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 Initial Evaluation Problem Formulation Tiered Risk Assessment Controlled Experiments and Gathering of Information Risk Evaluation Regulatory requirements, scientific inquiry, and scientific responses to public concerns

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 resistance of the two-spotted spider mite to various chemicals 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 Tier 2: 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 Hypothesis testing Assess potential ecological and biosafety risks (must be environmentally benign) Determine performance under real agronomic conditions (economic benefits) Compared with the isogenic or parent variety, must perform as well or better (RR soybeans)

Case of the Monarch Butterfly 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. 20 May 1999 Nature © Macmillan Publishers Ltd 1999 Registered No. 785998 England. Slide courtesy of D. Bartsch

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

Diamondback Moth Plutella xylostella 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

2 ha 4 ha Field East Field West 500m 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 6 ISO 7 8 INS Bearbeitunsrichtung 178 m 162 m 141 m 186 m Bt 5 ISO 3 INS 4 2 1 Bearbeitunsrichtung 237 m 248 m 162 m 182 m ca. 500 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 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 Plutella xylostella Xanthorhoe flucata Pieris rapae Slide courtesy of D. Bartsch

Broad spectrum pesticides 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 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) Regulatory requirements, scientific inquiry, and scientific responses to public concerns

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 What would be a good hypothesis? Bt Canola Brassica rapa pollen What would be a good hypothesis? F1 hybrid

Halfhill et al. 2005, Molecular Ecology, 14, 3177–3189. 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 GT 1 69% 81% 38% 62% 34% 25% 41% 33% GT 2 63% 88% 77% 23% 35% 31% 30% GT 3 50% 65% 24% 10% 20% GT 4 56% 7% 36% 26% GT 5 75% 79% 39% 17% GT 6 54% 51% 12% 21% GT 7 19% GT 8 67% 22% GT 9 48% 27% 28% GFP 1 71% 18% 32% GFP 2 100% 86% 57% GFP 3 11% 15%

Insect bioassay of hybrids First-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Impact Hazard Consequence

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

Soybean biomass Wet biomass (g) CC CC CT CT TT TT

Field level hybridization Third-tier Risk = Pr(GM spread) x Pr(harm|GM spread) Exposure Frequency 43

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 34/56,845 0.060% 44/50,177 0.088% B. rapa total 78/107,022 0.073% Halfhill et al. 2004. Environmental Biosafety Research 3:73 45

Backcrossing conclusions Backcrossing occurs under field conditions Backcrossing rates to B. rapa are low (1 out of 1,400 seeds) 46

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?