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General information Notification Number B/IT/24/02 Member State to which the notification was sent Italy Date of acknowledgement from the Member State Competent Authority 13/05/2024 Title of the Project Field trials of TEA/NGT-1 tomato lines for resistance to broomrape (Orobanche spp.) Proposed period of release: 13/05/2024 to 31/12/2027 Name of the Institute(s) or Company(ies) Council for Agricultural Research and Economics - Research centre for Vegetable and Ornamental Crops, Via Cavalleggeri, 51 Pontecagnano (Salerno) ITALY
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
tomato	solanaceae	lycopersicon	lycopersicon esculentum	esculentum	Ailsa Craig

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: The tomato genes D27 (Solyc09g065750) and CCD7 (Solyc01g090660) encode for the first two genes of the strigolactones (SLs) biosynthesis pathway. The tomato plants d27-KO1 and ccd7-KO1 derive from sited directed mutagenesis mediated by CRISPR-Cas9 with the aim of reducing the release of SL in root exudates, thus preventing the germination of parasitic plant seeds (Phelipanche ramosa, Orobanche, broomrape) already present in the soil.The lines d27-KO1 and ccd7-KO1 have been already analyzed in laboratory for their ability to induce germination of P. ramosa seeds. Moreover morphological, productive and qualitative traits have been also analyzed. The lines d27-KO1 and ccd7-KO1 show a reduced germination activity vs. P. ramosa seeds. The line ccd7-KO1 shows increased branching and hairy root development and fruit weight reduction, whereas the line d27-KO1 does not show significant alterations in yield and quality. Genetic modification Type of genetic modification: Insertion;Deletion; In case of insertion of genetic material, give the source and intended function of each constituent fragment of the region to be inserted: The genetic variation introduced in the gene D27 consists of an insertion of n.1 bp (+A) in the 5’UTR (untranslated region) that determines a frame shift in the coding sequence with the consequent production of a non-functional protein In case of deletion of genetic material, give information on the function of the deleted sequences: The genetic variation introduced in both the CCD7 and D27 genes consists of a deletion of n. 1 bp (-T) in the exon 1 and exon 2, respectively, that determines a frame shift in the coding sequences of the two genes, with the consequent production of a non-functional proteins. Brief description of the method used for the genetic modification: The lines d27-KO1 and ccd7-KO1 have been produced by classical Agrobacterium tumefaciens transformation of tomato and further independent segregation of the entire T-DNA constituted by Cas9, NPTII, and the single guide RNAs specific for the D27 and CCD7 genes. The d27-KO1 and ccd7-KO1 are T-DNA (transgene) free and biallelic homozygous for the specific mutation in the target gene. Off-target analysis excluded alteration in other possible sites. 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: Test in a field experiment the resistance of d27-KO1 and ccd7-KO1 to Phelipanche ramosa and evaluate the performance of such lines as rootstock for a commercial hybrid variety. Geographical location of the site: Azienda Agraria Sperimentale Stuard S.r.l. Strada Madonna dell’Aiuto, 7/a 43126 – Parma. Coordinates: 44°48'19.3"N 10°16'23.0"E https://maps.app.goo.gl/9XVbt4VzFdocCumw8 Size of the site (m2): 500 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 benefitsConventional and TEA/NGT-1 tomato plants have specific cultural needs that make them not suitable for growing in a wild environment becoming weeds.The mechanism of resistance introduced in the d27-KO1 and ccd7-KO1 prevents the germination of parasitic plant seeds already present in the soil but does not prevent infection if nearby conventional plants allow germinations. Moreover, the genetic modifications introduced in the D27 and CCD7 genes (i.e. d27-KO1 and ccd7-KO1, respectively) confer a recessive resistance in case of accidental outcross with conventional tomato plants. Tomato wild relatives are almost absent in Italy. For the weed Solanum nigrum (black nightshade) the risk of introgression from cultivated tomato is negligible.The lines d27-KO1 and ccd7-KO1 do not contain transgenes, so that it is unlikely that they may have any effect on the natural phenomena of horizontal gene transfer.Concerning non-target organisms, the genes D27 and CCD7 are already present in the tomato gene pool, so that adverse effects against human or animals due to accidental fruit consumption are not likely. The lack of release of strigolactones in root exudates could reduce the root colonization by arbuscular mycorrhizal fungi (AMF). However, this particular interaction will only be relevant for tomato itself rather than for the AMFs that can likely interact with other species Brief description of any measures taken for the management of risks: Tomato is a self-fertilizing species and the TEA/NGT-1 plants, as potential pollen donors, represent about 33% of the entire plants of the parcel field. Moreover, the entire experimental field is surrounded by two buffer zones constituted by conventional plants that will be monitored to capture rare events of crossing and dispersion of pollen. The experimental area is protected by fence and surveyed. Harvesting of tomato fruits will be carried out by hands. Crop residues will be destroyed, and the field excluded by further cultivations for 18 months since the ending of the experiment. A field register will be compiled, concerning all the experimental phases. In case of emergency, all the plants can be easily pulled out by hands and/or treated with a systemic herbicide. Competent authorities will be notified about any problems that may arise during the experiment. 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: Not known