1. 1
Name – Manish Kumar Choudhary

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Breeding for Salinity
tolerance

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• Salinity is one of the most serious factor, limiting the
productivity of agricultural crops, with adverse effects on
germination, plant vigour and crop yield (R Munns & Tester,
2008).
• It is estimated that 6 per cent
of world´s total land and 20 per
cent of the world´s irrigated
areas are affected by salinity.
There is a deterioration of
about 1 per cent of world
agricultural lands because of
salinity each year. (Turan et al. 2012)
Introduction

4. •Saline soils refers to a saturated soil paste
extract has an electrical conductivity of more than
4 ds/m (earlier mMhos/cm), ESP <15% and low
pH < 8.5
4
• Excess salt in the soil, reduces the water content
potential of the soil and making the soil solution
unavailable to the plants (Physiological Drought)
What is Salinity?
•Salinity is caused due to high accumulation of calcium,
magnesium as well as sodium and their anions such as
SO4
-2 , NO3
- , HCO3
- , CI- , CO3
-2 etc.
•In a salt affected soils , there is an excess
accumulation of soluble salts in the root zone leading
to detrimental effect on plant growth and development.

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Salinity tolerance is strongly influenced by other
factor i.e. Growth stage of the crop,
temperature, moisture stress etc.
Salt Tolerance
•The relative growth of plants in the presence of
salinity is termed their salt tolerance.

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Soil Salinity Class
Conductivity of the
Saturation Extract
(dSm-¹)
Effect on Crop Plants
Non saline 0 - 2 Salinity effects negligible
Slightly saline
2 - 4
Yields of sensitive crops may
be restricted
Moderately saline
4 - 8
Yields of many crops are
restricted
Strongly saline
8 - 16
Only tolerant crops yield
satisfactorily
Very strongly saline
> 16
Only a few very tolerant crops
yield satisfactorily
FAO, (2012)
Table 1 : Soil Salinity Classes and Crop Growth

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Country Total land
Area
cropped
Mha
Area irrigated Area of irrigated land
that is salt-affected
Mha % Mha %
China 97 45 46 6.7 15
India 169 42 25 7.0 17
Russia 233 21 9 3.7 18
United States 190 18 10 4.2 23
Pakistan 21 16 78 4.2 26
Iran 15 6 39 1.7 30
Thailand 20 4 20 0.4 10
Egypt 3 3 100 0.9 33
Australia 47 2 4 0.2 9
Argentina 36 2 5 0.6 34
South Africa 13 1 9 0.1 9
Subtotal 843 159 19 29.6 20
World 1,474 227 15 45.4 20
FAO, (2007)
Table 2: Global Estimate of salinisation in the world’s irrigated lands

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• Poor water management
• High evaporation
• Heavy irrigation
• Previous exposure to seawater
Causes of Soil Salinity

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Adverse effects of salt stress
•Interferes with plant growth and development.
• Leads to physiological drought conditions and ion toxicity.
• Nutritional Disorder
• High salt deposition - Leads to formation of low water potential
zone in the soil.
• Higher concentrations of Na+ (above 100 mM)
 Toxic to cell metabolism
 Can inhibit the activity of many essential enzymes, cell
division and expansion and osmotic imbalance which leads to
growth inhibition.

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Adverse salt affect

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Reclamation and Management of Salt affected Soil
1. Salt leaching
•Scraping
•Flushing
•Leaching
2. Drainage
3. Choice of crop – Rice in saine soil
4. Salt Tolerant varieties

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Rice –Arya33, Pokkali, CSR-1, CSR-2, CSR-3, CSR-30
(Basmati type) CSR-49, Jhona 349, CR Dhan 405,
Lunishree, CSR-36, CSR-43 (new)
Wheat- Kharchia, Rata, Raj -3077,KRL1-4, KRL-213, 210
Salt Tolerant Varieties
Indian mustard – CS-56, CS-54, CS-52
Chick pea – Karnal Chana - 1
Soya bean – S-100, Lee, Tiefeng 8, Wenfeng 7, Jindou
Barley – Selection from Composite cross XXl

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Salinity Resistance
Resistant to salinity – Induced Water Stress
Crop Adaptation To Salt Stress

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Mechanism Of Salt Resistance
SALT RESISTANCE
Primary stress Secondary stress
Osmotic stress tolerance
(dehydration avoidance)
Nutrient deficiency
stress
Avoidance Tolerance
Salt
exclusion
Salt
excretion
Salt
dilution
Avoidance of
ion balance
strain
Tolerance of
ion balance
strain
Accumulation
of salt in
vacoule
Accumulation
of organic solutes
Avoidance Tolerance
Low salt
permeability
Na+ extrusion
pump
Secretion of
salt into
vacoule
Replacement
of K+ by Na+

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Classification of Plants Based on Salt Tolerance
1. Highly Tolerant Crops
Sugar beet, Barley, Datepalm, Asparagus
2. Moderately Tolerant
Barley, Rye, Sorghum,Wheat, Safflower, Soyabean
3. Moderately Sensitive
Rice, Corn, Foxtail millet, Cowpea, Pea nut,
Sugarcane, Potato, Radish, Tomato, Cabbage
4. Extremely Sensitive
Citrus, Strawberry, Melon, Peas, Crrot, Okra, Onion

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Why is it necessary to develop salt
tolerant plants?
• The world population is increasing rapidly and may
reach 7 to 9.3 billion by the year 2050,whereas the
crop production is decreasing rapidly because of the
negative impact of salinity and other environmental
stresses (Varshney et al., 2011).
• Rapid shrinking agricultural land due to
industrialization and Urbanisation are major threat to
sustainable food production.
• So, it is almost necessary to raise salt tolerant plants
to effectively use salt affected lands for sustainable
crop production.

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Salt tolerant varities

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Approaches in Salt
Tolerance
Conventional Approach
1.Introduction
2.Selection( Mass selection /Pure selection)
3.Hybridization
• Pedigree
• Bulk method
• SSD method
• Back cross
• Multiple crossing

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Achievement Through Conventional
Approach
• To improve the crop yield and quality improving the
resistance of crops against abiotic stresses, especially
salinity stress through conventional method develop
salt tolerant variety.
For example,
•Some lines/cultivars of alfalfa (Medicago sativa L.)
such as AZ-Germ Salt II (Dobrenz et al., 1989), AZ-
90NDC-ST (Johnson et al., 1991), AZ- 97MEC and
AZ-97MEC-ST (Al-Doss and Smith,1998), ZS-9491 and
ZS-9592 (Dobrenz, 1999)
•Two salt-tolerant lines/cultivars of bread wheat (Triticum
aestivum L.) such as S24 (Ashraf and O'Leary, 1996) and
KRL1-4 (Hollington, 2000) were evaluated on natural salt-
affected soils.

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Drawback of Conventional
method
Low magnitude of
genetically based variation
in the gene pools of most
crop species
Reproductive barriers
Time Consuming

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Non Conventional Approach
 Transgenic Approach
 Molecular Approach (MAS)
 Mutation breeding
 TILLING (Targeting Induced Local Lesions IN
Genomes)
•Transgenic plants for ion transporters
•Transgenic plants for compatible organic solutes
•Transgenic plants for enhanced antioxidant production
•In Tomato fruit crop a vacuolar Na+/H+ antiport
(AtNHX1) from A. thaliana lead to salt
accumulating in the leaves of the plants, but not
in the fruit and allowed them to grow more in
salt solutions than wildtype plant
For example

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Molecular Approach
•In response to high salinity stress, various genes get
upregulated, the products of which are involved either
directly or indirectly in plant protection against salt stress.
• Some of the genes encoding osmolytes, ion channels,
receptors, components of calcium signaling and some
other regulatory signaling factors or enzymes are able to
confer salt tolerant phenotypes when transferred to
sensitive plants.
Allele mining : finding of superior allele from the natural
population.
Introgression of novel or superior
Allele from wild relatives into Cultivated one
OR

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Problems In Breeding For Salinity
Resistance
1. There is no simple to score, reliable and
dependable selection criterion for salinity
resistance.
2. Creation of reliable, dependable and controlled
salinity environment for selection work is tedious,
costly and beyond reach of many breeders.
3. The genetic control of salinity resistance is
generally complex and polygenic, which makes
transfers from germplasm lines and especially,
related species a very difficult task.
4. The basis of salinity resistance is poorly
understood, this makes genetic analysis and
breeding efforts considerably difficult.

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Fig. 1 : Genes associated with the salt tolerance mechanism

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1. Osmolytes synthesis
2. Ion Pumps, Calcium
3. Helicases
4. Reactive Oxygen Species (ROS)
5. LEA (Late Embryogenesis Abundant ) Protein
6. Signalling Molecules
7. Abscisic Acid and Transcription Factors
Several mechanisms of Salt tolerance

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Sugars and Sugar
alcohols
Sucrose
Trehalose
Sorbitol
Inositol
Mannitol
Glycerol
Arabinitol
Pinitol
Other polyols
Nitrogenous compound
Proline
Glycine Betaine
Glutamate
Aspartate
Choline
Polyamine
Organic acid
Oxalate Malate
1. Osmolyte synthesis
Osmolytes-“Compatible metabolites” or “Compatible solutes”

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2. ION PUMPS, CALCIUM, AND SOS PATHWAYS
Fig. 2 : Regulation of ion(e.g.,Na+, K+and Ca2+) homeostasis by SOS and related
pathways in relation to salt stress tolerance.
HKT-histidine kinase transporter
CAX1-H+/Ca+ antiporter
SOS1-Na+/H+ antiporters
NHX-vacuolar Na+/H+ exchanger

31. 3. HELICASES
• Helicases catalyze the unwinding of energetically stable duplex DNA or duplex
RNA secondary structures.
• Most helicases are members of the protein superfamily that play essential roles in
basic cellular processes regulating plant growth and development.
• RNA helicases are the best candidates for RNA chaperones because these
proteins can actively disrupt misfolded RNA structures in ATP dependent manner.
• There could be two possible sites of action for the helicases:
• At the level of transcription or translation to enhance or stabilize protein
synthesis
• In an association with DNA multisubunit protein complexes to alter gene
expression.

32. 4. Reactive Oxygen Species
• Reactive Oxygen Species produced in response to oxidative stress can
cause permanent damage to the cellular apparatus.
• Reactive oxygen intermediates (ROI) typically result from the excitation
of O2 to form singlet oxygen (1O) or the transfer of one, two, or three
electrons to O2 to form superoxide radical (O2
- ), hydrogen peroxide (H2O2),
or a hydroxyl radical (OH-), respectively.
• The ROIs react spontaneously with organic molecules and cause
membrane lipid peroxidation, protein oxidation, enzyme inhibition, and
DNA and RNA damage.
• The major ROI-scavenging enzymes of plants include superoxide
dismutase (SOD), ascorbate peroxidase, catalase, and Glutathione
reductase.

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5. Signalling molecules
• Exposure of any stress to plants causing
change in normal plant development is
sensed by sensor which leads to a
signaling cascade resulting in a stress
response by plant.
• Mitogen-activated protein kinases -
serine/ threonine protein kinases that play a
central role in the transduction of various
extracellular and intracellular signals,
including stress signals.
Kaur and Gupta, (2005)
Fig. 9 : MAPK cascade
Stimuli
External ( Environmental
stress)
Internal (Growth Factor)
Receptors
MAPK
Substrates : Transcritional Factors
Cellular Response

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6. LEA-Type Proteins
• Late embryogenesis Abundant proteins are a group of
hydrophilic proteins produced late during embryo development,
and constitute about 4 per cent of the total cellular proteins.
• Functions-
Acting as hydrating buffers
Sequestering ions
Helping in renaturation of proteins
Act as chemical chaperones
• These proteins can be induced by ABA and various water-
related stresses including salinity.

35. ZEP- zeaxanthin epoxidase;
NCED- 9-cis-epoxycarotenoid dioxygenase;
AAO-ABA-aldehyde oxidase;
MCSU- molybdenum cofactor sulfurase.
7. Abscisic Acid and Transcription Factors
Fig. 10 : ABA biosynthesis
pathway and its
regulation by osmotic
stress
Tuteja, (2007)

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Screening Techniques For Tolerance to Moderate Salinity
(50 – 150 mM NaCl)
Measurements of growth:
Root elongation
Leaf elongation
Biomass and Yield
Measurements of injury:
Leakage from leaf discs
Chlorophyll content
Chlorophyll fluorescence
 Specific traits:
Na+ exclusion
K+/Na+ discrimination
Cl− exclusion

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S9
Less chlorophyll loss in S9
Fig. : Leaf disk senescence assay for salinity tolerance in transgenic
tobacco plants (T0)

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Screening For High salinity
(200–300 mM NaCl)
Germination
Survival
Fig. : The grain size of
wildtype Hesheng 3
and transgenic line
B53(AtNHX1)
grown in normal and
saline soil in the
field.

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Fig. : Effects of salt on
the growth of AtNHX1
transgenic wheat lines
(T2) in absence and
presence of 150mM
NaCl for 30 days.
Less growth retardation and
longer root length
Fig. : Whole plants after 30-days of salt treatment.

40. Name of gene Source species Gene product Function Harbourg
species
betA E. coli Choline
dehydrogenase
Betaine synthesis Tobacco
BADH E.coli
S. oleracea
Betaine
dehydrogenase
Betaine synthesis Tobacco
CodA A. globiformis Choline oxidase Betaine synthesis Arabidopsis
Rice
COX A. pascens Choline oxidase Betaine synthesis Arabidopsis
Tobacco
TUR1 A. polyrriza Inositol synthase InsP3 synthesis Arabidopsis
IMT M.crystalinum Myo-inositol O-
methyltransferase
D-ononitol
synthesis
Tobacco
MtlD E.coli Mannitol 1-
phosphate
dehydrogenase
Mannitol synthesis Arabidopsis
Tobacco
P5CS V. aconitifolia Pyrroline-5-
carboxylate
Proline synthesis Tobacco, Rice
HVA1 H. vulgare LEA protein Protein protection Rice
DREB1A A. thaliana Transcription factor Improve gene
expression
Arabidopsis
CaN S. cereviseae Calcineurin Improve Ca2+
signaling
Tobacco
Table 3 : Salt resistant genes and their functions

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CONCLUSION
•Salt tolerance is a complex trait that involves coordination of
many signalling pathways involving action of various genes.
• Understanding molecular mechanisms are helpful in over
expression of existing gene and introduction of various salt
tolerant genes from bacteria and halophytes into salt sensitive
plants.
• Majority of salt tolerant plants produced by transformation
are tested for tolerance under laboratory control condition.
• Molecular approaches for modification of ion pump regulator
and synthesis of osmo-protectant, LEA protein, ROS and
helicases are efficient to produce salt tolerant plants.

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FUTURE PROSPECTIVES
•Role of endogenous RNA in regulating the salinity tolerance has to
be determined.
• Use of promoters that direct the expression at proper time and
location will maximize salt tolerance
• Use of more advanced and less time consuming technologies like
multi SNP analysis are required.
•Identifying and validating novel genes in crops that will significantly
improve its performance under salinity stress.
•salt stress tolerance of plants is a complex trait and involves
multiple physiological, biochemical mechanisms and a wide array of
genes.

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Thank You