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Critical Reviews in Plant Sciences

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Gene Flow, Risk Assessment and the Environmental Release of Transgenic Plants
Stephen Chandler & Jim M. Dunwell
a b a b
Florigene Pty. Ltd., 1 Park Drive, Bundoora, Victoria, 3083, Australia School of Biological Sciences, University of Reading, Reading, RG6 6AS, UK
Available online: 19 May 2008
To cite this article: Stephen Chandler & Jim M. Dunwell (2008): Gene Flow, Risk Assessment and the Environmental Release of Transgenic Plants, Critical Reviews in Plant Sciences, 27:1, 25-49 To link to this article: http://dx.doi.org/10.1080/07352680802053916
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Critical Reviews in Plant Sciences, 27:2549, 2008 Copyright c Taylor & Francis Group, LLC ISSN: 0735-2689 print / 1549-7836 online DOI: 10.1080/07352680802053916
Gene Flow, Risk Assessment and the Environmental Release of Transgenic Plants
Stephen Chandler1 and Jim M. Dunwell2
1 2
Florigene Pty. Ltd., 1 Park Drive, Bundoora, Victoria 3083, Australia School of Biological Sciences, University of Reading, Reading RG6 6AS, UK
Table of Contents 1. 2. INTRODUCTION ................................................................................................................................................26 MECHANISMS OF GENE FLOW IN PLANTS ...................................................................................................27 2.1. Asexual Propagation .......................................................................................................................................27 2.2. Seed Dispersal ................................................................................................................................................27 2.3. Pollen-Mediated Gene Flow .............................................................................................................................28 GENE FLOW AND AGRICULTURE ...................................................................................................................28 3.1. Crops That Have Escaped Cultivation ...............................................................................................................28 3.2. Crop-to-Crop Gene Flow .................................................................................................................................29 3.3. Gene Flow Between Crops and Wild Species .....................................................................................................29 IDENTIFICATION OF TRANSGENES IN PLANTS ...........................................................................................29 MEASURING GENE FLOW IN TRANSGENIC PLANTS ...................................................................................30 5.1. Experimental Trials .........................................................................................................................................30 5.2. Isolation Distances ..........................................................................................................................................30 5.3. Post-Release Mechanisms of Gene Dispersal .....................................................................................................31 ASSESSING THE PROBABILITY OF GENE FLOW FROM TRANSGENIC PLANTS ......................................32 6.1. Factors Affecting the Probability of Gene Flow ..................................................................................................32 6.1.1. Knowledge of the Biology of the Target Crop ........................................................................................32 6.1.2. Knowledge of the Distribution of Any Related Wild Populations .................................................................................................................................32 6.1.3. Centers of Biodiversity ........................................................................................................................33 6.1.4. Knowledge of the Compatibility to Related Species ................................................................................33 6.1.5. Reproductive Biology of the Transgenic Plant ........................................................................................33 6.1.6. Survivability .......................................................................................................................................33 6.2. Quantication of Gene Flow ............................................................................................................................33 6.3. Transgenic Crops and Gene Flow .....................................................................................................................34 6.3.1. Brassica napus ....................................................................................................................................34 6.3.2. Maize .................................................................................................................................................35 6.3.3. Soybean .............................................................................................................................................35 6.3.4. Cotton (Gossypium hirsutum) ...............................................................................................................35 6.3.5. Rice ...................................................................................................................................................35 6.3.6. Sugar Beet ..........................................................................................................................................35
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4. 5.
6.
Address correspondence to Stephen Chandler, Florigene Pty. Ltd., 1 Park Drive, Bundoora, Victoria 3083, Australia. E-mail: schandler@ origene.com.au
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26 6.3.7. 6.3.8. 6.3.9. 6.3.10. 6.3.11. 6.3.12. 6.3.13. 7.
S. CHANDLER AND J. M. DUNWELL
Sunower ...........................................................................................................................................36 Sorghum (Sorghum vulgare) .................................................................................................................36 Cucurbits ............................................................................................................................................36 Wheat ................................................................................................................................................36 Papaya (Carica papaya) .......................................................................................................................36 Alfalfa (Medicage sativum) ..................................................................................................................36 Trees ..................................................................................................................................................37
ASSESSING THE RISK OF GENE FLOW EVENTS ...........................................................................................37 7.1. Food and Pharmaceuticals ................................................................................................................................37 7.2. Fitness Traits ..................................................................................................................................................38 7.3. Insect-Resistant Crops .....................................................................................................................................38 7.4. Herbicide-Resistant Crops ...............................................................................................................................38 TECHNIQUES TO PREVENT GENE FLOW ......................................................................................................39 CONCLUSIONS ...................................................................................................................................................40
8. 9.
REFERENCES ............................................................................................................................................................41
1.
The release of genetically modied plants is governed by regulations that aim to provide an assessment of potential impact on the environment. One of the most important components of this risk assessment is an evaluation of the probability of gene ow. In this review, we provide an overview of the current literature on gene ow from transgenic plants, providing a framework of issues for those considering the release of a transgenic plant into the environment. For some plants gene ow from transgenic crops is well documented, and this information is discussed in detail in this review. Mechanisms of gene ow vary from plant species to plant species and range from the possibility of asexual propagation, short- or long-distance pollen dispersal mediated by insects or wind and seed dispersal. Volunteer populations of transgenic plants may occur where seed is inadvertently spread during harvest or commercial distribution. If there are wild populations related to the transgenic crop then hybridization and eventually introgression in the wild may occur, as it has for herbicide resistant transgenic oilseed rape (Brassica napus). Tools to measure the amount of gene ow, experimental data measuring the distance of pollen dispersal, and experiments measuring hybridization and seed survivability are discussed in this review. The various methods that have been proposed to prevent gene ow from genetically modied plants are also described. The current transgenic traits in the major crops confer resistance to herbicides and certain insects. Such traits could confer a selective advantage (an increase in tness) in wild plant populations in some circumstances, were gene ow to occur. However, there is ample evidence that gene ow from crops to related wild species occurred before the development of transgenic crops and this should be taken into account in the risk assessment process. Keywords genetic modication, regulation, pollen, hybridization
INTRODUCTION Along with the effects of consumption of food from genetically-modied (GM) plants, the issue of gene ow cuts to the heart of the concerns about their use in horticulture, agriculture, and forestry. Debates about super-weeds and genies escaping from bottles are addressed by an understanding of gene ow and applying that knowledge to risk assessment (Glaser, 2003; Clark, 2006). There are many excellent papers that provide recent perspectives and literature reviews on gene ow in the context of genetically modied plants (Andow and Zwahlen, 2006; Chapman and Burke, 2006; Davis, 2006; Eastham and Sweet, 2002; Ellstrand, 2006; Felber et al., 2007; Gealy et al., 2007; Glover, 2002; Hails and Morley, 2005; Jenczewski et al., 2003; Kwon and Kim, 2001; Messeguer, 2003; Pilson and Prendeville, 2004; Poppy, 2004; Raybould and Wilkinson, 2005; Sanvido et al., 2007a; Sharma et al., 2002; Smith-Kleefsman et al., 2005). Those publications and the scientic literature cited in this present review provide a comprehensive knowledge base that may be put in the context of the history of regulation of gene ow and GMOs (genetically modied organisms) through reference to Andow and Zwahlen (2006). In this document we provide an overview of the literature on gene ow in GM plants with thought to those contemplating applying to regulatory authorities to release a genetically modied plant into the environment. It is hoped this review will impart a better understanding of the major regulatory issues to be considered and may allow information on gene ow to be arranged in the form of a risk assessment, suitable for use by regulatory
GENE FLOW AND RELEASE OF TRANSGENIC PLANTS
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authorities. Our focus is on gene ow and genetic modication and we do not address the wealth of literature (Bass et al., 2006) that considers the ecology, biology and genetics of gene ow in relation to weeds, invasive plants (Dehnen-Schmutz et al., 2007) and evolution. These areas of plant biology all provide valuable background on gene ow. The reviews of Richards (2003, 2005) and Ramsay (2005) provide background on apomixis, hybridization (see also Wissemann, 2007) and pollen transmission respectively whilst Jrgensen and Wilkinson (2005) outline methods for hybrid identication. The paper by Tiffney (2004) on vertebrate dispersal of seed plants gives important ecological considerations for anyone considering developing an indepth assessment of the factors likely to affect gene ow from a GM plant. Additionally, the role of bacterial and animal gene ow in the context of gene ow in plants has been reviewed by Hails and Timms-Wilson (2007), and the presence of plant DNA in soil should also be considered (Pot et al., 2007). An e unusual additional route that may be relevant in a small number of cases is the transfer of mRNA from host plants to associated parasitic weeds such as dodder (Cuscuta sp.) (Roney et al., 2007). Throughout this review the emphasis will be on environmental and ecological effects (Gurian-Sherman, 2006; Thies and Devare, 2007) and not on any possible effects on human health associated with the presence of the transgene(s) (Ramessar et al., 2007). This issue, however, is central to the regulation of GM crops. Typically, a transgenic plant has one or a few inserted genes, and so may have only one or two new traits. The situation is therefore not completely analogous to the whole plant situation of exotic (Barbour et al., 2005a,b) or invasive species, but there are common lessons to be learned from those researching this area, such as the importance of hybridization, the potential importance of single genes (Lee, 2002; Kolar and Lodge, 2001) and the critical distinction between nonindigenous species and invasive species (Lodge and Shrader-Frechette, 2003). Distinction should also be made between the regulatory requirements for small scale eld tests (Koehler, 2006; Rose et al., 2006) and any subsequent breeding (Lamkey, 2002) or commercialization (USDA, 2007). The precise regulations and their context also differ between countries (Barratt et al., 2006; Flannery et al., 2005; Linacre et al., 2006; Stanley et al., 2006) and different agricultural systems (Cleveland and Soleri, 2006). 2. MECHANISMS OF GENE FLOW IN PLANTS What is gene ow? From the perspective of those working with GM plants, and wishing to cultivate and market them in the same way as any other plants, the question may be reiterated as the following two more specic questions: 1. First, what is the probability that the GM plant will move out of cultivation and establish a wild population? and 2. Second, what is the probability that there will be introgression between the cultivated GM plant and other plants, such that
the introduced transgene(s) move out of the cultivated GM plant population? These two questions will almost certainly need to be addressed in any application to release a GM plant. There is a third, interrelated question, which may or may not have to be addressed, depending on the GM crop/trait in question, and may not involve gene ow per se. 3. What is the probability that seed and/or pollen from the GM plant will move from the site of cultivation or its distribution chain and contaminate non-GM seed or food production? With these three questions in mind the sections below outline the routes of gene ow that should be considered in any risk assessment to varying degrees, depending on the biology of the crop and variety used for transformation. Gene ow routes are also summarized in Wolfenbarger and Phifer (2003). 2.1. Asexual Propagation Gene ow as a result of asexual propagation would typically result in the long-term survival and establishment of a plant in an abandoned cultivation area, or the spread of a plant to areas outside of cultivation through expanding, new growth. Some plants are only propagated vegetatively (many ornamental plants and cut-owers, for example) and are unlikely to survive outside cultivation. Other species may produce tubers, epicormic shoots, root-derived shoots, stolons or runners as a way of population establishment. A comparison of vegetative capacity between a transgenic variety and the parent it is derived from may therefore be appropriate in a crop with a propensity for vegetative spread, a trait, which is often associated with weediness. Amsellem et al. (2001) reported a switch in reproductive biology when a plant was moved from its native range to a new environment, demonstrating both the complexity of predicting the mechanism of gene ow and the need to have a comprehensive understanding of the biology and ecology of the transformed crop. 2.2. Seed Dispersal Many GM plants are cultivated for the eventual harvest of seed. As the seed is the nal product, from a risk assessment perspective gene ow issues will relate to the probability of seed dissemination, and consequent risks of wild population formation and/or the persistence of volunteer plants. An excellent example of gene ow by seed dissemination is Brassica napus (oilseed rape, canola), where the spread of wild populations can be correlated to transportation routes (Garnier et al., 2008; Yoshimura et al., 2006a), as a result of seed spillage (Claessen et al., 2005a,b; Saji et al., 2005). Seed can be formed after selfor cross-pollination. In a crop situation, the potential for crosspollination is of the greatest concern to regulators (see pollenmediated gene ow below).
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2.3. Pollen-Mediated Gene Flow Pollen-mediated gene ow has become a primary focus of gene ow risk assessment because there is also the possibility within a transgenic crop of pollination with nontransgenic pollen of the same species from outside sources, and vice versa, especially when GM and non-GM varieties are grown in close proximity. This has implications in an assessment of gene ow because of regulations that may stipulate thresholds of GM content in non-GM or organic seed crops, which will be discussed later in this review. An additional concern of regulators is that pollen from transgenic crops may pollinate surrounding plants and lead to the production of hybrids between transgenic and nontransgenic plants. Where there is a possibility of pollen-ow from a transgenic crop, there may be the possibility of hybridization with
ability of a fertilization or hybridization event). For example, the collection of pollen from GM plants is a concern to organic honey producers, and the possibility that transgene expression in pollen grains (if it occurs) may harm nontarget organisms can also be relevant (Feil et al., 2003; Mohr and Tebbe, 2007). 3. GENE FLOW AND AGRICULTURE Plants have been cultivated for thousands of years and knowledge of the extent of gene ow in the traditional agricultural setting is a critical part of an assessment of the potential for gene ow from a transgenic plant. For those new to the regulation of GM crops the importance of gene ow in the evolution of plants is useful background information, as is the potential rapid, signicant impact that gene ow can have on evolution (Ellstrand, 2003; Franks et al., 2007). Any assessment of the potential impact of a GMO on the environment should take into account the history of cultivation of the parental crop and whether the crop has become a weed or invasive in any environment outside of cultivation. Highly bred cultivars of maize, for example, do not exist outside of cultivation (Raven, 2005) whereas sugar beet (Beta vulgaris L.) occurs in Europe as a crop, wild populations and weedy forms (Desplanque et al., 1999). Many ornamental species have become severe weeds when introduced by man into environments outside their normal range, often as garden plants (Knowler and Barbier, 2005; Li et al., 2004). On occasion, such introductions can then freely hybridize intergenerically with native, related, species (Amsellem et al., 2001; Abbott et al., 2003). In some cases, knowledge of the history of use and cultivation of comparable non-GM varieties can be included in a baseline for assessment of the potential impact of a particular GM plant, if the trait imparted by the genetic modication is also available in non-GM varieties. An example is the conventionally bred herbicide-resistant varieties available in some crops (Tan et al., 2005). 3.1. Crops That Have Escaped Cultivation Crops escape cultivation through colonization of nonagricultural habitats and the persistence of volunteer plants in rotated crops, which may then establish seed banks. An example is the establishment of oilseed rape seed banks in elds rotated with cereals (Gruber and Claupein, 2007). Seed loss during harvest may have a role in establishing seed banks (Colbach et al., 2008; Lutman et al., 2005; Jrgensen et al., 2007) and imported seed may become established as a weed in countries where the crop itself is not grown (Saji et al., 2005). Disturbance is an important ecological factor in establishing a population in nonagricultural habitat, as cultivated plants can typically not compete successfully in undisturbed sites (Claessen et al., 2005a). The propensity of a crop to volunteer, and the conditions under which it occurs, should be taken into account in assessing the probability of gene ow from a transgenic plant. As has been mentioned, introduced ornamental plants are particularly prone to escape from cultivation, as they are grown over wide areas, in many
Other crops of the same species. Crop-to-crop gene ow has been quantied in B. napus (Weekes et al., 2005) and fodder maize (Zea mays) (Weekes et al., 2007) and more examples will be given later in this review; Wild populations of the same species; Related wild species; Breeding lines, pedigree lines or into any variety where seed purity is important; and Organic crops, which are grown to standards that require no or minimal GM content.
For risk assessment purposes, the basic reproductive biology of the crop must therefore be very well understood. Comparison of parental and transgenic lines (in terms of number and size of reproductive organs, for example, or the amount and viability of pollen produced) provides useful information to regulators, but it is also necessary to have a good baseline knowledge of the many factors that can affect pollen-mediated gene ow. These include whether the crop is wind- or insect-pollinated (Ramsay, 2005), likely distance of pollen dispersal, and the probability of fertilization (Smith-Kleefsman et al., 2005). More factors affecting pollen dispersal will be discussed when specic examples are cited later in this review. Though gene ow between plants is a common phenomenon, the formation of a hybrid plant does not mean a wild population will be established. Establishment depends on many factors, and is often enhanced by cultivation (Mack, 2000), the chance of which may itself be related to the distance of hybridization from cultivated crops. However, where hybridization is possible, the tness of any theoretical hybrid needs to be evaluated in a risk assessment, in order to estimate the possibility of introgression, or genetic drift. The potential for introgression to lead to speciation (Hails and Morley, 2005; Eastham and Street, 2002) is very difcult to quantify. As a nal note on pollen-mediated gene ow, there are situations where pollen ow per se may need to be considered in a risk assessment, in the absence of gene ow (i.e., no prob-
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locations as a result of use in home gardens and landscaping (Dehnen-Schmutz et al., 2007). 3.2. Crop-to-Crop Gene Flow Crop-to-crop gene ow occurs in agricultural crops (Ellstrand, 2003) and this is relevant to any situation where the genetic purity of seed produced by a crop is desirable. In crops where gene ow can happen over long distances (for example, canola or sugar beet) farm management practices can be used, such as isolation distance (discussed later in the context of transgenic plants) along with options for barrier crops, farm-to-farm communication and volunteer control during rotation (Eastham and Sweet, 2002). The same principles can be used in crops grown in proximity of wild stands of the same species, such as can happen in the case of sunower (Helianthus annuus) (Ureta et al., 2008). The organic industry has determined that certied organic growers may not use GM seed for planting and have used the consequent fact that organic food is nontransgenic as a marketing tool. Organic growers are therefore also concerned about crop-to-crop gene ow, in terms of pollen from GM plants fertilizing organic crops. Segregation of organic crops from transgenic crops is a very difcult issue, bringing to the fore as it does several social, political, and even ethical questions (Bruce, 2003). In the case of organic crops, protection from external pollen sources relies on both the application of scientically based management practices as well as the willing cooperation of adjacent growers. 3.3. Gene Flow Between Crops and Wild Species Hybridization between crops and their wild relatives (either the same, or different species) has occurred frequently in agricultural history (Ellstrand and Schierenbeck, 2000; Raybould and Wilkinson, 2005). Chapman and Burke (2006) have produced an excellent recent review on the subject and Ellstrand (2003) has tabulated a list of cultivated plants that are known to have hybridized with wild relatives in the course of history. Ellstrand et al. (1999) and Eastham and Sweet (2002) provide a detailed review of gene ow in the major food crops. In a more specic study, Armstrong et al. (2005) made a comprehensive review of hybridization between a large number of crops grown in New Zealand and their wild relatives, nding more than half of over one hundred crops were reproductively compatible to related wild species. Hoenicka and Fladung (2006) have conducted a similar review with an emphasis on tree species. Gene ow to wild species from crops occurs at higher frequency than might be expected from natural mutation rates (Ellstrand et al., 1999) and the majority of the most important crop plants have wild relatives (Ellstrand et al., 1999; Gealy et al., 2007). Introgression of crop genes (Ellstrand et al., 1999) has importance in evolution of weeds, the evolution of invasive species and extinction of rare species (Jarvis and Hodgkin, 1999; Gealy et al., 2007). The probability of gene ow from crop to related weed species is
habitat dependent and so variable between populations (Cureton et al., 2006). From a risk assessment perspective, though the presence of wild relatives increases the probability of gene ow, it does not necessarily infer hybridization is inevitable (Gealy et al., 2007). Any wild or weedy species needs to be sexually compatible. Though crops may have compatibility with wild species (Richards, 2005) if there are genetic barriers to fertilization these will be a major obstacle to gene ow.
IDENTIFICATION OF TRANSGENES IN PLANTS For evaluation of gene ow from transgenic plants it is necessary to have available techniques for reliable identication of the transgene in large numbers of plants. This is the only way to measure very low levels of hybridization in recipient plants or to identify putative escapes from cultivation in large numbers of wild plants. In some cases a novel phenotype resulting from expression of the transgene can be readily identied, such as herbicide resistance, ower color, leaf color (Reimet al., 2006) or pollen color (Moon et al., 2007). Like antibiotic resistance, herbicide resistance can be detected by spraying during seed germination or painting leaves with herbicide (Bae et al., 2008; Johnson et al., 2006; Llardi and Barba, 2001; Lim et al., 2007). This simple assay is an advantage of using herbicide resistance as a selectable marker gene. Johnson and Galloway (2008) describe the use of a dye analog to track pollen movement, which may be a useful way to measure the extent of pollen spread ahead of embarking on a search for hybrids. The use of an easily identiable marker gene, such as uorescence protein, to assess pollenmediated gene ow has also been used (Halfhill et al., 2001) and later extended to include identication of hybridization events and measurement of copy number and zygosity (Halfhill et al., 2003; Moon et al., 2006). Where the phenotype is less easy to identify, molecular methods such as real-time PCR may be used (Zeitler et al., 2002; Weekes et al., 2005; Pla et al., 2006). The cost of these techniques and the occurrence of false positives is however a potential problem when looking for very rare events (Begg et al., 2007; Berdal and Holst-Jensen, 2001). A very low frequency of transgenes in populations may lead to inconclusive or inconsistent data or results (Mercer and Wainwright, 2008). Molecular markers were used by Desplanque et al. (1999) to assess the large scale pattern of gene ow in sugar beet populations in France, by Burczyk et al. (2006) in oak and pine, and by Petti et al. (2007) in potato (Solanum tuberosum). Floras are an invaluable tool in gathering information of the distribution of wild populations and related species, but they may be less useful for identication of hybrids (Armstrong, 2005), which may be very similar, morphologically, to either parent, and may be very rare in the wild. The issue of identifying and measuring rare hybrids in the wild or over much larger areas than typical eld trials has been reviewed by Jrgensen and Wilkinson (2005). The authors suggest a more focused approach, based on a systematic review of distribution overlap between weed habitats
4.
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and cultivation, presence of common pollinators, owering time synchrony, common reproductive system and cross compatibility, in order to target searches to those sites where sympatric populations are most likely to occur (Wilkinson et al., 2000). This is not only a very sensible strategy for approaching a search for hybrids but also in post-release monitoring studies, as it is logical to look for gene ow in wild populations most likely to hybridize to the transgenic plants under cultivation. A renement of the strategy might be to concentrate on those sympatric populations most likely to exhibit increased tness if they were to receive a transgene, such as high-density weed patches subject to high natural selection pressures (Vacher et al., 2004). Another application of molecular techniques is to determine whether genes specic to cultivated crops occur in related wild populations (Kuroda et al., 2005). This is a useful method to determine whether there has ever been an introgression event in a species related to crops. For example, Guadagnuolo et al. (2001) identied species-specic markers for sea barley (Hordeum marinum L.) in wheat, even though no hybrids were found in the wild populations assessed near wheat elds. Luby and McNicol (1995) looked for genes conferring commercial traits in wild raspberries and extrapolated from the absence of such genes to the assumption that hybridization of wild raspberries with transgenic raspberry would be unlikely. 5. MEASURING GENE FLOW IN TRANSGENIC PLANTS
of pollen ow measurements from different trials for the same species (Halsey et al., 2005). Data gleaned from such trials are indicative, and depend on many factors. Major factors are whether the crop is windor insect-pollinated. For example, Kuparinen (2006) stated the need to take into account rare wind conditions and reviewed how to incorporate wind as a variable in gene dispersal models interpreting eld trial data. Wind was also an important factor in trials designed to measure transgene ow in rice (Oryza sativum) (Messeguer et al., 2004; Yuan et al., 2007). For wind-pollinated crops, wind direction and average and maximum speeds during eld trials are important factors to consider in nalizing a decision on hybridization rates with distance (Hoyle and Cresswell, 2007a). For insect-pollinated crops, variability in foraging behavior and pollinator population size is difcult to model and control (Hoyle and Cresswell, 2007b). Isolation is more difcult to reliably manage by distance in such crops, as insects may travel many kilometers (Ramsay, 2005). The presence of high numbers of insects, for example where hives are near trials, may also compromise isolation precautions (Llewellyn et al., 2007). 5.2. Isolation Distances The indicative information on hybridization as a function of distance from source transgenic plants can be used to determine isolation distance, a distance designed to reduce the probability of hybridization between adjacent crops to below pre-set limits. Ellstrand (2003) provides tables of hybridization at typical isolation distances for some of the major food crops. Isolation distance guidelines, if available, will be of critical importance in applications for trials of a transgenic crop. Separation of different varieties of a crop by separation distance is a well established practice to manage thresholds of purity, such as pedigree seed lots (Messeguer, 2003). Friesen et al. (2003) showed that more than half of 27 non-GM pedigree seed lots of Brassica napus examined in Canada had signicant (more than 0.25%) contamination with GM seed. This contamination could be due to reasons other than hybridization from external pollen sources (human error in sampling or bagging, for example), but in the case of transgenic plants established isolation distance does provide guidance to regulators issuing licenses for eld trials. Isolation distance may also provide an important component of farm management practice guidelines after release of the GM plant. Eastham and Sweet (2002) provide practical measures for European farming practices. Coexistence of conventional, GM, and organic crops is a sensitive subject (Pew Initiative, 2006; Demont et al., 2008; Levidov and Boschert, 2008) and in Europe, maximum (threshold) concentrations of GM seed in non-GM seed batches have been proposed. Isolation distance is one component of ensuring that these thresholds can be met (Van de Wiel and Lotz, 2006; Weber et al., 2006; Langhof et al., 2008). Table 2 summarizes some of the research designed to develop isolation distance parameters in several transgenic crops.
5.1. Experimental Trials In the past 10 years or so a considerable amount of research has been carried out to measure gene ow in various sizes of eld trials of transgenic plants. Typically, these experiments measure the frequency of hybridization to non-GM recipient plants, from plots of transgenic plants. The recipient plants may be male-sterile (Darmency et al., 2007; Saeglitz et al., 2000; Wang et al., 2006). This simplies the analysis, but hybridization event frequency may be affected by the lower amount of background pollen production (Walklate et al., 2003). The results from some of these studies are shown in Table 1. A recent experiment with transgenic maize has evaluated gene ow using large (ha) size plots, in which the pollen density of the receptor plants was altered. Out-crossing was enhanced when local (receiving plant) pollen load was low, suggesting this information should be incorporated into any future models of gene ow in this species (Goggi et al., 2007). Jia et al. (2007) measured gene ow in plots of rice (a species with relatively low distance gene ow) that were a comparable size to cultivation plot sizes. Although the majority of hybridization events occurred at under a few meters, events were detected up to 150 meters away onto male-sterile recipient plants. Belcher et al. (2005) used modelling to show the importance of spatial arrangement of GM and adjacent non-GM crops in determining the amount of gene ow. The actual and relative sizes of the transgenic eld and the recipient plant populations are therefore important factors to bear in mind when comparing the results
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TABLE 1 Summary of result from eld trials measuring pollen dispersal distance from transgenic plants Crop Brassica napus Observation Showed gene ow to no more than 3 km from 25100 ha elds of non-GM HT crops. Glyphosate-resistant plant detected 500 m from a 0.5 ha transgenic plant plot. Pollen-mediated gene ow to up to 800 m and persistence of seed volunteers, combining to lead to survival of double herbicide-resistant varieties. Measured pollen ow from individual plants. 50% of pollination occurred in less than 3 m. Showed in a 100-square-kilometer survey that pollination can occur at distances of over 1 km. From a central transgenic plot surrounded by recipient plants, no transgene detected beyond 200 m. No gene ow detected at distances greater than 12 m from small plots. Measured sunower gene ow to wild sunower, in Argentina. High levels of hybridization at a few meters and detectable at up to 500 m. No evidence for gene ow in Chinese cabbage grown adjacent to transgenic Chinese cabbage. Compatibility proven by hand-pollination. No evidence for any hybridization, even in adjacent plots. Gene ow detected primarily at under 2 km and in sentinel plants up to 20 km away from transgenic source. Hybrids identied in recipient plots up top 3.8 km down-wind from transgenic HT control area. Up to nearly 300 m using bait plants. Gene ow at detected at 200 m, using bait plants. Evaluated gene ow using ha size plots. Out-crossing occurred at 0.030.1% at a distance of 100 m. 0.5% transmission of a HT gene at a distance of 1 m and 0% at 10 m. No gene ow at distances over a few meters in transgenic soybean, most probably due to insect pollinators. Negligible transgene frequencies at less than 10 m. Relatively little pollen ow beyond 10 m. Measured gene ow in cultivation size plots of rice. Though majority of hybridization at under a few meters hybridization was detected at up to 150 m. Gene ow measured by ower color was virtually zero beyond a few meters (study used non-GM crops). Citation Rieger et al. (2002) Hall et al. (2000) Beckie et al. (2003)
Lavigne et al. (1998) Devaux et al. (2005) Wang et al. (2004b) Gatford et al. (2006) Ureta et al. (2007)
Tall fescue
Barley, wheat Sunower
Chinese cabbage
Lim et al. (2007)
Tomato Bentgrass (Agrostis stolonifera L.)
Llardi and Barba (2001) Watrud et al. (2004) Reichmann et al. (2006) Darmency et al. (2007) Saeglitz et al. (2000) Goggi et al. (2007) Abud et al. (2007) Yoshimura, (2006b) Rong et al. (2007) Messeguer et al. (2001, 2004) Jia et al. (2007)
Sugar beet
Maize Soybean
Rice
Common bean (Phaseolus vulgaris)
Ferreira et al. (2007)
5.3. Post-Release Mechanisms of Gene Dispersal Data collected from a eld trial can only be indicative of the potential for gene ow after commercial release of a GM plant. As Wilkinson et al. (2003) very validly point out, release per-
missions are usually made at a national, or at least geographical, level meaning regulators extrapolate from eld trials to a situation in which additional geneow mechanisms may appear. An example of such a mechanism of gene ow is distribution
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TABLE 2 Summary of recommended isolation distances for GM crops Crop Brassica napus Experimental data 1% at a distance of 50 m. Literature review. Recommended isolation distance Isolation distance of 150 m and use of buffer zone to meet EU thresholds. 100 m plus a 5-m buffer zone. Suggest a plant-free isolation of 10 m for Brassica napus. 20 m, based on EU threshold levels. 50 m, based on EU threshold levels. 3-m separation adequate to meet EU thresholds. Citation Weekes et al. (2005) Damgaard and Kjellsson (2005) Husken and DietzPfeilstetter (2007) Sanvido et al. (2008) Sanvido et al. (2008) Devos et al. (2005); Weekes et al. (2007) Halsey et al. (2005) Gustafson et al. (2006)
Silage maize Grain maize Fodder maize
Able to detect gene ow at distances up to 200 meters. From 1 ha elds, modelled gene ow at less than 0.9% at 20 meters.
Maize
Cotton
750 m required for complete isolation. Combinations of isolation distance of 20 m and border rows. 20 m or more are predicted to result in gene ow of less than 0.9%, as a blended average for receptor elds 1 ha or larger. A 20 m buffer of non-GM cotton is likely to be effective for isolation.
Llewellyn et al. (2007)
of seeds imbedded in vehicle tyres. Von de Lippe and Kowarik (2007) found seed of wheat (Triticum aestivum), rye (Secale cereale), and oilseed rape in motorway vehicles far away from sources of cultivation. Like the dispersal of oilseed rape along roads and around ports, this is a nonbiological mechanism of dispersal, which is experimentally impractical to estimate ahead of any large-scale release. 6. ASSESSING THE PROBABILITY OF GENE FLOW FROM TRANSGENIC PLANTS Armed with a technique for identifying transgenes and knowledge of the potential for seed and pollen dispersal it is possible to provide an assessment of the probability of gene ow. 6.1. Factors Affecting the Probability of Gene Flow It is in the interest of an applicant to provide information on the factors, summarized here, which affect the probability of gene ow, as comprehensively as possible. 6.1.1. Knowledge of the Biology of the Target Crop The mechanism of pollination in a crop will have a primary effect on the probability of gene ow. Pollen-mediated gene dispersal from self-pollinating crops, with very low out-crossing, such as soybean (Glycine max) (Ramsay, 2005) is likely to be low (Gealy et al., 2007) relative to a wind-pollinated out-crossing crop like maize (Jorgensen and Wilkinson, 2005). For reference, predictive modes and levels of out-crossing probability have been compiled by many authors (Angevin et al., 2008; Arritt et al., 2007; Kuparinen et al., 2007; Smith-Kleefsman
et al., 2005). If the target (transformed) plant has any weedy characters an assessment should also take into account factors determining plant invasiveness, such as likely settlement habitats and seed dispersal ecology (Bass et al., 2006). In assessing the probability of gene ow from a crop to a related weed species it is important to know whether the wild species exists in habitats close to cultivation, or in habitats shared by volunteers from cultivation, and to understand the ecology of any related weed species (Wilkinson et al., 2003). A lesson from studies with invasive plants is to review growth habits in native habitats as well as in new habitats that may be exposed (Hierro et al., 2005). Hooftmannn et al. (2007), using lettuce (Lactuca sativa) and a related wild species, collected data over several generations of eld tests, in order to assess the capacity for adaptation to the environment. 6.1.2. Knowledge of the Distribution of Any Related Wild Populations Floras, herbaria, plant databases, pollen proles and plant surveys are resources that can be used to determine the likely ora in the area that a transgenic plant is to be released (Wilkinson et al. (2003). If this research identies related species that are recent, invasive introductions, the characteristics of that plants history in a particular region (preferred habitat, means of reproduction, etc.) is relevant supplementary information (Pyek, s 2001). Related species may provide a genetic bridge to plants more remotely related to the transgenic crop, so a comprehensive survey is required, to identify any plants in the taxonomic group that may be noxious weeds. Satellite imagery has been used in
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the UK (Wilkinson et al., 2003; Elliott et al., 2004) to identify the distribution of Brassica napus elds, in order to correlate distribution to the habitat of a related weed species, Brassica rapa. It is appropriate to make an assessment of the distribution of related species at this scale (Wilkinson et al., 2003). Arriaga et al. (2006) mapped the distribution of local species compatible to cultivated Cucurbita species (squash) in Mexico, to identify those areas where hybridization would be most likely to occur. 6.1.3. Centers of Biodiversity Introduction of a transgenic crop into a center of biodiversity for that crop (Nedoluzhko and Dorokhov, 2007) or a related species (Meier, 2007) is likely to lead to more scrutiny from regulators. Engels et al. (2006) have reviewed centers of biodiversity in the context of GM crops, suggesting conservation practices to preserve genetic resources in centers of crop biodiversity, especially in Central America and Mexico (Martnez-Castillo et al., 2007). Heinemann (2007) has made a similar review for the Food and Agriculture Organization with respect to the preservation of genetic resources. The controversy surrounding the initial detection of transgenes in land races of Zea mays in Mexico has led to calls for a precautionary approach to the release of transgenic plants in their centers of biodiversity. One suggestion is the use of only male-sterile varieties for transformation, for example, in potato (Buijs et al., 2005; Celis et al., 2004; Conner, 2007; Green et al., 2005; Scurrah et al., 2008). Counter-arguments are presented by Raven (2005) who provides reasons why release of transgenic maize in Mexico, a center of origin for Zea mays, does not provide a threat to local landraces, and spontaneous hybridization between maize and teosinte has recently been described (Ellstrand et al., 2007). Though not necessarily centers of biodiversity, isolated habitats such as islands may also be considered a special case by regulators. This possibility is reviewed, in an Hawaiian context, by Munster and Wieczorek (2007). 6.1.4. Knowledge of the Compatibility to Related Species The demonstration of reproductive compatibility or incompatibility to related species and varieties of the same crop is important information for assessment of the probability of gene ow (Halsey et al., 2008). This information may need to be gathered experimentally (Armstrong, 2005). Such experiments should be embarked on only after identication of the wild species most likely to be exposed to pollen from the transgenic crop. As compatibility measured under controlled conditions may be different to that in a eld situation, experiments should be carried out under optimal fertilization conditions, as a means to negate this potential criticism. Combining information on proximity to wild populations, owering time overlaps, fertility of experimental hybrids and compatibility will give an indication of the probability of hybridization (Chapman and Burke, 2006). Comprehensive experiments may be required if rates of hybridization are to be tested experimentally since Pallett et al. (2006) measured wide variation in hybridization rate for a particular transgenic speciesnontransgenic species combination.
6.1.5. Reproductive Biology of the Transgenic Plant A critical part of a regulatory dossier will be a comparison of the transgenic line and the line used for transformation. Measurements such as adventitious root formation, survival in soil, ower number, owering time, anther number, pollen viability and longevity (Wilkinson et al., 2003), seed number, seed dormancy, and seed germination will all provide information on whether there are any morphological or physiological changes likely to have had an affect on asexual or sexual reproduction rates. Ramsay (2005) has written an excellent summary of adaptations and traits that may favor insect or wind pollination, which may also be used as a guide to characters to measure. There are few studies that look directly at the transformation process in this respect, though Hokanson et al. (1997), using Cucumis melo (squash), showed gene dispersal rates were the same for transgenic and nontransgenic plants of that species. 6.1.6. Survivability Survivability is a measure of how well propagules (seed, tubers, stem segments, root pieces, etc.) from a transgenic plant line will survive, in comparison to the non-GM variety that the transgenic plants were derived from. Such experiments are especially relevant in the EU, where there is still much debate about the cultivation of transgenic crops and concern over threshold levels and co-existence (Demont et al., 2008; Devos et al., 2005; Levidov and Boschert, 2008). Persistence and survival information is required to determine whether the transgenic line has an increased tness, measured by survival, should it escape cultivation. If the crop is one that seed can remain dormant for a long time, the experimental data required to answer the question needs to be collected over several years. Examples of experimental approaches that have been taken are to plant seed in wild habitats and check survival (Eastick and Hearndon, 2006; Hails et al., 1997) or to set up eld plots of transgenic and nontransgenic plants, with and without cultivation, to assess relative vigor and survivability (Stewart et al., 1997). Claessen et al., (2005b) through both trials and literature review, have predicted that persistence of oilseed rape would be elevated in transgenic plants with altered seed-oil content and reduced in transgenic plants tolerant to the herbicide glufosinate. Table 3 summarizes the observations made from some experiments measuring persistence and survival of transgenic plants. 6.2. Quantication of Gene Flow Once the base information is assembled, quantication of the probability of gene ow may be difcult, because of the multitude of factors that can affect the degree of gene ow under different situations. There is also the obvious problem of extrapolation from eld trials to often national scale production (Devaux et al., 2007; Messeguer et al., 2006). For this reason it is quite a valid approach to prepare a matrix, assigning simple levels of probability (low, medium, and high, for example) against each factor likely to be important for gene ow (Glover, 2002).
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TABLE 3 Summary of result from eld trials measuring survivability of transgenic plants Crop HT-oilseed rape, HT-sugar beet, HT maize, and Bt-potato Brassica napus Oilseed rape Planted in 12 natural habitats, without spraying, in the UK and compared survival to non-GM crops for over 10 years. In no case were the GM plants more persistent or invasive than the non-GM controls. Compared seed survival after two winters in a multi-site trial. Transgenic lines showed a lower survival rate. Planted seed of various transgenic lines, and their parents, at sites on cultivated land, uncultivated land and marginal sites. After 7 years no transgenic seed persisted, though conventional varieties and weed species planted at the same time did. Measured population survival over 5 years on several sites in the UK, compared to conventional cultivars. No clear differences between the cultivars. Planted Bt-transgenic and control seed in a number of habitats in Northern Australia and found no difference in ability to establish a population in the wild during a 4-year study. Concluded there was no possibility of establishment of populations in the wild. Citation Crawley et al. (2001)
Hails et al. (1997) Walker et al. (2004)
Lutman et al. (2005)
Cotton
Eastick and Hearnden (2006)
Though the literature modelling gene ow can be a useful source of information on the factors affecting gene ow, it is very difcult to model all factors accurately, such as the complex movement of pollinators such as bees (Osborne et al., 1999; Hayter and Cresswell, 2006) or the many factors affecting seed dormancy and sprouting of volunteers (Pekrun et al., 1998). Another factor that is difcult to model is the density of weeds in crops, as this is lower in HT (herbicide tolerant)transgenic crops (Heard et al., 2003). Despite the difculties, there has been extensive work on modelling gene ow (Andow and Zwahlen, 2006; Claessen et al., 2005a; Gruber and Claupein, 2007; Lanchier and Neuhauser, 2007). A very well developed model is the GENSYS model for B. napus (Colbach et al., 2001a, b). Other resources include Hoyle and Cresswell (2007b), who describe information suitable for modelling animal and insect mediated gene ow in a GM crop, and Lee and Natesan (2006) who provide an overview of models used for predicting gene ow in the major crops. Epperson (2007) and Smouse et al. (2007) describe a theoretical relationship between isolation by distance and spatial genetic structure for modelling seed and pollen dispersal, using oilseed rape as an example in the latter case. There are also a number of models of gene ow that have been developed for non-GM crops such as those for tree plantations (Wang, 2004; Williams et al., 2006), azuki bean (Wang et al., 2004) and chickpea (Toker et al., 2006). Whitlock and McCauley (1999) have described a mathematical model of gene ow and gene migration, with emphasis on genetic variation while there are various models for pollen ow from wind-pollinated species (Angevin et al., 2008; Arritt et al., 2007; Meagher et al., 2003).
6.3. Transgenic Crops and Gene Flow There is now signicant experience in the large-scale cultivation of transgenic plants. For the major crops it is therefore possible to include real-life data and observations in a probability assessment. 6.3.1. Brassica napus Research on gene ow in both canola and oilseed rape has been extensive, with studies under European, North American and Australian conditions. Some of this research is summarized in the tables of this review and is also summarized by Eastham and Sweet (2002) and by Fitzjohn et al. (2007), who set up a database of research results. H sken and Dietz-Pfeilstetter u (2007) have written a literature review of gene ow from transgenic B. napus, which is self- and insect-pollinated (Ramsay, 2005) and has a moderate level of out-crossing. There are several avenues for gene ow to occur in this species (Armstrong, 2005; Gealy et al., 2007) and transgenics have both escaped cultivation as volunteers (Warwick et al., 2003) and as discussed above, begun to introgress with wild related plants. Gruber and Claupein (2007) modeled return of seed to seed banks in lowand high-risk scenarios, providing suggestions to reduce gene ow through this route. Pivard et al. (2008) identied seed banks and seed immigration from elds adjacent to wild habitat as the main origin of feral oilseed rape populations. Around 15% of populations were due to transport related seed dispersal. Seed bank survival and dormancy (Claessen et al., 2005b) also have a signicant impact on population growth and persistence in this species (Mess an et al., 2007). Another specic issue in this e
35
crop is the incidence of feral populations with multiple herbicide resistance (Hall et al., 2000; Aono et al., 2006; Knispel et al., 2008). Detailed modelling of transgenic gene ow in Brassica napus has been developed by Claessen et al. (2005a, 2005b). Weekes et al. (2005) generated a model of gene ow in B. napus in a commercial eld situation and showed that hybridization decreased rapidly with distance from pollen source. Similarly, a mathematical model for pollen-mediated gene ow from B. napus by wind or insects showed that target crop fertility and source-crop size are the most important factors affecting distance of pollen dispersal (Walklate et al. 2003). With over a decade of large-scale cultivation, the stable introgression of transgenes from GM crops of B. napus to related species is well documented (Johannessen et al., 2006; Warwick et al., 2008; Weis, 2005). In a comprehensive study it had previously been concluded that inter-specic hybridization between B. napus and common weed species was either rare or unlikely (Warwick et al., 2003). Some of these related species are weeds and share habitat along the crop edges, such as eld-side waterways (Wilkinson et al., 2003). It is known, for example, that B. napus will hybridize with B. rapa (Pallett et al., 2006) and Wilkinson et al. (2003) estimated 32,000 hybrids form annually between B. napus and B. rapa in the UK. Hybridization to wild radish (Ch` vre et al., 2007) and cabbage (Ford et al., 2006) has e also been investigated. It should be stressed, however, that such gene ow from B. napus has occurred historically during the cultivation of the crop and is not a specic issue related to transgenic varieties. Because of the propensity for gene ow, several management practices have been suggested to try to reduce pollen ow to neighboring plants of B. napus. Reboud (2003) suggested that a gap such as a road is no barrier to gene ow, and simply increased the required isolation distance. He recommended instead the removal of eld borders after owering. Other specic management practices that have been suggested for transgenic HT oilseed rape (Ceddia et al., 2007; L g` re, 2005) ine e clude managed crop rotations (Hall et al., 2000; Graef et al., 2007).
6.3.3. Soybean Soybean is a self-pollinating crop (Ramsay, 2005). It exhibits a very low out-crossing level because cross-pollination is facilitated by insects. Although there is a limited probability of gene ow in this species and volunteers are unlikely to survive (Gealy et al., 2007), some concern has been expressed about the cultivation of transgenic crops in the centre of diversity for Glycine (Nedoluzhko and Dorokhov, 2007). 6.3.4. Cotton (Gossypium hirsutum) Cotton is a species with a low out-crossing frequency. In a study of bromoxynil resistant plants in California it was found that pollen gene ow (PGF) declined exponentially with increasing distance from 7.65% at 0.3 m to less than 1% beyond 9 m when there was high pollinator activity. In the absence of high pollinator (honeybee, Apis mellifera L.) populations, PGF was less than 1% beyond 1 m. Pollen ow in commercial elds was consistent with the experimental plot data, with only 0.04% PGF detected at 1625 m (1 mile) (Van Deynze et al., 2005). Bt (insectresistant)-cotton is the only commercial transgenic crop grown in Australia, and there have been two extensive studies of gene ow in that country. Llewellyn et al. (2007) describe detailed results of a number of eld trials in Eastern and Northern Australia, showing the relatively short distance that pollen dispersal was likely to occur over, though this distance could be increased if the incidence of insect pollinators was greater. Eastick and Hearnden (2006) conducted long-term tests of survivability and showed no increased survival in the transgenic line (Table 3). 6.3.5. Rice Though rice is a very low out-crossing species it is grown over a very wide area and in many parts of the world (Wang and Johnston, 2007). Accordingly the species is a primary target for genetic modication and several studies have focused on accurate measures of pollen-mediated gene ow from trial plots (Table 1). Rong et al. (2005) concluded that gene ow will occur in rice, but only at a very low frequency beyond a few meters, conrming that separation distance strategies would be effective for a self-pollinating crop like rice (Messeguer, 2003). A study of gene transfer from imidazolinone-herbicide-resistant CleareldTM (CL) rice to the weedy red rice showed most hybrids were found within 1 m of the CL plants, with a few up to 6 m away (Shivrain et al., 2007). Yuan et al. (2007) using a male-sterile line as the detector plants, looked at gene ow as a function of wind direction and while nding an increased distribution with the prevailing wind direction, also found gene ow was largely conned to 10 m from the source plants. The implied conclusions that gene ow from transgenic rice is unlikely is somewhat contradicted by the identication of a gene from cultivated rice in related wild species in Laos, indicating gene ow occurs extensively (Kuroda et al., 2005). 6.3.6. Sugar Beet Sugar beet is a major crop in Northern Europe and the potential for the development of transgenic varieties for production
6.3.2. Maize Gene ow in maize has been summarized by Eastham and Sweet (2002) and Gealy et al. (2007). Gustafson et al. (2006) have provided a model of gene ow in maize, showing that the effect of pollen source eld area was minimal above 4 ha. Pollen-mediated gene ow in this species has been estimated using natural color markers (Bannert, 2006; Bannert and Stamp, 2007) and has also been the subject of several mathematical models (Angevin et al., 2008, Arritt et al., 2008; Fricke et al. 2004). Maize is a moderately out-crossing crop (Watanabe et al., 2006) with relatively short-lived pollen, and from a transgenic crop perspective is unlikely to escape by hybridization, though volunteers do appear in cultivation (Gealy et al., 2007).
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there has encouraged research on gene ow in this species. It occurs as a volunteer, and is also established in the wild as wild beet populations, which interbreed (Dietz-Pfeilstetter and Kirchner,1998). Cold tolerance is important, as a winter season is necessary to induce owering. Pohl-Orf et al. (1999) showed there was no difference in survival rate for transgenic and nontransgenic varieties exposed to winter conditions. In eld trials carried out over several years, Darmency et al. (2007) showed gene ow from sugar beet was due primarily to seed formation in bolting plants, as well as hybridization to weed beets. Gene ow from sugar beet to sea beets occurs at a low level and has long been recognized in seed propagation areas (Andersen et al., 2005). Triploids were only identied in the rst generation, the authors suggesting introgression beyond the rst generation is not extensive. However, other studies (Viard et al., 2004; Fernart et al., 2007) show gene ow from beet to weed species is frequent and also raise the prospect of weed-to-weed gene ow subsequent to introgression, as gene ow in weedy Beta species was demonstrated at up to nearly 10 km. 6.3.7. Sunower Like sugar-beet, sunower (Helianthus annuus var. macrocarpus) has escaped from cultivation. Hybridization to the selfincompatible wild sunower (H. annuus) (Marshall et al., 2001; Poverene et al., 2004; Ureta et al., 2007) also occurs and is a good example of crop-wild species hybridization (Weis, 2005). Cantamutto and Poverene (2007) have reviewed gene ow from transgenic sunower and concluded there would be a very high risk of gene ow in areas where naturalized populations exist in growing areas, and also recommended that transgenic sunower should not be released in centers of biodiversity. Whitton et al. (1997) had previously showed cultivar (nontransgene) alleles in wild sunower populations ve generations after hybridization using an RAPD marker and suggested that neutral or favorable transgenes would be likely to persist in this species. Most recently, an assessment of seed bank formation concluded that increased seed production of individual sunowers resulting from crop transgenes would be unlikely to increase population growth on the scale of small patches. However, increased seed production could result in dispersal of seeds across larger areas, and thus lead to larger seed banks in disturbed environments (Moody-Weis and Alexander, 2007). 6.3.8. Sorghum (Sorghum vulgare) Sorghum is a crop that transgenes could ow to related weed species by hybridization, given both eld trial evidence and presence of crop genes in wild populations (Schmidt and Bothma, 2007; Snow and Ejeta, 2007). 6.3.9. Cucurbits Transgenic varieties of the genus Cucurbita sp. (squash) have been developed and experimentally it has been shown that pollen-mediated transgene gene ow occurs from variety to variety (Spencer and Snow, 2001) and that these hybrids are fertile
(Fuchs et al., 2004a). The potential of gene ow from transgenic Cucurbita has been reviewed by Arriaga et al. (2006), with a particular emphasis on the related species found in Mexico. 6.3.10. Wheat There are several theoretical and practical studies of gene ow in wheat. For example, Gustafson et al. (2005) and Br l -Babel ue et al. (2006) have modeled the risk of gene ow from conventional and GM herbicide-resistant wheat respectively. Although transgenic wheat has not been commercialized the authors suggest gene ow would be inevitable, by pollen dispersal and/or seed dispersal. Traits conferring a selective advantage would be likely to rapidly increase in volunteer populations outside the GM crop. Two studies used the blue aleurone marker to quantify gene ow (Hanson et al., 2006; Matus-C diz et al., 2004), a whereas others have measured hybridization with related species of Aegilops, either in the eld (Loureiro et al., 2007) or using transgenic wheat under glasshouse conditions (Schoenenberger et al., 2006). The only eld study with transgenic wheat is that measuring hybridization between imidazolinone-resistant plants and Aegilops cylindrica (Gaines et al., 2008). 6.3.11. Papaya (Carica papaya) Papaya resistant to the papaya ringspot virus (PRSV) is a deregulated crop in the United States and the only transgenic fruit widely consumed. This crop occurs in dioecious and gynodioecious forms and this property affects the frequency of outcrossing. A recent study used GUS expression as a method to quantify gene ow studies from transgenic plants and concluded that cross-pollination occurred in 70% of female plants (43% of assayed seeds), compared with only 13% of the predominantly self-pollinating hermaphrodite plants (7% of seeds) segregating in the gynodioecious border rows (Manshardt et al. 2007). 6.3.12. Alfalfa (Medicage sativum) Glyphosate resistant Roundup Ready (RR) alfalfa has proved to be a controversial crop. It was initially deregulated by the USDA in June 2005, and commercialized in August of that year. However, in late 2006 the Center for Food Safety and others led a suit against USDA claiming deciencies in the environmental assessment process used during the deregulation process. The judge ruled that the USDA environmental assessment should have included an assessment of the potential impact of RR alfalfa on the development of glyphosate-resistant weeds and on the economic environment, including potential economic impact on organic alfalfa producers (a consequence of gene ow). The ruling required USDA/APHIS to conduct an Environmental Impact Statement (EIS) and stopped any new sale or planting of RR alfalfa after April 1, 2007, pending the completion of the EIS and a new deregulation decision by the agency. In January 2008, USDA/APHIS published a Notice of Intent to prepare an Environmental Impact Statement and proposed scope of study for RR alfalfa. Prior to the withdrawal of the product, alfalfa had been used in a number of theoretical (St Amand
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fects are related to the harm part of the equationif a transgene becomes established in the wild will it potentially harm the environment? For reference, in a recent review Weis (2005) has provided an assessment of the ecological tness of recipients of 6.3.13. Trees One of the most active areas of GM research on a global scale transgenes. Fitness is a critical issue with respect to evaluation of trans(FAO 2004, Convention on Biological Diversity, 2007) is the gene gene ow, and largely determines whether a hybrid may production of trees modied for herbicide tolerance, to improve pathogen tolerance or for use as biofuels (Firbank, 2008; Low become invasive (Andow and Zwahlen, 2006). Hails and Morley and Booth, 2007). Some of the relevant reviews are focused on (2005) have tabulated the relative tness of hybrids in crop-wild regulatory issues and biosafety (Sedjo, 2006; Valenzuela et al., species hybridizations. Campbell et al. (2006) demonstrated that 2006) related to specic countries such as Canada (Finstad et al. though crop-weed hybrids may initially have poor tness, t2007) and the United States (Irwin and Jones, 2006). Genetic ness and fecundity may subsequently increase, especially when containment of GM trees and plantations has been considered hybrids are exposed to a new environment. Crop-wild species by several authors, either in general terms (Brunner et al., 2007; introgression in sunower may be enhanced in a more stressful DiFazio et al., 2004; Farnum et al., 2007) or in the context of environment, in which the hybrids express a higher tness level specic molecular approaches to reducing gene ow (Li et al., (Mercer et al., 2007). If the initial transfer of a transgene imparts 2008; Wei et al., 2006). A particularly interesting issue is raised a signicant selective advantage it may only need a very infreby the research designed to improve the disease tolerance of cer- quent hybridization event for introgression to result (Chapman tain heritage trees in the United States (Merkle et al., 2007). and Burke, 2006). It is also possible that any acquired genes As these authors state, is the fact that, rather than restricting will move to weed species related to the initial hybrid popuspread of the transgenes from the engineered trees to wild rel- lation (Zelaya et al., 2007). Alternatively, initial detection of a atives, many of the stakeholders involved believe that crossing transgene in wild populations may be followed by later elimiof the resistance genes into wild populations should actually be nation (Mercer and Wainwright, 2008), which is the process of a goal of these programs, as it presumably would lead to estab- genetic drift, or elimination of the transgene hybrid from the wild lishment of resistance genes in these populations, accelerating population. This could occur if a transgene imparts no selective the restoration of these species. This is certainly the case with advantage, or imparts a genetic cost. To assess this possibility, American chestnut (Castanea crenata), where planning is un- one strategy is to transform a weed species most likely to overlap derway not only to release fertile transgenic trees, but to begin with the cultivated transgenic crop with the transgene of interest a program of crossing with non-engineered genotypes to begin and then evaluate tness in greenhouse and eld trials (Moon et al., 2007). spreading the resistance genes more quickly. For introgression to occur, transferred genes must be stably expressed, as in the case of B. rapa, where Bt-toxin levels re7. ASSESSING THE RISK OF GENE FLOW EVENTS mained as high or higher than the B. napus lines, with which A decision on the probability of gene ow is only one part they hybridized (Zhu et al., 2004). In transgenic rice, it was of the various risk analysis equations used for regulators, an shown that gene dosage was one factor affecting long-term staexample is this one, from Hails and Timm-Wilson (2007): bility of gene expression (James et al., 2002). Not all transgenes are stably expressed, which is a potential additional factor to Risk = P(Transgene escape) P(Transgene spread/Escape) document, as suppression of expression will reduce the risk of P(Harm/Exposure) transgene introgression (Eastham and Sweet, 2002). and this one from Poppy (2004): Risk = consequence likelihood Further information on risk assessment frameworks is provided by Wolfenbarger and Phifer (2000) and useful analogous frameworks are those provided for invasive species (Stohlgren and Schnase, 2006) and exotic plant pests (Cook and Proctor, 2007; Finnoff and Shogren, 2004). We have discussed in the previous section methods for judging the probability of transgene escape. In this section we discuss factors likely to affect the stable introgression of a transgene, which is primarily a function of the tness associated with the expression of the transgene, which is itself a function of environmental factors. Fitness ef7.1. Food and Pharmaceuticals Before discussing potential tness effects of transgenics, it is important to emphasize that even in the absence of any introgression, or absence of any gene ow to wild populations, there may be regulatory risks and/or issues associated with transgenic plants thatwill still require strict containment. A good example of this is pharmaceuticals produced in transgenic plants, where control of gene escape is arguably more important than the inadvertent presence of approved GM seed in non-GM seed food supplies (Gealy et al., 2007; McHughen, 2006; Moschini, 2006; Murphy, 2007). Ellstrand (2003) also makes the point that while crop-to-crop gene ow has not received sufcient attention in the gene ow debate it is just as important with respect to plantderived pharmaceuticals. The probability of gene ow has been
et al., 2000) and eld-based pollen ow studies (Fitzpatrick et al., 2003; Van Deynze et al., 2004).
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measured in safower, one target species of plant molecular farming (McPherson et al., 2004). 7.2. Fitness Traits Gealy et al. (2007) have compiled a very useful table of agricultural traits that could improve the tness of any recipient weeds after introgression. The work by Muir and Howard (1999) on enhanced tness in transgenic sh also includes principles that can be applied to the development of transgenic plants. The conclusion of Gealy et al. (2007) is that herbicide tolerance (discussed separately below), insect tolerance, and disease tolerance (Latham and Wilson, 2008; Prins et al., 2008) are the only current commercial traits likely to affect tness. Burke and Rieseberg (2003), using sunower as the experimental system, describe systematic studies to determine whether introduction of a disease resistance gene would increase seed production, as one measure of potential tness changes. This issue was also considered by Moody-Weis and Alexander (2007). Spencer and Snow (2001) showed that hybrids of wild and virus-tolerant transgenic (cultivated) Cucurbita pepo had increased tness, measured by enhanced seed production in comparison to the wild species. An increase in seed shattering or any changes benecial to seed dispersal (such as resistance to digestion or increased dormancy) would also be potentially advantageous traits in a weed species (Gealy et al., 2007). Transfer of disease or insect resistance traits impart tness because most plants in the wild are under continuous disease and herbivory pressure. Accordingly, expression of resistance would confer a selective advantage. Fuchs et al. (2004b) demonstrated that virus-resistance genes could introgress in wild Cucurbita species and impose a selective advantage in eld conditions. Raybould and Cooper (2005) created a model for assessment of potential tness, also using virus resistance introgression as an example. 7.3. Insect-Resistant Crops Insect-resistant transgenic crops, transformed with the Bt gene, are widely grown. Theoretically, when transferred to wild species this transgene could lead to increased vegetative growth and seed production due to reduced insect herbivory. In some of the earliest eld trials with Bt-transgenic B. napus, Stewart et al. (1997) were able to show an advantage of Bt expression on survival in wild habitats, particularly in cultivated areas, which were then allowed to naturalize. Vacher et al. (2004) grew mixed stands of Bt-B. napus and a related wild species (B. rapa) and examined survival in the next seed generation. They found the transgenics to be less t in the absence of insect-herbivory pressure, but to survive far better than the wild species (measured by seed production) under high herbivory pressure. The effect was enhanced at high plant densities. Sutherland et al. (2006) measured quantitatively the relative resistance of B. napus, the related weed species B. rapa, and their hybrids, to defoliation. The authors did not see an increase in tness in the hybrids
but speculated that the assumptions may not be valid should transgene introgression occur. Snow et al. (2003) reported an increased seed production in wild sunower populations carrying the Bt resistance gene. 7.4. Herbicide-Resistant Crops One of the earliest types of GM crops were the now widely grown herbicide-tolerant varieties. As herbicides are used to control weeds it is inherently obvious that the potential for transfer of HT to weed or wild populations might increase the tness of those populations. This conclusion is reinforced by the fact there are a large number of conventionally-bred herbicidetolerant crops and that with the increased use of herbicides a signicant number of HT weeds species have been identied, as a result of mutation and natural selection (Park and MallorySmith, 2004; White et al., 2003; Tranel et al., 2004). This situation raises two important risk assessment issues. The rst is why it is, or whether, GM-HT crops should be regulated in a different way to conventional HT varieties. For example, in Australia, pollen-mediated gene ow from B. napus was measured at several kilometers from a conventional-HT eld, albeit at low frequency (Rieger et al., 2002). The second issue is the fact that not all weed species have acquired herbicide tolerance through introgression. Many have evolved resistance through mutation, particularly to the imidazolinone group of herbicides (Tan et al., 2005). Kwon and Kim (2001) provide a review of herbicideresistant crops and gene ow, emphasizing the herbicide resistance occurs naturally in weeds. In B. napus use of varieties with different herbicide resistances in the same cultivation areas has led to development of multiple-resistant volunteers (Aono et al., 2006; Hall et al., 2000; Knispel et al., 2008). A level of background mutation and natural selection confuses the interpretation of very low frequency apparent hybridization events detected in very large scale screening for herbicide resistance. Rieger et al. (2002) have made this point, and advocate that the baseline for theoretical hybridization in weed populations needs to be the natural mutation rate. There is evidence of the introgression of HT-resistance genes from crops to related species in several B. napus (canola, or oilseed rape) varieties. For example, Warwick et al. (2008) showed the stable introgression of glyphosate resistance has occurred into B. rapa from transgenic B. napus. Initial hybrids were characterized by reduced male fertility (measured as lower pollen viability) but over a six-year period an individual with HT resistance and no impaired fertility became established, overcoming the initial reduced tness. Similarly, Metz et al. (1997) showed stable expression of phosphinothricin tolerance in successive generations of hybrids between B. rapa and transgenic B. napus. In the presence of a HT-transgene in a wild population, herbicide spraying imposes a selection pressure, which strongly favors any hybrid plant, especially if it propagates vegetatively (Reichman et al., 2006). However, in the absence of selection pressure HT plants may not compete well. Lutman et al. (2006) compared conventional and GM HT oilseed rape
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and found no difference in persistence of seeds in the seed bank. Over three years, Harker et al. (2006) measured volunteer level and seed persistence after planting glyphosate-resistant canola in dened areas and monitoring survival when the land was used for normal agricultural practice, rotated with cereal crops. They found volunteers could be controlled through agronomic practice. Crawley et al. (2001) suggests HT resistance confers no tness benets in natural habitats, whereas Guadagnuolo et al. (2006) evaluated the tness of GM HT (glyphosate) maize X teosinte hybrids and found there was no change in tness in the absence of selection pressure. Roux et al. (2004) determined that there was a herbicide resistance cost in transgenic plants of the model species Arabidopsis thaliana (arabidopsis). Most weed species can be controlled not only through crop management but by a selection of herbicides. Unless multipleherbicide resistance occurs chemicals can most probably be used to control a HT-wild plant population or weed, should introgression occur. This is an important point in assessing the risk of gene ow. There are crops such as rice (Kwon and Kim, 2001), which herbicide-resistant volunteer plants pose a high risk, because they cannot easily be controlled.
TECHNIQUES TO PREVENT GENE FLOW For completeness, it is necessary to summarize the fact that there are a number of techniques available for the excision or deactivation of transgenes, or to induce sterility in lines used for transformation. These have been reviewed in a comprehensive report (http://www.defra.gov.uk/science/ project data/DocumentLibrary/CB02036/CB02036 3629 FRP. doc). The use of these techniques has not reached commercial fruition yet, though there are examples where the Cre/loxP auto-excision method has been used to remove selectable marker genes (Zhang et al., 2006; Luo et al., 2007). One compelling reason for the lack of uptake of these techniques may be that where the method involves genetic modication a risk assessment will need to be carried out and it may be very difcult to provide sufcient experimental evidence that the transgenes will function correctly (Haygood et.al., 2004) on a long-term basis, in the commercial environment. From a regulatory perspective, if a transgenic plant had such a high potential risk to the environment or health (in the case of pharmaceuticals for example) that gene ow from production would need to be completely contained by genetic means (Murphy, 2007), then evidence of proof of concept would need to be provided (Sp k, 2007). The recent discovery that a o contribution to plastid DNA by the paternal parent (pollen) may occur at a low frequency (Azhagari and Maliga, 2007; Svab and Maliga, 2007) adds additional complexity to the concept that plastid transformation (Okumura et al., 2006) may prevent gene transfer from pollen (Daniell, 2007; Ruf et al., 2007; Wills et al., 2005). Methods for the excision of selectable marker genes have been reviewed recently (Lee and Natesan, 2006; Natarajan and
8.
Turna, 2007), while Hills et al. (2007) reviewed the two Genetic Use Restriction Technologies (GURTs), T-GURT (trait-GURT) and V-GURT (varietal-GURT). The former technique allows seed formation but controls the expression of the transgene and the latter prevents transmission of the gene in the seed. Both strategies have proved extremely controversial in the context of international trade (Bustos, 2008) and agriculture (KameriMbote and Otieno-Odek, 2006). Singh et al. (2007) have outlined a concept of gene containment based on epigenetic imprinting and RNAi-based post-transcriptional gene silencing, which would prevent pollen-mediated gene ow while allowing seed set to occur normally. Kuvshinov et al. (2001, 2004) have described RBF (recoverable block of function), based on the barnase/barstar co-function system. There are examples of the effective use of this system to confer bisexual sterility (Kobayashi et al., 2006). Genetic modication may also be used to inhibit ower formation (Eastham and Sweet, 2002), which is only a feasible approach for non-seed products. Lemmetyinen et al. (2001) and L nnenp a et al. (2005) identied a MADS gene essential for a a owering and used a barnase construct driven by the promoter from this gene to prevent owering. The Glu-1Dx5DNA fragment from wheat can be used to reduce pollen viability in maize (Scott et al., 2007). In bentgrass Luo et al. (2005) engineered pollen sterility by suppression of a rice tapetum-specic gene, and expression of a stilbene synthase gene was shown to induce male sterility in Pinus radiata (H g et al., 2006). Dio rected elimination of transgenes from microspores is described by Mlyn rov et al. (2007) and an alternative approach for rice a a is suggested by studies using a gene that determines cleistogamy (Yoshida et al., 2007). Transgenic mitigation (Gressel and AlAhmad, 2006) is a concept that the tness of any recipient of the transgene is lower than that of the wild type as a result of expression of tandemly linked mitigating genes, whose expression is benecial in the transgenic crops but deleterious to any recipients of the transgene grown in competition. Al-Ahmad et al. (2005) showed that tobacco plants carrying a dwarng (mitigation gene) in tandem with a herbicide resistance gene grew far less well in competition with wild type tobacco. Similar results have been reported in Brassica (Al-Ahmad and Gressel, 2006; Al-Ahmad et al., 2006). Kuparinen and Schurr (2008) have, however, modeled how this mitigation strategy may break down, using modied trees as an example (Li et al., 2008; Wei et al., 2006). There are simpler, nonmolecular, containment strategies. Geographical isolation may be possible (Wang et al., 2004) and Wilkinson et al. (2000) propose avoidance of growing crops in areas where compatible species are known to exist. Use of malesterile varieties for transformation (Celis et al., 2004), such as in potato (Green et al., 2005) prevents pollen-mediated gene ow and it may be possible to regenerate L1 chimeric-transgenic plants, which do not carry the transgene in the male gametes. A better understanding of interspecic hybridization could lead to selection of target varieties for transformation that are not able to
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fertilize with the most common related weeds (Gueritaine and Darmency, 2001). Another approach is to use for transformation varieties, which have a lower inherent risk. For example, Darmency et al. (2007) suggest using sugar beet varieties that do not bolt, to reduce volunteer formation. Alternatively other authors suggest the use of non-owering, or non-food crops such as the alga Chlamydomonas (Manuell et al., 2007) or the duckweed Spirodela (Vunsh et al., 2007). CONCLUSIONS The evidence is that hybridization to wild relatives has occurred in most food crops and is implicated to weed evolution in rice, soybean, sorghum, millet (Setaria italica), beans, and sunower (Ellstrand et al.,1999). Gene ow from at least some transgenic plants should therefore be expected and for some species, in some circumstances, gene ow is inevitable (Glover, 2002; Dlugosch and Whitton, 2008). It is not a surprise to see long-distance gene ow in a wind-pollinated species such as creeping bent grass (Agrostis stolonifera) (Pfender et al., 2007), where pollen-mediated gene ow over several kilometers has been shown (Watrud et al., 2004) and transgenic populations have now become established in nonagronomic environments (Reichman et al., 2006; Zapiola et al., 2008). It should therefore also not be a surprise to groups wishing to release genetically modied plants that regulators will seek a realistic and thorough evaluation of the probability and potential impact of any gene ow. Our nal advisory points summarize the key components of what should be included and considered in any assessment of the probability and risk of gene ow from a transgenic plant. Some very useful papers that provide an overview of the principles of gene ow risk assessment to transgenic plants are the landmark paper of Wilkinson et al. (2003) who described hybridization of nontransgenic B. napus on a national scale; Espinoza-Esquivel and Arrieta-Espinoza (2007) who outlined the steps that were taken in evaluation of HT-rice for release in Costa Rica; and Arriaga et al. (2007) who assessed the release of Cucurbita in Mexico. 1. Fully understand and document the biology of the target species and related wild populations and species. One of the most useful sources of information on the basic biology of crops is the set of Biosafety Consensus Documents prepared by the OECD (http://www.oecd.org/biotrack). They are intended to be a snapshot of current information on a specic host organism or trait, for use during regulatory assessments and to date 28 have been published, the most recent being that on North American Larches (OECD, 2007). It is also useful to consult two additional texts. The rst, entitled An Introduction to the Biosafety Consensus Document of OECDs Working Group for Harmonisation in Biotechnology, explains the purpose of the Consensus Documents and how they are relevant to risk/safety assessment. The second text is Points to Consider for Consensus Documents on the Biology of Cul9.
2.
3. 4.
5.
6.
7.
tivated Plants. This is a structured checklist of points to consider for authors when drafting or for those evaluating a consensus document. Amongst other things, this text describes how each point is relevant to risk/safety assessment. Determine whether the various models that have been developed for gene ow from genetically modied plants are applicable to the specic application in question. If so, recognize the limitations of such models. A model cannot cover all possible scenarios for gene ow however much detail and number of factors are considered (Ellstrand, 2003). More generalized models such as those for predicting out-crossing probability (Smith-Kleefsman et al., 2005) will provide useful background information for dossier preparation. Rate the probability of gene ow using common riskassessment methods. Understand the environment where the transgenic plant will be released. The history of invasive plants in a particular region may be a predictor of the potential capacity of a theoretical hybrid to become invasive (Maillet and Lopez-Garcia, 2000). With the development of bio-fuel crops (Low and Booth, 2007), which are grown on a wider range of more marginal land, the potential exposure to wild populations has increased, as has the potential for selection of tter individuals, from which transgenes could be sourced (DiTornaso et al., 2007). Consider non-biological routes of seed and pollen dispersal. Seed dispersal is a very important gene ow mechanism (Andow and Zwahlen, 2006) and may be the main route in some cases (Baack, 2006). Illegal trade in GMOs and plant and seed smuggling is another avenue for gene ow (Gealy et al., 2007) famously exposed in the Brazilian soybean growing areas. The issue of adventitious presence, or a low level of seed present in a supposedly 100% pure seed lot (Gealy et al., 2007) is also of particular importance to organic growers, but is more an identity preservation issue, rather than one of gene ow. Consider the relative risk of transgenic crop cultivation to the environment, in comparison to similar conventionally bred varieties, if they are available. Consider the potential tness characteristics that could be imparted by the transgene. The common theme from many researchers on gene ow in transgenic plants is that as gene ow will and always has happened (Burke and Rieseberg, 2003; Chapman and Burke, 2006; Ellstrand, 2003; Messeguer, 2003; McHughen, 2006; Richards, 2005; Vacher et al., 2004), the focus of any assessment of a potential GM plant release should be on risk assessment of a theoretical hybridization event, rather than the probability of gene dispersal. Unless there is an extremely low (this may be the case for some intensively cultivated plant species) probability of gene ow, it is reasonable to include some assessment of the potential for introduced genes to impart tness traits. If a conclusion is that hybrid plants are likely to occur in some situations the most useful rst step of evaluation should focus on
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characterization of the hybrids in comparison to the parental controls (Poppy, 2004). If there are scenarios where tness could be increased in a theoretical hybrid, and there are management practices (Feil et al., 2003) that could be employed to reduce the amount of crop-to-crop or crop-wild gene ow, these should be elucidated. 8. Finally, evaluate and assess risk (Raybould, 2007). If the transgenic plant has no wild relatives and the gene does not confer tness it may be considered a low-risk product, whereas a higher risk might be allocated if wild relatives were present and the gene was likely to confer tness to hybrids (Lee and Natesan, 2006). A scheme for assessing potential environmental risks due to gene ow in a risk assessment framework, typically employed by regulators, has been discussed by Raybould and Wilkinson (2005) and Wilkinson and Ford (2007). There are situations (for example the production of pharmaceuticals) where transgenic plant release should only occur under conditions where gene ow can be strictly controlled (Andow and Zwahlen, 2006). These are perhaps the only situations where genetic modication gene restriction technologies should be considered, and then only when there is no possibility to use male sterile, vegetatively propagated or non-food crops. While there is a probability of some transgenic varieties escaping from cultivation, or hybridizing to wild species, the risk to the wild ora is not as great as the risks that have faced the natural environment from nontransgenic commerce and agriculture. Gealy et al. (2007) for example, rate the risk of invasive plants (including weed seeds exported in commercial grain shipments) as being much greater than the risk of introgression of transgenes into weeds or volunteer GM crops. It is beyond the scope of this review to debate the history of transgenic plant regulation and why it is that conventionally bred HT-resistant crops are not regulated to the same level of scrutiny as the same phenotypes produced by genetic modication. Whether reasonable or not, it is a fact that transgenic plants are highly regulated and whatever the detail and extent of the scientic information presented, there are nonscientic issues that will be decided by the policy makers, not the regulators. In Europe (Sanvido et al., 2007b), the enshrinement of the precautionary principle (Cocklin et al., 2008; Ervin et al., 2003; Morris, 2007) and application of conservation principles and risk-benet analysis to transgenic plants (Aslaksen and Myhr, 2007) must be considered by all applicants to release a GMO. Unnecessary or precautionary identity preservation and segregation, perhaps to meet very strict threshold levels, can be very costly Gealy et al. (2007). Additionally, concerns about the legal liabilities associated with transgene ow have been raised (Boadi, 2007; Clapp, 2008). This leaves anyone wishing to develop a transgenic product for the world market in a complex situation. In North America, transgenic crops such as maize, soybean, cotton, and canola are widely grown commercially with no need for borders or extensive isolation distances, and with relatively high comfort
levels in that respect (Esposito and Kolodinsky, 2007), especially when weighed against the environmental benets (Gealy et al., 2007). In contrast, in the EU, GM crops are still only grown in a few countries with a small number of varieties, despite very extensive research on measurements of gene ow (Gray, 2004, 2005; Demont et al., 2007). As a developer of genetically modied varieties, one has no choice but to make an intelligent decision about the key factors likely to be important for gene ow in any particular crop/trait combination, using resources such as those cited in this review. REFERENCES
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26 6.3.7. 6.3.8. 6.3.9. 6.3.10. 6.3.11. 6.3.12. 6.3.13. 7.

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Barley, wheat Sunower

Lim et al. (2007)

Tomato Bentgrass (Agrostis stolonifera L.)

Common bean (Phaseolus vulgaris)

Ferreira et al. (2007)

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Silage maize Grain maize Fodder maize

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Llewellyn et al. (2007)

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Hails et al. (1997) Walker et al. (2004)

Lutman et al. (2005)

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Eastick and Hearnden (2006)

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