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

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. Ecological Risks
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

7. Environmental Risk Assessment
Scientific Method: Observe, Create Hypothesis, Perform Experiments, Collect Data, Report
Initial Evaluation
Problem Formulation
Tiered Risk Assessment
Controlled Experiments and Gathering of Information
Risk Evaluation
Regulatory requirements, scientific inquiry, and scientific responses to public concerns

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

9. Tier 1: Lab Based Experiments
Examples of insect bioassays
Bioassays to determine the resistance of the two-spotted spider mite to various chemicals
A healthy armyworm (right) next to two that were killed and overgrown by B. bassiana strain Mycotech BB (K9122-1)

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

11. Goals of Field Research
Hypothesis testing
Assess potential ecological and biosafety risks (must be environmentally benign)
Determine performance under real agronomic conditions (economic benefits)
Compared with the isogenic or parent variety, must perform as well or better (RR soybeans)

12. Case of the Monarch Butterfly
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 England.
Slide courtesy of D. Bartsch

13. Slide courtesy of D. Bartsch
Monarch Butterfly Larvae Photo:

14. Diamondback Moth Plutella xylostella
In October 2001 PNAS– 6 papers delineated the risk for monarchs. Exposure assumptions made by Losey were far off.
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 , August 2006
Diamondback Moth Plutella xylostella
Cabbage Moth
Pieris rapae

15. Bt and Monarch Risk Model
cls.casa.colostate.edu/.../images/larva.jpg
Sears et al. (2001)

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

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

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

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
Xanthorhoe flucata
Pieris rapae
Slide courtesy of D. Bartsch

23. Broad spectrum pesticides
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
Initial Evaluation (Bt Pollen Could Spread to Neighboring Plants: Milkweed)
Problem Formulation (Bt Pollen Harms Non-Target Insects)
Tiered Risk Assessment (Lab Field)
Controlled Experiments and Gathering of Information (Unbiased Report of Data)
Risk Evaluation (Create Regulations Based on Actual Scientific Data)
Regulatory requirements, scientific inquiry, and scientific responses to public concerns

25. Tritrophic Interactions: Non-target Insect Model
Wilkinson et al 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. Gene flow from transgenic plants
Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Impact Frequency Hazard Consequence
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
Brassica napus – canola
contains Bt
Diamondback moth larvae.
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

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 Trends Plant Sci 8: 208

35. Pollination method What would be a good hypothesis? Bt Canola
Brassica rapa
pollen
What would be a good hypothesis?
F1 hybrid

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

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

38. Hybridization frequencies—
Hand crosses– lab and greenhouse
First-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency F1
Hybrids
BC1 Hybrids CA
QB1
QB2
Total
GT 1
69%
81%
38%
62%
34%
25%
41%
33%
GT 2
63%
88%
77%
23%
35%
31%
30%
GT 3
50%
65%
24%
10%
20%
GT 4
56% 7%
36%
26%
GT 5
75%
79%
39%
17%
GT 6
54%
51%
12%
21%
GT 7
19%
GT 8
67%
22%
GT 9
48%
27%
28%
GFP 1
71%
18%
32%
GFP 2
100%
86%
57%
GFP 3
11%
15%

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

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

41. herbivory
+herbivory TT CC

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

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

44. Field hybridization experiment

45. Field level backcrossing
Maternal Parent
F1 hybrid
Transgenic/germinated
Hybridization rate per plant
Location 1
983/1950
50.4%
Location 2
939/2095
44.8%
F1 total
1922/4045
47.5%
B. rapa
34/56,845
0.060%
44/50,177
0.088%
B. rapa total
78/107,022
0.073%
Halfhill et al Environmental Biosafety Research 3:73 45

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

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?