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General information Notification Number B/RO/19/01 Member State to which the notification was sent Romania Date of acknowledgement from the Member State Competent Authority 25/02/2019 Title of the Project Field trial of some HoneySweet hybrids Proposed period of release: 01/01/2020 to 15/12/2029 Name of the Institute(s) or Company(ies) Fruit Research and Development Station Bistrita, Drumul Dumitrei Nou Sreeet, no. 3, Bistrita, Romania
Is the same GMPt been notified elsewhere by the same notifier? No Has the same GMPt been notified elsewhere by the same notifier? Yes If yes, notification number(s): B/RO/07/04; B/RO/11/01; Genetically modified plant Complete name of the recipient or parental plant(s):

Common Name	Family Name	Genus	Species	Subspecies	Cultivar/breeding line
plum	rosaceae	prunus	prunus domestica		Honey Sweet hybrids

Description of the traits and characteristics which have been introduced or modified, including marker genes and previous modifications: -the Plum pox virus coat protein gene (PPV-CP);
-the neomycin phosphotransferase II gene (nptII) and
-the beta-glucuronidase gene (GUS);
The insert also contains a promoter (35S) and a terminator (NOS). 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 components of the inserted foreign DNA and their functions are the following:
a) coat protein (CP) gene from PPV genome, D strain. It was engineered for targeting the pathogen derived resistance approach. The mechanism of resistance in HoneySweet-resistant plum, and of the selected hybrids - derived from it carrying the HoneySweet transgene insert, is based on post-transcriptional gene silencing (PTGS) in which both transgene mRNA and homologous viral RNA are degraded (Scorza et al., 2001 - Transgenic Research; Hily et al., 2004 - Transgenic Research; Hily et al., 2005 - Molecular Plant – Microbe Interactions; Kundu et al., 2008 - Virus Genes);
b) npt II gene from Escherichia coli confers resistance to kanamycin. It was included as a selection marker in the transformation process.
C) beta-glucuronidase gene from Escherichia coli is a chromogenic marker.
D) 35S promoter from Cauliflower mosaic virus regulates gene expression.
E) NOS terminator from nopaline synthase gene, isolated from Agrobacterium tumefaciens, terminates the transcription of the PPV-CP transgene. Brief description of the method used for the genetic modification: Plum hypocotyl slices were transformed with the coat protein (CP) gene of Plum pox virus (PPV-CP) following cocultivation with Agrobacterium tumefaciens containing the plasmid pGA482GG/PPV-CP-33. This binary vector carries the PPV-CP gene construct, as well as the chimeric neomycin phosphotransferase and beta-glucuronidase genes (Scorza et al., 1994 - Plant Cell Reports). The C5 clone, called HoneySweet, proved to be resistant to PPV (Malinovki et al., 2006 – Plant Disease). Subsequently, it was used in conventional breeding to obtain male sterile hybrids resistant to PPV. 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 field trial of HoneySweet male sterile plum hybrids is to gain information relating to the agronomical and phenotype performance and compatibility of this event under PPV endemic area and geo-climatic conditions of Romania. The study will be performed in the context of Plum pox virus-free plum crops and environmental protection. The obtaining of a limited number of new hybrids resistant to PPV, derived from HoneySweet transgenic plum, is a secondary goal. Geographical location of the site: Experimental plot at Fruit Research and Development Station Bistrita (Statiunea de Cercetare-Dezvoltare pentru Pomicultura Bistrita), Bistrita-Nasaud county, Bistrita Hilly, Romania. Size of the site (m2): The size of the site plot proposed is 3500 sq.m 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: HoneySweet transgenic plum was tested in experimental orchards under conditions of high PPV inoculum pressure in Spain (notification numbers B/ES/96/16, B/ES/05/14), Poland (ref DOPgmo 4301/02-4/2002), Czech Republic (B/CZ/06/03) and Romania (B/RO/11/01). HoneySweet was highly resistant to graft-inoculation with PPV and no tree became infected via aphid-mediated transmission of PPV in the endemic areas (Malinowski et al., 2006 - Plant diseases; Jarosova et al., 2010 – Julius -Kuhn-Archiv, Zagrai et al., 2011 – Acta Horticulturae.). These data revealed the durability of resistance to PPV of HoneySweet plum trees. The engineered resistance to PPV in HoneySweet was also stable in the presence of heterologous viruses (Zagrai et. Al., 2008 – Journal of Plant Pathology; Polak et. Al., 2017 – Journal of Integrative Agriculture).
Because the transgenic plums carry the PPV CP gene, the environmental safety issues have been expressed on potential hazards concerning the emergence of PPV variants. The potential impact of transgenic plums expressing PPV CP gene construct on the diversity and dynamics of PPV populations was assessed in the frame of a research program sponsored by the European Commission (TRANSVIR QLK3-CT-2002-02140). Across field trials conducted in different locations (Romania and Spain) and environments (continental and Mediterranean), transgenic plums had no detectable effect on the emergence of recombinant PPV species over ten years. Also, these transgenic plums had a neutral impact on non-target organisms over extended time (Capote et al., 2008 - Transgenic Research, Zagrai et al., 2011 – Journal of Plant Pathology). Environmental Impact and Risk Management Summary of the potential environmental impact from the release of the GMPts: The release of GM-European plums carrying the coat protein gene of PPV is considered not to have any adverse effect on human, animals or plants. PPV resistance will not impart weediness to this plum. There is not possibility of an increase of the transgenic trees population nor the GM-trees have selective advantage compared to conventional ones, since they became resistant to PPV. The transgenic plum trees were exhaustively studied along 20 years period. Risk assessment studies demonstrated that the release of this GMO does not constitute an environmental risk. It was concluded that HoneySweet transgenic plum and any progeny derived from hybrid crosses with non-transformed plum varieties will be no more of a plant pest than plum varieties in traditional breeding (USDA/APHIS -Federal Register Doc. E7-13649, July 12, 2007). In addition, there is a history of another GMO carrying a similar insert (transgenic papaya exhibiting „pathogen-derived resistance”) that attest the safe consumption and no adverse effect on environment. The safety of HoneySweet is indicated by its deregulation in the U.S. (Scorza et al., 2013- Journal of Plant Biotechnology).
An Environmental Risk Assessment study has been completed and submitted with this application. In summary, no immediate or delayed adverse effects as a result of the direct and indirect interaction of the transgenic HoneySweet hybrids with the environment have been identified, especially since they are male sterile
The reducing pesticide use is among human targets to contribute to the conservation of the environment. Due to its resistance to PPV, HoneySweet transgenic plum and the hybrids derived from it can contribute to the decreasing of the number of insecticide treatments against aphid vectors and thus to protect the environment. Brief description of any measures taken for the management of risks: • The experimental field trials are focused on male sterile HoneySweet hybrids and therefore, isolation to minimize the pollen dispersal is not applicable. However, the experimental plot will be surrounded by non-transgenic border trees: apple, pear and cherry (which are sexually incompatible with plums);
• Planting and harvesting will be conducted by SCDP Bistrita personnel trained on precaution measures;
• Carrying out regular visits to inspect field trail site, making observations, samples collecting and verifying that adequate measures are applied for preventing pests and diseases;
• No fruit products resulting from the field trail will be used as food or feed;
• Potential plum seedlings will be monitored and removed.
• All GMP debris will be removed from the test area and destroy. At the end of the experiment, the plants will be herbicide-killed and then the trees eliminated by bulldozing into large piles and burning. Summary of foreseen field trial studies focused to gain new data on environmental and human health impact from the release: The use of male sterile transgenic hybrids will contribute to the avoiding of any controversy on coexistence issues. Final report - European Commission administrative Information Consent given by the Member State Competent Authority: Yes
05/29/2019
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