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BREEDING FOR ABIOTIC STRESS RESISTANCE

Minimizing losses due to abiotic stress
• Crop management
• Development of resistant varieties
Indirect breeding for stress resistance
Direct breeding for stress resistance
Selection for survival
Selection for yield
Selection for traits contributing to stress resistance
Common stresses
a. Drought b. Heat
c. Cold d. Salinity
e. Mineral toxicity e. Oxidation
f. Water logging

Criteria for the development and use of screening test

• Genetic variation in the germplasm pool
• Trait with high heritability than yield per se
• Trait should be correlated with a yield based stress resistance index
• Trait should enhance the yield
• Screening test should be easy, rapid and economical to apply

Focus on breeding for drought resistance should be on water use rather than water
saving
= × ×
Where,
T = Total seasonal crop transpiration
WUE = Crop water use efficiency
HI = Crop harvest index

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BREEDING FOR DROUGHT RESISTANCE

• Drought: inadequacy of water availability, including precipitation and soil moisture storage
capacity, in quantity and distribution during the life cycle of a crop to restrict expression of its
full genetic yield potential
• Moisture stress: the inability of plants to meet the evapo-transpirational demand
Is likely to develop to a different degree in different plant organs along this gradient
• Drought resistance
Is the mechanism causing minimum loss of yield in a drought environment relative the
maximum yield in a optimal environment of the crop
Is sum total of avoidance and tolerance
Refers to the genetic ability of plants to give good yield under moisture stress conditions

Mechanisms of drought resistance

• Drought escape
• Dehydration avoidance
• Dehydration tolerance

Drought escape
• Describes the situation where an otherwise drought susceptible variety performs well in a
drought environment simply by avoiding the period of drought
• Early maturity is an important attribute of drought escape and is suitable for environments
subjected to late season drought stress
• Maturity duration may be more deeply involved in the plant-water relations than its mere
effects on total evapotranspiration
Dehydration avoidance
• Is the ability of a plant “to retain a relatively higher level of hydration under conditions of soil
or atmospheric water stress”
• Protects various physiological, biochemical and metabolic processes of plants involved in
growth and yield from being exposed to water stress and thereby, protects the plants from
water stress
• Can be achieved by
a. Reduced transpiration: water saver (xerophytes)
Stomatal sensitivity to water stress

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Stomatal closure is likely to interfere with photosynthesis and drought resistance

mechanism based on stomatal sensitivity and reduced transpirations are generally opposed
to maintenance of a higher yield potential
b. Osmotic adjustment
Refers to the active accumulation of solutes (viz. organic acids – sugars, fructans; Inorganic
ions – K+) in cells during the period in which water stress develops
Osmoregulation is positively associated with yield under stress conditions as it allows
growth and results in delayed leaf death by maintaining turgor pressure
c. Abscisic acid (ABA): stress hormone (concentration increases in response to stresses)
 Plays a major role in water stress avoidance by effecting stomata closure, reducing leaf
expansion an promoting root growth
 Various components like ABA receptors, G-proteins, protein kinases, etc. are involved in
regulation of stomata closure by ABA
d. Cuticular wax
Amount of transpiration depend mainly on the wax deposited within and over the cuticle
Shape and angle of wax deposition may affect leaf reflectance within the spectrum range of
400 to 700 mm, which in turn may affect net radiation and leaf temperature

e.g., increased glaucousness in wheat and sorghum reduce net radiation and leaf temp and
improved yield under stress
Effect of cuticular wax on transpiration is small
e. Leaf characteristics
Leaf pubescence: increases leaf reflectance and reduces net radiation resulting in lower leaf
temperature under high irradiance
Leaf angle: net radiation can also reduce by altering leaf angle from horizontal to erect leaf
Paraheliotropic movement of leaf in legumes
Leaf rolling: induced by loss of turgor and hence is delayed by osmotic adjustment
f. Increased water uptake: root characteristics
Deep root system: when soil moisture is unlimited at deeper soil horizons
Large root length density and small root (hydraulic) resistance: if no additional moisture
reserves at deeper soil layers
Dense root system and/or a low root resistance: in transient soil drying and wetting
In stored moisture environments: minimize evapotranspiration either by reducing leaf area
index or by development of a greater hydraulic resistance in root system

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Dehydration tolerance
• When cells lose turgor and dehydration, there is
a) Reduced chemical activity of water
b) Increased concentration of solutes and macromolecules
c) Removal of water of hydration from macromolecules and
d) Alternation in cellular membranes
• Dehydration tolerance means that a significantly lower level of change are induced in
genotypes than those in another genotypes when both of them are subjected to same level of
dehydration
• Measurements of dehydration tolerance
a. Maintenance of membrane integrity:
Determined by leakage of solutes (amino acids, electrolytes, hormones, organic acids,
saccharides) from cells
b. Plant growth
Measure using seedling by assessing either a) growth or b) survival
 Seedling survival or recovery after stress is a useful index

 Seedling growth under stress: serve the same purpose as seedling survival
 Seed germination under osmotic stress created by mannitol or PEG
 Stem reserve mobilization/translocation appear to be related to drought tolerance/resistance
and could be signaled by ABA accumulated in response to water stress
 Presence of a large amount of awns is a drought adaptive attribute in cereals as awns are
more water use efficient than flag leaf and even glumes
 Proline accumulation appears to be involved in tolerance to water and other stresses as
large amounts of proline accumulation contributes to osmotic adjustment
Sources of drought resistance
Crop Wild relative Resistant to
• Cultivated varieties Wheat Aegilops variabilis Drought
• Land races Ae. speltoides Drought
• Related wild species Ae. umbellulata Drought
• Transgenes Ae. squarrosa Drought
Agropyron pontica Salinity
Sugarcane S. spontaneum Drought and salinity
Tomato L. cheesmanii Salinity

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ABA mediated stomatal response

• Stress proteins encoded by stress responsive genes, which in turn, induced by one or more
stresses (like heat, cold, salinity, drought, etc.)
• Early-responsive to dehydration (ERDS) genes induced ~1hr after dehydration begins
• Many compounds, e.g., osmolytes, chaperones, proteinase inhibitors, proton ATPases, etc. get
accumulated in response to water stress tolerance
• Many of the stress proteins like dehydrin, osmotion, Lea proteins, etc. are produced in
response to ABA; genes encoding such proteins are called ABA-responsive (ABAR) genes
• ABAR genes have in their promoters an ABA response element (ABARE), which has ACGT
as its core sequence
• ABA induced proteins are hydrophilic and are involved in a variety of functions, including
polyamine biosynthesis
• ABA activates transcription of genes for polyamine biosynthesis and, in turn, the polyamine
putrescine activates ABA biosynthesis
• Polyamine promote the production of NO and H2O2, a reactive oxygen species (ROS)
• The polyamines, NO and ROS act synergistically to promote ABA regulated closure of
stomata

Fig: Water, abiotic and oxidative stress and signaling

interaction between ABA, H2O2 and NO
Fig: Kinetics of ABA through plant

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Factors affecting genotypic difference in ABA

• Several factors determine ABA accumulation and the stomatal response to a given
concentration of ABA
water status of plants, leaf water potential, leaf turgor, soil water status, pH, soil
compaction
environmental factors such as temperature, relative humidity, light or time of day,
changes in water flux through the xylem and previous exposure to ABA flux
• ABA is mobile within the plant moving up and down the plant; from roots to leaves through
xylem and from leaves to roots through phloem
• ABA concentration reaching guard cells at any given time can vary due to sequestration,
remobilization and degradation and/or conjugation
• ABA accumulation in response to drought can be confounded effect of two or more of the
above factors
• Stomata are controlled by several feedback loops involving external and internal factors such
as light, VPD, intercellular CO2 concentration, leaf turgor and soil water status

Stomatal conductance and photosynthesis

• As stomata serve as a portal through which water exits the leaf and CO2 diffuses into leaf
tissue for photosynthesis, stomatal regulation to limit water use during water deficit is at the
expense of CO2 diffusion into leaf tissue which subsequently reduces photosynthesis
• In addition to the reduced CO2 influx, soil water deficits reduce mesophyll conductance of
CO2 limiting photosynthesis
• As the relative water content (RWC) decreases due to soil water deficit, decreased ATP
synthesis and consequent RuBP synthesis causes metabolic limitation of photosynthesis
• Interestingly, the rate of reduction of CO2 assimilation is comparatively less than the
reduction in transpiration
• In well-adapted plants, the stomatal role in controlling photosynthesis is not more than 20%
of the total photosynthetic inhibition
• So the positive effect of reduced transpiration may outweigh the negative effect of decreased
photosynthesis under terminal drought conditions
ℎ ℎ − ℎ
=
ℎ − ℎ

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Q. The following maize genotypes were grown normal condition. Calculate RWC and interpret
which genotype(S) is/are tolerance or sensitive to drought and why?
Genotypes Leaf weight(g)
Fresh Turgid Dry
H1 0.88 0.92 0.25
H2 1.11 1.18 0.30
H3 0.84 0.88 0.22
H4 1.47 1.55 0.41
H5 0.97 1.04 0.29
H6 0.59 0.62 0.16
H7 0.91 1.02 0.29
H8 0.80 0.91 0.25
H9 1.21 1.32 0.41
H10 1.00 1.05 0.28
Hints: Genotypes with the highest RWC value is most tolerant and with the lowest RWC value is the most
sensitive

Some important formulae

− !" Where, T = transpiration rate; H2Oint = vapour concentration inside stomatal cavity;
=
#+ % H2Oext = vapour concentration in the air; Rs = stomatal resistance to water vapour;
Rb = boundary layer resistance to water vapour

&' = # − ( Where, VPD = vapour pressure deficit es = maximum vapour pressure in air at vapour
saturation; ea = actual vapour pressure

' )' = ' − '

∴ '= '
' )' = ' − '
TP = 0 in fully plasmolysed cell and hence, SP = OP
Where, Ys = yield in stress condition; Yp = yield in
= +,- . − /.01 2× # non-stress condition; 7# = mean yield over all
) =
) = − ( 72 )2 genotypes under stress condition; 72 = mean yield
2 #
# over all genotypes under non-stress condition
2 + #
1− 7#
3' = 2 Higher STI, MP, GMP value, more the resistance
2 )) = ,) = 1 −
) 72 Lower ST value, more the tolerance
53' = 2 × # SSI≤0.5 = highly tolerant; >0.5-≤1.0 = moderately tolerant; >1.0 = susceptible

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Q. The following green gram genotypes were grown under irrigated condition and moisture
stressed condition. Calculate STI, SSI and ST. Identify the most resistant and susceptible
variety according to STI, SSI and ST.
Genotypes Seed yield/plant (g)
Irrigated condition Moisture stress condition
UM 37 8.46 7.59
UM 38 5.81 5.29
UM 40 6.68 5.07
UM 41 6.47 4.96
UM 44 6.44 5.25
UM 45 6.09 6.01
UM 46 5.93 5.41
UM 47 6.58 5.08
UM 48 6.74 4.60
UM 50 5.78 5.56

Proline biosynthesis Glutamate

• In plants, proline is preferentially produced from
P5CS
ornithine under normal condition
• However, it is made directly from glutamate under γ-Glutamyl phosphate
stress, the first two reaction of the pathway being
catalyzed by a single enzyme P5CS P5CS
• Over-expression of P5CS in plants enhances their
tolerance to osmotic stress Glutamic-γ-semialdehyde
• Accumulation of proline and other solutes, e.g., glycine Spontaneous
betaine, appears to be regulation of intracellular water cyclization
activity ∆< Pyrroline-5-carboxylate
• Under water stress, they may induce formation of strong
H-bonded water around proteins, thereby preserving the P5CR
native state of cell biopolymers
Proline
*P5CS = ∆< Pyrroline-5-carboxylate synthetase; Fig: The preferred route of proline biosynthesis in
P5CR = ∆< Pyrroline-5-reductase plants under stressful conditions

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SELECTION CRITERIA
Dehydration avoidance Dehydration tolerance
a) Leaf rolling: 0 = no rolling to 5 = tightly a) Seedling growth under PEG stress
rolled in rice in morning or at mid day b) Growth under stress in the field
b) Combination of leaf rolling and leaf c) Plant phenology: drought stress delays or
firing in maize and sorghum accelerates flowing depending on growth
c) Canopy temperature: negatively stage at which stress occurs and on stress
correlated with yield intensity
d) Leaf attributes: dense pubescence, heavy d) Grain filling by translocated stem reserve:
glaucousness, epicuticular wax load 1000 grain weight
e) Leaf water retention e) Cellular membrane stability under stress
f) Root characteristics f) Water use efficiency
g) Seed germination in an osmoticum
h) Presence of awn
i) Canopy temperature

DROUGHT TRAITS
Morphological Physiological
a) Earliness a) Photosynthesis under stress:
b) Reduced tillering photosynthesis efficiency reduced due to
c) Leaf characters: leaf rolling, leaf folding, chloroplast damage
leaf shedding, leaf reflectance b) Reduced transpiration and reduced
d) Reduced leaf area: erect leaf, narrow respiration loss
leaf, changes in leaf angle c) Stomatal behaviour: closure of stomata,
e) Hairiness: lower leaf temp and also change in size and number
transpiration d) Osmotic adjustment
f) Colors of leaves e) Leaf enlargement: thickness
g) Wax content f) Leaf cuticle wax: increases
h) Awns
i) Root system: rooting depth and intensity

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Biochemical Drought hardening

a) Accumulation of proline and betaine
• Refers to improved resistance of a genotype
b) Increase in ABA (barley) and ethylene to drought as a consequence of suitable
(maize & wheat) seed/seedling treatments
c) Protein synthesis: increases
• Hardening treatments are
d) Nitrate reductase activity
a. Pre-sowing treatments: sunflower, barley
Others
a) Leaf canopy temperature • Soaking of seed for 24hr in water and sun
drying them, and then sown these seed in the
b) High CT depression
field
c) STI: higher
d) SSI: lower b. Post-sowing treatments: oats, wheat
e) RWC: higher • Are applied to young seedlings
f) Grain filling rate and duration: better • Mild moisture stress applied to young
g) Flag leaf duration and senescence: longer seedlings is reported to improve their drought
duration resistance during later stages of growth

BREEDING METHODS AND APPROACHES

1. Adaptation to a specific environments 2. Adaptation to a variable environments
• Varieties are developed specifically for • Attempts to combine high yield potential with
adaptation to moisture deficit drought resistance
environments and selection and • Selection is based on yield and yield
evaluation are carried out under moisture components
stress • Evaluate yield stability of selected lines under
• Most useful in an environment where several environments ranging from stress to non
crop is gown on moisture stored during stress conditions
the previous season • Individual plant progenies are evaluated under a
• But have several pitfalls range of optimal to moisture stressed and
Reduced effectiveness of selection selection among progenies is based on their
Need for extreme care in field average performance over all the test
preparation environments
• Selected progenies are grown under optimal
condition and within progeny selection are
carried out for yield and yield attributes

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3. Combining selection for drought 4. Marker assisted back-crossing

resistance traits and high yield potential • MABC has been used to transfer QTLs
• Selection for yield potential and drought associated with drought resistance
resistance traits are integrated into a single
5. Genomics-based integrated approach
scheme
• Uses information and tools from several
• F1 is advanced to F4 under optimal
disciplines
moisture
• Use of omics technology
• In F4, a large number of individual plants
Functional genomics
are selected and harvested separately
• In F5, individual plant progenies are Transcriptomics
grown under moisture stress and Proteomics
progenies are evaluated for drought Metabolomics
resistance; superior progenies are • Bioinformatics
harvested separately
• Selected progenies are grown under
optimal moisture in F6 and selection is
based on yield, yield attributes and quality

WATER USE EFFICIENCY (WUE)

• Most important component of drought adaptation
• Physiologically, WUE is the relationship between the CO2 photosynthetic assimilation rate
(A) and the plant’s transpiration rate
'= − '>
=
1.6 &'> − &'=
Where,
WUE = water use efficiency, physiologically
PA = partial pressure of CO2 in the air
PI = partial pressure of CO2 inside the leaf
VPI = vapour pressure of the water inside the leaf
VPA = vapour pressure of the water in the air

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• Agronomically, WUEa is the relationship between dry mass produced and the volume of water
used in the cycle (precipitation +irrigation) in the cultivated area
5
( =
&
Where,
WUEa = water use efficiency, agronomically
GY = grain yield or dry mass produced
V= total water volume used in the cycle by the culture
• WUE measured through its components or related characteristics such as specific leaf area
(SLA), isotopic carbon discrimination, leaf chlorophyll level (SPAD), and others
• Under water stress conditions
Y= × ×
Where, Y= yield, E = transpired water, WUE = water use efficiency, HI = harvest index
• Large increases in productivity through breeding programs occurred by increasing the harvest
index
• Increased biomass production must be combined with lower water consumption or increased
WUE

• Reducing the stomatal conductance, increasing the photosynthetic capacity or even combining
both factors may lead to a higher WUE
• A reduction in stomatal conductance is not favorable in breeding programs aimed at
increasing productivity because it entails less CO2 influx and lower photosynthetic rates and,
therefore, less biomass accumulation
• The great challenge is to increase the photosynthetic capacity under conditions of low
stomatal conductance
• Several strategies have been proposed to increase the photosynthetic capacity of cultivars
to involve CO2 concentrator mechanisms, such as the one found in species that utilize C4
metabolism
to increase the CO2 specificity of the enzyme ribulose-1,5-bisphosphate
carboxylase/oxygenase (Rubisco), thus reducing losses related to photorespiration in C3
plants
increase in the mesophyll conductance is directly associated with increases in
photosynthetic rates without the need for increased stomatal conductance
increasing the specific leaf mass because such an increase represents a greater amount of
photosynthetic apparatus per leaf unit area

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BREEDING FOR HEAT STRESS

• Threshold temperature: refers to the daily average temperature that cause a reduction in
growth
• Temperature increase above the upper limit for a period of time sufficient to cause irreversible
damage to plant growth and development is defined as heat stress, which is a complex
function of the intensity, duration and rate of temperature increase
• Plants are subjected to heat stress under following conditions
a) The air temperature is high and plant receive energy by transfer of sensible heat
b) Solar radiation incident on the ground raises the temperature above the air temperature
c) Solar radiation induced heating and the inability to dissipate heat can rapidly warm the
leaves (upto 15oC above the air temperature or more) particularly in leaves that have a
low transpiration rate and are frequently subjected to high temperatures
• High temperature impact on final harvestable crop, if happen
Time around flowering – when the number of grains per land area is established
During the grain filling stage – when average grain weight is determined
• Heat would affect cell and tissue survival, growth and development and physiological
processes of plants

Morpho-physiological alternation to high temperature

a. Increase membrane permeability and decreased thermostability: loss of electrolytes
• Thermal stress increases the kinetic energy and movement of molecules composing biological
membranes, leading to relaxation of chemical bonds and providing fluidity to the lipid bilayer
• Also accompany by denaturation of proteins and /or increase of unsaturated fatty acids
b. Synthesis of heat shock proteins (HSPs)
• Proteins are easily denatured by high temperature
• Organisms synthesized HSPs in response to high temperature and designed to prevent or
reverse the effects of heat on protein denaturation
• Expression of HSPs and heat shock factors increase thermal tolerance
c. Pre- and post- harvest damage
• Leaf, twigs and stem burns, leaf senescence and abscission, root growth inhibition and fruit
discoloration and damage, which eventually reduce production
• Sugarcane grown in high temperature contains short internodes, greater tillering, early
senescence and reduction in total biomass

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d. Effect on development of anther and pollen, production, filling and quality of grains
• Reduce in number of pods and in seed production in bean
• Reduction in grain weight, number of grains per spike, grain filling period, grain size
• Reduces growth period but increases growth rate
e. Limitation to photosynthesis
• Photosynthesis is limited at temperature about 35oC due to decreased activity of rubisco,
stomatal closure, and so on
• Higher photorespiration rate are frequent at high temperature which ultimately reduce carbon
gain
• Lower regeneration capacity of ribulose-1,5-biphosphate (RuBP) limits photosynthetic rate
f. Accumulation of osmolytes: sugars, sugar alcohols, proline and betaines
g. Effect on days to anthesis/flowering
• Crop grown in higher temperature flowers earlier as compared to crop grown in lower
temperature
• Temperature sum or thermal unit (day degrees, d oC): product of days and temperature

• Maize crop grown at a constant temperature of 25oC and

has a threshold temperature 10oC will start flowering after
50 days. The temperature sum (TS) required for this crop
is 750 d oC. Days to anthesis (DA) is calculated as
) = +( B − ℎ B )
Where, TS = temperature sum, DA = days to anthesis, FTo
= field temperature, ThTo = threshold temperature
Q. When maize grown at a constant 35oC, then when will be
the flowering start?
h. Effect on respiration
• Total respiration increase with temperature and more
resistant to heat than photosynthesis
• Heat may lead to utilization of greater amount of
photosynthate than that produced by photosynthesis,
resulting in a progressive depletion of photosynthates

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Temperature coefficient, Q10

/ 0E /F 0. / G + 10 0 )
C<D =
/ 0E /F 0. / G 0
For chemical reaction Q10 = 2 to 3

Why C4 plants have a higher Amax at a higher optimum

temperature than C3 plants?
• Presence of PEP-carboxylase in C4 plants becoming
active at a higher To
• Photorespiration in C3 plants rises with increasing
To since the affinity of rubisco for O2 increases more
than that for CO2
• When photorespiration is suppressed (i.e., O2 poor
environment), C3 plants show a slower decline of
Amax with inversing To

Mechanism of heat stress resistance

• Heat stress resistance: the ability of some genotypes to perform better than others when they
are subjected to the same level of heat stress
Mechanisms of heat stress resistance
1. Heat avoidance
• Ability of a genotype to dissipate the radiation energy and therby, avoid a rise in plant
temperature to a stress level
a) Transpiration: mechanism of energy dissipation (cooling effect)
b) Reflective properties of leaves: determine the proportion of incident solar radiation absorbed
by leaves; presence of pubescence, glaucousness, etc. reduced light interception by leaves
c) Insulation by bark: reduce organ and twig temperatures
2. Heat tolerance
• Ability of genotypes to withstand/perform better than others when their internal temperatures
are comparable and in the realm of heat stress
• Largely associated with cellular and sub-cellular components and its expression is highly
dependent on heat hardening or heat acclimation

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• Heat hardening: an improved ability of a genotype to withstand a period of high temperature

as a consequence of an earlier exposure to high temperatures for given period of time
Exposure may vary from 20 sec at 50oC to 4-6hr, <1hr being the most common
Lower the hardening temperature, the lower is the level and slower is the process of
hardening
• When heat hardened plants are subjected to lower temperatures, they lose their hardening; this
is called dehardening
• Involved the following components
a) Membrane stability: tolerance usually improve membrane stability under stress
• Determined as 1) lipid fluidity or 2) electrolyte leakage; compared heat hardened and non-
hardened plant tissues
• A fair index of genetic variation in heat tolerant that bears a reasonable relationship with
plant performance under stress
b) Thermo-sensitivity of photosystem II: effect of heat stress on photosynthesis and chloroplast
damage can be assayed as variable chlorophyll fluorescence at 685nm
• Genotypes adapted to hot climates show reduced sensitivity of photosystem II to heat stress

• Photosynthesis in awns of cereals is more heat tolerant (Topt = 320C) than in leaves and
glumes (Topt = 250C)
• Awns are a xerophytic organ and lack transpirational cooling
c. Photosynthate translocation: callose formation in phloem sieve tubes is the major cause fro
inhibition of photosynthate translocation by heat in tomato
d. Stem reserve mobilization: better mobilization of stem reserves at all temperatures regime is
desirable
e. Osmoregulation: osmoregulators like proline and glycine-betaine may have a protective role
in heat stress as they protect several enzymes from heat inactivation in vitro

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Selection environment for heat stress resistance

a) Normal field environment
• Natural field environment is the simplest and cheapest to use
• Effectiveness depends mainly on repeatability of the heat stress profile over years and on
nature of heat tolerant being selected for
• Unsuitable for selection of such traits which require a critical temperature during specific
stage of developments
b) Abnormal field environments
• Use of abnormal field environments available at certain locations or during the off season
• e.g., growing wheat in summer
c) Programmed environments
• Available in either growth chambers or greenhouses
• Temperature programmed should be such that the plants are subjected to appropriate heat
hardening before their heat tolerance is evaluated
d) In vitro environments
• Performed in test tubes
• Membrane thermostability assay by electroconductivity method

<K Breeding methods

/ .I % = 1− × 100 1. Selection
<K
2. Introduction
Where, 3. Hybridization followed by
T1 = mean conductivities of the treatment vials backcrossing
for a genotype before autoclaving 4. Mutation
T2 = mean conductivities of the treatment vials 5. Somaclonal variation
for a genotype after autoclaving 6. Genetic engineering
C1 = mean conductivities of the control vials for 7. Germplasm collection
a the same genotype before autoclaving
C2 = mean conductivities of the control vials for
a the same genotype after autoclaving

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Selection criteria for heat stress resistance

a) Growth under heat stress: reflects the integrated Table: Different selection criteria for heat
consequences of various cellular components involved tolerance in crops
in heat tolerance Characteristics Measured as
b) Yield under heat stress: reflects effects of heat on Germination Per cent germination
reproductive processes like flower development, under heat stress
pod/fruit/seed set, pollen fertility, grain filling, etc. Growth during heat Yield, biomass
stress
c) Flower, fruit, seed, etc. formation and pollen fertility:
Membrane stability Solute leakage
when heat stress occurs during reproductive phase (conductivity test)
d) Seed germination under heat stress: all the seed Photosynthesis Chlorophyll
samples being compared should have been produced sensitivity fluorescence at
under comparable temperature regimes 685nm

e) Recovery after heat stress: Recovery after heat Yield, biomass

stress
f) Sensitivity of the photosynthetic process and of Sensitivity of Flower/bud/fruit/
chloroplasts to heat stress reproductive phase seed production;
g) Membrane stability following heat shock: measured by pollen fertility
electrical conductivity test

Impact of terminal heat

a) Photosynthesis
• Disruption of structure and function of chloroplasts
• Reduction in chlorophyll content
• Inactivation of chloroplast enzymes, mainly induced by oxidative stress
• Deactivation of Rubisco, losses Rubisco activities
• Increased rate of photorespiration: solubility of oxygen decrease to a lesser extent than CO2
• Damage of photosynthetic apparatus
b) Leaf senescence: chlorophyll biosynthesis is inhibited
c) Water relations: higher VPD which drive higher evapotranspiration
d) Grain growth and development
i. Grain number and size: speed up development of spike reducing spikelet numbers, floret
death, poor fertilization
ii. Starch synthesis: decreases the activity of sucrose synthase, soluble starch synthase and
granule bound starch synthase
iii. Grain filling rate and duration: accelerates rate of grain filling, whereas duration is
shortened, supply of assimilated may be limited and then grain weight is limited

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iv. Assimilate translocation: substantially reduced assimilation translocation occurs through

both symplastic and apoplastic pathways
v. Grain quality: determined by grain protein content and grain size
vi. Earlier maturity: affect the growth period, growth rate, development and developmental
rate of a crop variety and then accelerates earlier maturity
Growth period (GP)
• Refers to the length of a period enabling crop production as determined by environmental
conditions and crop genetic characteristics together governing crop development
0/ ,/ 1 0 F 0.
5' =
5 0 ℎ /
• Mainly affected by growth rate
0/ 0 ℎ1 0 =L / L 0 ℎ1 0 + 1 0 F L 0 ℎ1 0
• e.g., growth period in rice
Vegetative GP: emergence, seedling development, tillering, booting, heading
Reproductive GP: anthesis, flowering, pollination, grain setting, grain filling, grain
ripening

Growth rate (GR)

• Is the total dry matter production divided by growth period
0/ ,/ 1 0 F 0.
5 =
5 0 ℎ1 0
• Mainly affected by light, temperature and crop characteristics
• e.g., crop characteristics
Canopy not closed: leaf area expansion, exponential growth rate
Canopy closed: constant growth, linear growth
• Growth is increase in volume or weight of total plant or organ
Higher the temperature, higher growth rate/developmental rate followed by shorter growth
period and consequently lower yield
Higher temperature affects in days to anthesis/flowering
A grown in higher temperature flowers earlier as compared to a crop grown in lower
temperature region

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Development
• Means plant passing through consecutive phenological phase
• Dry matter partitioning is strongly related to crop development
• Influenced mainly by a) temperature and b) day length
Many crops needs a period of low temperature to induce flowering; e.g., winter wheat, rye
and need vernalization
Summer crops in temperate and tropical crops do no need a period of low temperature to
induce flowering
Threshold temperature: 0-10oC
25 25 25

20 GR 20 DR 20

15 15 15
GR

DR

GP
10 10 10

5 5 5 GP
0 0 0
5 10 15 20 25 30 35 5 10 15 20 25 30 35 5 10 15 20 25 30 35
Temperature Temperature Temperature

Breeding strategies Q. In normal condition if a crop variety

1. Growth period (GP) matures in 110 days and yields 5 and
• Shorter VGP but long RGP 10t/ha for EY and BY, respectively. If
• Longer grain filling period it takes 80 days to reach anthesis
from emergence and 30 days to
2. Growth rate (GR) and developmental rate (DR) reach maturity from anthesis.
• GR and DR are mainly affected by temperature Then,
• Higher temperature leads increase in growth rate 1
+L / 0E 1 − /. ℎ =
and developmental rate that ultimately enhance 80
earlier maturity and lower yield 1
+L / 0E 10 − /. ℎ =
• Generally constant or linear GR is preferable 30
1 1
• Higher DR during pre-anthesis but linear DR after 0E ℎ/ L/ + =
80 30
anthesis 10
5 0E O =
• Higher growth rate is most preferable during grain 110
filling period which is the goal of plant breeder 5
5 0E =
• Production is the function of GR × GP, which must 30
5
be more beneficial in grain filling period =
10

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Q. A variety of given crop in normal condition matures in 115 days after emergence taking 80
and 35 days pre and post anthesis duration, respectively with yields 10 and 4.5t/ha for BY
and EY, respectively. In heat stress condition, it matures in 97 days after emergence taking
70 and 27 days pre and post anthesis duration with yield 8.5 and 3.5t/ha for BY and EY,
respectively.
a) Estimate GR of BY and EY and HI of variety in both situations
b) Calculate DR of pre and post anthesis duration and maturity of that variety in both
conditions
c) Interpret the results
Q. Maize is grown at a constant temperature of 25oC and characterized by a temperature sum of
990 doC until anthesis and a temperature threshold value of 10oC. Find days to flowering of
this crop under such situation. If, it is grown at a constant 40oC (heat stress condition), how
many days does it takes to start flowering. Interpret the result based on this output.

BREEDING FOR COLD STRESS

• In many environments, crop productivity is limited by low temperatures
• When temperature remains above freezing, i.e., >0oC, it is called chilling
• When temperature remains below freezing, i.e., <0oC, it is called freezing
Chilling stress
• Chilling sensitive plants are typically tropical plants
1. Effect of chilling stress at plant level
a) Reduced germination
b) Poor seedling establishment
c) Decline in rood growth & Increase in root hydraulic resistance
d) “Locked open” state of stomata
e) ABA accumulation
f) Production of abnormal flower/fruits
g) Pollen sterility & failure of seed and fruit set
h) Stunted growth
i) Wilting, chlorosis, necrosis

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2. Effect of chilling stress at sub-cellular level

a) Membrane damage or reduced membrane stability
b) Conformational changes in proteins
c) Decline in net photosynthesis: affects electron transport, RuBISCO, PEP carboxylase
d) Interferes chlorophyll synthesis and chloroplast function
e) Interfere assimilate translocation
f) Suffer from toxicity injuries: H2O2 accumulation due to prevention to degrade
Chilling tolerance
• Ability of genotypes to survive/perform better under chilling stress than other genotypes
• Is the consequence of chill hardening, i.e., an earlier exposure to a near chilling temperature
for a specified period, as a result of which chilling tolerance of the concerned plants increases
• Tolerance involves
a) Membrane lipid unsaturation b) Reduced sensitivity of photosynthesis
c) Increased chlorophyll accumulation d) Improved germination
e) Improved fruit/seed set f) Pollen fertility

Sources of chilling tolerance

a) Well adapted breeding populations b) Germplasm c) Cold tolerant mutant
d) Somachlonal variant e) Transgenes
Selection environments
a) Field environments: selection at germination, seedling emergence and seedling
establishment
b) Controlled environments: created in germination cabinets, growth chambers; selection at
seedling growth, adult plant growth, seed/fruit set, pollen fertility, etc.
Selection criteria
a) Germination b) Growth under chilling stress
c) Chlorophyll loss under chilling stress d) Membrane stability
e) Photosynthesis f) Seedling mortality
g) Seed/fruit set h) Pollen fertility

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Freezing stress
• Development of a complex array of stresses and strains within plants when plants are
subjected to subzero temperatures
• Dormant state is conducive to freezing resistance, while resistance in actively growing tissues
is rare
• Largely involves surviving freezing stress in such a manner as to enable subsequent regrowth
when the temperature rises
Effect of freezing stress
a) Ice formation
• Intracellular ice formation: cause lethality i.e. physical disruption; a major and terminal
freezing stress
• Extracellular ice formation: increase the concentration of extracellular solutes
b) Membrane disruption
• Alter semipermeable properties of plasma membrane leading to a loss of solute from cells
• Cells remain plasmolyzed even after thawing which is often called as frost plasmolysis
• Cells with intact plasma lemma may take up excess water and become usually turgid

c) Supercooling
• Cooling of water below 0oC without ice crystal formation
• water may cool down to -1 to -15oC in most herbaceous species and to -40 to -45oC in hardy
trees
• Possible because of absence of internal ice-nucleators
d) Stress due to external factors
• Ice sheet formation in field above and below the ground causing reserve depletion, anoxia etc.
• Tissues killed during freeze-thaw are highly prone to pathogen attacks
• Auto-toxicity may occurs due to hydrolytic enzymes release
Freezing resistance
• Ability of a genotype to survive freezing stress and to recover and regrow after thawing
• A complex trait involves various physiological, chemical and physical processes
• Resistance involves
1. Freezing avoidance
• Ability of plant tissues/organs, but not the whole plant, to avoid ice formation at subzero
temperatures

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• Supercooling is mechanisms of freezing avoidance because of

Lack of ice nucleators (nucleating protein)
Small cell size
Little or no intercellular space
Low moisture content
Barriers against external nucleators
Presence of antinucleators
• Common in meristematic tissues of flower buds of hardy plants like plums, apricots, etc.
2. Freezing tolerance
• Ability of plants to survive the stresses generated by extracellular ice formation and to
recover and regrow after thawing
• Markedly affected by freeze hardening, which describes the increased tolerance to freezing
due to an earlier exposure to low temperatures for a critical duration, e.g., 3-6 weeks for
winter cereals
• Components of freezing tolerance
a) Osmotic adjustment b) Bound water c) Plasma membrane stability
d) Cell wall properties e) Cold- responsive proteins

Sources of freezing tolerance

a) Cultivated varieties b) Germplasm c) Induced mutant
d) Relative wild species e) Transgenes
Selection environments
a) Field environments: suitable site where desirable subzero temperatures occur as regularly
over the years as is possible; useful in nonwinter-hardy crops
b) Controlled environments: field evaluations should be supplemented with studies under
controlled environments
Selection criteria
a) Field survival
b) Freezing test: viability of thawed tissues, degree of browning of tissues, chlorophyll
fluorescence after a freeze-thaw cycle, electrolyte leakage, etc.
c) Freezing of isolated crowns
d) Osmoregulation: after hardening and before freezing

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BREEDING FOR SUBMERGENCE TOLERANCE/FLOODING

• Flooding is the presence of water in soil excess of field capacity
• Leads to deficiency of O2 and build up of CO2, ethylene and other toxic gases and this lead to
reduction in aerobic respiration
• Excessive amount of water quickly creates anoxic (oxygen deficit) soil condition causing
flood sensitive plants to suffer anoxia or hypoxia
• Fermentation occurs in plant root under such conditions
• Photosynthetic capacity of plant is significantly inhibited
• Some plants well adapted to water logged condition (flooded rice) while most plant need well
drained soils to grow properly
• Flood tolerant species have certain adaptive mechanisms such as the formation of aerenchyma
and adventitious roots
• Root tissues survival under hypoxia depends on the fermentation rate and sufficient sugar
supply to maintain cell energy and membrane function

Effect of water logging

a) Decreases in O2 but increases in CO2, ethylene and other toxic gases in soil
b) Slow diffusion of atmospheric O2 into waterlogged condition, thus O2 replacement is very
inefficient
c) Suddenly plunged root system into an aerobic condition; switching from aerobic to
anaerobic respiration disrupts root metabolism
d) Carbohydrate level get depleted because of
• Dissipation of metabolism
• High water temperature
• Low light
Characteristics of plants in response to water logging stress
a) Reduced growth/elongation
b) Chlorosis, senescence and abscission of lower leaves
c) Wilting and leaf curling
d) Hypertrophy (increase in size of organ due to increase in cell size)
e) Epinasty (downward growth of petioles)

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Mechanism of submergence tolerance

a) Adventitious root formation in the lower part of stem (close to surface so that O2 tension is
quickly restored after transient water logging): tomato
b) Lenticel formation: raised pores in the stem of plants
c) Aerenchyma formation in the cortex: provide canal parallel to axis of the root through
which gases can diffuse longitudinally; e.g., rice
d) Elongation capacity: in rice, best elongation response give 100% recovery from
submergence and poorest elongation gives 49% recovery
Scoring of elongation can be done between booting and flowering stage after flooding the
crop to varying depth
e) Carbohydrate concentration: observe a strong positive correlation between carbohydrate
concentration prior to submergence and tolerance to submergence
f) Alcoholic fermentation (increase): major metabolic adaptations that plants assume when they
are submerged or faced with lack of oxygen

Ideotype for flooded area

a) Capacity to carry out functional activity at low O2 concentration i.e. high cytochrome
activity
b) Ability for photosynthesis under low light intensity
c) Capacity to synthesis food rapidly
d) Regeneration capacity of shoots when damaged by flood
e) Ability to withstand drought at later growth stage
f) Deep root system
g) Narrow, medium long and dark green leaves with high sugar and protein content
Breeding methods
a) Selection
b) Introduction
c) Germplasm
d) hybridization: pedigree selection, back cross selection
e) Mutation breeding
f) Somaclonal variation
g) Genetic engineering

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BREEDING FOR SALT TOLERANCE

• Refers to the ability of plants to prevent, reduce or overcome injuries effects of soluble salts
present in their root zone
• Is a global problem as saline and alkali soils are found in almost all the countries
• Starts with assembling and screening germplasm for salinity tolerance
• Selected genotypes are used as parents to transfer the trait to desired cultivar, followed by
selecting desirable recombinants from segregating population
• Got some success through this approach: rice, wheat, Lucerne
• Problem is how to measure salinity tolerance
• Screening is commonly based on growth of plant under salt stress
• Two mechanism exist for salinity tolerance
1. Tolerance to the osmotic effect of the saline solution: osmotic effect makes it harder for
plants to extract water from soil
2. Tolerance to salt specific nature of the soil saline solution: a high sodium concentration
makes it difficult for the plant to exclude NaCl while taking up other ions

• Screening for salinity tolerance is a long process and requires a large amount of space to
screen progeny from crosses
• Screening for specific traits is quicker and more effective as they are less effective by
environment than growth rate are
• Most successful traits for assessing salt specific effects is the rate of Na+ or Cl- accumulation
in leaves which is measured as the increase in salt in a given leaf over a period of time
• Traits for osmotic effect are related to growth: leaf elongation, root elongation, shoot biomass,
leaf area expansion
• Molecular and genetic engineering techniques are most used in salt tolerance breeding
• Salt tolerance have been found in wild relatives of crops like tomato, pigeon pea
Problem of salinity can be overcome by two ways
a) Soil reclamation: costly, time consuming, short lived
b) Resistant varieties: less costly, more effective, long lasting, require longer period to develop

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Behaviour of plants to salt

a) Land races are more tolerant than high yielding varieties. Tolerant plant varieties are found
in affected area.
b) Salt tolerance capacity differ from species to species or cultivars to cultivars
c) Higher ploidy level crops are more tolerance than lower ploidy level: hexaploid wheat more
tolerant than tetraploid, tetraploid brassica more tolerant than diploid
d) In rice: tall, coarse grained, late maturing varieties are more tolerant
e) Different crop plants show differential response to salinity

Table: List of some important crops differing in their tolerance to salinity

Categories Crops
Sensitive Apple, bean, carrot, okra, onion, cabbage
Moderately sensitive Alfalfa, cabbage, maize, cowpea, cucumber, peanut, pepper, potato, radish, rice,
sugarcane, sweet potato, tomato, vetch
Moderately tolerant Barley (forage), broccoli, safflower, sorghum, soybean, wheat
Tolerant Barley (grain), cotton, sugarbeet

Effects of salinity stress

a) Water stress generated by osmoticum (i.e., K+, Na+, Cl-)
Glycophytes: not adapted to saline condition
Halophytes: adapted to saline conditions and are wild species, barley haplotypes show
tolerance by ion accumulation mechanism
b) Mineral toxicity stress caused by salt
Adversely affect activity of enzymes of glycolytic pathway
Affects oxidative phosphorylation, ATP/ADP ratio in root tissue, protein synthesis,
membrane integrity
Enhance proline content
c) Disturbances in mineral nutrition in plants
Mechanism of salinity resistance
1. Resistance to salinity induced water stress
• Osmoregulation: achieved by sugars (in tomato), various acids, glycine-betaine (in sugarbeet),
myoinositol (in tomato), proline (in barley, pea), K+ ion (in sorghum)

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2. Resistance to salinity induced ion toxicity

• Ion toxicity avoidance involves a mechanism which maintains a low nontoxic level of slats in
the cytoplasm
a) Ion exclusion: genotype take up smaller quantities of injurious ions i.e., Na+, Cl– so that
concentrations of these ions in their tissues is much lower than those of other genotypes
b) Salt tolerance: a differential effect on various life process of the same tissue concentration
of salt in different genotypes of a species
• Haplotypes usually dispose of the excess salt either by excretion through specialized salt
glands or by cellular compartmentation
Breeding strategies
• Emphasis on breeding for yield potential
• Do selection in stresses target environments
Sources of salinity resistance
a) Cultivated varieties b) Germplasm collection
c) Related species d) Somaclones
e) Transgenes

Selection environments
a) Non-saline field with saline environment: 3000, 6000, 9000 mg L-1 salt
b) Microplots
c) Greenhouse environments
Techniques used for selection/screening
1. Assessment of Germination
a) Filter papers b) antibiotic agar plate c) pots filled with saline soil
d) Pots filled with sand/gravel e) Saline hydroponic culture
2. Seedling survival and growth
• Seeds are germinated under non-saline condition and seedling may expose to saline
condition at desired growth stage
Selection criteria
a) Cell survival b) Germination c) Dry matter accumulation
d) Leaf death or senescence e) Leaf ion content f) Leaf necrosis
g) Root growth h) Osmoregulation i) Yield

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Breeding approaches
a) Salinity resistant root stocks b) Selection c) Hybridization
d) Interspecific hybridization e) Cell selection f) Genetic engineering

BREEDING FOR MINERAL DEFICIENCY/TOXICITY STRESS

• Mineral stress: when plant performance is adversely affected by an excess of mineral or
deficiency of an essential nutrient in soil
• Plants respond mineral stress by enhancing their root growth relative to that of shoot and by
increasing permeability of their membranes
• Plants also secrete organic acids which increase the availability of P, Zn and Mn particularly
in deficient soils
Mineral deficiency resistant genotypes and mineral deficiency resistance
• Genotypes that show markedly lower detrimental effects of mineral deficiency than do other
genotypes of the same species are called mineral deficiency resistant genotypes and the
phenomenon is called mineral deficiency resistance

• Avoidance of mineral deficiency: ability of genotypes to absorb and/or transport to the

appropriate sinks more quantities of the deficient mineral than are other genotypes
• Tolerance of mineral deficiency: refers to a lower level of injury to essential life processes
in some genotypes than in others when their tissues contain a comparable and a lower than
critical concentration of the given element
Mechanism of mineral deficiency resistance
a) Mineral redistribution b) Efficient mineral uptake
c) Increased mineral transport d) Increased root/shoot ratio
e) Increased root hair density/length
Sources of mineral deficiency resistance
a) Cultivated varieties b) Land races c) Obsolete varieties
d) Mutant e) Related species
Selection criteria
a) Visible deficiency symptoms b) Mineral contents of plant tissues
c) Biochemical assays d) Yield

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Mineral toxicity resistance

• Mineral toxicity stress is generated by the presence of toxic concentrations of available
minerals in the root zone
• Acidic soils contains toxic concentration of Al and Mn
• Al toxicity occurs when its concentration exceeds 2-3 ppm or even 4 ppm
• Al and Mn toxicity occur at any soil pH below 5.5
• At pH 5 or below, Al inhibits plant growth by interfering with cell division in root tips and
lateral roots, increasing cell wall rigidity, reducing DNA replication, decreasing respiration
and other effects
• Excess Al induces iron deficiency in some crops (i.e. rice, sorghum, wheat)
• Visual symptoms of Al toxicity is root pruning whereby root growth is severely inhibited
Breeding for Al tolerance
• On the basis of patterns of Al accumulation in plant tissue, three groups of Al-tolerant pants
may be identified
1. Those with an apparent exclusion mechanism allowing lower accumulation of Al in their
roots than Al-sensitive plants (e.g., wheat, barley, soybean)

2. Those with less Al accumulation in the shoot but more in the roots (e.g., wheat, barley,
potato)
3. Those with high Al accumulation in the shoot (e.g., pine trees)
Selection criteria for Al toxicity
a) Shoot dry matter b) Root length c) Root weight
d) Root deformation and discolouration
e) Roots stained with haematoxylin: roots of resistant seedling do not uptake the stain
f) Yield
Selection criteria for Mn toxicity
a) Shoot dry matter b) Root length c) Root weight
d) Root deformation and discolouration e) Leaf chlorosis and necrosis
f) Leaf crinkling and cupping g) Yield
Development of resistant varieties
a) Selection b) Introduction c) Hybridization: pedigree, back-cross
d) Mutation e) Somaclonal variation f) Genetic engineering
g) Germplasm collection and evaluation

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