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General information Notification Number B/ES/23/36 Member State to which the notification was sent Spain Date of acknowledgement from the Member State Competent Authority 16/01/2024 Title of the Project Development of a gene-edited tobacco line with high anatabine content. Proposed period of release: 01/03/2024 to 31/10/2024 Name of the Institute(s) or Company(ies) Asociación Empresarial de Investigación Centro Tecnológico Nacional Agroalimentario Extremadura - CTAEX, Ctra. Villafranco a Balboa, km 1.2, Badajoz Spain
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 mutations (deletions and insertions) in endogenous MPO genes. The mutations have been generated using the CRISPR / Cas9 system. These plants do not2 de 4contain any transgene. Genetic modification Type of genetic modification: Other; Brief description of the method used for the genetic modification: Tobacco transformation. Generation of transgenic T0 lines. The tobacco lines to be released have been obtained via CRISPR/Cas9. Tobacco cv K326 plants were transformed by Agrobacterium rhizogenes with a transformation vector that contain the transcriptional units of the DsRed and NptII proteins (selection markers), the transcriptional unit of the Cas9 protein, and the transcriptional units for the expression of gRNAs complementary to the target genes.Generation of T1 lines. T1 lines were obtained by self-pollination of T0 lines.Selection of T-DNA free T1 lines. The analysis of presence of T-DNA in the selected T1 lines was carried out by PCR amplification with two pairs of specific primers that amplify a fragment of the 35S promoter, a fragment of the Tnos terminator or a fragment of the RolD gene. Analysis of the presence of vector backbone fragments was performed by PCR with two pairs of specific primers that amplify two regions of the vector adjacent to the right and left borders of the T-DNA.Generation of T2 lines. T2 lines were obtained by self-pollination of T1 lines devoid of T-DNA.Generation of T3 lines. The T3 lines of the proposed release were obtained by self-pollination of a T2 line devoid of T-DNA.Identification of mutations present in the lines of the proposed release. Characterisation of the mutations present in the T1 and T2 lines was carried out by PCR amplification and sequencing of the gRNAs target regions. 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 proposed release is to determine the optimal field growth conditions of the edited line for use as an anatabine biofactory line. Geographical location of the site: CTAEX Experimental field, Villafranco del Guadiana, Badajoz. Size of the site (m2): 1550 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: Not applicable Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: Note especially if the introduced traits could directly or indirectly confer an increased selective advantage in natural environments; also explain any signifant expected environmental benefitsThe mutations present in the plants of the proposed release do not affect the survivability or confer any selective advantage to the plant. There is no risk to human health derive from the modified plants other than the risks associated to conventional tobacco plants.With respect to the potential of gene transfer to other plants, tobacco has no compatible wild species in Europe. There is a potential risk of cross-pollination of modified plants with commercial tobacco crops. The control measures described in the following section, however, eliminate the risk of transfer of genetic material.No negative environmental effects of cultivation, management and harvesting techniques are foreseen since they are not different from those used during commercial production Brief description of any measures taken for the management of risks: The following measures will be taken:• Seeds will be transported to the greenhouse in labelled closed containers• Plants will be topped before the opening of the first flower buds 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 100 km will be maintained between the experimental plot and any other tobacco production area.A second tobacco release trial is planned to be carried out at in the province of Badajoz by the same notifier. The distance between the two trials will be4 de 4approximately 40 km.• The release site will be regularly monitored (at least once a week) by personnel from CTAEX to register any unexpected adverse environmental effect. Should this happen, the competent authorities will be immediately notified.• Plant material left after harvest will be crushed with a disc harrow and buried in the ground, which will prevent vegetative propagation. The experimental plot will be monitored the year following the release allowing 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