Euphytica 96: 331–337, 1997.

331

c 1997 Kluwer Academic Publishers. Printed in the Netherlands.

Production of interspecific hybrids in the genus Delphinium via ovule culture

Kazushige Honda1  & Kiyoshi Tsutsui2

;

Faculty of Agriculture, Hokkaido University, Sapporo 060, Japan; 1 present address: Department of Forestry &
Landscape Architecture, Hokkaido College, Senshu University, Babai 079-01, Japan; 2 present address: Research
Section, Hokkai Sankyo Co., Ltd., Kitanosato, Kitahiroshima 061-11, Japan; ( author for correspondence)

Received 2 July 1996; accepted 8 April 1997

Key words: Delphinium, dwarfness, flower color, interspecific hybridization, ovule culture

Summary

Reciprocal crosses were made to introduce the red flower color of Delphinium cardinale Hook. and D. nudicaule
Torr. & A. Gray into the dwarf species, D. grandiflorum L., and an ovule culture was applied to keep hybrid
embryos from aborting. Interspecific hybrid plants of D. cardinale  D. grandiflorum and D. grandiflorum 
D. nudicaule were successfully obtained, and their hybridity was confirmed by isozyme analyses. Morphological
studies indicated that these hybrids were almost intermediate between the female and male parents in leaf shape and
flower color. Dwarf plants with red flowers were not obtained. All hybrids showed low pollen fertility and failed to
yield viable seeds by self- or backcross-pollination using fertile pollen grains of their parents. The applicability of
the ovule culture technique for genetic improvement of the genus Delphinium through interspecific hybridization
is discussed.

Abbreviations: DTT – dithiothreitol; MS – Murashige & Skoog (1962); PVPP – polyvinyl polypyrrolidone

Introduction in flower uses. At present, D. grandiflorum L. and D.

The genus Delphinium is a member of the family
Ranunculaceae and consists of about 300 species. Wild
species of Delphinium are widely distributed from the
North Temperate Zone to the North Frigid Zone as well
as in the African Highlands (Wilde, 1931). Delphinium
includes some horticulturally important species, such
as D. elatum L., D. cheilanthum Fisch., D. formosum
Boiss. & Heut., and their interspecific hybrids.
The breeding of Delphinium species has been car-
ried out mainly in Europe by means of intra- and inter-
specific hybridization in allied species (Royal Horti-
cultural Society, 1949). While those varieties formerly
consisted of mostly white, blue and violet flowers,
Legro (1961) succeeded in introducing red, orange
and yellow colors. Thus, many varieties are currently
raised. These have been improved primarily for gar-
den planting, which are tall with long and dense flower
spikes. Moreover there are plans to breed cultivars suit-
able for potting in response to the recent diversification
tatsienense Franch. and their varieties are used for pot-
ting. Both are dwarfs and have branched spikes which
are desirable traits for potted flowers. Present hues are
limited to blue, violet and white with red, yellow and
orange still unavailable.
In the present study, we tried by means of interspe-
cific hybridization to raise dwarf strains of different
colors appropriate for potted flowers. For this purpose,
D. grandiflorum, which has blue flowers, was used
as a source of dwarfness and crossed with D. cardi-
nale Hook. and D. nudicaule Torr. & A. Gray, both of
which have red flowers. Ovule culture was applied to
raise interspecific hybrids.
332
Materials and methods
Plant materials and cross-pollination
Three perennial Delphinium species, D. grandiflorum,
D. cardinale and D. nudicaule, were used as plant
materials. All these species have 2n = 16 chromosomes.
Seeds of D. grandiflorum 13317 were introduced from
Jardin Botanique de Bordeaux in France and those of
D. nudicaule 10308 from Hortus Submontanus Found,
N.J. Subrt, in Czechoslovakia. Seeds of D. cardinale
were obtained from Daiichi Engei Co., Ltd. in Japan.
One accession was used of each species. D. gran-
diflorum has branched spikes and bluish violet flow-
ers, while D. cardinale and D. nudicaule have sparse
spikes, and scarlet and orange-red flowers, respective-
ly. The seedlings from those seeds were grown in pots
in the greenhouse under standard conditions.
Reciprocal crosses were carried out between D.
grandiflorum and D. cardinale or D. nudicaule. Flow-
ers of female parents were emasculated one or two days
before anthesis, and hand-pollinated with fresh pollen
collected from greenhouse-grown plants. Styles were
collected 24 h after pollination, and pollen tube growth
in the style was observed by an aniline blue-staining
method (Linskens & Esser, 1957; Martin, 1959; Kho
& Baer, 1968). Thirty days after pollination, the result-
ing capsules were harvested before dehiscence, and the
seeds collected from them were sown to test for fer-
tility. These seeds were surface-disinfected by sodium
hypochlorite solution containing 1% active chlorine for
5 min. and rinsed twice with sterilized distilled water.
They were sown in a petri dish containing moistened
filter paper and incubated at 20  C in the dark. Germi-
nated seeds were potted in sterilized soil and cultivat-
ed in the greenhouse under standard conditions. This
study continued for 90 days.
Ovule culture
Ovaries were collected 20, 25, 30, and 35 days after
pollination. They were surface-disinfected by dipping
in 70% ethanol for a few seconds, then burning out
the ethanol sticking to the surface and rinsing three
times with sterilized distilled water. Six to eigtheen
ovules were excised from the ovaries and placed on
MS (Murashige & Skoog, 1962), a half-strength MS
(1/2 MS), or a quarter-strength MS (1/4 MS) medi-
um. Each medium contained 3% sucrose and 0.8%
agar at pH 5.7. Plant growth regulator was not added.
The cultures were maintained at 20  C under a 16 h
photoperiod of about 2000 lux supplied by fluores-
cent lamps. Germinated ovules were subcultured onto
a fresh medium. Healthy seedlings thus obtained were
potted in sterilized soil and kept in a moist environ-
ment. Thereafter, they were cultivated in the green-
house, and their morphological characteristics such as
plant form, plant height, leaf shape, and flower color
were compared with those of parental plants.
Isozyme analyses, pollen viability, and fertility of the
hybrid plants
To confirm the hybridity of these seedlings, isozyme
analyses were carried out. About 1 g of leaves in
fresh weight collected from greenhouse plants were
homogenized in an ico-cooled mortar with 1.5 ml of an
extraction buffer containing 756 g/l glycerol, 157 g/l
Tween-80, 31.4 g/l DTT, and 169 g/l Preset pH 7.5
Crystal (Sigma); 1.5 g PVPP was then added to the
homogenate as a phenol adsorbent. The homogenate
was centrifuged twice: 15000  g for 15 min., and
then for 45 min. The supernatant was subjected to elec-
trophoresis on 7.5% polyacrylamide gels which were
then stained for esterase, phosphoglucoisomerase and
phosphoglucomutase enzymes (Wetter & Dyck, 1983;
Jonathan & Weeden, 1989).
To determine pollen viability, mature pollen grains
were stained in 2% acetocarmine solution in accor-
dance with Alexander (1969). Frequencies of stained
pollen grains were observed under a light microscope.
Fertility of hybrid plants was further verified by
means of self-and backcross-pollination.
Results
As shown in Table 1, no seedlings were obtained from
any of the four combinations by conventional crosses,
although normal fruit set was observed in all com-
binations, and a few seeds from D. nudicaule  D.
grandiflorum had germinated. Microscopic observa-
tions revealed that pollen germination on the stigma
occurred normally, and that numerous pollen tubes
reached the upper part of the ovary in all crosses (Fig-
ure 1A).
Interspecific hybrids were obtained by preventing
hybrid embryos from aborting through ovule culture
of the samples collected at various intervals after pol-
lination (Table 2). From 20 days after the beginning
of culture, some explanted ovules germinated direct-
ly without passing through callus formation. Some of
 333

Figure 1. (A) Pollen tubes growing in the style of D. grandiflorum pollinated with D. nudicaule, bar = 1 mm; (B) Phosphoglucoisomerase

isozyme patterns of D. cardinale D. grandiflorum hybrid and its parents, CAD: D. cardinale, F1 : hybrid, GRA: D. grandiflorum; (C) The
morphological picture of D. cardinale, their hybrid and D. grandiflorum (from left), bar = 50 mm; (D) The leaf shape of D. cardinale, their
hybrid and D. grandiflorum (from left), bar = 10 mm; (E) The morphological picture of D. grandiflorum, two types of their hybrids and D.
nudicaule (from left), bar =50 mm; (F) The leaf shpae of D. grandiflorum, their hybrids and D. nudicaule (from left), bar = 10 mm.

Table 1. Results of interspecific crosses

Cross Number of Number of set Germination Number of

pollinated ovaries ovaries (%) (%)a seedlings obtained


D. cardinale D. grandiflorum 110 40 (36.4) 0.0 0

D. grandiflorum D. cardinale 108 11 (10.2) 0.0 0

D. nudicaule D. grandiflorum 120 32 (26.7) 7.5 0

D. grandiflorum D. nudicaule 170 56 (32.9) 0.0 0
a Incubated at 20 C in the dark for 90 days.
334
Table 2. Responses of cultured ovules of interspecific crosses, the values of reciprocal combinations are pooled together

Cross Days after MS medium Number of Germination Number of

pollination concentration cultured ovules (%) seedlings obtained

D. cardinale  D. grandiflorum 20 1 37 0 0
1/2 39 7.7 0
1/4 37 5.4 0
25 1 45 8.9 0
1/2 48 6.3 0
1/4 47 4.3 0
30 1 36 0 0
1/2 39 5.1 0
1/4 36 0 0
35 1 24 0 0
1/2 24 0 0
1/4 24 20.8 1
D. grandiflorum  D. nudicaule 20 1 46 15.2 0
1/2 46 19.6 5
1/4 44 20.5 5
25 1 54 25.9 3
1/2 54 13.0 3
1/4 51 15.7 6
30 1 6 0 0
1/2 18 5.6 0
1/4 18 0 0

Table 3. Results of self- and backcross-pollination

Cross Number of Number of Number of

pollinated ovaries set ovaries (%) viable seeds obtained

D. grandiflorum self 144 106 (73.6) 1614

D. cardinale self 17 6 (54.5) 80
D. nudicaule self 32 29 (90.6) 262

D. cardinale D. grandiflorum self 29 0 (0.0) 0

D. grandiflorum D. nudicaule self 186 0 (0.0) 0

(D. cardinale D. grandiflorum) 12 0 (0.0) 0

D. cardinale

(D. cardinale D. grandiflorum) 12 0 (0.0) 0

D. grandiflorum

(D. grandiflorum D. nudicaule) 75 0 (0.0) 0

D. grandiflorum

(D. grandiflorum D. nudicaule) 45 0 (0.0) 0

D. nudicaule

them expanded their cotyledons and developed into
seedlings, but others failed to grow due to morpho-
logical abnormalities or lack of shoot development to
match root growth. Especially in D. cardinale  D.
grandiflorum, many germinated ovules did not form
shoots but only roots. In D. nudicaule  D. grandiflo-
rum and D. grandiflorum  D. nudicaule, a correlation
was observed between MS strength and the character
of ovule growth, i.e., higher MS concentrations (MS,
1/2 MS) induced abnormal growth. No apparent cor-
relation was observed between medium strength and
germination rate in any of the combinations.
Seedlings were successfully produced from D.
cardinale  D. grandiflorum and D. grandiflorum

 D. nudicaule through ovule culture. In the latter
cross, in particular, the germination rate was markedly
increased by ovule culture and a total of 22 seedlings
was obtained. On the other hand, no seedlings were
obtained from D. grandiflorum  D. cardinale or D.
nudicaule  D. grandiflorum, although the germina-
tion rate was increased by using ovule culture. As to the
relation between the maturity of ovules at explanting in
terms of days after pollination and the germination rate
or number of seedlings obtained in D. grandiflorum 
D. nudicaule, these rates and numbers were markedly
greater at 20 or 25 days after pollination than at 30 days.
In the other crosses, no such relation was observed.
The seedlings from D. cardinale  D. grandiflorum
and D. grandiflorum  D. nudicaule grew to maturity
in the greenhouse. To confirm their hybridity, isozyme
analyses were carried out for esterase, phosphoglu-
coisomerase and phosphoglucomutase. In D. cardi-
nale  D. grandiflorum, zymograms for phosphoglu-
coisomerase were distinctly different from the parental
species, and seedlings from this cross had not only the
common bands of both parents but also the hybrid band
(Figure 1B). In esterase, isozyme banding patterns of
parents and hybrid were found to be distinct. These
hybrids displayed the same bands from both parents.
Similarly, in D. grandiflorum  D. nudicaule hybrids,
hybridity was confirmed by their banding patterns of
phosphoglucoisomerase and phosphoglucomutase.
The hybrid from D. cardinale  D. grandiflo-
rum resembled D. cardinale in plant height, but had
branched spikes like those of D. grandiflorum (Fig-
ure 1C). The flower color of deep reddish purple was
intermediate between that of D. grandiflorum (bluish
violet) and D. cardinale (scarlet). The leaf shape of
this hybrid was also intermediate between the parents
(Figure 1D).
On the other hand, hybrids from D. grandiflorum
 D. nudicaule were slightly taller than either parent,
and had branched spikes like those of D. grandiflo-
rum (Figure 1E). As for flower characteristics, there
were two strains. One strain had rather closed flow-
ers similar to D. nudicaule, and the other had rather
open flowers similar to D. grandiflorum. Both of them
bore vivid reddish or bluish purple flowers intermedi-
ate between those of D. grandiflorum and D. nudicaule
(orange-red). The shape of their leaves was intermedi-
ate between the parents (Figure 1F).
The percentage of viable pollen as determined by
aceto-carmine staining was 76.0, 94.8, and 91.4% in
the parental species, D. grandiflorum, D. cardinale,
and D. nudicaule, respectively, whereas that of the
335
D. cardinale  D. grandiflorum hybrid was 4.1% and
that of the D. grandiflorum  D. nudicaule hybrid was
13.5%. Although the parents produced many viable
seeds by self-pollination, hybrids from both D. car-
dinale  D. grandiflorum and D. grandiflorum  D.
nudicaule failed to yield viable seeds either by self-
pollination or backcross-pollination with fertile pollen
from each parent (Table 3).
Discussion
Stebbins (1950) classified the cross-incompatibility
barriers between distantly related species into pre-
and post-fertilization types. In all crosses made in the
present study, pollen germination normally occurred
on the stigma, and numerous pollen tubes reached
the upper part of the ovary. In addition, fructifica-
tion was observed in all crosses. These observations
revealed that although fertilization itself might occur
normally, a post-fertilization barrier arose. Hybrid
embryos from these crosses probably aborted due to
the lack of endosperm development as suggested in
several hybridization studies between distantly related
species (Cooper & Brink, 1940). Thus, while seedlings
were successfully obtained by preventing abortion of
embryos through ovule culture, none were obtained in
cross combinations without such intervention.
In ovule culture, the time of explanting influences
the success rate (Collins & Grosser, 1984; Hu & Wang,
1986). It is known that ovules beyond the globular
embryo stage can be cultured. In the present study,
we noticed a relation between the germination rate
or the number of seedlings obtained and the time of
ovule excision. In D. grandiflorum  D. nudicaule,
a relation was observed between these rates or num-
bers and the time of excision. It thus appears that 20
or 25 days after pollination was a more appropriate
time for culture than 30 days. This may be explained
by the fact that ovules collapsed more rapidly beyond
20–25 days with the result that few could have germi-
nated. In the other crosses, the germination rate and
number of seedlings obtained were small as compared
with D. grandiflorum  D. nudicaule. Since it is con-
sidered that, in these combinations of crossing, the
collapse of ovules begins earlier, ovule culture may
be more successful by explanting 20 days after pol-
lination. Marubashi & Nakajima (1985) observed this
process of collapse in Nicotiana through a microscopic
examination of paraffin sections and applied the results
to decide the most appropriate time to explant ovules.
336
In the present study, embryonic development was not
observed. A more detailed examination will be neces-
sary to raise the efficiency of ovule culture.
The effects of medium strength on germination
rates and numbers of seedlings obtained were also
studied. While no consistent correlation was observed
between medium strength and germination rate in all
crosses, medium strength contributed to the growth
of germinated ovules in some cross-combinations. In
addition, the responses of cultured ovules differed to
some extent according to the cross-combinations. The
above reasons suggest the necessity to further exam-
ine the appropriate medium strength and/or composi-
tion for the culturing of hybrid ovules for each cross-
combination.
Pontovich & Svenshnikova (1966) stated that when
ovules were cultured along with the placenta, the pla-
cental tissue exerted a beneficial effect. This was actu-
ally verified by Wakizuka & Nakajima (1975), and
similar instances were reported in Petunia and Cycla-
men (Niimi, 1973; Ishizaka & Uematsu, 1990, 1992,
1995). Likewise, many researchers succeeded in pro-
ducing interspecific or intergeneric hybrids of lilies
through ovary-slice culture (Hayashi et al., 1986; Van
Tuyl et al., 1991), and of Brassica, etc. through ovary
culture (Thengane et al., 1986; Gundimeda et al., 1992;
Nomura et al., 1994). There is a need to examine the
effectiveness of such methods also in Delphinium.
The fact that dwarfness of D. grandiflorum was not
introduced into its interspecific hybrids suggested that
tallness is dominant over dwarfness in this case. On the
other hand, the branching habit of the D. grandiflorum
spike was introduced, especially into hybrids with D.
cardinale, indicating that branching may be dominant
over non-branching. A similar result has already been
reported in the genus Matthiola; tallness and branch-
ing were dominant over dwarfness and non-branching
(Saunders, 1928). In contrast, flower colors of interspe-
cific hybrids were intermediate between the parents.
Further examination is needed on the inheritance of
flower color and plant shape to succeed in introducing
red color into the dwarf cultivars.
Since hybrid plants from both D. cardinale 
D. grandiflorum and D. grandiflorum  D. nudi-
caule were sterile, it is necessary to overcome the
sterility if dwarf strains are to be raised and further
improved. Their fertility could probably be restored
by chromosome duplication using colchicine treat-
ment as was already described for interspecific hybrids
between Iris laevigata and I. ensata (Yabuya, 1985)
and those between Cyclamen persicum and C. hederi-
forium (Ishizaka & Uematsu, 1994).
Despite our attempts in the present study to intro-
duce the red flower color of D. cardinale or D. nudi-
caule into the dwarf species D. grandiflorum by means
of interspecific hybridization, desirable hybrids were
not obtained. However, we conclude that the ovule
culture technique may be used to rescue hybrid seeds
from the danger of aborting which arises in the inter-
specific crosses of Delphinium. In addition, the ovule
culture technique will extend the future possibilities of
Delphinium breeding.
Acknowledgements
This work was carried out at the Laboratory of Flori-
culture and Landscape architecture, Faculty of Agri-
culture, Hokkaido Univ., Japan. We would like to thank
the members of the laboratory for their encouragement.
Thanks are also due to Dr. Y. Shimamoto and Dr. J.
Abe for their technical assistance on isozyme analyses.
The help of Dr. H. Nakajima, Dr. T. Kanazawa and Mr.
S. Akino in the experiment on pollen tube growth in
the style is also gratefully acknowledged.
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