Today in AgBioView:
* Joint statement from Scientists?
* Environmentalist Biofraud?
* Editorial: No Credible Scientific Evidence is Presented to Support Claims that Transgenic DNA was Introgressed into Traditional Maize Landraces in Oaxaca, Mexico
From: "Alex Avery"
Date: 21 Feb 2002
Subject: Joint statement from Scientists?
Has anyone else picked up on the ìJoint Statement on the Mexican GM Maize Scandalî being whored around by the anti-biotech activists? (see foodfirst.org, for example) Theyíre running WAY SCARED now that is becoming clearer and clearer that the ìstudyî by Chapela and Quist published in Nature, supposedly finding bits of CaMV 35S promoter in traditional ìlandracesî of maize in Oaxaca, is junkscience that shouldnít have made it past a rudimentary peer review process. Already, there are several groups who have sent formal challenges to Nature about the C & Q results and methods, as well as the lack of controls and confirming studies.
For anyone who didnít catch Ron Baileyís great piece in Reason Magazine (http://reason.com/rb/rb021202.shtml) on the on-going saga, check it out, as it is a great synopsis of events up to this point. Bailey uses a scathing analysis of the C & Q work that was recently published in Transgenic Research. Essentially, all of the challenges charge that C & Q didnít know what they were doing when they used inverse polymerase chain reaction (iPCR) to attempt to find biotech gene sequences in farmer-bred varieties of corn from the Mexican highlands. It is now widely believed that C & Q found false positives from contamination or homologous native sequences.
The Joint Statement desperately attempts to shore up the credentials of Chapela and Quist by repeatedly calling them ìrespected scientists.î It should be noted though that Quist is still a student, although he has been a highly vocal activist in a campaign against a contract Berkeley has with Novartis, and, according to an article in the Chronicle of Higher Education (http://chronicle.com/free/v47/i41/41a02401.htm), he was accused by faculty and students in another department of destroying biotech crop research plots. Quist denied the charge and was never charged, but apparently the vandalism stopped after Quist was confronted.
Chapela is an activist assistant professor of microbiology and only recently learned the iPCR technique so he could do this one ìstudy.î He isnít a geneticist, but he is on the board of Pesticide Action Network North America (an anti-pesticide activist group) and in 1999 signed an anti-biotech statement calling for a global moratorium on GM crops. Theyíre both far from the ìrespected scientistsî that the Joint Statement claims. (Then again, they do their darndest to paint Arpad Putzai as a ìwidely respected scientistî in the statement, despite the drubbing Putzaiís research and methodology took from the Royal Society experts.)
With this new joint statement the antis are attempting some MAJOR pre-emptive damage control before Quist and Chapelaís study is thoroughly refuted. The level of rhetoric in the statement is absurd, claiming that any scientist who dares to call into question the results of Q&Cís so-called study are engaging in ìacademic intimidation.î Pro-industry scientists (isnít it interesting that anyone who disagrees with them is pro-industry?), they claim, ìare engaging in a highly unethical mud-slinging campaign against the Berkeley researchers.î
They are also attempting to drive CIMMYT away from biotech by painting the center as a tool of corporations. Itís all quite entertaining in some sad way.
Fellow scientists, perhaps we should get out front on this and post a ìjoint statementî from academics noting that this type of debate and truth-testing is not unethical mudslinging, but exactly the type of rigorous debate over the truth that is the hallmark of the scientific process and discourse? Any ideas would be greatly appreciated.
Alex Avery, Director of Research and Education
Center for Global Food Issues
A new report challenges research published in the respected journal, Nature.
By Ronald Bailey
"DNA Contamination Feared," declared the Washington Post last fall. "Gene-altered DNA may be 'polluting' corn," warned USA Today. Both papersóas well as many other media outlets around the world--were reporting the results of a scientific study published in the prestigious journal Nature. Anti-biotech activists at Greenpeace, Friends of the Earth, and the Union of Concerned Scientists immediately seized on the results to press for a ban on planting and exporting genetically enhanced crops. It now appears that that study's conclusions are completely bogus.
Last fall, Ignacio Chapela and David Quist, two researchers from the University of California, claimed that they had tested a number of samples of local "creole" corn taken from farms located in remote areas of Oaxaca, Mexico. They claimed that by using a very sensitive genetic test called inverse polymerase chain reaction (IPCR), they had found the cauliflower mosaic virus 35S (CaMV35S) promoter in the local varieties of corn. (Such local, noncommercial varieties are sometimes known as "landraces.")
CaMV35S is regularly incorporated in many genetically enhanced crops as a way to get the genes added by biotechnologists (transgenes), such as those for insect resistance, to express themselves. The Berkeley researchers suggested that perhaps native Mexican corn varieties had by chance crossbred with genetically enhanced varieties brought in from the United States, despite a Mexican government ban since 1998 on planting bioengineered corn. Perhaps some Mexican farmers had used corn imported for feed as seed instead, they speculated. "The probability is high that diversity is going to be crowded out by these genetic bullies," asserted Chapela in USA Today.
Although Ignacio Chapela is an assistant professor of microbiology at Berkeley, he is not exactly the model of a dispassionate scientist. For example, in 1999 Chapela signed the "World Scientists' Statement Calling for a Moratorium on GM Crops and Ban on Patents," organized by a variety of anti-biotech activist groups. The Statement called for a five-year ban on planting all genetically enhanced crops and a permanent ban on patenting crops, cell lines, and genes. Chapela is also a board member of the activist group Pesticide Action Network of North America, which is also campaigning against plant biotechnology. In other words, Chapela is a well-known anti-biotech activist.
Two questions arise from the Nature study: Is it true? And does it matter?
Earlier this year, the Center for the Improvement of Maize and Wheat (CIMMYT) in Mexico (the research center that launched the Green Revolution) checked its extensive collections of corn and could find no evidence that local varieties and bioengineered varieties had crossbred. At the time CIMMYT released the results of its analysis, its researchers had asked Chapela for samples of his materials so that they could check them, but he had not sent them.
Now a comprehensive review prepared for the editors of the journal Transgenic Research, which will be published in its February 2002 issue, finds that "no credible scientific evidence is presented in the [Chapela and Quist] paper to support claims made by the authors that gene flow between transgenic maize and traditional maize landraces has taken place." The review finds, "It is most likely that the report by Quist and Chapela is a testimony to technical failure and artifacts which are common with PCR and IPCR." Typically IPCR false positives occur because samples can be easily contaminated with the material being tested for.
Furthermore, according to the review, "most frustrating" is that Chapela and Quist did not use other, more reliable, techniques to determine the presence of the transgenes.
The Transgenic Review article also soundly condemns Nature for rushing to publish this flawed study. "What is very surprising, however, is that a manuscript with so many fundamental flaws was published in a scientific journal that normally has very stringent criteria for accepting manuscripts for publicationÖ.It is very disappointing that the editors of Nature did not insist on a level of scientific evidence that should have been easily accessible if the interpretations were true. Consequently, no evidence is presented to justify any of the conclusions presented in the paper."
So are genetically enhanced varieties "genetic bullies" that threaten corn biodiversity as Chapela claims? Not at all.
"There is no scientific basis for believing that out-crossing from biotech crops could endanger maize biodiversity," said Luis Herrera-Estrella in a statement issued in December. Herrera-Estrella is a noted plant scientist and director of the Center for Research and Advanced Studies in Irapuato, Mexico. "Gene flow between commercial and native varieties is a natural process that has been occurring for many decades. Nor is there reason to believe that these genes will become fixed into landraces unless farmers select them for their increased productivity," added Herrera-Estrella. "In the end, that would result in improving the native varieties."
What's the bottom line? Chapela's "research" is probably not a case of witting fraud, just another activist "scientist" finding what he desperately wanted to find. But the serious question is, Why did the editors of one of the world's leading scientific journals choose to abet him in his anti-biotech campaign by publishing his sloppy work? One has to wonder if perhaps their scientific judgments are being clouded by their ideological concerns. The least the editors of Nature can do now is withdraw the paper formally with apologies to the scientific community.
Ronald Bailey is Reason's science correspondent and the editor of Earth Report 2000: Revisiting the True State of the Planet(McGraw-Hill)
Editorial: No Credible Scientific Evidence is Presented to Support Claims that Transgenic DNA was Introgressed into Traditional Maize Landraces in Oaxaca, Mexico
Transgenic Research 11: iiiñv, 2002.
By Paul Christou (on behalf of the Editorial Board)
Because maize and its progenitor are wind-pollinated and capable of outcrossing, the eventual introgression of transgenes from commercial hybrids into landraces and wild relatives is likely should they be grown in close proximity. On 14th November 2001, a paper was published in the journal Nature which claimed for the first time to present evidence that transgene DNA had introgressed from commercially-released transgenic maize varieties into traditional landraces. It is not surprising that a scientific paper with such a strong claim in the title would be seized upon by the media and the public, including those who have been working with transgenic plants for many years. What is very surprising, however, is that a manuscript with so many fundamental flaws was published in a scientific journal that normally has very stringent criteria for accepting manuscripts for publication. Members of the Editorial Board of Transgenic Research, and a number of other scientists with many decades of experience in the area of transgenics, have provided comments that indeed demonstrate that the data presented in the published article are mere artifacts resulting from poor experimental design and practices. Consequently, this editorial focuses strictly on a purely scientific analysis of the data presented in the manuscript. We will not address implications or consequences if such an event had actually happened, as this is beyond the scope of this analysis. Our conclusion following detailed analysis of the results presented in this paper is that no credible scientific evidence is presented in the paper to support claims made by the authors that gene flow between transgenic maize and traditional maize landraces has taken place.
A careful analysis of the data presented in the paper strongly suggest the following:
* Sample contamination is the most likely explanation for the observed results.
* Rather than rely on questionable PCR results, plants that were alleged to contain introgressed DNA should have been grown out and subjected to more reliable confirming studies.
* The inverse PCR results are technically flawed.
* Cross pollination and introgression would not produce these results.
To avoid any confusion about what was being tested, it is important to clarify that this was maize, Zea mays, a landrace perhaps, but maize nonetheless, and not a progenitor species of maize. The experimental data Quist and Chapela cite as evidence for their conclusions is based solely on PCR and inverse PCR (IPCR). All those working with these two techniques know very well that they are prone to artifacts and we have all learned in the course of many years to exercise caution in designing and interpreting ex-periments based on methodology involving PCR and IPCR. All evidence in the present study is based on PCR for various sequences (the CaMV35S promoter, nos terminator and Bt cryIAb). Among these, only the CaMV35S sequence was analysed by both PCR and IPCR. It was necessary to use nested PCR (two con-secutive PCR reactions) to detect an obvious product. This is a particularly risky approach, since extremely low levels of contamination introduced during the handling of samples can be the cause of a positive result. The authors claim the Diconsa store sample gave a strong product in the first round of PCR but have cut out this track of the gel, without explanation. The Criollo samples represented a bulk of 150-400 grains. If the claimed presence of the transgene was due to pollination from commercial transgenic maize, one would not need PCR to prove it, given the level of 'contamination' in this relatively small number of ker-nels. An old-fashioned, but more reliable Southern blot would have sufficed, and would have provided more clear and unequivocal data. Indeed, the progeny of such pollinations would be very evident in terms of the dramatic change in the phenotype of the plant and cobs. Given that the authors relied on PCR, 40 PCR cycles would have been enough to amplify a single template molecule ñ a molecule which could have very easily been present because of contamination from a source other than plant DNA. The authors do not employ measures to eliminate any source of contamination, and therefore do not rule out the most likely explanation for the results they observed.
Further questions remain regarding the data actually presented. (1) Why did the Criollo maize samples A2, A3, B2 and B3 give weak signals while K1 gave a signal comparable to the Bt1 and RRI positive controls? Indeed, a close examination of Figure 1 shows that it does not support the statements made in the text, since the RR1 signal is strongest, and lanes for K1 and Bt1 have each been reconstructed. (2) Why are data from the historic control population never shown, when this is in fact the most appropriate control? The Mexican government results are stated without data or protocols and without citation. Given the uncertainties created by two consecutive PCRs and the likelihood of ground sample contamination, the authors should surely have grown out samples, even if more had to be collected, to prove beyond doubt that these were the result of cross-pollinations. None of this critical evidence is reported. If these really were cross-pollination events, it would be very easy to growplants and test for kanamycin resistance, BT protein expression, or glyphosate resistance. The plants certainly would not grow true-to-type for the landrace, as mentioned above. The interpretation of the inverse PCR results in this article are flawed as well. The short fragments that in some cases abut unidentified DNA are interpreted by the authors to be the result of rearrangement of the sequences either during transformation or by recombination (during introgression?).
Transformation is not a likely explanation, since the sequences in these transformation events are well-accounted for, and stray CaMV promoters just are not present in these events. So, where did these CaMV sequences come from? Most likely not from transgenic corn, but again most probably from contamination of the sample or some other technical artifact. The fact that the authors have not been able to show the presence of intact inserts, which are more likely to be present than fragments of unknown origin, casts further doubt that the results observed come from a transgenic plant source. Recombination is not a satisfactory explanation either, since multiple generations of crossing have been done with all these constructs, and they have been shown to be stable ñ or else they would not have made it through the regulatory system. It is highly improbable that these genes would experience a high degree of rearrangement upon crossing into a Criollo maize background. Finally, the authors show that not all CaMV positive samples were positive for the nos terminator. The absence of the nos terminator in some samples further casts doubt on the presence of intact, functional genes, which would have been expected if they were of transgenic plant origin. Furthermore, the lack of intact functional genes means that speculation about any effects would be scientifically unwarranted. Introgression through pollen is most likely to bring in the complete cassette (promoter-coding region-terminator) along with any flanking plasmid DNA that integrated into the original transgenic line. The IPCR products would, therefore, yield a sequence comprising partly of the gene and partly of the vector in almost all the cases. In two cases where the authors detected an adh1 gene sequence flanking the CaMV 35S promoter, IPCR reveals that the promoter is located within the adh1 gene sequence and not at one end as in the original construct. This calls for a most unlikely recombination event. There is no indication that the adh1 gene sequence was exactly that used in the construct. Adh is a complex gene family with a number of introns and exons. The authors did not investigate if this was an endogenous maize sequence, rather than the one normally used in trans-formation constructs. In fact, when the sequence they submitted (AF434757) is compared against the maize genome, it has 89% plus/ plus identity in 91 bases and 86% plus/ plus identity in 107 bases to the maize chro-mosome 9s bz locus. It also has 84% plus/ plus identity in 108 bases to the 22-kD alpha zein. It, of course, has an 86% plus/ minus identity in 89 bases to the adh1 gene. In all three cases approximately the same region of their submitted sequences is considered (ca 26ñ 130). The second 'adh1' gene sequence flanking the CaMV 35S promoter (AF434755) is very similar if not identical, and is homologous to the bz and zein sequences. It is not clear why the authors chose to report homology to the adh1 gene and not to the bz locus sequence or to alpha zein.
The inverse PCR result is said to show diverse adjacent sequence and the authors claim this implies insertion of the 35S sequence into multiple loci in the Criollo samples. In fact, this is not what would be expected from pollination from commercial transgenic maize. The likelihood of recombination very close the 35S promoter is infinitesimally small. The authors interpret their data as indicating that introgression events are relatively common. However, these results actually indicate technical failure in the authors' experiments. There are few transgenic events in commercial maize and all the inverse PCR results would be expected to conform to one or other of these.
While some degree of sequence rearrangement is expected, the sequences obtained by PCR are rather strange: none of the sequences contains an EcoRV site (which would be expected from the religation event). Why are the amplified elements for A3 different/ longer than those for K1? That requires additional point mutations (to delete or create EcoRV sites) or further unexplained scrambling of sequences. The sequences that have been pulled out would generally be expected to contain vector sequence elements (for iCMV1 and iCMV2 amplicons) or transgene sequences (iCMV3 and iCMV4 amplicons) rather than zein, transposon and other unrelated sequences. Most strikingly, the sequences between the primer iCMV1 and the putative location of the EcoRV site are not well conserved at all! This implies that the original sequence between the 3 end of iCMV1 and the EcoRV site is lost/ altered in all cases, immediately after the primer.
It is most likely that the report by Quist and Chapela is a testimony to technical failure and artifacts which are common with PCR and IPCR. The PCR results are likely due to minute contamination of the ground sample powders. The inverse PCR results are problematic, internally inconsistent and not what is expected from cross-pollination by commercial transgenic maize. Most frustrating is the total failure of the authors to do the easy and incontrovertible ex-periment of growing out the suspected contaminated lines. Hybrids between Mexican landraces and trans-genic commercial maize would be very obvious. It is disappointing that the editors of Nature did not insist on a level of scientific evidence that should have been easily accessible if the interpretations were true. Consequently, no evidence is presented to justify any of the conclusions presented in the paper.
Reference Quist D and Chapela IH (2001) Transgenic DNA introgressed into traditional maize landraces in Oaxaca, Mexico.