New Plant Breeding Techniques

Transcription

1 Induced Hypomethylation Accelerated Breeding Centromere-mediated Genome Elimination Cisgenesis Zinc Finger Nuclease -Technique Methyltransferase-Technique Virus-induced Gene-Silencing Oligonucleotide-directed Mutagenesis RNA-directed DNA-Methylation Grafting on GM Rootstocks Intragenesis Agroinfiltration Meganuclease-Technique Reverse Breeding TALEN-Technique New Plant Breeding Techniques Groundwork for the Clarification of Outstanding Questions on the legal Regulation of New Plant Breeding Techniques Commissioned by the Swiss Federal Office for the Environment December 2012

2 New Plant Breeding Techniques Groundwork for the Clarification of Outstanding Questions for the Legal Regulation of New Plant Breeding Techniques. Federal Office for the Environment (FOEN), Biotechnology Section, Bern Building Department of the Canton of Zurich, Office for Waste, Water, Energy and Air (WWEA), Biosafety Section (SBS) Author Benno Vogel, Building Department of the Canton of Zurich, WWEA, SBS Project Team Khaoula Belhaj Fragnière, BAFU, Biotechnology Section Daniel Fischer, AWEL, SBS Sara Restrepo-Vassalli, BAFU, Biotechnology Section Albert Spielmann, BAFU, Biotechnology Section Barbara Wiesendanger, AWEL, SBS Anne-Gabrielle Wust Saucy, BAFU, Biotechnology Section English Translation John Barrett Cover Photo Rape Blossom Friedrich Böhringer, Wikimedia This report is based upon data and information that had been published prior to June The English translation was completed in December Address for Correspondence AWEL Amt für Abfall, Wasser, Energie und Luft Sektion Biosicherheit (SBS) Walcheplatz Zürich Switzerland Tel: Fax: biosicherheit@bd.zh.ch

3 Summary Background: Newly bred plants are regulated differentially in Switzerland. If they are deemed as genetically modified organisms (GMOs), then any handling them falls within the scope of the Gene Technology Act. If, however, they are not deemed as GMOs, the Agriculture Act and the Environmental Protection Act are decisive for any dealings with them. Which newly bred plants are legally classified as GMOs was defined in Since then, several new plant breeding techniques have been developed that employ genetic engineering in a manner that could not have been foreseen in These techniques confront the responsible authorities with a challenge, because their application calls into question the current legal definition of GMO, and moreover create legal uncertainty with regard to the regulation of plants derived from the techniques. Objective: In order to dispel the current legal uncertainty, the Federal Office for the Environment (FOEN) requires basic information with which steps for regulating the new techniques can be initiated. This report aims to provide this basic information. Twenty new plant breeding techniques: This report identifies and describes twenty plant breeding techniques, about which it remains to be clarified how the resulting plants are to be regulated by law. For eleven of the identified techniques, it is expected that the first plant varieties will come onto the global market by Particularity of the new Techniques: While the techniques identified exploit genetic engineering methods, their use may yet result in plants or products free from foreign genes. For fifteen of the new techniques, plants can be obtained that are entirely free from the genes introduced during the breeding process. GMO Classification: Whether the plants derived from the identified techniques are to be classified as GMOs is currently unclear and will need to be determined individually for each technique. Control Options: Plants can be obtained from the majority of the new techniques, which under current detection methods cannot be identified as GMOs. Should such plants be classified as GMOs, there would be an absence of control options during implementation of genetic engineering legislation. Freedom of Choice: If plants derived from one of the new techniques were not to be classified as a GMO, they would not have to be labelled as a GMO. Hence, the application of genetic engineering methods in the breeding process would remain indiscernible for consumer and farmer alike. Whether this would encroach upon freedom of choice, is currently unclear and would need to be clarified. Biosafety: It remains to be answered how to assess the biosafety of the twenty new techniques compared with conventional methods. This report provides the groundwork for nine of the techniques. With regard to the approval of plants derived from the new techniques, it needs to be clarified whether the evaluation of biosafety should be subject to strict state supervision, or be mandated to the responsibility of the individual manufacturers and importers. International Trade Relations: With regard to the import and export of plant products, further investigation is required into how the plants derived from the new techniques are regulated abroad. To date, no decisions regarding regulating have been taken in the EU Switzerland s primary agricultural trading partner. The EU Commission, however, have issued several evaluations on the topic.

4 Overview Table of Contents Illustration and Table Contents Abbreviations I VI VII Abridged Version IX Introduction 1 GMO Legal Definition 5 New Plant Breeding Techniques Agroinfiltration 7 Virus-induced Gene-Silencing 12 Oligonucleotide-directed Mutagenesis 16 RNA-directed DNA-Methylation 22 Reverse Breeding 29 Accelerated Breeding 35 Cisgenesis 42 Intragenesis 51 Grafting on GM Rootstocks 58 Zinc Finger Nuclease-Technique 63 Additional New Plant Breeding Techniques 69 Synopsis 76 Regulatory Aspects 96 Literature 102

5 Table of Contents Table of Figures... V List of Tables... VII Abbreviations... VIII Abridged Version... IX 1. Introduction Background Objective Techniques and Issues Discussed Detection and Identification with PCR Methods Biosafety Issues GMO Classification Issues Structure of the Report GMO Legal Definition New Plant Breeding Techniques Agroinfiltration Description of the Technique Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Agro-infiltrated Plants Cuttings from Agro-infiltrated Plants Seeds from Agro-infiltrated Plants Offspring from Plants from Seeds of Agro-infiltrated Plants Virus-induced Gene Silencing (VIGS) Description of the Technique Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues VIGS-treated Plants Seeds from VIGS-treated Plants Plants from Seeds of VIGS-treated Plants Oligonucleotide-Directed Mutagenesis (ODM) Description of the Technique Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues New Plant Breeding Techniques December 2012 I

6 3.3.7 Detection and Identification with PCR Methods GMO Classification Issues ODM in light of Art. 3 para. 1 let. d of the RO ODM in light of Annex 1 para. 3 let. a of the RO RNA-directed DNA-Methylation (RdDM) Description of the Technique Induction of RdDM with the aid of Transformation Induction of RdDM by means of Transient Transfection Induction of RdDM by means of VIGS Induction of RdDM by Grafting Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Induction of RdDM through Transformation Induction of RdDM by means of transient Transfection Induction of RdDM by means of VIGS Induction of the RdDM by means of Grafting Reverse Breeding (RB) Description of the Technique Suppression of Meiotic Recombination by means of Transformation Suppression of Meiotic Recombination by means of VIGS Suppression of Meiotic Recombination by means of Grafting Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Accelerated Breeding Description of the Technique Induction of Early Flowering by Transformation Inducing of Early Flowering by means of VIGS and VAGE Inducing Early Flowering by Grafting Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Induction of Early Flowering by Transformation Inducing Early Flowering by means of VIGS / VAGE Inducing Early Flowering by Grafting New Plant Breeding Techniques December 2012 II

7 3.7 Cisgenesis Description of the Technique Definition-related Aspects Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Cisgenic Plants in Light of Art. 3 para. 1 let. d of the RO Annex 1 para. 3 RO Intragenesis Description of the Technique P-DNA Concept Intragenic Vector Concept Definition-related Aspects Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR methods GMO Classification Issues Grafting on GM Rootstock Description of the Technique Potential Applications in Plant Breeding Current State of Development Intended Changes and Effects Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Scion Cuttings and Seeds Zinc Finger Nucleases Technique Description of the Technique Transformation Transient Transfection VAGE Agroinfiltration Potential Applications in Plant Breeding ZFN ZFN ZFN ZFN Current State of Development Intended Changes and Effects New Plant Breeding Techniques December 2012 III

8 Unintended Changes and Effects Safety Issues Detection and Identification with PCR Methods GMO Classification Issues Additional New Plant Breeding Techniques TALEN Technique Description of the Technique Potential Applications in Plant Breeding Current State of Development Meganuclease Technique Description of the Technique Potential Applications in Plant Breeding Current State of Development Centromere-mediated Genome Elimination Description of the Technique Potential Applications in Plant Breeding Current State of Development Induced Hypomethylation Description of the Technique Potential Applications in Plant Breeding Current State of Development Targeted Mutagenesis with T-DNA Description of the Technique Potential Applications in Plant Breeding Current State of Development Targeted Chemical Mutagenesis Description of the Technique Potential Applications in Plant Breeding Current State of Development Seed Production Technology Description of the Process Potential Applications in Plant Breeding Current State of Development Virus-Aided Gene Expression Transformation with wild-type Agrobacterium Rhizogenes Methyltransferase Technique Synopsis of Chapter Categorisation of New Plant Breeding Techniques Categorisation by Manner of Application of the Genetic Engineering Methods Direct Application of Genetic Engineering Methods Indirect Application of Genetic Engineering Methods Categorization by Genetic Engineering Methods Manner of Application Categorisation according to Objective of Use of the Genetic Engineering Methods Creating Genetic Variation Creating Epigenetic Variation New Plant Breeding Techniques December 2012 IV

9 Support for Traditional Breeding Approaches Current State of Development of the New Techniques Detection and Identification with PCR methods Safety Issues Unintended Effects Evidence of the Absence of Extracellular Introduced Sequences Issues concerning GMO Classification Direct Application of Genetic Engineering Methods Indirect Application of Genetic Engineering Methods Regulatory Issues Biosafety Protection of Freedom of Choice Protection of Production without GMOs Respect for the Dignity of Living Beings Detection and Identification of Plants derived from the New Techniques International Trade Relations Evaluations by the EU Commission Other Issues Repeated Use of Techniques on the same Plant Verification of the Absence of Extracellular Introduced DNA Literature Table of Figures Table 1: Twenty identified Plant Breeding Techniques in the Literature which employ Genetic Engineering Methods or Methods similar to Genetic Engineering in an unfamiliar manner, and their respective appraisals in Lusser et al. (2011), Schaart & Visser (2009), and in this present work VII Table 2: Transgenesis and Cisgenesis - Two different Approaches for the Transformation of Plants Fehler! Textmarke nicht definiert. Table 3: Transgenesis and Intragenesis - Two different Approaches for the Transformation of Plants Fehler! Textmarke nicht definiert. Table 4: Listing and brief Description of the twenty identified New Plant Breeding Techniques Fehler! Textmarke nicht definiert. Table 5: Potential Combinations of the twenty identified Techniques 78...Fehler! Textmarke nicht definiert. Table 6: Classification of the twenty identified Techniques into four Groups according to Lusser & Rodriguez-Cerezo Fehler! Textmarke nicht definiert. Table 7: Classification of sixteen of the twenty identified Techniques into four Categories according to Schaart & Visser and Tait & Barker. 80Fehler! Textmarke nicht definiert. Table 8: Manner of Application of Genetic Engineering Methods in the twenty identified Techniques Fehler! Textmarke nicht definiert. Table 9: Objective for the Use of Genetic Engineering Methods in the twenty identified Techniques Fehler! Textmarke nicht definiert. Table 10: Current level of Development of the New Techniques Fehler! Textmarke nicht definiert. New Plant Breeding Techniques December 2012 V

10 Table 11: Assessment of Detectability and Identifiability of Plants produced with the twenty New Techniques using PCR Methods Fehler! Textmarke nicht definiert. Table 12: Examples of Publications addressing Biosafety-aspects of the New Plant Breeding Techniques Fehler! Textmarke nicht definiert. Figure 1: Agroinfiltration of Nicotiana Benthamiana leaves. Source: Chandres, Wikimedia..8 Figure 2: Simplified schematic representation of ODM-technique for the case in which RNA- DNA oligonucleotides (RDOs) are delivered into protoplasts. Colour code for the RDO: green = DNA sequences which are homologous to the target gene; light green = RNA sequences which are homologous to the target gene; red = mismatch; orange = loops consisting of thymidine residues and GC clamps. Adapted from Hohn & Puchta (1999) Figure 3: Simplified schematic representation of RdDM. A: To trigger RdDM, genetic constructs (IR constructs or hairpin constructs) are introduced into plant cells, which exhibit homologies to the target sequence and are so structured that they are transcribed into dsrna. The dsrna are converted by the cell into so-called sirnas, which, in turn, cause certain cell proteins to methylate the target sequence. B: Consequences of methylation. If methylation occurs in a promoter sequence, it leads to the transcriptional silencing of the corresponding gene. If methylation occurs in a silencer, the transcription rate of the corresponding gene may increase Figure 4: Schematic representation of Reverse Breeding technique according to Wjinker & de Jong (2008). The starting plant is a fictive F1 hybrid with three chromosomes pairs (2n=2x=6). Meiotic recombination is suppressed by transforming the F1 with an RNAi construct. The RNAi construct leads to silencing one of the genes required for the formation of crossovers. The red dots represent the inserted RNAi constructs. The achiasmatic meiosis leads to spores, which carry non-recombinant chromosomes. Most of the resulting spores are unbalanced (not shown in the diagram); some spores, however, are balanced (several options drawn). Doubled-haploid plants are produced from balanced spores. Among the doubled haploids plants, reciprocal genotypes (Parent 1 and Parent 2), which no longer carry the RNAi construct and which yield the original F1 hybrid after crossing can be selected. Parent 1 and Parent 2 are derived from two different primary transformants, which carry the RNAi-construct on different chromosomes. Source image by: Wijnker & de Jong (2008) Figure 5: Simplified breeding schema where early flowering is induced by means of stable transformation to introgress a desirable genetic trait from a wild species in an elite variety (as per Flachowsky et al. 2009). The breeding schema consists of the following steps: (1) The plant variety, into which the desirable genetic traits are to be introgressed are genetically engineered to flower earlier than they would naturally do. (2) The GM plant is crossed with the plant containing the desirable genetic trait. (3) The crossbred offspring (filial generation 1, or, in short, F1) are selected, wherewith those individuals chosen for further breeding will contain the inserted genes for early flowering as well as the desirable genetic traits. (4) The selected F1 plants are crossbred with the genetically non-modified parental plant, in which the desirable genetic traits should are to be introgressed. (5) The offspring of the pseudo-backcross generation (BC1) are selected, whereby, in turn, those selected for further breeding contain both the inserted genes for early flowering as well as the desirable genetic trait. (6) The selected BC1 plants are, in turn, crossbred with the genetically non-modified parent plant, in which the desirable genetic traits are to be introgressed. (7) The pseudo-backcross is repeated so often until the undesirable genetic traits (linkage drag) that have been introduced along with the desirable traits, are gradually removed. (8) Once the linkage drag is sufficiently removed by backcrossing, plants having the desirable genetic traits, and not the genetically engineered constructs, are selected in the final stage New Plant Breeding Techniques December 2012 VI

11 Figure 6: Structure of a typical native gene. The gene is part of a chromosome and consists of a promoter and terminator, 5'- and 3'-untranslated regions (UTR), as well as exons and introns. The promoter and terminator are regulatory DNA sequences. During the transcription process the exons will be fused together and subsequently get translated into a protein. Figure as per Schaart & Visser (2009) Figure 7: The three possible species of chimera, which may result by combining grafting and genetic engineering; GM = genetically modified. Image Source: Schouten Figure 8: Schematic representation of possible strategies aimed at introducing ZFN-encoding genes into plant cells (Following the TALEN presentation by Mahfouz & Li 2011). ZFN genes can be transiently expressed in cells if they are transfected into protoplasts or suspensions cells. The transfected cells can be regenerated. Alternatively, a transient expression can also be achieved if the ZFN genes are introduced by agroinfiltration into leaf cells. The agro-infiltrated cells can, in turn, be regenerated by tissue culture. If ZFN genes are not inserted in the genome of plant cells during transfection or agroinfiltration, plants free of ZFN genes can be regenerated in these ways. The stable expression of ZFN genes is possible if they are inserted via Agrobacterium-based transfer methods into the genome of the plant. In this case, plants without ZFN genes may be obtained if the ZFN genes are segregated out during the further breeding process. The use of viral vectors (VAGE technique) provides another option to introduce ZFN genes into plant cells. If the virus cannot be transferred via seeds, plants free of the viral vector, and hence free of ZFN genes can be germinated. Source of images: Mahfouz & Li (2011) Figure 9: Simplified schematic representation of ZFN-1, ZFN-2, ZFN-3 and ZFN-4. For a more detailed description of the variants refer to the main text Figure 10: Simplified representation of Centromere-mediated Genome Elimination technique. An F1 line is crossed with a genetically engineered haploid inducer line. Following fertilisation the inducer line chromosomes (light blue) in the progeny can get lost during zygotic mitosis in a process known as genome elimination. Some of the progeny may therefore be haploid and carry only F1 line chromosomes. Haploid plants can spontaneously be converted into doubled haploid plants, or be transformed into doubled haploid plants following chemical treatment, so that in the end a doubled haploid F1-line originates. According to Chan (2010) Figure 11: Simplified diagram of four possible approaches with which the genetic material, and, hence the desirable function can be temporarily introduced into plants, plant cells or protoplasts. The approach with which genetic material is introduced via agroinfiltration is not presented here. Image Sources: Plants are from the NTWG (2011); petri dishes from Mahfouz & Li (2011); injections from Unver & Budack (2009), and the plasmid from Magnus Manske, Wikipedia List of Tables Table 1: Twenty identified Plant Breeding Techniques in the Literature which employ Genetic Engineering Methods or Methods similar to Genetic Engineering in an unfamiliar manner, and their respective appraisals in Lusser et al. (2011), Schaart & Visser (2009), and in this present work....3 Table 2: Transgenesis and Cisgenesis - Two different Approaches for the Transformation of Plants (as per Molesini et al. 2012) Table 3: Transgenesis and Intragenesis - Two different Approaches for the Transformation of Plants (As per Molesini et al. 2012; Rommens et al. 2011) Table 4: Listing and brief Description of the twenty identified New Plant Breeding Techniques New Plant Breeding Techniques December 2012 VII

12 Table 5: Potential Combinations of the twenty identified Techniques (with no claims to being exhaustive) Table 6: Classification of the twenty identified Techniques into four Groups according to Lusser & Rodriguez-Cerezo (2012) Table 7: Classification of sixteen of the twenty identified Techniques into four Categories according to Schaart & Visser (2009) and Tait & Barker (2011) Table 8: Manner of Application of Genetic Engineering Methods in the twenty identified Techniques (without sub-division into variants, and without consideration of possible combinations of techniques; in alphabetical order) Table 9: Objective for the Use of Genetic Engineering Methods in the twenty identified Techniques (without subdivisions into variants and without consideration of possible combinations of the techniques mentioned) Table 10: Current level of Development of the New Techniques: Information on Feasibility studies on Crop Species in the Technical Literature; applications in Breeding Programmes, and an Assessment of the Possibility that the first Varieties will reach the Market in the EU by 2017 (excluding variants and combinations) Table 11: Assessment of Detectability and Identifiability of Plants produced with the twenty new Techniques using PCR Methods depending on whether Prior Knowledge concerning Genetic Modification is available (without taking into account potential combinations of the techniques) Table 12: Examples of Publications addressing Biosafety-aspects of the New Plant Breeding Techniques Abbreviations ACRE Advisory Committee on Release to the Environment AgricA Agricultural Act [LwG] APHIS Animal and Plant Health Inspection Service BAC Biosafety Advisory Council CBD Convention on Biological Diversity COGEM Commissie genetische modificatie [The Netherlands Commission on Genetic Modification] ContainO Containment Ordinance [ESV] DgT Desirable genetic trait DH Double haploid DNA Deoxyribonucleic acid dsrna Double-stranded Ribonucleic acid EFSA European Food Safety Authority ENGL European Network of GMO Laboratories EPA Environmental Protection Act [USG] EU European Union F 1 FDHA First filial generation Federal Department of Home Affairs [EDI] New Plant Breeding Techniques December 2012 VIII

13 FOEN Swiss Federal Office for the Environment [BAFU] GMO Genetically Modified Organism GT Gene Targeting GTA Gene Technology Act [GTG] HR Homologous recombination IR Inverted repeat JRC Joint Research Centre LB Left border sequence LNA Locked nucleic acid mrna Messenger RNA NHEJ Non-homologous end joining NRE New restriction enzymes NTWG New Techniques Working Group ODM Oligonucleotide-directed Mutagenes Oligo Oligonucleotide PCR Polymerase Chain Reaction P-DNA Plant-DNA PEG Polyethylene glycol PNA Peptide nucleic acid PTGS Post-transcriptional Gene-Silencing QTL Quantitative Trait Locus RB Reverse Breeding RdDM RNA-directed DNA-Methylation RDO RNA-DNA-Oligonucleotide RB Right border sequence RNA Ribonucleic acid RNAi RNA-Interference RO Release Ordinance [FrSV] SBS Biosafety Section at WWEA sirna Small interfering RNA SPT Seed Production Technology TALE Transcription Activator-Like Effector TALEN Transcription Activator-Like Effector Nuclease T-DNA Transfer DNA TFO Triple helix forming oligonucleotides TGS Transcriptional Gene-Silencing USDA United States Department of Agriculture VAGE Virus-aided Gene Expression VGVL FDHA Ordinance on Genetically Modified Foodstuffs VIGS Virus-induced Gene-Silencing Wt Wild-type New Plant Breeding Techniques December 2012 IX

14 WWEA ZFN ZKBS Office of Waste, Water, Energy and Air [AWEL] Zinc Finger Nuclease Central Commission for Biological Safety [Zentrale Kommission für die Biologische Sicherheit] New Plant Breeding Techniques December 2012 X

15 Abridged Version Background and Objectives Seeds and seedling are among the most important means of agricultural production. In order to produce them in a sustainable and market oriented manner, a steady replenishment of newly bred crops is required, which are adapted to the constantly changing conditions in agricultural practices. In Switzerland, the manner in which newly bred plants are legally dealt with is subject to diverse regulations, dependent upon the breeding methods in question. Should the plants derive from genetic engineering methods, they shall then fall under the scope of the Swiss Gene Technology Act (GTG/GTA). Should, however, the plants derive from a technique which is not classified as a genetic engineering method, the Agriculture Act (LWG/AgricA) and the Environmental Protection Act (USG/EPA) shall be decisive. In Switzerland, organisms deemed as genetically modified organisms (GMOs) are defined in the GTA, and concretised in the Release Ordinance (FrSV/RO). Both the legal definition as well as its concretisation are more than twenty years old. At present, an array of new plant breeding techniques are being developed, which use genetic engineering methods in a hitherto unfamiliar manner, rendering it more challenging to establish a clear dividing line between genetic engineering methods and other breeding techniques. Hence, it is increasingly a matter of interpretation whether plants resulting from the application of the new techniques are to be deemed GMOs or not under currently applicable laws. It must be assumed given the progress in the development of new plant breeding techniques that the new techniques in Switzerland will at some point be applied in practice, and that products derived from the techniques will appear on the local market via imports. This report shall serve as a basis with which the Swiss Federal Office for the Environment (BAFU/FOEN), as leading enforcing agency of the GTA, can assess the need for action arising from the development of new breeding techniques, and decide on well-informed basis, whether and, if so, what further steps need to be initiated to regulate the new techniques. Techniques and Aspects Covered The focus of this report is on the ten plant breeding techniques that are currently also being evaluated in the EU by working groups which were already known at the outset of this work. These ten techniques are: - Accelerated Breeding - Grafting on GM Rootstocks - Agroinfiltration - Reverse Breeding (RB) - Cisgenesis - RNA-directed DNA-Methylation (RdDM) - Intragenesis - Virus-induced Gene Silencing (VIGS) - Oligonucleotide-directed Mutagenesis (ODM) - Zinc-finger Nucleases Technique (ZFN) New Plant Breeding Techniques December 2012 IX

16 Each of the above-mentioned techniques is accompanied by a technical description; a depiction of potential applications in plant breeding, a presentation of the current state of the development as well as an answer to the question whether the resulting plants can be detected and identified with PCR methods. In addition, for nine of the ten techniques, aspects are presented that could play a role in safety assessment, and the GMO classification of the resulting plants. During this assignment, a further ten plant breeding techniques have been identified, which use genetic engineering methods in an unfamiliar way. These techniques are briefly described in this report and listed below: - Centromere-mediated Genome Elimination - Targeted Mutagenesis with T-DNA - Induced Hypomethylation - TALEN Technique - Meganuclease Technique - Targeted Mutagenesis - Methyltransferase Technique - Transformation with wild-type Agrobacterium - Seed Production Technology (SPT) - Virus-aided Gene Expression (VAGE) Sixteen of the twenty identified techniques can be combined with at least one of the other new techniques. Furthermore, for several of the above-mentioned techniques different variants are currently being tested. Categorization of Techniques according to Application of Genetic Engineering Methods With regard to the manner of application of the genetic engineering methods, the twenty identified techniques may be classified into two categories: (1) Techniques, which use genetic engineering methods directly, and which result in plants, in which the genetic material introduced during the breeding process is stably integrated into their genome, and (II) techniques, which use genetic engineeering methods indirectly, and result in plants, in which the genetic material introduced during the breeding process is no longer present in the end product. In five of the techniques, the genetic engineering methods are deployed exclusively in a direct manner: Cisgenesis, Grafting on GM Rootstock, Intragenesis, Targeted Mutagenesis with T-DNA and Transformation with wild-type Agrobacterium. With the ZFN-, TALEN- and Meganucleases techniques, the genetic engineering methods are used in either a direct or an indirect manner, depending on the variant. In the remaining twelve techniques, genetic engineering methods are exclusively applied in an indirect manner. In these cases, the deployment of genetic engineering methods serves the purpose of transiently introducing a particular function into plants, or into plant-cells. Categorisation of Techniques according to Purpose of the Application of Genetic Engineering Methods With regard to the purpose of use of the respective genetic engineering methods, it comprises three categories: (I) use for the generation of genetic variation, (II) use for the generation of epigenetic variation, and (III) use as a means to support traditional breeding approaches. New Plant Breeding Techniques December 2012 X

17 In ten of the twenty identified techniques, the purpose is to generate genetic variation. The nature of the intended genetic modification differs depending on the technique in question, or of its variants. Distinctions can be made between the transfer of genes (e.g. Cisgenesis and Intragenesis); nucleotide exchanges (e.g. ODM); generation of indels (e.g. variants of ZFN technique) as well as gene deletion (e.g. variants of ZFN technique). In three techniques, genetic engineering methods are deployed to generate new epialleles in the genome of plants via DNA methylation. With the Methyltransferase Technique and RdDM Technique, a targeted methylation of a predetermined genomic sequence is achieved. With the Induced Hypomethylation Technique, on the other hand, non-targeted methylation is achieved. Seven techniques serve the purpose to support traditional breeding approaches. Agroinfiltration, VIGS and VAGE can be employed as a selection method; Accelerated Breeding shortens time for crossbreeding; both the RB-technique and SPT facilitate hybrid breeding, and the Centromeremediated Genome Elimination techniques supports haploid breeding. Current Status of Development New plant breeding techniques are developed in basic research, in applied as well as in breeding research, and ultimately employed in breeding programmes by private companies and/or public institutions to produce new plant varieties. It is known that fourteen of the twenty identified techniques are employed in breeding programmes. Over the coming five years, a commercial launch of new plant varieties derived from eleven of the twenty techniques is to be anticipated, if the respective varieties are not regulated as GMOs, but instead as conventionally bred varieties. Detection and Identification of Plants from the new Techniques The availability of test methods to detect and identify GMOs plays a vital role in enforcing GMO legislation. Given that detection of GMOs is at present conducted primarily with PCR methods, the plant breeding techniques covered herein will be evaluated so as to establish whether plants derived therefrom can be detected and identified by PCR methods. Detection: In the thirteen new techniques in which genetic or epigenetic variation is generated, the existence of a modification in the genetic material in plants should be detectable by PCR methods in relation to a comparator plant, if data concerning the modification is available. Identification: In techniques in which genetic engineering methods are directly employed, the existence of a modification in the plants is likely to be identified by PCR methods as a modification, which has been deliberately introduced by means of the technique in question. Unambiguous identification should only be possible if data concerning the modification is available. In techniques in which genetic engineering methods are indirectly used, an unambiguous identification by PCR methods is, as a general rule, not possible. New Plant Breeding Techniques December 2012 XI

18 In those techniques used to support traditional breeding approaches, and, in which no modifications in genetic material are intended, the resultant plants can neither be detected nor identified by PCR methods. Safety Issues Whether a plant derived from the new breeding techniques exhibits unintended traits that might impact on humans, animals or the environment in an undesirable manner, depends on the plant s phenotype, and, can only be assessed on a case-by-case basis. Whether a newly bred plant, in turn, poses a risk to human beings, animals and the environment, does not uniquely depend on the plant s phenotype, but also on exposure factors such as the extent to which a plant is bred, or the amount of which is consumed. Given that hardly any concrete data concerning the traits of individual plants derived from the new techniques is currently available, the subject of discussion in the literature focuses mainly on how the safety of the new techniques can be categorised in comparison with traditional breeding approaches, and with transgenesis. A central strand in this discussion is the possible occurrence of unintended effects. For nine of the twenty identified techniques, the present study demonstrates whether, and how, the respective breeding process may lead to unintended effects in the resulting plants. Aspects of GMO Classification Whether plants derived from the twenty techniques described herein are to be classified under current law as GMOs or not, needs to be investigated in each individual technique, taking into account the multiplicity of possible variants and combinations. In the present work, facets and questions will be discussed, which could play a role in GMO classification. Cisgenesis and certain variants of the Zinc Finger Nuclease -, TALEN- and Meganucleases techniques could meet the criteria for self-cloning, which gives rise to the question whether the plants resulting from these techniques or variants thereof could fall outside the scope of the GTA or not? In the case of Grafting on GM Rootstock, it needs to be determined whether the offspring of a scion, which has been grafted on to a GM rootstock, should be classified as a GMO or not. With techniques that could lead to an allele exchange in the genome of plants, the question arises, which of the following two aspect of GMO classification predominates: Is it the aspect of inserting nucleic acids into the genome of the organism? Or, is it the aspect whereby the modifications in genetic material are mutations? It may also be necessary to clarify the question whether the number of exchanged nucleotides in a particular endogenous sequence could be decisive with regard to GMO classification? In the event of Transformation with wild-type Agrobacterium, it remains to be clarified whether the technique shall be legally deemed a genetic engineering method or not. New Plant Breeding Techniques December 2012 XII

19 In cases where genetic engineering methods are indirectly applied, plants free of extracellular introduced genetic material may be obtained through use of the techniques. In terms of GMO classification of these plants, the following five questions come primarily to the fore: (I) Are the offspring of a stably transformed plant considered as GMOs, if they are free of the originally transformed genetic material? (II) Are the offspring of plants, which have been regenerated from transfected cells, considered as GMOs, if they no longer contain the originally transfected genetic material? (III) Are the offspring of plants, which contained recombinant viruses in a number of their cells, considered as GMOs, if they are free of the recombinant virus? (IV) Are the offspring of a scion, which is grafted onto a GM rootstock considered as a GMO? (V) Is it relevant in answering the first four questions whether the offspring exhibit modifications other than insertions of extracellular nucleic acids in their genetic material, which trace back to the use genetic engineering methods? Regulatory Aspects Aspects and issues that could play a role in the regulation of plants derived from the new techniques will herein be presented and discussed. Focus is on GMO legislation. Biosafety: Whether, and how, a plant derived from one of the new techniques impacts upon protected goods, can only be determined on a case-by-case basis. Irrespective of the GMO classification of a newly bred plant, questions arise from a regulatory perspective: Who should undertake and be responsible for the case-by-case assessment? Depending on the technique involved, is it justifiable that the assessment falls to strict State supervision? Or, depending on the technique involved, is it appropriate to make the assessment a matter of self-responsibility for the producer and for the importer concerned (as with non-gmos under the EPA and the AgricA). Freedom of Choice: Should it be decided that plants deriving from one of the new techniques be classified as a GMO, the plants must then be labelled as a GMO if and when placed on the market. This legal obligation to label also holds for food and feed products derived from those plants. Compulsory labelling of plants and products produced therefrom enables the freedom of choice for consumers (food) as well as the freedom of choice for farmers (seed, feed). New challenges concerning the implementation could result in cases in which plants, and hence products derived therefrom, are materially not clearly identifiable as GMOs. If, however, it has been determined that those plants derived from a certain new technique need not to be classified as a GMO, these plants need not then be labelled as GMOs when launched on the market. In this particular case, the question does nonetheless arise of whether freedom of choice is impaired if the applications of genetic engineering methods in the breeding process remain indiscernible for consumer and farmer alike. Respect for the Dignity of Living Beings: The activity through which the dignity of living beings could be violated in plants would be the genetic modification of the genome in a contained system. The question of how the new techniques are to be administered with respect for the dignity of the plant remains to be answered: Is the dignity of the plant only to be respected when application of the new techniques results in a plant with newly inserted genes be it as an end product, or in an interim stage New Plant Breeding Techniques December 2012 XIII

20 of the process. Or, is the dignity of a plant to be respected in every single plant, in which modifications are caused in the genetic material through use of these new techniques. Verification of the Absence of extracellular introduced DNA: For techniques in which genetic engineering methods are indirectly employed, plants free of extracellular introduced genetic material may emerge. From a regulatory perspective, the question could here be asked of who, and which methods proves the absence of extracellular introduced materials before field trials take place, or prior to placing on the market of the plants. Should the proof of absence be subject to the breeders duty of care and self-regulation, or should it be a matter of State control? International Trade Relations: Switzerland both imports and exports plant varieties and products derived therefrom. Whether new regulatory issues will arise concerning the import and export of plants from the new breeding techniques is contingent upon how these plants are regulated in the individual jurisdictions, and, whether, and how, the respective regulations differ from Swiss regulations. The approaches pursued internationally to regulate the new techniques lie beyond the scope of the present work. It is known that regulatory issues are a matter of discussion in different jurisdictions by the responsible officials and commissions. New Plant Breeding Techniques December 2012 XIV

21 1. Introduction 1.1 Background Seeds and seedlings are among agriculture s most vital means of production. In order to produce in a sustainable and market-oriented fashion, farmers must rely upon the steady supply of new crop varieties. Plant breeding companies and public breeding programmes cater for the supply of new varieties. They constantly produce genetically improved varieties, which are adapted to agriculture s everchanging conditions and requirements. Nowadays, plant breeding has at its disposal a wide spectrum of techniques to create new varieties. Since the mid-1980s these also include genetic engineering methods, which enable the transfer of isolated DNA. In Switzerland, the production, testing in the environment and the placing on the market of newly bred plants are regulated differently depending on the type of breeding techniques deployed. If the plants derive from genetic engineering methods, then the resulting genetically modified plants come under the scope of the relevant Swiss Gene Technology Act (GTG/ GTA). Should, however, the plants derive from techniques not deemed as genetic engineering methods, the Swiss Agriculture Act (LwG/AgricA) and the Swiss Environmental Protection Act (USG/ EPA) are decisive regarding their oversight. In Switzerland, organisms which are classified as genetically modified organisms (GMOs) are defined in the GTA, and formulated in concrete terms in the Release Ordinance (RO). The legal definition and concretisation are now over 20 years old, as both originate from EU directives brought into force in While both the legal definition and the concretisation of the concept of GMOs have in substance remained unaltered since their origins, new plant breeding techniques have meanwhile been developed that apply genetic engineering methods in a way that was not foreseeable in To date, the use of genetic engineering methods in plant breeding has led to plants with the following two attributes: (1) the genome of the plant contains DNA prepared outside the plant, and (II) the newly inserted DNA derives, wholly or partially, from foreign organisms. However, the use of the newly developed techniques employing genetic engineering methods can result in plants which either solely exhibit the first attribute (namely, the insertion of DNA supplied from outside) or, neither of the abovementioned attributes. Hence, a salient characteristic of the new techniques is: While employing genetic engineering methods, they nonetheless lead to plants free of foreign DNA. Since the resulting plants could only have genes in their genome that are already part of the gene pool of the respective plant species, they can in principle also be bred with conventional, and hence non-genetic engineering methods. With the development of new plant breeding techniques it has become increasingly difficult to establish a clear dividing line between genetic engineering methods and other breeding techniques. Given the characteristic differences between GMO and non-gmo are thereby diminishing, it is more and more a matter of interpretation whether the plants resulting from the new techniques are considered to be GMOs or not according to the legal definition as set out in the GTA. Questions still remain, whether the legal definition of the concept GMO needs to be reassessed, and, how the emerging plants derived from the new techniques are to be regulated? New Plant Breeding Techniques December

22 It is to be assumed that with the progress in the development of new plant breeding techniques that the new techniques will be employed in Switzerland, and that products derived therefrom will arrive on the Swiss market by way of imports. In order to be in a position to evaluate the ensuing need for action, the Federal Office for the Environment (BAFU/FOEN) commissioned the Biosecurity Section [SBS] of the Office of Waste, Water, Energy and Air (AWEL/WWEA) that is responsible for enforcing the Ordinance on the Release of Organisms (RO]) in Zurich Canton, to compile a baseline report concerning the new plant breeding techniques. 1.2 Objective This report shall serve as a basis with which the FOEN can assess the need for action arising from the development of new breeding techniques, and decide on well-informed basis, whether and, if so, what further steps to regulate the new techniques need to be initiated. In order to serve as a basis for any further decision-making, this report wants not only to present the substantive issues, but also to raise questions, which could arise on a legal or a regulatory level in connection with the new techniques. 1.3 Techniques and Issues Discussed This report shall focus on the ten techniques which are presented in Lusser et al (2011) and/or Schaart and Visser (2009), and, which were known at the outset of this study (refer to Table 1). These techniques are: Accelerated Breeding; Agroinfiltration; Cisgenesis; Grafting on GM rootstock; Intragenesis; Oligonucleotide-directed Mutagenesis; Reverse Breeding; RNA-directed DNA Methylation; Virus-induced Gene Silencing and Zinc-Finger Nuclease technique. For each of the above mentioned techniques the following will be provided: a technical description; a description of potential applications in plant breeding; an account of the current state of development as well as a response to the question whether the varieties derived from the techniques can be detected and identified with PCRmethods. Furthermore, for each of the respective techniques aspects that could play a role in safety assessment and GMO classification of the resulting varieties will be presented and discussed. The Zinc Finger Nuclease technique constitutes an exception with respect to both last aspects. This particular technique includes several variants requiring separate treatment with regard to safety issues and GMO classification, something that was not possible within this project s framework. Besides the techniques known at the outset, an additional ten plant-breeding techniques, which employ genetic engineering methods in an unfamiliar way, and, hence which could also pose legal and regulatory questions (refer to Table 1), were discovered in the literature over the course of assignment. These techniques are: Centromere-mediated Genome Elimination; Induced Hypomethylation; Meganuclease Technique; Methyltransferases Technique; Targeted Chemical Mutagenesis; Targeted Mutagenesis with T-DNA, Seed Production Technology; TALEN Technique; Transformation with wild-type Agrobacterium and Virus-aided Gene Expression. For each of these ten techniques, a technical description, a description of potential applications in plant breeding along with an account of the current stage of development will follow. New Plant Breeding Techniques December

23 Table 1: Twenty identified Plant Breeding Techniques in the Literature which employ Genetic Engineering Methods or Methods similar to Genetic Engineering in an unfamiliar manner, and their respective appraisals in Lusser et al. (2011), Schaart & Visser (2009), and in this present work. Technique Lusser et al Schaart & Visser 2009 This Report Accelerated Breeding * Agroinfiltration * Centromere-mediated Genome Elimination Cisgenesis * Grafting on GM rootstock * Induced Hypomethylation Intragenesis * Meganuclease-Technique Methyltransferase-Technique Oligonucleotide-directed Mutagenesis * Reverse Breeding * RNA-directed DNA-methylation * Seed Production Technology TALEN-Technique Targeted Chemical Mutagenesis Targeted Mutagenesis with T-DNA Transformation with Wt. Agrobacterium Virus-aided Gene Expression Virus-induced Gene-Silencing * Zinc Finger Nuclease-Technique * : These techniques have been taken into account and at least technically described. *: These techniques were known when this report was initiated, and are with the sole exception of the Zinc Finger Nuclease Technique treated differently in this report, also in relation to those aspects that could play a role in safety evaluation and GMO classification. Wt.: Wild-type Detection and Identification with PCR Methods The availability of methods, with which GMOs can be detected and identified, plays an important role in the enforcement of legislation covering genetic engineering. Hence, this report will, as previously mentioned, address the question of whether plants derived from the new plant breeding techniques New Plant Breeding Techniques December

24 could be shown to be GMOs by means of PCR methods. Focus is on PCR methods, because they allow a routine application, and are currently the method of choice in enforcement practice. With regard to the question of detectability, this report will distinguish between the possibility of detection, and, the possibility of identification. Detection refers to the possibility of determining the existence of a change in the genetic material of a plant, specifically with reference to an appropriate comparator. Identification, in turn, refers to the possibility of detecting the existence of a change in the genetic material of a plant as a modification that has been deliberately introduced through the use of a particular technique. This report does not examine to what extent other methods for the detection and identification of plants derived from new plant breeding techniques are available, besides PCR methods Biosafety Issues As has been outlined above, certain issues will be addressed for nine of the twenty identified techniques, which could play a role in assessing the safety of the individual techniques and the plants arising from them. Thereby, processes will be identified in the individual techniques, which could lead to unintended effects in the resultant plants. Unintended effects may be neutral, undesirable, or again, desirable, and are a constant feature in all plant breeding techniques. By demonstrating whether the identified processes also occur in conventional plant breeding methods, one can answer whether the processes are specific to each technique or not. The objective of this approach is to establish an initial base, with which to compare the biosafety of the new techniques with respect to conventional methods GMO Classification Issues As mentioned above, aspects are described which could play a role under current law in GMO classification of the resulting plants in nine of the twenty identified techniques. Thereby differing perspectives and interpretations, which could be taken or exist in the GMO classification, are in each case shown in a hermeneutic fashion. This approach is employed to identify potential uncertainties in GMO classification as well as to clear up the question of whether there is a need to further substantiate existing legal norms. Hence, the present report will neither offer an answer to the question whether plants derived from the new techniques are GMOs under current law or not, nor does it intend to answer the question whether the resulting plants should be GMOs or not. 1.4 Structure of the Report Chapter 2 briefly presents which organisms are considered as GMOs in Switzerland under current law. This presentation will serve as a basis to facilitate further discussion in the following chapters about aspects of GMO classification regarding plants derived from the new techniques. In Chapter 3, the above-described aspects of each technique will be discussed. Chapter 4 will provide a synopsis, in which issues discussed in Chapter 3 are comprehensively reviewed. The synopsis primarily introduces a categorisation of the new techniques. What follows are brief overviews concerning the detection and identification of plants derived from the new techniques as well as an update on their current state of development. Subsequently, questions and issues that might play a role in safety assessment and GMO classification of plants arising from the new techniques will be recapitulated. In Chapter 5, questions New Plant Breeding Techniques December

25 are raised and issues are presented which could play a role in how plants derived from the new techniques be regulated. Herein, focus is on the Swiss Gene Technology Act. Among the issues therein presented are biosafety, freedom of choice, plant dignity and international trade relations. 2. GMO Legal Definition In Switzerland, organisms legally considered as GMOs were defined initially in 1995 following an amendment to the Environmental Protection ACT (USG/EPA). In 1999, with the introduction of the Release Ordinance (RO), there followed a concretisation and detailed definition of the term GMO. Lawmakers extensively referred to the then valid EU directives 90/219 /EEC and 90/220 /EEC both in terms of the legal definition as well as in the concretisation and paraphrasing (EDI 1997). Those organisms currently considered as GMOs, were thus dealt with at the end of the 1980s; both EU directives were first put into force in in Under current Swiss law, GMOs are organisms whose genetic material has been changed in a way that does not occur under natural conditions by crossbreeding or natural recombination (Art. 5. para. 2 GTA; Art 7. para. 5ter EPA). Hence, GMOs are characterized by the fact that their genetic material has been modified by a genetic engineering method in such a way that does not occur under natural conditions as with crossbreeding, or by natural recombination (Errass 2006; Keller 2002). On the statutory ordinance level, the term GMO is more concrete and described in detail (Art. 3 para. 1 let. d RO and Annex 1 RO; see Box 1). With this detailed description, lawmakers shed light on the term GMO from two sides: On one hand, techniques are identified, which usually lead to GMOs; on the other hand, techniques are also described, which, as a rule, do not usually result in GMOs (see Box 1). Hence, a distinction is drawn between genetic engineering and non-genetic engineering methods. According to the Federal Department of Home Affairs (FDHA; EDI 1997), genetic engineering methods are those techniques leading to GMOs, in other words, to organisms whose genetic material has been modified in such a way as not to be expected under natural conditions according to current knowledge. Appendix 1 of the RO presents a non-exhaustive list of the following techniques as genetic engineering methods: recombinant nucleic acids techniques, techniques, in which genetic material is directly introduced, as well as certain cell fusion or hybridisation techniques. The self-cloning of pathogenic organisms shall be regarded as a genetic engineering method. In accordance with the FDHA (EDI 1997), non-genetic engineering methods are those techniques, in which genetic material remains unaltered, or, in which modifications to the genetic material are generated, as it might occur in nature. These techniques include, inter alia, plant cell fusion, mutagenesis and techniques in which an organism's chromosome set is modified. Organisms generated as a result of these techniques may be considered as non-genetically modified. However, this rule does not apply if the techniques are used in association with recombinant nucleic acid techniques. New Plant Breeding Techniques December

26 Box 1: Wording of the definition and concretisation of the term GMO in the RO Art. 3 Definitions 1. For the purpose of this Ordinance the following are considered: d. genetically modified organisms means organisms in which the genetic material has been altered by methods of gene technology in accordance with Annex 1, in a way that does not occur under natural conditions by crossing or natural recombination, as well as pathogenic or alien organisms that have also been genetically modified; Annex 1 (Art. 3 let. d) Definition of Gene Technology Methods 1. Gene technology methods means, in particular: a. recombinant nucleic acid techniques, in which nucleic acid molecules synthesised outside the organism are inserted into viruses, bacterial plasmids or other vector systems to produce novel combinations of genetic material, which are then transferred to a recipient (host) organism in which they would not naturally occur but are capable of continued propagation; b. techniques in which genetic material produced outside the organism is inserted directly into an organism, in particular by microinjection, macroinjection and microencapsulation, electroporation or on microprojectiles; c. cell fusion or hybridisation techniques in which cells with novel combinations of genetic material are produced by the fusion of two or more cells through processes that do not occur under natural conditions. 2. Self-cloning of pathogenic organisms shall be regarded as a method of gene technology. This consists of the removal of nucleic acid sequences from one cell of an organism and the complete or partial insertion of this nucleic acid or a synthetic equivalent (possibly after a previous enzymatic or mechanical treatment) into cells of the same species or cells which are closely related phylogenetically and which can exchange genetic material by natural physiological processes. 3. Self-cloning of non-pathogenic organisms and the following methods shall not be regarded as methods of gene technology, as long as they are not used in association with recombinant nucleic acid molecules or genetically modified organisms: a. mutagenesis; b. cell and protoplast fusion of prokaryotic microorganisms that exchange genetic material by natural physiological processes; c. cell and protoplast fusion of eukaryotic cells, including the production of hybridoma cell lines and the fusion of plant cells; d. in-vitro fertilisation; e. natural processes such as conjugation, transduction and transformation; f. changes in ploidy level, including aneuploidy and the elimination of chromosomes. New Plant Breeding Techniques December

27 The legal definition and concretisation of the GMO concept comprise process-related as well as product-related components. The process-related component is the designation of techniques that as a rule lead to GMOs. The itemised list of genetic engineering methods annotated in Annex 1 of RO is not exhaustive. Product-related designates that a GMO is characterised by the fact that if compared to the original plant it comprises a modification in its genetic material. What remains open to interpretation refers to the relative clause contained in the legal definition in a way that does not occur under natural conditions by crossing or natural recombination. If this relative clause refers to the technique, the focus of the legal definition and its concretisation is on the process. If it, however, refers to modifycation to the genetic material, then focus falls more on the product 3. New Plant Breeding Techniques 3.1 Agroinfiltration Agroinfiltration is a technique that uses recombinant Agrobacterium in order to achieve transient expression of genetic constructs in plant tissues. The integration of the constructs in the germ cells is not intended. Agroinfiltration is mainly employed in research, but it can also be used in breeding programmes (Lusser et al. 2011; Schaart & Visser 2009). Given that cuttings and seeds of infiltrated plants can be used for further crop improvement, the question arises as to how their regulatory status is to be determined (COGEM 2006a). If recombinant Agrobacterium, which include viral vectors, are used in Agroinfiltration, the method is called Agroinfection (Grimsley et al. 1986), or Agroinoculation (Elmer et al. 1988). Agroinfection and Agroinoculation can be assigned to the Virus-induced Gene Silencing (VIGS; Section 3.2), or the Virus-aided Gene Expression (VAGE; Section ). The floral dip method, in which flowers of a plant are dipped in an Agrobacterium suspension, can also be attributed to Agroinfiltration (Lusser et al. 2011). This particular technique, however, is not addressed here, because it leads to stably transformed plants, and hence to GMO products Description of the Technique During Agroinfiltration, the tissue of a plant, mainly the leaves, is infiltrated with a liquid suspension of recombinant Agrobacterium. The infiltration can be carried out with the aid of a syringe (see Figure 1) or with toothpicks, as well as by applying a vacuum. If the roots are the target tissue, the infiltration can also succeed by merely immersing it into an Agrobacterium-suspension (Agrodrench Method, Ryu et al. 2004). If the recombinant Agrobacterium are in the plant cells, their T-DNA will be transported into the cell nucleus, where it will lead to the transient expression of recombinant genes. The genes on the T-DNA can thereby be active as free DNA molecules; as a consequence they need not be integrated into the genome of plant cells so as to be expressed (Schaart & Visser 2009). Depending on the genetic construct infiltrated via recombinant Agrobacterium into plant tissues, two types of Agroinfiltration can be differentiated (Lusser et al ): New Plant Breeding Techniques December

28 1. Agroinfiltration sensu stricto : Infiltration takes place with Agrobacterium which contains non-replicative gene constructs. The expression of the introduced genes is confined locally to the section of the plant, which has been infiltrated. 2. Agroinoculation or Agroinfection : Infiltration takes place with Agrobacterium containing replicative gene constructs. The genes to be inserted are first incorporated into a viral vector, which is then, in turn, integrated into the T-DNA. Since the viral vector replicates and spreads within the cells, gene expression takes place throughout the entire plant. Depending on the design of the replicative gene construct, it can lead to the formation of a protein, or to the silencing of an endogenous gene. The former case can also be attributed to VAGE (Section ), and the latter case to VIGS (Section 3.2). Figure 1: Agroinfiltration of Nicotiana Benthamiana leaves. Source: Chandres, Wikimedia Potential Applications in Plant Breeding Various applications of Agroinfiltration are possible in plant breeding. Hence, the technology might be an interesting tool to test plants for possible disease resistance (Lusser et al. 2011; Schaart & Visser 2009). In such cases, Agroinfiltration serves as a means of selecting plants that could be used for further breeding. In the breeding of genetically modified plants, Agroinfiltration can be employed to test possible transgenes for a stable transformation in plant tissue (Leckie & Stewarts 2011). Recently, Agroinfiltration (particularly Agroinoculation/Agroinfection) is also being discussed as a possible tool for the RNA-directed DNA methylation (Section 3.4), for Reverse Breeding (Section 3.5), New Plant Breeding Techniques December