1. Field Testing of Transgenic Plants PS 353: Plant Genetics, Breeding and Biotechnology April 8, 2008 www.pictopia.com

2. Discussion Questions What are the two overarching objectives for the testing of transgenic plants? What are lower-tiered and upper-tiered testing? Examples? What controls are needed?

3. Discussion Questions Continued What factors would be needed for the risk assessment of a non-agronomic trait, such as pharmaceuticals? How much testing or risk assessment is necessary for a new transgenic crop to be considered “safe”?

4. What is Risk? Risk is defined as a function of the adverse effect (hazard or consequence) and the likelihood of this effect occurring (exposure).

5. What is Being Regulated? Why? Presence of the transgene…How does it affect the plant? Phenotype? Performance? Transgenic event Biosafety Concerns– human and environmental welfare “Protect” organic agriculture “Precautionary principle”

6. Non-target effects– killing the good insects by accident Transgene persistence in the environment– gene flow –Increased weediness –Increased invasiveness Resistance management– insects and weeds Virus recombination Horizontal gene flow Ecological Risks

7. Environmental Risk Assessment Scientific Method: Observe, Create Hypothesis, Perform Experiments, Collect Data, Report 1.Initial Evaluation 2.Problem Formulation 3.Tiered Risk Assessment 4.Controlled Experiments and Gathering of Information 5.Risk Evaluation

8. Tiered approach—mainly non-targets Wilkinson et al. 2003 Trends Plant Sci 8: 208

9. Tier 1: Lab Based Experiments www.ces.ncsu.edu/.../resistance%20bioassay2.jpg Bioassays to determine the resistance of the two-spotted spider mite to various chemicals www.ars.usda.gov/.../photos/nov00/k9122-1i.jpg A healthy armyworm (right) next to two that were killed and overgrown by B. bassiana strain Mycotech BB-1200. (K9122-1) Examples of insect bioassays

10. Tier 3: Field Studies Tier 2: Semi-Field/Greenhouse Greenhouse Study: Transgenic Tobacco Field Trials: Transgenic Canola Photo courtesy of C. Rose Photo courtesy of R. Millwood

11. Goals of Field Research 1.Hypothesis testing 2.Assess potential ecological and biosafety risks (must be environmentally benign) 3.Determine performance under real agronomic conditions (economic benefits)

12. Transgenic pollen harms monarch larvae JOHN E. LOSEY, LINDA S. RAYOR & MAUREEN E. CARTER Although plants transformed with genetic material from the bacterium Bacillus thuringiensis (Bt ) are generally thought to have negligible impact on non-target organisms, Bt corn plants might represent a risk because most hybrids express the Bt toxin in pollen, and corn pollen is dispersed over at least 60 metres by wind. Corn pollen is deposited on other plants near corn fields and can be ingested by the non-target organisms that consume these plants. In a laboratory assay we found that larvae of the monarch butterfly, Danaus plexippus, reared on milkweed leaves dusted with pollen from Bt corn, ate less, grew more slowly and suffered higher mortality than larvae reared on leaves dusted with untransformed corn pollen or on leaves without pollen. 20 May 1999 Nature © Macmillan Publishers Ltd 1999 Registered No. 785998 England. Case of the Monarch Butterfly Slide courtesy of D. Bartsch

13. Monarch Butterfly Larvae Photo: http://www.news.cornell.edu/releases/May99/Butterflies.bpf.html

14. Impact of Bt maize pollen (MON810) on lepidopteron larvae living on accompanying weeds ACHIM GATHMANN, LUDGER WIROOKS, LUDWIG A. HOTHORN, DETLEF BARTSCH, INGOLF SCHUPHAN* Molecular Ecology: Volume 15 Issue 9 Page 2677-2685, August 2006 In October 2001 PNAS– 6 papers delineated the risk for monarchs. Exposure assumptions made by Losey were far off. Diamondback Moth Plutella xylostella www.agf.gov.bc.ca/.../images/diamondback3.jpg Cabbage Moth Pieris rapae www.butterfliesandmoths.org/pic/Pieris_rapae.jpg

15. Bt and Monarch Risk Model Sears et al. (2001) http://www.geo-pie.cornell.edu/issues/monarchs.html cls.casa.colostate.edu/.../images/larva.jpg www.smartcenter.org/ovpm/babymonarch-09.jpg

16. Experimental Goals Does growing of Bt-maize harm non-target Lepidoptera under field conditions? Compare growing of Bt-maize with conventional insecticide treatment Is the presented experimental design a useful approach for monitoring non-target Lepidoptera? * Note: this study did not specifically look at how Bt pollen effect monarch larvae. Examined other lepidopteron larvae native to Germany which are commonly found within corn fields Slide courtesy of D. Bartsch

17. Farmer Field East Field West 500m 4 ha 2 ha Slide courtesy of D. Bartsch

18. Experimental Design: Field Study Bt 6 ISO 7 ISO 8 INS 8 INS 6 Bt 8 INS 7 ISO 6 Bt 7 Bearbeitunsrichtung 178 m 162 m 141 m 186 m Bt 5 ISO 5 ISO 3 INS 5 INS 3 Bt 4 ISO 2 Bt 1 Bt 2 INS 1 ISO 1 INS 2 INS 4 ISO 4 Bt 3 Bearbeitunsrichtung 237 m 248 m 162 m 182 m ca. 500 m Bt = Bt-maize Mon 810 INS = Isogenic variety with insecticide treatment ISO = Isogenic variety, no insecticide treatment (Control) Slide courtesy of D. Bartsch

19. Lepidopteron Larvae Exposure to Bt cry1Ab Slide courtesy of D. Bartsch Insect collectionSpecies Identification

20. Field Test Results Lepidopteron larvae were not affected by the pollen of Mon 810 under field conditions Sometimes pollen shed and development of lepidopteron larvae barely overlapped 2001 2002 Slide courtesy of D. Bartsch

21. Field Test Results Choice of a lepidopteron monitoring species will be difficult because –species must be abundant –theoretical prediction of the presence of abundant species is not easy –occurrence and abundance of species depends on alot of variables ( e.g. climatic conditions, landscape structure around the fields, management options) Slide courtesy of D. Bartsch

22. Abundant Species Autographa gamma Plutella xylostella Pieris rapae Xanthorhoe flucata Slide courtesy of D. Bartsch

23. Monarch butterfly What’s riskier? Broad spectrum pesticides or non-target effects?

24. ERA: Case of Bt Corn and the Lovely Butterfly Scientific Method: Observe, Create Hypothesis, Perform Experiments, Collect Data, Report 1.Initial Evaluation (Bt Pollen Could Spread to Neighboring Plants: Milkweed) 2.Problem Formulation (Bt Pollen Harms Non-Target Insects) 3.Tiered Risk Assessment (Lab Field) 4.Controlled Experiments and Gathering of Information (Unbiased Report of Data) 5.Risk Evaluation (Create Regulations Based on Actual Scientific Data)

25. Tritrophic Interactions: Non-target Insect Model Wilkinson et al. 2003 Trends Plant Sci 8: 208

26. Detlef Bartsch Geobotany Institute of the University of Gottingen (BS, MS, PhD) The first ecologist in Germany to study competitiveness and out-crossing with GMO sugar beets He was first opposed to GMOs, but now is pro-GMO Decided to leave academia and in 2002 became a regulator for the Federal German Agency Now is an independent expert for the European Food Safety Authority

27. Risk = Pr(GM spread) x Pr(harm|GM spread) ExposureImpact Frequency Hazard Consequence Gene flow from transgenic plants Intraspecific hybridization Interspecific hybridization

28. Discussion question What factors would be needed for the risk assessment of a nonagronomic trait, such as a pharmaceutical? Where would the risk assessor begin? How would we know when the risk assessment is over—that is, a decision between safe and not safe?

29. Gene flow model: Bt Cry1Ac + canola and wild relatives Diamondback moth larvae. http://www.inhs.uiuc.edu/inhsreports/jan-feb00/larvae.gif Brassica napus – canola contains Bt Brassica rapa – wild turnip wild relative

30. Brassica relationships Triangle of U

31. Bt Brassica gene flow risk assessment Is it needed? What kind of experiments? At what scale?

32. Ecological concerns Damage to non-target organisms Acquired resistance to insecticidal protein Intraspecific hybridization Crop volunteers Interspecific hybridization Increased hybrid fitness and competitiveness Hybrid invasiveness www.epa.gov/eerd/BioTech.htm

33. Experimental endpoints Hypothesis testing Tiered experiments– lab, greenhouse, field Critical P value Relevancy Comparisons– ideal vs pragmatic world HYPOTHESES MUST BE MADE— WE CANNOT SIMPLY TAKE DATA AND LOOK FOR PROBLEMS!

34. Tiered approach Wilkinson et al. 2003 Trends Plant Sci 8: 208

35. Pollination method Bt Canola Brassica rapa pollen F 1 hybrid What would be a good hypothesis?

36. Crossing method Halfhill et al. 2005, Molecular Ecology, 14, 3177–3189.

37. Brassica napus, hybrid, BC1, BC2, B. rapa B. napus F 1 BC 1 BC 2 B. rapa

38. Hybridization frequencies— Hand crosses– lab and greenhouse F 1 Hybrids BC 1 Hybrids CAQB1QB2TotalCAQB1QB2Total GT 169%81%38%62%34%25%41%33% GT 263%88%81%77%23%35%31%30% GT 381%50%63%65%24%10%30%20% GT 438%56% 50%7%30%36%26% GT 581%75%81%79%39%17%39%31% GT 650% 54%51%26%12%26%21% GT 731%75%63%56%30%19%31%26% GT 856%75%69%67%22% 21%22% GT 981%31% 48%27%28%23%26% GFP 150%88%75%71%18%33%32%27% GFP 269%88%100%86%26%20%57%34% GFP 319%38%19%25%10%22%11%15% First-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency

39. Insect bioassay of hybrids First-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Impact Hazard Consequence

40. Greenhouse Bt “superweed” experiment l S Soybean l C Brassica rapa l BT BC3 Bt transgenic Brassica rapa Assess transgenic weediness potential by assaying crop yield. Second-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Impact Hazard Consequence

41. -herbivory+herbivory TT CC

42. Soybean biomass Wet biomass (g) CC CT TT

43. Field level hybridization Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency

44. Field hybridization experiment

45. Field level backcrossing Maternal Parent F 1 hybridTransgenic/germinatedHybridization rate per plant Location 1983/195050.4% Location 2939/209544.8% F 1 total1922/404547.5% Maternal Parent B. rapaTransgenic/germinatedHybridization rate per plant Location 134/56,8450.060% Location 244/50,1770.088% B. rapa total78/107,0220.073% Halfhill et al. 2004. Environmental Biosafety Research 3:73

46. Backcrossing conclusions Backcrossing occurs under field conditions Backcrossing rates to B. rapa are low (1 out of 1,400 seeds)

47. Field experiment: Brassica hybrid herbivory damage Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Impact Hazard Consequence

48. Field experiment: Brassica hybrid productivity

49. Brassica hybrid field results Hybridization frequencies are low Hybrids have lower productivity in all cases More third-tier experiments need to be performed – such as competition experiments

50. Features of good risk assessment experiments Gene and gene expression (dose) –Relevant genes –Relevant exposure Whole plants Proper controls for plants Choose species Environmental effects Experimental design and replicates Andow and Hilbeck 2004 BioScience 54:637.

51. Discussion question Which is more important: that a field test be performed for grain yield or environmental biosafety?