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General information Notification Number B/BE/20/V1 Member State to which the notification was sent Belgium Date of acknowledgement from the Member State Competent Authority 08/01/2020 Title of the Project Scientific field evaluation of maize with modified growth characteristics Proposed period of release: 15/04/2020 to 31/10/2022 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 B104
Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The genetically modified maize plants have modified growth characteristics resulting from the additional expression of the AN3 gene under the control of the EF1a promoter. This gene is a transcriptional co-activator that is involved in the regulation of cell proliferation. As a result of the modification the duration of growth is elongated which results in the formation of larger plant organs such as larger leaves. It is also results in the formation of more biomass.

The plants also contain the bar gene which produces the Phosphinotrycine acetyl transferase (PAT) protein. The gene functions as a selection marker gene during the transformation process. Phosphiotrycine-based herbicides will not be used in the field.

Two of the three lines in the field additionally contain the aadA-gene under the control of a bacterial promotor, which provides resistance to the antibiotics streptomycin and spectinomycin when present in the correct bacterial genetic background. The presence of this aadA-gene is caused by the presence of vector-backbone sequences in these two lines.
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: The following elements have been inserted into the genome of the recipient maize plants:

All three GM lines contain:
Element Function Origin
Left T-DNA-border T-DNA insert border Agrobacterium tumefaciens
(= bar -TNOS) Phosphinotrycine acetyl transferase gevolgd door de nopaline synthase terminator Streptomyces hygroscopicus and Agrobacterium tumefaciens
P35S Transcription promoter Cauliflower mosaic virus
AttB2 Recombination site* Lysogenic E.coli
PEF1α Promoter of the EF1α gene Brachypodium distachyon
AttB1 Recombination site* Lysogenic E.coli
AN3 Coding sequence of the ANGUSTIFOLIA3 gene Zea mays
AttB4 Recombination site* Lysogenic E.coli
T35S Transcription terminator of the Cauliflower mosaic virus 35S gene Cauliflower mosaic virus
Right T-DNA-border T-DNA insert border Agrobacterium tumefaciens

Two of the three GM lines contain additionally:
Element Function Origin
pVS1sta Vector stability Pseudonomas
pVS1rep Vector replication Pseudonomas
pBR322bom ‘basis of mobility’; involved in conjugation Escherichia coli
pBR322ori Origin of replication Bifidobacterium longum
aadA** Spectomycine/streptomycine resistance Escherichia coli
Brief description of the method used for the genetic modification: Immature maize embryos have been co-cultivated with genetically modified Agrobacterium tumefaciens. During this co-cultivation the genes of interest (see table above) are transferred to cells of the immature embryo generating transformed cells. The transformed cells have then been selected using a positive screen (based on herbicide tolerance) and induced to regenerate whole plants. 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 confirm the maize’s modified growth characteristics under normal field conditions and to measure the effect of the modification on the cob formation and cob filling which is very difficult to measure in greenhouse conditions. Geographical location of the site: The field trial will take place on grounds belonging to the ILVO research institute in the municipality of Wetteren Size of the site (m2): The trial plot, including non-modified controls, non-modified fertilizer lines and non-modified buffer rows is 1100 m2. 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: The GM lines have not been field tested before. 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 modified characteristics are not expected to lead to 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. Any concerns about a possible unexpected change in the allergenicity of the maize pollen is also not relevant as the male flowers (the tassels) of the modified maize will be removed before they will be able to shed any pollen. By doing this it is also prevented that any pollen would spread to the environment. 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.
Also the presence of the aadA gene in two of the three lines does not result in negative impacts. Resistance to the strep/spec antibiotics is widespread in nature and the aadA gene itself is already present in animals for human consumption and in humans. The chances of the aadA gene being picked up by bacteria is low, the field trial is very local, small scale and temporary, and taken together this will not lead to a negative impact. EFSA is of the opinion that the presence of the aadA gene is allowed in plants for field trials.
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 removing 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. Experience with such field trials in the past 8 years has shown that the way the cobs are harvested effectively prevents any volunteers being formed. The field trial plot is surrounded by a 1.80 m high wire 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 and growth characteristics of the maize. Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
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