breeding for long vase life in dahlia (dahlia variabilis
TRANSCRIPT
Breeding for Long Vase Life in Dahlia (Dahlia variabilis) Cut Flowers
Takashi Onozaki* and Mirai Azuma**
Institute of Vegetable and Floriculture Science, NARO, Tsukuba 305-0852, Japan
Flower vase life is one of the most important traits for ornamental plants. The vase life of cut dahlia (Dahliavariabilis) flowers is very short, and genetic improvement of this trait is desirable. We started a breedingresearch program in 2014 to improve the vase life of dahlia flowers using conventional cross-breedingtechniques. We found large significant differences in flower vase life among 24 dahlia cultivars: Nine cultivarshad long vase life (e.g., ‘Syukuhai’, ‘Rinka’, and ‘Micchan’); eight had normal vase life (e.g., ‘Kamakura’,‘Agitate’, and ‘Benifusya’); and seven had short vase life (e.g., ‘Gin-Ei’, ‘Port Light Pair Beauty’, and‘Yumesuiren’). We used 22 cultivars as initial breeding materials, repeatedly crossed them, and selectedpromising offspring with long vase life for three generations from 2014 to 2018. Two cycles of selection andcrossing led to a 1.7-day increase in vase life (population mean) from the first to the third generation, clearlyshowing that this approach can extend the vase life of dahlia flowers. The mean vase life of ‘Kamakura’, aleading white dahlia cultivar in Japan, was 5.0–6.2 days in distilled water, 6.0–6.8 days in an isothiazolinicantibacterial agent CMIT/MIT solution (5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one) and 6.0–7.6 days in a GLA solution (10 g·L−1 glucose, 0.5 ml·L−1 CMIT/MIT, and 50 mg·L−1 aluminumsulfate), whereas in six finally selected lines it was 6.2–12.0 days in distilled water, 6.6–10.2 days in CMIT/MITsolution, and 9.4–13.6 days in GLA solution (1.4–2.1 times that in ‘Kamakura’). In particular, the selectedsecond-generation line 606-46 showed a stably longer vase life than ‘Kamakura’. ‘Micchan’, which has a longvase life, was a common progenitor used for breeding of parental lines in cross combinations with long vase lifein the second generation and all cross combinations in the third generation. The final six selected lines withlong vase life were all progeny of ‘Micchan’. Our results strongly suggest that ‘Micchan’ has genes related tolong flower vase life, and that the trait is heritable.
Key Words: crossing and selection, dahlia, flower longevity, flower senescence.
IntroductionThe vase life of cut ornamental flowers determines
their quality and ability to satisfy consumer preferences,thereby stimulating repeat purchasing. It is thereforeone of the most important breeding targets (Onozaki,2018a). Flower distributors want to reduce the deterio‐ration in flower quality during the long transportationtime from producer to consumer (Shibuya, 2018).Therefore, the ability to control petal aging and flower
Received; March 27, 2019. Accepted; June 27, 2019.First Published Online in J-STAGE on August 31, 2019.This work was supported by a grant from a project study on“Breeding of floricultural plants adapted for high practical needs anddevelopment of low cost cultivation techniques” commissioned by theMinistry of Agriculture, Forestry and Fisheries, Japan (projectnumber 15653424).
* Corresponding author (E-mail: [email protected]).** Present address: College of Bioresource Sciences, Nihon
University, Fujisawa, Kanagawa 252-0880, Japan.
senescence is of great interest to breeders (Zuker et al.,1998).
The main breeding targets for ornamentals used to bevisual qualities such as appearance, flower color, type,size, and stem length (Boxriker et al., 2017). Vase life isa highly complex quantitative trait that involves multi‐ple genes with additive effects (Boxriker et al., 2017,2018), and few attempts to improve flower longevitywere made before the 1990s (Harding et al., 1981; VanEijk and Eikelboom, 1976). Breeding for extended vaselife is challenging because vase life is affected by manydifferent deterioration processes (Van Geest et al.,2017). The assessment of vase life is labor-intensive(Boxriker et al., 2018) because each genotype must begrown and harvested, and cut flowers must be evaluatedevery day for senescence symptoms. However, the lon‐gevity of cut flowers has now become an importantquality factor because short-lived flowers have limitedconsumer appeal and therefore limited marketability.Breeding to improve vase life has been carried out in
The Horticulture Journal 88 (4): 521–534. 2019.doi: 10.2503/hortj.UTD-091
JSHS
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Horticultural Sciencehttp://www.jshs.jp/
© 2019 The Japanese Society for Horticultural Science (JSHS), All rights reserved.
snapdragon (Weber et al., 2005), chrysanthemum (VanGeest et al., 2017), gerbera (Wernett et al., 1996a, b),carnation (Onozaki, 2018a; Onozaki et al., 2001, 2006a,b, 2011, 2015, 2018), Asiatic hybrid lily (van derMeulen-Muisers et al., 1999), and rose (Carvalho et al.,2015; Fanourakis et al., 2012).
The Institute of Vegetable and Floriculture Science(NIVFS, NARO, Japan) has bred carnations (Dianthuscaryophyllus L.) for long vase life since 1992 (Onozaki,2018a). We crossed and selected promising offspringwith long vase life for seven generations from 1992 to2008. The mean vase life was improved from 7.4 daysin the first generation to 15.9 days in the 7th generationthrough conventional crossing techniques. Three devel‐oped cultivars, ‘Miracle Rouge’, ‘Miracle Symphony’(Onozaki et al., 2006a), and ‘Kane Ainou 1-go’ (Hottaet al., 2016), with a genetically determined long vaselife, and two lines, 532-6 and 806-46b, with an ultra-long vase life (>27 days) under standard conditions,commonly produce only trace amounts of ethylene dur‐ing natural senescence and show neither petal inrollingnor rapid wilting. Our nearly two-decade-long breedingand research program revealed a close correlation be‐tween vase life and ethylene production in cut carna‐tions (Onozaki et al., 2018).
Dahlia (Dahlia variabilis; Asteraceae), an importantbulb crop sold as cut flowers, garden ornamentals andpotted plants in many countries, has become a popularcut flower in Japan in recent years (Onozaki, 2018b).Dahlias are not surveyed in the nationwide Statistics ofAgriculture, Forestry and Fisheries in Japan, but thenumber of cut dahlia flowers handled in the Tokyo met‐ropolitan market increased from 1,024,124 in 2002 to4,733,901 in 2018 (a 362% increase; http://www.shijou-tokei.metro.tokyo.jp/). Demand for cut dahlia flowershas been increasing year by year. Dahlias show hugevariation in flower traits such as color, shape, and size,owing to their high polyploidy (Okumura and Fujino,1989; Wegner and Debener, 2008), likely autoalloocto‐ploidy (2n = 8x = 64) (Gatt et al., 1998). However, thevase life of cut dahlias without chemical treatment isusually only about four days at room temperature, withsome differences among cultivars (Ichimura et al.,2011). This very short vase life has curtailed the expan‐sion of demand for cut dahlia flowers (Shimizu-Yumotoand Ichimura, 2013). Although continuous exposure toethylene (2 or 10 μL·L−1) significantly accelerated petalabscission in cut flowers of ‘Kokucho’, a leading dark-red dahlia cultivar in Japan, silver thiosulfate complex,an inhibitor of ethylene action, did not extend its vaselife (Shimizu-Yumoto and Ichimura, 2013). Dole et al.(2009) reported that the vase life of the dahlia ‘KarmaThalia’ was unaffected by exogenous ethylene or anti-ethylene treatments (silver thiosulfate complex or 1-methylcyclopropene). These reports suggest thatethylene is not a very important factor in inducing flow‐er senescence in dahlia.
Three Japanese dahlia breeders informed us that longvase life of dahlia flowers may be related to a lack ofstem cavities, i.e., a smaller stem diameter (personalcommunications: Koji Washizawa and Yusaku Konishi,September 2014; Yoshiki Amano, July 2015). There‐fore, we investigated the relationship between stem di‐ameter and flower vase life in the first and secondgenerations.
Tsujimoto et al. (2016b) showed genetic variation inthe vase life of cut flowers of 27 dahlia cultivars andreported that several commercial dahlia cultivars (e.g.‘Rinka’, ‘Syukuhai’, ‘Moon Waltz’, ‘Benifusya’,‘Micchan’, and ‘Akebono-Temari’ in winter; ‘Rinka’,‘Syukuhai’, ‘Micchan’, ‘Akebono-Temari’, ‘PinkSapphire’, and ‘Moon Waltz’ in summer) have ex‐tended vase life. This genetic variation should make itpossible to breed lines with a genetically determinedlong vase life. In 2014, we started a breeding researchprogram to improve the vase life of dahlia flowersusing conventional cross-breeding techniques that hadproved to be very effective in carnation breeding(Onozaki, 2018a). The aim of this program is to pro‐duce commercially successful dahlia cultivars with along vase life. Here, we report the results of crossingand selection over three generations to improve the vaselife of dahlia.
Materials and MethodsPlant materials
The vase life of 24 commercial dahlia cultivars usedas cut flowers (Table 1) was evaluated. Bulbs were pur‐chased in April 2014 from Akita International DahliaPark (Akita, Japan); Takii & Co., Ltd. (Kyoto, Japan);Kokkaen & Co., Ltd. (Osaka, Japan); or FukukaenNursery & Bulb Co., Ltd. (Nagoya, Japan). Plants weregrown in an open field of NIVFS, Tsukuba, Japan, fromMay to November. Commercial fertilizer (Cyclo-Di-Urea (CDU) S222; Zen-Noh, Tokyo, Japan) was ap‐plied at N:P2O5:K2O = 20:20:20 kg/10a just beforeplanting each year. Two bulbs per cultivar were planted40 cm apart on 13 May 2014 and 12 May 2015. Liquidcompound fertilizer (Yoeki-Dokou No. 1, N:P2O5:K2O = 15:8:17; OAT Agrio Co., Ltd., Tokyo, Japan, 1:1000dilution) was applied once a week from June to Octoberin both years. The shoots of each plant were pinched totwo or three nodes on 17 June 2014 and 16 June 2015,and the plants were grown following standard produc‐tion methods in the open field for seasonal flowering(Yamagata, 2018).
Vase life evaluationVase life of dahlia cultivars was evaluated from late
July to October in 2014 and from September to earlyNovember in 2015. As an antibacterial agent, we used aCMIT/MIT (Kathon CG, formerly Legend MK; Rohmand Haas Japan K.K., Tokyo, Japan) solution contain‐ing 11.3 g·L−1 5-chloro-2-methyl-4-isothiazolin-3-one
522 T. Onozaki and M. Azuma
Tabl
e 1.
Va
se li
fe (d
ays)
of c
ut fl
ower
s of d
ahlia
cul
tivar
s in
disti
lled
wat
er (D
W),
antib
acte
rial a
gent
(CM
IT/M
IT),
or G
LA so
lutio
n.
Vase
life
in
dex
Cul
tivar
Flow
er
type
z
Dis
tille
d W
ater
(DW
)C
MIT
/MIT
GLA
(CM
IT/
MIT
)/DW
GLA
/D
WG
rand
M
ean
n20
14n
2015
Mea
nn
2014
n20
15M
ean
n20
14n
2015
Mea
n
1Sy
ukuh
aiFD
66.
2 ± 0.
5 ab
c6
7.0 ±
0.6
ab6.
66
6.3 ±
0.6
abc
68.
7 ± 0.
3 a
7.5
69.
3 ± 0.
9 ab
c6
11.3
± 1.
2 ab
10.3
1.1
1.6
8.1
1R
inka
FD6
5.8 ±
0.9
abc
68.
0 ± 0.
8 a
6.9
36.
3 ± 1.
3 ab
c4
5.3 ±
0.3
cdef
g5.
86
10.0
± 1.
0 ab
611
.8 ±
0.7
a10
.90.
81.
67.
9
1M
icch
anB
A6
6.0 ±
0.6
abc
67.
3 ± 0.
4 ab
6.7
66.
8 ± 0.
7 ab
66.
8 ± 0.
6 ab
cdef
6.8
610
.2 ±
0.3
a6
8.5 ±
0.3
abcd
9.3
1.0
1.4
7.6
1Su
per G
irlFD
65.
3 ± 0.
8 ab
c6
5.8 ±
0.9
abcd
5.6
67.
2 ± 0.
5 ab
66.
7 ± 0.
4 ab
cdef
6.9
69.
7 ± 0.
6 ab
c6
10.8
± 1.
2 ab
c10
.31.
21.
87.
6
1M
izou
Noi
rSC
66.
8 ± 0.
5 ab
66.
0 ± 0.
5 ab
cd6.
46
7.5 ±
0.6
ab6
6.7 ±
0.5
abcd
ef7.
16
9.5 ±
0.6
abc
68.
8 ± 0.
5 ab
cd9.
21.
11.
47.
6
1Je
ssy
Rita
FD6
7.7 ±
0.4
a6
6.8 ±
0.3
abc
7.3
66.
5 ± 0.
3 ab
c6
7.7 ±
0.4
abc
7.1
68.
5 ± 0.
2 ab
cde
67.
8 ± 0.
3 bc
de8.
21.
01.
17.
5
1Yu
kits
ubak
iB
A6
6.8 ±
1.0
ab6
6.0 ±
0.5
abcd
6.4
67.
5 ± 0.
6 ab
66.
3 ± 0.
7 bc
def
6.9
68.
7 ± 1.
1 ab
cd6
8.2 ±
0.3
bcde
8.4
1.1
1.3
7.3
1K
okuc
hoSC
64.
8 ± 0.
5 ab
c6
6.0 ±
0.8
abcd
5.4
68.
5 ± 0.
2 a
68.
0 ± 0.
0 ab
8.3
68.
8 ± 0.
3 ab
cd3
7.3 ±
1.9
bcde
8.1
1.5
1.5
7.3
1Pe
arl L
ight
BA
65.
5 ± 0.
7 ab
c6
5.5 ±
0.2
abcd
e5.
56
6.3 ±
0.3
abc
67.
5 ± 0.
3 ab
cd6.
96
9.0 ±
0.4
abcd
68.
7 ± 0.
6 ab
cd8.
81.
31.
67.
1
2K
yuen
FD6
5.2 ±
0.5
abc
65.
3 ± 0.
2 ab
cde
5.3
67.
2 ± 0.
3 ab
67.
0 ± 0.
3 ab
cde
7.1
67.
8 ± 0.
2 ab
cdef
68.
3 ± 0.
8 ab
cd8.
11.
31.
56.
8
2M
oon
Wal
tzW
L6
3.8 ±
0.7
bc6
7.3 ±
0.2
ab5.
66
6.7 ±
0.4
ab6
6.0 ±
0.5
bcde
fg6.
36
9.0 ±
0.4
abcd
67.
7 ± 0.
4 cd
e8.
31.
11.
56.
8
2K
amak
ura
FD6
6.0 ±
0.6
abc
64.
8 ± 0.
4 bc
de5.
46
7.7 ±
0.2
ab5
5.6 ±
0.7
bcde
fg6.
66
8.8 ±
0.4
abcd
67.
3 ± 0.
5 cd
e8.
11.
21.
56.
7
2Po
m-P
om C
hoco
lat
BA
65.
5 ± 0.
4 ab
c6
5.7 ±
0.2
abcd
5.6
66.
2 ± 0.
2 ab
c6
6.0 ±
0.4
bcde
fg6.
16
7.7 ±
0.2
abcd
ef6
8.2 ±
0.3
bcde
7.9
1.1
1.4
6.5
2A
gita
teSC
63.
8 ± 0.
7 bc
54.
8 ± 0.
9 bc
de4.
36
5.7 ±
0.5
bc6
6.5 ±
0.7
abcd
ef6.
16
7.3 ±
0.3
bcde
f6
9.2 ±
0.8
abcd
8.3
1.4
1.9
6.2
2B
enifu
sya
FD6
6.5 ±
0.6
ab6
5.2 ±
0.3
bcde
5.8
65.
5 ± 0.
3 bc
66.
2 ± 0.
2 bc
defg
5.8
66.
3 ± 0.
6 de
f6
7.5 ±
0.7
cde
6.9
1.0
1.2
6.2
2K
onat
suID
64.
0 ± 0.
5 bc
65.
7 ± 0.
7 ab
cd4.
85
6.2 ±
0.6
abc
46.
0 ± 0.
9 cd
efg
6.1
67.
3 ± 0.
4 bc
def
57.
0 ± 1.
3 de
7.2
1.3
1.5
6.0
2B
lack
Cat
ID6
6.2 ±
0.4
abc
65.
2 ± 0.
5 bc
de5.
76
6.7 ±
0.3
ab6
5.5 ±
0.6
cdef
g6.
16
6.5 ±
0.2
def
66.
2 ± 0.
3 de
6.3
1.1
1.1
6.0
3Zu
ihou
FD6
4.5 ±
0.4
bc6
6.3 ±
0.6
abc
5.4
66.
2 ± 0.
3 ab
c6
5.0 ±
0.3
efg
5.6
66.
5 ± 0.
4 de
f6
7.0 ±
0.6
de6.
81.
01.
25.
9
3D
ream
Wal
tzW
L6
3.8 ±
0.3
bc6
5.7 ±
0.4
abcd
4.8
35.
0 ± 1.
5 bc
66.
5 ± 0.
3 ab
cdef
5.8
65.
7 ± 0.
6 f
67.
2 ± 0.
2 de
6.4
1.2
1.4
5.6
3Sh
ishu
WL
64.
2 ± 0.
7 bc
64.
2 ± 0.
3 cd
e4.
26
5.5 ±
0.2
bc6
5.7 ±
0.5
bcde
fg5.
66
7.2 ±
0.5
cdef
66.
5 ± 0.
6 de
6.8
1.3
1.6
5.5
3R
ed S
tar
BA
65.
5 ± 0.
6 ab
c5
3.4 ±
0.2
de4.
5no
t tes
ted
65.
2 ± 0.
4 ef
g5.
26
6.3 ±
0.3
def
66.
3 ± 0.
8 de
6.3
1.2
1.4
5.3
3G
in-E
iFD
64.
0 ± 0.
4 bc
65.
5 ± 0.
6 ab
cde
4.8
not t
este
d6
4.7 ±
0.3
fg4.
76
5.8 ±
0.5
df6
6.5 ±
0.7
de6.
21.
01.
35.
2
3Po
rt Li
ght P
air B
eaut
yFD
64.
2 ± 0.
4 bc
35.
0 ± 0.
2 ab
cde
4.6
not t
este
d6
4.0 ±
0.3
g4.
06
5.3 ±
0.2
f6
6.8 ±
0.3
de6.
10.
91.
34.
9
3Yu
mes
uire
nW
L6
3.3 ±
0.6
c6
2.8 ±
0.3
e3.
16
4.2 ±
0.4
c6
5.3 ±
0.4
defg
4.8
65.
5 ± 0.
3 f
64.
7 ± 0.
6 e
5.1
1.5
1.6
4.3
Tota
l5.
25.
65.
46.
56.
26.
27.
87.
97.
81.
21.
56.
5
z Flo
wer
type
: FD
, for
mal
dec
orat
ive;
BA
, bal
l; SC
, sem
i-cac
tus;
WL,
wat
er li
ly; I
D, i
nfor
mal
dec
orat
ive.
Valu
es a
re m
eans
± S
E. V
alue
s with
diff
eren
t let
ters
indi
cate
sign
ifica
nt d
iffer
ence
s at P
< 0.
05 b
y Tu
key’
s tes
t. n,
num
ber o
f flow
ers t
este
d.Fl
ower
vas
e lif
e w
as e
valu
ated
at 2
3°C
and
70%
rela
tive
hum
idity
und
er a
12-
h ph
otop
erio
d.C
ultiv
ars a
re li
sted
in d
esce
ndin
g or
der o
f the
gra
nd m
ean
valu
e.Th
e hi
ghes
t and
low
est v
alue
s in
each
trea
tmen
t are
in b
old.
Vase
life
inde
x: 1
(lon
g va
se li
fe),
gran
d m
ean
of ≥
7 da
ys; 2
(nor
mal
vas
e lif
e), g
rand
mea
n of
≥ 6
to <
7 da
ys; 3
(sho
rt va
se li
fe),
gran
d m
ean
of <
6 da
ys.
Table 1. Vase life (days) of cut flowers of dahlia cultivars in distilled water (DW), antibacterial agent (CMIT/MIT), or GLA solution.
Hort. J. 88 (4): 521–534. 2019. 523
(CMIT) and 3.9 g·L−1 2-methyl-4-isothiazolin-3-one(MIT) as active ingredients. Flowers were harvestedwhen the first row of petals from the outside hadopened at right angles to the stem. The flowers werethen cut to a stem length of 40 cm and all leaves exceptthe top ones were removed. Two or three cut flowerswere placed in 300- or 500-mL conical beakers contain‐ing distilled water (DW) or 0.5 mL·L−1 CMIT/MIT so‐lution or GLA solution (10 g·L−1 glucose, 0.5 mL·L−1
CMIT/MIT [Legend MK], 50 mg·L−1 aluminum sulfate;Ichimura et al., 2006). As a result, we used three differ‐ent solutions (DW, CMIT/MIT, and GLA) to evaluate24 cultivars.
Flowers were maintained at 23°C, 70% relative hu‐midity, under a 12-h photoperiod provided by cool fluo‐rescent lamps (10 μmol·m−2·s−1 irradiance), and wereevaluated daily. The solutions were not exchanged, butif the level decreased to less than two-thirds of the ini‐tial level, more was added.
Flower senescence was classified visually as wilting,wilting with browning, browning, or petal abscission(Fig. 1). The vase life of each flower was determined asthe number of days from harvest until one-third of all ofpetals showed one of the four patterns.
Finally, the grand mean (total average of DW, CMIT/MIT, and GLA) was calculated and assigned an indexas follows: 1 (long vase life), ≥ 7 days; 2 (normal vaselife), ≥ 6 to < 7 days; 3 (short vase life) < 6 days.
Crossing and selectionAs initial breeding materials, we chose 22 of the 24
cultivars (‘Gin-Ei’ and ‘Black Cat’ were excluded; see
A B
DC
Fig. 1. Morphology of flowers with four senescence patterns: (A)wilting, (B) wilting with browning, (C) browning, (D) petal ab‐scission.
Table 1) with large differences in vase life. In autumn2014, crosses among these cultivars were made in avinyl house and open field (Table 2) using the standardcrossing method for dahlias (Konishi, 2009; Onozaki,2018b). All ray florets were removed from the wholeflower and bagged with water-resistant paper bags.After the disk florets had opened, the bags were re‐moved and the flowers were pollinated by hand. Afterpollination, they were bagged again to prevent pollina‐tion by insects. About 45 days later (just before the firstfrost in mid-November), the crossed flowers were cutfrom the plants and seeds were collected.
We then crossed and selected promising offspringwith long vase life for three generations from 2014 to2018. On 23 April 2015 and 14 May 2015, the 1,109seeds obtained were sown and 439 plants germinated by29 June 2015; 419 of these were grown in the openfield. Plants that did not flower until 18 September2015 (the last day of evaluation) were discarded, andthe remaining 314 plants were considered as the firstgeneration (Table 2). In October 2015, 64 plants withthe longest mean vase life (> 5.1 days) were selectedand used as the next crosses. From these crosses, 571seeds were obtained. These were sown on 4 April 2016,and 355 plants germinated by 12 June 2016; 349 ofthese were grown in the open field. Plants that did notflower until 16 September 2016 (the last day of evalua‐tion) were discarded, and the remaining 308 plants wereconsidered as the second generation (Table 3). InOctober 2016, 73 plants with the longest mean vase life(> 6.0 days) were selected and used for the nextcrosses. From these crosses, 764 seeds were obtainedand were sown on 3 April 2017 or 29 March 2018 and253 plants in total germinated by 25 May 2017 or 4June 2018; 247 of these were grown in the open field.Plants that did not flower until 15 September 2017 or10 September 2018 (the last days of evaluation) werediscarded, and the remaining 155 plants were consid‐ered as the third generation (Table 4). In October 2017and 2018, a total of 58 plants with the longest meanvase life (> 6.1 or > 6.2 days, respectively) were se‐lected.
The initially selected first- and second-generationlines were propagated vegetatively, and 11 lines out of64 in 2016 and 10 lines out of 73 in 2017 were furtherselected. We examined the vase life of the cut flowersof the initially selected lines in detail in two differentseasons and using three different cultivation styles, thatis, a winter–spring season in a greenhouse heated above12°C with daylength extension to 14.5 h, and/or asummer–autumn season in an open field or a vinylhouse (no heating, natural daylength). Lines with a vaselife of 10 days or more in GLA solution in at least onecultivation method were further selected. The secondselection was based mainly on the vase life but alsopartly on other important traits (e.g., flowers are fullydouble with no open center at any time, high productiv‐
524 T. Onozaki and M. Azuma
Table 2. Cross combinations and results of crosses for the first generation.
Cross No.
Cross combination Number of seedlings obtained
Number of flowered seedlings
Mean vase life of
progeny (days)
Range(days)
Number of primary
selected plants
Number of secondary selected plants
Number of final selected plants♀ ♂
533 Kokucho (1) × Red Star (3) 15 12 5.2 4.0–8.0 5 1 0525 Micchan (1) × Konatu (2) 10 9 5.1 4.3–6.0 4 0 0514 Kokucho (1) × Super Girl (1) 5 5 5.0 4.0–7.0 1 1 0516 Micchan (1) × Pom-Pom Chocolat (2) 8 8 5.0 4.0–6.0 3 0 0502 Micchan (1) × Mizou Noir (1) 18 12 4.9 3.7–6.0 4 1 0544 Port Light Pair Beauty (3) × Dream Waltz (3) 13 11 4.9 2.0–7.0 3 0 0569 Zuihou (3) × Yumesuiren (3) 4 4 4.8 3.8–5.6 2 0 0560 Zuihou (3) × Super Girl (1) 5 5 4.8 4.3–5.4 1 0 0512 Micchan (1) × Benifusya (2) 11 11 4.8 3.0–8.0 3 1 1519 Pearl Light (1) × Benifusya (2) 6 6 4.7 3.0–6.7 2 1 0528 Mizou Noir (1) × Red Star (3) 12 4 4.7 4.0–5.2 1 0 0507 Yukitsubaki (1) × Pearl Light (1) 5 5 4.7 3.5–6.7 1 0 0520 Super Girl (1) × Moon Waltz (2) 6 5 4.7 3.0–6.0 1 1 0505 Micchan (1) × Kokucho (1) 28 16 4.6 3.0–6.8 4 2 1534 Kokucho (1) × Konatu (2) 11 8 4.6 3.7–6.0 1 0 0537 Pom-Pom Chocolat (2) × Red Star (3) 8 8 4.6 3.0–6.7 2 0 0557 Pearl Light (1) × Kamakura (2) 4 4 4.6 4.0–5.7 1 0 0526 Kokucho (1) × Pom-Pom Chocolat (2) 20 11 4.5 3.0–7.0 3 1 0506 Kokucho (1) × Micchan (1) 11 9 4.5 4.0–5.6 1 0 0542 Agitate (2) × Port Light Pair Beauty (3) 5 3 4.4 4.0–5.0 0 0 0575 Mizou Noir (1) × Shishu (3) 14 12 4.2 2.0–6.0 3 0 0521 Kokucho (1) × Benifusya (2) 17 15 4.2 2.0–7.0 5 0 0541 Konatu (2) × Port Light Pair Beauty (3) 15 14 4.2 2.8–6.3 3 0 0527 Pom-Pom Chocolat (2) × Kokucho (1) 11 8 4.1 3.3–5.0 0 0 0524 Micchan (1) × Shishu (3) 6 6 4.0 2.8–5.9 1 1 0538 Yumesuiren (3) × Kokucho (1) 55 40 3.9 2.0–7.0 4 0 0517 Pom-Pom Chocolat (2) × Yukitsubaki (1) 12 9 3.8 1.0–5.0 0 0 0558 Pom-Pom Chocolat (2) × Rinka (1) 3 3 3.8 3.5–4.0 0 0 0515 Micchan (1) × Moon Waltz (2) 12 6 3.8 3.0–5.0 0 0 0532 Konatu (2) × Super Girl (1) 27 16 3.8 3.0–6.0 1 0 0539 Port Light Pair Beauty (3) × Pom-Pom Chocolat (2) 6 6 3.6 3.0–5.0 0 0 0543 Agitate (2) × Yumesuiren (3) 9 3 3.5 2.0–5.0 0 0 0
503 Yukitsubaki (1) × Rinka (1) 2 2 — 3.0–4.0 0 0 0508 Jessy Rita (1) × Micchan (1) 4 2 — 4.0–5.0 0 0 0511 Micchan (1) × Kyuen (2) 2 2 — 4.8–8.0 1 1 0513 Super Girl (1) × Pearl Light (1) 3 2 — 3.0–5.5 1 0 0529 Pearl Light (1) × Zuihou (3) 3 2 — 5.0 0 0 0548 Super Girl (1) × Mizou Noir (1) 2 2 — 3.0–4.3 0 0 0566 Yumesuiren (3) × Mizou Noir (1) 2 2 — 4.8 0 0 0540 Kamakura (2) × Yumesuiren (3) 1 1 — 4.0 0 0 0553 Jessy Rita (1) × Syukuhai (1) 1 1 — 3.8 0 0 0561 Super Girl (1) × Konatu (2) 1 1 — 3.0 0 0 0567 Red Star (3) × Agitate (2) 1 1 — 4.7 0 0 0570 Yumesuiren (3) × Agitate (2) 2 1 — 7.0 1 0 0578 Zuihou (3) × Mixed pollen (Pearl Light
(1) and Moon Waltz (2))3 1 — 7.5 1 0 0
Total 419 314 4.4 1.0–8.0 64 11 2
Mean vase life of progeny was calculated for at least three seedlings per cross combination.Cross combinations are listed in descending order of the mean vase life of progeny.Numbers in parentheses indicate vase life index: 1 (long vase life); 2 (normal vase life); 3 (short vase life).
Table 2. Cross combinations and results of crosses for the first generation.Hort. J. 88 (4): 521–534. 2019. 525
Tabl
e 3.
Cr
oss c
ombi
natio
ns a
nd re
sults
of c
ross
es fo
r the
seco
nd g
ener
atio
n.
Cro
ss N
o.C
ross
com
bina
tion
Num
ber o
f se
edlin
gs
obta
ined
Num
ber o
f flo
wer
ed
seed
lings
Mea
n va
se li
fe
of p
roge
ny
(day
s)
Ran
ge(d
ays)
Num
ber o
f pr
imar
y
sele
cted
pla
nts
Num
ber o
f se
cond
ary
sele
cted
pla
nts
Num
ber o
f fin
al se
lect
ed
plan
ts
Orig
in(C
ultiv
ars u
sed
for b
reed
ing
of p
aren
tal l
ines
)♀
♂
608
505-
47×
578-
412
107.
15.
0–12
.06
20
Mic
chan
, Kok
ucho
, Zui
hou,
Pea
rl Li
ght,
Moo
n W
altz
607
505-
46×
578-
44
46.
85.
0–8.
33
10
Mic
chan
, Kok
ucho
, Zui
hou,
Pea
rl Li
ght,
Moo
n W
altz
605
505-
18×
578-
411
106.
13.
0–8.
05
00
Mic
chan
, Kok
ucho
, Zui
hou,
Pea
rl Li
ght,
Moo
n W
altz
606
513-
53×
512-
212
116.
03.
7–9.
05
21
Supe
r Girl
, Pea
rl Li
ght,
Mic
chan
, Ben
ifusy
a62
252
4-29
×52
5-48
55
6.0
5.3–
7.3
21
0M
icch
an, S
hish
u, M
icch
an, K
onat
su61
954
4-42
×52
4-29
44
5.9
5.0–
7.0
10
0PL
PB, D
ream
Wal
tz, M
icch
an, S
hish
u60
951
1-1
×51
6-58
3734
5.6
2.0–
9.0
110
0M
icch
an, K
yuen
, Mic
chan
, Pom
-Pom
Cho
cola
t63
152
5-21
×56
9-63
1111
5.5
4.0–
7.5
50
0M
icch
an, K
onat
su, Z
uiho
u, Y
umes
uire
n61
452
1-6
×54
1-55
2117
5.5
3.5–
7.5
30
0K
okuc
ho, B
enifu
sya,
Kon
atsu
, PLP
B62
354
4-42
×51
6-50
1414
5.4
3.7–
6.7
30
0PL
PB, D
ream
Wal
tz, M
icch
an, P
om-P
om C
hoco
lat
610
538-
61×
512-
212
115.
33.
0–7.
04
00
Yum
esui
ren,
Kok
ucho
, Mic
chan
, Ben
ifusy
a62
950
5-46
×56
9-63
76
5.3
3.5–
7.3
22
1M
icch
an, K
okut
yo, Z
uiho
u, Y
umes
uire
n62
050
5-46
×54
1-41
3227
5.2
3.0–
8.0
51
1M
icch
an, K
okuc
ho, K
onat
su, P
LPB
616
505-
18×
541-
4110
105.
24.
0–8.
02
00
Mic
chan
, Kok
ucho
, Kon
atsu
, PLP
B61
250
5-47
×54
4-10
44
5.1
3.0–
6.3
10
0M
icch
an, K
okuc
ho, P
LPB
, Dre
am W
altz
601
537-
40×
512-
230
285.
02.
0–8.
06
00
Pom
-Pom
Cho
cola
t, R
ed S
tar,
Mic
chan
, Ben
ifusy
a62
856
9-63
×52
4-29
1816
4.8
2.0–
6.9
31
1Zu
ihou
, Yum
esui
ren,
Mic
chan
, Shi
syu
613
533-
62×
578-
48
84.
73.
5–5.
80
00
Kok
ucho
, Red
Sta
r, Zu
ihou
, Pea
rl Li
ght,
Moo
n W
altz
602
544-
42×
512-
24
34.
74.
0–6.
01
00
PLPB
, Dre
am W
altz
, Mic
chan
, Ben
ifusy
a61
853
8-25
×52
0-28
3124
4.3
2.0–
7.0
30
0Yu
mes
uire
n, K
okuc
ho, S
uper
Girl
, Moo
n W
altz
621
538-
25×
557-
497
64.
23.
5–5.
00
00
Yum
esui
ren,
Kok
ucho
, Pea
rl Li
ght,
Kam
akur
a62
554
1-55
×54
1-41
3831
4.1
1.0–
7.0
10
0K
onat
su, P
LPB
, Kon
atsu
, PLP
B62
454
4-42
×54
1-55
66
4.1
3.0–
5.5
00
0PL
PB, D
ream
Wal
tz, K
onat
su, P
LPB
603
524-
29×
512-
21
1—
5.3
00
0M
icch
an, S
hish
u, M
icch
an, B
enifu
sya
611
505-
46×
544-
102
2—
4.0–
5.7
00
0M
icch
an, K
okuc
ho, P
LPB
, Dre
am W
altz
615
519-
14×
557-
491
1—
6.4
10
0Pe
arl L
ight
, Ben
ifusy
a, P
earl
Ligh
t, K
amak
ura
626
505-
46×
521-
204
1—
7.0
00
0M
icch
an, K
okuc
ho, K
okuc
ho, B
enifu
sya
627
533-
24×
505-
461
1—
5.3
00
0K
okuc
ho, R
ed S
tar,
Mic
chan
, Kok
ucho
630
569-
63×
557-
492
2—
4.0–
4.8
00
0Zu
ihou
, Yum
esui
ren,
Pea
rl Li
ght,
Kam
akur
aTo
tal
349
308
5.2
1.0–
12.0
7310
4
Mea
n va
se li
fe o
f pro
geny
was
cal
cula
ted
for a
t lea
st th
ree
seed
lings
per
cro
ss c
ombi
natio
n.C
ross
com
bina
tions
are
list
ed in
des
cend
ing
orde
r of t
he m
ean
vase
life
of p
roge
ny.
PLPB
, ‘Po
rt Li
ght P
air B
eaut
y’.
Table 3. Cross combinations and results of crosses for the second generation.
526 T. Onozaki and M. Azuma
Tabl
e 4.
Cr
oss c
ombi
natio
ns a
nd re
sults
of c
ross
es fo
r the
third
gen
erat
ion.
Cro
ss N
o.C
ross
com
bina
tion
Num
ber o
f se
edlin
gs
obta
ined
Num
ber o
f flo
wer
ed
seed
lings
Mea
n va
se
life
of
prog
eny
(day
s)
Ran
ge
(day
s)
Num
ber
of p
rimar
y se
lect
ed
plan
ts
Orig
in (C
ultiv
ars u
sed
for b
reed
ing
of p
aren
tal l
ines
)♀
♂
823
609-
4×
631-
234
48.
16.
0–10
.33
Mic
chan
, Kyu
en, M
icch
an, P
om-P
om C
hoco
lat,
Mic
chan
, Kon
atsu
, Zui
hou,
Yum
esui
ren
813
628-
32×
601-
493
36.
95.
0–8.
82
Zuih
ou, Y
umes
uire
n, M
icch
an, S
hisy
u, P
om-P
om C
hoco
lat,
Red
Sta
r, M
icch
an, B
enifu
sya
828
609-
10×
614-
716
56.
95.
3–8.
33
Mic
chan
, Kyu
en, M
icch
an, P
om-P
om C
hoco
lat,
Kok
ucho
, Ben
ifusy
a, K
onat
su, P
LPB
820
631-
23×
606-
464
46.
55.
0–8.
23
Mic
chan
, Kon
atsu
, Zui
hou,
Yum
esui
ren,
Sup
er G
irl, P
earl
Ligh
t, M
icch
an, B
enifu
sya
826
628-
48×
614-
7118
116.
44.
0–10
.03
Zuih
ou, Y
umes
uire
n, M
icch
an, S
hisy
u, K
okuc
ho, B
enifu
sya,
Kon
atsu
, PLP
B82
463
1-23
×60
9-4
44
6.1
5.9–
6.5
0M
icch
an, K
onat
su, Z
uiho
u, Y
umes
uire
n, M
icch
an, K
yuen
, Mic
chan
, Pom
-Pom
Cho
cola
t81
262
8-32
×63
1-56
3621
6.0
3.0–
8.6
10Zu
ihou
, Yum
esui
ren,
Mic
chan
, Shi
syu,
Mic
chan
, Kon
atsu
, Zui
hou,
Yum
esui
ren
814
601-
49×
606-
4629
206.
03.
7–9.
07
Pom
-Pom
Cho
cola
t, R
ed S
tar,
Mic
chan
, Ben
ifusy
a, S
uper
Girl
, Pea
rl Li
ght,
Mic
chan
, Ben
ifusy
a80
362
8-32
×60
7-6
77
6.0
4.0–
10.0
1Zu
ihou
, Yum
esui
ren,
Mic
chan
, Shi
syu,
Mic
chan
, Kok
ucho
, Zui
hou,
Pea
rl Li
ght,
Moo
n W
altz
811
601-
49×
606-
737
155.
93.
0–12
.04
Pom
-Pom
Cho
cola
t, R
ed S
tar,
Mic
chan
, Ben
ifusy
a, S
uper
Girl
, Pea
rl Li
ght,
Mic
chan
, Ben
ifusy
a82
260
1-49
×62
8-48
379
5.6
3.0–
9.0
3Po
m-P
om C
hoco
lat,
Red
Sta
r, M
icch
an, B
enifu
sya,
Zui
hou,
Yum
esui
ren,
Mic
chan
, Shi
syu
709
609-
4×
631-
566
65.
54.
5–6.
72
Mic
chan
, Kyu
en, M
icch
an, P
om-P
om C
hoco
lat,
Mic
chan
, Kon
atsu
, Zui
hou,
Yum
esui
ren
710
614-
20×
606-
174
35.
55.
3–5.
70
Kok
ucho
, Ben
ifusy
a, K
onat
su, P
LPB
, Sup
er G
irl, P
earl
Ligh
t, M
icch
an, B
enifu
sya
805
628-
32×
606-
1727
205.
43.
0–8.
06
Zuih
ou, Y
umes
uire
n, M
icch
an, S
hisy
u, S
uper
Girl
, Pea
rl Li
ght,
Mic
chan
, Ben
ifusy
a82
162
8-32
×61
5-55
1210
5.1
3.5–
7.3
2Zu
ihou
, Yum
esui
ren,
Mic
chan
, Shi
syu,
Pea
rl Li
ght,
Ben
ifusy
a, P
earl
Ligh
t, K
amak
ura
719
623-
73×
622-
212
2—
6.2–
7.4
2PL
PB, D
ream
Wal
tz, M
icch
an, P
om-P
om C
hoco
lat,
Mic
chan
, Shi
shu,
Mic
chan
, Kon
atsu
808
606-
17×
601-
491
1—
10.0
1Su
per G
irl, P
earl
Ligh
t, M
icch
an, B
enifu
sya,
Pom
-Pom
Cho
cola
t, R
ed S
tar,
Mic
chan
, Ben
ifusy
a80
962
9-18
×60
6-46
11
—7.
00
Mic
chan
, Kok
utyo
, Zui
hou,
Yum
esui
ren,
Sup
er G
irl, P
earl
Ligh
t, M
icch
an, B
enifu
sya
810
609-
10×
606-
171
1—
9.0
1M
icch
an, K
yuen
, Mic
chan
, Pom
-Pom
Cho
cola
t, Su
per G
irl, P
earl
Ligh
t, M
icch
an, B
enifu
sya
815
628-
32×
609-
102
2—
5.8–
8.2
1Zu
ihou
, Yum
esui
ren,
Mic
chan
, Shi
syu,
Mic
chan
, Kyu
en, M
icch
an, P
om-P
om C
hoco
lat
816
606-
7×
614-
712
2—
5.5–
9.5
1Su
per G
irl, P
earl
Ligh
t, M
icch
an, B
enifu
sya,
Kok
ucho
, Ben
ifusy
a, K
onat
su, P
LPB
819
628-
32×
614-
712
2—
7.6–
12.0
2Zu
ihou
, Yum
esui
ren,
Mic
chan
, Shi
syu,
Kok
ucho
, Ben
ifusy
a, K
onat
su, P
LPB
825
605-
53×
615-
551
1—
8.0
1M
icch
an, K
okuc
ho, Z
uiho
u, P
earl
Ligh
t, M
oon
Wal
tz, Y
umes
uire
n, K
okuc
ho, S
uper
Girl
, Moo
n W
altz
829
623-
73×
618-
431
1—
6.0
0PL
PB, D
ream
Wal
tz, M
icch
an, P
om-P
om C
hoco
lat,
Yum
esui
ren,
Kok
ucho
, Sup
er G
irl, M
oon
Wal
tz
Tota
l24
715
56.
13.
0–12
.058
Mea
n va
se li
fe o
f pro
geny
was
cal
cula
ted
for a
t lea
st th
ree
seed
lings
per
cro
ss c
ombi
natio
n.C
ross
com
bina
tions
are
list
ed in
des
cend
ing
orde
r of t
he m
ean
vase
life
of p
roge
ny.
PLPB
, ‘Po
rt Li
ght P
air B
eaut
y’.
Table 4. Cross combinations and results of crosses for the third generation.
Hort. J. 88 (4): 521–534. 2019. 527
ity, not extremely late flowering). Trial production ofthe lines selected in the second round was conducted torelease cultivars at three sites (NIVFS, Akita Agricul‐tural Experiment Station, and Nara AgriculturalResearch Center) from 2017 to 2019 and at the KochiAgricultural Research Center from 2018 to 2019, andsix lines were finally selected on the basis of their vaselife and other important traits.
Vase life evaluation in seedlings and selected linesIn the seedling trials (the first year of each genera‐
tion), all harvested flowers were evaluated for vase lifein the CMIT/MIT solution. The average numbers forvase life surveyed per flowered seedling in the first,second, and third generations were 3.0, 3.4, and 3.3, re‐spectively. The number of days from planting to flower‐ing was recorded, and the diameters of the stem baseand stem neck of the first harvested cut flower weremeasured in the first and second generations with aVernier caliper.
In the 2017–18 winter–spring season, rooted cuttingsof four control cultivars (‘Kamakura’, ‘Kokucho’,‘Micchan’, and ‘Port Light Pair Beauty’) and six finallyselected lines (Table 5) were prepared and planted in21-cm-diameter pots with 4 L of standard culture soil(Royal Culture Soil; Tachikawa Heiwa Nouen Co.,Ltd., Tochigi, Japan) on 31 October 2017. Plants weregrown until April in a greenhouse at NIVFS heatedabove 12°C, with artificial lighting (daylength exten‐sion to 14.5 h) using incandescent light bulbs (K-RD110V75W/D; Panasonic Co., Ltd., Osaka, Japan)from 05:00 to 07:00 and from 16:00 to 19:30. Vase lifewas evaluated from January to April 2018.
In summer and autumn 2018, four control cultivars(‘Kamakura’, ‘Kokucho’, ‘Micchan’, and ‘Port LightPair Beauty’) and six finally selected lines (Table 5)were grown in the open field at NIVFS as described be‐fore, or in a vinyl house with a shade net (cool white,45%–50% shade; Dio Chemicals, Ltd., Tokyo, Japan)under 50%–55% sunlight (natural daylength, no heat‐ing). Rooted cuttings of each cultivar or line wereplanted 40 cm apart in the open field on 17 May 2018or in 24 cm-diameter pots with 5 L of the standard cul‐ture soil described above in the vinyl house on 24 April2018. The shoots from each plant were pinched to 2 or3 nodes on 18 June 2018, and the plants were grownuntil November following standard production methods(Yamagata, 2018). Vase life was evaluated fromSeptember to October 2018.
Statistical analysesThe results shown in Tables 1 and 5 were analyzed
with BellCurve for Excel software (Social SurveyResearch Information Co., Ltd., Tokyo, Japan) usingTukey’s test (P < 0.05).
ResultsGenetic variation in vase life among dahlia cultivars
We found large significant differences in flower vaselife among 24 dahlia cultivars (Table 1): ‘Syukuhai’,‘Rinka’, ‘Micchan’, ‘Super Girl’, ‘Mizou Noir’, ‘JessyRita’, ‘Yukitsubaki’, ‘Kokucho’, and ‘Pearl Light’ hadlong vase life (Index 1); ‘Kyuen’, ‘Moon Waltz’,‘Kamakura’, ‘Pom-Pom Chocolat’, ‘Agitate’,‘Benifusya’, ‘Konatsu’, and ‘Black Cat’ had normalvase life (Index 2); and ‘Zuihou’, ‘Dream Waltz’,‘Shishu’, ‘Red Star’, ‘Gin-ei’, ‘Port Light Pair Beauty’,and ‘Yumesuiren’ had short vase life (Index 3). Theresponses of the cultivars to the CMIT/MIT and GLAsolutions varied (Table 1). Although ‘Yumesuiren’ hadthe shortest vase life in DW (2.8–3.3 days) in bothyears, it showed the greatest response to CMIT/MIT(ratio of CMIT/MIT to DW = 1.5). The effect of GLAtreatment was strong in ‘Agitate’ (ratio of GLA to DW= 1.9) and in ‘Super Girl’ (1.8).
Crossing and selectionIn all three generations, continuous normal frequency
distributions of vase life were observed (Fig. 2). Themean vase life of individual seedlings increased slowlyand steadily as generations progressed. Although thefrequency of flowers with superior vase life (≥ 8 days)was 1.0% in the first generation and 4.2% in the secondgeneration, it rose to 19.4% in the third generation. Theproportion of flowers with inferior vase life (< 4 days)decreased from 29.3% in the first generation to 6.5% inthe third generation. The mean vase life was 4.4 days inthe first generation, but after two cycles of crossing andselection it increased to 6.1 days (a net total increase of1.7 days; Fig. 2). The increase was 0.8 days betweenthe first and second generations and 0.9 days betweenthe second and third generations. Therefore, the effectof crossing and selection between generations remainedalmost constant.
In the first generation, when flowers lost ornamentalvalue, 94.5% showed wilting (Fig. 3). The proportion offlowers with browning (browning plus wilting withbrowning) increased from 5.4% in the first generationto 19.3% in the third generation. Only 0.1%, 0.9%, and0% of individual flowers showed petal abscission in thefirst, second, and third generations, respectively.
Relationships between vase life and other characteris‐tics
No correlation between vase life and stem base diam‐eter was found (Fig. 4B). However, there was a weakbut significant positive correlation between vase lifeand the number of days from planting to flowering orstem neck diameter (Fig. 4A, C).
Mean vase life of progeny per cross combinationIn the first generation, the difference between the
528 T. Onozaki and M. Azuma
Tabl
e 5.
Fl
ower
vas
e lif
e of
dah
lia c
ultiv
ars a
nd se
lect
ed fi
rst-
and
seco
nd-g
ener
atio
n lin
es in
dist
illed
wat
er, a
ntib
acte
rial a
gent
(CM
IT/M
IT) o
r GLA
solu
tion
unde
r sta
ndar
d co
nditi
ons (
23°C
, 12-
h ph
otop
erio
d,70
% re
lativ
e hu
mid
ity).
Cul
tivar
or
sele
cted
line
z Flo
wer
ty
pe
2018
Win
ter a
nd sp
ring
in g
reen
hous
e20
18 L
ate
sum
mer
and
aut
umn
in o
pen
field
2018
Lat
e su
mm
er a
nd a
utum
n
in v
inyl
hou
seG
rand
M
ean
Dis
tille
d W
ater
CM
IT/M
ITG
LAD
istil
led
Wat
erC
MIT
/MIT
GLA
Dis
tille
d W
ater
GLA
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Vase
life
(d
ays)
%y
Con
trol c
ultiv
arK
amak
ura
FD 6
.2 ±
0.2
ab10
0 6
.8 ±
0.5
ab10
0 7
.6 ±
0.4
a10
05.
0 ± 0.
3 ab
100
6.0 ±
0.3
bc10
0 6
.8 ±
0.3
ab10
0 5
.4 ±
0.2
ab10
0 6
.0 ±
0.4
a10
06.
2Po
rt Li
ght P
air B
eaut
yFD
5.4
± 0.
2 a
87
4.8
± 0.
2 a
71
8.6
± 0.
2 ab
113
3.8 ±
0.2
ab 7
64.
0 ± 0.
0 a
67
5.6
± 0.
4 ab
82
4.0
± 0.
0 a
74
7.3
± 0.
3 ab
122
5.4
Kok
ucho
SC 8
.0 ±
0.6
bc12
9 7
.2 ±
0.4
b10
610
.0 ±
0.6
abc
132
4.9 ±
0.4
ab 9
84.
1 ± 0.
3 a
68
7.7
± 0.
6 ab
c11
3 7
.4 ±
0.9
abc
137
10.2
± 0.
2 ab
170
7.4
Mic
chan
BA
8.2
± 0.
5 bc
132
7.8
± 0.
4 bc
115
10.2
± 0.
7 ab
c13
46.
0 ± 0.
5 ab
c12
05.
4 ± 0.
3 ab
90
9.1
± 0.
6 ab
cd13
4 9
.6 ±
0.7
abc
178
10.6
± 1.
0 ab
177
8.4
Fina
l sel
ecte
d lin
e51
2-2
(1st
)xFD
10.8
± 0.
7 de
174
10.2
± 0.
9 c
150
12.8
± 0.
6 c
168
6.2 ±
0.7
abc
124
6.9 ±
0.5
cd11
5 9
.4 ±
0.8
bcd
138
10.8
± 1.
2 c
200
12.8
± 1.
6 b
213
10.0
505-
13 (1
st)
SC12
.0 ±
0.6
e19
410
.0 ±
0.8
c14
713
.4 ±
1.6
c17
66.
9 ± 0.
6 bc
d13
87.
3 ± 0.
3 cd
e12
211
.3 ±
0.7
d16
611
.4 ±
1.3
c21
111
.4 ±
1.7
ab19
010
.562
9-18
(2nd
)FD
9.0
± 0.
7 cd
145
7.0
± 0.
8 b
103
11.2
± 0.
4 ab
c14
77.
3 ± 0.
4 cd
e14
66.
9 ± 0.
4 cd
115
9.8
± 0.
5 bc
d14
4 8
.2 ±
0.9
abc
152
11.0
± 0.
7 ab
183
8.8
620-
29 (2
nd)
FD 6
.4 ±
0.4
ab10
3 7
.8 ±
0.6
bc11
511
.0 ±
0.7
abc
145
7.6 ±
0.4
cde
152
7.6 ±
0.3
de12
710
.6 ±
1.0
cd15
610
.8 ±
1.0
c20
011
.6 ±
1.8
ab19
39.
262
8-32
(2nd
)FD
9.6
± 0.
2 cd
155
8.6
± 0.
4 bc
126
13.6
± 1.
6 c
179
8.5 ±
0.5
de17
08.
7 ± 0.
3 e
145
11.8
± 0.
7 d
174
8.2
± 0.
6 ab
c15
211
.6 ±
1.3
ab19
310
.160
6-46
(2nd
)FD
10.0
± 0.
3 cd
e16
110
.0 ±
0.3
c14
712
.6 ±
0.4
bc16
69.
2 ± 0.
5 e
184
6.6 ±
0.3
bcd
110
10.5
± 0.
7 cd
154
9.8
± 1.
5 bc
181
11.8
± 1.
2 b
197
10.1
z Flo
wer
type
: FD
, for
mal
dec
orat
ive;
BA
, bal
l; SC
, sem
i-cac
tus;
ID, i
nfor
mal
dec
orat
ive.
Valu
es o
f vas
e lif
e ar
e th
e m
eans
± S
E of
the
data
for 3
–10
flow
ers.
Valu
es w
ith d
iffer
ent l
ette
rs a
re si
gnifi
cant
ly d
iffer
ent a
t P <
0.05
by
Tuke
y’s t
est.
y %, p
erce
ntag
e of
the
valu
e fo
r the
con
trol c
ultiv
ar, ‘
Kam
akur
a’.
x 1st
, sel
ecte
d fir
st-g
ener
atio
n lin
e; 2
nd, s
elec
ted
seco
nd-g
ener
atio
n lin
e.
Table 5. Flower vase life of dahlia cultivars and selected first- and second-generation lines in distilled water, antibacterial agent (CMIT/MIT) or GLA solution under standard conditions (23°C, 12-h photoperiod, 70% relative humidity).
Hort. J. 88 (4): 521–534. 2019. 529
cross combination with the highest mean vase life(cross no. 533; ‘Kokucho’ × ‘Red Star’; 5.2 days) andthat with the lowest mean vase life (cross no. 543;‘Agitate’ × ‘Yumesuiren’; 3.5 days) was 1.7 days (Table2). In the second generation, the difference between thecross combination with the highest mean vase life(cross no. 608; 505-47 × 578-4; 7.1 days) and that withthe lowest mean vase life (cross no. 624; 544-42 × 541-55; 4.1 days) was 3.0 days (Table 3). In the thirdgeneration, the difference between the cross combina‐tion with the highest mean vase life (cross no. 823;
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12 13
Freq
uenc
y(%
)
Vase life (days)
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12 13
Freq
uenc
y(%
)
Vase life (days)
n = 314mean = 4.4S.D = 1.15
n = 308mean = 5.2S.D = 1.45
First generation (2015)
Second generation (2016)
0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12 13
Freq
uenc
y(%
)
Vase life (days)
Third generation (2017–2018)
n = 155mean = 6.1S.D = 1.78
Fig. 2. Frequency distributions of flower vase life in the three gen‐erations of dahlia breeding populations. Vertical bar representsthe mean.
0%
20%
40%
60%
80%
100%
First Second Third
Freq
uenc
y
Genera�on
Wil�ng Wil�ng with browning Browning Petal abscission
Fig. 3. Frequency distribution of senescence patterns in the threegenerations of dahlia breeding populations.
609-4 × 631-23; 8.1 days) and that with the lowestmean vase life (cross no. 821; 628-32 × 615-55; 5.1days) was 3.0 days (Table 4). Tracing back to the originof each cross combination in the third generation re‐vealed that ‘Micchan’ was a common breeding materialin all cross combinations (Table 4).
Vase life of cultivars and selected linesWe examined the vase life of the cut flowers of four
control cultivars and six finally selected lines (Fig. 5) in
A
B
C
1
3
5
7
9
11
13
30 50 70 90 110
Flow
er v
ase
life
(day
s)
Number of days from planting to flowering
First genera�onSecond genera�onThird genera�on n = 777
0
1
2
3
4
5
6
7
8
9
10
04020
Flow
er v
ase
life
(day
s)
Stem base diameter (mm)
First genera�on
Second genera�on
0
1
2
3
4
5
6
7
8
9
10
510150
Flow
er v
ase
life
(day
s)
Stem neck diameter (mm)
First genera�on
Second genera�on
r = 0.18**
n = 590
n = 600
r = 0.14**
r = 0.03NS
Fig. 4. Relationships between flower vase life and (A) number ofdays from planting to flowering, (B) stem base diameter, and(C) stem neck diameter.
530 T. Onozaki and M. Azuma
detail in two different seasons and using three differentcultivation styles, and found a wide range of variation(Table 5). The mean vase life of ‘Kamakura’, a leadingwhite dahlia cultivar in Japan, was 5.0–6.2 days in DW,6.0–6.8 days in CMIT/MIT, and 6.0–7.6 days in GLA,whereas in the six finally selected lines it was 6.2–12.0days in DW, 6.6–10.2 days in CMIT/MIT, and 9.4–13.6days in GLA (1.4–2.1 times that in ‘Kamakura’). Inparticular, line 606-46 showed a stably and significantlylonger vase life than ‘Kamakura’, except in DW in thevinyl house and CMIT/MIT in the open field (Table 5).The outer petals of the control cultivar ‘Port Light Pair
Beauty’ began to wilt after four days (Fig. 6B) andcompletely lost ornamental value after six days(Fig. 6C), whereas lines 512-2 and 606-46 showed nowilting until eight days (Fig. 6D).
DiscussionSince dahlia have long been grown in home gardens
rather than commercially, there are no reports of dahliabreeding aimed at improving useful traits such as flow‐er longevity or disease resistance. Here, we report thefirst attempt to extend the vase life of dahlia flowersusing cross-breeding techniques. Two cycles of selec‐
629-18 620-29 628-32 606-46
‘Micchan’ 512-2 505-13
Selected first-generation lines
Selected second-generation lines
Fig. 5. Dahlia cultivar ‘Micchan’ and finally selected first- and second-generation lines with long vase life.
0 days 4 days
8 days6 days
A B
C D
Fig. 6. Vase life of two selected lines, 512-2 and 606-46, and the control cultivar ‘Port Light Pair Beauty’ in distilled water. Flowers were photo‐graphed at (A) 0, (B) 4, (C) 6, and (D) eight days after harvest (experimental period: 24 April to 2 May 2018). Left: selected first-generationline 512-2; center: ‘Port Light Pair Beauty’; right: selected second-generation line 606-46. The flowers were kept at 23°C, 70% relativehumidity, under a 12-h photoperiod.
Hort. J. 88 (4): 521–534. 2019. 531
tion and crossing led to a 1.7-day increase (Fig. 2). Theresults clearly show that the vase life of dahlia flowerscan be extended by crossing and selection.
The seed germination percentage in dahlia crosseswas relatively low. For example, the germination rate ofthe first generation at 67 days after sowing (23 April to29 June 2015) was 39.3%. The seeds from crossestended to germinate slowly and gradually, with someseeds germinating two months after sowing. Vivar-Evans et al. (2006) reported that seeds of wild Dahliacoccinea, one of the progenitors of cultivated dahlia inthis genus (Okumura and Fujino, 1989), showed phys‐iological dormancy. Dormancy inherited from a wildDahlia species may be responsible for the low germina‐tion rate. In dahlia breeding, it is considered necessaryto select an appropriate germination period to take intoaccount seed dormancy.
Although we examined the relationship between vaselife and stem base diameter in the seedlings of the firstand second generations, according to the recommenda‐tions of three Japanese dahlia breeders (see Introduc‐tion), we found no correlation between them (Fig. 4B).Harvested flowers were evaluated in CMIT/MIT solu‐tion, but not in DW because accidental stem rot waslikely to occur in summer. In DW, this correlation maybe observed because of a decrease in water absorptiondue to vascular occlusion. Breeding of dahlias with nohollows or small hollows in the stems to further im‐prove the vase life is our future objective.
Naka et al. (2015) investigated seasonal variations inthe vase life in three dahlia cultivars (‘Kamakura’,‘Kokucho’, and ‘Shukuhai’). The vase life in DW de‐creased from July to October and increased fromDecember to February, whereas there was little seasonalvariation in vase life in GLA solution and it was almostconstant throughout the year. In our study, to developcultivars with a long vase life in summer, when the vaselife of dahlia declines, seedlings were selected fromJuly to mid-September. After the initially selected lineswere vegetatively propagated, the vase life was exam‐ined in detail in two different seasons and using threedifferent cultivation styles (winter–spring in a green‐house and/or summer–autumn in an open field or vinylhouse). This procedure is reliable for selecting lineswith a genetically long vase life.
Senescence patterns observed when the flowers lostornamental value changed gradually with generations(Fig. 3). After two cycles of crossing and selection, therate of browning senescence patterns (browning pluswilting with browning) increased to 19.3%. In carna‐tion, the relationship between senescence patterns andvase life has been well characterized: petal inrolling atthe onset of wilting is a well-known characteristic ofethylene-dependent senescence of normal carnationflowers (Iwazaki et al., 2004; Otsu et al., 2007; Satoh,2011). In contrast, desiccation and browning of petalsare characteristics of ethylene-independent senescence
in carnation cultivars with low ethylene production. Ingerbera breeding for long vase life, changes in the pro‐portion of senescence patterns (bending, folding, andwilting) were also observed (Wernett et al., 1996a). Be‐fore breeding, gerbera flowers senesced due to bendingrather than wilting. After only one cycle of selectionand crossing, the proportion shifted dramatically frombending to wilting (Wernett et al., 1996a). We plan tocontinue studying the relationship between the senes‐cence patterns and vase life using future generations indahlias.
Dahlia flowers come in various flower types, such asstraight cactus, incurved cactus, water lily, and formaldecorative (Okumura and Fujino, 1989). The breedingmaterials shown in Table 1 had five different flowertypes. However, the finally selected lines had only twotypes, formal decorative and semi-cactus (Fig. 5; Table5). The flower type of 58 lines initially selected in thethird generation was almost exclusively formal decora‐tive (data not shown). Therefore, a loss of flower typediversity was caused by selection and crossing for longvase life. Further studies are needed to clarify the rela‐tionship between flower type and flower vase life.
As mentioned in the Introduction, Tsujimoto et al.(2016b) reported that ‘Rinka’, ‘Syukuhai’, ‘MoonWaltz’, ‘Benifusya’, ‘Micchan’, ‘Akebono-Temari’, and‘Pink Sapphire’ have very long vase life. Azuma et al.(2019) reported that the vase life of outer florets in DWwas longer in ‘Kokucho’, ‘Micchan’, and ‘Moon Waltz’than in seven other cultivars examined. The vase life ofcut dahlia flowers is affected by bacterial proliferationin the vase solution and carbohydrate level in petals(Azuma et al., 2019). Furthermore, we found that theCMIT/MIT treatment extended the vase life of threecultivars, all of which had relatively high numbers ofbacteria in their vase solutions (Azuma et al., 2019).Ichimura et al. (2011) showed that continuous treatmentwith GLA effectively extended the vase life of‘Kokucho’ dahlia flowers. Therefore, we used three dif‐ferent solutions (DW, CMIT/MIT, and GLA) over twoyears (2014 and 2015), and found that ‘Syukuhai’,‘Rinka’, and ‘Micchan’ were the top three Index 1 culti‐vars (Table 1), in agreement with the result ofTsujimoto et al. (2016b), although ‘Moon Waltz’ and‘Benifusya’ were classified as Index 2 (normal vaselife), unlike in Tsujimoto et al. (2016b).
Among 24 cultivars, the effect of GLA was highestin ‘Agitate’ and ‘Super Girl’ (Table 1). In a previousstudy, co-treatment with glucose and CMIT/MIT signif‐icantly increased the fresh weight of ‘Agitate’ flowersbecause the carbohydrate content in their petals wasparticularly low (Azuma et al., 2019). These results in‐dicate that low levels of carbohydrates in ‘Agitate’ mayhave contributed to the strong effect of GLA on vaselife.
We found a close relationship between the crosscombination and flower vase life. In the first genera‐
532 T. Onozaki and M. Azuma
tion, the difference between the cross combination withthe highest mean vase life and that with the lowestmean vase life was 1.7 days (Table 2). However, therewas no relationship between the vase life of the parentcultivar and that of the progeny. In the second genera‐tion, the difference between the cross combination withthe highest mean vase life and that with the lowestmean vase life was 3.0 days (Table 3). ‘Dream Waltz’,‘Port Light Pair Beauty’, and ‘Yumesuiren’ (all Index 3;Table 1) were used to breed parental lines of four crosscombinations with the shortest vase life (crossesno. 618, 621, 625, and 624; Table 3). In the third gener‐ation, ‘Micchan’ (Index 1; Table 1) was used to breedparental lines of all cross combinations. The six finallyselected lines with long vase life (Table 5) were allprogeny of ‘Micchan’.
For ‘Syukuhai’ and ‘Rinka’ (top two Index 1 culti‐vars; Table 1), 55 seeds were obtained in eight of the 11cross combinations in which these two cultivars wereused as seed parents or pollen parents (data not shown).All obtained seeds were sown, but the germination ratewas low. Only six seedlings were evaluated for vaselife: three from cross no. 558 (‘Pom-Pom Chocolat’ × ‘Rinka’), two from cross no. 503 (‘Yukitsubaki’ × ‘Rinka’), and one from cross no. 553 (‘Jessy Rita’ × ‘Syukuhai’). The mean vase life of these six seedlingswas 3.0–4.0 days, and none of them were selected ini‐tially (Table 2).
In each generation, we selected lines with long vaselife for use as breeding materials for the next genera‐tion. Plants were not initially selected for any othercharacteristics. Our results strongly suggest that‘Micchan’ has genes related to long flower vase life andthat the trait is heritable. ‘Micchan’, a pink ball-typecultivar (Fig. 5) bred by the famous Japanese dahliabreeder Koji Washizawa in 2005, had the largest marketshare in 2017 at the Ota Floriculture Auction Co., Ltd.,the biggest flower market in Japan (Onozaki, 2018b).One of the major factors that allowed this cultivar todominate the market seems to be its long vase life.
In Japanese morning glory (Ipomoea nil (L.) Roth),a NAC (NAM/ATAF1,2/CUC2) transcription factor,EPHEMERAL1 (EPH1), is a key regulator of ethylene-independent petal senescence (Shibuya, 2018; Shibuyaet al., 2014). As dahlia flowers also show ethylene-independent petal senescence, an EPH1 homolog maybe involved in petal aging in dahlia. Although there wasno correlation between vase life and stem base diameter(Fig. 4), Tsujimoto et al. (2016a) reported a highly sig‐nificant positive correlation between cell density in thesecond nodes of the stem cross-section and flower vaselife. We plan to measure cell densities in our selectedlines with long vase life to clarify the relationship be‐tween these traits.
In conclusion, using conventional cross-breedingtechniques, we extended the flower vase life of dahliaflowers and developed six lines with genetically deter‐
mined long vase life. We are currently evaluating otherimportant characteristics in these lines at four sites inJapan (NIVFS, Akita Agricultural Experiment Station,Nara Agricultural Research Center, and Kochi Agricul‐tural Research Center) with the aim of releasing themas high-value-added pilot cultivars in the near future.
AcknowledgementsWe sincerely thank Dr. M. Mato, Ms. A. Yamagata
(Akita Agricultural Experiment Station), Dr. T. Naka,Mr. T. Nakajima, Mr. K. Inda (Nara AgriculturalResearch Center), Mr. A. Kataoka, and Mr. S.Yamashita (Kochi Agricultural Research Center) fortheir contributions to the trial production of selectedlines. We also thank the members of the Tsukuba Tech‐nical Support Center, Fujimoto-Owashi OperationsUnit, NARO, for their technical support in field andgreenhouse management.
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