Last Updated on 12/4/00
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Part 1: Squaring Up Roundup Ready Crops
Alan Felsot The only problem with this seemingly miraculous product is that it kills just about any plant onto which it is directly sprayed. Thus, until recently, this synthetic amino acid, known as glyphosate (N-phosphono-methyl glycine) has had limited utility in agricultural production. And then along came genetic engineering. In just the last five years, Monsanto has commercialized soybeans, corn, cotton, and rape (canola) genetically engineered to resist the toxic effects of glyphosate. Monsanto trademarked these transgenic cultivars as Roundup Ready (RR), in reference to their commercial formulation of glyphosate. So everyone should be cheering about the decreased numbers of pesticide applications (or at least the potential to decrease them) on the large U.S. acreages of soybeans, corn, cotton and canola, right? Well, if you read any newspaper today it's clear that everyone is not cheering. In fact, as with all transgenic technology, RR crops have not escaped the wrath of advocates who seem hell bent to trash biotechnology in total rather than judge each development on its own merits or faults. So, in keeping with the National Academy of Sciences recommendation that each transgenic crop involving traits useful for protection from pests be judged individually (14), I will review the biochemistry of glyphosate tolerance and address the validity of critics' concerns. Biochemical Basis for Glyphosate Resistance All Roundup Ready crops contain an enzyme known as EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) that is resistant to the effects of glyphosate. EPSPS is naturally found in all plants, fungi, and bacteria but is absent in animals (18). The enzyme is an important catalyst in the biochemical pathway for synthesis of the aromatic amino acids phenylalanine, tryptophan, and tyrosine. Because animals do not contain EPSPS, they must ingest these aromatic amino acids in their diets. EPSPS is localized in the chloroplasts of plants, the cell organelle responsible for photosynthesis. Glyphosate latches on to EPSPS, inhibiting its synthetic activity. The inability to produce the aromatic amino acids eventually leads to cell death. The glyphosate-tolerant form of EPSPS has a low affinity for binding glyphosate yet it still helps synthesize the amino acids just as efficiently as the glyphosate-susceptible EPSPS. Roundup Ready canola plants have also been engineered to contain an enzyme called glyphosate oxidoreductase, or GOX. GOX, normally found in a common soil bacterium, Ochrobactrum anthropi strain LBAA, quickly metabolizes glyphosate into glyoxalate and aminomethylphosphonic acid (AMPA). Glyoxalate is a naturally occurring plant biochemical involved in carbon cycling and AMPA is of no toxicological concern in food (17). Genetic Basis for Glyphosate Resistance Plant species have long been known to be highly variable in their response to herbicides. For example, grasses are very tolerant to 2,4-D and other growth hormone mimics, but dandelions exposed to it wither and die. Soybeans can tolerate trifluralin, but corn never gets big enough to produce an ear. Furthermore, weed populations can become resistant to herbicides. During the 1980s, agricultural scientists tried in vain to take advantage of plants' natural variability to herbicide toxicity and their penchant to develop resistance. Attempts to conventionally breed glyphosate-tolerant crops failed (18). Such failure is not surprising; after twenty-five years of glyphosate use, plant resistance in the field has been noted in only two grass species (10). As molecular manipulation technologies developed (i.e., the ability to purposefully transfer specific genetic sequences from one organism to another), the stage was set for engineering plants resistant to glyphosate. So how does one "make" a plant resistant to glyphosate? Mimic Mother Nature. As with all cases of resistance evolution, two main mechanisms are responsible for herbicide tolerance in plants-an increased ability to detoxify the pesticide and/or an altered biochemical site of interaction with the pesticide (17). Both mechanisms involve altered protein functioning and/or production. In the case of detoxification, the proteins involved are enzymes that possess an enhanced capacity for breaking down the herbicide. Biochemical sites attacked by a herbicide may also be enzymes or alternatively receptors that trigger a cascade of physiological reactions. Altered enzymes and receptors have less affinity than their "normal" counterparts for binding the herbicide. Whatever the mechanism of herbicide tolerance, genes ultimately determine the characteristics of the proteins. Researchers either search for the genes of an organism which already possesses a detoxification mechanism (such as GOX from O. anthropi), or they add chemical reagents to plant cells in vitro (i.e., in cell culture) that change the genetic code and produce an "altered" enzyme (i.e., one with less affinity for glyphosate). Presently, only canola plants have been successfully engineered to contain a functional GOX enzyme (1). However, all the commercial RR crops contain a tolerant EPSPS gene. For soybean, cotton, and canola the glyphosate-resistant EPSPS was obtained from a soil bacterium in the genus Agrobacterium (strain CP4) (1, 15, 18). For corn, the source of EPSPS was its own cloned gene that had been mutagenized in vitro (i.e., in cell culture) (20). This technique involves changing the DNA bases of cultured plant cells by adding mutagenic chemical reagents. Resulting changes in DNA bases could slightly affect the amino acid composition of the host (i.e., corn) enzyme. Normally, mutagenesis will produce nonfunctional enzymes, but in some cases a few changes in amino acid sequence can still produce a functional enzyme. With the mutagenized corn line, the resulting EPSPS was 99.3% similar to the nonmutagenized EPSPS and still functional (i.e., it produced the aromatic amino acids), but it was resistant to the effects of glyphosate (20). The development of RR corn using a mutant version of its own EPSPS gene followed research nearly a decade earlier where petunia EPSPS was successfully altered and then reintroduced into the plant to effect tolerance to glyphosate (1,13). Preparing the Genes for Transfer to Plants Scientists have honed to a fine art the isolation of tolerant GOX or EPSPS genes. Before transfer to recipient plant cells, however, the genes must be modified to be capable of translation into proteins. Basically, the genes are linked to other pieces of DNA that serve as start and stop signals (promoter and terminator sequences, respectively) for "reading" the herbicide-tolerant gene. Modification of the desired-trait gene is accomplished in an intermediate organism or host known as a vector. The most commonly used gene vector is a nonpathogenic strain of the E. coli bacterium that we all carry in our intestines. The genetics and structure of the E. coli chromosome are very thoroughly understood. More importantly, E. coli, like many other bacteria, contain in addition to their chromosome a smaller piece of double stranded DNA called a plasmid. Plasmids have the unique ability to replicate themselves independently of cell division. When they replicate, they can make numerous copies of desirable genes. Thus, E. coli can serve as a factory for gene synthesis or cloning; therefore it makes an excellent vector for transferring genes from one host to another. Using various tricks of the trade, the molecular biologist piece-by-piece links the desirable sets of promoter and terminator DNA to the E. coli plasmid that will allow translation of the herbicide-tolerant gene into the EPSPS enzyme. These "translator" sequences of DNA come from other plants and their naturally associated viruses. For example, the source of the promoter for soybean and cotton was the cauliflower mosaic virus (15, 18); a rice promoter DNA sequence was used for corn (1) (Table 1). A terminator sequence, which signals the end of the gene message, was supplied by attaching part of an Arabidopsis gene called nopaline synthase to the plasmid vector. Neither the promoter nor terminator sequences are translated into a protein product. |
| TABLE 1 | ||||
| Source of trait genes and ancillary genetic elements in Roundup Ready Crops | ||||
| Canola | Corn | Cotton | Soybean | EPSPS | Agrobacterium CP4 (1, 15, 18) |
Corn (20) (mutagenized) |
Agrobacterium CP4 (1, 15, 18) |
Agrobacterium CP4 (1, 15, 18) |
| CTP (17) | Arabidopsis | Sunflower & Corn | Arabidopsis | Petunia |
| GOX | Ochrobactrum anthropi strain LBAA (1) |
Not Present | Not Present | Not Present |
| Promoter Sequence |
Figwort Mosaic Virus |
Rice (1) | Cauliflower Mosaic Virus (15, 18) |
Cauliflower Mosaic Virus (15, 18) |
| Terminator Sequence |
Pea | Agrobacterim tumefaciens |
Arabidopsis | Arabidopsis |
| Antibiotic Resistance Marker Gene (15) | Streptomycin (not expressed) | Beta-lactamase (not expressed) | Neomycin phosphotransferase II (expressed) | Neomycin phosphotransferase II (not expressed) |
Other DNA sequences and/or genes are spliced onto the vector plasmid to aid proper functioning of the herbicide tolerant gene after it is transferred to the plant cells. For example, plant EPSPS is synthesized with a small, attached protein called the chloroplast transporter peptide (CTP). This peptide helps carry the EPSPS from its site of synthesis in the cytoplasm to the chloroplast. The peptide is cleaved from the EPSPS at this point to make it a functional enzyme. The source of the CTP DNA is the petunia plant for soybeans, the Arabidopsis plant for cotton and canola, and a combination of sunflower and corn itself for corn (1, 15, 18) (Table 1). Building the plasmid vector with all the appropriate genes and DNA sequences is not a matter of simply throwing DNA at a bacterial cell. Not all E. coli cells will contain the right combination of elements on its plasmid. To help select only the E. coli cells containing the plasmids with the right combination of genes, marker sequences of DNA are also linked to the plasmid. Some common markers are genes for antibiotic resistance (Table 1). For example, the plasmid used to make RR cotton and soybean contain a gene coding for an enzyme (NPII) that makes bacteria resistant to neomycin. Such resistance is already widely disseminated among bacteria in the environment (7). When bacterial cells are exposed to neomycin, plasmids without the linked EPSPS and NPII gene will die. The remaining living cells will be further cultured to build up large amounts of the vector plasmid. Gene Transfer Techniques The bacterial plasmids can be introduced into plant cells in one of two ways. The oldest way of transferring DNA is to allow the vector bacteria to "mate" with a plant parasitic bacterium called Agrobacterium tumefasciens. A. tumefaciens is normally responsible for crown gall disease, but this strain's DNA is disarmed of disease traits without affecting its natural ability to transfer its plasmids directly into the plant cells (1, 15). The recipient plant cells (embryonic-like plant tissue known as a callus) are co-cultured with A. tumefaciens containing the engineered plasmids, which are then "injected" into the cells. Canola and cotton cells were transformed using Agrobacterium, but the technique does not work well with grasses. A more recent method for transferring genes is to shoot them into the plant cells. The E. coli cells are broken apart to recover the engineered plasmids. The plasmids are coated on miniscule tungsten or gold particles and fired from a gun-like device into a plant callus culture. Some of the DNA moves to the nucleus of the calli cells where it is incorporated into the genome. Soybeans and corn were transformed using this ballistic technique (18). Regardless of which gene transfer technique is employed, not all of the DNA will be successfully incorporated into the plant genome. Thus, another round of selection is imposed on the cultured plant tissue. Basically, the plant tissue is exposed to different doses of glyphosate, and the tissue showing no signs of toxicity is grown up into a whole plant. The resulting plants are allowed to flower, pollinate, and produce seed for further testing. Technology Critics Are Skeptics, Too As a proponent of skepticism in scientific research and teaching, I find it perfectly logical for gene technology critics to pose fanciful "what if" questions and worst case scenarios. In essence, these hypotheses are addressed by the Federal regulatory agencies when assessing the safety of transgenic crops (see "Regulating Herbicide Tolerant Plants," AENews No. 172, August 2000). The broad concerns about herbicide-tolerant genes are essentially the same as those of the insecticidal Bt transgenic technology (5)--food safety and ecological effects. For herbicide tolerant genes, however, safety of the herbicides is also questioned. Therefore, focusing specifically on Roundup Ready crops, five questions immediately come to mind. Do engineered RR genes have unintended effects on other plant genes or traits? Is plant metabolism sufficiently affected to produce new toxic proteins or allergens? Are RR crops nutritionally equivalent to traditionally bred crops? What do we know about the safety of glyphosate herbicide? Can RR crop genes escape to other plants and create superweeds? Epistasis and Pleiotropy When the herbicide tolerance gene is transferred to plants, no one knows exactly where in the plant genome the DNA sequences are inserted, even though the gene is completely functional. One concern has been that random insertion of genes may either adversely affect or alter the expression of other genes or traits. Single genes are known to affect the expression of other unrelated genes (epistasis), while the protein produced by a single gene can have effects on multiple plant traits (pleiotropy). Thus, not knowing exactly where engineered genes are located in the plant genome makes some people nervous because of the possibility of abnormal epistatic and/or pleiotropic effects. Some envisioned problems include poor agronomic performance, susceptibility of crops to disease, production of new toxins or allergens, and nutritional differences from conventionally bred crops. When the U.S. Food and Drug Administration (FDA) implemented its 1992 "new" plant variety foods policy (6), it examined the possible consequences of epistatic and pleiotropic effects in RR crops (11). Unintended gene effects and plant traits can be tested directly and indirectly. Direct tests include studies of the inheritance and expression of the new genes in the recipient crops. Indirect tests include studies for plant agronomic performance, toxicity, allergenicity, and nutritional equivalence. Despite not knowing exactly where the engineered genes are located on the plant chromosomes, scientists are able to measure directly the number of insertion points in the plant. The number of insertion points is important, because a high number of random insertions would have a greater probability of causing unpredictable epistatic or pleiotropic effects, assuming the plant survived the genetic engineering event in the first place. Knowing that there are only one or two insertion points in the genome of a plant that can then be bred through several generations of fertile seed production, scientists can confidentally predict a very low likelihood of unintended genetic effects. Pertinently, RR corn and soybean have been shown to have only one or two copies of the glyphosate-tolerant gene at a single chromosomal insertion point (18, 20). A second direct way to determine the probability of epistatic and pleiotropic effects is to backcross (i.e., mate) the transgenic variety with its conventionally bred (i.e., isogenic) cultivar from which it was derived or to mate it with other cultivars. In these experiments, the breeder is ensuring that the inheritance of the herbicide tolerant gene trait is stable over numerous generations and that the resulting plants grow, yield, and reproduce normally in the field. If the engineered gene had unpredicted effects, then you might expect agronomic failure when each generation of seeds is grown under a variety of environmental conditions in numerous locations. Genetic backcrossing studies with RR cotton, corn, and soybean show that the engineered EPSPS gene segregates during pollination in a manner consistent with typical dominant gene inheritance rules (15, 18, 20). One indirect method for testing unintended epistatic and pleiotropic effects is to ensure that the gene expresses itself similarly among each cropping cycle. Levels of EPSPS enzyme were found to be similar in RR cotton and soybean leaves and seeds grown in two successive growing seasons prior to commercialization (15, 18). More importantly, agronomic performance of RR crops was measured repeatedly in many plot locations around the United States over several growing seasons. For example, RR soybeans were tested in about twenty locations around the United States during each growing season from 1992 to 1994. Different rates of Roundup were applied to different growth stages at one or two different times; furthermore, other registered soybean herbicides were tested for comparison to glyphosate. Visible crop injury and yield of RR soybeans were not significantly different than the isogenic controls at nearly every single site during all three years of the study (3). Thus, the stable field performance of RR soybean at least over three seasons lead to the conclusion that the transgenic EPSPS was not behaving any differently than the EPSPS of the isogenic line. Unintended Byproducts: Toxic Proteins and Allergenicity The concern over the possibility that novel gene insertions might cause plants to unleash production of toxic proteins or allergens leads to tests that also indirectly address the issue of epistatic and pleiotropic effects. Even before conducting the tests, however, the specific biochemistry of gene and plant metabolism can be examined to glean some answers. For example, while concern over toxic/allergenic byproducts of RR crops is genuine, it seemingly implies that the conventionally bred crops do not naturally contain toxicants and allergens. Yet, naturally occurring soy lecithin can cause severe nausea, vomiting, and diarrhea if not removed and destroyed by proper soaking and cooking (6). Plants in the family Cruciferae (which includes canola) contain glucosinolates that can impair thyroid function (6). While risk, or the probability of an adverse toxicological or allergenic reaction, can never be zero, close examination of RR technology suggests that the transgenic versions of crops should essentially have no more risk for toxic or allergenic effects than the conventionally bred versions. Recall that all plants contain the EPSPS enzyme. The difference between the plant enzyme and the bacterial source is in the amino acid sequence of the protein, not in its physiological functions (9). The changes in amino acid sequence greatly reduce the tendency of glyphosate to bind to the enzyme, but do not completely negate binding at extremely high doses of glyphosate. Given that variations in EPSPS protein among different food sources is also due to differences in amino acid sequence, it is unlikely that humans would have any trouble handling the RR EPSPS (9). In fact, our intestinal tract has a wonderful ability to digest many plant proteins into either their constituent amino acids or small chains of amino acids called peptides. In the extreme acid environment of the stomach, many proteins are at least partially degraded. A common method for testing allergenicity is to place the isolated protein extract into a simulated gastric environment that contains the stomach enzyme pepsin and has a pH of 1.2. RR EPSPS degraded in fifteen seconds under such conditions, an amount of time similar to many common plant proteins (12). Furthermore, there were no detectable protein fragments resulting from the digestion. Known allergenic proteins persist much longer under simulated gastric conditions. Another method for testing for allergenicity is to determine whether the amino acid sequence of the transgenic protein has any similarity to known allergens. RR EPSPS also passed this test (9). Soybean extracts from RR seeds were tested for their ability to react with soybean-specific antibodies taken from the blood of individuals allergic to soy products (2). The reactions were identical between transgenic soybeans and their conventional parent cultivars. Nutritional Equivalence Another indirect method to test for unintended consequences of inserting new or altered genes is to study the nutritional equivalence between parental lines from which a transgenic crop was bred and several generations of the transgenic cultivars. The harvested seed can also be fed to animals to examine for toxic effects and more subtle effects on growth. The concept of substantial nutritional equivalence between new food varieties and their conventional counterparts is a principle adopted internationally by the World Health Organization (WHO), the United Nations Food and Agricultural Organization (FAO), and the Organization for Economic Cooperation and Development (OECD) (20). The principle asserts that if a new food or feed derived from conventional breeding or genetic engineering is substantially equivalent in standard nutritional parameters to its conventional counterpart, then the new food should be considered equally safe. Nutritional parameters were studied for several generations of RR crops, and results from RR corn, cotton, and soybean have now been published in the peer-reviewed literature (16, 19, 20, 21). Commonly measured parameters include content of protein, oil, ash, fiber, carbohydrates, and amino acids. No statistically significant differences were found between the transgenic cultivars and the parental strain of any of the crops, nor were differences found between different years of cultivation. Critics have pointed out that perhaps RR crops may not show glyphosate injury symptoms when sprayed directly with Roundup, but the plants may still be under enough stress to alter their normal nutritive value. Nutritional compositional analysis has been studied comparing RR soybeans sprayed directly with glyphosate and untreated RR soybeans (21). Furthermore, the crops were grown in soil that had been treated the previous growing season with glyphosate prior to soybean seedling emergence, controlling for the possibility that old residues might enter the crop through the root system. When compared to the untreated nontransgenic parental soybean cultivar, no statistically significant differences were noted in nutritional composition of two generations of RR seeds. In addition to standard nutritional composition analysis, recognized beneficial plant chemicals like phytoestrogens in the biochemical group known as isoflavones have also been measured in soybeans (19, 21). Again, no differences were discovered in isoflavone content between RR soybeans sprayed with glyphosate, unsprayed RR soybeans, and the unsprayed glyphosate-susceptible parental cultivar. Of course, conducting a nutrient analysis may still miss subtle biochemical effects. So the question becomes whether animals grow normally when fed RR crops. Livestock are perfect subjects for testing this hypothesis because a major part of their diet is made up of the ground grain. A diet of 50-60% (by weight) corn was fed to broiler chickens from two to forty days old. No differences were found in growth, feed efficiency, and fat pad weights between chickens fed RR corn and the parental nontransgenic control grain (20). Similarly, growth, feed efficiency, and muscle and fat tissue were not affected in rats, broiler chickens, catfish, and dairy cows fed conventional or RR soybeans (8). Compositional analysis of cow milk revealed no significant nutritional differences (8). In summary, no problems with RR crops related to agronomic performance, toxicity, allergenicity, or nutritional and phytochemical equivalence surfaced during several years of pre-commercial testing. Given the sound understanding that the glyphosate-tolerant EPSPS gene has a single insertion site on the plant genome, and the gene is stably inherited in backcrosses to parent cultivars as a typical dominant character, the probability of unintended crop and human safety concerns seems remote. Given widespread RR soybean cultivation in the United States without reported problems over five additional commercial growing seasons (4), one wonders how long to wait before declaring epistatic and pleiotropic effects a nonissue. Substantial Equivalence and Ecological Concerns The principle of substantial nutritional equivalence might be analogously applied to the two remaining concerns about RR crops--glyphosate safety and potential for superweeds. Is the widespread implementation of the technology doing anything to the environment that conventional agriculture has not already wrought? Might there, in fact, be environ-mental benefits from RR technology that surpass conventional crop management? Does more widespread use of glyphosate pose a substantially different risk than the amount currently used for weed control? Is glyphosate perhaps "greener" than other herbicides? Get ready for subsequent issues of this newsletter; I will round up the answers to these burning questions. Dr. Allan S. Felsot is an Environmental Toxicologist with Washington State University's Food and Environmental Quality Laboratory. He can be reached at (509) 372-7365 or afelsot@tricity.wsu.edu. REFERENCES
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Last Updated on 12/4/00 Email: information@biotech-info.net |
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