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Risk Assessment
Record information and status
Record ID
Date of creation
2017-05-19 12:41 UTC (marco.gielkens@rivm.nl)
Date of last update
2020-04-08 10:56 UTC (marco.gielkens@rivm.nl)
Date of publication
2020-04-08 10:56 UTC (marco.gielkens@rivm.nl)

General Information
  • Netherlands
Title of risk assessment
Application to import of cut flowers of colour modified carnation variety SHD-27531-4 (C/NL/13/01)
Date of the risk assessment
Competent National Authority(ies) responsible for the risk assessment
Ministry for Infrastructure and Water Management
NL-2500 EX
The Hague
The Netherlands, P.O.Box 20901
Phone:+31 6 25684418
Url:Rijksoverheid NL Biotechnologie (Dutch),General governmental webpage biotechnology (English)
Contact details of the main responsible risk assessor
Dr. Marco Gielkens
National Focal Point BCH
GMO Office
Ministry for the Environment
PO Box 1
Netherlands, 3720 BA
Phone:+31 30 274 4179
Risk assessment details
Living modified organism
SHD-27531-4 - Moontea™ carnation
Dr Yukihisa Katsumoto Changes in quality and/or metabolite content - Pigmentation / Coloration Resistance to herbicides - Chlorsulfuron, Sulfonylurea
Show detection method(s)
Scope of the risk assessment
  • Import of cut flowers for ornamental use
Methodology and points to consider
Potential adverse effects identified in the risk assessment
The following adverse effects have been taken into account, based on the expression of the Dfr and F'3'5'H genes and the SuRB gene:
- Selective advantage and potential for increased weediness or persistence
- Effects on non-target organisms
- Effects on the soil organisms
- Toxicity and allergenicity
- Change in agricultural practice
Likelihood that the potential adverse effects will be realized
Molecular characterization
- Inserts
Genomic DNA isolated from the transgenic line SHD-27531-4 and the non-transformed line Cinderella were compared using Southern analysis and sequencing to identify integrated sequences and copy number of the introduced genes. Southern analysis with EcoRI and BglII digested DNA indicates that a single integration of the T-DNA has occurred at a single locus in the carnation nuclear genome. A schematic of the arrangement of the inserted T-DNA is provided in Attachment A6. The sequence of the locus, including flanking regions, is provided in Attachment A7.
- Flanking sequences
The flanking sequences of both ends of the locus are sequenced (150 bp). The flanking sequences were analysed for putative open reading frames (ORFs). All ORFs were included (no minimal size, from stop to stop codon). Eleven new ORFs in the junctions insert/plant were identified. None of the ORFs showed biologically significant homology to known toxins or allergens.
- Absence of tetracycline resistance gene (tetA)
Southern analysis was conducted to demonstrate the absence of backbone vector sequences. The results prove the absence of any backbone vector sequences, including tetA sequences encoding a resistance gene to the antibiotic tetracycline.  In addition, PCR analysis with primers directed against the tetA gene confirmed the absence of this gene.
- Gene expression
Northern analysis conducted on RNA isolated from petal leaves showed that all three newly introduced genes are expressed in SHD-27531-4, whereas no signals could be detected in parental line Cinderella.
Except for flowers, delphinine production has not been observed in other tissues of the transgenic plant, such as stems, nodes, leaves and roots. Due to the petal specific promoter (CHS), production of delphinine is confined to the petals. Moreover, the biochemical pathway leading to anthocyanin biosynthesis is induced to coincide with flower development.
The concentration of delphinine was determined in flower samples of line SHD-27531-4 and of the non-transformed recipient strain by TLC and HPLC. The delphinine concentration amounts 1.18 mg/g fresh weight petal. Due to the genetic modification also cyanidine is produced in petal leaves with a concentration of 0.51 mg/g fresh weight.

Selective advantage and potential for increased weediness or persistence
- f3'5'h and dfr genes
There is no reason to assume that carnation plants from spilled or discarded carnation exhibit an increased potential to survive, as a result of the modified colour of flowers resulting from expression of the f3'5'h and dfr genes. The gene products of f3'5'h and dfr are involved in the biosynthesis of the pigment delphinine in petals. Accumulation of these pigments in petals results in a purple/red flower colour and does not alter the biological characteristics of carnation. Therefore it is highly unlikely that the genetically modified carnation line SHD-27531-4 exhibits a selective advantage over non-modified carnation, based on the presence of the f3'5'h and dfr genes.
- suRB gene
Carnation is not considered to be a weed in Europe. Carnation plants resistant to sulfonylurea herbicides can only exhibit a selective advantage after application of such herbicide. However, sulfonylurea herbicides are not designed/registered for use with ornamentals. Sulfonylureas are not effective against grasses, the major weeds of concern in the flower industry. The notifier prohibits use of sulfonylureas on their crops by their contract growers. The herbicide is not generally used for wide scale control of weeds outside agriculture.

Effects on non-target organisms
The environment in which the imported flowers will be used, the relatively small number of flowers imported, their dispersal across Europe, and the short longevity of the flowers are all factors that preclude any direct or indirect interaction between the genetically modified carnation and non-target organism.
Therefore it is highly unlikely that non-target organisms will be affected as a result of import of cut flowers of line SHD-27531-4.

Effects on the soil ecosystem
Because the products are to be imported as cut flowers, no cultivation takes place. As the genetically modified carnation plants have similar production requirements as other carnations, any impact is no different to that of conventional carnation. Flowers imported to the EU will eventually be discarded in domestic and commercial waste, but the volume of the flowers and the fact that the products will be widely dispersed mean the organic mass is negligible. In addition, the compounds responsible for the colouration of the flowers are natural compounds which are widely present in the environment.
Therefore it is highly unlikely that any adverse effect on the soil ecosystem will occur as a result of imported or discarded genetically modified carnation.

Toxicity and allergenicity
- Delphinine
Carnation has been used safely by humans for ornamental purposes for centuries. The modification in line SHD-27531-4 (production of delphinine) is novel for carnation, but there are many flowers and other ornamental species that produce delphinine, such as Gentiana, Petunia, Centaurea and Delphinium. Delphinine is also present in many common foods, such as red grapes, black currants, eggplant and blueberry. Toxicity studies of delphinidins indicate very low levels of toxicity. Humans are commonly exposed to and ingest delphinidins in fruits and vegetables at similar or greater concentrations than are found in genetically modified carnation, without adverse effects.
- f3'5'h and dfr proteins
Possible negative effects on human and animal health as a result of incidental consumption of petal leaves of carnation, for example as garnishing for food, were considered. The proteins for modified flower colour expressed in genetically modified carnation (f3'5'h and dfr) are similar to those found in purple-coloured fruits and vegetables that are commonly consumed, and in ornamental flowers. No significant homology was found between the inserted genes and known toxins or allergens.
Reports of allergenicity to carnations are rare and there are no reports of allergenicity to genetically modified carnation.
- SuRB protein
ALS enzymes are widely distributed among bacteria, yeast and higher plants. The suRB gene codes for an alternative form of the acetolacetate synthase enzyme. This enzyme is not a known toxin or allergen and related enzymes are expressed in a variety of edible plants (e.g. soybean and rice).
No homology was found between the suRB gene and known toxins or allergens.

Based on the nature of the inserted genes and the history of safe use of similar genetically modified carnation lines, it is concluded that it is highly unlikely that the genetically modification in carnation line SHD-27531-4 will cause an adverse effect on the human health with respect to incidental human consumption or allergenicity, as compared to conventionally bred carnation.

Change in agricultural practice
Since the notification covers only import, distribution and retailing of the genetically modified carnation, possible adverse environmental effects by changes in agricultural practice are not considered of importance for the risk analysis.
Possible consequences:
Carnation line SHD-57531-4 is unlikely to have adverse effects on human and animal health or the environment.
Estimation of the overall risk
The overall risk is negligible.
Not applicable.
Need(s) for further information on specific issues of concern
Not applicable.
Receiving environment(s) considered
Not applicable.
LMO detection and identification methods proposed
EU detection methods validated by the EU Reference Laboratory for GM Food Feed (EU-RL GMFF).

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