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General information Notification Number B/CZ/22/01 Member State to which the notification was sent Czech Republic Date of acknowledgement from the Member State Competent Authority 11/01/2022 Title of the Project Optimization of purification of LL-37 peptide producedby transgenic barley - extension of approved application see SNIF B/CZ/18/01 Proposed period of release: 01/04/2022 to 31/12/2029 Name of the Institute(s) or Company(ies) Usovsko a.s.
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
barley	poaceae	hordeum	hordeum vulgare	vulgare

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: Modification bHOR:DAMP4LL-37It contains peptide LL-37, hpt, B1 hordein promoter (bHOR) from barley and NOSterminator, KDEL, ZmCKX1 N-terminal signal peptide, KS – Kozak sequence,10xHis, DAMP4, linker, DPSModification AsGLO:DAMP4_LL-37It contains LL-37, hpt, NOS, Globuline1 promoter (AsGlo) from Avena sativa, KDEL,ZmCKX1 N-terminal signal peptide, At1G 5´-UTR, 10xHis, DAMP4, linker,DPS 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: Modification bHOR:DAMP4_LL-37Peptide LL-37 - human gene coding Cathelicidin, commercially synthesized.hpt (YP_006952299), the hygromycin phosphotransferase gene derived fromEscherichia coli. Source - binary vectors pBRACT209 and pBRACT214B1 hordein promoter (bHOR) - endosperm specific promoter from barley, used forexpression of LL-37 in barley endosperm. Source - commercially synthesized.ZmCKX1sp - the N-terminal signal of peptide cytokinin dehydrogenase 1 from Zeamays L., responsible for the translocation of the peptide through the endoplasmicreticulum. Source - commercially synthesized.NOS terminator - nopaline synthase terminator is from Agrobacteria tumefaciens. Partof a binary vector.35S promoter of cauliflower mosaic virus - hpt expression is controlled by the 35Spromoter and the NOS terminator (A. tumefaciens nopaline synthase terminator).DAMP4 - synthetic sequence, commonly used for the preparation of fusion proteinsand their purification. It is an artificial biosurfactant protein that increases thethermal stability of LL-37.10xHis - A polyhistidine-tag is for the purification of LL-37 peptide. Source -commercially synthesized.DPS - acid cleavage site; to facilitate purification of the fusion proteinKozak sequence (KS) - sequence for initiation of translationLinker (GGGGS)2, which affects the properties of the fusion protein, such asexpression level, solubility and its biological functionsModification AsGLO:DAMP4_LL-37It contains, as in previous modifications, LL-37, hpt, NOS, KDEL, ZmCKX1 Nterminalsignal peptide, At1G 5´-UTR, 10xHis, DAMP4, linker, DPSGlobuline1 promoter (AsGlo) from Avena sativa - endosperm specific promoter usedfor expression of LL-37 in barley endosperm. Source - commerciallysynthesized.At1G 5´-UTR - from A. thaliana to initiate transcription Brief description of the method used for the genetic modification: Transformation of barley immature grains (Golden promise) by Agrobacteriumtumefaciens strain AGL1. 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 introduction transgenic plants into the environment is tostudy the stable production of LL-37 peptide in field conditions, assess theyield of the recombinant product, and to optimize the purification of thispeptide from mature transgenic barley grains and also from immature grains(in milk-wax maturity). Geographical location of the site: ÚSOVSKO a. s., ÚSOVSKO AGRO s.r.o. 33, 789 73 Klopina, CzechRepublic. Cadastral territory Mohelnice, plot 2502/4 Size of the site (m2): 14919 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: Transgenic plants producing the LL-37 peptide (T1-T3 generation) were grownin closed phytotron and in the greenhouses of the Palacký University(CATRIN-CRH) in Olomouc for closed GMO plant management. No selectiveadvantages or disadvantages, no interactions with control plants or other targetorganisms, modified spring barley does not pose any risks to the environment,nor to any risks to human health or animal health were observed for selectedmodifications (bHOR:DAMP4_LL-37 and AsGLO:DAMP4_LL- 37). Brief description of any measures taken for the management of risks: All work on the experimental field and manipulation with GM plant materialwill be done by trained workers of Úsovsko a.s. All handling of GM with plantmaterial will be monitored and recorded. All personnel will be instructed aboutthe emergency plan and the operating rules of the experimental field prior tosowing.The monitoring plan will be implemented during and after the experiments, asdescribed in the monitoring plan. Monitoring will take place on the experimentalplot and in its immediate vicinity. Monitoring will also be carried out at the siteof an accident and in its immediate vicinity. 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: Yes
04/07/2022