ORIGINAL ARTICLES
Selection of Carnation Line 806-46b with Both Ultra-long Vase Life and Ethylene Resistance
2015 Volume 84 Issue 1 Pages 58-68
Details
2015 Volume 84 Issue 1 Pages 58-68
As the vase life of cut flowers is one of the most commercially important characteristics, genetic improvement of this trait is very desirable. Therefore, we started a breeding research program in 1992 to improve the vase life of flowers in carnation (Dianthus caryophyllus L.) using conventional cross-breeding techniques. We repeatedly crossed and selected promising progeny for three generations, from 2003 to 2010, in order to select lines that exhibit extremely long vase life and low ethylene sensitivity. In 2010, we finally selected a line, 806-46b, with both ultra-long vase life and ethylene resistance out of 50 progeny derived from a cross between 606-65S and 609-63S. The mean vase life of line 806-46b was 27.1 days (444% the value of a control cultivar, ‘White Sim’) at 23°C and 70% RH under a 12-h photoperiod, without chemical treatment, which was the longest vase life among the 6 cultivars and 7 lines tested. Line 806-46b also showed reduced ethylene sensitivity. The response time to 10 μL·L–1 ethylene of this line was 21.8 h, whereas that of ‘White Sim’ was 5.8 h. Line 806-46b did not show brownish discoloration of petal edges during senescence, which was a typical senescence symptom of other selected lines with low ethylene production, when the flower lost its ornamental value. Instead, its petals exhibited gradual reduction of surface turgor by moisture loss as senescence proceeded. This unique senescence pattern is shared by our previously reported line 532-6 with an ultra-long vase life. Line 806-46b showed extremely low ethylene production at senescence, which was also true after ACC treatment. Exogenous ethylene treatment to flowers of this line induced ethylene production without petal wilting, indicating that autocatalytic ethylene production functions normally.
Carnation (Dianthus caryophyllus L.) is one of the most commercially important floricultural crops in Japan and around the world. At present, the world’s major production areas are cool highlands with suitable climates for carnation growth and affordable labor, such as those in Colombia and China (Onozaki, 2006). The number of cut carnation flowers imported into Japan from these two countries has increased steadily each year. Of cut carnations sold in Japan, 52.4% were imported in 2012 (Onozaki et al., 2013). Therefore, breeding of high-value-added or distinctive cultivars is required for Japanese growers in order to compete with imported carnations.
The vase life of cut ornamental flowers, including carnation, is one of the most important characteristics determining their quality and their ability to satisfy consumer preferences, thus stimulating repeated purchasing. Recently, several Japanese supermarket chains have started to offer consumers a guaranteed vase life for some cut flowers (Ichimura, 2013). For these reasons, genetic improvement of flower vase life is very desirable.
Carnation is classified into the group of typical ethylene-sensitive flowers (Woltering and van Doorn, 1988). Senescence of carnation flowers is characterized by autocatalytic ethylene production from petals and the subsequent wilting of the petals (Satoh, 2011). Exposure of fully open carnation flowers to ethylene induces autocatalytic ethylene production and wilting in petals (Halevy and Mayak, 1981); hence, ethylene is an important determinant of flower vase life of carnation.
The vase life of carnations can be significantly extended by treatment with post-harvest chemicals. The most widely used and effective chemical for carnation is silver thiosulfate complex (STS), which is an inhibitor of ethylene action (Reid et al., 1980; Serek et al., 2006; Veen, 1979). However, as STS is harmful to the environment (Satoh, 2011), concerns about pollution due to waste STS solutions have increased in recent years (Klee and Clark, 2004) and the use of STS has already been prohibited in several countries (Serek et al., 2007). In addition, the use of chemicals incurs costs for the growers, such as those due to purchasing them and additional labor time for the treatment of flowers. Therefore, the development of alternative methods for improving the vase life of carnations is anticipated.
The breeding of carnation cultivars with a genetically long vase life would be the best approach, since the improved cultivars would require no chemical treatment to attain a longer vase life. Therefore, we started a breeding research program in 1992 to improve the vase life of carnation flowers using conventional cross-breeding techniques (Onozaki et al., 2001). In previous papers, we described the development of many carnation lines with increased vase life as a result of reduced ethylene biosynthesis achieved by crossing and selection (Onozaki et al., 2001, 2006b, 2011). Two cultivars, ‘Miracle Rouge’ and ‘Miracle Symphony’, with a genetically determined long vase life of 17.7 to 20.7 days under standard conditions (23°C and 70% RH under a 12-h photoperiod), were developed in 2005 (Onozaki et al., 2006a). Their flowers produce a very small amount of ethylene during senescence (Onozaki et al., 2006a), and their long vase life is correlated with the low expression of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase genes (DC-ACS1, DC-ACS2, and DC-ACO1) in the gynoecium and petals (Tanase et al., 2008). Furthermore, we developed an ultra-long-vase-life line, 532-6, with a flower vase life of over 27 days (Onozaki et al., 2011). However, these cultivars and line 532-6 remained sensitive to exogenous ethylene and their ethylene sensitivity at anthesis is generally equal to that of normal, ethylene-sensitive Sim-type cultivars (Onozaki et al., 2006a, 2011).
Although carnation flowers are generally highly sensitive to the exogenous application of ethylene (Woltering and van Doorn, 1988), Wu et al. (1991) reported that two long-vase-life cultivars, ‘Chinera’ and ‘Epomeo’, had lower ethylene sensitivity than the standard-vase-life cultivar ‘White Sim’. Similarly, our two selected lines (64-13 and 64-54) with a long vase life also showed lower ethylene sensitivity (Onozaki et al., 2001). We demonstrated that the ethylene sensitivity of carnation can be reduced by conventional cross-breeding techniques (Onozaki et al., 2008) and successfully developed a total of 13 lines with low ethylene sensitivity (Onozaki et al., 2008, 2011) with the aid of our video evaluation system (Onozaki et al., 2004a). These ethylene-insensitive lines are expected to be useful for reducing the effects of exogenous ethylene that may occur during storage and transportation. However, the mean vase life of these 13 ethylene-resistant lines ranged from 7.6 to 15.2 days under standard conditions, which was not as long as the values of the two ethylene-sensitive but extremely long-vase-life cultivars (‘Miracle Rouge’ and ‘Miracle Symphony’) and other selected lines, such as line 532-6, as described above.
Here, we evaluated the vase life and ethylene sensitivity of many carnation seedlings derived from crosses using 10 ethylene-resistant lines and 3-sensitive lines with a long vase life in order to combine an extremely long vase life and low ethylene sensitivity. In this paper, we describe the successful development of a new line, 806-46b, which has both an ultra-long vase life and ethylene resistance. Ethylene production and sensitivity of line 806-46b were also compared with those of six other cultivars under senescence, after ethylene or ACC treatment.
The breeding materials used in this study include 10 ethylene-resistant lines (902-48a, 092-31, 118-64S, 120-69S, 234-36S, 234-43S, 234-47S, 234-48S, 234-52S, and 204-41S) (Onozaki et al., 2008, 2011) and three lines (104-3, 108-40, and 104-47) out of 28 lines selected for a long vase life at the fourth generation in our previous study (Onozaki et al., 2006b) (Table 1). In the spring of 2003, 2004, and 2005, we made 10 cross combinations among these lines as shown in Table 2 using standard procedures (Onozaki et al., 2006a) in order to combine a long vase life with low ethylene sensitivity. All seeds obtained from these crosses were sown on 14 July, 2003, 30 June, 2004, and 30 June, 2005, respectively, and seedlings were grown in a greenhouse at NARO Institute of Floricultural Science (NIFS). Progeny that did not flower by 28 May, 2004, 24 May, 2005, and 15 May, 2006, which were the last days of evaluation, respectively, were discarded. In the seedling trials, all harvested flowers were evaluated for flower vase life or ethylene sensitivity. In the crosses between ethylene-resistant lines, we initially selected 8 lines out of 46 progeny in total with a longer mean response time to the 10 μL·L–1 ethylene treatment (≥ 55.0, 56.0, or 41.0 h, respectively) in June 2004, 2005, and 2006, and multiplied them vegetatively. In the crosses between an ethylene-resistant line and a long-vase-life line with ethylene sensitivity, we initially selected 18 lines out of 127 progeny in total with a longer mean vase life (≥ 16.0 days) or with a longer mean response time (≥ 10.0 or 16.0 h, respectively) in June 2004 and 2005, and multiplied them vegetatively. In 2006, 4 lines (307-51a, 404-21a, 427-26a, and 408-28a) were finally selected for the next crosses.
Flower color, vase life and ethylene sensitivity in 13 lines used for breeding materials.
Results of crosses and seedling evaluation for flower color, phenotype, ethylene sensitivity and vase life in four different cross groups.
In spring 2006, crosses were made among these 4 lines. All seeds obtained from the crosses were sown on 30 June, 2006, and were grown in the greenhouse. Progeny that did not flower until 25 May, 2007 (the last day of our evaluation), were discarded. In the seedling trials, all harvested flowers were evaluated for flower vase life or ethylene sensitivity. In June 2007, we initially selected 14 lines out of 58 progeny in total with a longer mean vase life (≥ 16.0 days) or with a longer mean response time (≥ 13.5 h) and multiplied them vegetatively. In 2008, 2 lines (609-63S and 606-65S) were finally selected for the next crosses.
In spring 2008, a cross was made between 606-65S and 609-63S. All seeds obtained from the cross were sown on 3 July, 2008, and were grown in a greenhouse. Progeny that did not flower until 17 May, 2009 (the last day of our evaluation), were discarded. In the seedling trials, all harvested flowers were evaluated for flower vase life or ethylene sensitivity. In June 2009, we initially selected 10 lines out of 50 progeny with a longer mean vase life (≥ 17.0 days) or with a longer mean response time (≥ 18.0 h) and multiplied them vegetatively. In 2010, we finally selected a line, 806-46b, with the longest mean vase life and ethylene resistance out of the 10 primarily selected lines. The pedigree of line 806-46b is shown in Figure 1, along with that of line 532-6, which was previously selected as an ultra-long-vase-life line (Onozaki et al., 2011).
Pedigrees of selected lines 532-6 and 806-46b with an ultra-long vase life. Ethylene-resistant lines are shown in italics and underlined.
Flower color was evaluated visually and flower phenotype was classified according to visual appearance; a normal flower with more than 5 petals as a double type and a proliferating flower with split calyxes as a bullhead type (or super-double type). Bullhead seedlings were discarded and excluded from the evaluation of ethylene sensitivity and vase life.
Vase life evaluationCarnation cultivars and lines were grown in the greenhouse at NIFS following standard production methods for carnations (Onozaki et al., 2006a) and harvested at the flower-opening stage (i.e., when the outer petals became orientated at right angles to the stem). Stems of the freshly harvested flowers were trimmed to 50 cm, and the two or three lowest pairs of leaves were removed. The flowers were then placed in 4-L jars containing ca. 1 L of distilled water. The water was replaced every 3 or 4 days.
The vase life of each flower was determined by the number of days from harvesting until the petals showed in-rolling, browning or other morphological changes to lose their ornamental value. Flowers were evaluated daily in an inspecting room, which was kept at 23°C, 70% RH, and 12-h photoperiod (08:00 to 20:00 h) provided by cool fluorescent lamps (10 μmol·m–2·s–1 irradiance). The mean vase lives of 6 cultivars and 7 lines were evaluated in 2009 and 2010 with 10–13 replications.
Morphological characteristics during senescence in three cultivars and line 806-46bTo compare the vase life and morphological characteristics during senescence, flowers of ‘Excerea’, ‘Miracle Rouge’, ‘Miracle Symphony’, and line 806-46b were harvested at the flower-opening stage on March 18, 2010. Flower stems were trimmed to 50 cm, and the two or three lowest pairs of leaves were removed. The cut flowers were kept in distilled water at 23°C, 70% RH, under a 12-h photoperiod as described above. They were photographed at 0, 8, 10, 20, 30, and 40 days after the start of the experiment.
Video evaluation of ethylene sensitivityEthylene sensitivity was measured using a time-lapse video recording system at 10 μL·L–1 ethylene concentration and 23 ± 1°C in a 50-L acryl chamber, as described previously (Onozaki et al., 2004a). On day 0, the stems of freshly harvested flowers were trimmed to 20 cm and kept individually in 100-mL Erlenmeyer flasks containing distilled water. The relative humidity in the chamber was kept between 40% and 60% by including about 100 g of silica gel. The time to the onset of petal in-rolling, referred to as ‘response time’ hereafter, was determined by fast-forwarding the image data files. The response times of 6 cultivars and 7 lines to ethylene treatment were evaluated in 2010 with 5 replications.
Measurement of ethylene production from whole flowers at senescence, after ethylene treatment or after ACC treatmentTo investigate the cause of the ultra-long vase life in line 806-46b, we measured ethylene production from whole flowers at senescence, after ethylene treatment, and after ACC treatment in six cultivars and line 806-46b, as described previously (Onozaki et al., 2011).
Ethylene measurements at senescence were carried out when senescence symptoms first appeared. Flowers were harvested at the stage of flower opening (i.e., when the outer petals became orientated at right angles to the stem) and their stems were trimmed to 5 cm and placed individually in a test tube containing distilled water. Flowers were kept under the conditions described above until petals showed in-rolling, browning or losses of surface turgor and ornamental value. When senescence was first observed, individual flowers were weighed (fresh weight), and then put in a 148-mL glass jar and kept at 23°C. After incubation for 1 or 2 h, a 0.5-mL sample of the headspace gas was withdrawn and injected into a gas chromatograph (model GC-13A; Shimadzu, Kyoto, Japan) equipped with an alumina column and a flame ionization detector to determine ethylene concentration.
Measurements of autocatalytic ethylene production were carried out using flowers on day 0 treated with ethylene at 2 μL·L–1 for 16 h at 23 ± 1°C in a 50-L chamber. After ethylene treatment, flowers were left for 8 h at 23°C and 70% RH and exposed to fresh air. The flowers were then individually enclosed in a 148-mL glass jar and held at 23°C. After 1-h incubation, each gas sample was analyzed for ethylene concentration as described above.
We measured ethylene production after the treatment with ACC, which is the immediate precursor of ethylene in higher plants (Abeles et al., 1992). Flower stems on day 0 were trimmed to 5 cm. The stem end of flowers was placed individually in a test tube containing 0.3 mM ACC solution for 24 h under the conditions described above. The flowers were then individually put in a 148-mL glass jar and kept at 23°C. After 1-h incubation, each gas sample was analyzed for ethylene concentration as described above.
All data of ethylene measurement are presented as the means ± SE of five or six flowers.
Statistical analysesThe results shown in Table 3 and Figure 6 were analyzed with Excel-Toukei 2012 (Social Survey Research Information Co., Ltd., Tokyo, Japan) using Tukey’s test (P < 0.05).
Flower color, vase life, and ethylene sensitivity in control cultivars, NIFS cultivars, and finally selected lines.
The 29 progeny derived from the crosses between ethylene-resistant lines (A group) showed a very wide distribution in terms of the response time to the exogenous ethylene application, from 10.0 to 83.0 h (Table 2; Fig. 2A), with a mean of 38.4 h. On the other hand, the 58 progeny obtained from the crosses between a resistant line and a sensitive line (B group) and the 51 progeny from the crosses between selected lines of B group progeny (C group) showed a narrow distribution in terms of the response time, with a single peak (Fig. 2B, C). The 31 progeny of the crosses between selected lines of C group progeny (D group) showed an intermediate range distribution, from 8.0 to 32.0 h (Table 2; Fig. 2D), with a mean of 12.2 h. The population mean response time increased by 3.3 h with the progress of generation from C to D group (Fig. 2C, D).
Frequency distribution of ethylene sensitivity in four different cross groups. Detailed cross combinations of each group are shown in Table 2.
The frequency distribution of flower vase life in the 6 progeny of ethylene-resistant crosses (A group) was very narrow, ranging from 8.7 to 11.0 days (Table 2; Fig. 3A). On the other hand, the 90 progeny of the B group showed a continuous normal distribution, ranging from 4.0 to 22.0 days (Table 2; Fig. 3B). The 46 progeny of C group and the 18 progeny of D group showed a wide and bimodal distribution (Table 2; Fig. 3C, D). The population mean vase life increased with the progress of generation by 2.6 days from B to C group and by 1.0 days from C to D group (Fig. 3B, C, D).
Frequency distribution of flower vase life in four different cross groups. Detailed cross combinations of each group are shown in Table 2.
We finally selected 7 lines in total, including 4 lines (307-51a, 404-21a, 427-26a, and 408-28a) out of 127 progeny in B group, 2 lines (609-63S and 606-65S) out of 58 progeny in C group and 1 line (806-46b) out of 50 progeny in D group.
Segregation of flower colorsAmong the 13 lines used for breeding materials, 10 ethylene-resistant lines have a yellow or orange flower color with red or pink stripes, whereas 3 long-vase-life lines have a pink or red flower color. A total of 29 flowered progeny derived from the crosses between ethylene-resistant lines (A group) showed a yellow tint in their base flower colors (Table 2). A total of 111 flowered progeny of the B group (cross between ethylene-resistant line and -sensitive line with red or pink flowers) showed a pink or red phenotype at an approximate ratio of 1:1, indicating that yellow or orange colors were controlled by recessive alleles. A total of 54 flowered progeny of C group showed pink or yellow in their base flower colors, while 32 flowered progeny of the D group produced only pink flowers.
Vase life and ethylene sensitivity in tested cultivars and selected linesSix cultivars and seven finally selected lines exhibited a wide range of variation in terms of mean vase life (Table 3). The mean vase life of ‘White Sim’, which has been used as a control cultivar in many flower senescence studies of carnation, was 5.9–6.1 days, whereas those of seven selected lines ranged from 13.8 to 27.1 days (2.3–4.4 times longer than ‘White Sim’). The mean vase life of our two bred cultivars with a long vase life, ‘Miracle Rouge’ and ‘Miracle Symphony’ (Onozaki et al., 2006a), was 16.1 to 18.4 days (2.7–3.1 times longer than that of ‘White Sim’). The mean vase life of ‘Karen Rouge’, our bred cultivar with resistance to bacterial wilt (Yagi et al., 2010), was 11.4–12.0 days. In particular, line 806-46b showed significantly longer vase life than other cultivars and lines tested (Table 3). The mean vase life of 806-46b was 27.1 days, which was 444% of the value of ‘White Sim’.
There were large differences in ethylene sensitivity among the six cultivars and seven selected lines (Table 3, Fig. 4). The six tested cultivars were all sensitive to ethylene, with response times of 5.6 to 7.2 h. The response time of line 806-46b was significantly longer than those of the other cultivars and lines tested (21.8 h), whereas those of the other six selected lines, which were parental and grandparental lines of 806-46b, ranged from 7.4 to 12.2 h (Table 3).
Variation in ethylene sensitivity between line 806-46b and ‘Karen Rouge’ flowers. A, B, and C were photographed at 0, 6, and 12 h after the start of 10 μL·L–1 ethylene treatment, respectively. Left: ethylene-sensitive cultivar, ‘Karen Rouge’. Right: ethylene-resistant line, 806-46b.
Figure 5 shows variation in the vase life and morphological differences during senescence among three cultivars and line 806-46b. Senescence of ‘Excerea’ was characterized by petal in-rolling and rapid wilting of whole flower, which are well-known characteristics of ethylene-dependent senescence in carnation flowers (Fig. 5B, C; Iwazaki et al., 2004; Otsu et al., 2007; Satoh, 2011). In contrast, the petals of ‘Miracle Rouge’ and ‘Miracle Symphony’ started browning from their edges at around 20 days (Fig. 5D), then slowly desiccated, faded and turned brown, which are characteristics of ethylene-independent senescence in carnation flowers (Fig. 5E, F; Iwazaki et al., 2004; Otsu et al., 2007; Satoh, 2011). On the other hand, the flower of line 806-46b did not exhibit visible signs of senescence until 30 days and showed no brownish discoloration in this experiment (Fig. 5E). Petals of line 806-46b gradually lost internal water, wrinkled slowly and finally lost their ornamental value (Fig. 5F).
Variation in the vase life of selected line 806-46b with an ultra-long vase life and three control cultivars, ‘Miracle Symphony’, ‘Miracle Rouge’, and ‘Excerea’. Flowers were harvested at the flower-opening stage. They were photographed at 0, 8, 10, 20, 30, and 40 days after the start of the experiment (Experimental period: March 18–April 27, 2010). ‘Miracle Symphony’, ‘Miracle Rouge’, line 806-46b, and ‘Excerea’ (left to right). The cut flowers were kept in distilled water at 23°C, 70% RH, under a 12-h photoperiod.
Whole-flower ethylene production at senescence (A), after ethylene treatment (B) and after ACC treatment (C) in six cultivars and line 806-46b with an ultra-long vase life. Values represent the means ± SE of five or six flowers, and those with different letters differ significantly at P < 0.05 by Tukey’s test.
At senescence, ‘White Sim’ and ‘Excerea’ evolved a large amount of ethylene (Fig. 6A). In particular, ‘Excerea’ produced the largest amount of ethylene among tested cultivars and lines. Our bacterial-wilt-resistant cultivar, ‘Karen Rouge’, which was derived from a cross of the long-vase-life cultivar ‘Miracle Rouge’ and the bacterial-wilt-resistant line 4AZ31-5 (Yagi et al., 2010), showed moderately low ethylene production at senescence (about half that of ‘White Sim’). On the other hand, ‘Miracle Rouge’, ‘Miracle Symphony’, and line 806-46b showed extremely low ethylene production. Exogenous application of ethylene induced autocatalytic ethylene production in line 806-46b (Fig. 6B), although the petal did not show wilting symptoms. There were large differences in ethylene production after ACC treatment (Fig. 6C). ‘White Sim’, ‘Excerea’, and ‘Sandrosa’ produced ethylene abundantly, whereas line 806-46b showed the lowest ethylene production after ACC treatment among the tested cultivars and lines.
In previous studies, we selected a total of 13 ethylene-resistant lines (Onozaki et al., 2008, 2011) by a video evaluation method (Onozaki et al., 2004a). However, none of lines had an extremely long vase life in comparison with our two long-vase-life cultivars (‘Miracle Rouge’ and ‘Miracle Symphony’) and other selected lines with extremely low ethylene production, such as line 532-6 (Onozaki et al., 2008, 2011). The vase life of these 13 ethylene-resistant lines ranged from 7.6 to 15.2 days under standard conditions (Onozaki et al., 2008, 2011). In the present study, the mean vase life in the 6 progeny of ethylene-resistant crosses (A group) was 9.9 days (Fig. 3A), even though the mean response time to 10 μL·L–1 ethylene in the 29 progeny of the cross was 38.4 h (Fig. 2A). Although we primarily selected 8 lines with a longer response time among the progeny of the crosses between ethylene-resistant lines (Table 2), we were not able to select lines with an extremely long vase life despite a replicated test in the second year (data not shown). Another problem is that the progeny of this cross tend to show late flowering (in other words, tend to delay flower bud differentiation), which is generally unsuitable for practical cultivation compared with early-flowering cultivars of carnation (Sparnaaij et al., 1990). Therefore, we finally concluded that crosses between ethylene-resistant lines were ineffective to produce lines with both ethylene resistance and an extremely long vase life.
Another strategy for achieving our breeding goal was as follows. Step 1: Conduct crosses between an ethylene-resistant line and a sensitive line with an extremely long vase life and select progeny with a longer vase life or with a longer response time. Step 2: Conduct crosses between selected lines from step 1 and select by longer vase life or longer response time. As a result, we succeeded in selecting line 806-46b with both ultra-long vase life and ethylene resistance, which had a vase life of 27.1 days (4.4 times the vase life of a control cultivar, ‘White Sim’) without chemical treatment.
Line 806-46b showed extremely low ethylene production at senescence (Fig. 6A) and even after ACC treatment (Fig. 6C). Since ACC is the direct precursor of ethylene and ACC oxidase catalyzes the reaction from ACC to ethylene (Abeles et al., 1992), it can be considered that the activity of ACC oxidase is low in line 806-46b. On the other hand, exogenous treatment of ethylene to whole flowers induced autocatalytic ethylene production in line 806-46b (Fig. 6B) without petal wilting, indicating that ethylene receptor genes function normally. Therefore, the flowers of line 806-46b are not completely insensitive to ethylene, but seem to have normal ethylene receptors and to produce ethylene autocatalytically.
In a previous study, we succeeded in producing line 532-6, which has an ultra-long vase life and a low level of ethylene production at senescence. However, it is sensitive to exogenous ethylene application like ordinary cultivars such as ‘White Sim’ (Onozaki et al., 2011). In contrast, line 806-46b produced in the present study as a strain with an ultra-long vase life showed low ethylene sensitivity (Fig. 4) and significantly longer vase life than other tested cultivars and lines (Table 3). These results show that the breeding of lines with both an extremely long vase life and ethylene resistance is possible using conventional cross-breeding techniques.
The control cultivar ‘Excerea’ showed senescence symptoms such as petal in-rolling and rapid wilting (Fig. 5B, C). On the other hand, long-vase-life cultivars, ‘Miracle Rouge’ and ‘Miracle Symphony’, did not show these symptoms, but exhibited petal fading and browning from the edges (Fig. 5E). These two types of senescence in carnation flowers are morphologically designated as wilting and fading, respectively (Satoh, 2011). In contrast, line 806-46b showed gradual reduction of surface turgor by moisture loss without brownish discoloration as senescence progressed. This unique senescence pattern is shared by line 532-6, which was previously reported to have an ultra-long vase life (Onozaki et al., 2011). In the present study, we demonstrated the existence of a third senescence pattern in carnation flowers, which was specific to the lines with an ultra-long vase life.
Figure 1 shows the pedigrees of lines 532-6 and 806-46b, with an ultra-long vase life. In the process of producing these two lines, three lines, 903-45, 908-43, and 908-19, which belong to selected third-generation lines, were used for breeding in common and the latter two lines, 908-43 and 908-19, were full-sib lines of ‘Miracle Rouge’. Therefore, it is possible that lines 532-6 and 806-46b have some common genes responsible for the trait of an ultra-long vase life derived from these three lines.
Lines 903-45, 908-43, and 908-19 were all shown to be ethylene-sensitive lines with response times of 7.5, 7.0, and 6.8 h, respectively, on day 0 (Onozaki et al., 2006b, and unpublished data). Moreover, line 532-6 was also ethylene-sensitive, with a response time of 6.4 h (Onozaki et al., 2011). These results imply that genes conferring an ultra-long vase life correlated poorly with ethylene sensitivity at anthesis. Our previous results showed that ethylene sensitivity after anthesis in ‘Miracle Rouge’ and ‘Miracle Symphony’ rapidly decreased with age, and became completely ethylene-insensitive or developed extremely low sensitivity by the end of senescence (Onozaki et al., 2006b). Further studies are required on the change of ethylene sensitivity after anthesis in lines 532-6 and 806-46b, both of which have an ultra-long vase life but different sensitivity to exogenous ethylene.
Line 806-46b showed slightly weak ethylene resistance (21.8 h) compared with 10 other ethylene-resistant lines used for breeding materials, with a response time of more than 24 h (Table 1). In particular, the most ethylene-resistant line, 234-43S, showed the longest response time of 57.9 h. All ethylene-resistant lines that we selected in previous studies have a yellow or orange tint in their base flower colors and their petals have pink or red visible stripes without exception, whereas line 806-46b has a solid pink flower color without visible stripes. In our previous studies, we assumed that genes conferring low ethylene sensitivity were linked with the genes for yellow or orange color and involved with a transposable element (Onozaki et al., 2008, 2011). Further studies are needed to clarify the relationship among ethylene resistance, flower color and a transposable element.
The results of our previous study suggested that ethylene resistance is inherited in a recessive manner (Onozaki et al., 2008), which was confirmed in the present study. Line 806-46b derived from the cross between lines 606-65S and 609-63S (Table 2; Fig. 1) showed a response time of 21.8 h (Table 3), whereas parental lines of this line had nearly half this values, that is a response time of 12.2 h for line 606-65S and 11.0 h for line 609-63S (Table 3), which were classified as moderately sensitive based on our criteria for the degree of ethylene sensitivity (Onozaki et al., 2008). These results suggest that both parental lines 606-65S and 609-63S carry recessive genes responsible for ethylene resistance in the heterozygous state.
In conclusion, although carnation flowers are generally highly sensitive to exogenous ethylene (Woltering and van Doorn, 1988), we succeeded in developing line 806-46b with both an ultra-long vase life and ethylene resistance by conventional cross-breeding techniques. Line 806-46b would be useful in research on genes involved in ethylene synthesis and response to exogenous ethylene. Recently, we compared expression levels of senescence-related genes involved in ethylene biosynthesis in flowers of 3 long-vase-life cultivars and lines including ‘Miracle Symphony’, and clarified the mechanism behind long vase life at the level of gene expression (Tanase et al., 2013). We are planning further research to clarify the molecular biological mechanism behind ultra-long vase life using lines 532-6 and 806-46b.
We have constructed genetic linkage maps for carnation and used these maps to identify quantitative trait loci (QTL) responsible for resistance to bacterial wilt (Yagi et al., 2006, 2012). Furthermore, we developed DNA markers tightly linked to bacterial wilt resistance (Onozaki et al., 2004b; Yagi et al., 2012) and flower type (Onozaki et al., 2006c; Yagi et al., 2014). In particular, the marker linked to bacterial wilt resistance is now being used in practical breeding programs to develop resistant commercial cultivars. However, DNA markers linked to flower vase life have not yet been developed. Improvement of flower vase life using conventional breeding techniques requires a great deal of time and effort. By combining conventional breeding techniques with the molecular genetics of flower vase life, the development of more reliable breeding methods of carnations can be achieved. Currently, we are planning to develop DNA markers tightly linked to flower vase life for marker-assisted selection by producing a new mapping population using line 806-46b with an ultra-long vase life.
