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General information Notification Number B/GB/19/52/02 Member State to which the notification was sent United Kingdom Date of acknowledgement from the Member State Competent Authority 14/01/2019 Title of the Project High iron wheat Proposed period of release: 01/04/2019 to 30/09/2021 Name of the Institute(s) or Company(ies) John Innes Centre
Is the same GMPt been notified elsewhere by the same notifier? No Has the same GMPt been notified elsewhere by the same notifier? No Genetically modified plant Complete name of the recipient or parental plant(s):

Common Name	Family Name	Genus	Species	Subspecies	Cultivar/breeding line
wheat	poaceae	triticum	triticum aestivum

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: Increasing the intrinsic nutritional quality of crops, known as biofortification, is viewed as a sustainable approach to alleviate micronutrient deficiencies. In particular, iron deficiency anaemia is a major global health issue, but the iron content of staple crops such as wheat has been difficult to improve using conventional breeding. We have shown that the wheat VACUOLAR IRON TRANSPORTER 2 gene (TaVIT2) functions as an iron transporter in wheat (Connorton et al 2017). Overexpression of TaVIT2 under the control of a wheat endosperm-specific promoter increases iron in white flour fractions by greater than 2-fold, in controlled environment grown plants. The antinutrient phytate was not increased and the iron in the white flour fraction was bioavailable in vitro, suggesting that food products made from the biofortified flour could contribute to improved iron nutrition. The single-gene approach did not affect plant growth as defined by several phenotypic measurements including plant height, tillers per plant, grain number per plant nor grain weight in controlled environment grown plants (Connorton et al 2017).
The plasmid also contained the nptI kanamycin resistance gene for selection of bacteria, and the Hyg gene for selection of plants (under control of the cauliflower mosaic virus 35S promoter).
This application seeks authority to investigate the effects on enhancing micronutrient accumulation in the grain by over-expressing the wheat TaVIT2 gene in the endosperm of wheat plants in the field. Genetic modification Type of genetic modification: Insertion; In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted: A sequence encoding wheat Vacuolar Iron Transporter TaVIT2-D (TraesCS5D02G209900) was cloned into vector pRRes14_RR.301 containing the promoter sequence for the endosperm-specific wheat high molecular weight glutenin subunit 1Dx5 (nucleotides -1,187 to -3 with respect to the ATG start codon of the GLU-1D-1 gene). The promoter-gene fragment was then cloned into vector pBract202 containing a hygromycin resistance gene and left and right border elements for insertion into the plant genome (Smedley and Harwood, 2015). The intended function of expressing TaVIT2-D in the endosperm was to increase iron levels in this tissue (confirmed in Connorton et al 2017). The plasmid also contained the nptI kanamycin resistance gene for selection of bacteria, and the Hyg gene for selection of plants (under control of the cauliflower mosaic virus 35S promoter). Brief description of the method used for the genetic modification: Transgenic wheat plants were produced using the standard protocol by Agrobacterium-mediated transformation described in Rey et al 2018. The constructs were introduced to T. aestivum cv. Fielder by Agrobacterium-mediated inoculation of immature embryos. Whole plants were regenerated and selected from somatic embryos induced in tissue culture. If the recipient or parental plant is a forest tree species, describe ways and extent of dissemination and specific factors affecting dissemination: Not applicable. Experimental Release Purpose of the release: The purpose is to investigate the effects of the ectopic over-expression of the wheat TaVIT2 gene in the endosperm of wheat grains and determine its effect on micronutrient accumulation and agronomic performance in the field. Specific questions to be examined are:
1) Does the over-expression of TaVIT2 lead to increased iron accumulation in the endosperm in field grown plants (i.e. as seen in greenhouse/controlled environment conditions)?
2) Are there any effects on phytate accumulation and distribution in field grown plants?
3) Is there any change to the content of other micronutrients in the grain when the transgenic lines are grown in the field (i.e. manganese, zinc)?
4) Is there any effect of over-expressing TaVIT2 on basic agronomic performance (phenology, yield components, etc)
5) Is there any difference in agronomic performance between the different transgenic lines?
6) Are the effects consistent across years? Geographical location of the site: The plants will be released at the John Innes Centre (JIC, Ordnance Survey map grid reference TG 179 075) in Norwich, Norfolk, United Kingdom. Size of the site (m2): The plants will be released on an area of arable land no larger than 75 metres squared. Each year the area planted with the genetically modified plants will be approximately 25 metres squared. In accordance with wheat planting practice, the plot will rotate within the release site each year of the trial. Relevant data regarding previous releases carried out with the same GM-plant, if any, specifically related to the potential environmental and human health impacts from the release: Events containing the TaVIT2 gene have not previously been released. Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: Summary
Based on the analyses provided below, the overall risk of harm to human health or the environment arising from this trial is assessed as very low.

Environmental risks
The probability of seeds escaping from the trial site or the transfer of inserted characteristics to sexually-compatible species outside the trial area is estimated as very low. Commercial wheat cultivars do not establish easily or thrive in uncultivated environments and are naturally self-pollinating with out-crossing being a rare event. Wheat seeds are relatively large and not normally dispersed by wind. Management procedures to minimise the spread of seeds or pollen will further reduce the probability of these events occurring. Appropriate physical barriers (fenced growing area and full height netted framework over experimental planting) will be employed to prevent access by mammals and birds. There will be no cereals grown for 20 metres from the boundary of the experimental plots and no sexually-compatible wild relatives of wheat exist in the vicinity.

It is highly unlikely that intended or unintended effects of the genetic modification of increased endosperm iron content will result in major changes in invasiveness or persistence. The gene introduced into the plants proposed for release do not confer characteristics that would increase the competitiveness of plants in unmanaged ecosystems.

Apart from the expected phenotype of increased iron content in the endosperm (checked by Perls’ staining and confirmed by ICP-OES analysis), plants from the three proposed events are indistinguishable from untransformed controls, when grown in glasshouses or in controlled environment rooms. No other changes to the plant morphology or development are apparent (Connorton et al 2017). Plants remain sensitive to all herbicides such as glyphosate or glufosinate. The introduced genes are thus not anticipated to confer any intrinsic advantage compared to conventional wheat cultivars with respect to persistence in agricultural habitats or invasiveness in natural habitats and no emergent hazard is predicted.

The risk of non-sexual, horizontal gene transfer to other species is extremely low. In the event of horizontal gene transfer to bacteria, neither the trait gene nor the selectable marker genes would be expected to confer a selective advantage in the field environment under consideration. The plasmid backbone sequences, nptI gene, origins of replication, border sequences etc. come originally from E coli and Agrobacterium tumefaciens, two common gut and soil bacteria respectively and these sequences are already widespread in the soil metagenome. Although this makes potential homologous recombination events more likely, we estimate the likelihood of horizontal gene transfer as low and the consequences, were it to occur, negligible. The area proposed to be planted with GMOs is small (total area <25 m2) and temporary lasting between 5 to 6 months during the three years (2019-2021).

Although the above-ground plant material will be cleared from the site, the nptI gene contained in the plant root DNA will decompose into the soil. The transgene is fully integrated into the plant DNA and the copy number is low thus the nptI gene represents a very small proportion (much less than one millionth) of the total DNA in any one cell of the transformed wheat plants. This excess of competing DNA will significantly dilute the rate of any nptI natural bacterial transformation. In addition, enzymatic degradation of free plant DNA in the soil and the low level of spontaneous bacterial competence to take up free DNA will significantly reduce the incidence of natural transformation. Although the transfer of functional gene units from plants to soil bacteria is accepted to be extremely low under natural conditions (Schluëter et al 1995, Nielsen et al 1997, EFSA, 2009), it cannot be completely discounted that some bacteria may successfully take up the nptI gene. However, there will be no antibiotics applied to the soil to provide additional selection pressure for the gene to persist in the environment. The source of the nptI gene is the gut bacterium E. coli carrying a plasmid containing the transposable element (Tn 903). R plasmids possessing resistance to aminoglycoside antibiotics are already naturally found in the soil and other environments. The nptI gene encodes the enzyme aminoglycoside 3’-phosphotransferase which confers resistance to kanamycin and related aminoglycoside antibiotics. Although these antibiotics still have some clinical applications, alternatives are readily available. Taken together, and bearing in mind the limited scope of this trial, the risk of generating of any additional antibiotic resistance within the soil microbial community or risks to human health or the environment if this were to occur as a result of the proposed trial is considered to be extremely low.

Human health risks
The gene donor organism is hexaploid wheat (Triticum aestivum) and both inserted sequences (promoter and TaVIT2 coding sequences) are already present in all modern wheat cultivars. These sequences are not known to be pathogenic or allergenic to humans, and none of the genes under investigation, or the selectable marker genes, are expected to result in the synthesis of products that are harmful to humans, other organisms or the environment. Any unknown hazards arising from the expression and ingestion of foreign proteins will not occur since the wheat grain harvested from the trial is not intended for general human or animal consumption.

Apart from the TaVIT2 gene, the only two other protein-coding genes present in the vector are the nptI and Hyg genes. The source organism for the gene encoding the hygromycin phosphotransferase (Hyg) enzyme (E. coli) is present in the large intestine of healthy humans and there have been no reports of its adverse effects on humans, animals or plants. The product of the Hyg gene, hygromycin phosphotransferase, has been evaluated on numerous occasions by EFSA and found to raise no safety concerns. According to EFSA (EFSA 2009) genes conferring resistance to hygromycin are included in the first antibiotic resistance marker genes (ARMG) group. They state that, “with regard to safety there is no rationale for inhibiting or restricting the use of genes in this category, either for field experimentation or for the purpose of placing on the market.” The neomycin phosphotransferase I (nptI) gene is under the control of a bacterial promoter and is used for bacterial selection only (i.e. before they are used to transform plant cells). The source organism for the gene encoding this enzyme (E. coli) is present in the large intestine of healthy humans and any NPTI ingested is expected to be broken down by digestive enzymes in the stomach and small intestine. The expression of NPTI in plant cells is very unlikely and the gene is already widely present in the environment.

Detailed evaluation of hazards, magnitude of exposure and management strategies to minimise risk. Brief description of any measures taken for the management of risks: Precautions to maintain the genetically modified plant at a distance from sexually compatible plant species, both wild relatives and crops: Wheat is a self-pollinating crop with very low rates of cross-pollination with other wheat plants. The only wild relatives of wheat commonly found in the UK are in the genera Elymus and Elytrigia (formerly Agropyron) although there are no reports of cross-hybridisation between wheat and these genera. The two most common inland species are Elytrigia repens (common couch grass = Agropyron repens) and Elymus caninus (bearded couch = Agropyron caninum). Other related species, such as Elytrigia juncea (Sand couch = Agropyron junceum), Elytrigia atherica (Sea couch = Agropyron pycnanthum) and hybrids are largely confined to coastal habitats.

E. repens is common in the JIC site whereas E. caninus is uncommon in Norfolk (National Biodiversity Network). E. repens propagates primarily by vegetative reproduction (rhizomes), rather than by sexual reproduction, and in any case, no reports of wheat x Elytrigia or Elymus spontaneous hybrids have been reported. E. repens will be controlled along with other weeds in and around the trial site using standard farm practices. No wheat or other cereals, including E. repens will be cultivated or allowed to grow within 20 meters from the trial.

Measures to minimise or prevent dispersal of any reproductive organ of the genetically modified plant (such as pollen, seeds, tuber): To avoid dispersal of seed while sowing, we will hand-transplant seedling plants grown in controlled environment conditions. Ears of all transgenic lines and controls for all ten replications will be hand-harvested, conditioned and threshed for phenotypic assessment and research purposes. Any remaining grain will be disposed by autoclaving and disposing in agreement with JIC standard operating procedures for transgenic material. All straw will be chopped and left on site.
Pollen will be allowed to be produced as we require grain but it’s short period of viability and the separation distance of the transgenic plants from other wheat crops (at least 20 metres) will minimise the risk of cross-pollination. Full height framework and netting will protect the planting from birds and mammals throughout the growing season (from transplant to harvest).
We have excluded the use of a pollen barrier given that its inclusion would more than quadruple the area under the release; a 2 m pollen barrier surrounding the release area would translate to a total area of 106.9 m2 compared to the 23.1 m2 of transgenic material. We are prepared to include a 2 m pollen barrier if requested (e.g the designated area is large enough, etc) but we propose that the separation distance of 20 m should minimise dispersal of any modified material.
Prior to planting, seedlings will be transported from JIC controlled environment rooms to the release site and the plants will not be mixed with either other plants or with equipment used for working on other agricultural land. Any equipment used during the growing season, including for planting and harvesting of transgenic material, will be thoroughly cleaned after use and before it is allowed to leave the release site.

Description of the methods for post-release treatment of the site or sites: Harvest will occur August/September depending on weather conditions at the time (if the plants senesce prior to this then harvesting will be brought forward). Ears (spike/inflorescences) of transgenic and control plants will be hand harvested, conditioned, and manually threshed in a separate designated area with seeds being stored in appropriate GM seed stores.

The plot will be monitored for volunteer plants immediately following harvest. This will include a shallow light tillage (minimum depth 5 cm) to encourage volunteers in autumn. The area will be left fallow over winter and in spring another shallow light tillage will be performed and glyphosate applied in case any volunteers are present. The area will be monitored for volunteers in this growing season (2019-2020) and the following season (2020-2021) during which time it will remain uncropped with wheat. Any volunteers detected in this two-year post-harvest period will be recorded and then destroyed by application of glyphosate or by autoclaving before ear emergence.

Description of the post-release treatment methods for the genetically modified plant material including wastes: Ears (spike/inflorescences) of transgenic and control plants will be hand harvested, conditioned, and manually threshed in a designated area. Seeds will be stored in appropriate GM seed stores.


Description of monitoring plans and techniques: The purpose of the monitoring plan is to enable early detection of any unintended effects related to the release of the transgenic wheat plants.
The release site will be visited by trained laboratory personnel who are working on the project at no less than weekly intervals. Visits will usually occur more frequently and records will be kept of each visit. Any unexpected occurrences that could potentially result in adverse environmental effects or the possibility of adverse effects on human health will be notified to the national inspectorate immediately. Should the need arise to terminate the release at any point the emergency plans detailed below will be followed.
Post-trial the release site will remain fallow to enable easy identification of volunteers. The site will be inspected fortnightly between harvest and September and any volunteers identified will be immediately destroyed either by application of a systemic herbicide or by hand pulling plants and digging out the root systems. These will then be autoclaved within JIC. If volunteers are found at the end of the 2-year period, DEFRA recommendations will be followed for the management of the release site.

Description of any emergency plans: In the unlikely event that the integrity of the site is seriously compromised, the trial will be terminated and all plants will be destroyed using a suitable herbicide or harvesting as deemed appropriate. All harvested material will be removed from the site and disposed of by incineration using our approved contractor. Transportation of waste materials will be in secure containers. The phone numbers of all key staff will be available to site security and field personnel.

Methods and procedures to protect the site: The release site will be fenced to protect against animal damage and entry by unauthorised persons. We will include additional measures for birds and rabbits by enclosing the planting in framework and netting throughout the growing season. The site will also be monitored by remote security cameras visible from the JIC reception which is manned throughout the day by JIC reception staff and by security guards out of normal working hours. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: Not applicable Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
04/09/2019
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