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General information Notification Number B/S/21/28 Member State to which the notification was sent Spain Date of acknowledgement from the Member State Competent Authority 21/01/2022 Title of the Project Molecular approaches to increase salinity and drought tolerance in broccoli - R17MOLBROC  Proposed period of release: 01/04/2022 to 28/02/2025 Name of the Institute(s) or Company(ies) Asociación Hortofrutícola Grupo Lucas
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
broccoli	brassicaceae	brassica	brassica oleracea	botrytis

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: It is a gene editing technique in broccoli using the CRISPR/Cas9 system. The main advantage of this system is that, unlike transgenesis, we can make a genetic modification to the plant's DNA (removing or inserting a few nucleotides from the DNA sequence, known as an indel), without incorporating foreign DNA.A list of the genes chosen for knockout is shown below:- ABI1. It codes for a type IIC phosphatase, which is a negative regulator of the abscisic acid signalling pathway. Its knockout gives drought tolerance and increases sensitivity to abscisic acid.- HAB1. Type IIC phosphatase which, like ABI1, is a negative regulator of the abscisic acid signalling pathway and its knockout makes the plant more tolerant to drought and more sensitive to ABA.- GSTU17. Glutathione S-transferase U17. Enzyme whose knockout gives tolerance to drought and salinity.- RPT2. Regulator of the potassium transporter KAT1 which, according to data from our laboratory, generates tolerance to drought.However, the phenotypic information described is based on information obtained from studies in the model plant Arabidopsis thaliana. Therefore, in previous phases we have studied the orthologous genes in broccoli and their expression levels. The broccoli genes that will not be expressed in the plants to be tested are:- ABI1 (GenBank accession, LOC106307446) has only one orthologue in the broccoli genome.- HAB1 (GenBank accession, LOC106312703) also has a single orthologue in the broccoli genome.- GSTU17 has two tandem orthologues (GenBank accessions, LOC106295499 and LOC106295500); therefore, the RNA guides were designed to complement in regions that are identical in the two orthologues. This allows gene editing to occur in both genes Genetic modification Type of genetic modification: Other; Other

Genetic edition using CRISPR/Cas Brief description of the method used for the genetic modification: It is a gene editing technique in broccoli using the CRISPR/Cas9 system. The main advantage of this system is that, unlike transgenesis, we can make a genetic modification to the plant's DNA (eliminating or inserting a few nucleotides from the DNA sequence, known as an indel), without incorporating foreign DNA. To do this, we will use the GoldenBraid cloning system, which, using two IIS-type restriction enzymes (BsaI and BsmBI) and the vectors mentioned below, allows us to obtain the necessary constructs to carry out this modification in the broccoligenome. The protocol followed to construct each vector was:The CRISPR/Cas9 protocol requires a scaffold and a tRNA that allows the RNA to self-cut. Between these two modules in the vector, a guide is inserted, which will indicate in which specific part of the genome the change is to take place. The sequence of the guide is obtained from two synthetic oligonucleotides designed in such a way that they can be ringed together. For this we will use the pUPD2 vector as an input vector. The three modules are inserted into the cleavage site of the BsmBI enzyme.Subsequently, a level 1 part (one for each target gene) will be inserted into the BsaI site with the previously mentioned level 0 parts plus a level 0 part corresponding to the RNA polymerase 3 promoter (U6-26) using the alpha2 vector as the target vector. After that, the final vector is constructed by BsmBI with the alpha2 vector with the guides and the promoter and an alpha vector with Cas9 in an omega 1 input vector.The description of the different vectors and the methodology used can be found in "Design and construction of multigenic constructs for plant biotechnology using the GoldenBraid cloning strategy" Alejandro Sarrion-Perdigones et al., Methods Mol Biol . 2014;1116:133-51.With this final construct, it is transformed into Agrobacterium tumefaciens. The culture of this bacterium, containing the plasmid with our construct, will be used to transform cotyledon explants, which will be selected thanks to the kanamycin resistance gene NptII incorporated in the final construct (to be confirmed by PCR). These initial explants (To) will be grown in the greenhouse and self-fertilised to obtain T1 plants. We will select those in which recombination has occurred and therefore the plant presents the two edited alleles in which the protein is not produced due to the elimination of a small sequence. On the other hand, in the plants that go to the field, the allele that incorporated the final CRISPR/Cas9 construction will not be present. Therefore, we would obtain a plant with a small modification that changes the reading pattern of the target protein without having incorporated foreign genetic material. 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 main objective of this project is to obtain broccoli lines with greater resistance to salinity and drought. With these field trials, to evaluate whether the edited material obtained using CRISPR/Cas9 technology is more resistant to salinity and drought, without repercussions on other commercial qualities. More specifically, to evaluate its agronomic suitability Geographical location of the site: The release will take place in a rented property of the company Grupo Lucas in the municipality of Santomera, province of Murcia. The plot has the cadastral reference Polígono 8 Parcela 464 and has a total size of 13,107 m2. Size of the site (m2): 13107 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: We are not aware of any studies in Spain carried out with genetically modified broccoli, nor any studies that aim to evaluate these genes. However, a search of European databases indicates that there is currently an ongoing trial with broccoli in Great Britain with notification number B/GB/19/52/01 and whose data can be consulted at the following link: https://gmoinfo.jrc.ec.europa.eu/gmp_report.aspx?CurNot=B/GB/19/52/01 Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: Likelihood that the GMHP will become more persistent than the recipient or parentalplants in agricultural habitats or more invasive in natural habitats.Minimal, there is no chance that the plant will ever be able to reproduce, survive and therefore persist in either the agricultural or natural environment.2. Any advantages or disadvantages gained by the GMHP.We expect the lines to be more resistant to drought and salinity than commercial cultivars but not so than wild species.3. Potential for gene transfer to the same or other sexually compatible plant species under GMHP planting conditions and any selective advantage or disadvantage gained by these plant species.Gene transfer with other plant material is not feasible as the plants are harvested prior to flowering, which prevents pollen/seed formation and avoids the risk of dispersal. On the other hand, the levels of resistance expected to be achieved are below the resistance levels of wild plants.4. Potential immediate and/or delayed environmental impact resulting from direct and indirect interactions between GMHP and target organisms such as predators, parasitoids and pathogens (if any).No potential impact is apparent.5. Potential immediate and/or delayed impact on the environment resulting from direct and indirect interactions between the GMHP and non-target organisms (also taking into account organisms interacting with target organisms), including impact on population levels of competitors, herbivores, symbionts (if any), parasites and pathogens. Brief description of any measures taken for the management of risks: Not applicable 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