Field Testing of Transgenic Plants PS 353: Plant Genetics, Breeding and Biotechnology April 8, 2008 www.pictopia.com
Dec 14, 2015
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?