1. NDSU Extension Analysis of Primary Biotechnology Literature Phil McClean Department of Plant Science North Dakota State University Biotechnology Education Forum NDSU Extension Inservice Training Bismarck, ND March 1, 2002

2. NDSU Extension Concerns for Biotech Products: Ecological Balance Will Be Altered Other organisms will be affected Monarch butterfly Maize Bt pollen falls on neighboring milkweed Milkweed a monarch butterfly food source Does the pollen affect the butterfly Losey et al. (1999) Nature 399:214 Yes!! Sears et al. (2001) PNAS 98:11937 No!!

3. NDSU Extension Concerns for Biotech Products: Inadvertent Gene Transfer Wild relatives receive transgene from biotech crops Important genetic diversity will be lost Has it been observed? Quist & Chapela (2001) Nature 414:541 Yes!! Has it been challenged? Christou (2002) Transgenic Research 11:iii Yes!!

4. NDSU Extension Transgenic DNA Introgressed Into Traditional Maize Landraces in Oaxaca, Mexico David Quist and Ignacio Chapela Nature 414:541-543 November 29, 2001 Let’s Review the Primary Literature

5. NDSU Extension What does the title tell us?

6. NDSU Extension What does the title tell us? Transgenes were found in landraces

7. NDSU Extension What does the title tell us? Transgenes were found in landraces What does this imply?

8. NDSU Extension What does the title tell us? Transgenes were found in landraces What does this imply? Biotech crops have contaminated nature

9. NDSU Extension Paragraph 1: What was analyzed?

10. NDSU Extension Paragraph 1: What was analyzed? Bulk seed from corn cobs from landraces Fields isolated from roads Six samples (A1-A3 and B1-B3) Store sample (K1) Controls Peruvian sample (P1) Oaxacan sample from 1971 (H1) Monsanto “Yieldgard” maize (Bt1) Monsanto “Roundup-Ready maize (RR1)

11. NDSU Extension Paragraph 1: How were samples analyzed?

12. NDSU Extension Paragraph 1: How were samples analyzed? DNA isolated from flour Scored for presence of 35S promoter Why??? Widely used transgene contruct element Polymerase chain reaction technique Presence of product = transgene promoter DNA is in sample

13. NDSU Extension Paragraph 2: What are the results?

14. NDSU Extension Paragraph 2: What are the results? Amplification observed in 5/7 samples Weak, but present Low copy # gave low signal Store sample gave strong signal Bt1 and RR1 gave strong signal Historical and Peruvian samples negative Positive control amplification observed in all samples

15. NDSU Extension Paragraph 2: Supporting evidence?

16. NDSU Extension Paragraph 2: Supporting evidence? Independent confirmation by Mexican gov’t Oaxaca and one other state Samples near authors site also positive at low levels Mexican experiment analyzed individual kernels Strong signal than pooled samples authors used

17. NDSU Extension Paragraph 3: Confirming Experiment?

18. NDSU Extension Paragraph 3: Confirming Experiment? Weak signal amplified with internal primers New fragment sequenced Sequence equal Monsanto 35S promoter sequence

19. NDSU Extension Paragraph 4: Other genes present?

20. NDSU Extension Paragraph 4: Other genes present? Samples asssayed for other transgenes NOS terminator sequence A3, B2 and K1 Bt toxin gene (cryIAb) B3 Conclusion: Multiple transgenes found in the Mexican landraces

21. NDSU Extension Paragraph 5: Clues to transgene origin?

22. NDSU Extension Paragraph 5: Clues to transgene origin? Samples assayed for genomic location of transgene Procedure: Inverse PCR Scores transgene and neighboring maize DNA Four samples contain known flanking genes A2, A3, B3, K1 Transgene located in same position as Novartis Bt11 Samples A3, K1 Conclusion: GMOs to landraces introgression occurring Despite GMO planting in Mexico (1998-now)

23. NDSU Extension The Challenge to Quist & Chapela Chistou (2002) Fundamental flaws (experimental design) in research Results can be explained by: Contaminated samples Flawed iPCR interpretation Cross pollination not supported by results Better experiment Grow plant samples out and: Score phenotype (Bt or Glyphosate tolerant) Score genotype (screening individual plants)

24. NDSU Extension What’s Next?? Funding becomes available Detailed experiments New results or interpretation What’s at stake? Integrity of biotech approaches to crop improvement