Field Testing of Transgenic Plants

PS 353: Plant Genetics, Breeding and Biotechnology

April 8, 2008
www.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”
 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
 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
Examples of insect bioassays

www.ces.ncsu.edu/.../resistance%20bioassay2.jpg www.ars.usda.gov/.../photos/nov00/k9122-1i.jpg

Bioassays to determine the A healthy armyworm (right) next to two

resistance of the two-spotted spider that were killed and overgrown by B.
mite to various chemicals bassiana strain Mycotech BB-1200.
(K9122-1)
 Tier 2: Tier 3: Field Studies
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
 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)
Case of the Monarch Butterfly
20 May 1999

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.

Nature © Macmillan Publishers Ltd 1999 Registered No. 785998 England.

Slide courtesy of D. Bartsch

Monarch Butterfly Larvae Photo: http://www.news.cornell.edu/releases/May99/Butterflies.bpf.html Slide courtesy of D. Bartsch
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 2677-2685, August 2006

Diamondback Moth
Plutella xylostella Cabbage Moth
Pieris rapae

www.agf.gov.bc.ca/.../images/diamondback3.jpg www.butterfliesandmoths.org/pic/Pieris_rapae.jpg
 Bt and Monarch Risk Model

cls.casa.colostate.edu/.../images/larva.jpg

Sears et al. (2001)

http://www.geo-pie.cornell.edu/issues/monarchs.html 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

 Field East

2 ha
Field West

4 ha

Farmer

Slide courtesy of D. Bartsch

 Experimental Design: Field Study
Bt = Bt-maize Mon 810
Bt ISO INS
INS = Isogenic variety with insecticide treatment
6 6 6
ISO = Isogenic variety, no insecticide treatment (Control) Bearbeitunsrichtung

ISO
178 m
INS Bt

7 7 7 162 m

INS Bt ISO
Bt INS ISO INS Bt
8 8 8
5 4 3 2 1 ca. 500 m
Bearbeitunsrichtung

182 m ISO Bt INS Bt ISO

141 m
5 4 3 2 1
162 m 186 m
INS ISO Bt ISO INS

5 4 3 2 1

237 m

248 m
Slide courtesy of D. Bartsch
Lepidopteron Larvae Exposure to
Bt cry1Ab

Insect collection Species Identification

Slide courtesy of D. Bartsch
 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 2001
sample 1 sample 2
flowering of maize 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 Plutella

xylostella
Xanthorhoe flucata

Pieris rapae 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 expert
for the European Food Safety Authority
Gene flow from transgenic plants

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

Exposure Impact
Frequency Hazard
Consequence
• 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
Brassica napus – canola
contains Bt

Diamondback moth larvae.

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

Brassica rapa – wild turnip

wild relative
 Brassica relationships

Triangle of U
 Bt Brassica gene flow risk
assessment

• Is it needed?
• What kind of experiments?
• At what scale?
 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
 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

What would be a good F1 hybrid

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
First-tier Risk = Pr(GM spread) x Pr(harm|GM spread)
Exposure
Frequency
F1 BC1 Hybrids
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%
 Insect bioassay of hybrids
First-tier Risk = Pr(GM spread) x Pr(harm|GM spread)
Impact
Hazard
Consequence
DBM Bioassay of Hybrids

80

70

60
Percent Defoliation

50

40

30

20

10

0
I

I
58

45

I
P1

45 1
C

C
C

C
xP
xP
U

xU

xU
xU

xU

xU

xU
W

W
pa

pa

48
45
ra

63
48

52

96

4
ra

O
W

12
B.

W
O

O
B.

O
Line
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.
-herbivory +herbivory

TT CC
Wet biomass (g) Soybean biomass

CC CC CT CT TT TT
 Field level hybridization
Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread)
Exposure
Frequency
Field hybridization experiment
 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%

Maternal Parent
B. 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?