Genetic improvement of vase life of carnation
¯owers by crossing and selection
Takashi Onozaki
*, Hiroshi Ikeda, Takashi Yamaguchi
1National Research Institute of Vegetables, Ornamental Plants and Tea, Kusawa, Ano, Mie 514-2392, Japan
Accepted 12 April 2000
Abstract
We used conventional cross-breeding techniques to develop many carnation lines with long vase life and either low ethylene production or low ethylene sensitivity. Two cycles of selection and crossing to improve vase life led to a 3.6-day increase in mean vase life. All 39 selected lines had signi®cantly longer vase life than the control cultivar, `White Sim'. In particular, second-generation lines 63-3, 63-12, 66-15, and 63-41 had a mean vase life of more than 15 days without chemical treatment. Measurements of ethylene production indicated that ¯owers of all second-generation selected lines had a greatly reduced capacity to produce ethylene. We screened three lines (515-10, 64-13, and 64-54) with low ethylene sensitivity. Evaluation by exposure to ethylene at high concentration showed that 64-13 and 64-54 were less sensitive to ethylene than `Chinera', which is known for it low sensitivity. The vase life of these low-sensitivity lines was about twice that of `White Sim'. The extended vase life of selected lines was related to low ethylene production at ¯ower senescence rather than to degree of ethylene sensitivity in young ¯owers. Ethylene sensitivity decreased with the age of the ¯ower in many selected lines. The results clearly show that vase life of carnation ¯owers can be extended by crossing and selection.#2001 Elsevier Science B.V. All rights reserved.
Keywords: Ethylene; Carnation (Dianthus caryophyllusL.); Breeding; Selection; Flower longevity; Vase life
*
Corresponding author. Tel.:81-59-268-4662; fax:81-59-268-1339.
E-mail address: [email protected] (T. Onozaki).
1
Present address: Fukkaen Nursery & Bulb Co., Ltd., Kitayama, Misato, Yokkaichi, Mie 512-1104, Japan.
1. Introduction
The potential vase life of cut ¯owers is one of the most important quality factors, because it strongly affects consumer satisfaction and repeat purchasing and it in¯uences the value of the cut ¯owers. However, as breeding has concentrated mainly on ¯oral traits such as ¯ower color, size, morphology, and duration of ¯owering, or on economic traits such as productivity, vase life had a much lower priority. The importance of vase life is now being recognized. Recently, inheritance and response to selection for vase life of ¯owers have been studied in gerbera (Wernett et al., 1996) and Asiatic hybrid lilies (Van der Meulen-Muisers et al., 1999).
Carnation is a major ornamental crop, ranking third in importance in Japan after chrysanthemum and rose. However, the carnation ¯ower is highly sensitive to exogenous ethylene (Woltering and Van Doorn, 1988), and its vase life is normally short without the use of preservatives. Vase life of carnation can be extended by postharvest chemical treatment. The onset of ¯ower senescence can be signi®cantly delayed by treatment with inhibitors of ethylene biosynthesis, such as aminooxyacetic acid (Fujino et al., 1980), aminoethoxyvinyl glycine (Baker et al., 1977), anda-aminoisobutyric acid (Onozaki and Yamaguchi, 1992; Onozaki et al., 1998), or with inhibitors of ethylene action, such as silver thiosulfate (STS) (Veen, 1979). In particular, STS is widely used by commercial carnation producers to extend the vase life of the cut ¯owers because of its outstanding effect. However, as concern about potential heavy-metal contamina-tion of the environment by waste STS solucontamina-tions has increased in recent years, alternative methods for improving the vase life of carnations must be developed. The breeding of carnation cultivars with genetically superior vase life may be the best approach because breeding and selection techniques that improve vase life can eliminate the use of chemicals.
Several researchers have studied genetic variation between carnation cultivars in the vase life of cut ¯owers. Several commercial carnation cultivars (`Sandra', `Chinera', `Killer', `Epomeo', and `Sandrosa') with extended vase life have been reported (Serrano and Romojaro, 1991; Wu et al., 1991a,b; Mayak and Tirosh, 1993; Woltering et al., 1993). These cultivars have a much longer vase life than most commercially grown standard carnations with climacteric ethylene production (e.g. `White Sim'). They feature either a low rate of ethylene production during senescence (`Sandra', `Killer', and `Sandrosa') or a reduced sensitivity to ethylene (`Chinera' and `Epomeo'). Woltering et al. (1993) have shown that reduced ethylene sensitivity is heritable. Therefore, improvement of vase life of carnation by crossing and selection seems possible.
on the results of crossing and selection over three generations to improve the vase life of carnation. We also investigated ethylene production and sensitivity in selected lines with a long vase life.
2. Materials and methods
2.1. Crossing and selection
To improve the vase life of carnation ¯owers, we repeated crossing and selection for three generations. We chose six commercial standard carnation (Dianthus caryophyllus L.) cultivars with large differences in vase life for breeding materials: four Mediterranean types (`Pallas', `Sandrosa', `Candy', and `Tanga') and two American Sim types (`White Sim' and `Scania') (Table 1). In the spring of 1992, crosses were made among these cultivars. On 15 August 1992, all obtained seeds were sown and grown. Plants that did not ¯ower until 12 July 1993 (the last day of evaluation) were discarded. We called the remaining 195 plants the parental-generation. In July 1993, 53 plants with the longest mean vase life (8.5 days) were primary-selected and multiplied vegetatively for further investigation. Six to eight rooted cuttings of selection were planted in sterilized soil beds in the greenhouse and grown. In 1994, 12 primary lines with the longest mean vase life were secondary-selected (parental-generation selected lines). In the spring of 1994 and 1995, crosses were made with 11 of these. All obtained seeds were sown on 12 August 1994 or 17 July 1995 and grown. Plants that did not ¯ower until 27 June 1995 or 24 June 1996 (the last day of evaluation) were discarded. We called the remaining 309 plants the ®rst-generation. In June 1995 and 1996, 82 plants with the longest mean vase life (9.2 days) were primary-selected and multiplied vegetatively. In 1996 and 1997, 17 primary lines with the longest mean vase life were secondary-selected (®rst-generation selected lines).
Table 1
In the spring of 1996, crosses were made with nine of these. All obtained seeds were sown on 24 June 1996 and grown. Plants that did not ¯ower until 30 May 1997 (the last day of evaluation) were discarded. We called the remaining 163 plants the second-generation. In June 1997, 58 plants with the longest mean vase life (11.0 days) were primary-selected and multiplied vegetatively. In 1998, 14 lines with the longest mean vase life were secondary-selected (second-generation selected lines).
2.2. Vase life evaluation
Carnation cultivars or lines grown in a greenhouse by standard production methods were harvested at commercial maturity (outer petals horizontal). The stems of freshly harvested ¯owers were cut to 50 cm, and the two lowest pairs of leaves were removed. The ¯owers were then placed randomly in 2.5 l jars containing about 800 ml of distilled water. The water was replaced for every 3 or 4 days.
The vase life of each ¯ower was determined by the number of days from harvest until the petals showed in-rolling or browning and had no decorative value. Flowers were evaluated daily in a temperature-controlled room with a constant air temperature of 238C, 70% RH, and a 12 h photoperiod (08:00± 20:00 h) provided by cool ¯uorescent lamps (10mmol mÿ2sÿ1irradiance).
In the seedling trials, all harvested ¯owers were used for vase life evaluation. For each selected line, 10 ¯owers were harvested for evaluation, and the mean vase life was determined (clonal test). Four other cultivars were also tested: `U Conn Sim', `Coral', `Chinera', and `Killer'. The vase life of individual selected lines was evaluated in 1999. The statistical signi®cance of mean vase life against `White Sim' or `Sandrosa' was evaluated byt-test (P0.01).
2.3. Measurement of ethylene production in senescencing ¯owers
Detailed study of ethylene production in aging carnation ¯owers has revealed that ¯ower senescence is normally characterized by a climacteric pattern of ethylene production, that biosynthesis of ethylene is associated with speci®c developmental stages, and that ethylene production peaks when petals showed in-rolling and slight wilting (Bu¯er et al., 1980; Lawton et al., 1989). To compare the ethylene production of a large number of materials during ¯ower senescence, we began measurements when senescence was ®rst observed.
and had no decorative value. When senescence was ®rst observed, individual ¯owers were weighed and then enclosed in a 470 ml glass jar and kept at 238C. After 1 h incubation, a 0.5 ml sample of headspace gas was withdrawn and analyzed for ethylene concentration with a gas-chromatograph, model GC-7A (Shimadzu, Kyoto, Japan) equipped with an alumina column and a ¯ame ionization detector.
2.4. Ethylene treatments at different ¯ower ages
We measured the sensitivity to exogenous ethylene by response to ethylene treatment. On an average, ®ve ¯owers of each cultivar or line were harvested at commercial maturity. The stems were cut to 20 cm and held in a 100 ml Erlenmeyer ¯ask containing distilled water. Flowers were aged under standard conditions (238C, 70% RH, 12 h photoperiod) for 0, 3, or 6 days. Then the ¯owers were exposed to 2 or 4ml lÿ1ethylene for 16 h at 238C in a 50 l sealed transparent acryl chamber into which pure ethylene gas was injected. A fan was used to ensure mixing of the gas during the treatment. After the treatment, the ¯owers were transferred to the temperature-controlled room and assessed daily for wilting symptoms. Finally, the vase life was determined.
3. Results
3.1. Crossing and selection
The frequency distributions of vase life in parental-, ®rst-, and second-generations were characteristic of continuous normal distribution (Fig. 1). Variation was large in parental- and second-generations (S.D.2.16 and 2.59), but relatively small in the ®rst-generation (S.D.1.76). The proportion of ¯owers with inferior vase life (vase life4±6 days) was high in the parental-generation but was markedly decreased in the ®rst-generation. The proportion of ¯owers with superior vase life was markedly increased in the second-generation. The population mean for vase life increased by 1.0 day from parental to ®rst-generation and by 2.6 days from ®rst- to second-®rst-generation. Thus, the effectiveness of selection was small from parental- to ®rst-generation but greater from ®rst to second-generation.
3.2. Vase life of cultivars and selected lines
cultivar, was 5.4 days. The mean vase life of `Sandrosa', which is known to lack a climacteric ethylene response (Mayak and Tirosh, 1993), was 10.1 days. `Killer' and `Chinera' had a mean vase life of about 11 days, in agreement with results reported by Serrano and Romojaro (1991) and Wu et al. (1991a).
Table 2
Flower vase life (daysS.E.) of carnation cultivars and selected lines under standard conditions (238C, 12 h photoperiod, 70% RH)
Cultivar or selected line Vase life Percentage of control
All 39 selected lines showed signi®cantly longer vase life than `White Sim'. Furthermore, 15 of the lines showed signi®cantly longer vase life than `Sandrosa', which had the highest vase life among the six parental cultivars (Table 2).
The mean vase life of second-generation selected lines ranged from 11.1 to 17.5 days. Lines 63-3, 63-12, 66-15 and 63-41 had a vase life of 15.0±17.5 days. In particular, 66-15 had 3.2 times the vase life of `White Sim' without chemical treatment.
3.3. Ethylene production in senescencing ¯owers
The 10 cultivars and 38 selected lines showed large differences in ethylene production by the ¯owers (Fig. 2). Ethylene production in `Pallas', `Candy', `White Sim', `Tanga', `Scania', `U Conn Sim' and `Coral' showed a typical climacteric pattern. The amount of ethylene produced ranged from 27.4 nl gfwÿ1hÿ1by `Scania' to 88.4 nl gfwÿ1hÿ1by `Tanga'. Ethylene production by `Chinera' was half of that by `White Sim'. In contrast, `Sandrosa' and `Killer' showed extremely low ethylene production. These results agree with those of Serrano and Romojaro (1991), Wu et al. (1991a) and Mayak and Tirosh (1993).
Several parental- and ®rst-generation selected lines showed low ethylene production, and 14-9, 945-25, and 515-13 showed high ethylene production.
All 14 second-generation selected lines showed low ethylene production. Ten lines (63-3, 63-12, 66-15, 63-41, 62-18, 63-35, 63-8, 62-48, 63-7, and 62-2) showed very low production and lacked the ethylene climacteric peak. In particular, lines 63-8, 62-48, 63-7, and 62-2 had a greatly reduced capacity to
Table 2 (Continued)
Cultivar or selected line Vase life Percentage of control
cultivar, `White Sim'
Signi®cance shown of difference against `White Sim': ** Ð 1% level.
b
n.s. Ð not signi®cant.
c
produce ethylene. These lines did not show petal in-rolling or rapid wilting at senescence, but faded and turned brown. Lines 64-56, 63-24, 64-13, and 64-54 showed low ethylene production but had a climacteric ethylene production pattern and senesced like the control cultivars, with in-rolling and wilting of the petals. Thus, the long vase lives of the second-generation selected lines were associated with the level of ethylene production.
3.4. Effect of ¯ower age at treatment on ethylene sensitivity
In general, the tested carnation ¯owers were highly sensitive to exogenous ethylene (Fig. 3). The vase life of all cultivars and lines was markedly reduced by ethylene exposure, owing to accelerated senescence of ¯ower petals, resulting in in-rolling or wilting. In contrast, `Chinera' and three selected lines (515-10, 64-13, and 64-54) were less affected than the other cultivars and lines exposed for 0 days and classi®ed as having low sensitivity to ethylene.
Ethylene sensitivity decreased with the age of the ¯ower. For example, lines 63-3, 63-12, 63-41, and 63-35 showed high sensitivity when exposed for 0 and 3 days but low sensitivity when exposed for 6 days, and lines 64-56, 63-7, 62-2, and 63-24 became less responsive to ethylene with age (Fig. 3).
Furthermore, `Chinera' and three selected lines (515-10, 64-13, and 64-54) were tested at a high ethylene concentration (4ml lÿ1
for 16 h). `Chinera' and 515-10 showed clear wilting after treatment for both 0 and 3 days. The vase life of 64-13 was 0.2 days after treatment for 0 days but 2.0 days after treatment for 3 days; that of 64-54 was 1.2 days after treatment for 0 days and 1.4 days after treatment for 3 days (Table 3). These results con®rmed that 64-54 had the lowest level of sensitivity, followed by 64-13, `Chinera', and 515-10.
4. Discussion
Rigid selection for vase life in the ®rst year seems ineffective, because one seedling produces only a few cut ¯owers and the number of replicates is restricted. About 30% of the seedlings were primary-selected for long vase life. In the second year, replicated tests were carried out after vegetative multiplication and selection to diminish the environmental variance, and about 20% of the population was further selected. This selection procedure is reliable in selecting lines with a genetically long vase life.
Plants were not selected on ethylene production or sensitivity, but on vase life of ¯owers. However, many selected lines had very low ethylene production, and three had low ethylene sensitivity from a young ¯ower age. Moreover, two cycles of selection and crossing to improve vase life led to a 3.6-days increase in the population mean from parental- to second-generation. These results suggest that the vase life of carnation is controlled by a few genes related to ethylene production and ethylene sensitivity.
Table 3
Effect of ethylene treatment (4ml lÿ1
for 16 h) at different ¯ower ages on ¯ower vase life (daysS.E.)
Cultivar or line 0 days 3 days
`Sandrosa' showed extremely low ethylene production among the six cultivars used for crossing. Fourteen second-generation lines with long vase life are all progeny of `Sandrosa' (crossing data not shown). This result indicates that the level of ethylene production in selected lines was reduced by the introduction from `Sandrosa' of genes related to low ethylene production, and that the trait of low ethylene production is heritable.
We developed three selected lines (515-10, 64-13, and 64-54) with lower ethylene sensitivity (after exposure for 0, 3, and 6 days) than the other cultivars and lines. In particular, 64-13, and 64-54 were less sensitive to ethylene than `Chinera', which is known for its low sensitivity (Table 3). However, these lines do not have longer vase life than the lines with very low ethylene production at ¯ower senescence (e.g. 63-3, 63-12, 63-41, and 63-35). These results suggest that low ethylene production during senescence is more effective than low ethylene sensitivity at a young ¯ower age for extending carnation vase life. However, as low sensitivity to ethylene at a young ¯ower age is an equally important trait in carnation breeding when we have to preserve the quality of cut ¯owers in ethylene-polluted environments (e.g. during transportation), breeding to combine low sensitivity and low production should be considered.
It is known that ethylene sensitivity of the ¯ower increases as the ¯ower ages from anthesis to senescence in many ethylene-sensitive species, such asPetunia hybrida(Whitehead and Halevy, 1989),Pelargonium(Deneke et al., 1990),Eustoma
(Ichimura et al., 1998),Portulacahybrid (Ichimura and Suto, 1998), andTorenia
(Goto et al., 1999). In carnation, `White Sim' ¯owers showed increased sensitivity to ethylene with age from bud stage until anthesis (Barden and Hanan, 1972; Camprubi and Nichols, 1978; Woodson and Lawton, 1988). To our knowledge, no report clari®es the changes in sensitivity of mature carnation ¯owers from anthesis to senescence except that of Mayak and Tirosh (1993); these authors reported that the senescence variant `Sandrosa' is unusual in the sensitivity of the ¯ower to ethylene diminishes with age, but they did not compare it with carnations with normal climacteric ethylene production (e.g. `White Sim'). The selected lines that we bred showed the same response as that reported by Mayak and Tirosh (1993): young ¯owers were more responsive to exogenous ethylene than older ¯owers (Fig. 3). Further study is necessary to clarify whether this change in sensitivity of our selected lines is unique to long-vase-life variants with low ethylene production, and why the sensitivity decreases in some lines as the ¯owers age.
Savin et al. (1995) reported that vase life of carnation was extended by the introduction of an antisense ACC oxidase gene. Their modi®ed plants, # 705 and # 2373B, had a vase life of 8±9 days at 218C. More recently, Bovy et al. (1999) reported that vase life of carnation was extended by the introduction of the
Arabidopsis etr1-1gene. Their bestetr1-1transgenic plants (Nos. 7086 and 8018) had a mean vase life of 24 days, nearly three times that of control ¯owers (8.3 days), at 208C. These two studies indicate that genetic engineering is a very powerful tool for breeding carnation with a long vase life. In contrast, our selected line 66-15 had a mean vase life of 17.5 days, 3.2 times that of `White Sim', at 238C. We assume that the lines created by the introduction ofetr1-1do not differ in vase life from the lines we produced by conventional breeding, at least at the temperatures at which we evaluated vase life. As carnation has a relatively short generation time (about 1 year), improvement by selection and crossing is not as time consuming as in bulb species such as tulip. Our results indicate that improvement of vase life of carnation by conventional cross-breeding is as practical as that by genetic engineering.
We conclude that vase life of carnations can be extended by selection and crossing, and that breeding can be an excellent alternative to the use of pollutant chemicals such as STS. We have obtained many lines with long vase life that show low ethylene production or low ethylene sensitivity.
References
Baker, J.E., Wang, C.Y., Lieberman, M., Hardenburg, R.E., 1977. Delay of senescence in carnations by rhizobitoxine analogue and sodium benzoate. HortScience 12, 38±39.
Barden, L.E., Hanan, J.J., 1972. Effect of ethylene on carnation keeping life. J. Am. Soc. Hort. Sci. 97, 785±788.
Bovy, A.G., Angenent, G.C., Dons, H.J.M., Van Altvorst, A.C., 1999. Heterologous expression of the Arabidopsisetr1-1allele inhibits the senescence of carnation ¯owers. Mol. Breed. 5, 301± 308.
Bu¯er, G., Mor, Y., Reid, M.S., Yang, S.F., 1980. Changes in 1-aminocyclopropane-1-carboxylic-acid content of cut carnation ¯owers in relation to their senescence. Planta 150, 439±442. Camprubi, P., Nichols, R., 1978. Effects of ethylene on carnation ¯owers (Dianthus caryophyllus)
cut at different stages of development. J. Hort. Sci. 53, 17±22.
Deneke, C.F., Evensen, K.B., Craig, R., 1990. Regulation of petal abscission in Pelargo-niumdomesticum. HortScience 25, 937±940.
Fujino, D.W., Reid, M.S., Yang, S.F., 1980. Effects of aminooxyacetic acid on postharvest characteristics of carnation. Acta Hort. 113, 59±64.
Goto, R., Aida, R., Shibata, M., Ichimura, K., 1999. Role of ethylene on ¯ower senescence of
Torenia. J. Jpn. Soc. Hort. Sci. 68, 263±268.
Ichimura, K., Suto, K., 1998. Role of ethylene in acceleration of ¯ower senescence by ®lament wounding inPortulacahybrid. Physiol. Plant. 104, 603±607.
Ichimura, K., Shimamura, M., Hisamatsu, T., 1998. Role of ethylene in senescence of cutEustoma
Lawton, K.A., Huang, B., Goldsbrough, P.B., Woodson, W.R., 1989. Molecular cloning and characterization of senescence-related genes from carnation ¯ower petals. Plant Physiol. 90, 690±696.
Mayak, S., Tirosh, T., 1993. Unusual ethylene-related behavior in senescing ¯owers of the carnation Sandrosa. Physiol. Plant. 88, 420±426.
Onozaki, T., Yamaguchi, T., 1992. Effect of a-aminoisobutyric acid (AIB) application on the prolongation of the vase life of cut carnation ¯owers. Bull. Natl. Res. Inst. Veg. Ornam. Plants Tea Ser. A 5, 69±79 (in Japanese with English summary).
Onozaki, T., Ikeda, H., Yamaguchi, T., 1998. Effect of calcium nitrate addition toa-aminoisobutyric acid (AIB) on the prolongation of the vase life of cut carnation ¯owers. J. Jpn. Soc. Hort. Sci. 67, 198±203.
Savin, K.W., Baudinette, S.C., Graham, M.W., Michael, M.Z., Nugent, G.D., Lu, C.Y., Chandler, S.F., Cornish, E.C., 1995. Antisense ACC oxidase RNA delays carnation petal senescence. HortScience 30, 970±972.
Serrano, M., Romojaro, F., 1991. Ethylene and polyamine metabolism in climacteric and nonclimacteric carnation ¯owers. HortScience 26, 894±896.
Van der Meulen-Muisers, J.J.M., Van Oeveren, J.C., Jansen, J., Van Tuyl, J.M., 1999. Genetic analysis of postharvest ¯ower longevity in Asiatic hybrid lilies. Euphytica 107, 149±157. Veen, H., 1979. Effects of silver on ethylene synthesis and action in cut carnations. Planta 145, 467±
470.
Wernett, H.C., Wilfret, G.J., Sheehan, T.J., Marousky, F.J., Lyrene, P.M., Knauft, D.A., 1996. Postharvest longevity of cut-¯ower Gerbera. I. Response to selection for vase life components. J. Am. Soc. Hort. Sci. 121, 216±221.
Whitehead, C.S., Halevy, A.H., 1989. Ethylene sensitivity: the role of short-chain saturated fatty acids in pollination-induced senescence ofPetunia hybrida¯owers. Plant Growth Reg. 8, 41±54. Woltering, E.J., Van Doorn, W.G., 1988. Role of ethylene in senescence of petals Ð morphological
and taxonomical relationships. J. Exp. Bot. 39, 1605±1616.
Woltering, E.J., Somhorst, D., de Beer, C.A., 1993. Roles of ethylene production and sensitivity in senescence of carnation ¯ower (Dianthus caryophyllus) cultivars white sim, chinera and epomeo. J. Plant Physiol. 141, 329±335.
Woodson, W.R., Lawton, K.A., 1988. Ethylene-induced gene expression in carnation petals. Relationship to autocatalytic ethylene production and senescence. Plant Physiol. 87, 498±503. Wu, M.J., Van Doorn, W.G., Reid, M.S., 1991a. Variation in the senescence of carnation (Dianthus caryophyllusL.) cultivars. I. Comparison of ¯ower life, respiration and ethylene biosynthesis. Sci. Hort. 48, 99±107.
