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General information Notification Number B/ES/22/01 Member State to which the notification was sent Spain Date of acknowledgement from the Member State Competent Authority 10/11/2021 Title of the Project Title of the project: Field test of tobacco cv Burley B5 plants derived (by self-pollination) fromlines NtB538061T0#1(T1#1T2#1T3#1), stable transgenic for ethanol-inducible thaumatin-2, forgrowth performance and recombinant protein production. Proposed period of release: 01/03/2022 to 31/12/2022 Name of the Institute(s) or Company(ies) Nomad Bioscience GmbH
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
tobacco	solanaceae	nicotiana	nicotiana tabacum	tabacum

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The tobacco plants to be released carry a transgene insertion, namely a T-DNA insertion for Ethanol-inducible expression of the sweet tasting protein thaumatin-2 from Thaumatococcus daniellii using a viral expression system. Kanamycin resistance was used as transgenic plant selection marker. Genetic modification Type of genetic modification: Insertion; Brief description of the method used for the genetic modification: Stable transgenic tobacco lines bearing a T-DNA transgene insertion were generated by Agrobacterium tumefaciens-mediated leaf disc transformation of N. tabacum cv Burley B5. For generation of transgenic T0 lines, plants were transformed using Agrobacterium tumefaciens strain GV3101 carrying the plasmid pNMD38061 following a slightly modified standard protocol5. Plasmid pNMD38061 bears the T-DNA described above, a neomycin phosphotransferase expression cassette (as kanamycin selection marker in bacteria) and origins of replication. Obtained T0 plants were selected for single copy T-DNA insertion by PCR- and DNA sequencing- based identification of genomic tobacco nucleotide sequences flanking T-DNA-insertions. Transgenic T1 lines were obtained by self-pollination of T0 lines and verified for single copy T-DNA insertion by segregation analysis of transgene using kanamycin resistance as plant selection marker. T1 plants homozygous for transgene were identified by PCR analysis of transgene insertion site and T2 plant lines were generated by self-pollination of homozygous T1 plants. T3 plant lines were generated by self-pollination of T2 plant lines and were used for production of T4 plant lines also by self-pollination. T2, T3 and T4 plant lines were also analyzed for homozygousity by test for transgene segregation using kanamycin resistance selection marker. Homozygous T4 lines with a single copy T-DNA insertion are intended to be used in the proposed release, namely NtB538061T0#1T1#1T2#1T3#1T4. 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 the optimization of the thaumatin-2 production under field conditions in relation to parameters such as plant age at the time of induction, ethanol concentration and way of application, or treatment of the plant material after harvest. Geographical location of the site: CTAEX experimental field, Villafranco del Guadiana, Badajoz Size of the site (m2): 1256 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 tobacco line NtB538061T0#1(T1#1T2#1T3#1) has been subject of a previous deliberate release (B/ES/21/03). In this trial, no difference was observed between the transgenic line and conventional tobacco with respect to its effect on the environment or human health. Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: The genetic modification introduced in the transgenic line does not affect the survivability or confer any selective advantage to the plants on the field (as kanamycin resistance instead of e.g. herbicide resistance is used as plant selection marker). There is no risk to human health or the environment posed by these modified plants other than the risks associated to conventional tobacco plants.Regarding the potential for gene transfer to other plants, tobacco has no compatible wild species in Europe, therefore, there is no risk of gene transfer to other species. Cross-pollination of the modified plants with commercial tobacco crops poses a potential risk which is eliminated by the control measures described in the next section.Cultivation, management, and harvesting techniques are not supposed to negatively affect environment as they do not differ from those used during commercial production. Induction of the recombinant protein or, in the case of induction, a spray with no more than 15% (v/v) Ethanol solution, no effects are expected as this is not toxic and easily evaporated.The same cultivation, management and harvesting techniques will be used as during tobacco commercial production, so its environmental impact will not differ from that of this. The induction of the production of the recombinant protein will be carried out by applying a spray of an ethanol solution at a concentration no higher than 15% (v/v). The application of this treatment does not pose a risk due to the absence of toxicity and rapid evaporation. Brief description of any measures taken for the management of risks: For risk control the following measures will be taken: Seeds will be transported to the site of release in labelled and closed containers. To exclude dispersal, plants will be topped before the opening of the first flower buds (in the event that flower buds appear before the time of harvest) preventing pollen and seed production. Growth of axillary buds will be inhibited by treatment with contact agents. Any axillary bud escaping chemical control will be manually eliminated. There will be, therefore, no production of pollen or seeds. An isolation distance of at least 100 km will be maintained between the experimental plot and any other tobacco production areas. Additional deliberate releases of genetically modified tobacco could be carried out at CTAEX facilities; in such case, these trials would apply the same risk control measures described in this section. In addition, a distance of 100 m would be maintained between trials. This distance ensures that, even in the hypothetical case of pollen production, there is no risk of cross pollination. CTAEX personnel will regularly monitor the release site to detect any unexpected adverse environmental effects in order to notify authorities immediately in that case.Vegetative propagation will be prevented by crushing plant material remaining on the field after harvest using a disc harrow and burying it into the ground. The experimental plot will be left fallow and monitored in the year following the release for the detection and removal of potential volunteer plants. 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: Not known