Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol87.Issue1-2.Jan2001:
Scientia Horticulturae 87 (2001) 107±120
Genetic improvement of vase life of carnation
¯owers by crossing and selection
Takashi Onozaki*, Hiroshi Ikeda, Takashi Yamaguchi1
National 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 caryophyllus L.); 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 5121104, Japan.
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 7 - 9
108
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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), and a-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 contamination of the environment by waste STS solutions 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.
We initiated a research breeding program in 1992 to improve vase life of
carnation ¯owers by conventional breeding techniques. The ultimate aim is to
produce commercially successful cultivars with a long vase life. Here, we report
109
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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 primaryselected 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
Parental cultivars used for crossing, and their vase life (daysS.E.) in standard conditions (238C,
12 h photoperiod, 70% RH)
Cultivar
na
Vase life
Pallas
Sandrosa
Candy
White Sim
Tanga
Scania
10
10
8
10
10
10
8.9
10.1
6.9
5.4
6.8
6.5
a
Number of ¯owers tested.
0.4
0.6
0.2
0.2
0.4
0.2
110
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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 (10 mmol mÿ2 sÿ1 irradiance).
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 by t-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 inrolling 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.
On an average, six ¯owers of each cultivar or line were harvested at
commercial maturity. Flower stems were cut to 5 cm and placed individually in a
test tube containing distilled water. Flowers were placed in standard conditions
(238C, 70% RH, 12 h photoperiod) until the petals showed in-rolling or browning
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
111
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 4 ml lÿ1 ethylene 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 secondgenerations 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 ®rstgeneration and by 2.6 days from ®rst- to second-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
In clonal tests, six cultivars and 39 selected lines exhibited a wide range of
variation (5.3±17.5 days) in mean vase life (Table 2). The mean vase life of
`White Sim', which has been used in many ¯ower senescence studies as a control
112
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
Fig. 1. Frequency distribution of vase life in parental-, ®rst- and second-generations.
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).
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
113
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
cultivar, `White Sim'
Control cultivars
White Sim
Sandrosa
5.4 0.2
10.1 0.6**a
100
187
Normal cultivars
U Conn Sim
Coral
6.1 0.2 n.s.b
5.3 0.2 n.s.
113
98
Long vase life variants
Killer
Chinera
11.1 0.7**
10.9 0.5**
Parental-generation selected line
4-50
5-20
2-16
4-14
8-13
4-1
8-51
9-53
14-9
12.4
12.2
10.2
10.7
8.7
8.9
11.1
8.4
8.1
0.5**Lc
0.6**
0.7**
0.5**
0.2**
0.2**
0.5**
0.3**
0.5**
230
226
189
198
161
165
206
156
150
First-generation selected line
945-1
945-2
941-5
945-32
945-7
945-17
945-25
945-24
945-15
508-22
507-2
515-13
511-15
510-23
501-6
515-10
13.1
11.8
10.6
11.9
9.9
12.9
11.9
12.2
9.4
13.2
13.1
9.7
9.2
10.7
13.2
11.9
0.8**L
1.0**
0.6**
0.8**
0.7**
0.6**L
0.8**
0.8**
0.7**
0.6**L
0.8**L
0.4**
0.9**
0.9**
0.6**L
0.9**
243
219
196
220
183
239
220
226
174
244
243
180
170
198
244
220
Second-generation selected line
63-3
63-12
66-15
63-41
15.4
15.1
17.5
15.0
0.5**L
0.5**L
1.1**L
0.3**L
285
280
320
278
206
202
114
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
Table 2 (Continued )
Cultivar or selected line
62-18
63-35
63-8
62-48
64-56
63-7
62-2
63-24
64-13
64-54
Vase life
Percentage of control
cultivar, `White Sim'
13.3
13.9
14.4
13.6
11.1
12.1
14.0
12.3
11.2
12.2
246
257
267
252
206
224
259
228
207
226
0.8**L
0.4**L
0.8**L
0.5**L
1.2**
0.6**
0.6**L
0.8**
0.7**
0.5**
a
Signi®cance shown of difference against `White Sim': ** Ð 1% level.
n.s. Ð not signi®cant.
c
L Ð signi®cantly longer than `Sandrosa' at 0.01 level.
b
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
gfwÿ1 hÿ1 by `Scania' to 88.4 nl gfwÿ1 hÿ1 by `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
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
115
Fig. 2. Ethylene production of carnation ¯owers at senescence in cultivars or selected lines.
Vertical bars represent S.E.
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.
116
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
Fig. 3. Effect of ¯ower age at treatment on ethylene sensitivity of carnation cultivars or selected
lines. Flowers of different ages were exposed to 2 ml lÿ1 ethylene for 16 h at 238C.
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, 6413, 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.
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
117
Table 3
Effect of ethylene treatment (4 ml lÿ1 for 16 h) at different ¯ower ages on ¯ower vase life
(daysS.E.)
Cultivar or line
Chinera
515-10
64-13
64-54
a
0 days
3 days
a
n
Vase life
na
Vase life
10
5
5
5
0.1 0.1
0
0.2 0.2
1.2 0.4
5
5
5
5
0.2 0.2
0
2.0 0.3
1.4 0.2
Number of ¯owers tested.
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 (4 ml 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.
118
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
`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 as Petunia
hybrida (Whitehead and Halevy, 1989), Pelargonium (Deneke et al., 1990), Eustoma
(Ichimura et al., 1998), Portulaca hybrid (Ichimura and Suto, 1998), and Torenia
(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.
Three selected lines (515-10, 64-13, and 64-54) with low ethylene sensitivity
are the progeny of `Candy' and `Sandrosa' (crossing data not shown). These two
parental cultivars are slightly less sensitive than `White Sim', `Tanga', and
`Scania' (Fig. 3). Woltering et al. (1993) have shown that reduced ethylene
sensitivity is heritable. It is possible that the ethylene sensitivity in 515-10, 64-13,
and 64-54 is low because genes related to low sensitivity from the two parental
cultivars are integrated.
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
119
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 etr1-1 gene. Their best etr1-1 transgenic 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 of etr1-1 do
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 crossbreeding 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 Arabidopsis etr1-1 allele 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-carboxylicacid 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 Pelargoniumdomesticum. 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 in Portulaca hybrid. Physiol. Plant. 104, 603±607.
Ichimura, K., Shimamura, M., Hisamatsu, T., 1998. Role of ethylene in senescence of cut Eustoma
¯owers. Postharvest Biol. Technol. 14, 193±198.
120
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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 to a-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 of Petunia 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
caryophyllus L.) cultivars. I. Comparison of ¯ower life, respiration and ethylene biosynthesis.
Sci. Hort. 48, 99±107.
Wu, M.J., Van Doorn, W.G., Reid, M.S., 1991b. Variation in the senescence of carnation (Dianthus
caryophyllus L.) cultivars. II. Comparison of sensitivity to exogenous ethylene and of ethylene
binding. Sci. Hort. 48, 108±116.
Genetic improvement of vase life of carnation
¯owers by crossing and selection
Takashi Onozaki*, Hiroshi Ikeda, Takashi Yamaguchi1
National 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 caryophyllus L.); 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 5121104, Japan.
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 6 7 - 9
108
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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), and a-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 contamination of the environment by waste STS solutions 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.
We initiated a research breeding program in 1992 to improve vase life of
carnation ¯owers by conventional breeding techniques. The ultimate aim is to
produce commercially successful cultivars with a long vase life. Here, we report
109
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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 primaryselected 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
Parental cultivars used for crossing, and their vase life (daysS.E.) in standard conditions (238C,
12 h photoperiod, 70% RH)
Cultivar
na
Vase life
Pallas
Sandrosa
Candy
White Sim
Tanga
Scania
10
10
8
10
10
10
8.9
10.1
6.9
5.4
6.8
6.5
a
Number of ¯owers tested.
0.4
0.6
0.2
0.2
0.4
0.2
110
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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 (10 mmol mÿ2 sÿ1 irradiance).
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 by t-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 inrolling 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.
On an average, six ¯owers of each cultivar or line were harvested at
commercial maturity. Flower stems were cut to 5 cm and placed individually in a
test tube containing distilled water. Flowers were placed in standard conditions
(238C, 70% RH, 12 h photoperiod) until the petals showed in-rolling or browning
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
111
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 4 ml lÿ1 ethylene 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 secondgenerations 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 ®rstgeneration and by 2.6 days from ®rst- to second-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
In clonal tests, six cultivars and 39 selected lines exhibited a wide range of
variation (5.3±17.5 days) in mean vase life (Table 2). The mean vase life of
`White Sim', which has been used in many ¯ower senescence studies as a control
112
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
Fig. 1. Frequency distribution of vase life in parental-, ®rst- and second-generations.
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).
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
113
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
cultivar, `White Sim'
Control cultivars
White Sim
Sandrosa
5.4 0.2
10.1 0.6**a
100
187
Normal cultivars
U Conn Sim
Coral
6.1 0.2 n.s.b
5.3 0.2 n.s.
113
98
Long vase life variants
Killer
Chinera
11.1 0.7**
10.9 0.5**
Parental-generation selected line
4-50
5-20
2-16
4-14
8-13
4-1
8-51
9-53
14-9
12.4
12.2
10.2
10.7
8.7
8.9
11.1
8.4
8.1
0.5**Lc
0.6**
0.7**
0.5**
0.2**
0.2**
0.5**
0.3**
0.5**
230
226
189
198
161
165
206
156
150
First-generation selected line
945-1
945-2
941-5
945-32
945-7
945-17
945-25
945-24
945-15
508-22
507-2
515-13
511-15
510-23
501-6
515-10
13.1
11.8
10.6
11.9
9.9
12.9
11.9
12.2
9.4
13.2
13.1
9.7
9.2
10.7
13.2
11.9
0.8**L
1.0**
0.6**
0.8**
0.7**
0.6**L
0.8**
0.8**
0.7**
0.6**L
0.8**L
0.4**
0.9**
0.9**
0.6**L
0.9**
243
219
196
220
183
239
220
226
174
244
243
180
170
198
244
220
Second-generation selected line
63-3
63-12
66-15
63-41
15.4
15.1
17.5
15.0
0.5**L
0.5**L
1.1**L
0.3**L
285
280
320
278
206
202
114
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
Table 2 (Continued )
Cultivar or selected line
62-18
63-35
63-8
62-48
64-56
63-7
62-2
63-24
64-13
64-54
Vase life
Percentage of control
cultivar, `White Sim'
13.3
13.9
14.4
13.6
11.1
12.1
14.0
12.3
11.2
12.2
246
257
267
252
206
224
259
228
207
226
0.8**L
0.4**L
0.8**L
0.5**L
1.2**
0.6**
0.6**L
0.8**
0.7**
0.5**
a
Signi®cance shown of difference against `White Sim': ** Ð 1% level.
n.s. Ð not signi®cant.
c
L Ð signi®cantly longer than `Sandrosa' at 0.01 level.
b
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
gfwÿ1 hÿ1 by `Scania' to 88.4 nl gfwÿ1 hÿ1 by `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
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
115
Fig. 2. Ethylene production of carnation ¯owers at senescence in cultivars or selected lines.
Vertical bars represent S.E.
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.
116
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
Fig. 3. Effect of ¯ower age at treatment on ethylene sensitivity of carnation cultivars or selected
lines. Flowers of different ages were exposed to 2 ml lÿ1 ethylene for 16 h at 238C.
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, 6413, 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.
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
117
Table 3
Effect of ethylene treatment (4 ml lÿ1 for 16 h) at different ¯ower ages on ¯ower vase life
(daysS.E.)
Cultivar or line
Chinera
515-10
64-13
64-54
a
0 days
3 days
a
n
Vase life
na
Vase life
10
5
5
5
0.1 0.1
0
0.2 0.2
1.2 0.4
5
5
5
5
0.2 0.2
0
2.0 0.3
1.4 0.2
Number of ¯owers tested.
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 (4 ml 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.
118
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
`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 as Petunia
hybrida (Whitehead and Halevy, 1989), Pelargonium (Deneke et al., 1990), Eustoma
(Ichimura et al., 1998), Portulaca hybrid (Ichimura and Suto, 1998), and Torenia
(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.
Three selected lines (515-10, 64-13, and 64-54) with low ethylene sensitivity
are the progeny of `Candy' and `Sandrosa' (crossing data not shown). These two
parental cultivars are slightly less sensitive than `White Sim', `Tanga', and
`Scania' (Fig. 3). Woltering et al. (1993) have shown that reduced ethylene
sensitivity is heritable. It is possible that the ethylene sensitivity in 515-10, 64-13,
and 64-54 is low because genes related to low sensitivity from the two parental
cultivars are integrated.
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
119
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 etr1-1 gene. Their best etr1-1 transgenic 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 of etr1-1 do
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 crossbreeding 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 Arabidopsis etr1-1 allele 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-carboxylicacid 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 Pelargoniumdomesticum. 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 in Portulaca hybrid. Physiol. Plant. 104, 603±607.
Ichimura, K., Shimamura, M., Hisamatsu, T., 1998. Role of ethylene in senescence of cut Eustoma
¯owers. Postharvest Biol. Technol. 14, 193±198.
120
T. Onozaki et al. / Scientia Horticulturae 87 (2001) 107±120
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 to a-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 of Petunia 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
caryophyllus L.) cultivars. I. Comparison of ¯ower life, respiration and ethylene biosynthesis.
Sci. Hort. 48, 99±107.
Wu, M.J., Van Doorn, W.G., Reid, M.S., 1991b. Variation in the senescence of carnation (Dianthus
caryophyllus L.) cultivars. II. Comparison of sensitivity to exogenous ethylene and of ethylene
binding. Sci. Hort. 48, 108±116.