Application to Develop in Containment Genetically Modified Organisms (Project Application) Under the Hazardous Substances and New Organisms Act 1996

APPLICATION FORM CONTAINMENT – GMO PROJECT

Application to develop in containment genetically modified organisms (project application) under the Hazardous Substances and New Organisms Act 1996

Send by post to: Environmental Protection Authority, PO Box 131, Wellington 6140 OR email to: [email protected]

Application number

APP201049

Applicant

The New Zealand Institute of Plant and Food Research Ltd

Key contact

Robin MacDiarmid The New Zealand Institute of Plant and Food Research Ltd. Private Bag 92169, Auckland 1142, NZ 120 Mt Albert Rd, Sandringham, Auckland 1025 Phone: 09 926 7000 Fax: 09 925 7001 Email: [email protected]

www.epa.govt.nz 2

Application to develop in containment genetically modified organisms (project application)

Important

This application form covers projects for the development (production, fermentation or regeneration) of genetically modified organisms (GMOs) which meet the criteria of 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. This application type is not publicly notified. The fee for this application can be found on our website at www.epa.govt.nz. This application form will be made publicly available so any confidential information must be collated in a separate labelled appendix. If you need help to complete this form, please look at our website (www.epa.govt.nz) or email us at [email protected]. This form was approved on 21 September 2011.

September 2011 APP201049 3

Application to develop in containment genetically modified organisms (project application)

1. Brief application description

Provide a short description (approximately 30 words) of what you are applying to do.

The project aims to understand how plants and plant viruses interact during infection.

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 to the viruses infecting them. Although all living things are diverse, they have similar responses to virus infection. We can use these similarities to help us understand exactly how plants protect themselves from viruses. We wish also to investigate if there are similarities in how mammals protect themselves against viruses when compared to plants. For instance, there is a protein that allows mammals to respond quickly to a multiplying virus. If we put this protein (for instance from rats or mice) into plants (for instance Thale cress or other non-native species) do the plants now respond more quickly? Or if we stop this protein from working in plants, do we see that the plant is now slow to respond to virus infection? When we put this protein into plants, are other plant functions changed? Alternatively, we will investigate proteins from viruses themselves. Some viruses invade their host and multiply without being detected. For example it has been demonstrated that some mammalian viruses make a protein, a cloaking device per se, to prevent detection. If we put such a protein (not the whole virus) into plants we can see whether the virus cloaking system works in plants too. Detailed analysis of these experiments will provide us with a lot of information about plants' defences against viruses. This knowledge will help solve problems with viruses in crop plants. As an additional benefit, this research may provide further insight into the mechanism of human response to virus infection without performing experiments on humans or other mammals.

3. Write a brief technical description of your project

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 genetically modified to express genes from mammals, insects, fungi, plants, bacteria or viruses that are involved in the virus-response pathways, including – but not exclusively - gene silencing, intron splicing, translation pathways, and/or pathways involving non-translated RNAs. We will infect these plants with virus and examine them for a change in response to a virus infection. Initially, experiments will be performed in Thale cress (Arabidopsis thaliana) or a Nicotiana species (e.g. Nicotiana benthamiana). The understanding we gain from these experiments will be confirmed by examining the effect of the same genes in other non-native species including other Nicotiana species, tomato, Kiwifruit and other Actinidia species, Blueberry and other Vaccinium species, Tamarillo, Petunia, Grapevine, Potato, and Rice.

This research involves use of small, well-defined pieces of DNA (genes) from mammals (including humans, but excluding genes derived from Maori), insects, fungi, plants, bacteria or viruses to be put into plants (non-native species). These genes have been previously isolated and characterized. This GMO-technique is done for research

September 2011 APP201049 4

Application to develop in containment genetically modified organisms (project application) only so that we can understand how plants naturally defend themselves from viruses. These modified plants will be kept in very secure enclosures and destroyed by autoclaving after they have been used.

4. The 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).

Plants Latin binomial, including full Arabidopsis thaliana (L.) Heynh (1842) taxonomic authority: Nicotiana benthamiana Domin Nicotiana tabacum L. Nicotiana clevelandii Gray Nicotiana glutinosa L. Solanum lycopersicum L. (Formerly known as Lycopersicon esculentum Miller (1768) Actinidia deliciosa C.F.Liang & A.R.Ferguson Actinidia chinensis Planch. Actinidia arguta (Siebold & Zucc.) Planch. ex Miq. Actinidia eriantha Benth. Vaccinium corymbosum L. Vaccinium ashei J.M.Reade Vaccinium angustifolium Aiton Vaccinium macrocarpon Aiton Malus domestica Borkh Cyphomandra betacea Cav. Petunia hybrida Vilm. Vitis vinifera L. Solanum tuberosum L. Oryza sativa L. Common name(s), if any: Thale cress Nicotiana species Tomato Kiwifruit Actinidia species Blueberry (Vaccinium species) Vaccinium species Apple Tamarillo Petunia Grapevine Potato Rice Type of organism and Catagory Whole plants Catagory B (as per Low-Risk Regulations) Tissue culture Catagory A Taxonomic class, order and Magnoliopsida, Capparales, Brassicaceae family: Magnoliopsida, Solanales, Solanaceae Ericales, Actinidiaceae Ericales, Ericaceae Rosales, Rosaceae Magnoliopsida, Solanales, Solanaceae

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Application to develop in containment genetically modified organisms (project application)

Solanales, Solonaceae, Petuniodeae Magnoliopsida, Vitales, Vitaceae Magnoliopsida, Solanales, Solanaceae Monocot, Poales, Poaceae Strain(s) if relevant: Any strain/variety/cultivar

Other information, including There are no known inseparable or associated with the listed organisms. presence of any inseparable or There are no known prohibited organisms involved with the listed associated organisms and any organisms. related animals present in New Zealand:

Microorganisms Latin binomial, including full Agrobacterium tumefaciens (Smith & Townsend 1907) Conn 1942 taxonomic authority: Escherichia coli Migula (1895) Common name(s), if any: Crowngall bacterium E. coli Type of organism and Catagory Bacterium (as per Low-Risk Regulations) Both Catagory A Taxonomic class, order and Alphaproteobacteria, Rhizobiales, Rhizobiaceae family: Gammaproteobacteria; Enterobacteriales; Enterobacteriaceae Strain(s) if relevant: Non-tumorogenic strains of Agrobacterium

Non-pathogenic laboratory strains of E.coli Other information, including There are no known inseparable or associated organisms associated with presence of any inseparable or listed organisms. associated organisms and any There are no known prohibited organisms involved with the listed related animals present in New organisms. Zealand: 5. 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 regulatory elements derived from bacterial,animal, fungal,plant and or virus genes: 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.

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Application to develop in containment genetically modified organisms (project application)

Insulators. Reporter genes such as colourimetric, bioluminescent or fluorescent genes. Transposable elements. Protein tags to determine transgene localisation and/or expression or aid purification.

Additional genetic material will contain one or more of the following elements:

Genes from living organisms Single genes (cDNA or genomic fragments) of mammalian (including humans, but excluding genes derived from Maori), insect, fungal, plant or bacterial origin encoding proteins that are involved in the virus-response, 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, nor will genetic material from culturally valued species sourced from New Zealand be used.

Genes from viruses Genetic material from viruses may include; coding, non-coding or regulatory regions of viral genes, or fragments thereof and may include variants with nucleotide substitutions or deletions to determine functional domains or to modify activity. However, the viral genetic material will be such that the single genes or gene constructs: do NOT comprise more than two thirds of the virus genome; are NOT infectious (self-replicating); and are NOT complemented to become infectious in any organism or cell culture. The donor genetic material will exclude: Genetic material that increases the pathogenicity, virulence, or infectivity of the host organism. Genetic material that results in the modified organism having a greater ability to escape from containment than the unmodified host.

Genes that encode for vertebrate toxins with an LD50 < 100 µg/kg.

Category A modifications Escherichia coli and Agrobacterium 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 (stable or transient), electroporation, PEG-mediated DNA uptake and/or biolistics of plants. Agrobacterium-mediated plant transformation will use disarmed hosts of A. tumefaciens containing a subsection of the vectors described above being plant transformation vectors that may carry additional genetic material (as described above).

6. Describe the proposed containment system (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.

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Application to develop in containment genetically modified organisms (project application)

We maintain MAF approved PC2 containment facilities, some of which complies with the MAF/ERMA Standard Containment Facilities for Plants 2007 and some that are MAF approved PC2 containment facilities which comply with the MAF/ERMA Standard Facilities for Microorganism and Cell cultures :2007a, and some both. Work with the organisms will be limited to those facilities that have been MAF approved 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 MAFBNZ, and includes procedure such as the requirement of staff to wear disposable lab-coats with no pockets, and disposable 'bootees', as a precaution against the transport of pollen and seeds out of the glasshouses.

The plant tissues require particular 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 PC1 laboratories to PC2 plant houses at this stage in their development.

Whole plants have the ability to establish outside of containment and form self-sustaining populations. These plants will be maintained in PC2 plant houses. These 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.

All transgenic material 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

7. What are the nature 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 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.

Despite the widespread use of A. tumefaciens in laboratory research there appear to be no risk assessments of Agrobacterium laboratory strains. 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

September 2011 APP201049 8

Application to develop in containment genetically modified organisms (project application) inhabit the plant rhizosphere. Therefore, we consider that there is uncertainty over whether the 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, Heitkamp et al. 1993). Therefore, the chances of E. coli being released to the environment are extremely low. Human Health and Safety

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 human health.

The Economy

There are limited benefits to the ecomony as this is for research only. 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. We attach the letter sent to local Iwi representatives from each of the Plant and Food sites covered under this application (Auckland, Palmerston North and Lincoln) (letter attached in Appendix 1). Society and Community

There are no international obligations concerning these organisms nor the modicifications that we propose.

8. 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.

9. Appendices(s) and referenced material (if any) and glossary (if required)

References

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Application to develop in containment genetically modified organisms (project application)

Heitkamp, MA., et al. (1993) Fate in sewage of a recombinant Escherichia coli K-12 strain used in the commercial production of bovine somatotropin. J. Ind. Microbiol. 11, 243-252.

Matzk et al. (1996) Localization of persisting Agrobacteria in transgenic tobacco plants. Molecular Plant Microbe Interactions 9:373-381.

Smith HW. (1975) Survival of orally administered E.coli K12 in alimentary tract of man. Nature 255, 500-502.

Appendix 1: Consultation letter sent to local Iwi of the Auckland, Palmerston North and Lincoln sites of Plant and Food Research Ltd.

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

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

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.

Signature Date

September 2011 APP201049