Field Testing of Transgenic Plants

PS 353: Plant Genetics, Breeding and Biotechnology

April 8, 2008www.pictopia.com

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?

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”?

What is Risk?

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

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”

• 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

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

Tiered approach—mainly non-targets

Wilkinson et al. 2003 Trends Plant Sci 8: 208

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

Tier 3: Field StudiesTier 2: Semi-Field/Greenhouse

Greenhouse Study: Transgenic Tobacco

Field Trials: Transgenic Canola

Photo courtesy of C. Rose

Photo courtesy of C. Rose

Photo courtesy of R. Millwood

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)

Transgenic pollen harms monarch larvaeJOHN E. LOSEY, LINDA S. RAYOR & MAUREEN E. CARTERAlthough 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

Slide courtesy of D. BartschMonarch Butterfly Larvae Photo: http://www.news.cornell.edu/releases/May99/Butterflies.bpf.html

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 MothPlutella xylostella

www.agf.gov.bc.ca/.../images/diamondback3.jpg

Cabbage MothPieris rapae

www.butterfliesandmoths.org/pic/Pieris_rapae.jpg

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

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

Farmer

Field East

Field West500m

4 ha

2 ha

Slide courtesy of D. Bartsch

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

Lepidopteron Larvae Exposure to Bt cry1Ab

Slide courtesy of D. Bartsch

Insect collection Species Identification

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

July August 26. 27. 28 29. 30. 31. 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 13. 14. 15. sample 1 sample 2 flowering of maize sample 1 sample 2 flowering of maize

2001

2002

Slide courtesy of D. Bartsch

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

Abundant Species

Autographa gamma Plutellaxylostella

Pieris rapae

Xanthorhoe flucata

Slide courtesy of D. Bartsch

Monarch butterfly

What’s riskier?

Broad spectrum pesticides

or

non-target effects?

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)

Tritrophic Interactions: Non-target Insect Model

Wilkinson et al. 2003 Trends Plant Sci 8: 208

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 expertfor the European Food Safety Authority

Risk = Pr(GM spread) x Pr(harm|GM spread)

Exposure Impact Frequency Hazard

Consequence

Gene flow from transgenic plants

• Intraspecific hybridization

• Interspecific hybridization

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?

Gene flow model: Bt Cry1Ac + canola and wild relatives

Diamondback moth larvae. http://www.inhs.uiuc.edu/inhsreports/jan-feb00/larvae.gif

Brassica napus – canolacontains Bt

Brassica rapa – wild turnipwild relative

Brassica relationships

Triangle of U

Bt Brassica gene flow risk assessment

• Is it needed?

• What kind of experiments?

• At what scale?

Ecological concernsEcological concerns

• Damage to non-target organisms• Acquired resistance to insecticidal

protein • Intraspecific hybridization

• Crop volunteers

• Interspecific hybridization• Increased hybrid fitness and

competitiveness

• Hybrid invasivenesswww.epa.gov/eerd/BioTech.htm

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!

Tiered approach

Wilkinson et al. 2003 Trends Plant Sci 8: 208

Pollination method

Bt Canola Brassica rapa

pollen

F1 hybridWhat would be a good hypothesis?

Crossing method

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

Brassica napus, hybrid, BC1, BC2, B. rapa

B. napus F1 BC1 BC2 B. rapa

Hybridization frequencies—Hand crosses– lab and greenhouse

F1

Hybrids

BC1 Hybrids

CA QB1 QB2 Total CA QB1 QB2 Total

GT 1 69% 81% 38% 62% 34% 25% 41% 33%

GT 2 63% 88% 81% 77% 23% 35% 31% 30%

GT 3 81% 50% 63% 65% 24% 10% 30% 20%

GT 4 38% 56% 56% 50% 7% 30% 36% 26%

GT 5 81% 75% 81% 79% 39% 17% 39% 31%

GT 6 50% 50% 54% 51% 26% 12% 26% 21%

GT 7 31% 75% 63% 56% 30% 19% 31% 26%

GT 8 56% 75% 69% 67% 22% 22% 21% 22%

GT 9 81% 31% 31% 48% 27% 28% 23% 26%

GFP 1 50% 88% 75% 71% 18% 33% 32% 27%

GFP 2 69% 88% 100% 86% 26% 20% 57% 34%

GFP 3 19% 38% 19% 25% 10% 22% 11% 15%

First-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency

Insect bioassay of hybrids

DBM Bioassay of Hybrids

0

10

20

30

40

50

60

70

80

B. rap

a P1

B. rap

a UCI

W58

W45

W45

x P1

W45

x UCI

O48 x

P1

O48 x

UCI

O52 x

UCI

W63

x UCI

O96 x

UCI

O124

x UCI

Line

Pe

rce

nt

De

folia

tio

n

First-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Impact Hazard

Consequence

Greenhouse Bt “superweed” experiment

S Soybean C Brassica rapa 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

-herbivory +herbivory

TT CC

Soybean biomassW

et b

iom

ass

(g)

CC CC CT CT TT TT

Field level hybridizationThird-tier Risk = Pr(GM spread) x Pr(harm|GM spread)

Exposure Frequency

Field hybridization experiment

Field level backcrossingMaternal 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%

Maternal ParentB. rapa Transgenic/germinated Hybridization rate per

plant

Location 1 34/56,845 0.060%

Location 2 44/50,177 0.088%

B. rapa total 78/107,022 0.073%

Halfhill et al. 2004. Environmental Biosafety Research 3:73

Backcrossing conclusions

• Backcrossing occurs under field conditions

• Backcrossing rates to B. rapa are low

(1 out of 1,400 seeds)

Field experiment: Brassica hybrid herbivory damage

Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread)

Impact Hazard

Consequence

Field experiment: Brassica hybrid productivity

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

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.

Discussion question

•Which is more important: that a field test be performed for grain yield or environmental biosafety?