Genomic DNA isolated from the transgenic line FLO-40685-2 and the
non-transformed parental line Cream Cinderella were compared using
Southern analysis and sequencing to identify integrated sequences
and copy number of the introduced genes. Southern analysis with
EcoRV and NdeI digested DNA indicated that integration of the T-DNA
has occurred at four loci in the carnation nuclear genome. Sequence
data confirmed the integration of two copies of the LB, two copies
of the SurB gene, four copies of the f3'5'h gene, two copies of the
dfr gene and five copies of the RB.
The flanking sequences of the four inserts are sequenced (150 bp).
The flanking sequences and inserts were analysed for putative open
reading frames (ORFs). All ORFs were included (no minimal size,
from stop to stop codon). A total of 64 new ORFs were
identified in the insert/plant junctions and a total of 2996 ORFs
were identified in the four inserts. None of the ORFs showed
biologically significant homology to known toxins or
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 the tetracyclin resistance
gene (tetA). PCR analysis confirmed the absence of this gene.
Northern analysis conducted on RNA isolated from petal leaves
showed that all three newly introduced genes are expressed in
FLO-40685-2, whereas no signals could be detected in parental line
Except for flowers, delphinidin 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
delphinidin is confined to the petals. Moreover, the biochemical
pathway leading to anthocyanin biosynthesis is induced to coincide
with flower development.
The concentration of delphinidin was determined in flower samples
of line FLO-40685-2 and of the non-transformed recipient strain by
TLC and HPLC. The delphinidin concentration amounts 1.79 mg/g fresh
weight petal. Due to the genetic modification also cyanidin is
produced in petal leaves with a concentration of 0.02 mg/g fresh
Selective advantage and potential for increased weediness or
f3'5'h and dfr genes
Carnation is not considered to be a weed in Europe. There is no
reason to assume that carnation exhibits 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
delphinidin in petals. Accumulation of these pigments in petals
results in a dark purple flowers and does not alter the biological
characteristics of carnation. Therefore it is highly unlikely that
the genetically modified carnation line FLO-40685-1 exhibits a
selective advantage over non-modified carnation, based on the
presence of the f3'5'h and dfr genes.
Carnation plants tolerant to sulfonylurea herbicides can only
exhibit a selective advantage after application of such herbicides.
However, sulfonylurea herbicides are not designed nor 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.
It is therefore highly unlikely that the carnation line FLO-40685-2
exhibits a selective advantage over non-modified carnation, based
on the presence of the suRB gene.
Effects on non-target organisms
There is no reason to assume that the new traits introduced
(modified flower colour, tolerance towards to sulfonylurea
herbicides) will result in adverse effects on non-target organisms.
In addition, 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 minimize direct or indirect interaction between
the genetically modified carnation and non-target organisms.
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
commercial cultivation takes place. 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. Also
in case of propagation of the genetically modified carnation
FLO-40685-2 by stem cuttings no adverse effects are foreseen.
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
Possible negative effects on human and animal health as a result of
handling flowers or as a result of incidental consumption of petal
leaves of the genetically modified carnation, for example as
garnishing for food, were considered.
Carnation has been used safely by humans for ornamental purposes
for centuries. The modification in line FLO-40685-2 (production of
delphinidin) is novel for carnation, but there are many flowers and
other ornamental species that produce delphinidin, such as
Gentiana, Petunia, Centaurea and Delphinium. Delphinidin is also
present in many common foods, such as red grapes, black currants,
eggplant and blueberry. Studies with 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
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.
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 FLO-40685-1 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.
The Dutch CA concludes that the provided information is sufficient
and is of the opinion that in the context of its intended use,
carnation line FLO-40685-2 is unlikely to have adverse effects on
human and animal health or the environment.