Table of Contents

1. Introduction
2. Stability of expression and inheritance of transgenes
3. Safety of consumption of the transgene
4. Effects of random transgene insertion on crop traits
5. The effect of transgenes on the nutritional value of transgenic plant products
6. The structural stability of the transgene construct 

I. Introduction and Background

Biotechnology is an important tool for improving the characteristics of crops. Like plant breeding during the last century, biotechnology has been instrumental in increasing yields, reducing damage by pests and improving the quality and usefulness of end products. Modern methods of biotechnology have allowed crop scientists to make these improvements more rapidly, precisely and predictably than is possible with conventional plant breeding.

Biotechnology also provides crop scientists with greater flexibility for improving crops than conventional breeding. In the last half of the 20th century, plant breeders developed a number of techniques that allowed them to breed crops with plants in different species and, for some crops, different genera. Broadened access to genes in other species increased the number of genes available to plant breeders, but limits remained. Only those plants related to the crop could serve as potential sources of crop improvement genes. With biotechnology tools, a gene found in any species can be incorporated into a crop.

A second aspect of the greater flexibility provided by biotechnology involves changes within the crop genome, not adding genes from other organisms. Biotechnology allows scientists to use a crop’s genetic regulatory elements to control gene expression very precisely. For example, using the tools of biotechnology, scientists can significantly increase or decrease the amount of protein a gene produces well beyond the amounts typically found in the crop. In addition to controlling protein amount, crop scientist can control, where in a crop, the protein is produced by using tissue specific promoters. This highly specific fine-tuning of gene expression is virtually impossible with conventional plant breeding.

Nonetheless, the goals of plant breeding and biotechnology are identical: creating genetically diverse populations of crop plants, most of which have new, beneficial traits. The techniques for generating that genetic diversity differ. In plant breeding, the genome is recombined with another plant’s genome through sexual reproduction. In biotechnology, single genes with known, useful functions, are inserted into the crop genome using two techniques not available to plant breeders until the mid 1980’s.

• First, DNA elements, such as structural genes and the sequences that control gene expression, are assembled in unique ways. Genes in nature consist of three elements: promoter sequences that control when, where and how much of the transcript product (mRNA) is produced from the second element, the structural gene, and transcription end sequences that control the length and other regulatory characteristics of the transcript. Biotechnology allows plant scientists to form unique combinations of these three gene elements to change how and in what tissues genes are expressed. These unique genetic units are called “transgenes.”
• Secondly, the transgene must be inserted into the existing genome of the plant through the process of transformation. Transformation occurs at the level of single cells and results in the random placement of a new gene into the cell’s genome. Using plant tissue culture techniques, the cells are then regenerated into whole plants. Only those plants that contain the transgene are retained.

The remaining steps for creating a new, transgenic line are those that are followed during product development through conventional plant breeding. The iterative testing and backcrossing process that has provided us with high yielding, high quality crops is the same whether the new trait was provided to a crop by a plant relative through crossbreeding or a transgene created and inserted with molecular techniques.

The genetically diverse plant populations created through either plant breeding or biotechnology contain both superior and inferior genotypes. The inferior types are discarded; the superior types are retained and crossed with existing, proven high performance, “elite” lines. Progeny that inherit the new trait, whether it was added to the parental genome through breeding or biotechnology, are tested to ensure they exhibit the growth and crop production traits required for commercial production. The progeny that exhibit these traits are retained and backcrossed to the elite line; the resulting progeny are tested for growth and performance, and so on. This repetitive cycle of testing and backcrossing has consistently produced a safe and abundant food supply.

Plant breeders have begun to rely more heavily on the more precise methods of biotechnology to create genetically diverse lines because they can better predict the outcome of attempts to provide existing crops with new traits. In addition, genetic improvement through biotechnology is faster. In biotechnology, one gene of known function is inserted into the genome of an existing, high performance line; plant breeding adds thousands of genes to the elite line. Often, many years of backcrossing are required to rid the elite line of undesirable genes added to the elite line through breeding. A thorough analysis comparing the differences, similarities and relative risks of biotechnology with traditional breeding is written by Conner and Jacobs, 1999.

Though the scientific methods used to develop products with biotech tools have been developed over the past 70 years, some perceive the techniques as new and are asking questions. Some of these questions are:

• Are transgenes inherently unstable in either expression or inheritance?
• Are transgenes safe and wholesome for consumption?
• Does random insertion of the transgene cause unexpected changes in the transgenic crop, especially in its nutritional quality or safety?
• Do plant viral promoters used in many transgene constructions have a high propensity to recombine with the host plant genome or with organisms that might be exposed to the DNA of transgenics?

This narrative will examine some of the methods of biotechnology that are used by scientists to improve crops, as well as the safety evaluations that must precede transgenic crop commercialization. The focus will be on what these methods are, on how they are used and on some of their potential shortcomings.

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II. Discussion of biotechnology methods used in crop development

Transgenes inserted into the crops often have no homologous counterpart in the host plant genome. Questions have been raised about whether the lack of a homologous gene will affect transgene expression or its stability of inheritance.

2.1   Transgene Expression

During the early stages of transgenic crop development, some plants containing the transgene do not exhibit the trait encoded by the transgene. This phenomenon is known as gene silencing, which is defined as loss of gene expression due to pre or post-transcriptional mechanisms and not to gene deletion¹. Some instances of gene silencing in transgenic crops are intentional because the goal of the genetic modification is suppression of the protein produced by a specific gene or genes. However, unintentional gene silencing can occur during the development of transgenic crops. These plants would exhibit characteristics that would not meet performance standards and would be discarded before commercialization.

The silencing of an introduced transgene is not the result of introducing non-homologous DNA to the crop genome. In fact, the opposite is true. Gene silencing actually depends upon the introduced gene having substantial sequence homology with an endogenous gene in the host plant (Vaucheret et al., 1998; Gura, 2000). This effect was discovered when a petunia chalcone synthase transgene caused suppression of the endogenous petunia chalcone synthase genes, resulting in changes in flower color (Napoli et al., 1990) Therefore, an introduced transgene with a homologue in the host plant genome is more likely to be silenced than a gene with no homologous counterpart.

Gene silencing depends upon the structure of the RNA produced by the introduced gene rather than the method of introduction (Grant, 1999; Selker, 1999). Normally, the RNA moving freely around a cell is single-stranded messenger RNA (mRNA), the intermediate between host genes and the proteins they encode. If the RNA transcribed from the new gene, introduced by biotechnology or breeding, has a sequence that is complementary to the endogenous mRNA produced by the host, then double - stranded RNA (dsRNA) will be formed (Baulcombe, 1996). Also, in transgenic crop development, if the integration of a transgene at a locus is complex, then dsRNA may unintentionally be generated (Grant, 1999; Selker, 1999). It is well documented that dsRNA and other aberrant nucleic acid structures can result in gene silencing (Fire et al., 1998; Waterhouse et al., 1998). Therefore, in both examples just described, the dsRNA’s effectively block translation of mRNA into the protein, leading to unintentional silencing of the introduced gene. In addition, if the introduced gene is homologous to part of the host’s genetic sequence, then both the host gene and introduced gene are silenced.

Even though some have claimed this does not occur in plant breeding (Hansen, 2000), gene silencing by dsRNA can be caused whether the gene causing the co-suppression is introduced into the plant by molecular techniques or breeding (Matzke et al., 1993; Neuhuber et al., 1994).

As mentioned above, in transgenic crop development, not all gene silencing is unintentional. Gene silencing can be used to produce a desired or improved trait that depends on inhibiting gene expression (Kinney and Knowlton, 1998). If dsRNAs are introduced intentionally to silence endogenous genes, then the phenomenon of silencing is independent of the integration site (Waterhouse et al., 1998). This precise and targeted suppression of protein production is not possible with plant breeding.

2.2   Stability of Inheritance

The lack of a homologous gene in transgenic crops can affect the stability of inheritance. During the pairing of homologous chromosomes in meiosis, lack of homology might result in the transgene being eliminated.

In the earliest stages of transgenic crop development, the population of transgenic plants contains both stable and unstable lines (Iglesias et al., 1997). As is true of the variation in phenotypic expression described above, only those lines that demonstrate stable inheritance of the transgene are retained. Once a stable line has been selected from this population, it normally remains stable over many generations and across many environments (Iglesias et al., 1997; Padgette et al., 1995; Delannay et al., 1995).

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Since transgenes represent new genetic constructs, questions have been raised about:

• the safety of consuming transgenic DNA, and
• the possible movement of the transgene out of the crop and into the genome of the animal or person that eats a transgenic crop or derivative food product.

Both the World Heath Organization and the U.S. Food and Drug Administration concluded that there is no inherent risk in consuming DNA, including that derived from transgenic crops (WHO, 1991; US-FDA, 1992). The basis for their conclusion is that humans and other animals have always consumed DNA from a wide variety of sources including plants, animals, bacteria, parasites and viruses. Both transgenic DNA and DNA from non-transgenic crops are made of the same 4 nucleotides - adenosine, guanosine, thymidine, and cytosine. The addition of transgenic DNA does not add any new chemical entity, so the digestive system is well adapted to processing transgenic DNA. DNA given directly to steers was degraded into mononucleotides by the animal’s digestive tract in 4 hours (McAllen, 1982).

DNA is an essential component of all living things, so it is present in nearly all foods humans consume, but in relatively small amounts. The total amount of DNA in food contributes only 0.02% to the total dry matter of the food (Watson and Thompson, 1998). Foods derived from transgenic crops contain only one part in 100,000 more DNA than traditional foods. To put this into perspective, a dairy cow eating transgenic corn would consume 0.006.8 mg/day of transgenic DNA compared to a daily total intake of 680 mg of non-transgenic DNA.

The specific sequence of nucleotides in a transgene that is responsible for the production of the novel protein is destroyed once the digestive process begins. Rasche (1998) conducted a direct analysis of the stability of transgenic DNA that encodes the PAT gene in canola. In this study the transgene was completely broken down into nucleotides in swine, chicken, and cows within one hour at 37oC and pH 1.5. This rate of degradation of transgenic DNA is similar to other non-transgenic DNA (McAllen, 1982).

For an ingested transgene to be integrated into the genome of the organisms that has consumed it, at least two, and often three events must occur. First, the transgene must be taken into cells as an intact gene rather than as individual nucleotides or very small oligonucleotides. While a number of studies have shown that fragments of purified plasmid DNA and plant chloroplast DNA have been intestinally absorbed at low levels in mice and cows (Schubbert et al., 1997; Schubbert et al., 1994; Schubbert et al., 1998; Doerfler and Schubbert, 1998; Klotz and Einspanier, 1998, 2000), there is no evidence that whole, intact genes are absorbed by cells.

To date, fragments of recombinant DNA from transgenic crops have not been detected in animal tissue. Klotz and Einspanier (1998) and Khumnirdpetch, et.al. (2001) showed that the gene responsible for glyphosate tolerance in soybeans was not detectable in either the blood or milk of cows or the liver, skin and meat of chickens, while other studies (Eispanier, et.al., 2001; Flachowsky, et.al., 2000) could not detect any fragments of the Bt gene in any tissue samples from cows, steers or chickens that were fed grain from Bt corn.

The two additional events that must occur for these observations to be of concern are the integration of the plant DNA into the mammalian genome and the expression of the plant DNA. There is no evidence that ingested plant genes are integrated into mammalian genomes and maintained in a stable manner (Hohlweg and Doerfler, 2001). Without stable integration into DNA that will permit replication of it, such as a chromosome, meaningful expression is not possible.

To determine whether DNA that is absorbed from the digestive tract can be expressed, scientists tested whether extensive feeding of DNA to mice resulted in detectable levels of mRNA and proteins in several organs (Holweg and Doerfler, 2001). Mice were fed 50ug of DNA per day for three weeks, but no mRNA or protein encoded by the DNA were detectable in liver, spleen, blood or intestinal epithelia.

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As described above, the current techniques for creating a transgenic plant do not allow for the specific placement of a transgene within the host genome. Because the site of integration of transgenes is random, some have wondered if the transgene could insert within a gene, disrupting the gene and/or gene regulation and, as a result, alter the plant’s composition and development. Others have also questioned whether the transgene promoter might activate adjacent genes at the insertion site, leading to the unintentional increase in protein production by those genes or activation of unexpressed genes.

Random insertion of transgenes can disrupt the expression and regulation of endogenous plant genes. Scientists are working on methods that permit the precise integration of transgenes in pre-determined chromosomal sites. In addition, there is evidence that the promoter used in most transgenic crops currently on the market, the 35S promoter from the cauliflower mosaic virus, can enhance the activity of adjacent genes (Unger, et.al.. 2000; Odell, et.al., 1998). If this effect were large and had very long-range influence within the genome, it could lead to unexpected effects due to the transgene. However, the transcriptional enhancement of endogenous plant genes associated with the 35S promoter in transgenes, the distance over which this enhancement is quite small. As a result, widespread changes of multiple genes are very unlikely to occur.

If random transgene insertion leads to plant with an undesirable phenotype, whether through disrupting or activating endogenous plant genes, those plants are discarded during the crop development process. As described above, in transgenic crop plant development plant breeders select stable, high performance transgenic plants whose food and feed products are similar or superior to their non-transgenic counterparts. Particular attention is paid to nutritional quality and food safety in these assessments. These tests are discussed in detail below.

It is important to reiterate that during crop development, unexpected changes occur whether molecular techniques or classical plant breeding methods are used to produce new crop varieties. The methods of plant biotechnology, in which one or a few genes with known functions are introduced into the plant genome, are less likely to lead to unexpected changes than traditional plant breeding methodology, in which thousands of genes, many of unknown function, are recombined (OECD, 1986; National Research Council, 1989).

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Nutrient content of transgenic plant products is of paramount importance to consumers, regulatory officials and the biotechnology industry. Because unexpected changes can occur during the early stages of transgenic crop development, we must confirm that those unexpected changes have not altered the nutritional value of the crop or products derived from it. We establish nutritional value with two types of studies: biochemical analysis and animal performance studies.

5.1   Biochemical Assasys

Before a transgenic crop is brought to market, companies must show that the transgenic variety is “substantially equivalent” to the comparable non-transgenic variety in terms of the amount and availability of nutrients. To standardize the evaluations of transgenic crops and foods derived from them internationally, FAO/WHO (1996) recommended that ‘assessments based upon the concept of substantial equivalence be applied in establishing the safety of foods and food components derived from biotech organisms.’ (Substantial equivalency must also be demonstrated for agronomic properties, such as growth rate, yield, susceptibility to disease and fruit size, but the details of these assessments are not discussed here.)

To establish substantial equivalency, compositional studies must prove that the biochemical components of the plant, such as total protein, fiber, and mineral content, do not differ between the transgenic and non-transgenic varieties. The major nutritional elements in plant products are well known, and their amounts can be measured very accurately, thereby establishing whether the transgenic plant is nutritionally equivalent to its progenitor. The nutrients that are measured include the following: concentrations of individual micronutrients, such as vitamins and minerals; total amount of protein and concentrations of individual amino acids; total amount of fat and concentrations of specific fatty acids; total amount of carbohydrates and concentrations of starch and sugars; percent fiber; percent ash; percent water and total calories. Not only are the amounts of these nutrients assessed but also their bioavailability.

Certain crops naturally contain compounds that can affect a crop’s nutritional value and potential physiological impacts. For example, some plants, such as potato, contain naturally occurring toxins. If the transgenic crop is one that contains natural toxins or other substances with physiological impacts, we must confirm that insertion of the trangene has not increased the concentrations of these substances.

Because environmental conditions can affect the concentration of some of these substances, crops are grown at multiple locations over multiple years to generate the plant material used for substantial equivalency testing.

Therefore, the term “substantial equivalence” means that the transgenic crop and its non-transgenic counterpart are the same in terms of nutritional value, except for the intended improvement brought about by the introduction of the transgene and the protein encoded by the transgene. This protein is then subjected to a series of tests to establish its dietary safety.

If the crop improvement trait is a trait associated with improved nutrition, such as healthier oils, we must establish nutritional equivalency for all measures discussed above, except the intended nutritional improvement. Several new, nutritionally improved product candidates are in research and development pipelines. These transgenic crops have nutrient contents that, by design, differ significantly from their progenitors and are being developed to enhance foods and feed. Additional testing of these transgenic crops is required before they can be commercialized such as test for nutrient stability and effects of processing on nutrient content.

Virtually all of the transgenic plants currently under commercial cultivation have been modified for agronomic enhancement and not nutritional characteristics. Research by both industry and independent scientists has demonstrated no change in nutritional content of these transgenic varieties. [Padgette et.al., (1996); Hammond et al., (1996)].

5.2   Animal Performance Studies

In addition to conducting biochemical analyses to establish the nutritional equivalency of transgenic and non-transgenic crops, animal performance studies are conducted. These studies are designed to establish nutritional equivalency by measuring such variables as feed intake, weight gain, feed efficiency and the nutritional value of food products derived from the animals, such as milk, meat and eggs.

To date, more than 30 animal performance studies comparing feed derived from transgenic and non-transgenic crops have been conducted; more than 20 of these have been published in scientific journals. The animals used in these studies are very diverse and include the following: lactating cows, beef cows, calves and steers, sheep, swine, laying hens, broilers and catfish. Transgenic corn, soybeans, canola and sugar beets have been compared to the non-transgenic varieties from which they were derived. The length of the studies varies from 5 days to 2 years. Variables measured include feed intake, body weight gain, feed conversion, mortality, milk production, milk composition, rumen fermentation characteristics, digestible organic matter, true metabolizable energy, amino acid digestibility, daily weight gain, carcass weight, abdominal fat, percent fat, breast meat weight and composition and size of fish fillet.

In summary, these studies showed that there was no difference in either animal performance or composition of animal products when animals were fed transgenic or non-transgenic varieties. These studies are cited and discussed in great detail in Crops Derived from Biotechnology in the Animal Production Industry, which can be found, along with a list of references, on this website.

In summary, the nutritional quality of all transgenic crops must be assessed prior to commercialization. To date, all of these studies have demonstrated that transgenic crops are nutritionally equivalent to the non-transgenic varieties from which they were derived.

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As we mentioned earlier, biotechnology allows crops scientists to join unique combinations of genetic elements from various species to form a transgene. Some people have expressed concern that the transgene may not be inherently stable because it is composed of elements that do not occur contiguously in nature.

6.1   Mutation Rates

Studies indicate that transgenes appear to be no more susceptible to mutation than endogenous genes in the plant genome (Meyer, 1993; Meyer et al., 1992; Peterhans et al., 1990). Although point mutations in a transgene leading to amino acid changes have been observed in mammalian cell culture (Harris et al., 1993), this has not yet been observed in transgenes of plants (Russell, 2000). Thus, there is no reason to believe unexpected changes will occur due to alterations in trangenes themselves.

6.2   The CaMV 35S Promoter

The promoter sequence that is used most often to control gene expression in the transgenic crop plants currently marketed was isolated from a virus that commonly infects plants in the cauliflower family, the cauliflower mosaic virus, and is called 35S. The 35S promoter has properties that make it particularly useful in transgenic crop development: high levels of gene expression; active in many plant cells; one of the best-studied elements controlling gene expression in plants. Therefore, it is very effective in producing plants that express the desired trait and its comparatively long history of study makes it a good choice in transgenic crop protection applications. However, some have expressed concern that the promoter is prone to recombination.

6.3   Recombination and the CaMV 35S Promoter

Based on the complex integration patterns of DNA introduced into transgenic rice, Kohli et al. (1999) identified a sequence with high potential for recombination in the CaMV 35S promoter. Ho et al. (1999) interpreted this to imply that the CaMV 35S promoter would therefore provide a recombinagenic site in the genome of transgenics. They claimed that this might allow the 35S promoter to recombine with and activate dormant viruses in the genome of transgenic plants that utilized this promoter.

Morel and Tepher (2000) discounted the interpretation of Ho et al., citing work by Vaden and Melcher (1990) showing the promoter of CaMV was not a gene sequence prone to unusually high rates of recombination (a recombination “hot spot”) compared to the rest of the virus. They interpreted the observed high recombination rate in this instance to be due to the presence of a high concentration of DNA fragments following particle bombardment, a temporary and artificial condition known to increase the activity of cells’ recombination and repair systems. They also pointed out that the CaMV 35S promoter was present in three copies in the introduced plasmid used by Kohli et.al. (1999). This alone might account for the relatively frequent presence in recombination junctions they observed.

Researchers routinely observe resolution of complex integration patterns into simpler patterns during cell and plant culture. These cases, however, are the result of:

• isolation of distinct transformation events at discrete loci in different callus sectors, or
• segregation of discrete transformation loci of a cell line among plant progeny.

However, it is typically seen that both simple, single integration events and complex, co segregating events are maintained over multiple generations without visible changes in the insertion structures, as judged by Southern analysis, whether or not the 35S promoter is present.

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