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Phenotyping Abiotic Stress Phenotyping Abiotic Stress Tolerance Traits in Mango Tolerance Traits in Mango
((Mangifera indica Mangifera indica L.)L.)
Sridhar Gutam, PhD, ARSSridhar Gutam, PhD, ARSSenior Scientist (Plant Physiology)Senior Scientist (Plant Physiology)
Central Institute for Subtropical Horticulture, LucknowCentral Institute for Subtropical Horticulture, Lucknow<[email protected]><[email protected]>
INSTITUTE SEMINARINSTITUTE SEMINAR
What is a Phenotye?What is a Phenotye?
The phenotype is the physical characteristics of the organism.
It is controlled by the genetic make-up of the organism and the environmental pressures the organism is subject to.
PhenotypePhenotype
http://www.plant-phenotyping-network.eu/
genotype (G) +
environment (E) +
genotype & environment interactions (GE) →
phenotype (P)
What is a Trait?What is a Trait?
A trait is a distinct variant of a phenotypic character of an organism that may be inherited, be environmentally determined or be a combination of the two.
Extended PhenotypeExtended Phenotype
A biological concept introduced by Richard Dawkins in a 1982 book with the same title.
The main idea is that phenotype should not be limited to biological processes such as protein biosynthesis or tissue growth, but extended to include all effects that a gene has on its environment, inside or outside of the body of the individual organism.
What is Phenomics?What is Phenomics?
Phenomics is an area of biology concerned with the measurement of phenomes ― the physical and biochemical traits of organisms ― as they change in response to genetic mutation and environmental influences.
CLIMATE CHANGECLIMATE CHANGE
TEMPERATURETEMPERATURE
Record of global average temperatures <http://data.giss.nasa.gov/gistemp/graphs/>
Abiotic StressAbiotic Stress
The term abiotic stress describes all kinds of environmental influences that may stress plants and thus reduce yield or growth, in comparison to optimum growth conditions.
It is an important aim of many breeding programmes to create increasingly stress tolerant plants.
For a better understanding of how plants are able to deal with abiotic stress, it is important to impose abiotic stress on the plants in a very precise and quantitatively graded way.
Abiotic StressesAbiotic Stresses
Extended droughtFloodsHeat wavesCold wavesNutrition stressSoil salinity
Objectives of Mango BreedingObjectives of Mango Breeding
DwarfnessRegular bearingFruit colourDisease resistancePest resistance
World market’s demand for specific qualities
Plant Adaptive TraitsPlant Adaptive Traits
Plants have evolved diverse adaptive strategies to cope with abiotic stresses.Cellular level tolerance Stress avoidance effects
Mechanisms that sustain growth under stress
Breeding towards tolerant plants requires precise phenotyping and characterization of adaptive traits
Improving AdaptationImproving Adaptation
Tolerant traitsPrecise phenotypingGenotypes with adaptive traitsGenes/QTL that regulate adaptive
mechanismsNovel and comprehensive breeding
strategies to pyramid traits
Drought adaptive traitsDrought adaptive traits
Primary traits (heritable)Constitutive
Phenology, Roots, WUE Waxes, Stomatal index
Induced/acquiredOsmolytes, Scavenging, ABA, Gas exchange etc.
Secondary traitsRWC, WP, Canopy Temp., Leaf senescence
Integrated traitsDrought susceptibility index, Yield, Plant
growth
Drought Susceptibility IndexDrought Susceptibility Index
Under defined moisture stress situationRain free field conditions
Non stress (FC), Stress I (25% FC), Stress II (50% FC), Stress III (80% FC)
Managed drought environments – Rainout sheltersSimulate rainfall – mist spray
DSI = [(1-Yd/Yp)]/DYd – Genotype yield under stressYp – Genotype yield under irrigatedD – Mean yield of all genotypes (under stress/under
irrigated)
Water relationsWater relations
Water uptake ability under variable soil moisture status, adaptive mechanisms to control transpiration loss, conservation by gas exchange
Relative water contentExpresses effect of osmotic adjustmentRWC % = [(Fw-Dw)/(Tw-Dw)]x100
Water potentialPotential energy of water/unit mass of waterDepends on the concentration of dissolved
substancePsychrometer
Osmotic AdjustmentAmount of solutes dissolved in plant tissuesOsmometer
Water MiningWater Mining
Phenotyping for root traitsPlant growth and biomass linked with
amount of water transpiredAbility to extract water from soilDeep rooted plants – better
productive
Root TraitsRoot Traits
Root lengthRoot to shoot biomass ratioDeep root biomassHard pan penetration abilityRoot growth responseRoot volumeRoot length densityNumber of lateral rootsRoot pulling forceRapid root growthRoot biomassRoot weight densityTap root lengthTotal root length
Root measurementRoot measurement
Genetic variabilityTubular containers
Mini rhizotrons/mini-lysimetersTubes – soil core
Root study structuresRaised wall structures
Hydroponics – to asses root growth characteristics
Oxygen isotope ratioOxygen isotope ratio
18 O/16 O as surrogate for root traitsAlteration in the stable isotopic composition of
water during evaporationRelationship between stomatal conductance
(transpiration) and leaf water 18 O enrichmentHigh throughput and very accurateΔ 18 O of leaf biomass X leaf area durationIsotope Ratio Mass Spectrometers (IRMS)
CCATDCCATD
Crop Canopy Air Temperature DifferenceInfrared thermometer.Canopies emit long wave infrared radiation
as a function of their temperature.Genotypes that maintain lower canopy
temp. are desired.Measurements should be made under well
developed drought stress.
Carbon Isotope DiscriminationCarbon Isotope Discrimination
Δ 13 CIsotope Ratio Mass Spectrometer
(IRMS)Alteration in isotopic composition of
end product compared with starting compound
Inverse relationship between Δ 13 C and WUE
Alternate surrogate WUE traitsAlternate surrogate WUE traits
Specific leaf area (SLA)Ratio of leaf area to leaf dry weightIndirect measurement of leaf thicknessHigher SLA represents larger surface area
for transpiration. Inversely related to WUEPositive correlation b/w SLA and Δ 13 CSPAD chlorophyll meter reading - SLA &
WUE
Intrinsic ToleranceIntrinsic Tolerance
Inherent ability to adjust at cellular levelMembrane integrity
Lipid peroxidation Higher level leads to higher the cell viabilityLeakage of cellular constituentsSullivan’s leaf disc & Evan’s blue Under stress, loss of membrane stability and
enhances the uptake of dyeSpectrophotometer/ELISA plate reader
Oxidative stress tolerance
Oxidative Stress ToleranceOxidative Stress Tolerance
When radiant energy exceeds the plants capacity to utilize, cytotoxic reactive oxygen species (ROS) are generated
AdaptationAvoidance of excess light interceptionDissipation (non photo chemical quenching)Management of excess photochemical energyUnder abiotic stress enhances ROS
Chlorophyll FluorescenceChlorophyll Fluorescence
Most powerful technique for assessing stress
Excess energy can be dissipated as heat (chlorophyll fluorescence)
Exposing to light of defined wavelength and measuring the amount of light re-emitted at lower wavelengths
Information on the state of PSII (Fv/Fm)Can be compared with well irrigated and
stresses plants
Phenotyping for oxidative stressPhenotyping for oxidative stress
Methyl viologen/Paraquat inducedParaquat inhibits photosynthesisAccepts electrons from PSI and transfers to
molecular oxygenROS formed
Superoxide radical quantificationNitroblue tetrazolium (NBT) staining
H2O2 staining agent 3.3’ diaminobenzidine (DAB)
Quantification of oxidative stressQuantification of oxidative stress
Estimation of antioxidant enzymesEstimation of reactive oxygen species in
tissues like H2O2
Estimation of cellular damage – membrane leakages, chloroplast degradation, lipid peroxidation
Scavenging ROSScavenging ROS
Antioxidant enzymesSuperoxide desmutase (SOD)Ascorbic acid (AsA)Glutathione reductase (GR)Ascorbase peroxidase (APX)Monodehydroascorbate reducatase (MDHAR)Dehydroasorbate reductase (DHAR)Catalase (CAT)PeroxiredoxinGlutaredoxinGlutathione peroxidase
Water conservation traitsWater conservation traits
Leaf surface waxHydrophobic barrierColorimetric assay (Ebercon et 1976)
Stomatal characters and conductanceStomatal frequency (stomata/unit area)Stomatal index (ratio of stomata per unit area to total
epidermal cells)Under stress, optimize the CO2 uptake & transpiration
rateIncrease in frequency and decrease in sizeHigher stomatal conductance, cooler canopies and
increased photosynthesisPorometer or IRGA
Water use efficiencyWater use efficiency
Gravimetric methodWhole plant level in potsLoss of weight due to transpirationYield = T x TE x HIFacilitate determination of root biomassPot weighing deviceWatering regimesNet assimilation rateMean transpiration rate
Stomatal ConductanceStomatal Conductance
Linear relationship with photosynthesisLinear relationshipAffected by droughtReflects changes in water use efficiencyKensington (polyembryonic) showed more
rapid decrease than Irwin (monoembryonic)
Not well understood
PhotosynthesisPhotosynthesis
Depends on the following:-Leaf nitrogen concentration
Associated with internal fruit disorders – jelly seedChlorophyll concentration
SPARD (Soil Plant Analysis Diagnostic) meterLeaf temperature
Quantum efficiency Fv/FmNon photochemical quenchingAdaptive mechanism
Xanthophyll cycleAllows excessive energy to be dissipated
Elevated COElevated CO2 2 - Photosynthesis- Photosynthesis
Net photosynthesis increases upto 1200 µmol CO2/mol
At 600 µmol CO2/mol More dry matter partitioning to rootsIncreased fruit dry weightPartition into mesocarp
Stomata close when CO2increasesCarboxylation efficiency reduced Elevated CO2 increases temperature
FLOODSFLOODS
Flooded Orchard <geograph.org.uk>
FloodingFlooding
Mango is considered to be a moderately flood-tolerant
Evolved mechanismStem lenticels for O2 diffusion
Adventitious roots
Whether or not they are formedHow quickly they are formed
Effects of FloodingEffects of Flooding
Enhanced movement of O
2 towards the
apex promotes deeper rooting in waterlogged soils.
Drew (1984); Colmer and Voesenek (2009)
DROUGHTDROUGHT
Australian drought by Peripitus
DroughtDrought
Deep root systemDesiccation-tolerant surface feeder rootsAvoidance mechanisms
Rapid stomatal closureResin canals distribution (turgor maintenance)Leaf water potential/leaf turgorKensington drought tolerant
effective water regulation and maintenance of leaf turgor.
Sap flow measurement – irrigation scheduling
WATER DEFICIT - SEEDLINGSWATER DEFICIT - SEEDLINGS
The effect of water deficit on the number of leaves of mango rootstock seedlings grown at Maseno, Kenya. W-Watering daily, X-Watering twice in a week, Y-Watering once in a
week, Z-Watering once in two weeks. Luvaha et., al. (2008) <DOI: 10.3923/ajpp.2008.1.15>
SOIL SALINITYSOIL SALINITY
Salt-affected soils in Colorado <http://www.nrcs.usda.gov/news/archive/2004newsroom.html>
SalinitySalinity
Greater salt tolerance in polyembryonic than in monoembryonicLower leaf conc. of K+, Cl-, Mg+13-1 root-stock
Tolerance to Cl- than ion exclusionExclusion of Na+ in shoots and accumulation in
root cell vacuoles
Sufficient genetic diversity and development of rootstocks.
SalinitySalinity
Adaptation to osmotic and ionic stress (Meloni et al., 2004)
– curtailment of Na+ influx and prevention of intracellular Na+ accumulation
– osmotic adjustment by uptake of inorganic ions (K+), osmoprotectants (glucopyranosylglycerol, sucrose trehalose, or glycine betaine)
Salinity - Growth ResponseSalinity - Growth Response
Source: Carillo et al., 2011. Adapted from Munns (1995)
GlycinebetaineGlycinebetaine
Glycinebetaine acts as a compatible solute by stabilizing the quaternary structures of proteins, cell membranes, and the oxygen-evolving complex of PSII
(Papageorgiou and Murata, 1995).
GlycinebetaineGlycinebetaine
Giri 2011. 10.4161/psb.6.11.17801
Signalling Pathways – Na+ ExtrusionSignalling Pathways – Na+ Extrusion
Source: Carillo et al., 2011. Adapted from Silva & Gerós (2009)
Mango - SalinityMango - Salinity
Grafting may also be helpful in withstand deleterious salinity effects.
In mango, certain rootstock-scion combinations can reduce uptake and root-shoot translocation/accumulation of dissolved salts (Na+ and/or Cl–).
(Schmutz and Lüdders, 1999)
TOOLSTOOLS
Leaf Sensor on Sugar Cane by Leafsen
POLYEMBRYONIC MANGOPOLYEMBRYONIC MANGO
Government EffortsGovernment Efforts
The Government of India has initiated a major national effort on climate resilient agriculture covering research and technology dissemination.
In 2011, National Initiative on Climate Resilient Agriculture (NICRA) has been launched with an outlay of Rs.350 crores for the XI Plan.
Government EffortsGovernment Efforts
Research infrastructure (Lemna Tech) is being developed at major national research institutes involved irrigated crops, rainfed crops, horticulture and fisheries.
Standardizing high through put Phenotyping protocols for mango by IIHR.
CREDITSCREDITS
The mango : botany, production and uses/edited by Richard E. Litz – 2nd ed.
Training course on 'Phenotyping and Molecular breeding for improving drought adaptive traits in crops' by M Udayakumar
Wikipedia Wikimedia commons Director, CISH, Lucknow
Thank you allThank you all
Thank You AllThank You All