1. Uwe Stolz NERL Postdoctoral Research Fellow Genomics Workshop, Chicago, IL April 29, 2005 Agricultural Biotechnology: Genomic Approaches to Monitoring the Effects of Genetically Modified Crops

2. Agricultural Biotechnology: Genetically modified crops

3. 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

4. Major environmental and health concerns regarding GM crops Non-target effects Gene flow / Transgene Escape Insect Resistance Allergenicity

5. 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.

6. 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

7. Agricultural Pesticides: UnintendedEnvironmentalImpact Intended and unintended effects Human Health Effects Effects Targeted Insect Pest

8. 80 million acres of corn planted per year (20% of total crop acreage) Western Corn Rootworm (WCR) is major pest of corn. Conventional insecticides applied to 14-18 million acres per year. WCR responsible for 1 out of 7 insecticide applications for all agricultural crops WCRdamage

9. 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

10. Bt-corn NonBt-cornNonBt-corn Bt-corn reduces the need for conventional pesticides:

11. The Evolution of Bt-Resistance is an Environmental Problem ??

12. 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

13. 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

14. 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

15. Non-Target Effects: The Agricultural Landscape ? ? ? Non-economic Economic

16. 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

17. 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

18. Bt Corn WCR Gene Expression Changes DIET +ROOT +BT STD DIET 1 3 4 5 6 2 bt DIET +ROOT

19. Results from QPCR for six clones identified through differential display.

20. 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

21. 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

22. Gene Flow / Transgene Escape

23. 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

24. 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

25. Case Study of EPA work on Transgene Flow Herbicide (Roundup) resistant bent grass Considered for turf management Improve golf courses

27. CD = managed bentgrass field Sentinel plants = potted plants Resident = naturally occurring

28. 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).

30. 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 by monitoring potential environmental problems.

31. Acknowledgements Ecological Exposure Research Division/ Molecular Ecology Research Branch Mark Bagley Jim Lazorchak Sobran Inc.