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October 2003 Updates

Transgenic Lines Proven Unstable

The insert in every commercially approved GM line has undergone rearrangement. The cauliflower mosaic virus promoter plays a major role. This should be the final nail in the coffin for GM crops, says Dr. Mae-Wan Ho, who has, for years, challenged scientific committees advising governments over this very issue.

The insert in every commercially approved GM line has undergone rearrangement. The cauliflower mosaic virus promoter plays a major role. This should be the final nail in the coffin for GM crops, says Dr. Mae-Wan Ho, who has, for years, challenged scientific committees advising governments over this very issue.

There is plenty of evidence that transgenic lines are unstable, which is why ISIS has long recommended that appropriate molecular methods must be used to document the stability of the GM insert before any transgenic line is released into the environment. The characterization of the insert must be "event-specific", which not only gives the structure of the insert, but also the host genome sequences flanking the insert, proving that the insert remains stable in successive generations. This recommendation has been incorporated into the current European Directive (2001/18 /EC) on deliberate release of GMOs.

Petri dish sprouts

But to this day, pro-GM scientists advising the UK and other governments have refused to acknowledge the evidence on transgenic instability, and worse. In its latest reply to ISIS, the UK Advisory Committee on Releases to the Environment (ACRE) has gone as far as to say that event-specific molecular characterization is not necessary, thus going against the European Directive (see ISIS' final response to ACRE: Let the people decide).

ISIS has reiterated 5 experiments which should be done to address the 'areas of uncertainty', one of which calls for full event-specific molecular characterization of all transgenic lines to establish uniformity and genetic stability of the transgenic DNA insert(s), and "comparison with the original data supplied by the biotech company to gain approval for field trials or for commercial release."

I am pleased to report that some effort has recently been made to do such experiments by French scientists from the Laboratory of Methods for Detecting GMOs in Versaille, and the Laboratory of Biometry and Artificial Intelligence, Domaine de Vilvert in Jouy-en-Josas. And they have presented their results in a poster at a conference in June 2003 [1].

The scientists recognized that, as labeling laws and thresholds are established for foods containing GMOs in Europe, Japan, Australia, New Zealand and elsewhere, "reliable GMO identification and quantification methods are needed to comply with the regulations." And "in order for these tests to be specific, the sequence and detailed characterization of the GMO inserts and their edges are required."

Five different commercially approved GMOs in Europe were analyzed: three from Monsanto, one from Bayer and one from Syngenta. All inserts were rearranged from their intended gene order. Moreover, all five inserts showed further rearrangements from the original structure submitted by the companies. In other words, either the companies were mistaken about the original structure, or more likely, further rearrangements had occurred after the crops had been commercially grown. The details are given in Box 1.

 
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Transgenic Lines Proven Unstable (continued)

Box 1

Scrambling and further scrambling of GM inserts

T25 maize LibertyLink (Bayer)

Modified for tolerance to herbicide glufosinate. Company data showed insert includes a truncated ampicillin resistance bla gene in the plasmid vector pUC18, a CaMV 35S promoter (hereafter referred to as P35S) driving a synthetic pat gene (glufosinate tolerance) terminated by CaMV 35S terminator (hereafter referred to as T-35S). On analysis, the insert was found to have undergone further rearrangement, so that a second, truncated and rearranged P35S has been joined to the 5' (left, or head) end of the insert, while additional pUC18 sequences were found at the 3' (right, or tail) end.

Edges flanking the insert show homologies (similarities) with Huck retrotransposons (a class of mobile genetic elements) in the maize genome.

Mon 810 maize YieldGard (Monsanto)

Modified for resistance to lepidopteran insects (butterflies & moths). Company data showed insert has a P35S driving a CrylAb synthetic gene with terminator T-nos. Analysis revealed however, that T-nos and part of the 3' (tail) end of the CrylAb gene have been deleted. T-nos has been detected elsewhere in the genome, indicating that it has moved from its original position.

The 5' (head) end of the insertion site shows homology to the long terminal repeats (LTR) of the maize alpha Zein gene cluster, but no homology to the maize genome was detected at the 3' site, indicating that there has been scrambling of the maize genome at the insertion site.

GTS 40-3-2 soybean (Monsanto)

Modified for tolerance to herbicide glyphosate (Roundup Ready). Company data showed insert with P35S driving a composite gene containing the N-terminal chloroplast transit peptide (CPT4) joined to modified epsps gene with T-nos terminator.Analysis revealed that a 254bp piece of DNA homologous to the epsps gene and 534bp of unknown DNA have been joined to the 3'end of the insert.

It was not possible to identify the insertion site at all, indicating substantial genome scrambling or deletion at the insertion site.

Bt 176 maize (Syngenta)

Modified for tolerance to herbicide glufosinate, male sterility and insect resistance. The structures of two inserts, originating from two GM constructs, were provided by the company. Only the simpler construct was analyzed. Company data showed insert contains P35S driving the bar gene (glufosinate tolerance) terminated by T35S, followed by the ampicillin resistance (bla) gene plus bacterial promoter, and plasmid origin of replication, ori. Analysis revealed several fragments, all containing CaMV 35S promoter, one with P35S joined to T35S, a second with P35S joined to an unknown sequence, and a third with P35S joined to the bar gene with the T35S deleted.

There were at least three insertion sites.

GA 21 maize (Monsanto)

Modified for tolerance to herbicide glyphosate (Roundup Ready). Company data indicated insert contains multiple copies of the cassette with the rice actin gene promoter (P-ract) driving the composite gene containing the N-terminal chloroplast transit peptide (CPT4) joined to modified epsps gene and T-nos. There were three complete cassettes flanked by a cassette with P-ract partially deleted at the 5' end, and one cassette with 3' deletion of epsps plus a lone P-ract at the 3' end. Analysis found partial deletion of P-ract and deletion of T-nos in two different cassettes.

The insertion site at the 3' end is flanked by sequences of pol polyprotein gene belonging to a PREM2-retrotransposon.

The results revealed that:

  • All GMO inserts had rearranged from the structure provided by the company.
  • Many of the breakpoints for rearrangement involve the CaMV 35S promoter, as can be predicted from its known recombination hotspot.
  • Scrambling of the genome at the site of insertion occurred in at least two out of five inserts.
  • GMO inserts appear to show a preference for mobile genetic elements (retrotransposons), with Long Terminal Repeats containing strong promoters, which would result in "altered spatial and temporal expression patterns of genes" nearby. In addition, it increases the chances that the inserts will move with the retrotransposons, resulting in further genome scrambling and horizontal gene transfer.

With considerable irony, whether intended or not is unclear, the authors conclude: "Studying GMO's structure is necessary to develop reliable quantification and detection tests complying with the different regulations, but it also leads [one] to ask fundamental questions about genome fluidity. Many of the mechanisms involved in recombinant DNA integration are similar to those underlying genome evolution. Therefore, characterized GMO inserts are a very good model to study the molecular system involved in DNA rearrangements in general."

1. Collonier C, Berthier G, Boyer F, Duplan M-N, Fernandez S, Kebdani N, Kobilinsky A, Romanuk M, Bertheau Y. Characterization of commercial GMO inserts: a source of useful material to study genome fluidity. Poster courtesy of Pr. Gilles-Eric Seralini, PrŽsident du Conseil Scientifique du CRII-GEN, www.crii-gen.org

http://www.i-sis.org.uk/TLPU.php

 

When Genes Escape
Does it matter to crops and weeds?

Susan Milius
Science News
Week of Oct. 11, 2003; Vol. 164, No. 15

This may not sound like boffo material, but genetic–engineering-policy specialist Michael Rodemeyer knows his crowd. "As I was coming out here, I thought about making bumper stickers that say, 'Gene flow happens.'" The line gets a good laugh; after all, Rodemeyer, a director of the Pew Initiative on Food and Biotechnology in Washington, D.C., is addressing a roomful of botanists. They routinely think about genes moving from plant to plant, and they get his reference to worries that engineered genes will jump from a crop to a wild cousin and create a real Godzilla of a weed.

Judging by the questions they ask and the eyebrows they arch, the folks at the Botany 2003 meeting in Mobile, Ala., in late July hold a range of attitudes about genetically engineered crops. Yet just about everyone laughs with Rodemeyer.

The discussion of gene flow has changed in the past decade. The question is no longer, Can genes move? By now, scientists have tested some of the basic scenarios and reported their observations. The current consensus is that genes certainly can flow, says Allison Snow of Ohio State University in Columbus. Her tests and others' have shown that much. "The important question now is, 'What are the consequences?'" she says.

Researchers are starting to examine that question. The answers may strongly influence the future of genetic engineering in agriculture.

Roots

When bioengineers first inserted foreign genes, or transgenes, into plants in the 1980s, the scientists generally expected crop-to-wild hybridizations to be only "rare and idiosyncratic," says Norman Ellstrand of the University of California, Riverside. However, interest in how cultivated plants consort with wildlings had started long before genetic engineering was even a glimmer in a test tube.

Ellstrand, a dedicated investigator of gene-flow questions, points out an 1886 treatise on domesticated plants that mentions their capacity for mating with wild relatives. Even the term superweed goes back at least to 1949, in a book on hybridizing plant species. The author raised the possibility that a traditional farm plant's wild side pairings might yield especially tough but undesirable offspring.

In a few cases, scientists have traced a trait moving from a conventional crop into the wild. For example, Ellstrand notes a 1959 report of a brainstorm that fizzled in India. Agriculturists encouraged farmers to plant a rice variety with red seedlings, easy to distinguish from a pale, weedy form that farmers had been removing from their paddies. The venture failed when the red color quickly migrated into the weed.

Scientists continue to examine conventional crops to gain insight into what genetic engineering might yield. For example, in 1998, Randal Linder of the University of Texas at Austin and his colleagues, including Snow, reported their study of wild sunflower patches that had grown near farmed sunflowers for up to 40 years. All of the 115 wild plants that the researchers tested carried at least one genetic marker characteristic of the commercial plants.

Tracking a rare genetic marker from a conventional alfalfa crop, Paul St. Amand of Kansas State University in Manhattan and his colleagues have documented the gene in stray plants outside farm fields. In some cases, the gene turned up as far as 230 meters away. "Data suggest that complete containment of transgenes within alfalfa-seed– or hay-production fields would be highly unlikely using current production practices," the researchers commented in their 2000 paper.

Ellstrand has built the case that opportunities abound for crossings of crops and weeds. In 1999, he reviewed the world's top 13 crops for human consumption (ranked by area harvested) and found reports that 12 crops hybridize with a wild relative somewhere in their range. Wheat, for example, has given rise to at least 21 natural hybrids, and certain crop-weed crosses of rice have yielded unusually fertile offspring. The exception was peanut plants, which typically self-fertilize.

Less-prominent crops, too, often mate with their wild relatives, Ellstrand says. He's added 31 plants, including grapes, avocados, lettuce, coffee, chocolate, and watermelons, to his list of crops that in some part of the world have hybridized with a wild mate.

Loose genes

The movement of genes from engineered plants has triggered more concern than gene flow from conventional crops ever did. Genetic engineering enables scientists to transplant a much wider range of genes than is available through traditional breeding.

Some experiments have observed neighboring barley picking up genes introduced into crops by genetic engineering. A marker from transgenic barley, for instance, traveled to up to 7 percent of conventional barley plants nearby that don't produce competing pollen. However, rogue pollination dwindled rapidly in frequency the farther researchers got from the source plants. Anneli Ritala of VTT Biotechnology in Espoo, Finland, reported in the January–February 2002 Crop Science.

Perhaps the most famous studies of transgene escapes aren't intentional experiments at all. For example, Mexicans are watching their traditional maize versions, or landraces, to see whether they'll pick up genes from the abundant U.S. crops of transgenic corn. Mexico itself has banned the growing of transgenic corn.

The ancestral home of corn lies in Mexico, where rich variety in the old landraces persists. Even today, the original lineage of crop corn survives in a lanky grass called teosinte, which has tiny stubs of seeds that only a botanist could love.

In 2001, California biologists reported traces of transgenes in landraces (SN: 12/1/01, p. 342: Available to subscribers at http://www.sciencenews.org/20011201/fob5.asp). Other researchers challenged some of the findings as artifacts of the genetic techniques, and Nature eventually took the unusual step of saying there hadn't been enough evidence to justify its publishing the paper
(SN: 4/13/02, p. 237: Available to subscribers at http://www.sciencenews.org/20020413/note12.asp).

Now, other labs have found signs of transgenes in maize landraces in Mexico. Sol Ortiz-García of the Ministry of Environmental and Natural Resources in Mexico City described the findings of two research teams at the July botany meeting. Farmers who bought U.S. corn as animal feed may have tried growing some of it, or the feed corn may have sprouted spontaneously.

The teams are gathering further data to confirm the presence of the transgenes, but Snow says, "I believe it."

Canadian scientists have described transgene movement from a different crop. Farmers grow canola for the oil in its seeds, and controlling weeds in the fields had ranked high among canola-grower headaches. Starting in 1996, strains genetically engineered to withstand treatment by one of two herbicides have become popular in Canada. These strains could then be doused with pesticide powerful enough to wipe out troublesome weeds. About 70 percent of the country's crop carry a transgene to aid in weed control.

Those transgenic plants are hybridizing with Brassica rapa, one of the weedy parents of crop canola, according to Suzanne Warwick at Agriculture Canada in Ottawa. She and her colleagues documented the first crop-wild hybrid from a regular commercial field in the August 2003 Theoretical and Applied Genetics.

Transgenes also move from one type of crop canola to another. A canola field planted with one variety sprouted hybrid volunteers that combined the herbicide resistances of their parents, Linda Hall of Agriculture Canada in Edmonton, Alberta, and her colleagues reported in 2001.

The canola-transgene movement can complicate life for farmers, Hall says. Canola seeds that stay in the ground after the farmer has rotated crops can pop up as weeds in a wheat or barley field. If those volunteers have picked up unexpected herbicide resistance, the farmer's herbicide regimen may be insufficient.

Bottom line

Is the canola-gene flow a lot or a little? It doesn't matter, says John Burke, now at Vanderbilt University in Nashville. In 2001, he and Loren Rieseberg of Indiana University in Bloomington published an analysis of what it takes for a new form of a gene to get established if it moves into a weed or other species. They reported that the rate at which a gene migrates makes little difference, compared with whether it helps the plant survive and reproduce.

According to Burke and Rieseberg, if a transferred gene supercharges a plant into leaving more offspring, the gene will spread. "If it's disadvantageous or neutral, it won't do much, no matter how high the rate of gene flow," he says.

Some scientists looking for benefits to plants that receive stray transgenes have studied crops instead of weeds. They pitted the engineered version of a crop against its old-fashioned counterpart in a survival marathon. In the first test of survival advantages conferred by a transgene in a natural setting, Mick J. Crawley of Imperial College in Berkshire, England, and his colleagues chose 12 habitats. In each, they planted adjacent patches of transgenic and traditional versions of several crops: rape, maize, beets, and potatoes. The researchers then left the plants to fend for themselves.

After monitoring the experiment for 10 years, Crawley and his team reported in 2001 that none of the transgenic-plant populations had lasted significantly longer than the conventional ones did, and none of the patches had gained ground.

The experiment made the transgenics look pretty tame. Yet Crawley cautions that the crops his team examined had been engineered to resist herbicides, moth and butterfly caterpillars, and perhaps those qualities didn't matter much in the wild. Transgenes that confer different advantages, such as tolerance to drought or to other pests, might make more of difference.

Snow and Burke are approaching the problem by inserting transgenes into wild relatives of commercial plants. They both used wild sunflowers but studied different genes and got different results.

Snow and her colleagues began with wild sunflowers engineered to make the Bt pesticide, a toxin named for the Bacillus thuringiensis bacterium, in which the gene originates. The researchers used traditional breeding methods to move the gene into the wild sunflowers, which they planted in contained fields.

The souped-up wildlings set 50 percent more seeds than the regular wild ones did. "We were surprised," says Snow. Her team's results appeared in the April Ecological Applications.

In a series of studies, Burke and his colleagues are tracking a genetic construct called OxOx, which fortifies commercial sunflowers against white mold. The pathogen's abundant oxalic acid, or oxalate, breaks down plant tissue, and the transgene OxOx encodes the amino acid sequence for oxalate oxidase. The transgene is "making an antacid," Burke explains.

Earlier work showed that about two-thirds of all commercial U.S. sunflower fields lie near wild sunflowers that bloom at the same time. He calls gene flow between commercial and wild sunflowers "a virtual certainty." To mimic this potential spread, Burke and Rieseberg bred OxOx into a wild species and planted the enhanced offspring in cages in California, Indiana, and North Dakota.

The researchers exposed all the offspring plus unenhanced wild plants to white mold. Burke says that the gene gave different levels of protection from mold in the different states.

In none of the three states, however, did the genetically enhanced plants set significantly more seeds than the wild ones did, the researchers reported in the May 23 Science. If results from more years confirm these findings, the gene probably won't create aggressive weeds, they conclude.

Burke says his work "provides a nice counterpoint" to the study on the Bt gene. The disparity in outcomes, he says, emphasizes that for transgenes, "we need to be assessing the risks and benefits on a case-by-case basis."

References and Sources

References

  1. Burke, J.M., and L.H. Rieseberg. 2003. Fitness effects of transgenic disease resistance in sunflowers. Science 300(May 23):1250. Abstract available at http://www.sciencemag.org/
  2. Crawley, M.J., et al. 2001. Transgenic crops in natural habitats. Nature 409(Feb. 8):682-683. Abstract available at http://dx.doi.org/10.1038/35055621.
  3. Ellstrand, N.C., and S.M. Scheiner, eds. 2003. Dangerous Liaisons: When Cultivated Plants Mate with Their Wild Relatives. Baltimore: Johns Hopkins University Press.
  4. Linder, C.R. . . .A. A. Snow, et al. 1998. Long-term introgression of crop genes into wild sunflower populations. Theoretical and Applied Genetics 96(March):339-347. Abstract available at http://dx.doi.org/10.1007/s001220050746.
  5. Ortiz-García, S., and E. Exequiel. 2003. Transgenic maize in Mexico: Risks and reality. Botany 2003. July 26-31. Mobile, Ala. Abstract.
  6. Ritala, A., et al. 2002. Measuring gene glow in the cultivation of transgenic barley. Crop Science 42(January-February):278-285. Available at http://crop.scijournals.org/cgi/content/full/42/1/278.
  7. Rodemeyer, M. 2003. Policy implications of transgenic crop gene flow. Botany 2003. July 26-31. Mobile, Ala. Abstract.
  8. Snow, A.A., et al. 2003. A Bt transgene reduces herbivory and enhances fecundity in wild sunflowers. Ecological Applications 13(April):279-286. Abstract.
  9. Warwick, S.I., et al. 2003. Hybridization between transgenic Brassica napus L. and its wild relatives: Brassica rapa L., Raphanus raphanistrum L., Sinapis arvensis L., and Erucastrum gallicum (Willd.) O.E. Schulz. Theoretical and Applied Genetics 107(August):528-539. Abstract.

Further Readings

  1. Ellstrand, N.C., H.C. Prentice, and J.F. Hancock. 1999. Gene flow and introgression from domesticated plants into their wild relatives. Annual Review of Ecology and Systematics 30(November):539-563. Abstract available at http://dx.doi.org/10.1146/annurev.ecolsys.30.1.539.
  2. Milius, S. 2002. Journal disowns transgene report. Science News 161(April 13):237. Available to subscribers at http://www.sciencenews.org/20020413/note12.asp.
  3. ______. 2001. Transgenes migrate into old races of maize. Science News 160(Dec. 1):342. Available to subscribers at http://www.sciencenews.org/20011201/fob5.asp.
  4. Stewart, C.N., M.D. Halfhill, and S.I. Warwick. 2003. Transgene introgression from genetically modified crops to their wild relatives. Nature Reviews Genetics 4(October):806-817. Available at http://dx.doi.org/10.1038/nrg1179.

Sources

  1. John M. Burke, Vanderbilt University, Department of Biological Sciences, VU Station B 351634, Nashville, TN 37235
  2. Norman C. Ellstrand, Biotechnology Impact Center, Department of Botany and Plant Sciences, University of California, Riverside, Riverside, CA 92521-0124
  3. Linda Hall, Alberta Agriculture, Plant Industry Division, Agronomy Centre, 2nd Floor, 6903 - 116 Street, Edmonton, AB T6H 5Z2, Canada
  4. Randal Linder, University of Texas, Austin, Section of Integrative Biology, 1 University Station C0930, Austin, TX 78712
  5. Sol Ortiz-García, Instituto Nacional de Ecología, SEMARNAT, Av. Periférico Sur 5000, 5° piso, Col. Insurgentes Cuicuilco. Deleg., Coyoacan México D. F. 04530, Mexico
  6. Allison Snow, Department of Evolution, Ecology, and Organismal Biology, Ohio State University, 1735 Neil Avenue, Columbus, OH 43210-1292
  7. Paul St. Amand, Kansas State University, Agronomy Department, 2004 Throckmorton Hall, Manhattan, KS 66506-5501
  8. Suzanne Warwick, Agriculture and Agri-Food Canada, Eastern Cereal and Oilseed Research Centre, 960 Carling Avenue, Ottawa, ON K1A 0C6, Canada

http://www.sciencenews.org/20031011/bob8.asp

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