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Analysis of Primary Biotechnology Literature Biotechnology Education Forum NDSU Extension Inservice Training Bismarck, ND March 1, 2002 Phil McClean Department of Plant Science North Dakota State University NDSU Extension Concerns for Biotech Products: Ecological Balance Will Be Altered NDSU 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!! 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!! NDSU Extension Let’s Review the Primary Literature Transgenic DNA Introgressed Into Traditional Maize Landraces in Oaxaca, Mexico David Quist and Ignacio Chapela Nature 414:541-543 November 29, 2001 NDSU Extension What does the title tell us? NDSU Extension What does the title tell us? Transgenes were found in landraces NDSU Extension What does the title tell us? Transgenes were found in landraces What does this imply? NDSU Extension What does the title tell us? Transgenes were found in landraces What does this imply? Biotech crops have contaminated nature NDSU Extension Paragraph 1: What was analyzed? NDSU Extension Paragraph 1: What was analyzed? NDSU 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) Extension Paragraph 1: How were samples analyzed? 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 NDSU Extension Paragraph 2: What are the results? 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 NDSU Extension Paragraph 2: Supporting evidence? 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 NDSU Extension Paragraph 3: Confirming Experiment? NDSU Extension Paragraph 3: Confirming Experiment? Weak signal amplified with internal primers New fragment sequenced Sequence equal Monsanto 35S promoter sequence NDSU Extension Paragraph 4: Other genes present? 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 NDSU Extension Paragraph 5: Clues to transgene origin? NDSU Extension Paragraph 5: Clues to transgene origin? NDSU 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) Extension The Challenge to Quist & Chapela NDSU 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) Extension What’s Next?? Funding becomes available •Detailed experiments •New results or interpretation What’s at stake? Integrity of biotech approaches to crop improvement NDSU Extension