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General information Notification Number B/ES/23/34 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 thaumatin-2-producing tobacco line. Proposed period of release: 15/02/2024 to 31/12/2024 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 a transgene insertion, namely a T-DNA insertion for seed-specific expression of the sweet tasting protein thaumatin-2 from Thaumatococcus daniellii. The plants also contain the phosphinothricin resistance gene, used as a selection marker in the genetic transformation process. 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 T-DNA region inserted to the genome is composed of right and left border of Agrobacterium tumefaciens T-DNA and 2 expression cassettes for: 1) constitutive expression of phosphinothricine resistance transgenic plant selection marker, 2) seed-specific expression of thaumatin-2 protein.Expression cassette 1) consists of Agrobacterium tumefaciens nopaline synthase gene promoter, Streptomyces hygroscopicus Phosphinothricin N-acetyltransferase gene and terminator of Agrobacterium tumefaciens nopaline synthase gene. Expression cassette 2) consists of Phaseolus vulgaris beta-phaseolin gene promoter, apoplast targeting presequence of Oryza sativa fused to sequence encoding thaumatin-2 mature peptide of Thaumatococcus daniellii (UniProtKB/Swiss-Prot: P02884.1), the 35S terminator of cauliflower mosaic virus and terminator of Agrobacterium tumefaciens octopine synthase gene. 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 pNMD52711 following a slightly modified standard protocol5. Plasmid pNMD52711 bears the T-DNA described above, a neomycin phosphotransferase expression cassette (as kanamycin selection marker in bacteria) and origins of replication. The T0 plants obtained were analysed by PCR and sequencing to identify the genomic sequences flanking the insertion point and to determine the T-DNA copy number, selecting plants that could be single copy. T1 lines were obtained by self-pollination of selected T0 lines, and T-DNA copy number was assessed by phosphinothricin resistance segregation analysis. These plants were analysed by PCR and T1 plants carrying a single copy of the transgene in homozygosity or plants with more than one copy of the T-DNA of which at least one was in homozygosity were identified. T2 lines were obtained by self-pollination of selected T1 plants. T3 lines were obtained by self-pollination of T2 plants. The plants in this test are derived from a line with multiple copies of the T-DNA of which one is in homozygosity. 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 develop a thaumatin-2 production line for use in molecular farming and, more specifically, to optimise the harvesting and the scale-up of the extraction and purification processes of the recombinant protein, for which cultivation on a pre-industrial scale is essential.Additionally, the release aims to collect data on the extent of dispersal of tobacco seeds under the tested trial conditions and their inability to germinate in the field in a feral form. This information will be used to determine, based on empirical data, the biosafety distance required in future trials to prevent the dispersal of genetically modified tobacco plants via their seeds. Geographical location of the site: Municipality of Mérida Size of the site (m2): 60 ha 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 plants subject to this release are a T4 generation derived (through two generations of self-pollination) from the T2 line NtB10-14/13. Plants of the previous generation (T3 generation) were the subject of voluntary release B/ES/22/28 and did not show any effect on the environment or human-health that differentiate them from conventional tobacco. 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 genetic modification introduced in the transgenic lines does not affect the survivability. The phosphinothricin resistance used as a selection marker might confer a selective advantage to the plants on the field in case of application of the herbicide. During the development of the field trial the herbicide will not be used. 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. Brief description of any measures taken for the management of risks: For risk control the following measures will be taken:• The transfer of seeds from the laboratory to the greenhouse will be carried out in properly labelled and sealed containers.• The trial plot will be surrounded with 2 guard rows of wildtype tobacco plants serving as a trap for transgenic pollen.• A minimum distance of 60 meters will be maintained between the transgenic plants and the edge of the release area.• An isolation distance of 100 km will be maintained between the release site and other commercially cultivated tobacco plantations. A second tobacco plant release trial is planned in the province of Badajoz by the same notifier. The distance between both trials will be approximately 40 km.• The trial will be regularly monitored to record any unexpected adverse effects on the environment, which would be immediately reported to the competent authority.• To prevent the possible regrowth of plant residues remaining in the field after harvest, disc harrowing passes will be conducted to crush the remains of roots and stems, burying them in the soil.• Additionally, the plot will be monitored during the following year after the release to control and, if necessary, eliminate potential regrowth in the release area. The appearance of tobacco plants in the non-cultivated area and their distance from the release site will be documented. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: N/A Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
05/28/2024