Editor’s Choice: Evaluating the Potential for Adverse Interactions within Genetically Engineered Breeding Stacks

Plant breeding has a long history of developing varieties with desirable traits in response to the needs of both growers and consumers. Although the bases for most of these traits are not known genetically or biochemically, conventional breeding combines these multiple traits to create new hybrids and stable varieties that are safe and not generally subject to safety assessment. With the advent of genetic engineering, a tool for incorporating additional traits has become available to plant breeders. The safe application of genetic engineering to food and feed crops is widely acknowledged as a useful tool in addressing global agricultural challenges, including population growth and climate change. As used here, the term genetically engineered (GE) stack refers to a plant in which two or more transgenic events (i.e. single-locus insertions) that have been separately assessed for safety have been combined by conventional breeding (Table I). In recent years, increasing numbers of GE stacks have been planted, the first of which offered combinations of insect and herbicide tolerance genes to combat a wider range of pests and weeds than covered by the single events (Que et al., 2010; James, 2011). Two main questions arise when considering the food and feed safety of GE stacks: (1) does incorporation of more than one event increase genomic instability, and (2) can potential interactions between the products of the combined events impact safety? A related paper considered the stacking of events in light of the plasticity of plant genomes and concluded that enhanced genetic instability from a transgene or from common sequences in two or more transgenes is remote (Weber et al., 2012). This paper addresses the second question of potential interactions between events and their products combined in a stack, reviews the basic principles of plant breeding and its history of safe use, and extends these principles to the feed and food safety of events combined through the same processes used in conventional breeding of non-GE plants. Potential environmental impacts are outside the scope of food and feed safety. The new varieties developed through modern biotechnology are identified by a number of terms, including genetically modified (GM), GE, transgenic, biotech, recombinant, and plants with novel traits. The term GE is used here as defined by Weber et al. (2012). For these reasons, the term GE is preferred over the term GM. There are many methods encompassed by the general term conventional breeding, including wide crosses and selection, mutagenesis, and somaclonal variation. When the parental species are not closely related, the cross may be facilitated by embryo rescue, somatic hybridization, or x-ray-induced translocations. The term interaction, as used in this paper, refers to an effect, such as a new or modified metabolic activity, resulting from a combination of transgenes. An example of an interaction is protein-protein binding resulting in a novel effect only seen with a specific combination of proteins, for instance, protein cofactors or subunits for the same enzymatic complex or subcellular metabolic binding reaction. Examples can also include a direct metabolic interaction that would inhibit or activate components in a metabolic pathway shared by the proteins newly combined in the GE stack or components of independent metabolic pathways that indirectly interact by way of a common metabolite. Thus, interactions within GE stacks generally refer to the metabolic or physiochemical interplay between the products of transgenes or between the product of one transgene and the second gene, rather than between the two genes themselves. Conventional breeding has a long history of safe use despite the presence of antinutritional factors, toxins, and allergens in crops. There is no evidence that a random genomic change in a crop has resulted in a novel food or feed safety issue (Weber et al., 2012). Historically, humans have selected desirable traits that arise from the crop’s genomic plasticity and interactions between genes. As breeding became more advanced, new methods were applied to select and combine desired traits, which also modify the genome as a consequence. Plants produce a multitude of metabolites that provide various functions. These include signaling activities in response to environmental stress or attack from plant pathogens and pests. Some metabolites have beneficial effects, while others are toxic when fed at high levels to sensitive animal species (Ames et al., 1990). Although particular metabolites tend to be specific to some plant families, there are metabolites of concern in a number of common food and feed crops, including apple (Malus domestica), apricot (Prunus armeniaca), Brassica spp., celery (Apium graveolens), cucumber (Cucumis sativus), lima bean (Phaseolus lunatus), potato (Solanum tuberosum), cherry (Prunus avium or Prunus cerasus), and sorghum (Sorghum bicolor; Beier, 1990; D’Mello et al., 1991; Stewart, 2009). Examples of such metabolites include alkaloids, lectins, glucosinolates, furanocoumarins, cyanoglucosides, nicotines, and phytoestrogens. Some of these can impart bitter taste, and conventional breeding has repeatedly reduced their levels to acceptable concentrations (Drewnowski and Gomez-Carneros, 2000). There are few documented cases in which breeding led to an unacceptable level of a metabolite. Following reports of bitter taste, the potato var Lenape, which was developed from a cross of potato to a wild Solanum species, was found to contain doubled levels of glycoalkaloids (Anonymous, 1970). The only other documented incident of unacceptable metabolite levels associated with plant breeding is that of a disease-resistant celery containing elevated levels of furanocoumarins, which may have contributed to dermatitis among grocery store personnel (Berkley et al., 1986; Seligman et al., 1987). The environment can also play a role in impacting furanocoumarin levels; a later report of dermatitis among celery harvesters in southern Israel was attributed to delayed harvest, and hence more mature plants being handled, due to the Gulf War (Finkelstein et al., 1994). Similarly, reports of toxic squash (Rymal et al., 1984) and zucchini (Herrington, 1983) appear to have been limited to individual plants within otherwise widely grown varieties, suggesting the higher toxin levels were due to environmental conditions or mutations. Despite these few instances, these crops remain safe for food or feed use. Although breeders recombine tens of thousands of genes with virtually infinite potential interactions, to our knowledge, there has never been a report of a completely novel toxin or allergen appearing in a genus as a result of conventional breeding. The development of an interspecific somatic hybrid of potato (S. tuberosum and Solanum brevidens) containing demissidine (Laurila et al., 1996) has often been cited as proof that novel toxins can arise during breeding. In fact, Jadhav et al. (1981) documented the presence of demissidine in potato more than a decade before it was found in the hybrid. To guard against unexpected increases in levels of known toxins, breeders have instituted screens for these compounds in new varieties before they are released. Examples include cotton (Gossypium hirsutum), potato, lima bean, and canola (Brassica napus), because they contain known compounds that can impact food or feed safety. However, breeders cannot and do not screen for de novo compounds. Hundreds of thousands of varieties have been bred without the emergence of any novel allergens or toxins, indicating that the likelihood of such events is virtually zero (Stevens, 1974). Improved crop varieties produced by conventional breeding are the cornerstone of food production in the world today. Thus, the ways in which risks are managed during conventional breeding of non-GE crops, which includes testing for any known concerns, sets the framework for the food and feed safety assessment of GE stacks. As is the case for any other new variety or hybrid, GE stacks are only considered for market introduction if the combined traits have met the intended thresholds for efficacy and stability. Newly developed GE events undergo rigorous safety assessments by the developer and by regulatory agencies prior to their commercial release. These assessments evaluate the impact of both intended and potential unintended effects on food, feed, and other aspects of crop safety. Assessment criteria and objectives are based on national priorities and on international standards, such as those of the Joint Food Standards Program of the Food and Agriculture Organization of the United Nations and the World Health Organization (FAO/WHO, 1996, 2000), as well as Codex Alimentarius Commission recommendations for conducting food safety assessments of GE crops (Codex Alimentarius Commission, 2009). Codex principles and guidance are widely followed for single events. The key components of GE crop safety assessments include (1) descriptive information on the transgenes and inserted recombinant DNA; (2) detailed characterization of the DNA insert relative to the native genome, in planta concentration and stability of the products of the transgene(s), analysis of plant phenotype, and descriptive information such as efficacy, mechanism or mode of action of the new trait(s), and crop management considerations; (3) evaluation of the safety of the products of the transgene(s) in the context of common commercial crop practices and uses; and (4) comparative safety assessment for suitability as food and/or feed. Such analyses might include a determination of expression levels of known allergens and toxins, and overall composition, including nutrients, antinutrients, and selected metabolites for that crop (FAO/WHO, 1996, 2000; Cellini et al., 2004; Codex Alimentarius Commission, 2009; Thomas et al., 2009). Assessments of nutrient composition and agronomic characteristics, as well as an assessment of potential allergenicity and toxicity, are widely recognized as significant components of GE crop safety assessments (FAO/WHO, 1996, 2000; Metcalfe et al., 1996; Kuiper et al., 2001; Cellini et al., 2004; Delaney et al., 2008; Codex Alimentarius Commission, 2009; Thomas et al., 2009). Differences between the GE crop and appropriate comparators are evaluated in terms of biological relevance, magnitude of difference, exposure, and impact on food or feed safety to determine the need for further investigation. The comparators may include a closely related non-GE variety and other commercial GE and non-GE varieties. The International Life Sciences Institute’s Crop Composition Database (http://www.cropcomposition.org; International Life Sciences Institute, 2006) may also serve as a reference for comparison for some crops (Ridley et al., 2004). These holistic analyses also take into account interactions of the transgenes with endogenous genes and their products. Agriculturally important plants express approximately 25,000 to 50,000 genes at any given time. The safety assessment evaluates the GE crop phenotype, which includes interactions between the event and the host genome that may impact safety. Once the safety assessment is completed, conventional breeding is used to incorporate the event into different genetic backgrounds, without undergoing additional assessment. The developer of the resulting food and feed products is, however, responsible for the safety of those products and for meeting all relevant statutory and regulatory requirements, as is the case for all foods and feeds. Even within a single genus having species that differ in ploidy level, the number of genes within a plant can vary. For example, some 400 genes are not common in the historically important maize (Zea mays) inbreds B73 and Mo17; each of these genes is found in B73 but not in Mo17 or vice versa (Springer et al., 2009; Lai et al., 2010). Similarly, of the approximately 46,000 of genes in soybean (Glycine max), 856 genes were present in some but not all of the soybean genotypes tested (Lam et al., 2010). Of the two potato genotypes with sequenced genomes, 275 genes are found in only one or the other et al., 2011). Thus, different non-GE genotypes in novel combinations in novel interactions, but no safety have been this of novel interactions is to be by the stacking of transgenes. Despite the genetic in crop most regulatory agencies do not new non-GE varieties developed by conventional breeding there is a known safety concern or associated with the new variety of and Food and of the Food on Organization and Health Food This is based on the that conventional breeding is as As defined conventional breeding traits from genetic interactions of genes and their products are in breeding to produce new crop varieties. In plant breeders through crosses that combine different different et al., in some different genes et al., 2011). 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