Drought Resistant CropsDrought Resistant CropsDrought Resistant Crops

Kiran K. SharmaP. Bhatnagar-Mathur, V. Vadez

Genetic Transformation Laboratoryk.sharma@cgiar.org

Kiran K. SharmaKiran K. SharmaP.P. BhatnagarBhatnagar--MathurMathur, V., V. VadezVadez

Genetic Transformation Laboratoryk.sharma@cgiar.org

International Crops Research Institute for the Semi-Arid Tropics Presented at: AsianBio2006, Manila

This presentationThis presentation

• Constraints to food security• Drought and agricultural crops• Biotechnology for crop improvement• Current Status of transgenics@ICRISAT• Future prospects

Agriculture and global food security by 2025

Agriculture and global food Agriculture and global food security by 2025security by 2025

• Food requirements will double with a global population of 8 billion.

• Malnourished people will be over 1 billion.

• 85% of global population will be in the developing countries.

• 60% of population in developing countries will live in cities.

• Hence, increased pressure to protect the environment and biodiversity, and conserve natural resources.

Major challenges Major challenges in agricultural sciencein agricultural science

• Generate new technologies that provide sustainable production systems.

• Create opportunities for diversificationin agricultural value-chains.

• Develop production systems for low potential areas.

Constraints to crop productivityConstraints to crop productivity

• Resource-poor farmers carry out 60% of global agriculture, but produce only 15-20% of world’s food. This tends to perpetuate rural poverty.

• Farm lands are in fragile environments that are low in fertilityand productivity.

• Crops face major challenges from biotic and abiotic stresses.

• Limited access to external inputs like pesticides, fertilizers, irrigation.

• Agriculture means growing marginal crops on marginal lands with marginal resources.

ISAAA

Constraints to crop productivityConstraints to crop productivity

Drought is the major constraint

Drought is the major constraint

Chickpea crop affected by drought

Breeding for drought toleranceBreeding for drought tolerance

Three approaches

• Escape

• Tolerance

• Avoidance• Better use of water

• Access more water

Drought escape: through early maturity

ICCV 2 matures in 85-90 days

Super-early chickpea lines developed: matures in 75-80 days vs 85-90 days.

A typical plant response curve under soil-drying conditions

FTSW

0.00.20.40.60.81.0

Nor

mal

ize d

tran

s pir a

t ion

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Stage IStage II

Stage III

Drought avoidance:through better water use

Transpiration Efficiency or Water Use efficiency

Variation in Transpiration Efficiency in groundnutVariation in Transpiration Efficiency in groundnut

Constraints to crop improvement

Constraints to crop improvement

1. Available germplasm may lack genes/switches for major biotic and abiotic stresses.

2. Limited variability in the available germplasm.

3. Future breakthroughs will depend on creating additional variability and inflow of desirable genes from related or unrelated species.

Role of biotechnologyRole of biotechnology

1. Provides modern ideas and techniques to complement, facilitate and speed-up breeding efforts.

2. To develop commercial processes and products.

3. Transforms agriculture from a resource-based to a science-based industry.

4. Generate social, economic and environmental benefits.

Potentials of Transgenic PlantsPotentials of Transgenic Plants

Gene(s) for desirable traits that are inaccessible to plant breeder can be cloned and functionally introduced into desired cultivar.

Gene dosage can be precisely controlled.

Tissue-specific expression of the transgene can be regulated.

Factors influencing Plant Factors influencing Plant response to stressresponse to stress

Genotype:Variations in the tolerance to stresses between

genotypes.

Timing: (the age and stage of development)Plants are very sensitive to desiccation during the

vegetative phase of their life cycle, where as desiccation is necessary for seeds.

Duration: (The length and severity of the stress)Type, length and severity of the stress have more

influence on the plant response to stress. (Lethal or sub-lethal )Resurrection plants can survive even with <5% of their

total water in the vegetative tissues (desiccation tolerance)

Intensity:

Perception of stress responsesPerception of stress responses

Ozone

Drought

Salt

Flooding

Stress recognition

Stress transduction

Altered gene expression

Altered metabolism

Physiological/ developmental

response

Temperature stress

APPROACHES FOR COMBATING APPROACHES FOR COMBATING DROUGHT STRESSESDROUGHT STRESSES

• Conventional– Agronomic Management Practices– Classical Breeding Methods

• Biotechnological– Molecular Markers & Marker Assisted

Breeding– Transgenic Technology

Major components of the Major components of the dehydration tolerancedehydration tolerance

• Tolerance is the result of a complex cascade of molecular events.

• Dehydration tolerance includes three major components:

Osmotic adjustment

Antioxidant capacity

Desiccation tolerance

KEY TOOLS FOR DEVELOPING KEY TOOLS FOR DEVELOPING TRANSGENICSTRANSGENICS

• STRUCTURAL GENES:– Osmoprotectants– LEA Proteins– Transporter genes– Heat shock genes– Detoxifying genes– Multifunctional genes for lipid

biosynthesis

• REGULATORY GENES– Transcription factors– Signal transduction component genes

Osmotic AdjustmentOsmotic AdjustmentGenes Affecting Compatible SolutesGenes Affecting Compatible Solutes

Osmotic adjustment results from the accumulation of compatible solutes within cells, which lowers the osmotic potential and helps maintain turgor.

Transport or uptake processes

Plasma membrane Na+/H+antiporter

Tonoplast Na+/H+ antiporter*RS allele*(PMB) Alllele*Water channels

Compatible solute accumulation

Mannitol-1-phosphate dehydrogenase (mtld)

Inositol methyl transferaseAldose reductaseP5C synthetaseP5C reductaseBetaine aldehydedehydrogenseCholine mono-oxygenase*

LEA proteins

• Major group of proteins that typically accumulate at: – Late stage of embryogenesis– Response to

• dehydration • low temperature, • salinity

• exogenous ABA treatment

• Play a role in protecting cytoplasmic structures during dehydration.

• Divided into groups based on predicted biochemical properties and motifs with sequence similarities.

Antioxidant capacityAntioxidant capacity

• Accumulation of Reactive oxygen species in several abiotic stresses including drought and salt stresses will damage cellular structures.

• Plants have evolved various enzymatic and nonezymaticprotective mechanisms allowing them to scavenge active oxygen species.

Gene encoding enzymes for removing reactive oxygen species (Antioxidant capacity)

• Superoxide dismutase (SOD) related [Sod2 (Pea);SODCc1 (Rice)]• Ascorbate peroxidases (AP) related [Apx1(Pea)]• Catalases(CAT) related [Catl-Cat3 (Maize, Arabidopsis)]• Glutathione reductase (GR)related [cDNA (Pea)]

StressStress--inducible promotersinducible promoters

Abiotic stress inducible promoters contain cis-acting elements and activated by Cis-trans interaction.

The rd29A promoter is shown to contain two cis-acting elements, one involved in the ABA-associated response and the other induced by changes in the osmotic potential.

Among the 22 genes that were expressed in all three-stress treatments, 16 genes (84%) contain DRE (TACCGACAT) or DRE-related CCGAC core motif in their promoters.

All these 16 genes were regulated by the DREB1/CBF or DREB2 transcription factors.

Transcription factorsTranscription factors

• Various transcriptional regulatory mechanisms function in the abiotic stress signal pathways

• Among the all stress inducible genes ~11% are regulatory genes/transcription factors.

• Several different classes of transcription factors are induced by water-deficit stress:– bZIP AREB1– HD-ZIP ATHB-6,-7 and -12– AP2/EREBP DREB1,2– MYB ATMYB2– MYC-like bHLH rd22BP1

• Many of these transcription factors are members of large multigene families.

Transcriptional regulatory networks (cis-acting elements and transcription factors) involved in osmotic and cold stress responsiveness in Arabidopsis

Yamaguchi-Shinozaki & Shinozaki 2006, ARPB

Molecular tailoring of Molecular tailoring of farnesylationfarnesylation

for drought tolerance and yield protectionfor drought tolerance and yield protection

• The ERA1 mutation in Arabidopsis increases sensitivity to ABA (tight closure of the guard cells with reduced wilting under drought stress) but has severe pleiotropic phenotypes.

• ERA1 encodes for farnesyl transferase b-subunit (farnesylation is a type of regulative protein modification where a lipid chain -15C- is attached to the -aa chain).

• A negative regulator of guard cell sensitivity to ABA signaling must be farnesylated to modulate ABA response.

A holistic approach for A holistic approach for enhancing drought toleranceenhancing drought tolerance

Plant BreedingOsmoregulation(codA, P5CF-129A, mtld)

-Anti-oxidative enzymes(Annexin)-Ion homeostasis(Na+-H+ antiport)

Drought Responsive Elements (rd29A :DREB1A)

Elite genotypes Applied Genomics

Transformation

Transgenics

MAS

Molecular Markers

Regulation of droughtRegulation of drought--responsive gene expression responsive gene expression

rd29A-P DREB1A

Promotor A Gene A

Promotor B Gene B

Promotor C Gene C

Promotor D Gene D

Regulatory protein[Transcriptional activator]

Drought

DRE

Transcription/Translation

Stress Response and Tolerance

Plant Cell

Signal Perception

Signal transduction

Gene Expression

rd29A:DREB1A Stress Response

rd29A:DREB1A Stress Response

rd29A:DREB1A CaMV 35S:DREB1A

Effect of DREB1A driven by 35S and rd29A Effect of DREB1A driven by 35S and rd29A promotorspromotorson germination and growthon germination and growth

CaMV 35S:DREB1A rd29A:DREB1A• 45 events with rd29A:DREB1A.• 15 event screened in dry-down

set-ups in greenhouse.• 3 promising events under

validation.• Contained field trials planned

for 2007

Three approaches:•Escape•Tolerance•Avoidance

•Better use of water•Access more water

Groundnut transgenics with DREB1A

rd29 CEffect of promoters on tissue culture response Effect of heat stress

on groundnut transgenics

Groundnut Dry-Down set-up

Dendrogram based on FTSW threshold where TR declines and days to reach FTSW=0

JL24 Dendrogram showing relative similarities in 15 transgenic events (including control JL 24) based on FTSW threshold values and the number of days to end point under water deficit conditions.

RD19

RD12

RD20

RD2RD11

Transpiration Efficiency & FTSW under drought stress

Variation in TE in groundnutVariation in TE in groundnut

RILsRILs v/sv/s DREB1ADREB1A transgenicstransgenics

**Larger range of variation for TE in DREB1A transgenics

Drought tolerance in groundnut

(Many !!!) Prospects(Many !!!) Prospects

• Recovery – Free radical• Comparison with 35S promoter• Re-exploration of the physiology of TE and Gs• Screen more events• Confirmation of these results in different drought

stages• Comparison with cultivated genotypes having high TE• Contained field trial

FUTURE PROSPECTSFUTURE PROSPECTS

Most abiotic stress resistance in plant is polygenic trait, but major genes can play an important role in the genetic control ofthe component traits:

Osmolyte accumulation- P5CS for Proline.Enzymes that scavenge reactive oxygen species.Transcription factors-DREB.Components involved in signal transduction.

Simultaneous transfer of several genes will be the likely next step allowing to achieve practical levels of plant resistance:

Introduction of sets of genes .Sequential transformation.Sexual crosses between transgenic plants harbouring transgenes of interest.

Switching on a transcription factor and abiotic stress inducible promoter:

Over expression of a transcription factor will activate the downstream genes such as LEA genes.Stress inducible promoters to drive the expression of transgenes has an additional advantage over single gene approach.

Our callOur callBiotechnology is a valuable tool in eliminating global hunger, poverty and malnutrition!

It is a strategic weapon in winning the next Green Revolution!

Let us harness biotechnology with due regard for consumer and environmental safety!

AcknowledgementsAcknowledgements

Dr. Kazuko Yamguchi-Shinozaki, JIRCAS, Japan

Thank youThank you