Feasibility of new breeding techniques for organic farming

This is a preprint of the paper later published and to be cited as: Andersen, M.M., X. Landes, W. Xiang, A. Anyshchenko, J. Falhof, J.T. Østerberg, L.I. Olsen, A.K. Edenbrandt, S.E. Vedel, B.J. Thorsen, P. Sandøe, C. Gamborg, K. Kappel, M.G. Palmgren, 2015: Feasibility of new breeding techniques for organic farming. Trends in Plant Science, 20, xxx-xxx. Online at: http://dx.doi.org/10.1016/j.tplants.2015.04.011 1 Feasibility of new breeding techniques for organic farming Martin Marchman Andersen1, Xavier Landes1, Wen Xiang2, Artem Anyshchenko2, Janus Falhof3, Jeppe Thulin Østerberg3, Lene Irene Olsen3, Anna Kristina Edenbrandt4, Suzanne Elizabeth Vedel4, Bo Jellesmark Thorsen4, Peter Sandøe5, Christian Gamborg4, Klemens Kappel1, Michael G. Palmgren3 1 Department of Media, Cognition and Communication, University of Copenhagen, Karen Blixens Vej 4, DK-2300 Copenhagen S, Denmark 2 Centre for Public Regulation and Administration, Faculty of Law, University of Copenhagen, Studiestræde 6, DK-1455 Copenhagen K, Denmark 3 Center for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark 4 Department of Food and Resource Economics and Center for Macroecology, Evolution and Climate, University of Copenhagen, Rolighedsvej 23, DK-1958 Frederiksberg C 5 Department of Large Animal Sciences, University of Copenhagen, DK-1870 Frederiksberg C, Denmark Address for correspondence: Michael Broberg Palmgren, Department of Plant and Environmental Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark. Tel.: +45 2398 8444 Email. palmgren@plen.ku.dk Keywords Rewilding; reverse breeding; organic agriculture; TALEN; CRISPR-Cas9; cisgenesis; 2 Organic farming is based on the concept of working ‘with nature’ instead of against it, but compared to conventional farming organic farming reportedly suffers from lower productivity. Ideally, the goal should be to narrow this yield gap. In this review, we specifically discuss the feasibility of new breeding techniques (NBTs) for rewilding, a process involving the reintroduction of properties from the crops’ wild relatives, as a method to close the productivity gap. The most efficient methods of rewilding are based on modern biotechnology techniques, which have not so far been embraced by the organic farming movement. Thus, the question arises of whether the adoption of such methods is feasible, not only from a technological perspective, but also from a conceptual, socio-economic, ethical, and regulatory perspective. Organic farming and biotechnology Although conventional agriculture is highly productive, it is often considered incompatible with the principles of alternative approaches to food production, such as organic farming. Traditional breeding methods have been exceptionally successful in creating crop plants with high yields and other desirable properties, but modern crops often require intensive management to avoid being outcompeted by weeds, infected by diseases, or eaten by insects. Organic farming is an agricultural system that aims to mimic processes in natural ecosystems for the provision of nutrients and pest control, instead of relying on chemical inputs. For this reason, chemical fertilizers, pesticides, and other agents used in conventional agriculture are restricted or prohibited in organic production. As a consequence, productivity is often lower in organic than conventional agriculture [1-4], and several strategies have been suggested to close this yield gap between high- and low-performing conventional systems [5-7]. A plea for merging organic agriculture and genetic engineering approaches has previously been published [8]. Here we discuss the feasibility – in a broad sense – of introducing new methods of plant biotechnology to allow for sustainable intensification of organic farming (i.e., increased production from existing cultivated land with minimal pressure on the environment). Organic farming excludes a number of practices due to sustainability, health, and safety concerns. These concerns are reflected in the four principles of health, ecology, fairness, and care, as defined by The International Federation of Organic Agriculture Movements (IFOAM) [9]. According to these principles, certain forms of biotechnology have been considered to be irreconcilable with organic agriculture, as stated by IFOAM: 3 ‘Organic agriculture should prevent significant risks by adopting appropriate technologies and rejecting unpredictable ones, such as genetic engineering’ [9]. However, as technologies evolve, it is not obvious that all forms of technology, and especially those involving some kind of genetic modification sensu lato, should be deemed incompatible with organic farming. IFOAM’s statement suggests that genetic modification of plants has unpredictable consequences. But whether, and to what extent, this is so, is a contingent empirical question that must be examined in detail for any crop developed. Moreover, the issue is complicated further by the fact that the term “genetic engineering” spans several strategies in modern plant biotechnology that cannot adequately be evaluated as one. For instance, reverse breeding (rewilding) aims to bring crops “back to nature” by furnishing them with lost properties that their ancestors once possessed. The most direct and predictable tools for (re)developing crops with such improved properties involve genetic engineering. Thus, the question is, how feasible is the use of new breeding techniques (NBTs) for organic farming? To address this, we conducted an evidence-based feasibility study based on available literature within the fields of natural and social science, law and ethics, and allowed for informed conjectures when evidence was incomplete. We stress the importance of a thorough cross-disciplinary approach, as the challenges faced are not resolved by referring to technological possibilities only. The three main questions addressed in this paper are: 1) could reverse breeding (rewilding) be considered useful to organic farming from a natural science point of view?, 2) what is the nature of the perceived incompatibility, and what might be possible grounds for rewilding to be considered congruent with basic principles of organic farming?, and 3) how feasible are such techniques from socio-economic, ethical, and legal perspectives? Technological feasibility and usefulness: Why rewilding? Traditional breeding – a tale of mutated plants In nature, mutations happen spontaneously all the time and are the basis of evolution [10]. They can result from internal mistakes in the functioning of the cellular machinery or from external factors such as UV radiation from the sun or background radiation from the universe. Breeders have always made use of such mutations by exploiting the resulting diversity of traits and have also accelerated the process by creating random mutations using radiation or mutagenic chemical treatment [11]. Consequently, important traits have been selected for, including ease of harvesting, increased yield, and reduced toxicity. This holds true for all modern crop plants, including those used in organic agriculture [12,13]. 4 Rewilding crops to reverse the loss of genetic diversity Wild relatives of crop plants have many beneficial traits that are not present in modern crops [14]. There may be two main reasons for this. First, evolutionary pressure, such as exposure to a previously unencountered pathogen, forces wild plants to adapt to changes in the environment. Thus, in the wild, evolutionary pressure ensures that plants with advantageous mutations survive [15,16] whereas in crops, progeny selection is breeder controlled, and has mainly been focused on traits that improve output in a high-input environment [13]. Second, it has been proposed that during the domestication process, mutations have occurred in genes responsible for traits other than those being selected for and such mutations may have caused loss of gene function and, as a result, the traits associated with these genes were weakened or completely lost during domestication [17]. Thus, unintended mutations in crop plants may have compromised the hereditary basis for plant survival during times of environmental stress, both biotic (such as herbivory and disease) and abiotic (such as drought, flooding, nutrient deficiencies, and salinity). Around 95% of the crop plants used in organic farming were bred for conventional agriculture approaches and are therefore not suited to organic farming techniques, which limit pesticide, herbicide, and fertilizer usage [12,13]. Many modern crop plants suffer from low genetic variation and we cannot rely on their present diversity to select for desired traits [18,19]. To close the yield gap between organic and conventional farming, organic farming is in need of more robust plants. By bringing back select lost properties of their ancestors, rewilding has the potential to increase genetic diversity and reintroduce wild traits that would benefit organic farming. How can we ensure that unintended mutations in our crop plants are repaired, and how can we help crop plants benefit from advantageous mutations that arise in wild plants? These are the challenges of rewilding. Available methods for rewilding The traditional method of transferring genes from wild plants to crop plants is to cross the crop plant with a wild relative and repeat the crossing process several times by a method known as introgression breeding (Figure 1). Introgression breeding strategies have already been demonstrated to expand the available gene pool in crops and increase overall genetic diversity (Table 1; [20,21]). This method has the advantage that the gene(s) responsible for a desired trait need not be known. However, it also has some important drawbacks. First, it takes a considerable amount of time 5 (typically more than ten years). Second, as introgression breeding initially involves mixing all genes present in two related organisms, it may lead to plants with major alterations in their genetic material, including genes associated with favorable traits. For example, unwanted genes close to the sought-after gene may be hard to eradicate [22]. Third, this method is not a viable option if more than one gene or allelic variant is required to establish a trait, as is the case for cold, salt, and drought resistance [23]. Modern biotechnological approaches, including NBTs such as cisgenesis and precision breeding (Figure 1; Table 1), offer precise alternatives to introgression breeding that are much faster, because they are not based on crosses. However, these approaches require that the gene or genes responsible for the desired wild trait be known. Only recently have we been able to map in detail the genetic variation in plant genomes and link genetic differences to specific traits [24-26]. However, with the advancement of next generation sequencing programmes, we can expect a massive increase in this type of information in the near future [27,28]. The first crop plants modified by precision breeding were loss-of-function mutants [29,30]; however, this method holds excellent promise for rewilding. From a biological perspective, rewilded plants generated through introgression breeding and precision breeding are in principle identical. In both cases, the end product is a crop plant carrying a wild-type allelic variant. Advisory bodies have concluded that crops resulting from precision breeding cannot be distinguished from conventionally bred crops [31,32]. Due to the techniques used to produce them, crop plants resulting from cisgenesis and precision breeding are nonetheless classified as genetically modified (GM) plants according to current legislation, at least in the EU [33]. This legislation was originally implemented to control the use of so-called transgenic plants where foreign gene material is transferred across species barriers. Given that organic farming would benefit from crops that are hardened by rewilding, and that the biotech approach to rewilding is feasible from a technological perspective, the question arises of whether rewilded plants bred using NBTs are compatible with organic farming principles. Conceptual feasibility: Are new breeding techniques compatible with the principles of organic farming? The first question to consider is whether rewilding is compatible with the core principles of organic farming. If it is, the second question is whether NBTs would be considered acceptable tools to realise these principles. Perhaps there are multiple answers to these questions, as there are several ways to define the essential features of organic farming. However, it makes sense to try to assess 6 the issue of compatibility through the organic farming principles of IFOAM [9], which serve as international standards and represent a widely accepted understanding of organic agriculture. Principle of health The formulation of this principle is open to many interpretations. However, IFOAM specifies that: “In particular, organic agriculture is intended to produce high quality, nutritious food that contributes to preventive health care and well-being. In view of this it should avoid the use of fertilizers, pesticides, animal drugs and food additives that may have adverse health effects.” This requirement is exactly what rewilding aims to meet. One of the aims of rewilding is to furnish crops with lost properties of their ancestors and thereby increase their resistance to pests and diseases. By enabling crops to utilize available natural resources more effectively, the use of fertilizers and pesticides can be minimized without harvest failure. Thus, rewilding is not only compatible with the principle of health, it is perhaps the most feasible way to promote it. Principle of ecology IFOAM specifies this principle as follows: “Inputs should be reduced by reuse, recycling and efficient management of materials and energy in order to maintain and improve environmental quality and conserve resources.” The essence of rewilding is to restore natural properties of plants that have been lost during traditional breeding. As a tool, rewilding therefore has a strong ecological potential, since it can effectively be used not only to sustain, but also to reinforce, ecological systems. IFOAM also specifies that: “Organic agriculture should attain ecological balance through the design of farming systems, establishment of habitats and maintenance of genetic and agricultural diversity.” As for diversity, NBTs can, of course, be used for different aims. Nonetheless, rewilding is potentially beneficial for diversity, because it may reduce the need for pesticides and fertilizers, which have an adverse effect on diversity. Principle of fairness IFOAM describes this principle as follows: “Fairness is characterized by equity, respect, justice and stewardship of the shared world, both among people and in their relations to other living beings.” This principle is open to multiple interpretations. None of the obvious ones appear to be inherently incompatible with NBT-based rewilding; however, the question of technology ownership requires attention. 7 Principle of care This principle aims to balance efficiency with health and well-being, and reveals that organic agriculture in principle is open to new technologies: “Practitioners of organic agriculture can enhance efficiency and increase productivity, but this should not be at the risk of jeopardizing health and well-being. Consequently, new technologies need to be assessed and existing methods reviewed.” However, significant risks should be avoided: “Organic agriculture should prevent significant risks by adopting appropriate technologies and rejecting unpredictable ones, such as genetic engineering.” Whether, and to what extent, this is so, must be examined for each method used and for each product, and hence remain largely an empirical question. We can conclude from the principle of care that the spirit of organic agriculture has a conservative risk profile. However, it is not obvious that rejecting new technologies like NBTs is the least risky strategy. Socio-economic feasibility: Likely reactions to NBTs by consumers and farmers Although consumers view the organic production process favorably [34], the total organic market share remains small [35]. Conversely, consumers disfavor GM breeding technology [36,37]. The following questions arise: Why do some consumers prefer (and buy) organic products? Why do many consumers prefer non-GM breeding technologies and how are rewilded products likely to be perceived? What are the potential market effects of classifying products based on crops rewilded by NBTs as organic foods? The consumers’ beliefs and attitudes toward organic food help explain their purchases. Personal gains, such as perceived health benefits and higher safety, are the strongest motivators for purchasing organic food [35,38,39]. To a lesser extent, organic food is perceived as being more nutritious and tastier. Interestingly, positive environmental impacts are often cited by consumers as a reason to purchase organic foods, whereas statistical analyses show that stated motivations rarely explain the actual purchases of consumers [35,40,41]. However, committed organic consumers find environmental aspects more important than occasional consumers [42]. The socio-demographic characteristics of the organic consumers revealed few clear patterns that could explain purchases [38,43]. However, women seem to purchase relatively more organic products than do men, and households with children or higher education levels are more likely to purchase organic food [34,41,44]. Further, country-specific food cultures, general values, and food policies can be linked to the significant differences in the organic market share among countries with similar income levels [43]. Similar to organic food, the consumers’ willingness to pay for non-GM over GM food 8 is largely explained by health and environmental concerns, while socio-demographics are less important [45,46]. Furthermore, the willingness-to-pay gap is higher in the EU than in the US, but significant in both regions [37,47,48]. Consumers are less averse to GMs when the transferred genes originate from a plant rather than an animal [42]; however, non-GM foods are still preferred. Given that consumers are more likely to accept cisgenesis than transgenesis [47,49], we also expect that precision mutagenesis will be preferred over transgenesis. We note that this extrapolation may not be valid for consumers of organic products. The premium for non-GM foods is lower when the corresponding GM product is associated with fewer pesticides [50]. However, consumers most in favor of organic food production are also generally against GM foods [42,51], and least willing to purchase GM products [52]. Thus, being non-GM is a non-trivial attribute of the organic brand itself, in both the EU and the US [53]. Committed organic consumers may find rewilded crops unacceptable, and if organic labels allow for rewilding, these consumers may demand stricter labeling practices, or seek other traditional or local products. Many consumers express a preference for organic over conventional, although the relatively small organic market share proves that most consumers are unwilling to pay the current premiums for organic products [39,54]. If rewilding enables lower production costs, and organic labels tolerate it, a broader consumer segment that is more price sensitive than the current organic purchasers may emerge as an important market. Most European studies on farmers’ attitudes are based on hypothetical scenarios, yet studies often do not report specifically on organic farmers’ preferences [55-57]. Organic farmers typically use conventionally bred seeds, and it remains an open question if they would view rewilded seeds as compatible with organic production if these seeds gave rise to plants with improved resistance to biotic or abiotic stress. Studies have found that organic farmers reject the use of GM crops [58], and organic and conventional farmers’ resistance to GM crops is well aligned with consumers’ views [59]. For instance, Canadian farmers (from organic to GM producers) all ranked the risks associated with herbicide-tolerant wheat (Triticum aestivum) as higher than the benefits [60], a pattern confirmed elsewhere [61,62]. In Austria, where 11% of all farmers practice organic farming, a restrictive co-existence policy for GM crops has been implemented, driven by both the public and organic farmers [63]. 9 Thus, organic farmers are unlikely to view rewilded crops as being compatible with their production practices and philosophy, even if these crops have enhanced performance. However, as more farmers convert to organic practices to take advantage of the associated price premiums, the average motivation of organic producers may change [64]. This could imply a higher potential for acceptance of rewilded crops among some organic producers in the long run. Ethical feasibility: Sustainability, naturalness, and integrity The ideal of sustainability has evolved over hundreds of years and has come to encompass a growing number of concerns, as a response to changing societal agendas [65]. In relation to breeding, sustainability refers to the tenet that environmental concerns, genetic diversity, ethical considerations, and social issues should be addressed and also that short-term and long-term economic value should be preserved [66]. Similar to proponents of organic farming, proponents of modern biotechnology claim to have means to achieve a higher degree of sustainability [67,68]. Especially when viewed in the context of developing countries, it has been argued that genetic modification techniques may be used to limit the environmental impact of farming and may therefore be viewed as compatible with the principles of organic farming [69]. However, such claims have long since been challenged by the organic agriculture movement and by advocates of other movements that see themselves as alternatives to industrial agriculture [70]. Biotechnology – and especially the production of GMOs – is generally considered to be in conflict with alternative approaches to food production, and thus perhaps some kind of coexistence of approaches is the best one could hope for [71]. The scientific and broader public debate have remained quite polarized [72], framing novel breeding techniques as friend or foe, and not allowing for discussions focusing on applications and outcomes rather than on the technology per se. The main objections to the use of biotechnology, and especially forms of genetic modification, in organic agriculture fall into four categories: (i) risks to human health and the environment (e.g., release of organisms that have never before existed in nature and that cannot be recalled; pollution of the gene pool of cultivated crops, micro-organisms and animals; and pollution of off-farm organisms); (ii) so-called socio-ethical objections (e.g., perceived loss of farmer autonomy; violation of farmers' fundamental property rights; and endangerment of their economic independence), (iii) incompatibility with the ideas of sustainable agriculture (e.g., different interpretations of sustainability concurrent with different views on humans’ relationship with nature), and (iv) the dynamic complexity of the ecosystem as a whole that may lead to 10 unpredictability and unintended effects (i.e., biotechnology is perceived as a technological fix for problems that are essentially systemic) [73, 74]. Furthermore, attempts have been made to rationalize doubts or justify rejection by focusing on the notion of naturalness [75] and on the purported intrinsic value and integrity of plants produced by organic farming [76-78]. Recently, the term ‘natural’ has been attached to food products whenever possible, as it is taken to have a pronounced positive connotation, essentially referring to an “original” state of things [79]. However, a recent study [80] identified five different arguments in favour of the conclusion that genetically modified organisms are unnatural, corresponding in substance to ones found in the literature on the concept naturalness [81]. Thus, the question of naturalness becomes much broader than to be just about biological similarity. These arguments are ‘history-based’ (stressing that GM entities are unnatural because they are a product of human interference); ‘substance-based’ (saying that different species are crossed − clearly not the case in rewilding); ‘feature-based’ (saying that GM entities have features that differ from those of their unmodified relatives); ‘harmony-based’ (saying that they may create imbalances in nature); and ‘acquaintance-based’ (saying that GM entities are unnatural because they are not well known). Combinations of these arguments may play out in ways favoring crops obtained by rewilding over transgenics, or equate them. Arguments that assume that crops obtained by NBTs are more natural, should be regarded with caution if the meaning of ‘natural’ is not defined. In a similar vein, factors that might trigger concern among the public include: crossing species boundaries – and for public perception, the distance between the gene recipient and gene donor is relevant; familiarity and habituation – e.g., rejecting hybrids that cannot easily be named and classified; and human intervention and the method used [82]. A lack of a shared understanding of the terms ‘integrity’ and ‘naturalness’, and the role these concepts should play, seems to have contributed to the stalemate in the debate about using technologies that could potentially narrow the yield gap between organic and conventional farming practices [83]. It is important to note that while organic agriculture can be defined by certain principles [9], which are formulated in a way that gives room for interpretation and hence for differences in resulting practices, market forces and hence expectations among potential consumers (and farmers’ perceptions of these consumers and markets) also exert a notable influence [84]. Moreover, IFOAM’s principle of care does not rule out the use of science and scientific knowledge produced in scientific institutions, which may serve to caution against steadfastly rejecting gene 11 technology [cf. ref. 85]. Thus, abandoning an outright rejection of technologies, especially those involving some kind of genetic modification, renders three questions that provide a useful basis for dialogue: (i) Is there anything to be gained by embracing certain forms of gene technology such as NBT-derived rewilding? (ii) Would consumers be able to understand and accept such forms of back-to-nature breeding? (iii) Could rewilding be considered compatible with the basic ideas of organic farming? Regulatory feasibility: Legal uncertainties of new breeding techniques Legally, organic farming excludes GMOs It makes little sense to use NBTs in organic agriculture if the food derived from the resulting plants could not be labelled as organic. Thus, the question arises of whether the use of NBTs would be in conflict with the rules and standards for labelling of organic food. Although legal definitions of organic production vary from region to region, we can address this question based on the elements that these definitions have in common. The Codex Alimentarius Commission, which was jointly established by the Food and Agriculture Organization (FAO) of the United Nations and the World Health Organization (WHO), aims to achieve international harmonization of the requirements for organic products. Its ‘Guideline of the Organically Produced Foods’ provides a collection of internationally adopted organic food standards and lists substances that are permitted in organic production [33]. However, several regions have their own definitions and principles for organic production. The Code of Federal Regulations (CFR) of the United States of America provides a definition of organic production and practice standards for controlling crop pests and weeds, and lists allowed and prohibited substances [86]. The EU’s Regulation No 834/2007 [87] and its implementing Regulation No 889/2008 mainly pertain to organic production and labelling of organic products. Organic products are commonly partly defined in these legislative acts by excluding the use of GMOs. For instance, Article 9(1) of the EU Regulation No 834/2007 reads as follows: “GMOs and products produced from or by GMOs shall not be used as food, feed, processing aids, plant protection products, fertilisers, soil conditioners, seeds, vegetative propagating material, micro-organisms and animals in organic production.” [87]. To be or not to be GMOs 12 There are two general legal approaches to regulating and defining GMOs, a process-based approach and a product-based approach [17,88]. According to the process-based approach, which has been adopted de facto by the EU, GMOs are defined as arising from the use of certain specific methods. According to the product-based approach, which is used in the US and Canada, a GMO is defined as possessing a new combination of genetic material that could not have occurred naturally. We will evaluate both approaches to determine whether NBTs necessarily lead to GMOs and thus to legal incompatibility with organic farming practices. Precision breeding results in point mutations, but does not create any new combinations of genetic material, and similar mutations occur naturally in conventional breeding. Such mutations are targeted in precision breeding, whereas they occur at random in conventional breeding and are often accompanied by other unknown mutations. Thus, precision breeding should arguably be legally distinguished from transgenic techniques and not be included among the techniques that lead to GMOs in both product-based and process-based jurisdictions [89]. Indeed, in the EU, precision breeding may already be specifically excluded from the techniques that result in GMOs according to Annex I B of the Directive 2001/18/EC. Article 3(1) of this Directive states that mutagenesis may be excluded under certain conditions: “Techniques/methods of genetic modification yielding organisms to be excluded from the Directive, on the condition that they do not involve the use of recombinant nucleic acid molecules or genetically modified organisms other than those produced by one or more of the techniques/methods listed below, are: (i) mutagenesis; (ii) cell fusion (including protoplast fusion) of plant cells of organisms which can exchange genetic material through traditional breeding methods” [90]. Precision breeding techniques do not insert recombinant nucleic acids into the genome and so would seem to be excluded from the class of processes that result in GMOs referred to in Article 3(1). However, the case is not clear-cut. First, this article was written before precision breeding techniques emerged. Second, in precision breeding, the enzyme (nuclease) that induces the formation of mutations is guided to its target site by recombinant nucleic acids (directly or indirectly) and, therefore, the process arguably involves the use of recombinant nucleic acid molecules. By contrast, according to a product-based approach, precision breeding should clearly be included among the techniques exempted in Article 3(1) of Directive 2001/18/EC. Cisgenesis makes use of recombinant nucleic acids and employs a GMO (i.e., Agrobacterium tumaefaciens) to introduce the material into the plant genome. Therefore, according to the process-based approach, the resulting plants would be defined as GMOs. The most likely conclusion is that precision 13 breeding would be considered to produce GMOs according to the process-based approach adopted in the EU, while it may not necessarily be considered to produce GMOs in the US. Need for rethinking the regulation of NBTs in the EU The legal uncertainties of how to classify NTBs and the increasing pressure from other trade partners within the WTO regime may prompt the EU to rethink its de facto process-based regulatory framework. Firstly, the definition of GMOs adopted by the EU might need to be modified in the light of other international regimes. Directive 2001/18/EC of EU describes GMOs as organisms “in which the genetic material has been altered”, whereas the Cartagena Protocol specifies that a GMO needs to have “a novel combination of genetic material” [91]. In either case, if plants obtained from NBTs cannot be distinguished from crops bred by conventional means, they should be exempted from the current GMO legal framework. Secondly, a derogation (i.e., a partial annulment) with respect to cisgenesis might also be considered in the EU Directive 2001/18/EC. Furthermore, cisgenesis should arguably be included in Annex I B, and thereby excluded from the Directive even if GM techniques have been used throughout the process, provided that, in the end product, no novel combinations of genetic material are obtained that could not have occurred naturally. Concluding remarks There seems to be growing recognition among breeders and farmers that valuable natural traits have been lost in both conventional and organic crops. A common understanding of the difference between organic farming and mainstream farming is that the former prohibits the use of soluble mineral inputs as well as synthetic herbicides and pesticides. Some NBTs seem to represent a feasible means of reducing the need for such chemicals. Contrary to transgenesis, where new genes are introduced to an organism, reverse breeding is a technique that brings crops “back to nature” by furnishing them with lost properties that their ancestors once possessed. Existing examples of organic principles as advocated by IFOAM reject genetic engineering on the premise that it is unpredictable. This, however, is not a defining property of genetic engineering, but a changeable empirical matter. Specifically, it would seem that successfully rewilded organisms are no more unpredictable or risky than their ancestors. Moreover, in debates concerning agriculture, it is often asserted that 'sustainability’, ‘naturalness’, and ‘integrity' express concerns that are inherently 14 incompatible with NBTs or rewilding. However, it is far from clear why this should be so, though it should be acknowledged that it might be so for some groups, depending on their interpretations. Finally, it should be noted that the potential use of NBTs in organic farming is limited in the EU, where the current regulatory framework is process-based and hence would classify products produced using NBTs as GMOs. 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Vanblaere, T. et al. (2011) The development of a cisgenic apple plant. J. Biotechnol. 157, 304–311 23 Figure legends Figure 1. Breeding techniques available for rewilding of crop plants and their legal feasibilities in the EU. (A) Introgression breeding is the standard method used to introduce genes and traits from wild plants into domesticated crops. This method employs an initial cross between the crop and the wild relative of interest followed by repeated backcrossing to the domesticated crop to erase as much genetic material from the wild relative as possible while keeping the trait of interest. Molecular markers can be used to track the trait of interest through the crosses, a process called ‘marker-assisted breeding’. However, introgression breeding is time consuming and technically challenging when more than one gene is being selected for, and it is often difficult to get rid of closely linked undesired genes. As introgression breeding does not involve GM techniques, the product is not classified as a GMO in the EU; (B) Transgenesis allows for the transfer of a desired gene from an unrelated organism into the domesticated crop. The process is based on the random genomic insertion into the crop plant of genetic material by the soil bacterium Agrobacterium tumefaciens. Typically, all of the inserted elements are transgenic. The gene in question is cloned into a binary vector system along with a selection marker and between short transfer DNA (TDNA) border sequences of bacterial origin. Subsequently, Agrobacterium inserts gene material between T-DNA border sequences into the crop genome. T-DNA insertion by Agrobacterium occurs widely in nature and the major crop potato was recently identified as naturally transgenic since it harbors T-DNA [92]. (C) Cisgenesis allows for the direct transfer of complete genes from wild relatives into domesticated crops. As the transfer process can be restricted to a single gene only, the method avoids co-transfer of genetically linked genes that could have undesired effects. The genes introduced by cisgenesis can be in their natural configuration and contain their own promoter, regulatory elements, and introns. As DNA is typically introduced by Agrobacteriummediated transformation, transgenic material is introduced concomitant with the gene of interest. However, selection markers that originate from rice and other plants are now available, and it is possible to remove the marker genes after they have served their purpose. Further, T-DNA sequences can be modified so that they are identical to sequences found in the plant (in which case they are known as P-DNAs). As cisgenesis involves the use of several GM techniques, the product will have to be classified as a GMO in the EU; (D) Precision breeding – also called ‘genome editing’ – is a new technique in plant breeding that offers even greater precision than cisgenesis [93-96]. This technique can be used to create specific mutations and has the potential to generate plants that differ from the original plant at a single nucleotide position and otherwise carry no signs 24 of the modification. Precision breeding can be used when the wild-type gene differs from the gene in the domesticated crop at a single nucleotide. Several variants of the technique exist, such as those involving zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases (RGENs) derived from the bacterial clustered regularly interspaced short palindromic repeat (CRISPR)–Cas (CRISPR-associated) system [96]. Generally, a system, which has been engineered to cleave a specific DNA sequence, is introduced into the nuclei of plant cells where it creates a DNA double-strand break at the exact position of the recognized DNA sequence. The break is subsequently repaired by the DNA repair machinery of the plant cell. This process can be imprecise (by the non-homologous end-joining pathway) or precise (by the homologous recombination pathway) [96,97]. In the second case, targeted specific mutations or insertions can be introduced. As precision breeding techniques are so new, there are still no examples of crops rewilded by this technique. Precision breeding involves the use of a GM technique, which implies that the plant product could be classified as a GMO. However, this is is still uncertain in the EU. 25 Table 1. Examples of genes that have been introduced into domesticated crop varieties rendering them less dependent on herbicides, pesticides, and fertilizers Recipient Wild relative donor Improved traits Gene Method Refs Rice (Oryza sativa) O. longistaminata Bacterial blight resistance Xa21 IB [98] O. minuta Blast resistance Pi9 IB [99,100] Fr13a (Landrace) Submergence tolerance Grain protein content Salt tolerance Sub1 IB [101] Gpc-B1 IB [102,103] Nax 1, Nax 2 IB [104] Ph-3 IB [105] S. pennellii Late blight resistance High yield Brix9-2-5 IB [106] Potato (Solanum tuberosum) S. stoloniferum S. venturii Late blight resistance Rpi-sto1, Rpivnt1.1 CG [107] Apple (Malus domestica) M. floribunda 821 Scab resistance HcrVf2 CG [108] Wheat (Triticum aestivum) T. turgidum spp. dicoccoides T. monococcum Tomato (Solanum lycopersicum) S. pimpinellifolium Abbreviations: CG, cisgenesis; IB introgression breeding. 26 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