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General information Notification Number B/BE/18/V8 Member State to which the notification was sent Belgium Date of acknowledgement from the Member State Competent Authority 16/08/2018 Title of the Project Scientific field evaluation of maize with an impaired DNA-repair mechanism and maize with modified growth characteristics Proposed period of release: 01/04/2018 to 31/10/2018 Name of the Institute(s) or Company(ies) VIB (Vlaams Interuniversitair Instituut voor Biotechnologie)
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
maize poaceae zea zea mays mays inbred line B104
Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The maize plants have been edited using the CRISPR/Cas9 system resulting in the mutation of either the ALT gene or the NGAL2 gene. In both cases one extra DNA base pair has been introduced into the target genes resulting in the knock-out of these genes by means of a frameshift mutation.
The ALT gene plays a role in the repair of DNA damage and in these plants faults in the DNA are believed to accumulate to a higher degree. The plants have thereby become a good biosensor for the type of DNA damage that is caused by different forms of abiotic stress such as drought, heat, or environmental pollution.
The NGAL2 is a transcription factor that has been shown to negatively affect the expression of the PLA1 gene. The PLA1 gene codes for a cytochrome P450 mono-oxygenase enzyme that is involved in the production of factors that control cell proliferation. By knocking out this gene the levels of PLA1 may go up resulting in altered growth characteristics, which are phenotypically shown by resulting in a significant larger leaf size.
Genetic modification Type of genetic modification: Other; Other

frameshift mutation
Brief description of the method used for the genetic modification: A CRISPR/Cas9 gene cassette, necessary to induce the desired mutation, was introduced into
maize plant by means of Agrobacterium tumefaciens mediated transformation. Transformants
containing the gene cassette and the desired mutations were selected, and by means of
conventional crossing with wild-type plants the CRISPR/Cas9 gene cassette was removed by
selecting T1 plants that only contained the desired mutation, but no longer contained the gene
cassette (null-segregants). The plants in the field trial therefore do not contain foreign genetic
material. They only contain the desired frameshift mutation.
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 of the release is to (1) assess the phenotype of the ALTKO plants under realistic environmental conditions and assess whether abiotic stress leads to measurable DNA damage, and (2) assess whether NGAL2KO plants show similar characteristics under realistic field conditions as plants in which the PLA1 gene is being additionally expressed. Geographical location of the site: The field trial will take place in the town of Zwijnaarde, near Ghent, Belgium. Size of the site (m2): The trial plot, including non-modified controls is approximately 120m2. 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 environmental impact from the release is expected to be zero. The mutations are not
expected to result in characteristics such as greater weediness or the ability of the maize to establish in non-agricultural habitats. The modified characteristics are also not expected to change the interaction of the maize with herbivores or other non-target organisms and also not to change the toxicity and allergenicity of the maize. But we have not tested the latter, as this is not necessary and not required for such a small-scale field trial of which the produced materials are not going to be consumed by humans or animals. The modified seeds that will be formed are well retained in the cobs and these cobs will be very carefully hand harvested, also the tiniest ones, to prevent any spread of seeds.
Brief description of any measures taken for the management of risks: The risk of spread of the modified properties to the environment is mitigated by performing the trial on an isolated location more than 1 km away from any cultivated maize fields and by in almost all cases by removing the tassel, or in exceptional cases hulling the tassel, thus preventing the spread of modified pollen to non-modified maize plants in the surroundings. The formed modified seeds are, as already stated above, well retained in the cobs and these cobs will be very carefully hand harvested, thus preventing any spread of seeds to the environment. In case some seeds would be lost during the harvest, they are not expected to result in the establishment of the maize outside the field. The field trial is surrounded by a 1.50 m high wired fence to prevent accidental trespassing and accidental removal or spread of GM material. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: There are no specific studies foreseen to gain new data on the environmental and human health impact from the release other than the study of the phenotype, genotype and growth characteristics of these maize mutants. Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Not known
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