zhang et al., (2009), Blum et al., (2009). These are
the surrogate measure for grain yield. Present work is undertaken to identify suitable
physiological traits which can be useful in wheat breeding under water stress conditions,
parameters related to grain yield and water use efficiency and further identification of
QTLs for selected RILs. Therefore the objectives selected for present work are-
1.14.1 Objectives:
The main objective of the proposed work is to identify suitable
physiological traits which can be useful in wheat breeding under water stress
environment.
Introduction and Review of literature
30
To analyze the relationship between carbon isotope discrimination,
ash content, grain yield and water use efficiency components in durum wheat.
Identification of QTLs for carbon isotope discrimination, ash content
and canopy temperature depression.
In order to achieve these objectives review of literature pertaining to carbon isotope
discrimination, ash content and canopy temperature depression and QTL analysis for
these traits was carried out. The work regarding these parameters has been organized in
subsequent chapters-
Chapter 1 Introduction and review of literature
Chapter 2 Utilization of carbon isotope discrimination and ash content to select
wheat under water stress condition.
Chapter 3 Relationship between carbon isotope discrimination, ash content, canopy
temperature depression and grain yield in wheat under water stress
condition.
Chapter 4 Analysis of the relationship between carbon isotope discrimination, ash
content,
grain yield and water use efficiency components in durum wheat.
Chapter 5 Identification of QTLs for carbon isotope discrimination, ash content and
canopy temperature depression.
Chapter 6 Summary and Conclusion
Bibliography
Curriculum vitae