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Notification report


General information

Notification Number
B/GB/19/52/01

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
Genetic regulation of Sulphur metabolism in Brassica oleracea

Proposed period of release:
01/04/2019 to 31/12/2021

Name of the Institute(s) or Company(ies)
John Innes Centre, ;


3. Is the same GMPt release planned elsewhere in the Community?
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 NameFamily NameGenusSpeciesSubspeciesCultivar/breeding line
cabbagebrassicaceaebrassicabrassica oleracea

2. Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications:
Small indels of a known glucosinolate biosynthesis regulator, Myb28, were introduced into the Brassica oleracea chromosomal DNA using CRISPR-Cas9 gene editing technology, as described in (Lawrenson et al., 2015). This region functions as the coding sequence of this gene, with the gene edits proposed to disrupt the translation of this gene, leading to a non-functional protein. This sequence change was characterised by PCR amplification of the gene region and subsequent Sanger sequencing.

Genetic modification

3. Type of genetic modification:
Insertion; Deletion; Fusion; Other;
Other
CRISPR Cas9 mediated gene editing (small indels)

In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted:
A small insertion resulting from the plants own repair mechanism following the Cas9 nuclease activity has led to a frameshift mutation at the target site.

In case of deletion of genetic material, give information on the function of the deleted sequences:
A small deletion resulting from the plants own repair mechanism following the Cas9 nuclease activity has led to a predicted amino acid change in the resulting protein which may disrupt its function.

6. Brief description of the method used for the genetic modification:
The CRISPR Brassica oleracea lines were produced via Agrobacterium-mediated transformation using 4-day old cotyledonary explants, as described in https://www.jic.ac.uk/app/uploads/2018/11/Brassica-Transformation.pdf. This is an updated version of the method published in (Sparrow et al., 2006a).

Details of CRISPR protocol use for Brassica oleracea gene editing published as (Lawrenson et al., 2015)


7. 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

1. Purpose of the release:
This is a research trial to determine the role of a gene, known as Myb28, in regulating sulphur metabolism, specifically the accumulation of aliphatic glucosinolates, in field-grown Brassica oleracea. Brassica plants of this type, when grown under glasshouse conditions, produce almost undetectable levels of these compounds, therefore this trial is required in order to better imitate the commercial interaction between these compounds and their environment and ultimately how this transcription factor Myb28 may mediate this interaction. Field evaluation of these traits allows for a better understanding on improvement of these crops in the future. This trial is being proposed purely for exploratory purposes and no future commercial use or feeding trials are intended at this time. No tests of hybridisation have been carried out on these lines.

2. Geographical location of the site:
The release site (Ordnance Survey map grid reference TG 179 075) is arable land located at the John Innes Centre (JIC). The land to be used is the area that was previously sown with GM potatoes under consent 10/R29/01 from 2010 – 2012.

3. Size of the site (m2):
No larger than 1000 m2

4. 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:
Not applicable

Environmental Impact and Risk Management

Summary of the potential environmental impact from the release of the GMPts:
The probability of B. oleracea seeds or pollen escaping from the trial site or the transfer of inserted characteristics to sexually-compatible species outside the trial area is estimated as very low. Plants grown in the trial will have their inflorescences removed at the early stages, prior to pollen exposure and therefore seed set. Primary inflorescences produced will be harvested for analysis, along with leaf material. Following this, all plants will be uprooted and destroyed in their entirety by autoclaving at the John Innes Centre to prevent further flowering. Less than ten individuals will be allowed to flower with their inflorescences contained within a pollen proof bag, preventing pollen or subsequent seed release. As B. oleracea is unable to clonally propagate and reproduces exclusively through sexual reproduction, it is unlikely any residual plant material will lead to further emergence of plants. Moreover, no Brassica plants will be grown within 20 metres of the trial site and surrounding areas will be monitored for the presence of species capable of crossing with B. oleracea. If any species are found which may cross pollinate within 20m of the B.oleracea plants in this study, they will be treated with a herbicide (glyphosate). The potential removal of defence compounds, glucosinolates, in trial plants suggests that they would not possess a selective advantage over any existing Brassica plants and will be very unlikely to outcompete any wild or ruderal plants.

The risk of non-sexual, horizontal gene transfer to other species is extremely low. Current data suggests an absence of the transgene used to generate the mutation in this line and therefore an absence of the plasmid (further analysis of vector components is currently underway), further reducing the risk of any potential gene transfer. In the event of horizontal gene transfer to bacteria, neither the trait genes nor the marker genes would be expected to confer a selective advantage in the field environment under consideration. The genes introduced in B. oleracea have been inserted via Agrobacterium tumefaciens-mediated gene transfer. We estimate the likelihood of horizontal gene transfer as low and the consequences were it to occur, as negligible.
We address the nine points of Annex II, D2 to Directive 2001/18/EC, following a classic six-step process of risk assessment detailed below.


Brief description of any measures taken for the management of risks:
Plants grown in the trial will have their inflorescences removed at the early stages, prior to pollen exposure and therefore seed set. Primary inflorescences produced will be harvested for analysis, along with leaf material. Following this, plants will be uprooted and destroyed in their entirety by autoclaving at the John Innes Centre to prevent further flowering. Less than ten individuals will be allowed to flower with their inflorescences contained within a pollen proof bag, preventing pollen or subsequent seed release. As B. oleracea is unable to clonally propagate and reproduces exclusively through sexual reproduction, it is unlikely any residual plant material will lead to further emergence of plants. Moreover, no sexually compatible Brassica plants will be grown within 20 metres of the trial site, with the exception of a barrier of Brassica napus plants, and surrounding areas will be monitored for the presence of species capable of crossing with B. oleracea. If any species are found which may cross pollinate within 20m of the B.oleracea plants in this study, they will be treated with a herbicide (glyphosate). Following harvest, the plot will be left fallow, monitored for remaining Brassica oleracea material during the remainder of the year and sprayed with a systemic broadleaf herbicide. Any Brassica oleracea identified will be destroyed by herbicide treatment (e.g. glyphosate) or removed by hand and destroyed by autoclaving at the John Innes Centre.

Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release:
There are currently no trials planned to gain new data on the environment and human health impact of the release.

Final report
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European Commission administrative information

Consent given by the Member State Competent Authority:
Yes
09/04/2019 00:00:00
Remarks: