2018 Volume 87 Issue 2 Pages 258-263
A new blue petunia cultivar, TX-794 bred by Takii & Co., Ltd, has a sweet floral fragrance different from conventional petunia cultivars that typically have no odor or have a somewhat unfavorable smell. Since fragrant bedding plants suitable for summer cultivation are rare, we expect that the fragrance will enhance the value of TX-794. The characteristics of emitted scent compounds from TX-794 were evaluated in this study. The major scent compound was phenylacetaldehyde, a C6-C2 aromatic compound that was emitted at higher levels in TX-794 compared to conventional petunia cultivars. iso-Eugenol, a C6-C3 aromatic compound, is the major scent compound in conventional petunia cultivars. These results suggest that TX-794 has a high capacity to biosynthesize C6-C2 aromatic compounds, resulting in a significantly different scent compound composition compared to conventional petunia cultivars. Time-course analysis of scent compound emission revealed that the largest release of scent compounds by TX-794 occurs during the switch from light periods to dark periods. Soon after flowering, that is, in the light period on the day of anthesis, the major scent compound was p-cresol. Thereafter, the primary scent compound was phenylacetaldehyde or methyl benzoate with 2-phenylethanol occupying the next position. Since a bed or a container of petunias has flowers that are primarily over 2-days old, the basic fragrance of TX-794 plants is derived from phenylacetaldehyde, which has a hyacinth-like scent, 2-phenylethanol, which has a rose-like scent, and methyl benzoate, which has a dry fruit-like scent.
Petunia cultivars with their diversity of color and morphology are popular summer bedding plants. The garden petunia, Petunia hybrida, was bred from moth-pollinated P. axillaris and a bee-pollinated species of the P. integrifolia clade that includes P. inflate (Segatto et al., 2014; Stehmann et al., 2009), producing a wide variety of cultivars. The white-flowered P. axillaris has a strong and sweet fragrance during the night. The major scent of P. axillaris is primarily composed of three aromatic compounds: methyl benzoate, iso-eugenol, and benzaldehyde. The emission of all P. axillaris scent compounds has a diurnal rhythm with maxima at night and minima at noon (Oyama-Okubo et al., 2005). In contrast, the purple-flowered species of the P. integrifolia clade have no fragrance. Petunia cultivars, the progeny of P. axillaris, could also be expected to be fragrant, but few cultivars have fragrances. Some blue petunia cultivars emit a spicy sweet scent with iso-eugenol as the major scent compound during the daytime (Nakamura et al., 2006). However, since the smell of these blue cultivars is somewhat unpleasant, fragrance is not generally considered in the commercial value of petunias.
Although Torenia and Impatiens are major bedding plants in summer, these plants do not have a fragrance. Fragrant bedding plants suitable for cultivation in the summer are rare, so fragrance is a factor that adds high value. The petunia breeding line, TX-794 (international cultivar name, ‘Evening Scentsation’; domestic cultivar name in Japan, ‘Blue Moon’), is a unique cultivar with a semi-creeping habit and blue corolla not found in conventional cultivars (Fig. 1). To breed TX-794, a petunia breeding line developed by Takii & Co., Ltd (Kyoto, Japan) and the wild species P. axillaris and P. altiplana, which have non-fragrant purple flowers with a creeping habit, were used. TX-794 has a sweet fragrance with notes of hyacinth, honey, and rose that is different from other fragrant P. axillaris accessions and conventional fragrant blue-flowered cultivars. Therefore, we expect that TX-794 could be a new petunia with high commercial value due to its fragrance.
Petunia TX-794 ‘Evening Scentsation’.
In this paper, we compared the composition of emitted scent compounds of TX-794 with those of three commercial blue-flowered cultivars and determined the sensory characteristics of the TX-794 fragrance based on compositional differences. In addition, a time-course analysis of scent compounds emitted by TX-794 identified changes in fragrance profiles with time. Furthermore, based on the structures of the major scent compounds, a model for the interrelatedness of biosynthetic pathways for fragrance compounds in TX-794 and other petunia cultivars is presented.
Seeds of TX-794, ‘Baccara Blue’, ‘Carpet True Blue’, and ‘Celebrity Blue’ were germinated in 200-cell plug trays filled with sowing soil (Takii & Co., Ltd) on June 20, 2015, and seedlings were grown in a greenhouse at Takii Plant Breeding & Experimental Station (Konan, Shiga, Japan). The seedlings were transplanted into plastic pots (9 cm in diameter and 10 cm deep) filled with horticultural soil (Takii & Co., Ltd) on July 13, 2015 and grown in a greenhouse. During the cultivation period, plants were managed under natural conditions, and flowering of all cultivars began in early August. All flowering cultivars were transported by package-delivery service to Institute of Vegetable and Floricultural Science, NARO (Tsukuba, Ibaraki, Japan) on August 25, 2015 and were grown in a greenhouse at NARO under natural conditions. Just before the experiments, plants were acclimated for at least a week in the laboratory at a constant temperature of 21°C ± 2°C under a photosynthetic photon flux density of about 150 μmol·m−2·s−1, and a 12 h·12 h (8:00 to 20:00 light·20:00 to 8:00 dark) photoperiod. Time-course experiments used the day of anthesis (full, open bloom) as the reference time point.
Collection and preparation of emitted volatilesEmitted volatile compounds were collected by the dynamic headspace method (Oka et al., 1999). Tedlar Bags (1L volume; GL Science, Tokyo, Japan) were placed over individual petunia flowers including the pedicels. A constant stream of air (approximately 300 mL·min−1) was filtered through activated charcoal and then piped through the bag, and volatiles were collected with a Tenax-TA tube (180 mg, 60 × 80 mesh; Gerstel Inc., Linthicum, MD, USA) (Oyama-Okubo et al., 2005). For quantitative analysis, the emitted compounds were collected for 24 h (starting at 8:00) from 2-day old flowers. For the time-course analysis, emitted compounds were collected every 4 h from 8:00 on the first day post-anthesis until 8:00 of the fifth day post-anthesis.
GC-MS analysisSamples collected in the Tenax-TA tubes were directly introduced onto the GC-MS using a thermal desorption system. GC-MS was performed using an Agilent 7890A gas chromatograph coupled to an Agilent 5975C Mass Selective Detector (Agilent Technologies, Wilmington, DE, USA) (Oyama-Okubo et al., 2005). The GC was coupled to a Thermal Desorption System 3 (TDS3; Gerstel Inc.) and a DB-WAX capillary column (30 m length, 0.25 mm i.d., and 0.25 μm film thickness). The thermal desorption conditions were heating from 30°C to 250°C at 60°C·min−1, holding for 10 min at 250°C, and cryofocusing at −50°C in the cooled injection system 4 (CIS4; Gerstel Inc.). Following tube desorption, the CIS4 was heated to 300°C at a rate of 12°C·s−1 in splitless mode to transfer the analytes to the GC column. The temperature program of the GC column oven was set to 40°C for 2 min, 4°C·min−1 up to 180°C, held at 180°C for 5 min, 15°C·min−1 up to 250°C, and held at 250°C for 10 min. The injection, interface, and ion source temperatures were 250°C, 280°C, and 250°C, respectively. Helium was used as the carrier gas. The mass scan range was m·z−1 30–300, and the electron potential was set to EI 70 eV. Compounds were identified with the Wiley 9th/NIST 2011 library search algorithm provided with the GC-MS software and crosschecked by comparing the mass spectra and retention times with authentic samples under the same conditions. The amount of each compound was calculated using calibration curves based on the peak area of authentic samples separated by total ion chromatography.
Emitted volatiles were collected from intact flowers on plants growing in the laboratory at 21°C ± 2°C under a 12-h photoperiod. Twenty-four aromatic compounds were identified by GC-MS analysis of the 24-h headspace of flowers on the second day after anthesis. The major scent compounds were as follows: TX-794: phenylacetaldehyde (48.7%), benzaldehyde (14.3%), and methyl benzoate (8.5%); ‘Baccara Blue’: benzaldehyde (25.8%), iso-eugenol (16.7%), and p-creosol (14.0%); ‘Carpet True Blue’: benzaldehyde (32.6%), methyl benzoate (23.7%), and iso-eugenol (13.6%); ‘Celebrity Blue’: iso-eugenol (21.1%), phenylacetaldehyde (19.6%), and benzaldehyde (16.3%) (Table 1).
Composition of scent compounds emitted per flower of Petunia cultivars (mol/mol %).
The total amount of major scent compounds emitted by TX-794 was about the same as that for ‘Baccara Blue’ and ‘Celebrity Blue’, about 0.75 times that of ‘Carpet True Blue’ (Table 2). TX-794 had a higher percentage of phenylacetaldehyde, which has a hyacinth-like scent (Burdock, 2010), compared to the three blue commercial cultivars. The amount of phenylacetaldehyde emitted by TX-749 was 35 times higher than that of ‘Baccara Blue’, 75 times higher than that of ‘Carpet True Blue’, and 3.5 times higher than that of ‘Celebrity Blue’. Likewise, the amount of rose-scented 2-phenylethanol emitted by TX-794 (Burdock, 2010) was about 8.5 times greater than that for ‘Baccara Blue’, 12 times greater than that for ‘Carpet True Blue’, and 2.1 times greater than that for ‘Celebrity Blue’.
Amounts of major scent compounds emitted per flower of Petunia cultivars.
In contrast, the level of iso-eugenol, which has a rich sweet spicy scent (Burdock, 2010), was lower in TX-794 compared to the three blue commercial cultivars. The amount of iso-eugenol emitted by TX-794 was 0.15 times that of ‘Baccara Blue’, 0.11 times that of ‘Carpet True Blue’, and 0.12 times that of ‘Celebrity Blue’. Similarly, TX-794 emitted vanillin, which has a vanilla-like sweet spicy scent, at about 0.17 times the level of ‘Baccara Blue’ and ‘Celebrity Blue’, and about 0.15 times that of ‘Carpet True Blue’. Furthermore, TX-794 emitted p-cresol, which has a characteristic disinfectant odor, at 1.8 times the amount of ‘Baccara Blue’, 2.2 times that of ‘Carpet True Blue’, and 1.3 times that of ‘Celebrity Blue’.
Time-course of floral scent emission by TX-794Scent compounds emitted from individual TX-794 flowers were collected from 8:00 on the day of anthesis until the end of 4 days post-anthesis at consecutive 4 h periods. The emission of scent compounds by TX-794 increased beginning at 12:00 and showed a pronounced diurnal rhythm maximum from 20:00 to 24:00 (Fig. 2). The major scent compound collected between 8:00 and 16:00 on the day of anthesis from TX-794 was p-cresol. Thereafter, the percentage of p-cresol decreased and the percentages of methyl benzoate and phenylacetaldehyde increased. From 2 days post-anthesis onward, the major scent compound of TX-794 between 8:00 and 16:00 was methyl benzoate, and those between 16:00 and 8:00 were phenylacetaldehyde and 2-phenylethanol.
Time-courses of scent compound emission levels from Petunia TX-794 over 4 days following anthesis. The flowers opened at approximately 8:00 on the day of anthesis. The emitted volatiles were collected at consecutive 4 h periods from 8:00 on the day of anthesis until 8:00 on the fifth day post-anthesis. Concentrations were determined by GC-MS. Petunias were grown in pots at constant temperature (21 ± 2°C), photosynthetic photon flux density (150 μmol·m−2·s−1), and a 12·12 h (8:00 to 20:00 light 20:00 to 8:00 dark) photoperiod, as indicated by the background shading. Results were averaged from three replicate experiments. These data including standard error are shown in Table S1.
The major scent compounds of TX-794 were aromatic compounds structurally dissimilar to those of P. axillaris and other garden petunias. Because there are no wild species or petunia cultivars that have phenylacetaldehyde as a major scent compound (Nakamura et al., 2006), the fragrance of TX-794 is novel.
The major scent compounds of P. axillaris and many other petunia cultivars are C6-C1 aromatic compounds biosynthesized from phenylalanine via trans-cinnamic acid and C6-C3 aromatic compounds biosynthesized via p-coumaric acid (Fig. 3; Boatright et al., 2004; Oyama-Okubo et al., 2013). In contrast, the major scent compounds of TX-794 are C6-C2 aromatic compounds biosynthesized by a different pathway, and the levels of C6-C3 aromatic compounds are low. These results indicate that TX-794 must have a high capacity for C6-C2 aromatic compound biosynthesis. Future breeding efforts may produce a petunia cultivar with a higher percentage of the C6-C2 aromatic compounds and a lower percentage of the C6-C1 and C6-C3 aromatic compounds than found in TX-794, thus yielding petunias with scents like roses or hyacinths.
Proposed biosynthetic pathways for scent compounds emitted by petunia cultivars. Continuous arrows are characterized biosynthetic steps and dashed arrows are less defined steps. The words in parentheses indicate the fragrance quality of the scent compound.
A previous study reported that the fragrances of blue petunias were emitted diurnally (Nakamura et al., 2006). The amount of scent compounds emitted by TX-794 was the highest during the switch from the light period to the dark period. Furthermore, scent compound levels were their lowest at the beginning of the light period. As a result, the fragrance of TX-794 was sensuously felt more strongly from afternoon to evening than in the morning. The fragrance of TX-794 is a sweet scent that includes a pungent odor on the day of anthesis that changes to a sweet and pleasant scent as the days go by. Since the amount of p-cresol was high on the day of anthesis, the irritating odor detected at that time is likely caused by p-cresol, which has a characteristic disinfectant odor. Unlike other benzenoids biosynthesized from phenylalanine, p-cresol is assumed to originate from tyrosine (Fig. 3; Oliva et al., 2015). The sweet and favorable fragrance that became stronger 2 days post-anthesis is thought to be caused by increases in the percentages of the C6-C2 aromatic compounds phenylacetaldehyde and 2-phenylethanol. The time-cause change in scent compounds suggests that on the first day of anthesis, a biosynthetic pathway leading to p-cresol through tyrosine is dominantly active. After anthesis, a biosynthetic pathway leading to other aromatic compounds through phenylalanine is gradually activated. Especially, biosynthetic activity leading to C6-C2 compounds without trans-cinnamic acid contributes to the fragrance of TX-794 flowers.
Two days post-anthesis, the compositions of emitted scent compounds in the light and dark periods were different. The level of the C6-C1 aromatic compound methyl benzoate was highest in the light period and the levels of the C6-C2 aromatic compound, phenylacetaldehyde and the C6-C3 aromatic compound, iso-eugenol, were highest during the switch from light periods to dark periods. Not only the emitted amount of TX-794 scent compounds, but also the quality of emitted compounds, changed with time. The biosynthetic pathway leading to these compounds through phenylalanine is activated during the switching term; even so, biosynthesis of each compound seems individually and finely regulated. Since the flowering period for an individual flower of TX-794 is usually about 7 days, most of the flowers are older than 2 days post-anthesis on bedding plants. Therefore, the fragrance sensed from whole plants is derived from combinations of phenylacetaldehyde and 2-phenylethanol, or methyl benzoate.
