Transcript Molecular genetics of gene expression

Lectures 20-21, Chapters 12-13
Regulations and risk assessment
Neal Stewart
Discussion questions
1. What are regulations supposed to achieve?
2. With GM crops being used so extensively, how are we
assured of their health and environmental safety?
3. How is genetic engineering (biotechnology) regulated?
4. When is plant genetic engineering not regulated?
5. How do the risks posed by products of biotechnology
compare to those posed by conventional
technologies?
6. How do different countries regulate products of
biotechnology?
Plant genetic modification
Any gene, any organism
The new plant will pass the transgene
to its progeny through seed.
Recall… progression of transgenic
plants
• Input traits– commercialized fast from
1996
• Output traits—commercialized slowly from
early 2000s
• Third generation– pharma, oral vaccines,
phytoremediation, phytosensors—
emerging gradually. How might regulating
these be more challenging.
Bt maize
Bt cotton
Golden rice
Engineered to deliver pro-vitamin A
GFP canola
Plants to detect landmines
No TNT
induction
Using inducible
promoter/GFP
fusions
+TNT
Agriculture and Nature
•
•
•
•
Are farms part of nature?
Of the environment?
Direct or indirectly?
Impacts on nature and agriculture might
be inter-related but the endpoints will be
different
Big picture—ecological impacts of
agriculture
• Major constraint is
agriculture itself
• Tillage and pesticide
practices
• Crop genetics (of
any sort) is
miniscule
ag v wild
tillage
pesticides
herbicides
crop
genetics
Amount of genetic information
less
added to ecosystems
Tran
s
gene
Conventiona
s
l breeding
Mutagenesi
s
Half genomes,
e.g., wide
crosses in
hybrids
Whole genomes, e.g.,
horticultural introductions
or biological control
Risk??
more
Figure 12.1
Domestication of corn
9000
years ago?
Teosinte
Corn
Domestication of carrot
Daucus carota
300 to 1000
years ago?
Queen Anne’s Lace
1700s
orange carrots
appear in Holland
Brassica
oleracea
Wild
cabbage
Ornamental kale
Late 1900s
Kohlrabi
Germany 100 AD
Cauliflower 1400s
Kale 500 BC
Broccoli Italy 1500s
Cabbage 100 AD
Brussel sprouts
Belgium 1700s
Regulations
What/why regulate
• Biosafety– human and environmental
welfare
• Recombinant DNA (rDNA) triggers
regulation in most countries
• Transgenic plants and their products are
pound for pound the most regulated
organisms on earth
• “Protect” organic agriculture
• “Precautionary principle”
US history of regulating
biotechnology
• Early 1970s recombinant organisms are
possible (microbes)—plants in 1980s
• Asilomar conference 1975
• NIH Guidelines 1976—regulating lab use
• OSTP Coordinated Framework—1986
• Set up the USDA, EPA and FDA to
regulate aspects of transgenic plants
Regulatory agencies provide safeguards and requirements to assure safety—
determination and mitigation of risks.
Roles of agencies in US regulation of
transgenic plants
• USDA: Gene flow, agronomic effects
• EPA: Gene flow, environmental/nontarget, toxicity when plants harbor
transgenes for pest control
• FDA: human toxicity/allergenicity
Ecological Risk Assessment of
Transgenic Plants
Problem formulation—assessment and
measurement endpoints
exposure assessment
hazard assessment
Objectives
At the end of this lecture
students should…
• Understand framework for assessing risks
• Be able to define short-term and long-term
risks for a transgenic plant application—i.e.,
define ecological endpoints
• Understand exposure assessment and
hazard assessments for today’s GM plants
• Critically think about exposure and hazard
assessments for upcoming GM plants
Methods of risk analysis
• Experimental approach (toxicology or
ecology)
– Controlled experiments with hypothesis
testing
– Cause and effect
• Theoretical modeling
• Epidemiological approach—association of
effects with potential causes
• Expert opinion
Adapted from 2002 NRC report: Environmental Effects of Transgenic Plants
Risk
Likelihood of harm to be manifested
under environmentally relevant
conditions
Joint probability of exposure and effect
Qualitative is more reasonable than
quantitative
Risk analysis
Johnson et al. 2007 Trends Plant Sci 12:1360
Ecological Risks
Risk = exposure x hazard
Risk = Pr(event) x Pr(harm|event)
• The example gene flow
• Exposure = probability hybridization
• Hazard = consequences of ecological or
agricultural change--severity of negative
impact
Ecological Risks
Risk = exposure x hazard
Risk = Pr(event) x Pr(harm|event)
• Transgene persistence in the environment–
gene flow
– Increased weediness
– Increased invasiveness
• Non-target effects– killing the good insects by
accident
• Resistance management– insects and weeds
• Virus recombination
• Horizontal gene flow
Public perception: Risk = visibility x hysteria
Stated another way and with terms:
Risk = Pr(GM spread) x Pr(harm|GM spread)
Exposure
Frequency
Impact
Hazard
Consequence
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!
Example endpoints
• H, insect death: toxicology of insect
resistance genes
• E, hybridization frequency: gene flow
What are some ideal features of end points?
Risk analysis
Johnson et al. 2007 Trends Plant Sci 12:1360
Balancing exposure and hazard
• R = E x H: an example from the
world of gene flow
• R= E x H: an example from the
world on non-targets
Johnson et al. 2007 Trends Plant Sci 12:1360
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?
Tiered approach—mainly nontargets
Wilkinson et al. 2003 Trends Plant Sci 8: 208
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
Brassica napus, hybrid, 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%
Gene flow model with insecticidal
gene
Wilkinson et al. 2003 Trends Plant Sci 8: 208
In the UK, Wilkinson and
colleagues predict each
year…
•32,000 B. napus x B. rapa waterside
populations hybrids are produced
•16,000 B. napus x B. rapa dry
populations hybrids are produced
But where are the backcrossed hybrids?
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
Genetic Load
 Negative effects of genetic load may hinder a
hybrid’s ability to compete and survive
 Negative epistatic effects of genetic load could
trump any fitness benefits conferred by a fitness
enhancing transgene
GM Crop
Weed
F1 Hybrid
Weed
BCX weed
Field level hybridization
Third-tier
Risk = Pr(GM spread) x Pr(harm|GM spread)
Exposure
Frequency
percentage of B. napus-specific markers
Genetic introgression
Bn
F1
100
BC1F1
BC2F1
BC2F2 Bulk
75
50
25
0
CA x GT1
2974 x GT1
2974 x GT8
Halfhill et al. 2003 Theor Appl Genet 107:1533
AFLPs
Generating transgenic “weeds”
testing the consequences
´
Brassica rapa
(AA, 2n=20)
B. rapa
B. rapa
´
´
Brassica napus
(AACC, 2n=38)
F1 Generation
(AAC, 2n=29)
BC1F1 Generation
(AAc, 2n=20 + 1 or 2)
BC2F1 Generation
(AA, 2n=20)
´
BC2F1 Generation
(AA, 2n=20)
BC2F2 Generation
(AA, 2n=20)
Competition field design
Competition results
b
a
a
150
NC
a
750
600
120
b
Wheat seed mass per m2 (g)
90
c
c
c
c
450
c
c
60
B. rapa
BC2F2
Bt
BC2F2
GT1
c
Wheat
Only
a
180
B. rapa
BC2F2
Bt
BC2F2
GT1
d
Wheat
Only
500
a
ab
GA
ab
400
150
bc
c
120
B. rapa
BC2F2
Bt
BC2F2
bc
c
bc
c
GT1
Wheat
Only
B. rapa
BC2F2
Bt
BC2F2
GT1
Halfhill et al 2005 Mol Ecol 14:3177
300
Wheat
Only
Wheat vegetative dry weight per m2 (g)
b
BMC
Biotechnol
2009
9:83
Figure 1. Genetic Load Study: Productivity. Average vegetative dry weight and seed
yield (2e +4 = 20,000 seeds, 1e + 5 = 100,000 seeds, etc.) of non-transgenic Brassica
napus (BN), Brassica rapa (BR) and transgenic BC1/F2 hybrid lines (GT1, GT5 and GT9)
grown under non-competitive (A and C) and competitive field conditions (B and D).
Columns with the same letter do not differ statistically (P < 0.0001). Error bars represent ±
standard error of the means. Note that different Y-axis scales are used among figure
panels.
Discussion question
•Which is more important: that a field test be
performed for grain yield or environmental
biosafety?
Monarch butterfly exposure to
Bt cry1Ac
Monarch butterfly
What’s riskier?
Broad
spectrum
pesticides
or
non-target
effects?
In October 2001 PNAS– 6 papers delineated the risk for monarchs.
Exposure assumptions made by Losey were far off.
Tiered approach—mainly nontargets
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)
Tiers of assessment &
tiers of testing
 level of concern
 degree of uncertainty
… arising from a lower tier of
assessment drives the need to
move toward a higher tier of data
generation and assessment
Tier IV
Tier III
Tier II
Tier I
Jeff Wolt
Lab
Microbial protein
High dose
Lab
PIP diet
Expected
dose
Long-term Lab
Semi-field
Field
Assessment
Testing
Non-target insect model
Wilkinson et al. 2003
Trends Plant Sci 8:
208
Examples…identifying
Endpoints for Risks, Exposure, Hazards
• Plant system (crop, weeds, communities,
etc)
• Phenotype
• Biotic interactions
• Abiotic interactions
Class to give examples—discussion—setting
up experiments
Expert knowledge is important
• Biotechnology
– Transformation methods
– Transgene
– Regulation of expression
• Ecology
–
–
–
–
–
–
Plant
Insect
Microbial
Populations
Communities
Ecosystems
• Agriculture
– Agronomy
– Entomology
• Regulator acceptance
– Developed world
– Developing world
• Public acceptance
– Finland and EU
– Where GM crops are
widely grown
– New markets
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.
Risk assessment links
research to risk management
Data Acquisition, Verification, & Monitoring
Risk Assessment
Problem
Formulation
Jeff Wolt
Exposure & effects
characterization
Risk Management
Risk
Characterization
An example of agricultural risk
that is not regulated
The evolution of weed resistance
to herbicides
Conyza canadensis
• Marestail or horseweed—found widely
throughout North America and the world
• Compositae
• First eudicot to evolve glyphosate resistance
• Resistant biotypes appeared in 2000,
Delaware—resistant Conyza in 20+ US states
and four continents, e.g. in countries such as
Brazil, China, and Poland
• 2N = 18; true diploid; selfer
Spread of glyphosate resistance
in Conyza
Fig. 1. The proportion of soybean acreage sprayed with glyphosate from 1991 to 2002 relative to other
herbicides
Baucom, Regina S. and Mauricio, Rodney (2004) Proc. Natl. Acad. Sci. USA 101, 13386-13390
Copyright ©2004 by the National Academy of Sciences
Resistant
biotype 1
14 DAT
rate in
lbs ae/Ac
Susceptible
biotype
C.L. Main
UTC
0.38
0.75
1.12
1.5
2.25
3
8
RR weed risk assessment research
•
•
•
•
Is it needed?
What kind of experiments?
At what scale?
Other weeds?
Environmental benefits of
transgenic plants
Big environmental benefits
Herbicide tolerant crops have increased and
encouraged no-till agriculture– less soil erosion.
Over 1 million gallons of unsprayed insecticide
per year.
When transgenic plants are
not regulated
The case of the ancient
regulations
USDA APHIS BRS
7 CFR Part 340.0 Restrictions on the Introduction of Regulated Articles
(a) No person shall introduce any regulated article unless
the Administrator is:
(1) Notified of the introduction in accordance with 340.3,
or such introduction is authorized by permit in accordance with
340.4, or such introduction is conditionally exempt from permit
requirements under 340.2(b); and
(2) Such introduction is in conformity with all other applicable
restrictions in this part. 1
1 Part 340 regulates, among other things, the introduction
of organisms and products altered or produced through
genetic engineering which are plant pests or which there
is reason to believe are plant pests. The introduction
into the United States of such articles may be subject
to other regulations promulgated under the Federal Plant
Pest Act (7 U.S.C. 150aa et seq.), the Plant Quarantine
Act (7 U.S.C. 151 et seq.) and the Federal Noxious Weed
Act (7 U.S.C. 2801 et seq.) and found in 7 CFR parts
319, 321, 330, and 360.
Transgenic plants would be regulated by the
USDA if they contain some of these vectors
Not regulated by USDA
http://www.aphis.usda.gov/biotechnology/downloads/reg_loi/Ceres_switchgrass_TRG108E_loi.pdf
http://www.aphis.usda.gov/biotechnology/downloads/reg_loi/Ceres_switchgrass_responses.pdf
What factors should trigger
regulation?