This document contains copies of three recent literature reviews. Hyperlinks to each review are provided in the table below
Scope of review EU agency to
which literature review sent
Date Related dossiers
A literature search on information relevant for the safety of GM carnation to humans,
including the safety of delphinidin and potential allergenicity⃰.
EFSA August
2014
C/NL/09/01 and
C/NL/09/02 Updated assessment of the probability of gene
dispersal from cut-flowers of the cultivated carnation (Dianthus caryophyllus) imported into Europe
Bureau GGO, NL October 2013
C/NL/13/02
Updated review of potential toxicity and allergenicity of the acetolactate synthase (ALS) protein
Bureau GGO, NL October 2013
C/NL/13/02
⃰ Copies of the papers cited in this review are provided.
An assessment of the probability of gene dispersal from cut-flowers of the cultivated carnation (Dianthus caryophyllus) imported into Europe
CONTENTS
Section Page
1. Baseline information; Dianthus biology 1
1.1 Taxonomy 2
1.2 Distribution 3
1.3 Reproductive biology 7
1.4 Inter- and intra-specific hybridisation 8
1.5 Weediness of Dianthus 10
2. Baseline information: carnation 11
2.1. Types of cultivated carnation 11
2.2. Carnation cultivation methods 12
2.3. Carnation utilization in Europe 13
2.4. Carnation import and distribution in Europe 13 3. Probability of gene dispersal from carnation 14
3.1. Introduction 14
3.2. Probability of gene dispersal by vegetative propagation 14 3.3. Probability of gene dispersal by seed set and seed distribution 14 3.4. Probability of gene dispersal by pollen distribution 15
4. Conclusions 16
5. Literature cited 18
1. Baseline information; Dianthus biology
Attachment B4 has been expanded from earlier versions included in previous marketing
applications to the EU. As part of the regulatory process for the release of transgenic carnation in Australia, the Australian government has also produced an assessment of the biology and ecology of carnation (OGTR, 2006).
1.1 Taxonomy Dianthus genus
The Dianthus genus is a member of the Caryophyllacea, or pink, family and contains about 300 species. The genus is native to Europe, Asia, North Africa and the Arctic region, where one species is found (Hickey and King, 1981; Tutin and Walters, 1993). The second edition of Flora Europaea (Tutin, and Walters, 1993) lists 115 species, and 91 sub-species within 32 of these species. Seventy seven of the species listed are endemic to Europe.
Recent studies have suggested that the Dianthus genus has one of the fastest rates of evolution in plants, possibly due to ployploidization (Weiss et al., 2002; Balao et al., 2009; Balao et al., 2011b).
Dianthus chromosomes number ranges from 30 (diploid) to 105 (Weiss et al., 2002; Andersson- Kotto and Gairdner, 1931). Dianthus species are largely perennial, though some species, such as D.
armeria, are annuals (Jurgens et al., 2002).
Dianthus caryophyllus
In its unimproved, single flower form, Dianthus caryophyllus is called, in English, the clove pink or Grenadine (Britannica, 1999) or the clove gillyflower (Harvey, 1978). The English translation of the Latin word “caryophyllus” is “cloves” and caryophyllus was a commonly used name in the early taxonomy of the Dianthus genus (Smith, 1794). Clove pink was grown in the middle ages for its clove like perfume (Harvey, 1978) and was named by Linnaeus in Species Plantarum (De Langen et al., 1984). Dianthus caryophyllus may be taken as the type species for Dianthus.
The taxonomy of Dianthus caryophyllus has a long history and there are many sub-species and varieties of this species in the scientific literature. Dianthus siculus, D. arrosti, D. gasparrinii, D.
virgatus, D. tarentinus and D. longicaulis were sometimes previously treated as subspecies or varieties of D. caryophyllus (Bacchetta et al., 2011). Bracchi and Romani (2010) include Dianthus caryophyllus var. inodorus L. and Dianthus caryophyllus var. orophilus (Jord.) Rouy & Foucaud in a recent checklist of Piacenza, Italy and Giardina et al. (2007) list a synonym of Dianthus
gasparrinii as D. caryophyllus subsp. Gasparrinii. A worldwide Dianthus species, D. sylvestris, is known by the synonym D. caryophyllus ssp. sylvestris or var. sylvestris (Flora Piacentina, 2001;
http://www.anthos.es). The e-flora of the French botany network (http://www.tela-
botanica.org/site:accueil) lists nine varieties and eight sub-species of D. caryophyllus, in addition to the species itself. A compilation of the sub-species and varieties of Dianthus caryophyllus are shown in Table 1. The confusion in nomenclature can lead to mis-identification in the field – a problem recognized by Smith (1794) two centuries ago.
Table 1. Sub-species and varieties of Dianthus caryophyllus recorded in the taxonomic literature Dianthus caryophyllus subsp. sylvestris (Wulfen)
Rouy & Foucaud, 1896 ( = Dianthus sylvestris Wulfen ).
Dianthus caryophyllus var. inodorus L., 1753
Dianthus caryophyllus var. collivagus (Jord.) Cariot &
St.-Lag., 1889
Dianthus caryophyllus var. juratensis Gren., 1865
Dianthus caryophyllus var. consimilis (Jord.) Rouy &
Foucaud, 1896
Dianthus caryophyllus var. orophilus (Jord.) Rouy & Foucaud, 1896 Dianthus caryophyllus var. guyetanii (Jord.) Rouy &
Foucaud, 1896
Dianthus caryophyllus var. saxicola (Jord.) Cariot & St.-Lag., 1889 Dianthus caryophyllus subsp. longicaulis (Ten.)
Arcang.
Dianthus caryophyllus subsp.
godronianus (Jord.) Sennen, 1932 Dianthus caryophyllus var. longicaulis (Ten.)
P.Fourn., 1936
Dianthus caryophyllus subsp. virgineus sensu auct. plur.
Dianthus caryophyllus subsp. siculus (C.Presl) Arcang., 1894
Dianthus caryophyllus subsp.
coronarius (Lam.) Bonnier, 1913 Dianthus caryophyllus var. coronarius L., 1753 Dianthus caryophyllus subsp.
coronarius (Lam.) P.Fourn., 1936
Information;http://www.florealpes.com/fiche_oeilletsauvage.php?photonum=4&PHPSESSID=98ka413mmbrqb38otk1c bh8n5egkvi97# (Fleurs des Hautes-Alpes, de montagne, de Provence et d'ailleurs) and
http://inpn.mnhn.fr/espece/cd_nom/133832/tab/rep (INPN, Inventaire National du Patrimoine Naturel, France.)
The chromosome number of Dianthus caryophyllus samples collected from the wild has consistently been counted at 30 (Jones and Cooper, 1968; Gadella and Kliphuis, 1970; Hassall, 1978).
Carnation
Carnations are double-flowered cultivars and in the general trade, botanical and horticultural literature carnation cultivars are considered to belong to the species Dianthus caryophyllus (Smith, 1794). The common name for Dianthus caryophyllus is carnation. However, the exact taxonomic and breeding history of carnation is not precisely known (Hughes, 1991; Harvey, 1978) and it is almost certain that carnation is a hybrid involving two or more Dianthus species, one of which is likely to be Dianthus caryophyllus (Hughes, 1991; Allwood, 1954). It is believed that carnation breeding began in the 1500’s in France (Holley and Baker, 1963). The double-flowered varieties of the carnation were known as an ornamental plant in Europe in the 15th century (Harvey, 1978). The herbarium specimen assessed by Linnaeus included a double-flowered carnation specimen (this may be viewed at http://www.linnean-online.org). More than one hundred years ago carnation breeding was well established in the USA, and today there are half a dozen large breeders in the world. The second edition of the International Dianthus register (1983) lists over 30,000 cultivars.
1.2 Distribution
Dianthus genus
The centre of biodiversity for Dianthus is southern Europe and the greatest range of Dianthus species are found in the south eastern European countries. Table 2 (adapted from Tutin and Walters, 1993) lists the number of species recorded in countries with the greatest diversity of Dianthus species. In Europe, most Dianthus species are found in the Balkan region and in the Mediterranean countries.
In North Europe Dianthus species are far less common, or even absent. For example, only six species are known in the British flora (Clapham et al., 1987), and five in Holland (Tutin and Walters, 1993). The majority of Dianthus species are not widely spread in Europe, and are confined to one or two countries, to specific mountain regions (Strid and Tan, 1997) or to alpine areas (Schwegler, 1979).
Table 2. European countries with the greatest diversity of Dianthus species
Country Number of Dianthus
species
Former Yugoslavia 44
Bulgaria 39
Greece 37
Romania 32
Central + S.W. European Russia 31
Spain 26
Italy 24
Albania 21
France 20
There are six Dianthus species that have been found throughout the world. They are described below using information from Tutin and Walters (1993):
• D. barbatus. Not a European species exclusively, D. barbatus is native to all Balkan countries and Eastern Europe. It has naturalized elsewhere after escape from cultivation.
• D. armeria. The most widely distributed Dianthus species in Europe, as far north as Southern Sweden. This species has a world-wide distribution.
• D. sylvestris. (Bacchetta et al., 2010). This species is highly polymorphic and closely related to D. caryophyllus. D. sylvestris is found in southern Europe and Mediterranean islands. There are six sub-species, three of these which are endemic to Europe, though the species itself is not.
Dianthus sylvestris is found “from SE Spain to Greece and Northwards to the Swiss Jura and the Alps”. The typical habitat is rocky places (Polunin, 1980).
• D. superbus. This species, which is not endemic to Europe, is found in all areas except much of the west and south of Europe (Tutin and Walters, 1993). There are three sub-species.
• D. deltoides. This species is not endemic to, but is distributed in, most of Europe. It is rarer in the south (Tutin and Walters, 1993). Experiments have shown this species capable of
establishment in restored meadows (Kalle et al., 2009).
• D. carthusianorum. The only one of the six species which is possibly endemic, this species is found in south, central and western countries and has a very variable form.
In northern European countries the majority of Dianthus populations, if present, will be represented by one or more of the above six species (e.g. Perring and Walters, 1976; Van der Meijden, 1990).
The distribution of these species in the North of Europe may be sporadic (Berten, 1990; Schonfelder and Ahlmer, 1990; Benkert et al., 1996). D. deltoides and D. carthusianorum are more common than the other four species listed above (Schonfelder and Ahlmer, 1990; Benkert et al., 1996).
Dianthus caryophyllus
Unimproved Dianthus caryophyllus is reportedly only found wild in Mediterranean countries, and is rare. Whilst this area encompasses Spain, France, Greece, North Africa and Italy, Tutin and Walters (1993) state that the species is possibly only native to Sicily, Sardinia, mainland Italy and Greece. The Flora Europaea, accessed in September 2012 (http://193.62.154.38/FE/fe.html) still identifies France, mainland Spain, Italy (including Sicily and Sardinia) and Greece as countries where the species may occur wild. (Bracchi and Romani (2010) identified Dianthus caryophyllus in the province of Piacenza in Italy. Polunin and Huxley (1967) suggest the species occurs in France, Algeria and Morocco. In European floras the species is listed in Italy, Sicily and Sardinia
(Zangheri, 1996), but not in floras of Greece (Strid, 1986; Strid and Tan, 1997; Turland et al., 1993), France (Guinochet and Vilmorin, 1973) and Andalucia (Valdes et al., 1987). In Mallorco Dianthus caryophyllus is only listed as a garden species (Barcelo, 1978). According to Ingwersen (1949) wild Dianthus caryophyllus can only be found commonly in specific coastal regions of Corsica. Figure 1 shows a photograph of Dianthus caryophyllus in the wild. This photograph is one of several recent collections of the species made in Southern France and posted on the website of the e-flora of the French botany network (http://www.tela-botanica.org/site:accueil).
The double-flower of cut-flower carnation varieties is now quite different to a flower from a wild Dianthus species, as shown in Figure 1.
Figure 1. Comparison of the morphology of a flower from Dianthus caryophyllus (left hand side) to a cut-flower “standard” carnation variety (right hand side). The image on the left is extracted from http://www.isatis31.botagora.fr/en/eflore31/eflore31-v1.aspx (eFlore31, la flore en ligne de
la Haute Garonne, France) Ornamental Dianthus species
The genus Dianthus contains several species which have been cultivated for hundreds of years for their ornamental value (Ingwerson, 1949). In the English language, these plants were called “pinks”
from the 16th century, a term used to describe the whole Dianthus genus from Elizabethan times (Harvey, 1978). Table 3 lists some of the more commonly grown ‘pinks’ species. Photographs of most of these species are available on-line (Jagel, 2012).
Table 3. Dianthus species commonly grown for their ornamental value
Species Common name Species Common name
D. gratianopolitanus Cheddar pink D .plumarius Cottage pink, grass pink D.carthusianorum Carthusian pink D. alpinus Alpine pink
D. superbus Fringed pink D. sylvestris Wood pink
D. armeria Deptford pink D. chinensis Chinese pink, Rainbow pink, Indian pink, Japanese pink
D. deltoides Maiden pink
Varieties of “carnation” have been reported very occasionally to have naturalized in the British Isles. These reports are summarized in Table 4. These accounts refer to garden plant varieties of pinks and carnation, which are not the same as cut-flower varieties and predate the development of modern cut-flower varieties by several centuries. Clement and Foster (1994) refer to carnation in the wild as "cultivars of this species (caryophyllus), native D. gratianapolitans and D. plumarius”.
Table 4. Summary of reports of naturalization of Dianthus caryophyllus and hybrids in British floras
Reference Dianthus species Comments by authors
Maby, 1996 D. caryophyllus Probably introduced by Normans and naturalized in old walls in Rochester castle and Beaulieu abbey
Perry and Ellis, 1994
D. caryophyllus “an escape of European origin” ( no further detail)
Walters, 1993 D. caryophyllus X D. barbatus
Single report from the year 1717 Stace, 1997 D. caryophyllus X
D. gratianopolitans, D. caryophyllus X D. gratianopolitans X D. plumarius
Parentages of escaped garden pinks, some of which are occasionally naturalized on old walls.
Clement and Foster, 1994
D. caryophyllus Known for more than three centuries on the walls of Rochester castle.
Clapham et al.,1987
D. caryophyllus Occasionally naturalized on old garden walls.
Preston et al., 2002
D. caryophyllus “D. caryophyllus has been cultivated in Britain since the 16th century and is very common in gardens. It was first recorded in the wild in 1778, when it was discovered on the walls of Rochester Castle (E. Kent)”.
”Some historical records may be referable to hybrids with other species, which are also commonly grown.”
“Not known with any certainty as a wild plant;
recorded as doubtfully native in S. Europe.”
D. caryophyllus X D. gratianopolitans
A single record in 1980.
D. caryophyllus X
D. plumarius A tufted perennial herb which “occurs as a casual garden throw-out on rubbish tips and roadside verges”.
D. caryophyllus X D. gratianopolitans, D. caryophyllus X D. gratianopolitans X D. plumarius
Parentages of escaped garden pinks, some of which are occasionally naturalized on old walls.
Due to the international trade in ornamental plants, ornamental Dianthus species can now been found in many plants of the world, and on occasion have escaped from cultivation in Europe (section 1.5).
1.3 Reproductive biology Dianthus genus
The flowering period of wild Dianthus species is limited to summer in Europe (Table 5).
Table 5. Flowering period for several Dianthus species in the wild. Alpine flowering periods are from Schwegler (1979) and Mediterranean data is from Strid and Tan (1997)
Alpine Mediterranean
Species Flowering months Species Flowering months
armeria June - August armeria June - August
arenarius June - September deltoides June - September
barbatus June - September elegans May - July
carthusianorum June - September giganteus June - August
glacialis July - August gracialis June - August
monspessulanus June – September integer July - August
pavonius July superbus July - August
sylvestris June - September sylvestris July - September Pollination of Dianthus in nature is facilitated by insects (Knuth, 1908; Frankel and Galun, 1977;
Erhardt, 1988; Jennersten, 1983 and 1984; Bloch et al., 2006) and is only effectively achieved by the Lepidoptera (butterflies, moths). Nocturnal hawk moths are one group of common insect pollinator (Balao et al., 2011a) and various Dianthus species are nocturnal- or diurnal-flowering, producing types and amounts of scents to attract specific types of pollinators (Jurgens et al., 2003, 2002; Jurgens, 2006). The chemical composition of several wild Dianthus species is described by Kishimoto et al. (2011). Lepidopteron insects are the only ones with proboscis long enough (up to 2.5 cm; Erhardt, 1990) to reach the nectaries, which are located right at the base of the flower in all Dianthus species (Hickey and King, 1981). Dianthus flowers are tubular (Figure 1), with strong bracts and calyx to exclude other insects (Knuth, 1908; Bloch and Erhardt, 2008). Table 6 lists pollinators of Dianthus observed in Europe.
Table 6. Reported Lepidoptera insect pollinators of Dianthus
Dianthus species Moth genera Butterfly genera
deltoides - Hesperia, Aphantopus, Aporia,
Cyaniris, Ochlodes, Mesoacidalia, Polyommatus,Thymelicus
superbus Macroglossum -
carthusianorum Macroglossum Hesperia, Plusia, Inachis, Melanargia, Papilio, Thymelicus
gratianopolitanus Macroglossum, Autographa, Hadena, Hemaris
Papilio, Aglais, Cynthia, Pieris
inoxianus Hyles
chinensis - Plusia
barbatus Macroglossum Pieris
sylvestris Macroglossum, Hadena -
glacialis Zygaena
corymbosus Thymelicus, Aporia, Pieris
monspessulanus Macroglossum -
From Knuth (1908), Jennersten (1983, 1984), Erhardt (1988), Erhardt (1990), Erhardt and Jaggi (1995), Collin et al.(2002), Bloch et al. (2006), Bloch and Erhardt (2008), Yurtsever et al. (2010) and Balao et al.
(2011a)
Dianthus species are largely protrandous, i.e. the anthers and pollen mature before the pistils (Knuth, 1908; Buell, 1952; Keane, 1989; Gargano et al., 2011). In some species gynomonoecious- gynodioecious mating systems also occur (Jurgens et al., 2002; Collin and Shykoff, 2003).
Typically, when Dianthus flowers first open, the styles remain short and smooth. At this time the flower sheds pollen, and the styles are non receptive. As the flowers age the styles elongate and become covered on their inner surface with many hairs. If a flowers is not successfully pollinated
the styles continue to grow and curve. This can be seen in some cut carnation flowers, when the styles of some varieties protrude beyond the petals when they are left in the vase. If fertilization in Dianthus has been successful the flower collapses and the styles quickly shrivel. Though protrandy largely prevents self-pollination, D. deltoides and D.barbatus are known to readily self seed. D.
armeria (Jurgens, 2006) and D. sylvestris (Jurgens et al., 2002; Collin and Shykoff, 2003) are also capable of selfing and the rare alpine species, D. glacialis is non-protrandous (Erhardt and Jaggi, 1995).
Jurgens et al. (2002) has provided information on pollen and ovule number in seven wild Dianthus species and supplemented this information with quantification of pollen grain size and style length in the same 7 species (Jurgens et al., 2012). Even within single anthers there are several populations of pollen grain size in Dianthus (Jurgens et al., 2012,Tejaswini, 2002). Bloch et al. (2006)
established that in D.carthusianorum successful seed set was dependent on the number of pollen grains deposited on the stigma, with no seed set occurring with less than approximately 25 pollen grains.
Seed from Dianthus species is relatively short-lived. Mondoni et al. (2011) established the seed longevity in storage of Dianthus carthusianorum, D. sylvestris and D.glacialis to be three months or less. The same authors showed in comparisons of lowland and alpine populations of same species that alpine populations had shorter lived seeds. Seed from Dianthus rupicola had an optimal and rapid germination, at 15-25ºC (Lantieri et al., 2012). Seed of Dianthus morisianus was shown to be non-dormant and to exhibit non-light dependent germination. This species is unable to form a persistent seed bank, indicating the low long-term survivability of the seed (Cogoni et al., 2012).
Dianthus caryophyllus
We are unaware of any scientific literature on the reproductive biology of wild Dianthus caryophyllus.
Viability of carnation pollen is varietal dependent. In varieties that produce low amounts of viable pollen, correct choice of anther stage for harvest is critical to optimize viability (Kim et al., 2005a).
Tejaswini (2002) measured pollen grain size in 5 varieties of carnation establishing medium size grains were more likely to germinate. Though there was no significant difference between varieties there was a difference in mean germination by variety and pollen tube length was proportional to percentage germination and grain size.
Seed of Dianthus caryophyllus is light and 3 – 5 mm in length. Seed provided from a wild
population collected in France is shown in Figure 2. This seed was collected in August 2009 and is partially viable; the seed was sown in August 2012 and approximately one-third of the seed
germinated. As of April 2013 these plants were still in a vegetative state (Figure 2).
Figure 2. Seed and seedling of Dianthus caryophyllus. Seed kindly provided by Jardin Botanique de Bordeaux, France. The red bar represents 5 mm. The lower plate shows a germinated seedling
established as a young plant 6 months after germination.
Additional information on the reproductive biology of carnation is provided in section 1.4.
1.4 Inter- and intra-specific hybridization
In nature the Dianthus genus is characterized by a capacity for interspecific hybridization (Pax and Hoffmann, 1934; Ingwersen, 1949; Demmink, 1978; Castroviejo et al., 1990). Inter-specific hybridization has been utilized in breeding ornamental Dianthus species (Lee et al., 2005; Fu et al., 2011) and in fact inter-specific hybridization in Dianthus was one of the first scientific reports of plant hybridization, in the early 1700’s (Andersson-Kotto and Gairdner, 1931; Zirkle, 1934). D.
knappii has been used as a genetic resource for yellow flower colour, D.superbus for its long feather like petals and D.barbatus for its multiple flower head. According to Tutin and Walters (1993)
natural hybridization in Dianthus is restricted to local regions where two species grow in high density and is particularly common in the Pyrenees. In these wild conditions there may be 4-5 flowering plants of any one species per square meter (Jennersten, 1984). The ploidy levels of inter- specific hybrids is not related to fecundity (Gatt et al., 1998). Carolin (1957) made 108 different interspecific crosses of Dianthus, and found 22% of crosses were fertile or sub-fertile, possibly due to embryo abortion (Buell, 1953). Bleeker et al. (2007) have stated Dianthus barbatus rarely hybridizes to the native D. deltiodes and D superbus in Germany. Andersson-Kotto and Gairdner (1931) have documented the compatibilities resulting from many inter-specific crosses within the Dianthus genus and indicate compatibility may not to be related to ploidy level.
Some carnation cultivars are self-sterile (Darwin, cited in Knuth, 1908 and Owens and Miller 2009 first observed this phenomenon in D. caryophyllus) and selfing, even under controlled conditions, produces either no seed or fewer viable seeds per capsule than cross-pollination (Mehlquist and Geissman, 1947; Zhou et al., 2013). Andersson-Kotto and Gairdner (1931) note that the self-fertility of carnation is markedly lower in double-flower varieties. Whilst there are some female sterile cultivars, (e.g. Copareve, Eolo) the cultivated carnation is not usually completely sterile (Silvy, 1978) and poor ‘selfer’ lines may produce seed after cross-pollination (Mehlquist and Geissman, 1947).
Interspecific hybridization with D. caryophyllus
Efforts to hybridize Dianthus caryophyllus and other Dianthus species have been made to introduce useful horticultural genes into the cultivated carnation. Table 5 lists the species reported to
hybridize to D. caryophyllus. Hybrids may be diploid, or triploid/tetraploid (Nimura et al., 2008). In crosses with D. caryophyllus Nimura et al. (2003) found that no seed set occurred when D.
japonicus was used as the female. Wen et al. (1995) used embryo rescue to recover hybrids with D.
chinensis and D.barbatus when D. caryophyllus was used as the female. As Table 7 shows, it has been possible to achieve a successful cross of D. caryophyllus to the widely spread European species D. barbatus, D. carthusianurom and D. sylvestris (this species is closely related to D.
caryophyllus). All four of these species are diploid (2n = 30).
Table 7. Dianthus species reported to hybridize with D. caryophyllus
Species Reference
arenarius Holley and Baker, 1963; Umiel et al., 1987
barbatus Pax and Hoffman, 1934; Umiel et al., 1987; Wen et al., 1995 carthusianorum Demmink, 1978; Segers, 1987; Sparnaaij and Koehorst, 1990
chinensis Mehlquist, 1945; Demmink, 1978; Sparnaaij and Koehorst, 1990; Wen et al., 1995
deltoides Umiel et al., 1987
gallicus Holley and Baker, 1963; Andersson-Kotto and Gairdner,1931 giganteus Demmink, 1978; Sparnaaij and Koehorst, 1990
hungaricus Kishimoto et al., 2013 japonicus Nimura et al., 2003, 2008
knappii Holley and Baker, 1963; Segers, 1987; Sparnaaij and Koehorst, 1990 monspessulanus Holley and Baker, 1963
plumarius Gatt et al., 1998
sinensis Holley and Baker, 1963; Umiel et al., 1987
sylvestris Holley and Baker, 1963; Umiel et al., 1987; Demmink, 1987; Andersson- Kotto and Gairdner,1931
seguieri Holley and Baker, 1963 versicolor Sparnaaij and Koehorst, 1990
Interspecific crosses using carnation can only be made in the glasshouse using manual intervention.
Where carnation is the female parent this entails preliminary petal removal, manual pollination, calyx opening, final petal removal and fruit ripening on the plant (Sparnaaij and Beeger, 1973;
Keane, 1989, Gatt et al., 1998).
The wild Dianthus species, Dianthus sylvestris, is found from South East Spain to Greece and Northwards to the Swiss Jura and the Alps. The typical habitat for this species is rocky places (Polunin, 1980). However, despite the fact carnation has been grown and traded in Europe on a large scale for decades there are no reports of the existence of hybrids between carnation and D. sylvestris in the wild in Europe (Tutin and Walters, 1993).
1.5 Weediness of Dianthus
In Europe, there have been escapes from cultivation of some Dianthus species (Tutin and Walters, 1993). These populations could be considered weeds because of their appearance in disturbed lands. However, they cannot be considered ecologically or economically important weeds (Holm et al., 1979; Guillerm and Maillet, 1982; Holzner and Immonen, 1982). Many Dianthus species are adapted to very specific geographical and climatic regions, such as alpine, rocky or sandy areas.
This restricts their capacity for weediness.
As outlined, several Dianthus species are and have been grown for ornamental purposes for many centuries. Rare escapes from cultivation have been recorded in floras of Europe, such as for Dianthus barbatus (Bracchi and Romani, 2009). Clement and Foster (1994) describe a single population of Dianthus sylvestris in the UK, established on the rocks near Whitby harbour, in Yorkshire. Marco et al. (2010) established that cultivated Dianthus barbatus and Dianthus plumarius have not escaped in abandoned agricultural lands of Lauris villages, near Marseille, France. Schlaepfer et al. (2010) compared the seed germination characteristics of Dianthus armeria, considered to be a relatively invasive species, to D. carthusianorumnon. Dianthus armeria, in comparison to Dianthus carthusianorumnon germinated faster, produced more biomass and had a higher proportion of flowering plants.
Dianthus caryophyllus
Dianthus caryophyllus is not a weed (Tutin and Walters, 1993). Specific references to the potential for escape from cultivation indicates very occasional survival, typically in rocky habitats like walls (Flora Piacentina, 2001; Dr César Delnatte, pers. comm.; National Botanic Garden of Belgium, pers. comm.; Societe Nationale des Sciences Naturelles et Mathematiques de Cherbourg, France.
pers. com.). Brachhi and Romani (2009) have reviewed the alien species of the Emilia Romagna region of Italy and list Dianthus caryophyllus as an ornamental species that may have escaped cultivation and naturalized within population centres.
Carnation
Despite decades of cultivation carnation has not become a weed, or escaped from cultivation, anywhere in the world. Each year over 10 billion flowers are produced for the world’s flower markets, and we have studied the floras of several areas with a significant area of cultivated carnation. There are no reports of naturalization of carnation in these floras:
• Japan. Flora-Kanagawa, 1988; Ohwi, 1965.
• Andean mountains, Ecuador. Jorgensen and Ulloa Ulloa, 1994.
• Uplands of Kenya. Agrew, 1974.
• Michoacan, Mexico. Garcia and Jimenez, 1993, Jimenez and Garduno, 1995.
• Israel. Weissmann-Kollmann, 1965; Zohary, 1966.
• Victoria and New South Wales, Australia. Harden, 1992; Willis, 1988
Cultivated clove pink (single flowered D. caryophyllus) is not winter hardy (MUS, 1996) as expected from the species natural distribution range on the coastal regions of the Mediterranean.
The carnation will not survive outdoors in northern Europe. Florigene/Suntory has spoken to many carnation growers and has our own experience of the large scale production of transgenic carnation in Israel, Holland, Australia, Japan and Ecuador. Carnation has never been found growing wild, even in the immediate vicinity of carnation flower growing areas. In surveys carried out around the sites of production of carnation in Colombia, including composting areas, no carnation populations were identified (Chandler et al., 2008; section 2.2.1).
The cultivated carnation has no capacity to escape from cultivation as the crop possesses no vegetative propagation mechanisms and there are no opportunities for seed-set (section 3).
It could be possible, using tissue culture or other propagation techniques, to eventually deliberately cultivate plants from imported flowers.
2. Baseline information; carnation 2.1 Types of cultivated carnation
Cut flower varieties of carnation grow to 60 – 120 cm high, depending on variety, and produce flowers with a diameter up to 80 mm. Carnation can have from 30 – 100 petals per flower and the reproductive tissues of the flower are enclosed by the petals. In contrast, wild Dianthus species have an open flower, with the stigma and style easily accessible. The distinctive calyx of the genus Dianthus is seen in both flowers shown in Figure 1. The long tubular calyx is a morphological adaptation to pollination by moths and butterflies in the wild. In wild Dianthus species, including D.caryophyllus, the calyx ranges from 5 – 30 mm (Strid, 1986) and in cut flower carnation varieties from 25 - 40 mm. The calyx is relatively thick in the larger cut flower varieties.
There are hundred’s of cut-flower varieties of cultivated carnation, categorized according to plant form, flower size and flower type. The two dominant groups, accounting for 96% of sales in the Dutch auctions, are standards and sprays, shown in Figure 3.
Figure 3. Spray type (left hand side) and standard type carnation (right hand side)
• Standards. These cultivars are grown under conditions in which a single large flower is produced per stem. Side shoots and buds are removed (a process called disbudding) to
increase the size of the terminal flower.
• Sprays (also called miniatures in the USA market). These cultivars are intended for
cultivation to give a large number of smaller flowers (one per side shoot) per stem. Only the central flower is removed, allowing the laterals to form a 'fan' of stems.
2.2 Carnation cultivation methods
The cultivated carnation is vegetatively propagated and to produce plants for cut flower production cuttings are taken from vegetative 'mother plants' which are continually pruned to produce a high number of vegetative cuttings from axillary buds. These cuttings are rooted in conditions of high humidity, after treatment with auxin containing rooting powder, gels or solutions. Rooted plants may be planted in soil or grown hydroponically, and are kept for 1-2 years. Flowers are produced in flushes, beginning 3-5 months after rooted cuttings are planted. Picking of all flowers is essential and flowers must be harvested in tight bud (or closed bud for spray types) for distribution and marketing. The correct pick stage is strictly enforced by flowers entering the Dutch auction system, to ensure a satisfactory vase life in the hands of the consumer. A major problem for growers is the fungal wilt disease Fusarium oxysporum (OGTR, 2006). The occurrence of this fungus in untreated soil has led to relocations of growing areas in countries such as Spain, and adoption of cleaner cultural practice by the majority of European growers. Major pests of carnation are thrips, aphids and mites (OGTR, 2006).
Disposal of waste materials
During carnation production, waste material is generated on a daily basis. This comprises vegetative material and flowers which are rejected due to quality problems, such as short length, or plants which have been uprooted when they reach the end of their productive life. In the latter case, irrigation is turned off to dry the plants before they are discarded. At both farms in South America where the transgenic carnation is grown, waste material is composted (Figure 4)
Figure 4. Composting areas in carnation production facilities in Colombia (left hand side) and Ecuador (right hand side)
As part of the regulatory compliance in Colombia, experiments have been carried out in which rooted carnation cuttings, uprooted cuttings and chopped plant material have been discarded for a period of up to 6 months in cleared plots and plots with existing vegetation. These experiments have shown carnation was unable to survive under these conditions.
The vicinity of the compost heaps have also been inspected on an at least annual basis for the presence of established carnation plants. None have been found. In Colombia the plants growing near the compost heaps have been identified (Chandler et al., 2008). 100 species from 35 families were identified across 8 sample sites. The dominant family at farm locations was the Asteraceae, followed by the Poaceae. The species found at the most sites in the farm environments were
Pennisetum clandestinum (kikuyu grass), Taraxacum officinale (dandelion) and Poa annua (annual bluegrass). No carnation (Dianthus caryophyllus) plants were found in any transect. The only Caryophyllaceae species found were Arenaria lanuginosa ( Michx.) Rohrb., Silene gallica L., Spergula arvensis L. and Stellaria media ( L.) Cirillo. Of these 4 species, only Stellaria media was identified at the composting area itself.
2.3 Carnation utilization in Europe
Historically, approximately 8 billion carnation flowers are consumed in Europe each year (Heinrichs and Siegmund, 1998). Approximately 20% of the European supply of cut carnation flowers is imported (largely into Holland, United Kingdom and Germany) from Colombia, Ecuador, Kenya, Israel, Morocco, Turkey and Zimbabwe (Heinrichs and Siegmund, 1998). Most of the flowers produced in Europe are grown in Italy and Spain. The data suggests that there have been in excess of 800 million carnation plants in cultivation on an annual basis in Europe for more than 30 years. On the basis of an average yield of 8 flowers this represents 6 to 8 billion flowers a year, the vast bulk of which would be consumed in Europe.
2.4 Carnation import and distribution in Europe
Information on the actual number of carnation flowers imported into Europe from outside the EU can be accessed from EUROSTATS. Table 8 provides an extract of the data, with details for
Colombia and Ecuador. Carnations are imported into Europe by air freight, or by truck from Turkey and Morocco. Typically imports are handled by specialist importers, who provide distribution to wholesale and retail flower outlets. At the wholesale level these outlets could be wholesale florists or flower markets, such as Covent Garden in the UK. There are many specialist importers affiliated to the Dutch auctions. Wholesale florists and markets provide access to flowers for florists.
Importers may also be, or may forward to, companies that specialize in provision of flowers for supermarket/ grocery/ garage chains. In these case final product (perhaps assembled in bouquets with other flowers), would be sleeved, labeled and distributed to individual stores. Flower longevity in this chain is summarized in Table 9.
Table 8. Number of carnation flowers imported into the Netherlands and the EU from 2009 - 2012. Units are million flowers
Importing country
Exporting country
Year Colombia Ecuador All Countries
Netherlands 2009 326 25 460
2010 313 19 439
2011 302 20 474
2012 326 17 592
All EU 27
2009 815 30 1,662
2010 780 26 1,587
2011 652 31 1,604
2012 631 28 1,593
Table 9. The process chain for cut carnation flowers imported into Europe
Step in process Duration (days) Maximum total
duration (days)
Harvest of flowers at farm 0 0
Processing, storage for flying 1- 14 14
Distribution to importers 1-3 17
Processing and distribution to arrival at final retail destination
1-3 20
Display at retail 5 25
Consumption “in the vase” –e.g. display and use by consumer before discarding
3- 21 46
Until the time flowers are displayed for consumers, distributors maintain flowers dry in boxes, or in buckets with a small amount of water. During this time flowers are refrigerated, typically in large walk in cool rooms, and the flowers are closed, as the refrigeration prevents flowers opening.
In the consumers hands, at ambient temperature, and in water, flowers will hydrate, fully open and eventually senesce and die. Carnation vase life is determined by the age of the flowers from harvest, variety, and how well the flowers have been treated after harvest with preservative chemicals.
Correct treatment prevents damage by ethylene, the compound that triggers senescence in carnation flowers which is produced 2 – 7 days after flowers are fully open. However, even treated carnation flowers will eventually dehydrate, senesce and die.
3. Probability of gene dispersal from carnation 3.1 Introduction
The dispersal of genes from an imported cut flower can be by three routes;
• Through vegetative spread of the discarded flower, leading to the formation of clonal populations. This possibility is discussed for carnation in Section 3.2.
• Through the formation and dispersal of seed from the flower, as a result of self fertilization or fertilization with pollen from an external source. This possibility is discussed for carnation in Section 3.3.
• Through the formation and dispersal of seed by a recipient plant, fertilized by pollen transferred from the flower. This possibility is discussed for carnation in Section 3.4.
3.2 Probability of gene dispersal by vegetative propagation
Carnation is vegetatively propagated (by cuttings) but the species does not spread vegetatively, i.e.
the plant does not produce organs such as stolons, rhizomes, root-borne shoots, tubers, bulbs, corms or runners. Cuttings have to be struck in optimized conditions, and roots will not form on discarded untended materials, i.e. cut-flowers or old plants disposed of by growers or florists. This has been confirmed experimentally and by observation (see section 2.2.1).
3.3 Probability of gene dispersal by seed set and seed distribution
For gene flow by seed dispersal to occur in a carnation flower, the following events must all occur:
• Arrival of viable pollen on the stigma.
• Pollen germination and pollen tube growth to the ovule.
• Fertilization.
• Growth and maturation of the embryo and seed maturation on the cut flower.
• Seed dispersal.
• Seed germination and plant establishment.
Whilst fertilization of carnation, even with pollen from other Dianthus species, is theoretically possible, the probability of natural fertilization in carnation, even under cultivation conditions is extremely low. This is because of the physical barrier of the multiple petals. This barrier presents a significant obstacle to any potential pollinating insects that might cross pollinate within the flower growing area or be carrying pollen from external sources. During production flowers are picked closed, and exported to Europe at this stage.
Notwithstanding the fact that successful pollination is unlikely to occur, no seed set could occur on the cut flower. This is because the process of seed development takes from 5 weeks (Sparnaaij and Beeger, 1973; Gatt et al., 1998) to 2 months (Arthur, 1981). Cut flowers of carnation, even if treated with silver for increased vase-life cannot be kept in the vase for longer than 3 weeks.
Separation of the flower from the plant would in any event deprive any developing embryos of essential hormones and nutrients, preventing maturation.
3 .4 Probability of gene dispersal by pollen distribution
For gene dispersal to be successful by pollen dispersal, viable pollen would have to be transmitted to a recipient plant and fertilization, seed set and seed dispersal occur. This sequence of events is extremely unlikely.
Standard and midi type cultivars of the cultivated carnation produce little or no pollen (Mehlquist et al., 1954; Kho and Baer, 1973; Nichols, 1976; Kim et al., 2005b) because in most commercial cultivars of this type anthers are converted to petals early in flower development (Arthur, 1981).
Kim et al. (2005b) identified poor filament growth as another cause of poor pollen production.
Spray type carnations produce more pollen than standards but amongst the spray types there are cultivar to cultivar differences in pollen production. In general, production of viable pollen by carnation is much lower than that of wild Dianthus species. It is known that water and nutrient stress can improve anther production in some standard cultivars and that temperature controls stamen and pollen production (Kho and Baer, 1973 Mehlquist et al., 1954). Carnations may be subject to high temperatures during the summer in southern Europe and this will reduce the
potential for pollen production. Carnation pollen can be stored for one week (Sparnaaij and Beeger, 1973; Otten, 1991), placing a limit on the time available for transfer to a potential recipient. In enclosed environments such as glasshouses and greenhouses high humidity reduces pollen longevity.
The carnation flower opens out in the vase, increasing accessibility to the reproductive structures.
In practice this is the only realistic opportunity for insect mediated pollen dispersal. However, at this stage of flower development any anthers are likely to have fallen from the stamens (Spaarnaaij and Beeger, 1973) and pollen would have significantly reduced viability, if any (Buell, 1952;
Keane, 1989). If there were any viable pollen, it would still need to be dispersed to a suitable recipient plant.
As outlined in section 1.3, Dianthus is insect-pollinated and carnation pollen cannot be spread by wind. Any pollen produced is heavy and sticky (Jennersten, 1983) and buried deep in the flower. A survey of the atmosphere in the Netherlands, a country with a large carnation industry, failed to detect any carnation pollen (Driessen and Derksen, 1988).
Only insects have the capacity to disperse carnation pollen, and only Lepidoptera could facilitate pollen transfer from carnation flowers to potential recipient plants. Common pests like thrips, aphids and spider mites are unlikely to move pollen and though ants can be found in flowers of all
types, ants are not likely to move much further than a few meters (Armstrong, 1979). Ants are also typical `nectar robbers' and their secretions usually kill pollen (Herrera et al., 1984; Gottsberger, 1989; Harriss and Beattie, 1991; Gomez and Zamora, 1992). Bees and wasps can be observed foraging in carnation flowers, but there is no evidence that this foraging may lead to cross pollination. As a precaution, bee movement is controlled in breeding houses, because petals are usually removed from flowers.
Assuming, theoretically, an insect were to access a carnation flower in a vase in someone’s home, and carry away any viable pollen that might be present, the probability of subsequently fertilizing a recipient, flowering, Dianthus plant is limited. There are several studies suggesting insect
pollinators only move pollen hundred’s of meters from source when feeding (Price, 1984; Nilsson et al., 1992). In the extremely remote case that a seed was formed as a result of fertilization of carnation pollen onto a wild Dianthus species, there are limited environments where a plant could become established. In nature, Dianthus caryophyllus, like many Dianthus species is only found in suitable habitats, such as rocky walls, sand or limestone hills and outcrops. The seed of the putative hybridization event would probably have to germinate in such environments in order to become established in the wild. Many Dianthus species are found at high altitude on mountains (Strid, 1986) or on islands, not in carnation growing areas. These species are very unlikely to receive pollen from carnation plants. When wild Dianthus species are not in flower, which is for 8 – 11 months, there is no risk of gene dispersal from carnation as a result of fertilization and seed set in recipient plants.
Probability of intergeneric hybridization
There are several species in the Caryophyllaceae which are widespread throughout Europe, and which are also considered serious weeds. These include Arenaria serpyllifolia, Polycarpon tetraphyllum, Sagina apetala, Stellaria media, Silene gallica, Silene vulgaris and Spergularia rubra. However, as far as we are aware, there are no reports of intergeneric hybridization in the Caryophyllaceae.
4. Conclusions
It is reasonable to conclude that the probability of unintentional gene dispersal from an imported carnation flower is nil because possible avenues for gene dispersal are not available.
There is no risk of gene dispersal by vegetative spread:
Carnation does not spread vegetatively, i.e. the plant does not produce organs such as stolons, rhizomes, root-borne shoots, tubers, bulbs, corms or runners. Roots will not form on discarded or old cut-flowers. Florigene/Suntory has experience of large scale production of carnation in
Australia, Japan, Colombia and Ecuador. Carnation has never been found growing wild, even in the immediate vicinity of carnation growing areas where waste material has been discarded or has been left for composting.
There is no realistic possibility that seed could form on an imported cut flower
For gene dispersal by seed formation to occur from a cut carnation flower, the following events would all need to occur successfully; arrival of viable pollen on the stigma of the carnation, pollen germination, pollen tube growth to the ovule of the carnation, fertilization, seed formation and seed dispersal. Notwithstanding the fact that successful pollination of a carnation flower in a vase is highly unlikely, no seed set could occur. This is because the process of seed development takes at least 5 weeks on a plant – where the growth of any developing embryo could be sustained. A cut flower will remain in a consumers hand for three weeks at most before dying.
There is no possibility that pollen could disperse from a cut flower and create a viable hybrid population
There are several mutually exclusive facts that, in combination, indicate that potential pollen spread is not a feasible avenue for gene dispersal.
Firstly, the potential for pollen spread from a cut flower is only theoretically possible;
• In general, production of viable pollen by carnation is much lower than that of wild Dianthus species.
• Hybridization of Dianthus in nature is facilitated by insect pollination and is only effectively achieved by the Lepidoptera (butterflies, moths). Pollen is not spread by wind.
• The only point in the chain where insects could be reasonably expected to access flowers is when on display or in consumers hands. The physical barrier of the multiple petals presents a significant obstacle to any potential pollinating insects in less open flowers.
• As a carnation flower opens out in the vase, any anthers are likely to have fallen from the stamens and any pollen would have significantly reduced viability.
Secondly, were viable pollen to actually be produced and successfully dispersed by an insect vector, the realistic chance of a successful fertilization resulting in a wild hybrid population is also
extremely unlikely.
• Dianthus caryophyllus is very rare.
• In commercial carnation production, flowers are removed. The high concentrations of carnation production in Europe are therefore not available as potential recipients.
• While interspecific hybridization is know in Dianthus, hybridization is restricted to local regions where two species grow in high density and freely intermingle and is particularly common in the Pyrenees. In these wild conditions there may be 4-5 flowering plants of any one species per square meter.
• The majority of Dianthus species are not widely spread in Europe, and are confined to one or two countries, to specific mountain regions or to alpine areas.
• The flowering period of wild Dianthus species is limited to summer in Europe. When wild Dianthus species are not in flower, which is for 8 – 11 months, there is NIL risk of gene dispersal from carnation as a result of fertilization and seed set in recipient plants.
• In the extremely remote case that a seed was formed as a result of fertilization of carnation pollen onto a wild Dianthus species, there are very limited environments where a plant could become established.
The long history of cultivation and utilization of carnation flowers in Europe supports the assertion that there is nil risk of gene dispersal. Carnation is not a weed in Europe. Despite hundreds of years of cultivation, and plantings in parks and gardens, it has not become a weed, or escaped from cultivation, anywhere in the world. Each year over 10 billion flowers are produced for the world’s flower markets. We have provided evidence for a lack of hybrids between cut flower carnation varieties and native Dianthus species in the wild in Europe, and for a lack of naturalized carnation in Europe. This is after decades in which very large numbers of carnation flowers have been imported into Europe. Large numbers of carnation are also grown in Europe. Compared to an imported cut flower, established plants of cut flowers varieties would theoretically be a more likely source material for gene dispersal than cut flowers. However, no hybrid between carnation and any other Dianthus species has ever been recorded in the wild
Expert opinion
In earlier submissions for marketing approval of transgenic carnation imported into Europe we have provided two expert opinions. These expert opinions support the conclusions outlined above.
Dr. Keith Hammett, an acknowledged expert on Dianthus breeding has concluded “that the likelihood of gene dispersal from cut flowers of fully double carnations to be highly improbable, if not inconceivable”
Dr. Flavio Sapia stated;
“The areas of the Coté d’Azur in France and of Riviera dei flora in Italy has been from 1920 to 1976 the biggest producer of carnation cut flowers in Europe. Even if this area has seen the growing of millions of plants and thousands of different varieties, never we had a cultivated carnation capable to survive in wild conditions”
“for this reason the dispersal of a gene inserted in a carnation variety is, from my point of view, impossible..”
The Australian government (OGTR, 2006) concluded;
“In Australia, gene transfer from carnations to any other plant species, even the most closely related naturalized Dianthus species, is unlikely due to the very low fertility of carnations.”
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