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<gutam2000@gmail.com><gutam2000@gmail.com>

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