APPLICATION FORM Containment – GMO Project

To obtain approval for projects to develop genetically modified organisms in containment

Send to Environmental Protection Authority preferably by email ([email protected]) or alternatively by post (Private Bag 63002, Wellington 6140) Payment must accompany final application; see our fees and charges schedule for details.

Application Number

APP203947

Date

www.epa.govt.nz 2

Application Form Approval for projects to develop genetically modified organisms in containment

Completing this application form

1. This form has been approved under section 42A of the Hazardous Substances and New Organisms (HSNO) Act 1996. It only covers projects for development (production, fermentation or regeneration) of genetically modified organisms in containment. This application form may be used to seek approvals for a range of new organisms, if the organisms are part of a defined project and meet the criteria for low risk modifications. Low risk genetic modification is defined in the HSNO (Low Risk Genetic Modification) Regulations: http://www.legislation.govt.nz/regulation/public/2003/0152/latest/DLM195215.html. 2. If you wish to make an application for another type of approval or for another use (such as an emergency, special emergency or release), a different form will have to be used. All forms are available on our website. 3. It is recommended that you contact an Advisor at the Environmental Protection Authority (EPA) as early in the application process as possible. An Advisor can assist you with any questions you have during the preparation of your application. 4. Unless otherwise indicated, all sections of this form must be completed for the application to be formally received and assessed. If a section is not relevant to your application, please provide a comprehensive explanation why this does not apply. If you choose not to provide the specific information, you will need to apply for a waiver under section 59(3)(a)(ii) of the HSNO Act. This can be done by completing the section on the last page of this form. 5. Any extra material that does not fit in the application form must be clearly labelled, cross- referenced, and included with the application form when it is submitted. 6. Please add extra rows/tables where needed. 7. You must sign the final form (the EPA will accept electronically signed forms) and pay the application fee (including GST) unless you are already an approved EPA customer. To be recognised by the EPA as an “approved customer”, you must have submitted more than one application per month over the preceding six months, and have no history of delay in making payments, at the time of presenting an application. 8. Information about application fees is available on the EPA website. 9. All application communications from the EPA will be provided electronically, unless you specifically request otherwise. Commercially sensitive information

10. Commercially sensitive information must be included in an appendix to this form and be identified as confidential. If you consider any information to be commercially sensitive, please show this in the relevant section of this form and cross reference to where that information is located in the confidential appendix. 11. Any information you supply to the EPA prior to formal lodgement of your application will not be publicly released. Following formal lodgement of your application any information in the body of this application form and any non-confidential appendices will become publicly available.

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Application Form Approval for projects to develop genetically modified organisms in containment

12. Once you have formally lodged your application with the EPA, any information you have supplied to the EPA about your application is subject to the Official Information Act 1982 (OIA). If a request is made for the release of information that you consider to be confidential, your view will be considered in a manner consistent with the OIA and with section 57 of the HSNO Act. You may be required to provide further justification for your claim of confidentiality. Definitions

Restricting an organism or substance to a secure location or facility to prevent Containment escape. In respect to genetically modified organisms, this includes field testing and large scale fermentation

Any obligation or restrictions imposed on any new organism, or any person in relation to any new organism, by the HSNO Act or any other Act or any Controls regulations, rules, codes, or other documents made in accordance with the provisions of the HSNO Act or any other Act for the purposes of controlling the adverse effects of that organism on people or the environment

Any organism in which any of the genes or other genetic material:  Have been modified by in vitro techniques, or Genetically Modified  Are inherited or otherwise derived, through any number of replications, from Organism (GMO) any genes or other genetic material which has been modified by in vitro techniques

A new organism is an organism that is any of the following:  An organism belonging to a species that was not present in New Zealand immediately before 29 July 1998;  An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar prescribed as a risk species, where that organism was not present in New Zealand at the time of promulgation of the relevant regulation;  An organism for which a containment approval has been given under the HSNO Act;  An organism for which a conditional release approval has been given under the HSNO Act; New Organism  A qualifying organism approved for release with controls under the HSNO Act;  A genetically modified organism;  An organism belonging to a species, subspecies, infrasubspecies, variety, strain, or cultivar that has been eradicated from New Zealand;  An organism present in New Zealand before 29 July 1998 in contravention of the Animals Act 1967 or the Plants Act 1970. This does not apply to the organism known as rabbit haemorrhagic disease virus, or rabbit calicivirus A new organism does not cease to be a new organism because:  It is subject to a conditional release approval; or  It is a qualifying organism approved for release with controls; or  It is an incidentally imported new organism

An individual or collaborative endeavour that is planned to achieve a particular Project aim or research goal

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Application Form Approval for projects to develop genetically modified organisms in containment

1. Applicant details

1.1. Applicant

Company Name: (if applicable) University of Canterbury

Contact Name: Dr Claudia-Nicole Meisrimler

Job Title: Lecturer in molecular plant biology

Physical Address:

Kirkwood Avenue, Ilam, Christchurch

Postal Address (provide only if not the same as the physical): University of Canterbury, Private Bag 4800, Christchurch 8140, New Zealand

Phone (office and/or mobile): +64 3 3691019

Fax:

Email: [email protected]

1.2. New Zealand agent or consultant (if applicable)

Company Name:

Contact Name:

Job Title:

Physical Address:

Postal Address (provide only if not the same as the physical):

Phone (office and/or mobile):

Fax:

Email:

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Application Form Approval for projects to develop genetically modified organisms in containment

2. Information about the application

2.1. Brief application description Approximately 30 words about what you are applying to do

To develop in containment low-risk genetically modified organisms for research and teaching purposes. Organisms will include low-risk microorganisms, plant originated cell lines and plants.

2.2. Summary of application Provide a plain English, non-technical description of what you are applying to do and why you want to do it

The research in this application involves understanding how plants respond on a molecular and organismal level to abiotic and biotic stress factors. Plants are diverse in their appearance and evolved diverse mechanisms to adapt to these stress factors and some of the mechanisms are conserved between species, whereas other mechanisms are unique to a species or even a cultivar. Nowadays, we know that plants have diverse mechanisms to perceive and transmit different stress signals and the translation of these signals is the basis for the physiological process called adaptation. We wish to investigate how different plants adapt to biotic, abiotic and combinatory stress. This is essential to understand how climate change, which consists of a variety of abiotic stresses, affects plant health and the interaction with plant pathogens. The general question is, how do we define plant health these days? For instance, how does drought, heat and flooding affect the plant immune system? Can some plant pathogens use such conditions for an improved infection? Do some microbes or pathogens target physiological pathways used by the plant to adapt to stresses as a loop-hole? Might even some beneficial microbes turn into pathogens under specific circumstances? If we put microbial “effector” proteins, that originally target specific plant proteins to facilitate infection or symbiosis, into plants (for instance Arabidopsis thaliana) do the plants now respond differentially to microbes or abiotic stress? Or if we stop the targetted plant protein from working in plants, do we see that the plant is now confined in defense mechanisms or stress adaptation? Detailed analysis of these experiments will provide us with a lot of information about plants' defences against microbial pathogens and symbiosis with beneficial microbes, adaptation to abiotic stresses and how abiotic stress might affect the interaction of the plant with the microbe(s).This knowledge will be the basis for breeding of more pathogen and climate change resilient crop species and to define strategies for more efficient ecosystem and crop protection strategies in the future.

2.3. Technical description

Briefly describe the host organism(s) and the proposed genetic modifications. Please make sure that any technical words used are included in a glossary. Note if any part of this research project is already covered by an existing HSNO Act approval that your organisation holds or uses.  Specifically, we will be examining plants and plant derived material modified to express genes from plants, fungi, or bacteria that are involved in physiological processes - including but not exclusively stress physiology pathways, immunity, developmental and infection processes.  Furthermore, we will examine genetically modified plants silenced, knocked out or knocked down (produced by miRNA, siRNA, CRISPR/CAS or similar techniques) for one or multiple genes.  We will make use of floral dipping with agrobacterium for a more efficient transformation of Arabidopsis plants. Arabidopsis plants will be grown in our PC2 plant facilities and contained as described in the Containment Manual, Containment Facility for Plant Species, Facility 559, university of canterbury. Floral dipping will be accomplished in a class II Biohazard flow hood and

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Application Form Approval for projects to develop genetically modified organisms in containment

plants kept in a closed container until harvest of the seeds. For other host organism described in table 2, plant cell lines, callus cultures, embryo cultures, plastids will be used for transformation by agrobacterium or particle bombardment. Transformed plants will be grown and propagated for screening of homozygous lines, which will be used for experiments on plant-microbe-interaction, abiotic stress effects or both combined.  Modified and non-modified plants will be compared for infection with pathogenic microbes (fungi, oomycetes or bacteria) and compared to non-infected control plants under similar conditions. We will use these experiments to examine the visual and molecular phenotype for control conditions, abiotic stressors and combinatory abiotic-biotic-stress effects.  Initially, experiments will be performed on whole plants or plant derived material with different pathogens indicated in table 1. Experiments will be accomplished with the following non-native species: Arabidopsis thaliana, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Capsicum annuum, Lactuca sativa, Cucurbita pepo (alternatively on Cucumis sativus) and Pinus radiata.

GMO-techniques are done for research only so that we can understand how plants interact with microbes, adapt to abiotic stress and how this abiotic stress might affect the plant-microbe-interaction. These modified plants and/or infected plants will be kept in very secure enclosures and destroyed by autoclaving after they have been used. Modified plants (Table 2) will be cultivated at all times under PC2 conditions (see also proposed containment section) and will be inoculated with microbes (Table 1) under PC2 conditions. All plants and tissue cultures save A. thaliana will be sourced from New Zealand sources, for example research institutes, universities or garden centers. A. thaliana wild types will be imported under permit and genetically modified A. thaliana developed elsewhere will be imported under APP201858 (e.g. The Arabidopsis Biological Resource Center (ABRC), the Nottingham Arabidopsis Stock Center (NASC), the RIKEN Bioresource Center (BRC)/ SENDAI Arabidopsis Seed Stock Center (SASSC), the INRA- Versailles Genomic Resource Center, or Lehle Seeds).

Table 1: Pathogens used in this project are all non-modified. There are no known inseparable or associated organisms associated with listed organisms. There are no known prohibited organisms involved with the listed organisms, all of the pathogens have been described as present in New Zealand and are not listed as unwanted or notifiable organisms. Organism Common name(s), if Phylogeny any: Botrytis cinerea Persoon (1794) Gray mold Leotiomycetes, Helotiales, Sclerotiniaceae

Pseudomonas cichorii (Swingle Bacterial blast, cancer, Gamma Proteobacteria, 1925) Stapp 1928 blight Pseudomonadales, Pseudomonadaceae

Pseudomonas syringae pv. Bacterial blast, cancer, Gamma Proteobacteria, syringae van Hall 1902 blight Pseudomonadales, Pseudomonadaceae

Pseudomonas syringae pv. Bacterial blast, cancer, Gamma Proteobacteria, tomato van Hall 1902 blight Pseudomonadales, Pseudomonadaceae

Sphaeropsis sapinea (Fries) Diplodia dieback Dothideomycetes, Botryosphaeriales, Dyko & Sutton (= Diplodia pinea Botryosphaeriaceae (Desmazieres) Kickx)

Pseudoperonospora cubensis Cucurbita downy mildew Oomycetes, , (Berk. & M.A. Curtis) Rostovzev

Pectobacterium carotovorum (or blackleg or soft rot Bacteria, Gammaproteobacteria, Erwinia carotovora Pectobacteriaceae pluvialis Reeser, Red needle cast Oomycetes, Peronosporales, Sutton, and E. Hansen Peronosporaceae

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Application Form Approval for projects to develop genetically modified organisms in containment

Phytophthora aleatoria sp. nov. Red needle cast Oomycetes, Peronosporales, Peronosporaceae Phytophthora nicotianae Breda Black Shank Oomycetes, Peronosporales, de Haan Peronosporaceae Phytophthora infestans(Mont.) Late blight or potato Oomycetes, Peronosporales, de Bary blight Peronosporaceae

Bremia lactucae Regel (1843) Lettuce downy mildew Oomycetes, Peronosporales, Peronosporaceae

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Application Form Approval for projects to develop genetically modified organisms in containment

3. Information about the new organism(s)

3.1. Identity of the host organism(s)

For each host organism:  Provide its taxonomic name and describe what type of organism it is.  Provide a description of the strain(s) being applied for (if relevant).  If the host organism is derived from humans (eg, cell lines) or may have cultural significance (e.g. sourced from native biota), provide details of its source. State the category (Category 1 or Category 2) of the host organism (as per the HSNO (Low Risk Genetic Modification) Regulations). Table 2: Plants host organisms. The project will rely on modification done in tissue cultures and whole plants. As per the HSNO (Low Risk Genetic Modification) regulations, whole plants with reproduction structures fall into category 2, therefore plants modified and used for propagation to screen for homozygosity of the insert or comparable will fall into category 2. Plant tissue cultures and whole plants with reproduction structures fall into category 1. Experiments of plant- pathogen-interaction and abiotic stress effects will be accomplished either with tissue cultures or whole plants without reproduction structures. There are no known inseparable or associated organisms associated with listed organisms. There are no known prohibited organisms involved with the listed organisms. Organism Common name Taxonomy Category Arabidopsis thaliana (L.) Heynh (1842) Thale cress Angiosperms, Dicotyledonae, Rosids, 2 whole plants Brassicales, Brassicaceae Nicotiana benthamiana Domin. Nicotiana Angiosperms, Dicotyledonae, Asterids, 2 whole plants Solanales, Solanaceae Solanum lycopersicum L. (Formerly Tomato Angiosperms, Dicotyledonae, Asterids, 2 known as Lycopersicon esculentum Solanales, Solanaceae Miller (1768) whole plants Solanum tuberosum L. Potato Angiosperms, Dicotyledonae, Asterids, 2 whole plants Solanales, Solanaceae Capsicum annuum L. Bell pepper and pepper Angiosperms, Dicotyledonae, Asterids, 2 whole plants Solanales, Solanaceae Lactuca sativa L. Lettuce Angiosperms, Dicotyledonae, Asterids, 2 whole plants Asterales, Asteraceae

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Application Form Approval for projects to develop genetically modified organisms in containment Cucurbita pepo L. ssp. Pumpkin and Zucchini Angiosperms, Dicotyledonae, Rosids, 2 whole plants Cucurbitales, Cucurbitaceae Cucumis sativus L. Cucumber Angiosperms, Dicotyledonae, Rosids, 2 whole plants Cucurbitales, Cucurbitaceae Pinus radiata D.Don Radiata pine Gymnosperms, Conifers, Pinales, Pinaceae 2 whole plants Arabidopsis thaliana (L.) Heynh (1842) Thale cress Angiosperms, Dicotyledonae, Rosids, 1 e.g. Tissue culture, explant, protoplast Brassicales, Brassicaceae Nicotiana benthamiana Domin. Nicotiana Angiosperms, Dicotyledonae, Asterids, 1 e.g. Tissue culture, explant, protoplast Solanales, Solanaceae Solanum lycopersicum L. (Formerly Tomato Angiosperms, Dicotyledonae, Asterids, 1 known as Lycopersicon esculentum Solanales, Solanaceae Miller (1768) e.g. Tissue culture, explant, protoplast Solanum tuberosum L. Potato Angiosperms, Dicotyledonae, Asterids, 1 e.g. Tissue culture, explant, protoplast Solanales, Solanaceae Capsicum annuum L. Bell pepper and pepper Angiosperms, Dicotyledonae, Asterids, 1 e.g. Tissue culture, explant, protoplast Solanales, Solanaceae Lactuca sativa L. Lettuce Angiosperms, Dicotyledonae, Asterids, 1 e.g. Tissue culture, explant, protoplast Asterales, Asteraceae Cucurbita pepo L. ssp. Pumpkin and Zucchini Angiosperms, Dicotyledonae, Rosids, 1 e.g. Tissue culture, explant, protoplast Cucurbitales, Cucurbitaceae Cucumis sativus L. Cucumber Angiosperms, Dicotyledonae, Rosids, 1 e.g. Tissue culture, explant, protoplast Cucurbitales, Cucurbitaceae Pinus radiata D.Don Radiata pine Gymnosperms, Conifers, Pinales, Pinaceae 1 e.g. Tissue culture, explant, protoplast

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Application Form Approval for projects to develop genetically modified organisms in containment Table 3: Laboratory strains for cloning and plant transformation purposes

Organism Common name Taxonomy Strain(s) if Category relevant: Agrobacterium tumefaciens (Smith Crown gall Alphaproteobacteria, Non-tumorigenic 1 & Townsend 1907) Conn 1942 bacterium Rhizobiales, Rhizobiaceae strains of Agrobacterium

Escherichia coli Migula (1895) E. coli Gammaproteobacteria, Non-pathogenic 1 Enterobacteriales, laboratory strains of Enterobacteriaceae E.coli

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Application Form Approval for projects to develop genetically modified organisms in containment

3.2. Regulatory status of the organism

Is the organism that is the subject of this application also the subject of:

An innovative medicine application as defined in section 23A of the Medicines Act 1981?

☐ Yes ☒ No

An innovative agricultural compound application as defined in Part 6 of the Agricultural Compounds and Veterinary Medicines Act 1997?

☐ Yes ☒ No

4. Information about the project

4.1. Describe the nature and range of the proposed genetic modifications

 Describe the nature and range of the proposed genetic modifications (e.g. the range of elements that the vectors or gene constructs may contain, and the type, source and function of the donor genetic material).  State the category (Category A or Category B) of the genetic modifications (as per the HSNO (Low Risk Genetic Modification) Regulations). We will use vectors that contain one or more of the following genetic elements derived from bacterial, animal, fungal, plant, bacteriophages and other viruses and or of synthetic origin:  Promoters (constitutive, endogenous or inducible).  Localisation signals.  Internal ribosome entry site.  Regulatory peptides.  Regulatory elements for inducible expression.  Polyadenylation signals.  Multiple cloning sites.  Origins of replication.  Splice acceptor/donor sites.  Transcriptional activators.  Transcriptional terminator sequences.  Secretory and targeting signals.  Recombination sites and flanking sequences.  Selection markers.  Insulators.  Reporter genes such as colorimetric, bioluminescent or fluorescent genes.

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Application Form Approval for projects to develop genetically modified organisms in containment

 Transposable elements.  Protein tags to determine transgene localisation and/or expression or aid purification.  CRISPR/CAS elements  DNA Vectors for siRNA, miRNA and generally RNA interference

Additional genetic material will contain one or more of the following elements: Genes from living organisms Single genes (cDNA or genomic fragments) of fungal, plant, oomycetes or bacterial origin encoding proteins that are involved in plant-microbe-interaction or stress responses, including gene silencing, intron splicing and/or translation pathways and/or pathways involving non-translated RNAs. Genetic material from native New Zealand species will not be used. Category A modifications E.coli and A. tumefaciens will be developed using standard cloning and transformation techniques and will employ cloning and expression vectors from commercial and reputable research laboratory sources. Category A (Plant tissue culture) Category B (Whole plants) Transformation vectors will be constructed for Agrobacterium-mediated plant transformation by floral dipping, incubation, infiltration or other means (stable or transient), electroporation, PEG-mediated DNA uptake and/or biolistics of plants. Agrobacterium-mediated plant transformation will use disarmed A. tumefaciens containing a subsection of the vectors described above being plant transformation vectors that may carry additional genetic material (as described above). Modifications will exclude: Production of infectious particles, toxins effective on vertebrates or other modifications that enhance its ability to escape containment are not permitted under this approval. Therefore, users of this approval must not use vector systems or donor genetic material that will enhance its toxicity to vertebrates and the ability to escape containment. Furthermore, modifications will exclude sequences derived from humans or New Zealand native flora and fauna, material from CITES-listed species, transgenes for proteins known to present a vertebrate disease risk; and genes that encode vertebrate toxins with an LD50 < 100 μg/kg.

4.2. Proposed containment of the new organism(s) (physical and operational)

 State which Containment Standard(s) your facility is approved to.  State the minimum containment level (PC1 or PC2 as per AS/NZS2243.3:2002) required to contain the GMOs (as per the HSNO (Low Risk Genetic Modification) Regulations).  Discuss whether controls in addition to the requirements listed in the Standard(s) are necessary to adequately contain the GMOs. The University of Canterbury maintains MPI approved PC2 containment facilities (#559), which meet the requirements of standards: Room 668 in building Pūtaiao Koiora has approval under the following standards • Containment Facility for Micro-organisms and Cell Cultures • Transitional Facility for Biological Products • Containment Facilities for Plants 2007 Plant Basement Facility in Building Pūtaiao Koiora is approved under standard

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Application Form Approval for projects to develop genetically modified organisms in containment

 Containment Facilities for Plants 2007 Work with the organisms will be limited to those facilities that have been MPI approved to the relevant standard for that organism. This includes an operational protocol and maintenance programme to prevent any release of the contained organisms and we consider the likelihood of escape to be minimal. This protocol also requires approval by MPI, and includes procedures such as the requirement of staff to wear lab-coats with no pockets and disposable 'bootees', as a precaution against the transport of pollen and seeds out of the plant facilities. The plant tissues require particular growth conditions, and human intervention to regenerate into whole plants. Plant tissues are approved hosts systems as described in subclause 8 of the HSNO (Low-Risk Genetic Modification) Regulations 1998, requiring PC1 containment unless allowed to develop reproductive structures or kept outside of a closed container in which case they require PC2 containment. Regenerating plantlets will be transferred from PC2 laboratories to PC2 plant facilities at this stage in their development. Whole plants have the potential to establish outside of containment and form self-sustaining populations. These plants will be maintained in PC2 plant facilities. Floral dipping of A. thaliana with A. tumefaciens for stable transformation will be accomplished in PC2 facilities under the Biohazard hood, followed by transfer of the dipped plants into closed vessels and kept under closure until seed harvest. Transgenic plants are separated from any other plants of that species and pollen and seed spread is reduced or prevented by bagging inflorescences when possible or placement of the inflorescences in seed collection tubes. Modified plants will be grown under in vitro conditions in closed vessels on defined, sterile media or sterile soil before they are inoculated with bacteria, fungi or oomycetes under PC2 conditions and further incubated in a PC2 containment facility. Plants used in pathogen infection assays will never have reproductive structures. This will effectively prevent the spread of pollen, seeds and pathogens so that the likelihood of escape of reproductive material and pathogens will be minimal. Plant organs infected with the above listed pathogens (Table 1) will be harvested under PC2 conditions. For microscopy, they will be transferred enclosed in at least two containers to a within house microscopy facility operating at PC2. For some experiments plant material will be frozen or used for extraction processes to analyse molecular content. All transgenic material and wild type plants, including growth media and soil, inoculated with pathogens will be autoclaved prior to disposal. Consequently, there will be no impact of this research on the environment, local flora and fauna or public health.

5. Risks, costs and benefits

Provide information of the risks, costs and benefits of the GMOs in the following areas of impact:  The environment.  Human health and safety.  The economy (e.g. the ability of people and communities to provide for their economic wellbeing).  The relationship of Māori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga, and the principles of the Treaty of Waitangi (The details of any engagement or consultations with Māori that you have undertaken in relation to this application should be discussed here).  Society and community.  New Zealand’s international obligations. The Environment All the organisms and their development will be in containment, and we have procedures to ensure they will not escape, therefore it is not expected that any of the organisms created will any adverse effects on the environment. The genetic modifications are all of a well described nature and can efficiently be tracked.

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Application Form Approval for projects to develop genetically modified organisms in containment

Agrobacterium tumefaciens associated to this application are “disarmed” strains (Gelvin, 2003). The native disease-causing T-DNA is altered or deleted in disarmed strains but the virulence operons required for gene transfer remain intact. Survival of A. tumefaciens strains on plants that have been genetically modified by them have been reported (e.g., Matzk et al. 1996), so that it appears that at least some laboratory strains of A. tumefaciens will be able to survive outside of containment. We did not find any studies that investigated whether laboratory Agrobacterium strains are able to colonize other plants once they are outside of containment. The A. tumefaciens strains would, however, have to compete with other Agrobacterium strains and species that naturally inhabit the plant rhizosphere. Therefore, we consider it unlikely that laboratory strains will be able to establish self-sustaining populations. The E. coli strains are derivatives of E. coli strain K12, which has been demonstrated to be unable to establish a self-sustaining population outside of laboratory culture (Smith 1975, Bogosian et al. 1996). Human Health and Safety All organisms will be in containment, and we have procedures to ensure they will not escape. Therefore, it is not expected that any of the organisms created will have adverse effects on human health. It is important to note that all proposed pathogens are non-vertebrate hosts. Thereby minimising health risks for experimenter and environment. The Economy All the organisms will be in containment, and we have procedures to ensure they will not escape. Therefore, it is not expected that any of the organisms created will have adverse effects on the economy. There are limited direct benefits to the economy as this is for research only. However, in the future the results of the planned studies will benefit New Zealand by enabling breeding of comparable genes for more pathogen and stress durable crops. The application covers Radiata pine, which is, with approximately 90% of New Zealand’s plantation forests being Pinus radiata, the dominant tree in New Zealand’s forestry at present. Forestry exports generated ~5.6 billion dollars in 2018, with approximately 5.2 billion of that coming from the export of Pinus radiata (https://www.nzffa.org.nz/farm-forestry- model/species/radiata-pine/). Furthermore, we will work on multiple significant vegetable crops, e.g. potato, capsicum, bell pepper and cucumber, which count as key vegetables grown in New Zealand (Horticulture New Zealand report 2017). Furthermore, outcomes might enable us to decrease the use of pesticides and develop improved strategies for agriculture in a changing climate. Culture This work is not expected to impact in any way on the relationship of Maori and their culture and traditions with their ancestral lands, water, sites, waahi tapu, valued flora and fauna and other taonga. Society and Community All the organisms will be in containment, and we have procedures to ensure they will not escape. Therefore, it is not expected that any of the organisms created will have adverse effects on society and the community. There are limited direct benefits for the society as this is for research only. The results of the planned studies associate with plant defence and climate change and therefore contribute to future agriculture, conservation and a more sustainable society. International Obligations There are no international obligations concerning these organisms nor the modifications that we propose.

6. Other information

Add here any further information you wish to include in this application including if there are any ethical considerations that you are aware of in relation to your application. One of the main risks of this project is the escape of a plant pathogenic microorganism from containment and its establishment on a plant species of cultural or economic value. However, the probability of this

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Application Form Approval for projects to develop genetically modified organisms in containment occurring is extremely low. The microorganisms (Table 1) will be grown in sealed petri dishes or on plants in PC2 laboratories. Pathogens used in this project are all non-modified. There are no known inseparable or associated organisms associated with listed organisms in Table 1. There are no known prohibited organisms involved with the listed organisms, all of the pathogens have been described as present in New Zealand and are not listed as unwanted or notifiable organisms. Aerial dispersion of spores is possible for fungi and oomycetes – to avoid the spread of spores, all work with these fungi will be carried out in a class II Biohazard flow hood in PC2 facilities and disease symptoms on plants observed before the formation of sporulation structures. In case of observation of sporulation structures by microscopy in PC2 facilities, microscopy slides will be prepared under class II Biohazard flow hood and coverslips closed off with glue or nail polish. This will reduce the additional risk of air-dispersion and escape of the fungi. Furthermore, the fungus Sphaeropsis sapinae, has a very specific and small host range – therefore spread in case of escape is very unlikely. The species listed in table 1 are spread aerially or are soil-borne and spores require dispersal in water, for aerial dispersal this would be rain droplets, mist or fog (Erwin & Ribeiro, 1996). This significantly reduces the ability of these organisms to escape the PC2 microorganism containment facility, especially in comparison to many fungal species which are easily aerially spread. In general all microbes will be propagated on plates. Transfer during propagation from one plate to another or infection of plants will be accomplished under the class II Biohazard flow hood and all material that was potentially in contact with microbes sterilized using alcohol and flames. Infected plants are kept in containment in PC2 facilities. At the end of the experiment infected plant material will be double packed and destroyed by autoclaving. Staff working in this project will follow all of the protocols described in University of Canterbury, School of Biology PC2 Containment Facility Manual.

Potential benefits: This project will determine how abiotic stress effects plant-pathogen-interaction. This work is important for understanding how climate change will affect plant health and pathogen behavior on plants. In long- term, this knowledge can be used in the future to develop breeding strategies, irrigation and pesticide treatment strategies or comparable with application agriculture and forestry.

7. Checklist This checklist is to be completed by the applicant

Application Comments/justifications

All sections of the application form completed ☒ Yes ☐ No or you have requested an information waiver (If No, please discuss with an under section 59 of the HSNO Act Advisor to enable your application to be further processed)

Confidential data as part of a separate, ☐ Yes ☒ No identified appendix

Supplementary optional information attached:

 Copies of additional references ☐ Yes ☒ No

 Relevant correspondence ☐ Yes ☒ No

Administration

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Application Form Approval for projects to develop genetically modified organisms in containment

Are you an approved EPA customer? ☐ Yes ☒ No If Yes are you an: Applicant: ☒ Agent: ☐

If you are not an approved customer, payment of fee will be by:  Direct credit made to the EPA bank ☐ Yes ☐ No account (preferred method of payment) ☒ Payment to follow Date of direct credit:

 Cheque for application fee enclosed ☐ Yes ☐ No ☐ Payment to follow

Electronic, signed copy of application e- ☒ Yes mailed to the EPA

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Application Form Approval for projects to develop genetically modified organisms in containment

Signature of applicant or person authorised to sign on behalf of applicant

☒ I am making this application, or am authorised to sign on behalf of the applicant or applicant organisation.

☒ I have completed this application to the best of my ability and, as far as I am aware, the information I have provided in this application form is correct.

04/03/2020 Signature Date

Request for information waiver under section 59 of the HSNO Act

I request for the Authority to waive any legislative information requirements (i.e. concerning ☐ the information that has been supplied in my application) that my application does not meet (tick if applicable).

Please list below which section(s) of this form are relevant to the information waiver request:

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Application Form Approval for projects to develop genetically modified organisms in containment

Appendices and referenced material (if any) and glossary (if required)

1. Gelvin, S. B. (2003). Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol. Mol. Biol. Rev., 67(1), 16-37. 2. Matzk, A., Mantell, S., & Schiemann, J. (1996). Localization of persisting agrobacteria in transgenic tobacco plants. MPMI-Molecular Plant Microbe Interactions, 9(5), 373-381. 3. Smith, H. W. (1975). Survival of orally administered E. coli K12 in alimentary tract of man. Nature, 255(5508), 500-502. 4. Bogosian, G., Sammons, L. E., Morris, P. J., O'Neil, J. P., Heitkamp, M. A., & Weber, D. B. (1996). Death of the Escherichia coli K-12 strain W3110 in soil and water. Appl. Environ. Microbiol., 62(11), 4114-4120. 5. https://www.nzffa.org.nz/farm-forestry-model/species/radiata-pine/ 6. Horticulture New Zealand report 2017: https://www.hortnz.co.nz/assets/HortNZAnnualReport17.pdf 7. Erwin, D. C., & Ribeiro, O. K. (1996). Phytophthora diseases worldwide. American Phytopathological Society (APS Press).

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