Uwe StolzUwe Stolz

NERL Postdoctoral Research FellowNERL Postdoctoral Research Fellow

Genomics Workshop, Chicago, IL Genomics Workshop, Chicago, IL

April 29, 2005 April 29, 2005

Agricultural Biotechnology: Agricultural Biotechnology: Genomic Approaches to Monitoring Genomic Approaches to Monitoring the Effects of Genetically Modified the Effects of Genetically Modified

CropsCrops

Agricultural Biotechnology:Genetically modified crops

Current and Future GM Crop Traits

1. Insect Resistance (Plant Incorporated Protectants) – ex: BT corn & cotton

2. Herbicide Tolerance – ex: Glyphosate Resistant Corn & Soybeans (i.e. Roundup Ready)

3. “Value Added” Crops – ex: Golden Rice containing vitamin A

4. Stress Tolerance – ex: drought, salt resistant varieties

5. “Biopharming” – Production of drugs, chemicals on agricultural scales

Major environmental and health concerns regarding GM crops

• Non-target effects

• Gene flow / Transgene Escape

• Insect Resistance

• Allergenicity

Issues for Regional Monitoring Efforts

• Non-target Effects Species, Communities, Ecosystems

• Beneficial species, threatened / endangered species

• Gene Flow / Transgene Escape Crop to Crop, Crop to Wild Relative, Crop

to distant relative• Insect resistance genes, herbicide tolerance

genes

• Future – vaccines, chemicals, drugs, etc.

Non-target effects of GM corn

1. Bt corn and non-target effects: Yieldgard Corn

2. ORD/NERL research approach / rationale

3. Research progress

4. Monitoring Applications

Agricultural Pesticides:

UnintendedUnintendedEnvironmentalEnvironmental

ImpactImpact

Intended and unintended effects

Human HealthHuman Health EffectsEffectsTargetedTargeted

InsectInsect

PestPest

80 million acres of corn planted per year(20% of total crop acreage)

Western Corn Rootworm (WCR) is major pest of corn.

Conventional insecticidesapplied to 14-18 million

acres per year.

WCR responsible for 1 out of 7 insecticide applications for all agricultural crops

WCRWCRdamagedamage

CRW will continue to be a problem…Infested acres of CRW expected to increase: 39 million acres by 2013

EPA identified 10 insecticides used in agriculture as the most toxic to birds - 3 currently used to control corn rootworm (carbofuran, phorate and methyl parathion).

CRW have adapted to crop rotation with soybeans and have evolved resistance to several chemical insecticides

Bt-cornBt-corn

Non Non Bt-cornBt-corn

Non Non Bt-cornBt-corn

Bt-corn reduces the need for conventional pesticides:

The Evolution of Bt-Resistance is an Environmental Problem

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Current Research of ORD -NERL in Cincinnati

• Monitor resistance genes in pest populations-Identify resistance/tolerance genes

-Develop molecular techniques for rapid and cost effective screening

• Monitor non-target species for effects of Bt exposure

-Identify gene expression markers for Bt exposure in target pests

-Develop assays to test for effects in related non-targets

Complements ecological monitoring by adding another level of analysis and increases the amount of information that can be gathered about the status and health of species

Advantages of Genetic Monitoring

Non-Target-Monitoring

• What is a non-target species?

• How do we choose non-targets?

• Molecular genetics to monitor non-targets Gene Expression

• Benefits of molecular genetic techniques

Non-Target Effects: The Agricultural Landscape

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Non-economicNon-economic

EconomicEconomic

Non-Targets

• Economic non-targets Non-target pests Beneficial predators Other beneficial Insects

• Non-Economic non-targets Threatened/endangered species Species of public concern Species whose primary habitat is not

agricultural

Gene Expression

• Detects exposure to pesticides/PIP• Detects biologically meaningful exposure• Detects non-acute effects• Permits study of non-lab species• Highly sensitive - ex: EDCs in streams

Bt CornBt Corn

WCRWCR

Gene Expression ChangesGene Expression Changes

DIET +ROOT +BTDIET +ROOT +BT

ST

D D

IET

ST

D D

IET

1 3 4 5 62

btDIE

T +

RO

OT

DIE

T +

RO

OT

Effect of Bt-corn diet on gene expression

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

7A(1.21) 7D(4.23) 4A4(6.85) 4B1(6.18) 6A1(2.82) 6A2(1.82)

clone name

Rel

ativ

e G

ene

Exp

ress

ion

cnt

bt

Results from QPCR for six clones identified through differential display.

Once Gene Expression Markers Confirmed

•Non-target species can be monitored for exposure to PIPs and/or pesticides (QPCR, RT-PCR)

•Several closely related species can be monitored at the same time to detect ecosystem level patterns

Proof of Concept Research

•We are examining ground beetle species across corn growing areas•Examine several species communities•Look at exposure to Bt corn

-ELISA assays using beetle guts to check for presence of Bt toxin-Gene expression assays

Gene Flow / Transgene Escape

• Contamination of food supply• Creation of “super-weed” crops• Transgene escape to native relatives

via hybridization, creation of super-weeds

• Genetic pollution

Major Issues of Transgene Escape / Gene Flow

• Use GM crops with few wild relatives (ex: Corn in the USA, Canada, Europe)

• Use GM crops with no weedy relatives (ex: Sorghum and Johnson Grass)

• Provide sufficient physical and physiological barriers to gene flow (ex: plant isolated patches, have appropriate borders, sterility)

• Monitor for the escape and movement of transgenes

Keys to Prevention of Transgene Flow

Case Study of EPA work on Transgene Flow

• Herbicide (Roundup) resistant bent grass

• Considered for turf management

• Improve golf courses

CD = managed bentgrass field

Sentinel plants = potted plants

Resident = naturally occurring

In addition to PCR, also used Elisa (checks for protein product of transgene) and used seedlings from sentinel plants to check for RoundUp resistance (phenotype of transgene).

Ultimate Goals

• Apply molecular monitoring methods to conserved, threatened, and endangered species (i.e. difficult to study in the lab).• Apply strategy to species / populations /

communities that are most at risk to changing crop practices.• Ensure the safety of biotech crops bymonitoring potential environmental problems.

Acknowledgements

Ecological Exposure Research Division/Molecular Ecology Research Branch

Mark BagleyJim Lazorchak

Sobran Inc.