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
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
Selective advantage and potential for increased weediness or
- 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
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
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
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
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.