J. Japan. Soc. Hort. Sci. 82 (2): 145–153. 2013. Available online at www.jstage.jst.go.jp/browse/jjshs1 JSHS © 2013
Analysis of Scents Emitted from Flowers of Interspecific Hybrids between Carnation and Fragrant Wild Dianthus Species
Kyutaro Kishimoto, Masafumi Yagi, Takashi Onozaki, Hiroyasu Yamaguchi, Masayoshi Nakayama and Naomi Oyama-Okubo*
NARO Institute of Floricultural Science, Tsukuba 305-8519, Japan
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.
Key Words: carnation, Dianthus, floral scent, GC-MS, interspecific hybrid.
however, in most modern carnations, scent variety and Introduction intensity have been lost, and methyl benzoate has become Fragrance is an important property of marketable the dominant scent component (Clery et al., 1999). ornamental flowers (Dudareva and Pichersky, 2006). Therefore, the commercial value of modern carnations In questionnaire surveys conducted in Osaka and could be improved by increasing the amount of total Wakayama, Japan in 1993 and 1994, 20% of consumers scent substances as well as the introduction of scents chose “fragrance” as an important quality of cut flowers other than methyl benzoate. for gifts (Tsuji, 2000). Carnations (Dianthus caryophyllus Dianthus hungaricus Pers is a fragrant wild species L.) are popular ornamentals and are mainly used as cut that produces various benzenoids (Kishimoto et al., flowers. Their scents are composed of benzenoids, 2011). We crossed D. hungaricus with a modern terpenoids, fatty acid derivatives, and other minor carnation cultivar and obtained an interspecific hybrid components (Clery et al., 1999; Hudak and Thompson, line. In addition, we studied interspecific hybrids that 1997; Schade et al., 2001; Zuker et al., 2002). Classical had been bred by Onozaki et al. (2011) and in our other fragrant carnations possess a spicy and clove-like odor works, derived from crosses of modern carnations and caused mainly by benzenoids (Clery et al., 1999); some other fragrant wild species. These hybrids lend themselves to the study of the heredity of fragrances. Received; August 27, 2012. Accepted; November 21, 2012. We analyzed the scents of the interspecific hybrids and * Corresponding author (E-mail: [email protected]). the parental lines by gas chromatography-mass
145 146 K. Kishimoto, M. Yagi, T. Onozaki, H. Yamaguchi, M. Nakayama and N. Oyama-Okubo spectrometry (GC-MS), and evaluated the usefulness of The trapped volatiles were analyzed using GC-MS wild species for fragrant carnation breeding. (Agilent 5973; Agilent Technologies, Wilmington, DE, USA) coupled to a Thermal Desorption System 2 Materials and Methods (Gerstel Inc., Linthicum, MD, USA) (Oyama-Okubo et Plant material al., 2011). The thermal desorption conditions were Dianthus caryophyllus cv. ‘Miracle Symphony’ heating from 30°C to 250°C at 60°C·min−1, holding for (Onozaki et al., 2006) and Carnation (D. caryophyllus) 10 min at 250°C, and cryofocusing at −100°C in the cold 229-1 (Onozaki et al., 2011) were bred in the Institute injection system (CIS; Gerstel Inc.). Following of Floricultural Science, National Agriculture and Food desorption of the Tenax column, the CIS was heated to Research Organization (NIFS) in Tsukuba, Japan. 300°C at a rate of 12°C·s−1 in splitless mode to transfer Dianthus hungaricus (accession number 59) is main- the analytes to the gas chromatograph (GC) equipped tained as a gene resource at the NIFS gene bank. with a capillary DB-WAX column (30 m length, 0.25 mm Dianthus superbus L. var. longicalycinus F. N. Williams i.d., 0.25 μm film thickness; Agilent Technologies). was sampled in Akita, Japan (Onozaki, 2001). Dianthus Helium was used as a carrier gas at a flow rate of sp. 4 (accession number 142A-2), Dianthus sp. 5 1.0 mL·min−1. The temperature program of the oven was (accession number 242B-1), and Dianthus sp. 6 set to 45°C for 2 min, then increased by 3°C·min−1 to (accession number 13) are unidentified wild species 220°C, and kept at this temperature for 10 min. Interface maintained as gene resources at the NIFS gene bank. and ion source temperatures were 280°C and 250°C, OB44 is an interspecific hybrid between ‘Miracle respectively. Ionization was performed in electron Symphony’ and D. hungaricus (pollen parent). Lines of impact mode at 70 eV, and a mass scan range of 30– 4K38 (4K38-2, -3, -5, -6, -10, -11, -14, and -15) are 350 m/z was monitored. Volatile compounds were interspecific hybrids between 229-1 and D. superbus identified using the NIST02 library search system var. longicalycinus (pollen parent) (Onozaki et al., provided with GC-MS software (Agilent Technologies) 2011). 10W02-1 is an interspecific hybrid between and crosschecked by comparing the mass spectra and D. caryophyllus cv. ‘Ai Pink’ (Fuji-Plants Co., Aichi, retention times with authentic samples analyzed under Japan) and Dianthus sp. 4 (pollen parent). Lines of the same conditions. The amount of each compound was 10W10-1 and -2 are interspecific hybrids between calculated based on the corresponding peak area of total D. caryophyllus cv. ‘Nagisa’ (Fuji-Plants Co., Aichi, ion chromatography of its authentic sample. The amount Japan) and Dianthus sp. 5 (pollen parent). Lines of of isoelemicin was calculated based on the peak area of 10W13 (10W13-1, -2, -3, -4, and -5) are interspecific elemicin’s total ion chromatography. The sensual feature hybrids between D. caryophyllus cv. ‘Shikibu’ (Fuji- of major scent compound of each flower scent was Plants Co., Aichi, Japan) and Dianthus sp. 6 (pollen presented by referring to a fragrance encyclopedia edited parent). These wild species were used as pollen parents by Burdock (2010). because carnation pollen is rarely obtained. Wild Results and Discussion Dianthus were grown in unheated greenhouses at NIFS. Carnations and their hybrids were grown in heated 1. An interspecific hybrid of carnation and D. hungaricus greenhouses at NIFS under standard growing conditions Methyl benzoate was the dominant scent compound for carnations. in carnation ‘Miracle Symphony’, contributing 97% of Hybridity of progenies between carnations and wild the total volatiles (Table 1). In contrast, D. hungaricus Dianthus was verified by SSR analysis. Genomic DNA (Fig. 1) emitted various kinds of benzenoids, with methyl was extracted from fresh young leaves using the DNeasy salicylate being one of the most important odor-active Plant Mini Kit (Qiagen GmbH, Hilden, Germany). Four compounds (Table 1) (Kishimoto et al., 2011). ‘Miracle SSR markers (DCB140, DCB135, DCB131, DCB109) Symphony’ and D. hungaricus had a weak fruity scent reported by Smulders et al. (2003) were used. PCR and a minty or medicinal scent derived from their amplification and the following electrophoresis were dominant volatiles, methyl benzoate and methyl performed according to the methods of Yagi et al. (2006). salicylate, respectively. We crossed the two plants to see which scent compounds were produced in the Analysis of emitted floral volatiles interspecific hybrids. The volatiles emitted from flowers on the second day Crossing was performed five times and several seeds of blooming were collected using a dynamic headspace were obtained (Table 2). One seed successfully sampling system (Oyama-Okubo et al., 2005). Freshly developed to the flowering stage (Table 2). We confirmed cut flowering branches were covered with a Tedlar Bag its hybridity (data not shown) and designated it OB44 (500 mL volume; GL Science, Tokyo, Japan). A constant (Fig. 1). The total volatile emission of OB44 was similar stream of air filtered through activated charcoal was to that of ‘Miracle Symphony’ and amounted to about pumped through the bag at a flow rate of 500 mL·min−1. 7% of that of D. hungaricus (Table 1). Methyl benzoate The volatiles were trapped on a Tenax column (180 mg; was the single dominant volatile in OB44, at about the GL Science) in a glass tube. same level observed in ‘Miracle Symphony’ (Table 1). J. Japan. Soc. Hort. Sci. 82 (2): 145–153. 2013. 147
Table 1. Amounts of scent compounds emitted from flowers of carnation ‘Miracle Symphony’, D. hungaricus, and their interspecific hybrid OB44.
Carnation Wild Dianthus Interspecific hybrid Compounds ‘Miracle Symphony’ D. hungaricus OB44 Benzenoids Benzyl acetate —z 1.2 ± 0.6y,x — Benzyl alcohol tracew 5.5 ± 1.2 — Benzyl benzoate trace 0.8 ± 0.2 — Benzyl tiglate — 0.7 ± 0.1 — Cinnamyl acetate — 0.8 ± 0.0 — Cinnamyl alcohol — 4.5 ± 0.6 — Cinnamyl aldehyde — 0.5 ± 0.1 — Elemicin — 1.5 ± 0.1 trace Eugenol — 0.6 ± 0.1 trace Hexyl benzoate — 0.6 ± 0.1 — Isoelemicin — 5.0 ± 1.3 0.1 ± 0.0 Isoeugenol trace 5.3 ± 0.7 — Methyl o-anisate — 0.6 ± 0.1 — Methyl benzoate 3.7 ± 0.6 16.6 ± 3.1 4.1 ± 0.3 Methyl eugenol trace 0.6 ± 0.0 — Methyl isoeugenol trace 2.5 ± 0.8 — Methyl salicylate trace 15.3 ± 6.1 trace Phenylacetaldehyde — 0.6 ± 0.1 — Others — 0.4 ± 0.0 — Terpenoids β-Ocimene — 0.8 ± 0.1 — Others — 0.2 ± 0.0 — Fatty acid derivatives (Z)-3-Hexenyl benzoate trace 0.1 ± 0.0 trace Total 4.1 ± 0.7 66.1 ± 2.8 4.3 ± 0.6 z not detected. y nmol·g FW−1·h−1. x Indicates SE (n = 3). w trace < 0.1. Bold values indicate principal scent compounds in each flower.
D. hungaricus as a useful hybridization partner for fragrant carnation breeding.
2. Interspecific hybrids of carnation and D. superbus var. longicalycinus Total volatiles emitted by Carnation 229-1 (Fig. 2) were dominated by methyl benzoate (89%) and lacked terpenoids (Table 3). Dianthus superbus var. longicalycinus (Fig. 2) had high amounts of β-ocimene and β-caryophyllene, amounting to 67% of the total volatiles (Table 3). Benzenoids were also present but methyl benzoate was not detected (Table 3). The total amount of volatiles emitted by D. superbus var. longicalycinus was about 103 times higher than that of 229-1 (Table 3). Carnation 229-1 and D. superbus var. longicalycinus had a weak fruity scent and a woody scent derived from their dominant volatiles, methyl benzoate and two terpenoids, respectively. We expected Fig. 1. An interspecific hybrid (OB44) between carnation ‘Miracle that interspecific hybrid lines between 229-1 and Symphony’ and D. hungaricus. Bar = 10 mm. D. superbus var. longicalycinus (4K38; Onozaki et al., 2011) would emit terpenoids as well as benzenoids including methyl benzoate. The sensual scent was weak and similar to that of The total amounts of volatiles in these lines were ‘Miracle Symphony’. These findings did not suggest about 2–15 times higher than in 229-1, equaling 2–15% 148 K. Kishimoto, M. Yagi, T. Onozaki, H. Yamaguchi, M. Nakayama and N. Oyama-Okubo
Table 2. Results of interspecific hybridization between carnation cultivars and fragrant wild Dianthus.
Cross combination No. of flowers No. of capsules No. of seeds No. of seeds No. of normal z Carnations (seed parent) Wild Dianthus (pollen parent) pollinated setting obtained germinated flowering ‘Miracle Symphony’ × D. hungaricus 51 511 ‘Ai Pink’ × Dianthus sp. 41 12411 ‘Nagisa’ × Dianthus sp. 51 12122 ‘Shikibu’ × Dianthus sp. 62 21575y z All seeds were sown. y Two other seedlings grew but produced no or few flowers.
methyl benzoate in 4K38-14. In the parent lines, the proportions of these volatiles were low, except for methyl benzoate. The amount of the principal benzenoid, except for methyl salicylate, in each hybrid line was higher than in the parental lines. Thus, several benzenoids, in particular benzyl benzoate, elemicin, and eugenol, often increased following hybridization with the wild species D. superbus var. longicalycinus. Line of 4K38-5 had a slightly different scent from the parental carnation. The sensual scent of 4K38-10 and -14 was similar to that of the parental carnation, probably derived from methyl benzoate. The sensual scent of other hybrid lines was weak.
3. Interspecific hybrids of various carnation cultivars and unidentified Dianthus species Methyl benzoate was the dominant volatile (75–85%) in ‘Ai Pink’, ‘Nagisa’, and ‘Shikibu’ (Table 4). Limonene was the second most abundant volatile (15%) in ‘Ai Pink’ (Table 4). In contrast, various benzenoids were Fig. 2. Interspecific hybrids between carnation 229-1 and D. superbus detected in the three wild Dianthus species (Table 4). var. longicalycinus. A, parental lines; B, interspecific hybrid These carnations had weak fruity scent derived from lines. Bar = 10 mm. methyl benzoate. The proportion of isoeugenol in the total volatile was the highest (16%) followed by methyl of that of D. superbus var. longicalycinus. All volatiles isoeugenol (11%) in Dianthus sp. 4 (Table 4). In detected in the parental lines were found also in the Dianthus sp. 5, benzyl alcohol was most abundant (32%) hybrids. β-Ocimene and β-caryophyllene were detected followed by methyl salicylate and limonene (18% and in all hybrid lines although their amounts differed 9%, respectively). Methyl o-anisate (30%) and methyl (Table 3). These terpenoids were the principal volatiles benzoate (21%) dominated in Dianthus sp. 6 (Table 4). in the three hybrid lines, 4K38-2, -11, and -15 (Table 3). The total amounts of volatiles emitted by these wild The total amounts of terpenoids in these hybrid lines species were 23- to 38-fold higher than those produced were less than 20% of those emitted by D. superbus var. by the carnations. Dianthus sp. 4 had a spicy scent longicalycinus, but this was fully sufficient to allow derived from eugenol and eugenol derivatives. Dianthus sensing their presence due to their characteristic woody sp. 5 and 6 had each characteristic scent probably derived scent. These results indicated that D. superbus var. from each principal benzenoid. We investigated whether longicalycinus was a promising hybridization partner for fragrant properties of the wild species were transferred the introduction of β-ocimene and β-caryophyllene into to interspecific hybrids. the spectrum of scent substances of carnation flowers. Dianthus sp. 4 was crossed with ‘Ai Pink’ and about Other hybrid lines produced certain benzenoids or the 20 seeds were obtained (Table 2). One plant, designated fatty acid derivative, 1-octen-3-ol, as the principal 10W02-1, matured to the flowering stage (Table 2) and volatiles (Table 3). In many cases, the amounts of a its hybridity was confirmed (data not shown; Fig. 3). particular substance emitted in the different lines The flower of 10W02-1 had a strong minty scent like a differed. The most abundant compounds were methyl winter green. The total amount of volatiles of 10W02- salicylate in 4K38-3, benzyl benzoate and elemicin in 1 was about 9 times higher than that of ‘Ai Pink’, but 4K38-5, 1-octen-3-ol and eugenol in 4K38-6, methyl only about 28% of that of Dianthus sp. 4 (Table 4). The benzoate and eugenol in 4K38-10, and 1-octen-3-ol and dominant volatiles were benzenoids, particularly methyl J. Japan. Soc. Hort. Sci. 82 (2): 145–153. 2013. 149
Table 3. Amounts of scent compounds emitted from flowers of carnation 229-1, D. superbus var. longicalycinus, and their interspecific hybrids.
Carnation Wild Dianthus Interspecific hybrid lines Compounds D. superbus 229-1 var. 4K38-2 4K38-3 4K38-5 4K38-6 4K38-10 4K38-11 4K38-14 4K38-15 longicalycinus Benzenoids Benzyl benzoate —z 0.1 ± 0.1y,x 0.2 ± 0.0 tracew 1.4 ± 0.5 0.1 ± 0.0 0.2 ± 0.1 0.1 ± 0.1 0.1 ± 0.0 0.2 ± 0.1 Elemicin — 0.5 ± 0.1 trace trace 5.6 ± 1.0 trace trace trace 0.2 ± 0.0 0.2 ± 0.1 Eugenol trace 0.2 ± 0.1 trace — 0.2 ± 0.1 0.3 ± 0.1 2.2 ± 0.6 trace trace trace Methyl benzoate 0.7 ± 0.3 —0.8 ± 0.2 trace 0.4 ± 0.1 trace 7.0 ± 0.2 0.4 ± 0.1 3.4 ± 0.7 trace Methyl salicylate trace 4.3 ± 0.6 0.3 ± 0.1 0.7 ± 0.1 0.2 ± 0.0 0.2 ± 0.0 0.9 ± 0.2 trace 0.6 ± 0.1 — Others trace 14.4 ± 0.2 0.2 ± 0.0 0.2 ± 0.0 1.1 ± 0.2 0.4 ± 0.1 0.8 ± 0.2 0.2 ± 0.1 0.4 ± 0.1 0.3 ± 0.0 Terpenoids a-Caryophyllene — 0.6 ± 0.0 — — trace — — — — — β-Caryophyllene — 10.4 ± 0.3 1.7 ± 0.3 trace 0.3 ± 0.0 0.2 ± 0.0 0.1 ± 0.0 0.6 ± 0.3 0.6 ± 0.3 0.5 ± 0.0 β-Ocimene — 44.5 ± 4.1 6.6 ± 0.9 0.3 ± 0.1 0.2 ± 0.1 trace 0.1 ± 0.0 0.7 ± 0.4 0.8 ± 0.2 0.1 ± 0.0 Others — 0.4 ± 0.1 0.1 ± 0.0 — 0.1 ± 0.0 trace — trace trace trace Fatty acid derivatives (Z)-3-Hexenol — 7.9 ± 0.3———————— (Z)-3-Hexenyl acetate — 8.9 ± 1.9 — trace — trace trace — trace trace 1-Octen-3-ol — 2.8 ± 0.2 0.4 ± 0.1 — — 0.5 ± 0.0 0.6 ± 0.1 — 4.5 ± 0.2 — Others trace 0.1 ± 0.0 trace trace 0.1 ± 0.0 trace trace trace trace trace Total 0.8 ± 0.3 82.1 ± 5.3 10.5 ± 1.3 1.4 ± 0.3 9.5 ± 1.1 1.9 ± 0.4 12.1 ± 0.1 2.2 ± 1.0 6.7 ± 1.4 1.4 ± 0.1 z not detected. y nmol·g FW−1·h−1. x Indicates SE (n = 3). w trace < 0.1. Bold values indicate principal scent compounds in each flower. benzoate (Table 4). Methyl salicylate was the second level of 10W10-2 was five-fold that of Dianthus sp. 5. most abundant volatile in 10W02-1 and exceeded the The scent of 10W10-1 and 2, dominated by methyl levels detected in the parental lines by 760- and two- benzoate and benzyl alcohol, was stronger than that of fold, respectively (Table 4), which explained its winter ‘Nagisa’ but was qualitatively similar. This result green-like minty scent (Burdock, 2010). This result is suggests that crossing carnations with Dianthus sp. 5 remarkable since methyl salicylate has not been reported could increase the floral emission of benzenoids, previously as a major scent compound of carnation especially benzyl alcohol and methyl benzoate. (Clery et al., 1999; Hudak and Thompson, 1997; Schade Methyl salicylate, which was the second most et al., 2001; Zuker et al., 2002). On the other hand, abundant scent compound in Dianthus sp. 5, was isoeugenol and methyl isoeugenol, principal volatiles in decreased by half in 10W10 lines (Table 4). Limonene, Dianthus sp. 4, were minor scent compounds in 10W02- the third principal volatile in Dianthus sp. 5, was absent 1 (Table 4). Benzyl acetate, benzyl alcohol, and veratrol, or present in small amounts in the 10W10 hybrids which were not detected in the parents, were emitted (Table 4). β-Caryophyllene and caryophyllene oxide, from 10W02-1 (Table 4). These benzenoids probably did which were not detected in the parental plants, were not contribute to the noticeable scent of 10W02-1 released by 10W10 flowers at levels higher than those because of the low emission levels. We concluded that found in 4K38-11 and -15 (Tables 3 and 4); however, Dianthus sp. 4 was a promising hybridization partner we could not sense β-caryophyllene in the 10W10 lines, enabling the introduction of methyl salicylate to the probably due to masking by the rich benzenoids. flower scent spectrum of carnations. Two crosses between Dianthus sp. 6 with ‘Shikibu’ Crossing of Dianthus sp. 5 and ‘Nagisa’ yielded about yielded 15 seeds, five of which succeeded in growing 20 seeds (Table 2). Two plants could be raised and to the flowering stage (Table 2). We confirmed their produced flowers (Table 2) and their hybridity was hybridity (data not shown) and designated them 10W13- confirmed (data not shown) and recorded as 10W10-1 1, -2, -3, -4, and -5 (Fig. 3). The total volatile amounts and -2 (Fig. 3). The total amount of volatiles produced of these lines were 3–8 times higher than that of by 10W10-1 was about eight times higher than that of ‘Shikibu’, corresponding to 14–36% of that of Dianthus ‘Nagisa’ and about 22% of that of Dianthus sp. 5 sp. 6 (Table 4). Methyl benzoate, which was a principal (Table 4). In 10W10-2, the amount of volatiles was 68 volatile in the parental lines, was a dominant volatile times higher than that of ‘Nagisa’ and about twice as also in most of the hybrid 10W13 lines (Table 4). The high as that of Dianthus sp. 5 (Table 4). Methyl benzoate most abundant volatile in Dianthus sp. 6, methyl o- and benzyl alcohol, which were the principal volatiles anisate, was detected at a high level in 10W13-4, but at in ‘Nagisa’ and D. sp. 5, were the dominant volatiles moderate levels in the other hybrid lines (Table 4). also in 10W10-1 and -2 (Table 4). The benzyl alcohol Methyl salicylate, eugenol, methyl eugenol, and benzyl 150 K. Kishimoto, M. Yagi, T. Onozaki, H. Yamaguchi, M. Nakayama and N. Oyama-Okubo 0.1 0.3 0.1 0.1 0.4 0.3 0.4 0.1 0.0 1.6
± ± ± ± ± ± ± ± ± ±
race 0.9 1.9 1.4 9.4
0.0 trace 0.0 1.1 0.1 0.9 0.00.4 trace 0.2 2.7 0.7 0.6 0.1 0.30.1 — 0.4 0.0 0.1 0.4 —
±
± ± ± ± ± ± ± ± ± ± ± ±
3.4 0.9 4.4 0.3 sp. 6 0.1 0.5 0.0 0.2 0.1 0.00.0 0.2 0.1 1.8 0.6 0.4 0.4 0.0 0.2 0.8 14.1
± ± ± ± ± ± ± ± ± ±
—0.9 Dianthus
×
3.8 1.8 0.5 0.1 1.0 0.2 0.1 0.3——— 0.0 0.1 trace0.10.1 0.4 0.0 —0.0——— — — 0.1 1.1 0.0 0.1 0.2 trace0.8 0.0 —0.7 — 9.2 — — trace
‘Shikibu’ ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.5 0.9 1.1 1.8 2.4 9.1
0.0———— 0.9 0.1 0.20.0 0.8 0.3 0.8 0.1 0.4 0.7 0.1 — 0.2 0.7 0.5 0.8
±
± ± ± ± − ± ± ± ± ± ±
trace — — — trace 3.1 3.5 species, and their hybrids. interspecific and species, Interspecific hybrid lines Interspecific hybrid 1.00.4 trace2.3 0.2 0.1 0.7 0.00.7 2.2 0.3 0.80.4 6.4 0.1 1.4 1.4 0.2 — 0.1 0.3 1.4 0.30.5————t —0.2 1.9 0.1 0.1 trace 4.5 24.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Dianthus ×
sp. 5 sp. 2.0 161.5 ‘Nagisa’
0.70.0 2.9 0.5 1.6 135.7 1.20.3 11.6 0.1 1.8 0.1—————— 0.9 0.1 0.9 0.2 0.8 0.10.1 1.4 1.8
±
± ± ± ± ± ± ± ± ± ± ± Dianthus
3.4 1.9
×
sp. 4 0.1 2.3 0.2 0.00.0 trace0.1 trace0.01.3 — — 0.6 0.4 — 7.6 0.0 0.7 1.1 — —0.80.1 trace — 0.6 0.2 0.1 — 1.0 0.1 —0.12.0 — trace trace 19.8 0.2 trace trace 0.9 trace 0.2
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.4 0.2 10W02-1 10W10-1 10W10-2 10W13-1 10W13-2 10W13-3 10W13-4 10W13-5 ‘Ai Pink’ Dianthus sp. 0.4 0.6 0.70.2 0.7 trace0.5 trace0.4 0.1 0.3 0.1 0.2 0.2 2.11.3 12.4 3.8 0.40.2 — —0.1 — 0.4 0.1 —1.7 — — — — 0.9 2.4 0.0 —8.4 — 0.2 25.7 — 0.5 trace trace 0.7
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6 1.7 8.8 14.4 20.3 Dianthus sp. 1.20.3 1.0 8.1 — trace0.60.1 0.3 1.6 0.6 3.0 0.7 1.1 0.7 —4.7 0.2 0.7 0.1 0.1 —0.7 — 3.4 — trace0.1 0.3 0.3 0.4 2.3 6.6
0.0 0.6 18.7 67.6
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5 ± ±
Dianthus 4.3 4.0 0.8 15.7 28.3 Dianthus y,x sp. 4.2 1.0 1.7 8.2
.— — —— ————— — —— ————— — ————— —— ————— 0.30.7—— trace0.5—— 0.1—— 3.8 0.12.1 1.0 0.5 6.5 3.2 1.2 0.10.6 5.9 1.5 3.8 0.21.3 0.6 2.4 1.2 1.1——trace—— 0.40.1 — — 0.6 0.1 — trace 0.1 trace 1.1
11.5 88.8 0.1 — 0.3 0.6
± ±
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
4 ± ± ±
2.6 14.5 10.2 Dianthus 0.6 0.1 13.4 0.5 90.5
± ± ±
0.1 2.5 0.1 —0.1 4.1 0.2 trace 3.0 0.2
± ± ± ±
—— —1.9 trace trace 4.9 Carnations Wild Amounts of scent compounds emitted from wild flowersAmounts of scent compounds unidentified cultivars, modern carnation of z 0.4 1.9 0.0 trace 0.2 0.1 0.2 0.5 2.4 w
± ± ± ±
2.1 ‘Ai Pink’ ‘Nagisa’ ‘Shikibu’ Table 4. 3).
= .
1 − ·h 1 − -anisate — — — 3.2 o 0.1.
Compounds <
)-3-Hexenyl acetate — — trace 4.3 -Caryophyllene — trace — 1.4 Z Benzyl benzoateBenzyl — — — 1.4 tesrctaerc——— —— ————— EugenolIsoeugenol — — eugenolMethyl trace — trace — 6.4 — —Otherstracetracetrace——— —( 5.7 Total 2.8 2-Phenylethanol — trace trace 0.4 earl— ———2.9 Cinnamyl alcoholCinnamyl aldehydeElemicin — — — — — — — — 2.8 1.3 —Veratrol——— 0.6 Cinnamyl acetate —Isoelemicin — salicylateMethyl Methyl — —Phenylacetaldehyde trace 1.7 — — isoeugenolMethyl traceVanillin — trace — — 3.0 2.4 — — oxideCaryophyllene — trace — — — trace 4.1 trace — 0.3 5.7 β Benzyl alcoholBenzyl — trace — — Limonene 0.4 Benzaldehyde trace Benzyl acetateBenzyl — benzoate Methyl Others 0.2 Others trace 0.2 not detected. not nmol·g FW SE (n Indicates trace Fatty acid derivatives acid Fatty Terpenoids Benzenoids z y x w Bold values indicate principal scent compounds in each flower. each in scent compounds principal values indicate Bold J. Japan. Soc. Hort. Sci. 82 (2): 145–153. 2013. 151
y sp. 6 Dianthus 10W13-5 10W13-1 10W13-4 10W13-5 10W13-2
y sp. 5 Dianthus 10W10-2 10W10-2
y sp. 4 Dianthus y Interspecific lines hybrid OB44 10W02-1 10W10-1 D. hungaricus y var. var. longicalycinus D. superbus 4K38-6 10W13-1 4K38-10, 4K38-10, 4K38-14 4K38-6 10W13-1 4K38-2, 4K38-11, 4K38-11, 4K38-2, 4K38-15 4K38-2 4K38-34K38-6 10W02-1 10W13-2 species, and interspecific species, and hybrids. var. var. var. sp. 4 4K38-6, 4K38-10 10W13-3 sp. 6 sp. 6 10W13-4 sp. 5 10W10-1 sp. 5 Dianthus Dianthus Dianthus Dianthus D. hungaricus Dianthus Dianthus D. hungaricus Dianthus longicalycinus D. superbus longicalycinus D. superbus Carnations Wild x z Principal scent compounds of carnations, wild of compounds scent Principal Table 5. Strong aromatic odor of clove aromatic odor Strong taste. In pungent a spicy, and and methyl isoeugenol addition, have a faint, orris- isoeugenol odor. like, green, sweet, woody odor and a sweet taste and odor melon.reminiscent of odor and a slightly pungent, pungent, a slightly and odor sweet taste. and a almond reminiscent of taste. pungent sharp, note reminiscent of banana. note wintergreen-like odor. wintergreen-like with a herbaceous strong, note reminiscentlavender- of hay. rose and lavandin, aroma. -anisate warm, Herbaceous, anise-like o )-3-Hexenyl acetate)-3-Hexenyl floral Powerful, fruity, green, -Ocimene odor. herbaceous Warm -Caryophyllene clove-like dry, Woody-spicy, Z Eugenols (eugenol, (eugenol, Eugenols methyl isoeugenol, and methyl eugenol isoeugenol) Methyl Methyl benzoate cananga. similar to odor, Fruity All the carnations Benzyl benzoateElemicin Light, balsamic odor Major factor of nutmeg scent. β Methyl salicylateMethyl β sweet, spicy, Minty, 1-Octen-3-ol Powerful, odor sweet, earthy Compounds scent the of Description Benzenoids Benzyl alcohol Characteristic fruity pleasant, Fatty acid derivatibes acid Fatty ( Terpenoids The descriptions are from “Flavor Ingredients”, sixth edition (Burdock, 2010), except for elemicin. 2010), (Burdock, are edition fromThe descriptions “Flavor Ingredients”, sixth Pollen parents. p. 202–214. of several phenylisopropylamines. related of nutmeg and The chemistrypsychopharmacology et al. (1967), and Shulgin z y x 152 K. Kishimoto, M. Yagi, T. Onozaki, H. Yamaguchi, M. Nakayama and N. Oyama-Okubo
Fig. 3. Interspecific hybrids between carnations and wild Dianthus species. Bar = 10 mm. benzoate were major volatiles in Dianthus sp. 6 and As we actually noted the fragrance of principal odor- some 10W13 lines (Table 4). Elemicin and (Z)-3-hexenyl active compounds from flowers of some hybrid lines, acetate, minor volatiles in the parents, were the most the suitability of interspecific hybridizations to give scent abundant volatiles in 10W13-1 and 10W13-2, respec- properties to carnation was confirmed. Dianthus tively (Table 4). β-Caryophyllene, which was not superbus var. longicalycinus and Dianthus sp. 4, 5, and detected in the parental lines, was present in low amounts 6 were identified as promising resources, enabling the in most 10W13 lines (Table 4). The scents of 10W13-1, addition of terpenoids and an increased variation of -4, and -5 were stronger than that of ‘Shikibu’ but were benzenoids, respectively, in carnations. qualitatively similar. The line of 10W13-2 had a different Our results did not indicate simple Mendelian scent from the parents. The line of 10W13-3 had a spicy inheritance patterns; progenies possessing qualitatively scent derived from eugenol and methyl eugenol. These and quantitatively distinct profiles were generated from findings suggested that cross-breeding with Dianthus sp. the same parent partners, and one hybrid, 10W10-2, 6 could increase the variety of benzenoids in carnation emitted higher amounts of some compounds than found floral scents. in any of the parents. This complexity might be due to the heterozygosity of ornamental carnations and/or 4. Conclusion heterosis. In hybrids between Freesia refracta and The principal odor-active compounds of the inter- F.hybrida, the complex inheritance pattern of flower specific hybrids and the descriptions of their scents are scent was also pointed out to be possibly involved in given in Table 4. The emission of these compounds the heterozygosity of the parents (Fu et al., 2007). In proved to be an inheritable trait transmitted from wild Cyclamen hybrids, it is suggest that each emission level Dianthus parents to most hybrid lines. Both the variety of some floral scent compounds is affected by the amount and the amounts of fragrant compounds tended to of the genome (gene) derived from its parents (Ishizaka increase in hybrids compared to the parental carnations. et al., 2002). In the inheritance analysis of flower scents J. Japan. Soc. Hort. Sci. 82 (2): 145–153. 2013. 153 using diploid segregating populations of rose, the var. longicalycinus and D. superbus in Mie prefecture and emission levels of some benzenoids and terpenoids were Hokkaido. Annual Report on Exploration and Introduction identified to be regulated by six QTLs (Spiller et al., of Plant Genetic Resources (Japan) 17: 49–54 (In Japanese with English abstract). 2010). These reports suggest that various genes Onozaki, T., H. Ikeda, M. Shibata, N. Tanikawa, M. Yagi, T. contribute to the inheritance of each flower scent, and Yamaguchi and M. Amano. 2006. Breeding and characteris- the emission of scent is affected by both of the amount tics of carnation norin No. 1 ‘Miracle Rouge’ and No. 2 and quality of their genes. As indicated in a study on ‘Miracle Symphony’ with long vase life. Bull. Natl. Inst. Flor. roses (Spiller et al., 2010), QTL analysis using genetic Sci. (Japan) 5: 1–16 (In Japanese with English abstract). markers seems a suitable technique to clarify the factors Onozaki, T., M. Yagi, Y. Fujita and K. Tanase. 2011. Characteristics involved in a complex phenomenon. Now we are of interspecific hybrids between carnation (Dianthus caryophyllus) lines with long vase life and D. superbus var. developing genetic markers of carnation (Tanase et al., longicalycinus, and their backcrossing progenies. Hort. Res. 2012; Yagi et al., 2006) and will apply them to the (Japan) 10: 161–172 (In Japanese with English abstract). analysis of scent traits. Oyama-Okubo, N., T. Ando, N. Watanabe, E. Marchesi, K. Uchida All hybrids obtained in this study by crossing and N. Nakayama. 2005. Emission mechanism of floral scent carnations with wild Dianthus possessing serrated single in Petunia axillaries. Biosci. Biotechnol. Biochem. 69: 773– petals developed double serrated petals but sometimes 777. in insufficient numbers (Figs. 1–4). Flowers of hybrids Oyama-Okubo, N., M. Nakayama and K. Ichimura. 2011. Control of floral scent emission by inhibitors of phenylalanine were smaller than those of the parental carnation but ammonia-lyase in cut flower of Lilium cv. ‘Casa Blanca’. J. larger than those of the parental wild Dianthus. These Japan. Soc. Hort. Sci. 80: 190–199. morphological inheritances are the same as previous Seo, R. and N. Soich. 1998. Studies of Raising of the varieties of reports (Nimura et al., 2003; Nimura et al., 2006; Seo carnation by the interspecific crossing 1. The interspecific and Soichi, 1998). Round and double petals are preferred cross with several wild species and the characteristics of morphological traits of ornamental carnations as well as progeny hybrid individuals. Kagawa Prefecture Agricultural larger flower size. Morphological improvement of Experiment Station-Research Report 50: 13–19 (In Japanese with English abstract). fragrant hybrids could be achieved by using back- Schade, F., R. L. Legge and J. E. Thompson. 2001. Fragrance crossing with carnation and so on to give an ornamental volatiles of developing and senescing carnation flowers. value of carnations. Phytochemistry 56: 703–710. Literature Cited Shulgin, A. T., T. Sargent and C. Naranjo. 1967. The chemistry and psychopharmacology of nutmeg and of several related Burdock, G. H. 2010. 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