Dianthus Species Research Articles

Dianthus barbatus-A New Host of Stolbur Phytoplasma in Serbia.

Sweet William (Dianthus barbatus, Caryophyllaceae) is a biennial or short-lived perennial plant native to southern Europe, from the Pyrenees to the Carpathians and the Balkans. During the summers of 2012 and 2013, phytoplasma-like symptoms were observed on D. barbatus plants on a Serbian plantation (Pancevo, 44°51'49″ N, 20°39'33″ E, 80 m ASL). Only seven symptomatic plants were observed in the summer of 2012. Disease incidence in 2013 was estimated to be less than 1% but increased during 2014 to 4%. Affected plants, showing symptoms of leaf reddening, malformation, and proliferation; flower bud deficiency; and abnormal shoot production, were tested for phytoplasmas. Samples were collected from seven symptomatic and three symptomless plants each year (20 samples), and total nucleic acid was extracted from midrib tissue using a method that includes a phytoplasma enrichment step and DNA purification by chloroform/phenol (3). Oligonucleotide primers specific to the phytoplasma 16S to 23S rRNA intergenic spacer region were used in polymerase chain reaction (PCR) assays on DNA extracted from Sweet William plants (1,3). Using phytoplasma universal primer pairs P1/P7 and P1/16S-Sr, phytoplasma-specific 1.8- and 1.5-kb amplicons were obtained from four and six symptomatic plants collected in 2012 and 2013, respectively. Nested PCR with R16F2n/R2 primers yielded ~1.2-kb amplicons from DNAs of all symptomatic plants (1). No amplicon was generated in PCRs conducted with DNA templates from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of amplified 1.2-kb fragments was performed using four endonucleases (AluI, Tru1I, HhaI, and HpaII). Comparative analysis was done using RFLP patterns of Stolbur (Stol), Aster Yellows (AY), Flavescence Doree-C (FD-C), Poinsettia Branch-Inducing (PoiBI), and Clover Yellow Edge (CYE) phytoplasmas. PCR-RFLP patterns from tested samples were identical to those of the Stol reference strain, indicating that diseased Sweet William was affected by phytoplasma belonging to the 16SrXII-A (Stolbur) group. The sequence of a 1.2-kb rDNA PCR product derived from sample Tk9 (deposited under accession number KM401436 in NCBI GenBank) showed the closest identity (100%) to those of Bulgarian corn (KF907506.1), Iranian 'Bois Noir' (KJ637208.1), and two Serbian phytoplasmas (KJ174507.1 from Calendula officinalis and KF614623.1 from Paeonia tenuifolia), all belonging to the 'Candidatus Phytoplasma solani' Stolbur subgroup. Previously, Aster Yellows Phytoplasma (16SrI) had been detected in two Dianthus species: D. barbatus (Sweet William) and D. caryophyllus (carnation) (2). This is the first record of the 16SrXII-A phytoplasma subgroup being associated with yellowing and reddening of D. barbatus in Serbia. The Stolbur phytoplasma occurrence on Sweet William is significant for the management of the disease in Serbia.

Presence of <I>Dianthus roseoluteus</I> Velen. (Caryophyllaceae) in Turkey and a New Species: <I>Dianthus macroflorus</I> Hamzaoğlu

Abstract The presence of Dianthus roseoluteus Velen. species in Turkey is based on specimens collected from Antalya province. After the publication of Flora of Turkey, many specimens have been collected from Thrace (Kirklareli and Edirne provinces) that belong to this species. A comparison of the specimens collected from Antalya and Thrace revealed that they were morphologically different. The literature survey verified that the specimens collected from Thrace were Dianthus roseoluteus. The species collected from Antalya province is a new species, Dianthus macroflorus Hamzaoglu, and its description, pictures, distribution, habitat, and threatened categories are given. Its general morphology and seed micromorphology are compared with the related species, Dianthus roseoluteus. In addition to phylogenetic relationships between some closely related Dianthus species that belong to section Dentati in Turkey, Dianthus macroflorus and 13 additional Dianthus species sequences obtained from GenBank were examined wi...

Td-DAMD-PCR assays for fingerprinting of commercial carnations

Dianthus L. is one of the highly valued plant species in the family Caryophyllaceae. More than 30,000 cultivars of commercial carnations have been recorded and there is a need for an effective and cheap method to reveal their genetic diversity and identify the cultivars. To the best of our knowledge this is the first report of implementation of the touchdown direct amplification of minisatellite-region DNA polymerase chain reaction (Td-DAMD-PCR) technique in the genus Dianthus. A total of 12 core minisatellite primers empirically selected from 22 primers were used in fingerprinting and phylogenetic studies of some commercial carnation cultivars. Analysis revealed that commercial carnations have a wide genetic base and they were probably obtained using inter-crosses between and among different species of the genus Dianthus. A total of 17 DNA markers were variety specific and most of the remaining markers obtained in the present study were useful in fingerprinting of the commercial varieties studied. Commercial varieties were differentiated in spray and standard carnation groups in a principal coordinate analysis and a Bayesian 50% majority-rule consensus tree. Td-DAMD-PCR markers reported in this study could be very useful in species identification, determination of genetic relationships, and phylogenetic studies of species of Dianthus.

First Report of Carnation vein mottle virus Infecting Dianthus amurensis in China.

Dianthus amurensis, commonly known as Amur Pink, is a species of the genus Dianthus in the family Caryophyllaceae. This perennial Dianthus is also a Chinese medicinal herb. In August 2012, mosaic, leaf cupping, leaf distortion, reduction in leaf size, and flower-breaking symptoms were observed on some D. amurensis plants in a Chinese herb nursery in Changchun, Jilin Province, China. Disease incidences ranged from 40 to 50% in different plots. Symptoms on diseased D. amurensis were similar to those on the diseased D. caryophyllus, D. barbatus, and D. japonicus. The symptoms resembled to those caused by Carnation vein mottle virus (CVMoV), a member of genus Potyvirus in the family Potyviridae (3). CVMoV infects carnations (D. caryophyllus) worldwide and causes a serious disease. To investigate the presence of CVMoV, leaf samples were collected from three symptomatic plants and tested by dot-ELISA using universal potyvirus group monoclonal antibody (Agdia, Inc., Elkhart, IN). The antibody reacted with the diseased samples, supporting the presence of a potyvirus. To identify the potyvirus species, total nucleic acids were extracted from the diseased samples by a CTAB method (2) and used as template in RT-PCR with potyvirus universal primers Sprimer and M4T (1). An amplicon of the expected size (~1.7 kb) was obtained from all three diseased samples. The amplicons were cloned into pMD18-T vector (TaKaRa, Dalian, China) and sequenced. Sequences obtained from all three samples were identical and deposited in GenBank (Accession No. KJ605654). BLAST search showed that the nucleotide sequence shared 97 and 98% identity with a Japanese CVMoV isolate infecting D. japonicus (AB017630) (3) and a Korean CVMoV isolate (AY512554), respectively. The results confirmed the association of CVMoV with the disease on D. amurensis. To determine the pathogenicity of CVMoV to D. amurensis plants, purified CVMoV from the diseased plants were used to inoculate to healthy Dianthus spp. plants. Inoculated Dianthus spp. plants also showed the symptoms of mottle, leaf cupping, and leaf distortion, and CVMoV could be detected by RT-PCR from these plants. The result confirms that CVMoV is the causal agent of the disease. To our knowledge, this is the first report of CVMoV infection in D. amurensis. Since D. amurensis is economically important in China, proper virus management strategies for the cultivation of this crop should be adopted.

Scientific Opinion on objections of a Member State to a notification (Reference C/NL/13/01) for the placing on the market of the genetically modified carnation SHD‐27531‐4 with a modified colour, for import of cut flowers for ornamental use, under Part C of Directive 2001/18/EC from Suntory Holdings Limited

The European Commission asked the Scientific Panel on Genetically Modified Organisms of the European Food Safety Authority (EFSA GMO Panel) to address the objections of Cyprus to the placing on the market of the genetically modified (GM) carnation SHD-27531-4. The GM carnation cut flowers, with a modified petal colour, are intended to be imported and distributed in the European Union for ornamental use only. The EFSA GMO Panel responded to the objections of Cyprus, taking into account the limited intended use of carnation SHD-27531-4 and the information available. First, the EFSA GMO Panel concludes that the propagation of carnation SHD-27531-4 by individuals cannot be excluded. However, should this occur, carnation SHD-27531-4 would not show any potential for increased survival, fitness or weediness compared with its parental line. Second, the EFSA GMO Panel is of the opinion that the potential spread of pollen of the GM carnation SHD-27531-4 by Lepidoptera to wild Dianthus species cannot be eliminated but is highly unlikely to occur and, if it did occur, it is very unlikely that viable hybrids would be produced, survive and result in adverse environmental effects. Third, considering the very low potentials for hybridisation and/or seed production of (GM) carnations, the EFSA GMO Panel concludes that plant-to-plant gene transfer of the introduced genes is very unlikely and, if it did occur, it is unlikely to result in viable seed production leading to adverse environmental effects.

A new serpentine‐adapted carnation (Caryophyllaceae) from Turkey: Dianthus serpentinus sp. nov.

In the framework of a revision of Turkish Dianthus, specimens collected from Kızıldağ (Derebucak, Konya) are described as a new species. The new species is named as Dianthus serpentinus Hamzaoğlu sp. nov., and a description, images, chorology and ecology, and threat category are given. It is compared with the closely related D. acrochlorus, based on its general morphology and seed micromorphology. In addition, the phylogenetic relationship among the closely related species, some Dianthus species belonging to section Dentati from Turkey, D. serpentinus and 14 additional Dianthus species obtained from GenBank are investigated based on DNA sequence data from the nuclear ribosomal internally transcribed spacer region. The results obtained from the molecular analyses supports the morphological data.

Mixed evidence for the cultivar vigour hypothesis: The case of calcareous grassland forbs in a matrix of Festuca rubra

According to the cultivar vigour hypothesis, cultivars are frequently selected for traits such as increased biomass and resource capture, which may hinder the establishment of non-cultivars. Despite this concern, cultivar seed material is still used in grassland restoration. An alternative is wild plant material, which is commercially produced and used in the respective region (‘regional seed’). We established a greenhouse experiment to examine the performance of cultivar and regional forb species with and without competition from cultivar and regional dominant grass species. This was conducted in order to understand the potential effects of sowing cultivar seed in grassland restoration projects. Three calcareous grassland forb species (Buphthalmum salicifolium, Dianthus carthusianorum and Linum perenne) and one dominant grass species (Festuca rubra ssp. commutata) were selected. One forb species was sown in the centre of each pot and either zero, two or four F. rubra ssp. commutata individuals were planted around it. Above-ground biomass, plant height, leaf length, leaf width, number of leaves and stems were measured. When grown without competitors, each forb species performed differently with only L. perenne exhibiting cultivar vigour. Cultivar forbs were less negatively affected by competition with F. rubra ssp. commutata than their regional counterparts, thus supporting the cultivar vigour hypothesis. Overall, regional F. rubra ssp. commutata suppressed the above-ground biomass of the forbs species more than the cultivar F. rubra ssp. commutata, thus highlighting the potential competitiveness of regional seed. Ultimately, the goals of the restoration project will determine the source of seed material to be used in the project. Due to the complex nature of the issues involved in restoration, it should be considered that the reliance on simple guidelines in relation to the source of seed material used in grassland restoration has its limitations.

The effectiveness of asexual and sexual reproduction in clonal species Dianthus superbus L. in different site conditions – the consequences for population development

The effectiveness of asexual and sexual reproduction of rare, clonal species Dianthus superbus L. were conducted in the years 2008-2010 in Kostrze (Southern Krakow). The observations were carried out in Molinietum caeruleae patches dominated by small species creating delicate, erect or procumbent stems (MC) , prevailed by tall grasses (GR), and overgrown by willows (SA). The number of above-ground units per ramet cluster were much greater in MC patch, than in sites GR and SA. Controversially, the number of fruit per generative shoot and seed production were greater in GR and SA patches, than in MC patch. The highest seedling recruitment, occurring only in artificially created gaps in plant canopy and litter was noted in patch MC, while the decline of genet emergence were found in SA and GR patches. In light of performed studies, it should be concluded, that population established in patch dominated by small meadow species has the greatest chance for survival, whereas populations inhabiting sites prevailed by tall grasses or overgrown by willows have much lower possibilities of persistence in occupied places

Micropropagation of the Endangered and Decorative Specie <i>Dianthus serotinus</i> Waldst. et Kit.

During past decades, great attention has been paid to propagation of endangered plant taxa in order to preserve biodiversity. The aim of this study was to optimize a protocol for in vitro propagation of the critically endangered and decorative species Dianthus serotinus Waldst. et Kit. The effects of different concentration of MS salt (Murashige and Skoog) of the culture, medium pH and different carbohydrates (sucrose, glucose, and fructose) on shoot multiplication were examined. The best results were obtained on half-strength MS (Murashige and Skoog) medium, whose pH was 5.8, with sucrose supplied at a concentration of 3%, when shoots with 1-2 nodes or shoot tips (with terminal buds only) were used as explants. The shoots were rooted (76.7%) on half-strength MS medium containing 0.5 mg∙L-1 NAA (1-naphthaleneacetic acid). The obtained plantlets were successfully acclimatized (89%) in a 4:1 mixture of peat and sand and they flowered the following year. Presented protocol enables successful in vitro propagation of D. serotinus.

The genus Aphidura (Hemiptera, Aphididae) in the collection of the Muséum national d’Histoire naturelle of Paris, with six new species

Specimens were studied of 65 samples of the genus Aphidura (Aphididae, Aphidinae, Macrosiphini) from the collection of the Muséum national d’Histoire naturelle (Paris). The possible synonymies of three pairs of species are discussed. New aphid host plant relationships are reported for Aphidura bozhkoae, Aphidura delmasi, Aphidura ornata, Aphidura pannonica and Aphidura picta; this last species is recorded for first time from Afghanistan. The record of Aphidura pujoli from Pakistan is refuted. The fundatrices, oviparous females and males of Aphidura delmasi are described. Six new species are established: Aphidura gallica sp. n. and Aphidura amphorosiphon sp. n. from specimens caught on species of Silene (Caryophyllaceae) from France and Iran, respectively, Aphidura pakistanensis sp. n., Aphidura graeca sp. n. and Aphidura urmiensis sp. n. from specimens caught on species of Dianthus, Gypsophila and Spergula (Caryophyllaceae) from Pakistan, Greece and Iran, respectively, and Aphidura iranensis sp. n. from specimens caught on Prunus sp. from Iran. Modifications are made to the keys by Blackman and Eastop to aphids living on Dianthus, Gypsophyla, Silene, Spergula and Prinsepia and Prunus (Rosaceae). An identification key to apterous viviparous females of species of Aphidura is also provided.

Ex situ Conservation of Three Endemic and/or Endangered Dianthus Species

Within the current context of declining biodiversity, the botanical gardens play an essential role in its conservation. Dianthus callizonus, D. glacialis ssp. gelidus and D. spiculifolius are the species that we seek to preserve in "Alexandru Borza" Botanical Garden of Cluj-Napoca (Romania). Several replicates were collected for each taxon from different populations in order to avoid the genetic uniformity. The material collected from the natural sites, was planted on a rockery, specially designed for this collection in the Botanical Garden. At the time of planting, each individual was sampled for setting up an in vitro collection and further biochemical and molecular analyses. In case of ex situ outdoor conservation of the three Dianthus species, 80.6% of the individuals collected in the field survived during the first year but the percentage decreased drastically after four years. In the case of in situ collected individuals, as well as in the case of in vitro individuals, D. spiculifolius had the best ability to acclimatize in the Botanical Garden, and D. callizonus presented the lowest number of surviving individuals. The ex vitro acclimatization of the plantlets had 80% efficiency at 10ºC, using three different substrates: soil and pearl stone mix 1/1, soil and sand mix 1/1 and pearl stone. All the three species are preserved in vitro, whereas the plantlets are acclimatized outdoors. Ex situ conservation of these species will have a positive impact on the biodiversity conservation.

Palatinose-hydrolyzing Activity and Its Relation to Modulation of Flower Opening in Response to the Sugar in Dianthus Species

Palatinose (isomaltulose) is an analog of sucrose and was regarded as non-metabolizable in plant tissues until recently. In the present study, we found that crude extracts from carnation petals had activity to hydrolyze palatinose. Preliminary characterization of this activity using a crude enzyme extract from ‘Lillian’ carnation petals revealed that hydrolyzing activity was exhibited by α-glucosidase, which uses isomaltose and palatinose, both being α-1,6-glucosides, as substrates. Exogenous application of palatinose stimulated flower opening of carnation cultivars (Dianthus caryophyllus ‘Lillian’, ‘Pure Red’, and ‘Light Pink Barbara’), but suppressed it in D. barbatus ‘Shin-higuruma’. Palatinose-hydrolyzing activity was much higher in the extract from carnation than that from D. barbatus. These observations suggested that palatinose stimulated flower opening in carnation by supplying glucose and fructose, but suppressed it in D. barbatus, probably through the inhibition of general metabolism, similar to the action of α-glucosidase, caused by its excess accumulation.

Analysis of Scents Emitted from Flowers of Interspecific Hybrids between Carnation and Fragrant Wild Dianthus Species

Most modern carnation (Dianthus caryophyllus L.) cultivars have weak fragrances dominated by the scent of methyl benzoate. Wild Dianthus species with strong or unique scents may be useful gene resources for the improvement of carnation fragrances. We investigated the scents of interspecific hybrids between carnations and fragrant wild species by gas chromatography-mass spectrometry (GC-MS), and evaluated the usefulness of wild species for fragrant breeding in carnations. Dianthus hungaricus, which produced large amounts of various benzenoids, was crossed with a carnation with a floral scent dominated by methyl benzoate, but benzenoid diversity was not increased in the interspecific hybrid. We also analyzed some existing interspecific hybrids. Dianthus superbus var. longicalycinus had high amounts of β-ocimene and β-caryophyllene. These terpenoids were acquired as principal scent compounds by some interspecific hybrids between this species and a carnation lacking terpenoids. Three unidentified wild species (Dianthus sp. 4, 5, and 6) emitted high amounts of benzenoids, including eugenol, benzyl alcohol, methyl o-anisate, and methyl salicylate. These benzenoids were also detected in interspecific hybrids between carnations and the wild species, and the amounts were increased compared to the parental carnation. The emission of these scent compounds of wild Dianthus species was inherited by most hybrids lines; the variety and amounts of scent compounds tended to increase compared to parental carnations, although there was no general hereditary pattern. As we actually sensed the fragrances of the principal compounds from some hybrid flowers, the usefulness of interspecific hybridizations for the improvement of flower fragrances was confirmed. Dianthus superbus var. longicalycinus and Dianthus sp. 4, 5, and 6 seemed promising resources regarding the addition of terpenoids and the increase in benzenoid variation in the floral volatiles of carnations.

The effectiveness of asexual and sexual reproduction in clonal species Dianthus superbus L. in different site conditions – the consequences for population development

The effectiveness of asexual and sexual reproduction in clonal species Dianthus superbus L. in different site conditions – the consequences for population development

Evaluation of ploidy level and endoreduplication in carnation (Dianthus spp.)

Carnation (Dianthus caryophyllus L.) is one of the fifth most important ornamental species worldwide. Many desirable plant characteristics, such as big size, adaptation under stress, and intra or interspecific hybridization capability, are dependent on plant ploidy level. We optimized a quick flow cytometry method for DNA content determination in wild and cultivated carnation samples that allowed a systematic evaluation of ploidy levels in Dianthus species. The DNA content of different carnation cultivars and wild Dianthus species was determined using internal reference standards. The precise characterization of ploidy, endoreduplication and C-value of D. caryophyllus ‘Master’ makes it a suitable standard cultivar for ploidy level determination in other carnation cultivars. Mixoploidy was rigorously characterized in different regions of several organs from D. caryophyllus ‘Master’, which combined with a detailed morphological description suggested some distinctive developmental traits of this species. Both the number of endoreduplication cycles and the proportion of endopolyploid cells were highly variable in the petals among the cultivars studied, differently to the values found in leaves. Our results suggest a positive correlation between ploidy, cell size and petal size in cultivated carnation, which should be considered in breeding programs aimed to obtain new varieties with large flowers.

Contrasting phylogenetic patterns of anther smuts (Pucciniomycotina: Microbotryum) reflect phylogenetic patterns of their caryophyllaceous hosts

Anther smuts in the genus Microbotryum often show very high host specificity toward their caryophyllaceous hosts, but some of the larger host groups such as Dianthus are crucially undersampled for these parasites so that the question of host specificity cannot be answered conclusively. In this study we sequenced the internal transcribed spacer (ITS) region of members of the Microbotryum dianthorum species complex as well as their Dianthus hosts. We compared phy- logenetictreesoftheseparasitesincludingsequencesofanther smuts from other Caryophyllaceae, mainly Silene ,w ith phy- logenies of Caryophyllaceae that are known to harbor anther smuts. Additionally we tested whether observed patterns in parasitesareduetosharedancestryorifgeographic separation is a factor that should be taken into consideration in delimitat- ing species. Parasites on Dianthus showed mainly an arbitrary distribution on Dianthus hosts, whereas parasites on other Caryophyllaceae formed well-supported monophyletic clades that corresponded to restricted host groups. The same pattern was observed in the Caryophyllaceae studied: morphological- ly described Dianthus species did not correspond well with monophyletic clades based on molecular data, whereas other Caryophyllaceae mainly did. We suggest that these different patterns primarily result from different breeding systems and speciation times between different host groups as well as difficulties in species delimitations in the genus Dianthus.

Green Roof Substrate Type and Depth Affect the Growth of the Native Species Dianthus fruticosus Under Reduced Irrigation Regimens

Extensive green roofs are a promising technology for reintroducing lost flora in degraded urban environments, but further research is needed for their application in semiarid regions. Therefore, research was undertaken to determine the effects of substrate type and depth and the amount of irrigation during a drought period on the establishment, growth, and physiology of the native species Dianthus fruticosus sub. fruticosus. Treatments included two substrate types [a soilless substrate (Pum50:Per20:C20:Z10) or a substrate with soil (S15:Pum40:Per20:C20:Z5), in which Pum = pumice; Per = perlite; C = compost; Z = clinoptinolite zeolite; and S = sandy loam soil, mixed in a volumetric proportion indicated by their subscripts], two substrate depths (7.5 cm or 15.0 cm), and two irrigation regimens during drought [15% or 30% of pan evaporation (Epan)]. Measurements included substrate characteristics such as particle size distribution, dry and saturated bulk density, water characteristic curves, and in situ determination of substrate moisture during drought stress. Plant growth was determined based on biometric measurements such as growth index (GI) and dry weight and physiological indicators such as SPAD, chlorophylla+b, and carotenoid contents. It was found that substrate moisture during drought was increased in the soil substrate compared with the soilless substrate as a result of its better water retention capacity in low tensions. Dianthus fruticosus sub. fruticosus growth was promoted by the deep substrate (15 cm) throughout the entire study, whereas substrate type and irrigation during the drought period did not have an effect. Similarly, leaf dry weight was increased in the deeper substrates, whereas shoot and root dry weights were similar in all treatments. SPAD was found to be a more sensitive method than chlorophyll and carotenoid analysis and revealed an interesting sequence of treatment influences on D. fruticosus sub. fruticosus physiology that depended on the climatic conditions and stress imposition. More specifically, during establishment, both substrate type and depth affected growth with the soil substrate and deep profiles yielding higher SPAD measurements. Soon after the initiation of drought, the deep profiles had higher SPAD values than the shallow ones, whereas in high-irrigation regimens and, to a less extent, deeper profiles provided increased SPAD values after the middle of drought imposition. Chlorophyll and carotenoid levels reduced during the drought stress period, but very limited differences were detected between treatments. It was concluded that D. fruticosus sub. fruticosus is a very promising native plant for use on extensive green roofs in the Mediterranean region, and its growth was better in a substrate depth of 15 cm. However, its growth was sufficient even with a 7.5-cm substrate depth and irrigation of 15% Epan.

A new species of Dianthus L. (Caryophyllaceae) from the West Sayan, Altai‐Sayan Mountains, Russia

AbstractA new species Dianthus mainensis (sect. Chinenses), which is endemic to the West Sayan is described and illustrated. The species is most close to D. versicolor but differs from the latter in a number of characters (e.g., leaf and stem indumentum; shape and pubescence of epicalyx scales; petal color). Similarly to the other members of this group, D. mainensis is an obligate petrophyte and grows on outcrops of carbonate rocks. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Effects of genotype and stigma development stage on seed set following intra- and inter-specific hybridization of Dianthus spp.

The caryophyllaceae is a family of flowering plants commonly known as the pink or carnation family. Members of this family are widely grown as ornamental plants. The species Dianthus chinensis, Dianthus barbatus, and Dianthus superbus include notable examples of such ornamental varieties. Intra- and inter-specific hybridization is important for the breeding of improved varieties and, therefore, it is desirable to optimize the efficiency of cross-pollination procedures within this family. Here, we conducted intra-specific pollination using four lines of D. chinensis, and inter-specific pollination between D. chinensis, D. barbatus and D. superbus genotypes. For each of these crosses we recorded information of pollen viability, stigma development stage at time of pollination, pollen germination on the stigma surface and pollen tube elongation within the stigma, and final seed set. Pollen viability levels were determined at the start of anther dehiscence, which occurred on day 1 of flower opening. The genotypes D. chinensis var. ‘Bz2’, D. barbatus var. ‘Xb’, and D. superbus wild species ‘Qm’ all displayed good levels of pollen viability (i.e. between 68% and 75%); D. chinensis vars. ‘H58III’, ‘H68I’ and ‘H6II’ showed lower levels of pollen viability (i.e. between 32% and 51%). In each Dianthus line, the stigma organs were observed to undergo morphological changes during the 1–7 d following flower opening. We have characterized these changes as five distinct development stages, namely, ‘column’ (day 1), ‘Y-like’ (day 2), ‘Y-shaped with slight curve’ (days 3–4), ‘Y-shaped horn-like’ (days 5–6), and ‘wilting’ (day 7). Pollen was artificially applied to stigma organs at each of these development stages and aniline blue staining was used to follow pollen tube germination on the stigma surface and pollen tube elongation down the length of the stigma. This showed that the ‘Y-shaped slightly curved’ stigma stage (3–4 d) was optimal for pollination. The ‘Y-shaped horn-like’ stage (5–6 d) was also receptive to pollination. By contrast, the earlier ‘Y-shaped’ (2 d) and ‘column’ (1 d) stages, and also the later ‘wilting’ (7 d) stage, were far less receptive. Parallel field experiments were conducted in which seed set was measured following the artificial pollination of flowers at different development stages. These data also indicated that the optimal time for pollination was during 3–4 d of flower opening. This conclusion held true for both intra- and inter-specific crosses of the various Dianthus lines. Thus, stigma receptivity in Dianthus does not become optimal until 3–4 d after flower opening. Since anther dehiscence occurs at 1 d (i.e. when stigma are at the unreceptive ‘column’ stage), this suggests that Dianthus employs a delayed self-fertilization strategy. We also found a significant effect of genotype on pollen tube growth and seed-set values. The highest numbers of seed per flower were achieved when the D. superbus wild species ‘Qm’ was the female parent in inter-specific crosses. Thus, genotype and stigma development are identified as key factors that determine the success of cross-pollination in Dianthus lines.

Possible Origin of Two Variants of a Carnation 1-Aminocyclopropane-1-carboxylate Synthase Gene, DcACS1a and DcACS1b, as Suggested by Intron Structure in Homologous Genes in Dianthus Species

The intron structures of two variants of 1-aminocyclopropane-1-carboxylate synthase (ACS) genes (DcACS1a and DcACS1b) in carnation (Dianthus caryophyllus) and genes homologous to them (ACS1 homologous genes) in other 10 Dianthus species (16 strains in total) were studied by comparing the sizes of the PCR amplificates and nucleotide sequence of the introns. All 16 sequenced homologous ACS1 genes, including DcACS1 genes themselves, had five exons and four introns. The exons had similar nucleotide sequences and consequently similar deduced amino-acid sequences. The sizes of three introns (intron-1, -2, -3) were variable among the homologous genes, whereas that of the fourth intron (intron-4) was almost identical. The variation in introns was probably caused by the insertion (and deletion) of nucleotide fragments of given lengths. Interestingly, the 3'-UTR of DcACS1a was different from that of DcACS1b, and the latter was similar to other 14 ACS1 homologous genes. Moreover, the length of Thr repeat in the C-terminal region was long in DcACS1a protein but short in DcACS1b protein, and the latter resembled ACS1 homologous proteins in other Dianthus species. The present findings suggest that (1) the variation in intron structure between two variants of carnation DcACS1 is reminiscent of the variation that occurred universally in Dianthus species, (2) DcACS1a is probably a gene intrinsic to carnation, and (3) DcACS1b was acquired from another, as yet unknown, Dianthus species, in the course of breeding modern carnation cultivars.