Floral Scent of Wild Gladiolus Species and the Selection of Breeding Material for This Character

Breeding Science 58 : 89–92 (2008)

Note

Floral scent of wild Gladiolus species and the selection of breeding material for this character

Kazunori Suzuki*1), Naomi Oyama-Okubo2), Masayoshi Nakayama2), Yasumasa Takatsu1) and Masakazu Kasumi1)

1) Plant Biotechnology Institute, Ibaraki Agricultural Center, 3165-1, Ago, Kasama, Ibaraki 319-0292, Japan 2) National Institute of Floricultural Sciences, 2-1, Fujimoto, Tsukuba, Ibaraki 305-8519, Japan

To select suitable genetic resources for scent breeding, we qualitatively and quantitatively analyzed floral scent emitted from 9 wild Gladiolus species. Dynamic headspace collection was performed and scent compounds emitted from the wild species were analyzed using gas chromatography-mass spectrometry, resulting in detection of twenty scent compounds. The analyzed species were divided into 4 groups;—Linalool/Benzenoid group, Nerol group, Ionone group or Ocimene/Caryophyllene group. Based on the kind and amount of scent compounds we selected G. orchidiflorus, G. recurvus, G. tristis and G. watermeyeri as potential genetic resources for fragrance. Temporal changes in emissions were investigated in 3 promising species that were selected. G. orchidiflorus emitted the maximum amount of scent compounds between 10:00 and 14:00 while the maximum output of G. recurvus and G. tristis was between 18:00 and 22:00. This differential release of scent compounds during the day is an important character to select for, in addition to the quality and quantity of scent compounds, in breeding program for Gladiolus.

Key Words: Gladiolus, floral scent, genetic resources.

The genus Gladiolus is a member of the family would attract consumers in flower markets. This conviction Iridaceae and is distributed in southern Africa, Madagascar prompted us to find suitable genetic resources in order to and Eurasia. The current number of species in the genus is breed scented Gladiolus cultivars. We qualitatively and 255 (Goldblatt and Manning 1998). It is thought that mod- quantitatively analyzed floral scent emitted from 9 wild ern cultivars of G. × grandiflora originated from a small Gladiolus species chosen as favorable scented flowers from number of wild species, viz. G. cruentus, G. natalensis, germplasm collections of 21 species maintained in the G. oppositiflorus, G. papilio and G. saundersii (Imanishi Ibaraki Agricultural Center. Temporal changes in emissions 1989); thus, most Gladiolus species did not participate in the were also investigated in the 3 representative species used in establishment of modern cultivars. These species may this study. possess useful characters for plant breeders. Nine scented species, G. gracilis, G. orchidiflorus, One particular trait found in wild species is floral scent. G. pritzelli, G. recurvus, G. trichonemifolius, G. tristis, Previously studied wild species and their major scent com- G. uysiae, G. venustus and G. watermeyeri, were planted pounds are: G. liliaceus – eugenol; G. alatus, G. maculatus, in pots in the beginning of October and grown in a green- G. recurvus and G. tristis – linalool; G. jonquilliodorus, house. Dynamic headspace collection was performed under G. orchidiflorus, G. patersoniae and G. scullyi – geraniol or daylight and dark conditions for 12 hours, respectively geraniol acetate, nerol and citronellol; G. carinatus and (06:00–18:00 light/18:00–06:00 dark) at 22°C on average G. virescens – β-ionone (Goldblatt et al. 1998, Goldblatt and and 25% relative humidity. Emitted scent compounds were Manning 2002). While these studies were performed to de- collected from one flower for 24 hours from the first flower- termine their attractiveness to pollinators, the data suggested ing day using the method of Oyama-Okubo et al. (2005). A large qualitative variations in floral scents, including ben- spike with one opening flower was covered with a 500 ml zenoids and terpenoids, in wild Gladiolus species. Tedlar bag (GL Science, Tokyo, Japan). A constant steam Currently no modern Gladiolus cultivar has floral of air filtered through charcoal was supplied through the bag scent. We believe that Gladiolus cultivars with floral scent and compounds were collected with Tenax-TA traps. Scent compounds extracted from the Tenax-TA traps were analyzed Communicated by K. Okuno using gas chromatography-mass spectrometry (GC-MS) with Received December 4, 2007. Accepted January 9, 2008. an Agilent 6890N gas chromatograph coupled to an Agilent *Corresponding author (e-mail: [email protected]) 5973N Mass Selective Detector (Agilent Technologies, 90 Suzuki, Oyama-Okubo, Nakayama, Takatsu and Kasumi 3.30 0.80

13.87 16.49

± ± ± ±

Ocimene/ Caryophyllene 1.8614.22 — — 16.06 39.53

± ± ±

0.200.84 — 23.85 — 0.65 25.57

± ± ±

G. uysiae G. watermeyeri G. gracilis 0.532.59 1.18 11.22 3.08 12.41

± ± ±

G. trichonemifolius 20.5420.54 — 9.33 — — —

± ±

hours are described.

0.54 59.70 2.022.55 59.70 — — — — 24.51

± ± ±

g/flower/24 µ

> 1.78 —6.69 — — — — — — — — — — —

iflorus G. pritzelli G. venustus 11.7419.23 — 1.40 34.82 3.63 — — — — —

± ± ± ± ±

.4———— — — — — — — — 4.64—————— 3.37—————— 4.68—————— 0.26—————— 3.76—————— 6.43—————— 5.42—————— 1.02——————

10.79 — —14.57 — —26.97 104.15 — — — — — — — — — —

3). Only compounds

± ± ± ± ± ± ± ± ± ± ± oral scent compounds

SD, n

0.76 9.39 5.77 3.13 1.02 4.42 0.51 — — — — — — — 13.87

34.76 3.32 40.91 62.21 82.15 166.60

± ± ± ± ± ± ±

species based on fl hours (Mean

G. recurvus G. tristis G. orchid Gladiolus g/flower/24 Species µ -ionone — — — — — 1.96 β Scent group Linalool/Benzenoid Nerol Ionone -Ocimene — — — 2.24 -Ocimene — — — — — — — — 1.15 -Ionone — — — — — — — 1.72 -Caryophyllene 2.71 -Ionone — — — — — 7.37 Dihydro- trans Methyl eugenol — 9.57 Nerol — — 48.47 β Geranyl acetate — — 41.00 β Geraniol — — 10.84 Total 280.23 cis Linalool 185.60 Benzyl acetateBenzyl alcoholBenzyl benzoateEugenol 76.48 Methyl benzoate 11.45 —Phenylacetaldehyde2-Phenylethylalcohol 4.28 — 2.65 — — 48.92 11.57 9.80 α Classification of wild 1.

Scent compound unit is Constituents Benzenoids Benzaldehyde 1.34 Table Terpenoids Citral — — 3.85 Floral scent of wild Gladiolus species 91

Wilmington, DE, USA). The GC was equipped with a split- resulting in an intense, rose-like fragrance which can be less injector and a DB-WAX capillary column (30 m in length, applied for scent breeding. We did not select G. venustus be- 0.25 mm i.d. and 0.25 µm film thickness). Helium was used cause this species is inferior to G. orchidiflorus in the kind as the carrier gas. GC analysis was carried out using an and amount of scent compounds. In the Ionone group we se- Agilent 6850 gas chromatograph monitored by FID (Flame lected G. watermeyeri, which emits α-ionone and has a strong, Ionization Detector). violet-and-freesia-like fragrance. The other two species in The floral scent of each plant was characterized using the group emit β-ionone and dihydro-β-ionone, which yield compounds occurring at a concentration of at least 1 µg/ an unsuitable woody note. G. gracilis belonging to the flower/24 hour, resulting in the detection of twenty scent Ocimene/Caryophyllene group is not a suitable gene pool compounds. These were 9 benzenoids: benzaldehyde, because of emitting only weak scent compounds. benzyl acetate, benzyl alcohol, benzyl benzoate, eugenol, Flowers of snapdragon, tobacco and petunia are report- methyl benzoate, methyl eugenol, phenylacetaldehyde and ed to change their scent emissions in a time-dependent man- 2-phenylethylalcohol; 7 monoterpenes: citral, geraniol, geranyl ner (Kolosova et al. 2001, Oyama-Okubo et al. 2005). To acetate, linalool, nerol, cis-ocimene and trans-ocimene; and examine the time-dependent changes in the scent emission 4 sesquiterpenes: β-caryophyllene, dihydro-β-ionone, α- of wild Gladiolus, the major compounds emitted from 3 spe- ionone and β-ionone. The analyzed species were divided cies with large scent outputs (G. orchidiflorus, G. recurvus into 4 groups based on their major scent components. The and G. tristis) were collected every 4 hours a day and sub- groups were: Linalool/Benzenoid group (G. recurvus and jected to GC-FID analysis. Within each species, each com- G. tristis); Nerol group (G. orchidiflorus, G. pritzelli and pound (benzenoid, monoterpen or sesquiterpene) exhibited G. venustus); Ionone group (G. trichonemifolius, G. uysiae the same diurnal or nocturnal fluctuations. G. orchidiflorus and G. watermeyeri) and Ocimene/Caryophyllene group (G. gracilis) (Table 1). Linalool/Benzenoid group. G. recurvus and G. tristis were the only species emitting both benzenoid and terpenoid compounds. The main compounds emitted from G. recurvus were linalool (185.6 µg/flower/24 hours, 66.2%) and benzyl alcohol (76.5 µg, 27.3%). G. tristis emitted many scent compounds but linalool was the dominant compound (62.2 µg, 37.3%) followed by methyl benzoate (48.9 µg, 29.4%), phenylacetaldehyde (11.6 µg, 6.9%), and then benzaldehyde, eugenol, methyl eugenol etc (Table 1). Nerol group. G. orchidiflorus emitted 4 scent compounds (citral, geraniol, geranyl acetate and nerol) while the other 2 species emitted 1 or 2 scent compounds. G. orchidiflorus emitted the largest volume of scent compounds (104.2 µg) in the group (Table 1). Ionone group. G. trichonemifolius and G. uysiae emitted β-ionone (7.4 µg and 11.2 µg, respectively) and its deriv- ative dihydro-β-ionone while G. watermeyeri emitted α- ionone (1.7 µg) and β-ionone (23.9 µg) (Table 1). Ocimene/Caryophyllene group. G. gracilis emitted ocimene and β-caryophyllene, both of which expressed weak scents (Table 1). Six species, G. gracilis, G. orchidiflorus, G. recurvus, G. trichonemifolius, G. tristis, G. venustus and G. watermeyeri, emit a relatively larger amount of total scent compounds, but the threshold of fragrance recogni- tion and the quality of fragrance differ in each scent compound; thus, based on the kind and amount of scent compounds released we selected 4 species as potential genetic resources for fragrance in Gladiolus. Both G. recurvus and G. tristis belong to the Linalool/Benzenoid group and are promising because they both emit a strong, sweet, clove-like fragrance abundant in both linalool and benzenoids. In the Nerol group, G. orchidiflorus emits 4 kinds of scent com- Fig. 1. Diurnal changes in floral scent compounds of 3 wild Gladiolus pound in quantity, citral, geraniol, geranyl acetate and nerol, species. 92 Suzuki, Oyama-Okubo, Nakayama, Takatsu and Kasumi emitted the maximum amount of scent compounds between Literature Cited 10:00 and 14:00 while the maximum output of G. recurvus and G. tristis was between 18:00 and 22:00 (Fig. 1). Goldblatt, P. and J. Manning (1998) “Gladiolus in Southern Africa” G. recurvus showed significant scent output throughout Fernwood Press, Vlaeberg. the day while low volumes were detected during the night in Goldblatt, P., J.C. Manning and P. Bernhardt (1998) Adaptive radiation G. orchidiflorus and during the day in G. tristis. This differen- of bee-pollinated Gladiolus species (Iridaceae) in southern tial release of scent compounds during the day is an important Africa. Ann. Missouri Bot. Gard. 85: 492–517. Goldblatt, P. and J.C. Manning (2002) Evidence for moth and butterfly character to select for, in addition to the quality and quantity pollination in Gladiolus species (Iridaceae: Crocoideae). Ann. of scent compounds, in the breeding program of Gladiolus. Missouri Bot. Gard. 89: 110–124. As many wild Gladiolus species flower during winter Imanishi, H. (1989) Collected data of plant genetic resources. In and modern cultivars of G. × grandiflora flower in summer, “Gladiolus” Matsuo, T. (ed.), Kodansya Scientific, Tokyo. pollen storage is necessary to facilitate interspecific hybrid- p.1077–1080 (in Japanese). ization. Takatsu et al. (2001) demonstrated that hybridiza- Kolosova, N., N. Gorenstein, C.M. Kish and N. Dudareva (2001) Regu- tion between a modern cultivar and G. tristis was efficient at lation of circadian methyl benzoate emission in diurnally and low air temperature (15 to 20°C) using pollens of G. tristis nocturnally emitting plants. Plant Cell 13: 2333–2347. stored at −20°C. This technique would be applicable to other Oyama-Okubo, N., T. Ando, N. Watanabe, E. Marchesi, K. Uchida and wild species selected in this study to breed scented cultivars. M.Nakayama (2005) Emission mechanism of floral scent in Petunia axillaris. Biosci. Biotechnol. Biochem. 69: 773–777. Takatsu, Y., M. Kasumi, T. Manabe and M. Hayashi (2001) Tempera- Acknowledgements ture effects on interspecific hybridization between Gladiolus × grandiflora and G. tristis. Hortscience 36: 341–343. We thank Dr. T. Gonai and Ms. M. Fujita (Plant Bio- technology Institute, Ibaraki, Japan) for help with plant growth and discussions.