Plant Cell Tiss Organ Cult (2006) 86:249–255
DOI 10.1007/s11240-006-9114-2
ORIGINAL PAPER
Flower bud culture of shallot (Allium cepa L. Aggregatum
group) with cytogenetic analysis of resulting gynogenic
plants and somaclones
Endang Sulistyaningsih Æ Youhei Aoyagi Æ
Yosuke Tashiro
Received: 6 October 2005 / Accepted: 21 April 2006 / Published online: 4 July 2006
Springer Science+Business Media B.V. 2006
Abstract Unpollinated flower culture was applied for induction of gynogenesis and somatic
organogenesis in three shallot strains, ‘Dili-white’,
‘Yogya’ and ‘Dili-red’, from Indonesia. Chromosome surveys were performed on the plants
obtained. From a total of 6,812 flowers, 89 plantlets were obtained by gynogenesis, of which 10
could be acclimated. Most of the plantlets were
induced from ‘Dili-white’. Of the gynogenetic
plants examined, two were haploid (2n = 8), four
were naturally doubled haploid (2n = 16) and the
remaining four were mixoploid (2n = 8, 16 or
2n = 16, 32, 64). ‘Dili-red’ showed the highest
frequency of somatic organogenesis. Fifty-nine
directly regenerated plants and 293 callus-derived
plants were obtained from somatic organogenesis
from the cultured flowers. Based on the chromosome number, frequencies of somaclonal variation
were high both in the directly regenerated plants
and the callus-derived plants. The frequency of
tetraploid plants (2n = 32) in the former (50%)
was higher than in the latter (33%). From these
E. Sulistyaningsih (&)
Faculty of Agriculture, Gadjah Mada University,
Bulaksumur, Yogyakarta, Indonesia
e-mail: [email protected]
Y. Aoyagi Æ Y. Tashiro
Faculty of Agriculture, Saga University,
Saga 840-8502, Japan
results we conclude that unpollinated flower
culture is an effective method for chromosome
doubling simultaneously with haploid induction in
shallot.
Keywords Haploid regeneration Æ Indirect
organogenesis Æ Liliaceae Æ Tetraploid
Introduction
Shallot (Allium cepa L. Aggregatum group) has
been a vegetatively propagated plant although it
can set seeds. Self-progenies of shallot and hybrid
plants between shallot and common onion (Allium
cepa L. Onion group) showed variation for many
characters (Tashiro et al. 1982). Therefore, shallot
is in a heterozygous state. This will hinder the
progress of the new breeding in this crop.
Haploid plants followed by chromosome doubling will offer pure lines and solve the limitation
of cross breeding. A haploid plant of shallot
(2n = · = 8) obtained from the crossing between
diploid and triploid plants was reported by Endang-Sulistyaningsih et al. (1997). For breeding, it
is necessary to obtain many haploid plants, however, a high frequency of haploid induction is not
available through crossing between diploid and
triploid plants. Androgenesis in anther or pollen
culture and gynogenesis in unpollinated flower,
ovary or ovule culture have been the main
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pathways for haploid induction in many higher
plants (Jain et al. 1996). In Allium, success of
haploid induction has been reported in common
onion through unpollinated flower culture
(Campion and Azzimonti 1988; Muren 1989;
Keller 1990b; Campion et al. 1992), while no
success has been reported using anther culture
(Campion et al. 1984; Roy 1989; Keller 1990a). In
addition, Endang-Sulistyaningsih et al. (2002) reported success in haploid induction from F1 hybrids between CMS shallot with Allium
galanthum cytoplasm and common onion by unpollinated flower culture. Therefore, unpollinated
flower culture seems to be an appropriate method
to produce haploid plants in shallot.
On the other hand, Luthar and Bohanec (1999)
reported that direct somatic organogenesis could
be induced in unpollinated flower or ovary culture
in common onion. Multiple shoot structures were
formed when flowers were cultured on induction
medium with 2,4 dichlorophenoxyacetic acid (2,4–
D 2 mg l–1) and 6-benzylamine (BA 2 mg l–1).
However, a chromosome survey of the regenerated plants was not reported. In this report we
produce and describe chromosome surveys of
plants obtained both from gynogenesis and somatic organogenesis in unpollinated flower culture of shallot.
Materials and methods
Flower culture
Shallot strains ‘Dili-white’, ‘Yogya’ and ‘Dili-red’
from Indonesia were used as donors of flowers.
All shallot plants were planted in pots with 15 cm
diameter and 17 cm height, and placed in the
greenhouse at Saga University, Japan. Pollen
fertility was greatest if anthesis occurred between
March and April (unpublished). Such flower buds
were collected 5 to 3 days before anthesis for
flower culture. The collected flowers were sterilized with 70% ethanol for 10 s and then with
0.1% HgCl2 solution for 5 min. They were then
rinsed 3 times in sterile water. After sterilization,
the flower pedicels were cut off and the flowers
placed on B5 induction medium containing
1–2 mg l–1 2,4-D, 1–2 mg l–1 BA, 7.5% sucrose,
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Plant Cell Tiss Organ Cult (2006) 86:249–255
Fig. 1 Gynogenesis in unpollinated flower culture of ‘Diliwhite’. Arrow: A plantlet from gynogenesis
and 0.7% agar (Muren 1989; Geoffriau et al.
1997). The medium was adjusted to pH 5.8 before
autoclaving at 121C for 15 min. The medium was
dispensed into 9 cm diameter petri dishes with
25 ml medium per petri dish. The flower buds
were then cultured at 25C under a 16 h photoperiod.
After 90 days in culture some gynogenetic
plantlets emerged from inside the ovaries (Fig. 1).
In parallel, callus formed and shoots emerged
directly at the base of the anther filament of cultured flowers. All the seedlings, calli and shoots
were subcultured onto MS germination media
containing 4% sucrose and 0.7% agar and cultured for 1 month. Then the gynogenetic plantlets, and two types of plantlets from somatic
organogenesis (directly regenerated plantlets,
Fig. 2 and callus-derived plantlets, Fig. 3) were
transplanted onto MS elongation media containing 3% sucrose and 0.7% agar. For MS germination and elongation media, 50 ml of each
medium solution was poured in a vessel with 9 cm
diameter and 13 cm height. When the plantlets
reached a height of about 10 cm, they were
planted in pots and acclimatized for 1 week
before placing in a greenhouse.
Karyotype analysis
Root tip cells, taken from pot-grown plants were
pretreated with 0.05% colchicine for 2.5 h at 20C
and fixed in a mixture of acetic acid and ethyl
Plant Cell Tiss Organ Cult (2006) 86:249–255
Fig. 2 Directly regenerated plantlet from somatic organogenesis in unpollinated flower culture of ‘Dili-red’
(arrow)
alcohol (1:3 v/v). After hydrolysis in 1N HCl at
60C for 7 min, they were stained with leucobasic
fuchsin and squashed in 45% acetic acid. Karyotype was analysed with Chantal karyotyping
software program (Leica co., Ltd., Germany).
Results and discussion
Gynogenetic embryos started to emerge from
inside the ovaries 40 days after culture initiation.
Each shallot strain responded differently. There
were 87 seedlings from 3,127 flowers of ‘Diliwhite’ (Table 1). However, only one embryo
emerged from inside of one ovary of 2,568 flowers
of ‘Yogya’ and 1,117 flowers of ‘Dili-red’,
respectively. Thus, ‘Dili-white’ showed a higher
Fig. 3 Callus-derived plantlet from somatic organogenesis
in unpollinated flower culture of ‘Yogya’ (arrow)
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Fig. 4 A haploid cell (2n = 8) of a seedling from gynogenesis of ‘Dili-white’. Ca. 1500x
frequency of gynogenesis compared to other two
shallot strains. Geoffriau et al. (1997) and Bohanec and Jakse (1999) reported that haploid
induction ability in unpollinated flower culture of
onion cultivars was significantly influenced by the
genotypes used. Therefore, it is plausible that
each shallot strain examined in this study differed
genetically for haploid induction ability. Most of
the gynogenetic embryos obtained from ‘Diliwhite’ could not convert into whole plants.
Moreover, the survival rate of the seedlings in
pots was low because 20% of the seedlings were
albino. Finally, nine seedlings of ‘Dili-white’ and
one seedling of ‘Dili-red’ survived.
Karyotype analysis of the nine seedlings of
‘Dili-white’ showed that two of them were haploid (2n = 8, Fig. 4), three were diploid (2n = 16)
and the remaining four were mixoploid
(Table 2). The mixoploid plants showed chromosome numbers of haploid and diploid or
those of diploid, tetraploid (2n = 32) and octoploid (2n = 64) in the same root or different
roots. The seedling of ‘Dili-red’ was observed to
be diploid. The presence of mixoploid plants
suggests the occurrence of spontaneous chromosome doubling. Spontaneous chromosome
doubling in onion was also observed by Campion
and Azzimonti (1988), Campion et al. (1992),
and Doré and Marie (1993). Therefore, it is
possible that the diploid plants obtained from
‘Dili-white’ and ‘Dili-red’ are doubled haploid.
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Plant Cell Tiss Organ Cult (2006) 86:249–255
Table 1 Number of plantlets from gynogenesis and somatic organogenesis in unpollinated flower culture of three shallot
strains
Strain
Dili-white
Yogya
Dili-red
Total
No. of flowers cultured
3,127
2,568
1,117
6,812
No. of plantlets from gynogenesis
No. of plantlets from somatic organogenesis
87
1
1
89
Directly regenerated
Callus-derived
11
17
31
59
82
86
125
293
Table 2 Chromosome numbers of seedlings from gynogenesis in unpollinated flower culture of two strains
Strain
Dili-white
Dili-red
Total
No. of plants observed
9
1
10
Frequency distribution of seedlings that showed different
chromosome numbers
2n = 8
16
8,16
16, 32, 64
2
3
1
4
3
1
3
1
2
Table 3 Percentages of florets that formed callus, callus that regenerated plantlets and florets that formed directly
regenerated plantlets
Strain
Percentage of
florets that
formed callus
Percentage of
callus that
regenerated plantlets
Percentage of
florets that
formed directly
regenerated plantlets
Dili-white
Yogya
Dili-red
7
7
10
15
18
42
1
1
1
To confirm this possibility, marker analysis will
be done in the future.
The plantlets derived by somatic organogenesis
were obtained both indirectly from callus and directly from explant tissue. The percentages of
florets that formed callus were: 7 from ‘Diliwhite’, 7 from ‘Yogya’ and 10 from ‘Dili-red’
(Table 3). The callus could regenerate plantlets in
percentages of 15, 18, and 42 from ‘Dili-white’,
‘Yogya’ and ‘Dili-red’, respectively. The percentages of florets that directly regenerated plantlets
from ‘Dili-white’, ‘Yogya’ and ‘Dili-red’ were the
same i.e., 1%. Total plantlets obtained by direct
somatic organogenesis in flower culture were 11,
17 and 31 and indirectly were 82, 86 and 125 from
‘Dili-white’, ‘Yogya’ and ‘Dili-red’, respectively
(Table 1). ‘Dili-red’ showed a greater tendency
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for somatic organogenesis in flower culture and
might reflect a genotypic influence.
In the chromosome survey of the directly
regenerated plants, we found that ‘Dili-white’ and
‘Dili-red’ produced diploid, tetraploid and mixoploid plants whereas in ‘Yogya’ there were only
diploid and tetraploid plants (Table 4). About
80% of the plants of ‘Dili-white’ and ‘Yogya’
were tetraploid. The chromosome survey of the
callus-derived plants showed that all three shallot
strains produced diploid, tetraploid and mixoploid plants (Table 4). ‘Dili-white’ produced a
single trisomic plant (2n = 17). Frequency of tetraploid plants in each strain was lower than 48%.
Karyotype analysis in detail demonstrated that all
the tetraploid plants were results of simple doubling of the somatic chromosome complement
Plant Cell Tiss Organ Cult (2006) 86:249–255
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Table 4 Chromosome numbers of directly regenerated and callus-derived plants
Strain
No. of plants
observed
Frequency distribution of plants that showed different chromosome
number
2n = 16
17
32
16, 32
Directly regenerated plants
Dili-white
9
Yogya
6
Dili-red
25
Total
40
1 (11%)
1 (17%)
15 (60%)
17
0 (0%)
0 (0%)
0 (0%)
0
7 (78%)
5 (83%)
8 (32%)
20
1 (11%)
0 (0%)
2 (8%)
3
Callus-derived plants
Dili-white
56
Yogya
47
Dili-red
125
Total
178
27 (48%)
34 (72%)
81 (65%)
142
1 (2%)
0 (0%)
0 (0%)
1
27 (48%)
.9 (19%)
38 (30%)
74
1 (2%)
4 (9%)
6 (5%)
11
Fig. 5 Karyotype of a tetraploid cell (2n = 32) of a directly regenerated plant from ‘Yogya’. Ca. 1000x
(Fig. 5) and that the trisomic plant had the extra
chromosome 8A (Fig. 6).
In a comparison between the results mentioned
above, there was no relationship between the
capacities for gynogenesis and somatic organogenesis in shallot. Moreover, frequencies of
somaclonal variations did not show the same tendency between the directly regenerated plants and
callus-derived plants. The frequency of tetraploid
plants was higher (50%) in the directly regenerated plants than in the callus-derived plants (33%).
High concentrations of plant growth regulators in
the induction medium and lengthy in vitro culture
may caused high frequencies of somaclonal variation (Reisch 1983; Tashiro & Miyazaki 1985).
In conclusion, unpollinated flower culture is an
effective method for chromosome doubling
simultaneously with haploid induction in shallot.
The doubled haploid plants generated from two
shallot strains will serve as pure lines for shallot
breeding. In addition, the tetraploid plants
obtained from somatic organogenesis of three
shallot strains may also be useful to generate
triploid plants after crossing with diploid shallot.
Triploid shallot was demonstrated in our previous
study (Endang-Sulistyaningsih and Tashiro 1999)
to be superior material promising high production
of larger bulbs. A trisomic plant obtained from
‘Dili-white’ is also an interesting material for genetic analysis of shallot.
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Fig. 6 Karytope of a trisomic cell (2n = 17) of a callus derived plant from ‘Dili-white’. Ca.1500x
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