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Unintended Effects of Genetic Manipulation
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Some Examples of Unintended Effects of Genetic Manipulation

Craig Holdrege


Many unintended (or, as I will usually refer to them here, "nontarget") effects of genetic manipulation have been recorded in the scientific literature. Below we present a few examples of different types in order to give an impression of the broad spectrum of nontarget effects, ranging from alterations at the level of DNA to impact on the broader environment. For more examples and further commentary on the issue of nontarget effects of genetic manipulation, click here.


Genes from GM crops escape into the wild

Creeping bentgrass is widely used on golf courses. Monsanto and The Scotts Company have developed an herbicide-resistant variety of creeping bentgrass that they hope to market on a large scale. In this case -- the only such to date -- the EPA and USDA requested an environmental impact report, since the GM creeping bentgrass could easily cross-pollinate with wild creeping bentgrass and other weedy grasses, possibly resulting in herbicide-resistant "superweeds."

In 2003 The Scotts Company planted 400 acres of herbicide-resistant creeping bentgrass in a field trial. EPA and university scientists then looked to see whether the herbicide-resistance gene spread into the wild (Reichmann et al. 2006; Watrud et al. 2004; Zapiola et al. 2008). They found that it did, through seeds and via pollen. The herbicide resistance transgene spread via pollen to an area up to 13 miles beyond the control area perimeter and had pollinated wild creeping bentgrass as well as a close relative (redtop). In the following three years, researchers found that a majority of the creeping bentgrass plants they investigated in an area up to 2.4 miles beyond the study area had become herbicide-resistant (93% in 2004 and 62% in 2006).

The university scientists who were part of this investigation concluded that it is "unrealistic to think that a transgene could be contained in an outcrossing, wind-pollinated, small-seeded, perennial crop, even with expanded isolation distances and stringent production practices," and the "elimination of transgenes is unlikely to be feasible" (Zapiola et al. 2008, pp. 5, 7). Although The Scotts Company was fined $500,000 in 2007 by the USDA for failing to comply with "performance standards and permit conditions" (USDA News Release No. 0350.07), even the most stringent practices would probably not have prevented the transgenes escaping into the wild. And now they are there and will most likely spread further.


Unexpected effects arising under field conditions

Sometimes the side-effects of genetic manipulation do not become apparent until the crops are grown commercially. In the late 1990s some farmers in Georgia complained about the poor performance of their Roundup Ready soybeans under conditions of drought and heat. Scientists then carried out a comparative laboratory study of transgenic and conventional soybeans (Gertz et al. 1999). They found that the transgenic plants were shorter, had a lower fresh weight, had less chlorophyll content, and, at high temperature, suffered from stem splitting.

In another case, genetically engineered Bt corn, which produces its own pesticide, was found -- after five years of commercial planting on millions of acres -- to contain substantially more lignin in the stalks than unmodified corn (Saxena and Stotzky 2001; Poerschmann et al. 2005). Lignin makes the stems woodier. No one has investigated how the higher lignin content might affect the corn's digestibility by the cattle or pigs that are fed on it. What other biochemical pathways may have been repressed or altered as a result of the increased production of lignin also remains unknown.


Adverse effects on animals fed genetically modified plants

To make peas more resistant to weevils, scientists in Australia genetically altered peas with a gene construct containing DNA from beans, which have a natural defense against weevils. Scientists isolated from beans the gene related to the production of a protein that blocks the breakdown of starch and thereby causes the weevils feeding on the beans to starve. The genetically altered peas gained the same kind of defense against weevils. To see if the transgenic peas might present a risk to human health, the peas were tested on mice (Prescott et al. 2005). To their surprise, the scientists found that the mice developed an immune response to the peas, meaning they produced antibodies against the genetically modified protein. The scientists discovered that the protein in the pea had, after initial formation, been altered (post-translational modification) and in this changed form elicited an inflammatory reaction in the mice.

Since peas are used as a component of animal feed, the scientists also investigated whether there were any marked effects on the animals fed with feed containing the GM peas (Collins et al. 2006; Li et al. 2006). They found that starch digestion in the small intestines was markedly decreased in pigs and chickens fed a diet including the transgenic peas. In addition, the growth rate of broiler chickens fed for forty days on a diet including the transgenic peas was reduced by eleven percent. Australia's Commonwealth Scientific and Industrial Research Organization, which is the governmental organization that carried out the research, decided on the basis of these results not to pursue further work with these GM peas.


Genetic manipulations can alter overall plant structure and metabolism

Researchers genetically engineered canola plants to produce seeds with increased amounts of carotene, so that eventually canola could be used as a commercial source of carotene (Shewmaker et al. 1999). Normally canola seeds do not accumulate carotene. The manipulation succeeded and the transgenic mature canola seeds contained up to a 50-fold increase in carotene (mainly alpha- and beta-carotene), making the embryo visibly orange. But there were many unintended effects:

  • The amount of chlorophyll produced by the plant in developing seeds was reduced.

  • Levels of gamma tocopherol (a form of vitamin E) were also decreased in seeds.

  • Some microscopic structures in the embryos were altered and thread-like bodies of unknown nature appeared.

  • The amount of phytoene in the plants was increased compared with controls. Phytoene is normally an intermediate product of metabolism and does not accumulate.

  • The amount of lutein and xanthophylls was not higher, although they also are end products of the carotene metabolic pathway and "should" have been produced in increased amounts.

  • The composition of fatty acids was altered: there was more oleic acid and less linoleic and linolenic acid. This change in fatty acid composition was wholly unexpected since there is no known link between fatty acid synthesis and carotene synthesis.

  • Germination in transgenic seeds was delayed by one or two days compared to control plants.

Evidently, it is naive to believe that we can change a single substance and nothing else. Changing one thing has multiple effects and sometimes these are a complete surprise.

In another experiment, researchers produced five lines of transgenic potatoes that were genetically altered to produce insecticidal substances (Birch et al. 2002). They wanted to determine whether the genetic transformation of potatoes to produce insecticide resulted in any unintended changes in the amounts of glycoalkaloids in the leaves of the plants. Glycoalkaloids are normally present in the leaves and stems of potato plants and are highly toxic to mammals. Ecologically, glycoalkaloid-containing leaves may be feeding deterrents to browsing mammals and some insects due to their toxicity and bitterness, so there is some concern about the possible unintended results of altered glycoalkaloid levels in genetically modified potatoes. The researchers realized that "it is incorrect to assume that the current methods of genetic engineering used to express single transgenes in plants are completely targeted and will have no, or minimal, effects on unrelated biosynthetic pathways in transformed plants" (p. 144).

They found a number of nontarget effects:

  • Four of five transgenic lines produced significantly less foliage than the control plants.

  • In three of the five transgenic lines, stem production was significantly reduced, while in one of the lines stem production was significantly higher, which was reflected in the bushy growth form of the plants in that transgenic line.

  • All transgenic lines had significantly lower levels of leaf-glycoalkaloids than both the normal controls and the tissue culture controls. For example, one line had a 24% reduction in glycoalkaloid levels, while three lines had on average 44% less leaf-glycoalkaloid content.

  • Four of five transgenic lines had significantly lower levels of glycoalkaloids in their stems.

  • In two of the five transgenic lines, the ratios of two different glycoalkaloids (alpha-chaconine and alpha-solanine) were significantly altered in the leaves.

In most studies the researchers do not investigate substances they assume to be unrelated to the metabolic pathway they aim to alter by means of the genetic manipulation. In this case, as the authors found, there were significant changes in an important group of substances (the glycoalkaloids). These changes could have significant implications, since widespread planting of transgenic potatoes that inadvertently have lowered amounts of the glycoalkaloids in their leaves could lead to an increase in potato pests, thereby counteracting the very benefit that was intended by the genetic manipulation. The authors conclude: "We think that it is as important to monitor unintended changes in the levels of such secondary plant compounds as it is to evaluate the potential risks and benefits of the intended transgene product (anti-insect gene products) in the agro-ecosystem" (p. 148).


What causes a change in characteristics is not necessarily the target gene

Scientists wanted to make potato plants resistant to the potato leaf luteovirus, which is transmitted by aphids that feed on potato plants (Presting et al. 1995). Potato leaf luteovirus infections can cause severe losses in potato crops. Previous experiments had shown that transgenic potatoes with the gene for a viral coat protein (CP) had some resistance to the virus, even though the potatoes did not actually make the CP protein. In this experiment the researchers created different transgenic lines of Russet Burbank and Ranger Russet potatoes. Some lines of each variety contained the CP gene, and some contained a modified CP gene, which the researchers hoped would cause plants to produce the viral coat protein and thereby gain increased resistance to the virus. Finally, they created control lines containing a gene construct that lacked a target gene (that is, lacked both the CP gene and the modified CP gene).

All Russet Burbank transgenic lines containing the CP gene or the modified CP gene showed significantly lower virus infection than unmanipulated potato plants. But this was not due to the coat protein, because none could be detected. What caused the increased resistance remained unclear. Only 4 of 15 transgenic lines of the Ranger Russet potatoes containing the CP transgene showed increased virus resistance.

Even more surprising was the discovery that the most resistant transgenic Ranger Russet line was the one that had been transformed with the control construct, which did not contain the CP gene or the modified CP gene. The target effect arose without the target gene! Finally, there were significant differences in virus resistance among the lines that had been transformed with the same construct, so that considerable variability in resistance was induced by the genetic transformation process. This study clearly illustrates how a target effect in a transgenic plant -- virus resistance in this case -- can appear, although the relation of that phenotypic change to the genetic manipulation remains a complete riddle.


Transgenes can be silenced and those that are not may not produce the desired effect

In an effort to make spring wheat resistant to scab (headblight), a major fungal disease in wheat, researchers inserted two genes into wheat that were known to be related to scab resistance (Anand et al. 2003). After the manipulation, 24 different lines were found to contain one or both of the transgenes and to express one or both of the proteins that were meant to convey scab resistance. Surprisingly, transgene activity in 20 of the 24 lines (80%) shut down after the initial generation. This is known as transgene silencing. In some unknown way, the plants respond to the manipulation by inhibiting the expression of the inserted genes.

The four other lines showed stable inheritance of the transgenes for up to four generations and produced one or both of the target proteins. While one of these lines showed reduced scab infection under greenhouse conditions, none of them was resistant to scab infection under field conditions. Moreover, plants of the line that had the highest transgene expression also suffered from a "lesion-mimic phenotype" in which the leaves developed patches of dying tissue during flower formation.


Integration of foreign genes into the host organism's DNA is unpredictable

In a study carried out with the mustard plant Arabidopsis, which is the most thoroughly studied plant from a genetic perspective, researchers wanted to investigate how a gene construct is incorporated into the plant's DNA (Forsbach et al. 2003). Gene constructs containing a marker gene and various reporter genes were inserted into Arabidopsis tissue via a bacterium (Agrobacterium) and numerous transgenic plants were generated from tissue cultures. The researchers found that in 80% of the transgenic lines the gene construct had been inserted at a single site (locus). However, in only 22% of these did the gene construct remain intact. From this group, 122 lines were selected for further investigation; in these lines the gene construct was of "sufficient length and quality to determine unambiguously the position of integration in the Arabidopsis genome" (p. 165). In connection with these 122 lines, the researchers found:

  • Only three of 71 lines investigated contained a gene construct that corresponded precisely to the full-length strand of DNA in the originally inserted construct; in all the others there were small deletions at one or both ends of the sequence.

  • In 88.7% of the lines, the integration of the gene construct resulted in deletions of plant DNA at the site of integration. Only 2.7% "inserted exactly into the target site" -- that is, without such deletions or other changes (p. 168).

  • In over half the lines, short "filler sequences" of DNA were found between the borders of the construct and the plant DNA flanking those borders. In those cases in which the origin of this filler DNA could be identified, it was found to consist of DNA fragments from either the plant or the transgenic construct.

  • In two of the lines "extensive chromosomal rearrangements associated with the integration" (p. 169) of the gene construct were found. Chromosomal fragments were swapped between different chromosomes (reciprocal translocation).

The researchers summarize: "In the majority of cases, single-copy T-DNA [i.e. the transgenic construct] insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences and gross chromosomal rearrangements such as translocations" (p. 161). The authors also point out that the frequency of DNA rearrangements "is likely to be even higher if transgenic lines containing multiple T-DNA inserts are analyzed" (p. 173), and they refer to other research.

In a different study, researchers investigated the integration of the foreign DNA construct shot into oat plants via microprojectile bombardment (Makarevitch 2003; for a description of this technique see Table 2 in "Understanding the Nontarget Effects of Genetic Manipulation" in this issue). In the majority of transgenic lines the construct was not integrated as a single unit into the host organism's DNA: only 7 of 36 lines appeared to show simple insertion. Two of these lines with simple integration sites were chosen for further examination. Closer investigation of one showed that, in addition to the major integration site with the functional gene, there were two smaller integration sites, which had gone undetected in previous studies. In addition, in both lines the gene constructs showed complex integration: "full-length and partially truncated copies of the delivered DNA were integrated in different orientations, interspersed with regions of extensively scrambled transgene and genomic [oat] DNA sequences, and, in two cases, flanked by rearranged genomic [oat] DNA" (p. 429).

Both these studies show that the integration of the foreign DNA into the genome of the host organism is more or less haphazard and can in no way be predicted. The desired result at the level of DNA, namely the integration of a simple copy of the gene construct without an effect on the host organism's DNA, rarely occurs.


Increased planting of glyphosate-resistant crops and application of glyphosate causes an increase in glyphosate-resistant weed species

Glyphosate-resistant crops -- especially soybeans, cotton, corn, canola -- are the most prevalent genetically modified crops and are grown on millions of acres of cropland in the U.S. and other countries. Ninety percent of soybeans grown in the U.S. in 2007 were genetically modified glyphosate-resistant varieties.

The wide-spectrum herbicide glyphosate has been sold by Monsanto Company under the name Roundup since 1974. In 1996 the first glyphosate-resistant weeds were reported and in the decade since then -- which coincides with the increased cultivation of genetically modified, glyphosate-resistant plants -- ten different glyphosate-resistant weed species have been found in fields planted with glyphosate-resistant crops (Cadeira and Duke 2006; Nandula et al. 2005; Owen and Zelaya 2005; www.weedscience.org). These include common waterhemp (Amaranthus rudis), common ragweed (Ambrosia artemisiifolia), and horseweed (Conyza canadensis). The latter has evolved increased resistance to glyphosate since resistant populations were first detected in fields planted with Roundup Ready soybeans in Delaware in 2000. Since then, resistant populations have been found in fourteen other states as well as in Brazil and China.

In addition to these weed species that have evolved resistance in relation to glyphosate use, at least 13 other species of weeds possessing natural resistance to glyphosate are now being observed in fields grown with glyphosate-resistant crops in the U.S., Brazil, and Argentina. These include common lambsquarters (Chenopodium album), velvetleaf (Abutilon theophrasti), and species of morning glory (Ipomeoa).

In a greenhouse experiment, researchers found that glyphosate-resistant horseweed could form hybrids with its nonresistant close relative, dwarf horseweed (Conyza ramosissima), which is also a common weed. The hybrids were fertile, had superior resistance to glyphosate, and the resistance was inherited as a semi-dominant trait (Zelaya et al. 2007). "Glyphosate-resistant crop systems are suggested to be simple and without great environmental consequences. However, we have demonstrated that there are major ecological and economic consequences from these presumed simple systems. . . . We propose that if hybridization of new taxa with glyphosate resistance as a semi-dominant trait can occur with relative ease, the current agroecosystem is at considerable jeopardy" (Zelaya et al. 2007, p. 669).

As one overview study concludes: "High levels of adoption of GR [glyphosate-resistant] crops by U.S. farmers have dramatically increased the use of glyphosate, with a concomitant decrease in use of other herbicides. This has impacted weed communities. . . . The problem of GR weeds is real, and farmers have to understand that continuous use of glyphosate without alternative strategies will likely result in the evolution of more GR weeds. Even in the short term, no one can predict the future loss of glyphosate efficacy due to weed species shifts and evolution of glyphosate resistance" (Nandula et al. 2005, p. 186).


References

Anand, A., T. Zhou, H. N. Trick, B. S. Gill et al. (2003). "Greenhouse and Field Testing of Transgenic Wheat Plants Stably Expressing Genes for Thaumatin-like Protein, Chitinase and Glucanase against Fusarium graminearum," Journal of Experimental Biology vol. 54, pp. 1101-11.
To see our report on this study, click here.

Birch, A. N. E., I. E. Geoghegan, D. W. Griffiths, and J. W. McNicol (2002). "The Effect Of Genetic Transformations for Pest Resistance on Foliar Solanidine-Based Glycoalkaloids of Potato (Solanum tuberosum)," Annals of Applied Biology vol 140, pp. 143-9.
To see our report on this study, click here.

Cadeira, A. L. and S. O. Duke (2006). "The Current Status and Environmental Impacts of Glyphosate-Resistant Crops: A Review," Journal of Environmental Quality vol. 35, pp. 1633-58.
To see our report on this study, click here.

Collins, C., P. Eason, F. Dunshea, T. Higgins, and R. King (2006). "Starch But Not Protein Digestibility is Altered in Pigs Fed Transgenic Peas Containing Alpha-Amylase Inhibitor," J. Sci. Food Agric. vol. 86, pp. 1894-9.
To see our report on this study, click here.

Forsbach, A., D. Schubert, B. Lechtenberg, M. Gils et al. (2003). "A Comprehensive Characterization of Single-Copy T-DNA Insertions in the Arabidopsis thaliana Genome," Plant Molecular Biology vol. 52, pp. 161-76.
To see our report on this study, click here.

Gertz, J. M., W. K. Vencill, and N. S. Hill (1999). "Tolerance of Transgenic Soybean (Glycine max) to Heat Stress," in: The 1999 Brighton Conference: Weeds. Farnham, Surrey, UK: The Council, pp. 835-40.
To see our report on this study, click here.

Holdrege, C. (2008). "The Nontarget Effects of Genetic Manipulation: An Introduction." Available online: http://natureinstitute.org/txt/ch/nontarget.php.

Li, X., T. Higgins, and W. Bryden (2006). "Biological Response of Broiler Chickens Fed Peas (Pisum sativum L.) Expressing the Bean (Phaseolus vulgaris L.) Alpha-Amylase Inhibitor Transgene," J. Sci. Food Agric. vol. 86, pp. 1900-7.
To see our report on this study, click here.

Makarevitch, I., S. K. Svitashev, and D. A. Somers (2003). "Complete Sequence Analysis of Transgene Loci From Plants Transformed Via Microprojectile Bombardment," Plant Molecular Biology vol. 52, pp. 421-32.
To see our report on this study, click here.

Nandula,V. K., K. N. Reddy, S. O. Duke, and D. H. Poston (2005). "Glyphosate-Resistant Weeds: Current Status and Future Outlook," Outlooks in Pest Management vol. 16, pp. 183-7.
To see our report on this study, click here.

Owen, M. D. K. and I. A. Zelaya (2005). "Herbicide-Resistant Crops and Weed Resistance to Herbicides," Pest Management Science vol. 61, pp. 301-11.
To see our report on this study, click here.

Poerschmann, J., A. Gathmann, J. Augustin, U. Langer et al. (2005). "Molecular Composition of Leaves and Stems of Genetically Modified Bt and Near-Isogenic Non-Bt Maize: Characterization of Lignin Patterns," J. Environ. Quality vol. 34, pp. 1508-18.
To see our report on this study, click here.

Prescott, V., P. Campbell, A. Moore, J. Mattes et al. (2005). "Transgene Expression of a Bean alpha-Amylase Inhibitor in Peas Results in Altered Structure and Immunogenicity," J. Agric. Food Chem. vol. 53, pp. 9023-30.
To see our report on this study, click here.

Presting, G. G., O. P. Smith, and C. R. Brown (1995). "Resistance to Potato Leafroll Virus Transformed with the Coat Protein Gene or with Vector Control Constructs," Phytopathology vol. 85, pp. 436-42.
To see our report on this study, click here.

Reichman, J., L. Watrud, E. Lee, C. Burdick et al. (2006). "Establishment of Transgenic Herbicide-Resistant Creeping Bentgrass (Agrostis stolonifera L.) in Nonagronomic Habitats," Molecular Ecology vol. 15, pp. 4243-55.
To see our report on this study, click here.

Saxena, D. and G. Stotzky (2001). "Bt Corn has a Higher Lignin Content than Non-Bt Corn," American Journal of Botany vol. 88: 1704-6.
To see our report on this study, click here.

Shewmaker, C., J. A. Sheehy, M. Daley, S. Colburn et al. (1999). "Seed-specific Overexpression of Phytoene Synthase: Increase in Carotenoids and Other Metabolic Effects," The Plant Journal vol. 20, pp. 401-12.
To see our report on this study, click here.

http://www.weedscience.org: this website tracks and documents herbicide-resistant weed species.

Watrud, L., E. Lee, A. Fairbrother, C. Burdick et al. (2004). "Evidence for Landscape-level, Pollen-mediated Gene Flow from Genetically Modified Creeping Bentgrass with CP4 EPSPS as a Marker," PNAS vol. 101, pp. 14533-38.
To see our report on this study, click here.

Zapiola, M., C. Campbell, M. Butler, and C. Mallory-Smith (2008). "Escape and Establishment of Transgenic Glyphosate-resistant Creeping Bentgrass (Agrostis stolonifera) in Oregon, USA: A 4-year Study," Journal of Applied Ecology doi: 10.1111/j.1365-2664.2007.01430.x.
To see our report on this study, click here.

Zelaya, I. A., M. D. K. Owen and M. J. VanGessel (2007). "Transfer of Glyphosate Resistance: Evidence of Hybridization in Conyza (Asteraceae)," American Journal of Botany vol. 94, pp. 660-73.
To see our report on this study, click here.

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