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: onozaki@affrc.go.jp).** 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

�e Japanese Society for

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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