Transcript Field Testing of Transgenic Plants

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
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)
Tier 2:
Semi-Field/Greenhouse
Tier 3: Field Studies
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
www.agf.gov.bc.ca/.../images/diamondback3.jpg
Cabbage Moth
Pieris rapae
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
INS = Isogenic variety with insecticide treatment
ISO = Isogenic variety, no insecticide treatment (Control)
Bt
INS
ISO
INS
Bt
5
4
3
2
1
ISO
Bt
INS
Bt
ISO
5
4
3
2
1
INS
ISO
Bt
ISO
INS
5
4
3
2
1
Bt
ISO
INS
6
6
6
ISO
INS
Bt
7
7
7
INS
Bt
ISO
8
8
8
Bearbeitunsrichtung
178 m
162 m
ca. 500 m
Bearbeitunsrichtung
182 m
141 m
162 m
186 m
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
26. 27.
28
sample 1
August
29. 30. 31. 01. 02. 03. 04. 05. 06. 07. 08. 09. 10. 11. 12. 13. 14. 15.
sample 2
2001
flowering of maize
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.
2.
3.
4.
5.
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)
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
Frequency
Impact
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
hypothesis?
F1 hybrid
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
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%
Insect bioassay of hybrids
Risk = Pr(GM spread) x Pr(harm|GM spread)
Impact
Hazard
Consequence
DBM Bioassay of Hybrids
80
70
60
50
40
30
20
10
xP
W
45 1
xU
C
I
O
48
xP
O
1
48
xU
C
O
I
52
xU
C
W
I
63
xU
C
O
I
96
xU
O
C
12
I
4
xU
C
I
W
45
W
45
W
58
C
U
pa
ra
B.
ra
pa
P1
I
0
B.
Percent Defoliation
First-tier
Line
Greenhouse Bt “superweed” experiment
Second-tier



S
C
BT
Risk = Pr(GM spread) x Pr(harm|GM spread)
Impact
Hazard
Consequence
Soybean
Brassica rapa
BC3 Bt transgenic Brassica rapa
Assess transgenic weediness potential by
assaying crop yield.
-herbivory
TT
+herbivory
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%
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%
Maternal Parent
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?