APP Application APP to develop, as a project, genetically modified organisms (as described in

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1 DECISION Date 20 June 2018 Application code Application type Applicants APP To develop in containment genetically modified organisms under sections 40(1) and 42A of the Hazardous Substances and New Organisms Act 1996 The New Zealand Institute for Plant & Food Research Limited Date application received 12 June 2018 Consideration date 20 June 2018 Considered by Purpose of the application The Acting General Manager Hazardous Substances and New Organisms of the Environmental Protection Authority (EPA) 1 To develop in containment, microorganisms and plant host species to explore the function of plant genes which encode proteins containing domains specifically rich in glutamine and aspargine amino acids. 1. Summary of decision 1.1 Application APP to develop, as a project, genetically modified organisms (as described in Tables 1 and 2 of this decision) in containment is approved, with controls. 1.2 I had sufficient information to assess the application. The application was considered in accordance with section 42A of the Hazardous Substances and New Organisms (HSNO) Act 1996 ( the Act ), the Hazardous Substances and New Organisms (Low-Risk Genetic Modification) Regulations 2003 ( the Regulations ), and the Hazardous Substances and New Organisms (Methodology) Order 1998 ( the Methodology ). 1.3 I note that in accordance with section 58(1)(c) of the HSNO Act, the Department of Conservation (DOC) and the Ministry for Primary Industries (MPI) were given the opportunity to comment on this 1 The Acting General Manager Hazardous Substances and New Organisms of the EPA has made the decision on this application under delegated authority in accordance with section 19 of the Act.

2 2 application. DOC indicated that this application did not appear to have any biodiversity implications, and had no comments on it. MPI did not comment on the application. 2. The approved genetically modified organisms (GMOs) and the controls imposed Purpose of the project 2.1 The purpose of this application is to develop, by genetic modification, microorganisms and plant host species to explore the function of plant genes which encode proteins containing domains specifically rich in glutamine (Q) and asparagine (N) amino acids, also known as Q/N rich regions. 2.2 I determined that this application is for a valid purpose; the development of any new organism as provided for in section 39(1)(a) of the Act. Description of the organisms to be developed 2.3 Pursuant to section 42A(1), the application describes a project for the development of genetically modified organisms, the identity of host organisms and the nature and range of the proposed genetic modifications. 2.4 As per section 42A(2) of the Act, I was satisfied that the host organisms and the proposed genetic modifications conform to the requirements of: Category 1/2 host organisms (as per clause 7(1) of the Regulations), and Category A/B genetic modifications (as per clause 5(1) of the Regulations) (as described in Tables 1 and 2). Table 1: Approved organism description Microorganisms Host organisms Escherichia coli (Migula, 1895) Castellani and Chalmers 1919, non-pathogenic laboratory adapted strains Agrobacterium tumefaciens (Smith and Townsend, 1907) Conn 1942, disarmed strains Saccharomyces cerevisiae Meyen ex EC Hansen (1883), non-pathogenic laboratory adapted strains Pichia pastoris Guillerm Phaff (1956), non-pathogenic laboratory adapted strains Ogataea angusta (Teun., H.H. Hall & Wick) Suh & Zhou 2010 (synonym Pichia angusta), non-pathogenic laboratory adapted strains Schizosaccharomyces pombe Lindner 1893, non-pathogenic laboratory adapted strains non-sporulating strains

3 3 Tissue culture plants, protoplasts and plant cell cultures (without reproductive structures and kept in closed containers) Category of host organism Nicotiana spp. L. and Graham Arabidopsis thaliana Heynh. Petunia spp. Juss. Marchantia polymorpha (marchantia) L. Host microorganisms are Category 1 host organisms because they: are clearly identifiable and classifiable are characterised to the extent that their main biological characteristics are known are not normally able to (or contain infectious agents normally able to) cause disease in humans, animals, plants or fungi do not normally infect, colonise or establish in humans, and do not produce desiccation-resistant structures such as spores or cysts that can be normally disseminated in the air. Modifications Tissue culture plants, protoplasts and plant cell cultures (without reproductiove structures and kept in closed containers) are Category 1 host organisms because they: are clearly identifiable and classifiable They are characterised to the extent that their main biological characteristics are known are not normally able to (or contain infectious agents normally able to) cause disease in humans, animals, plants or fungi do not normally infect, colonise or establish in humans, and do not produce desiccation-resistant structures such as spores or cysts that can be normally disseminated in the air will not have reproductive structures and will be kept in closed containers. Standard non-conjugative cloning, binary or expression plasmid vectors may be used to integrate DNA sequences into the plant, bacterial or yeast strains for protein expression, gene functions studies, and/or transformation of plants (either transient or stable transformation). Vectors such as pbin, pcambia, part, pmon, and onov may be used for expression studies; phannibal, pkannibal, phellsgate Gateway TM may be used for RNAi research, and ppopoff series may be used for inducible expression. Transformation might be effected by, but is not limited to, Agrobacterium transformation and biolistics. Vectors may consist of regulatory elements including promoters, multiple cloning sites, origins of replication, operators, binding sequences, inducible promoter elements, flanking sequences, recombination sites, sequences that facilitate recombination, protein purification tags, enhancer and silencer sequences, recombination sequences and recombinases, origin of conjugative transfer, suppressing sequences, polyadenylation signals, intron/exon slice sites, secretory and targeting signals, reporter and selectable marker genes, antibiotic resistance genes, ribosomal binding sites, transcriptional activators, transcriptional response elements,enhancers and transcriptional terminators and Agrobacterium, T-DNA and P-DNA border elements, T-DNA/P-DNA processing and transfer sequences, and other elements outside the T-DNA or P-DNA regions. These regulatory elements may be sourced from plant, bacterial, fungal (including yeast), viral or bacteriophage origin.

4 4 Donor genetic material may be sourced from the genomic DNA or complementary DNA of bacteria, fungi (including yeast), plant, viral and synthetic sources. The gene contents specifically sourced from plant species might contain genes with a high Q/N rich region. Target genes/cdna sequences may be incorporated in sense or antisense orientations. RNAi technology (excluding viral induced gene silencing) may also be used to limit the production of target proteins. Modifications may include heterologous gene expression and precision genome editing, involving the use of vectors carrying engineered DNA binding proteins, to facilitate gene knockout (mutation and deletion), transcriptional activation or repression, nucleic acid insertion (both sense and anti-sense and including nucleotide substitutions). Engineered DNA binding proteins include, but are not limited to Zinc Fingers (ZFs), Transcriptional activator like effectors (TALEs), and clustered regularly interspaced short palindromic repeat-crispr associated protein 9 nucleases (CRISPR-Cas9). Gene sequences involved in pigment binding of host organisms might be also studied. Molecular tools may contain human derived DNA sequences. Such standard molecular tools will be sourced from overseas and will not contain DNA of Māori origin. Some viral DNA from viruses that infect plants will be used, however, only defined segments of the viral genome that regulates transcription (eg. The 35S promoter) and transcriptional terminator sequences of the viral genome will be used. The modifications will exclude: Genetic material that is derived from CITES-listed species DNA that is directly derived from humans The production of infectious particles normally able to cause disease in humans, animals, plants, or fungi Genetic material that intentionally increases the pathogenicity, virulence, or infectivity of the host organism Genes that encode for vertebrate toxins with an LD50 < 100 µg/kg Modifications that result in the GMO having a greater ability to escape from containment than the unmodified host organism Genetic material derived from New Zealand native or taonga flora and fauna, unless consultation has been conducted with representatives of appropriate iwi Uncharacterised sequences from pathogenic microorganisms RNAi technology that includes viral induced gene silencing Category of modifications Minimum containment level required The modifications are Category A because they: PC1 involve category 1 host organisms, as defined in the Regulations are carried out under a minimum of PC1 containment as defined in the Regulations do not increase the pathogenicity, virulence or infectivity of the host organism to laboratory personnel, the community or the environment do not result in the GMO having a greater ability to escape from containment than the unmodified host organism.

5 5 Table 2: Approved organism description Microorganisms Host organisms Category of host organism Agrobacterium tumefaciens (Smith and Townsend, 1907) Conn 1942, armed strains Whole plants or tissue cultures (either with or without reproductive structres and kept or not kept in closed containers) Nicotiana spp. L. and Graham Arabidopsis thaliana Heynh. Petunia spp. Juss. Marchantia polymorpha (marchantia) L. Armed strains of Agrobacterium tumefaciens are Category 2 host organisms because they are: clearly identifiable and classifiable characterised to the extent that their main biological characteristics are known micro-organisms of risk group 2 that are or contain an infectious agent pathogenic to humans, animals, plants, or fungi Whole plants or tissue cultures of Nicotiana spp., Petunia spp., and Arabidopsis thaliana (either with or without reproductive structures and kept or not kept in closed containers) are Category 2 host organisms because they are: clearly identifiable and classifiable with or without reproductive structures and will not be kept in closed containers Modifications Whole plants or tissue cultures of Marchantia polymorpha (either with or without reproductive structures and kept or not kept in closed containers) are Category 2 host organism because they are: clearly identifiable and classifiable Risk Group 2 organisms as defined in the Regulations that produce desiccation-reistant structures such as spores or cysts that can be normally disseminated in the air, with or without reproductive structures and will not be kept in closed containers Standard non-conjugative cloning, binary or expression plasmid vectors may be used to integrate DNA sequences into the plant, bacterial or yeast strains for protein expression, gene functions studies, and/or transformation of plants (either transient or stable transformation). Vectors such as pbin, pcambia, part, pmon, and onov may be used for expression studies; phannibal, pkannibal, phellsgate Gateway TM may be used for RNAi research, and ppopoff series may be used for inducible expression. Transformation might be effected by, but is not limited to, Agrobacterium transformation and biolistics.

6 6 Vectors may consist of regulatory elements including promoters, multiple cloning sites, origins of replication, operators, binding sequences, inducible promoter elements, flanking sequences, recombination sites, sequences that facilitate recombination, protein purification tags, enhancer and silencer sequences, recombination sequences and recombinases, origin of conjugative transfer, suppressing sequences, polyadenylation signals, intron/exon slice sites, secretory and targeting signals, reporter and selectable marker genes, antibiotic resistance genes, ribosomal binding sites, transcriptional activators, transcriptional response elements,enhancers and transcriptional terminators and Agrobacterium, T-DNA and P-DNA border elements, T-DNA/P-DNA processing and transfer sequences, and other elements outside the T-DNA or P-DNA regions. These regulatory elements may be sourced from plant, bacterial, fungal (including yeast), viral or bacteriophage origin. Donor genetic material may be sourced from the genomic DNA or complementary DNA of bacteria, fungi (including yeast), plant, viral and synthetic sources. The gene contents specifically sourcing from plant species might be containing genes with high Q/N rich region. Target genes/cdna sequences may be incorporated in sense or antisense orientations. RNAi technology (excluding viral induced gene silencing) may also be used to limit the production of target proteins. Modifications may include heterologous expression and precision genome editing, involving the use of vectors carrying engineered DNA binding proteins, to facilitate gene knockout (mutation and deletion), gene transcriptional activation or repression, gene or nucleic acid insertion (both sense and anti-sense and including nucleotide substitutions). Engineered DNA binding proteins include, but are not limited to Zinc Fingers (ZFs), Transcriptional activator like effectors (TALEs), and clustered regularly interspaced short palindromic repeat-crispr associated protein 9 nucleases (CRISPR-Cas9). Gene sequences involved in pigment binding of host organisms might be also studied. Molecular tools may contain human derived DNA sequences. Such molecular tools will be sourced from oversease and will not contain DNA of Māori origin. Some viral DNA from viruses that infect plants will be used, however, only defined segments of the viral genome that regulates transcription (eg. The 35S promoter) and transcriptional terminator sequences of the viral genome will be used. The modifications will exclude: Genetic material that is derived from CITES-listed species DNA that is directly derived from humans The production of infectious particles normally able to cause disease in humans, animals, plants, or fungi Genetic material that intentionally increases the pathogenicity, virulence, or infectivity of the host organism Genes that encode for vertebrate toxins with an LD50 < 100 µg/kg Modifications that result in the GMO having a greater ability to escape from containment than the unmodified host organism Genetic material derived from New Zealand native or taonga flora and fauna, unless consultation has been conducted with representatives of appropriate iwi Uncharacterised sequences from pathogenic microorganisms RNAi technology that includes viral induced gene silencing

7 7 Category of modifications The modifications to whole plants or tissue cultures are Category B because they: involve category 2 host organisms, as defined in the Regulations are carried out under a minimum of PC2 containment as defined in the Regulations involves a host organism that is not normally able to cause disease in humans, animals, plants, or fungi do not increase the pathogenicity, virulence or infectivity of the host organism to laboratory personnel, the community or the environment do not result in the GMO having a greater ability to escape from containment than the unmodified host organism. The modifications to armed strains of Agrobacterium tumefaciens are Category B because they: involve category 2 host organisms, as defined in the Regulations are carried out under a minimum of PC2 containment as defined in the Regulations involves a host organism that is normally able to cause disease in humans, animals, plants, or fungi provided that the nucleic acid that is introduced is characterised to the extent that o Its sequence is known, and o Its gene function is understood; and o Its potential gene products are understood do not increase the pathogenicity, virulence or infectivity of the host organism to laboratory personnel, the community or the environment do not result in the GMO having a greater ability to escape from containment than the unmodified host organism. Minimum containment level required PC2 2.5 I considered that this project represents a particular line of scientific inquiry and has clearly defined objectives to develop the organisms specified in Tables 1 and 2, to develop in containment, microorganisms and plant host species to explore the function of plant genes which encode proteins containing domains specifically rich in glutamine and aspargine amino acids. This will allow the work described above. 3. Rapid assessment of adverse effects of the project 3.1 As the host organisms and genetic modifications meet the criteria of low-risk genetic modification (as described in the Regulations), I have made a rapid assessment of the adverse effects of carrying out the project (as per section 42A(2) of the Act). 3.2 I note that the GMOs would first need to escape from the containment facility into the environment to cause non-negligible adverse effects. However, as the GMOs will be developed within approved

8 8 containment facilities which have structural requirements and operational procedures to prevent the escape of the GMOs, I consider that it would be highly improbable that the GMOs will escape from containment. 3.3 I did not identify any non-negligible adverse effects on the environment, public health, market economy or New Zealand s international obligations. 3.4 I did not identify any non-negligible adverse effects on personnel handling the GMOs as exposure to the GMOs is voluntary and those personnel are trained to safely handle the GMOs. 3.5 I did not identify any non-negligible adverse effects on society and community as: The GMOs will be developed within approved containment facilities which have structural requirements and operational procedures to prevent the escape of the GMOs The GMOs do not involve host organisms or genetic modifications that I consider will adversely affect society and community. 3.6 I did not identify non-negligible adverse effects on Māori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna, and other taonga as: The GMOs will be developed within approved containment facilities which have structural requirements and operational procedures to prevent their escape The host organisms do not include native or valued flora and fauna Genetic material will not be derived from Māori. 4. The decision-making 4.1 I had sufficient information to assess the application as submitted by the applicant. 4.2 As per section 42A(3) of the Act, after completing a rapid assessment of adverse effects, I have decided to approve the application and impose controls providing for each of the matters specified in Schedule 3 as I think fit. 4.3 The matters to be addressed by containment controls for developing GMOs are listed in Part 1 of Schedule 3 of the Act. To address these, controls must be imposed to: limit the likelihood of any accidental release of any organism or any viable material exclude unauthorised people from the facility exclude other organisms from the facility and control undesirable and unwanted organisms within the facility prevent the unintended release of the organisms by experimenters working with the organisms control the effects of any accidental release or escape of the organisms, and specify inspection and monitoring requirements for the containment facilities.

9 9 4.4 I imposed the controls detailed in Table 3 to provide for the matters above and any other matters I considered necessary to give effect to the purpose of the Act. 4.5 I note that the applicant has detailed their containment regime in section 4.2 of the application. I consider that this containment regime is adequate, since the genetic modifications will be carried out in MPI-approved facilities at a minimum of a PC1 standard for category A genentic modifications, and at a minimum of PC2 for category B genetic modifications.

10 10 Table 3: Controls The approval holder must ensure compliance with the following controls 1) This approval is limited to develop in containment, microorganisms and plant host species described in tables 1 and 2, to explore the function of plant genes which encode proteins containing domains rich in glutamine and aspargine. 2) The approved organism must not escape containment. 3) The approved organisms must be developed within a containment facility that complies with: The MAF/ERMA New Zealand Standard: Facilities for Microorganisms and Cell Cultures 2 : 2007a; The Australian/New Zealand Standard AS/NZS :2002 Safety in laboratories: Part 3: Microbiological aspects and containment facilities 3 ; and Physical Containment level 1 (PC1) requirements of the above Standards (at minimum) for category A genetic modifications of microorganisms Physical Containment level 2 (PC2) requirements of the above Standards (at minimum) for category B genetic modifications of microorganisms 4) The approved organisms must be developed within a containment facility that complies with: The MAF/ERMA New Zealand Standard: Facilities for Plants The Australian/New Zealand Standard AS/NZS :2002 Safety in laboratories: Part 3: Microbiological aspects and containment facilities 5 ; and Physical Containment level 1 (PC1) requirements of the above Standards (at minimum) for category A genetic modifications involving tissue culture plants, protoplasts or plant cell cultures kept in closed containers and without reproductive structures Physical Containment level 2 (PC2) requirements of the above Standards (at minimum) for category B genetic modifications involving whole plants and tissue cultures kept or not kept in a closed container and with or without reproductive structures 5) Given asexual reproduction of Merchantia polymorpha liverworts is via gemma that form in splash cups on the thallus surface, water must not be directly applied to Merchantia polymorpha thalli. 6) Male and female Merchantia polymorpha thalli must be kept separated 7) The approval holder must ensure that within 24 hours of the discovery of any breach of containment (includes the escape of an organism(s) or a failure in the structural integrity of physical containment), 2 Any reference to MAF/ERMA New Zealand or AS/NZS Standards in these controls also refers to any subsequent version approved or endorsed by the EPA. 3 Any reference to MAF/ERMA New Zealand or AS/NZS Standards in these controls also refers to any subsequent version approved or endorsed by the EPA. 4 Any reference to MAF/ERMA New Zealand or AS/NZS Standards in these controls also refers to any subsequent version approved or endorsed by the EPA. 5 Any reference to MAF/ERMA New Zealand or AS/NZS Standards in these controls also refers to any subsequent version approved or endorsed by the EPA.

11 11 the Ministry for Primary Industries biosecurity inspector responsible for supervision of the facility, has received notification (written or verbal) 6 of the breach and the details of any remedial action taken. 4.6 The applicant is not, in this instance, required to provide progress reports as this application does not raise any novel issues. 4.7 I have not imposed an expiry date on this approval. 5. Summary of the decision 5.1 Application APP203565, to develop in containment GMOs (as described in Tables 1 and 2 of this decision), is approved, with controls under section 42A(3) (as described in Table 3 of this decision). This decision was based on the information supplied by the applicant and was considered in accordance with section 42A of the Act, the Regulations, and the Methodology. 20 June 2018 Gayle Holmes Acting General Manager, Hazardous Substances and New Organisms Environmental Protection Authority Date 6 The biosecurity inspector s contact details can be found in the facility containment manual.

12 12 Approval codes for organisms on application APP Host organisms Approval number GMD GMD GMD GMD GMD GMD GMD GMD GMD GMD Organism Escherichia coli (Migula 1895), Castellani and Chalmers 1919 nonpathogenic laboratory adapted strains Agrobacterium tumefaciens (Smith and Townsend, 1907) Conn 1942, armed and disarmed strains Saccharomyces cerevisiae Meyen ex EC Hansen (1883), nonpathogenic laboratory adapted strains Pichia pastoris Guillerm Phaff (1956), non-pathogenic laboratory adapted strains Ogataea angusta (Teun., H.H. Hall & Wick) Suh & Zhou 2010 (synonym Pichia angusta), non-pathogenic laboratory adapted strains Schizosaccharomyces pombe Lindner 1893, non-pathogenic laboratory adapted strains non-sporulating strains Marchantia polymorpha (marchantia) L., tissue culture plants, protoplast and plant cell cultures, whole plants or tissue cultures Nicotiana spp. L. and Graham, tissue culture plants, protoplast and plant cell cultures, whole plants or tissue cultures Arabidopsis thaliana Heynh., tissue culture plants, protoplast and plant cell cultures, whole plants or tissue cultures Petunia spp. Juss., tissue culture plants, protoplast and plant cell cultures, whole plants or tissue cultures