The Monsanto Co., now a division of Pharmacia Corp., recently admitted that its Roundup Ready soybeans contain two extra bacterial DNA sequences derived from the original transformation event about 10 years ago. The genetically modified (GM) plants are tolerant to Monsanto's herbicide Roundup, or glyphosate, so farmers can kill bothersome weeds without harming their crop.

Plants manufacture essential aromatic amino acids such as phenylalanine through the shikimic acid pathway, housed in their chloroplasts. Glyphosate blocks a key enzyme in the pathway, 5-enolpyruvylshikimate-3-phosphate synthase, or EPSPS. No enzyme activity means no aromatic amino acids--ergo no go.

Monsanto discovered a gene for glyphosate-resistant EPSPS in Agrobacterium species CP4 (U.S. patent #5633435), and through the wonders of genetic engineering, inserted it into soybean's nucleus along with appropriate controlling sequences. A transit peptide lets the cytoplasmically made protein enter chloroplasts. The original transgenic soybean line was called GTS 40-3-2, but the new findings affect all Monsanto Roundup Ready commercial cultivars, which were derived by cross-breeding with 40-3-2. The results don't apply to other tolerant crops such as canola, corn, and cotton, which were obtained in separate transformation events, but Monsanto scientists are checking these varieties too.

The U.S. Department of Agriculture (USDA) and the Food and Drug Administration (FDA), which have oversight authority in this case, approved Roundup Ready soybean in 1994 following public comment and review. USDA deemed the crop safe enough to deregulate it in 1996. That same year, the Environmental Protection Agency signed off on applying glyphosate in conjunction with tolerant soybeans, which paved the way for widespread commercial use of the plants.

According to Roy Fuchs , director of regulatory science at Monsanto's St. Louis headquarters, "We have contacted literally dozens of regulatory authorities around the world" about the extra inserts. Adds Fuchs, "The FDA and USDA have already seen these data and have agreed that there are no safety concerns." Canadian regulators feel the same. According to Health Canada in Ottawa, "The additional molecular characterization data have no impact on the safety of glyphosate tolerant soybeans available in the Canadian marketplace." Scientific Value

The scientific community will also judge Monsanto's findings. Fuchs says, "The information will be submitted for publication soon. It has value for the development of product-specific detection methods."

Monsanto claims it discovered the extra inserts while developing sensitive line identification methods in preparation for product labeling. The company is using those methods on its other crops too. According to Fuchs, the extra soybean DNA came from the EPSPS structural gene. But the situation is complicated because the vector used for transformation contained two copies of the gene. Because the copies were identical, "The original source of the EPSPS sequences ... is not known nor is it important," says Fuchs.

The question is, why didn't Monsanto discover the extra sequences when it first characterized the 40-3-2 line?1 Fuchs maintains they looked for other inserts but didn't detect them, despite the fact that "the initial molecular characterization used state-of-the-art methods at the time." In other words, those methods--mainly digesting DNA with restriction enzymes and probing the fragments on Southern blots--didn't have the resolution needed to find the extra inserts. That's why the team could emphatically state that "no other plasmid-derived DNA sequences are present."1

University of Georgia geneticist Richard Meagher , who has fathered useful transgenic plants of his own, is sympathetic: "It doesn't surprise me. For one thing, detecting anything below 150 base pairs is very difficult. The hybridization signal is too low."

Five to 10 years ago, sequencing around inserts to check transformation events wasn't routine. According to Mark Tepfer of the Institut National de la Recherche Agronomique (INRA) in Versailles, France, "It's quite true that not long ago, nobody considered it necessary to go looking for other changes that occurred during transformation." That's critical, because the larger of the two extra pieces is located just downstream of the functional EPSPS gene, on a 5,800 base pair fragment Monsanto scientists followed for several generations after transformation. Had they sequenced the fragment, they probably would have seen the hitchhiking piece of DNA. Now, having just done sequencing, they know it's there. Still, some wonder if Monsanto could have done more checking, even after it released Roundup Ready soybeans for commercial production.

How'd It Happen?

Monsanto still isn't certain how the inserts got into the soybean genome. "Our hypothesis is that these sequences resulted from separate portions of the plasmid vector being incorporated during the initial particle gun bombardment process," offers Fuchs, referring to the biolistic method used to shoot DNA into plant cells.

In hindsight, perhaps that's not surprising. Based on initial genetic evidence, the Monsanto team decided that "the original R0 plant [called 40-3] had received two DNA inserts located at different positions in the genome."1 In other words, they realized that incorporating the bacterial DNA into soybean wasn't a straightforward process. Researchers now know transformation can be messy--strange things happen when vector DNA enters plant chromosomes.2 But "those [initial extra] sequences were removed by plant breeding" by the time Monsanto got to line 40-3-2, concludes Fuchs, who doesn't think the newly discovered inserts are related to them. On the other hand, he also doesn't "expect that [they] are the result of any posttransformation process, such as recombination."

Whatever their origin, the two new sequences contain just 72 and 250 base pairs, small compared to the more than 1,360 base pairs in the EPSPS gene. That's probably why they're nonfunctional--Fuchs says Monsanto hasn't detected truncated messenger RNA or protein encoded by the inserts.

Should Monsanto be faulted because of the inserts? Tepfer believes "they are guilty of no wrongdoing."

Of more concern, however, is whether the discovery casts doubt on the reliability of GM technology in the eyes of the public. A May 28 article in Scotland's Sunday Herald on "rogue DNA" quoted a Greenpeace activist as saying, "Neither the industry or the regulators actually know what genes are in it. What don't we know?" Three days later, Britain's Guardian cited another critic as saying, "This shows ... that genetic modification is inherently unpredictable and will have all sorts of knock-on effects once released into the environment."

Obviously, U.S. and Canadian authorities disagree, and Fuchs concurs, recalling that the extra sequences have always been in Roundup Ready soybean, which went through environmental and health risk assessments. Says Jeffrey Bergau, manager of public affairs for Monsanto in Chicago, "The entire product was reviewed, and that's the important takeaway." Asked to comment on activist concerns, Bergau declined: "I wouldn't try to speculate on what others might say." Adds Fuchs, "We know the scientific answers--facts are facts."

Tepfer also discounts the inserts' significance: "I remain convinced that this issue should not be a big deal, or should not have been turned into one. In addition to knowing for decades that genetic transformation is mutagenic (as is regeneration of even nontransgenic plants), we should remember that intentional random mutagenesis is a not-unusual plant breeding technique to increase variability."

But Georgia's Meagher is more cautious: "I don't worry about it expressing anything. I worry more about it disrupting something." If that's the case, did the inserts hurt? It's difficult to say. Word around the farmhouse not long ago was that Roundup Ready soybeans suffered from yield drag, or somewhat lower productivity, cause unknown. But that may be disappearing in successive generations of seed and as more acres are planted. S

Barry A. Palevitz is a contributing editor for The Scientist.

References

1. S.R. Padgett et al., "Development, identification, and characterization of a glyphosate tolerant soybean line," Crop Science, 35:1451-61, 1995.

2. W.P. Pawlowski and D.A. Somers, "Transgenic DNA integrated into the oat genome is frequently interspersed by host DNA," Proceedings of the National Academy of Sciences, 95:12106-10, 1998.