Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol84.Issue1-2.Apr2000:
Scientia Horticulturae 84 (2000) 191±204
Interspeci®c hybrids between
Lilium nobilissimum and L. regale produced
via ovules-with-placental-tissue culture
Yumi Obata, Yoshiji Niimi*, Masaru Nakano,
Keiichi Okazaki, Ichiro Miyajima
Faculty of Agriculture, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan
Accepted 31 August 1999
Abstract
Reciprocal pollination was made between Lilium nobilissimum and L. regale. Pollen tubes
reached the base of style within 144 h after pollination, but no mature seeds were obtained in either
cross combination. Explants, or ovules-with-placental-tissue excised from each carpel 30 and 40
days after pollination (DAP), were cultured on a medium composed of major salts of B-5
macronutrient (Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Exp. Cell Res. 50, 151±158),
micronutrient, Fe-EDTA and vitamins of MS (Murashige, T., Skoog, F., 1962. Physiol. Plant. 15,
473±497), 5% sucrose and 0.2% gellan gum. In L. regale Lilium nobilissimum, 3% of ovules
excised at 30 DAP and 9% of those excised at 40 DAP developed into seedlings. In Lilium
nobilissimum L. regale, only 3.6% of ovules excised 40 DAP developed into seedlings, and none
of the ovules excised 30 DAP produced any seedlings. Bulbs of L. regale and hybrids transplanted
to soil showed some resistance to bulb-rot, leaf-top scorch, browning spots and/or streaking on
leaves, but those of Lilium nobilissimum were sensitive to these diseases. Flowering individuals
were nearly intermediate between parents in their morphological characteristics. All ¯owering
individuals (2n 24 chromosomes) were identi®ed as hybrids based on karyotype, isozyme and
random ampli®ed polymorphic DNA analyses. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Lilium spp.; Interspeci®c hybrids; Isozyme; Karyotype; Ovules-with-placental-tissue
culture; RAPD
*
Corresponding author. Tel.: 81-25-262-6614; fax: 81-25-262-6614.
E-mail address: himesa@agr.niigata-u.ac.jp (Y. Niimi).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 1 5 - 6
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
1. Introduction
The issue of solving problems with diseases in horticultural plants is an
important one, because the future use of pesticides and fungicides in agriculture
will be restricted (Jaap et al., 1992). To breed hearty lilies, interspeci®c
hybridization has been used to incorporate disease resistance, using the methods
of embryo rescue (North and Wilis, 1969; Asano, 1978, 1982; Okazaki et al.,
1992, 1994), culture of ovules-with-placental-tissue (Niimi et al., 1995), and
ovary slice culture (Hayashi et al., 1986; Kanoh et al., 1988; Niimi et al., 1996;
Van Tuyl and Van Holsteijn, 1996; FernaÂndez et al., 1996; Arzate-FernaÂndez
et al., 1998). Lilium nobilissimum Makino, a species native to Japan, where they
no longer grow naturally in the ®eld, has potential as an ornamental plant because
of its erect, funnel-shaped white ¯owers and pleasant fragrance although it is very
sensitive to fungal diseases, while L. regale Wilson, a species native to China, has
economically and horticulturally desirable traits as a commercial plant. The
introduction of the variable traits of L. regale into other Lilium spp. has been
attempted (Van Creij et al., 1992; Niimi et al., 1996).
The purpose of this study was to develop hybrids between Lilium nobilissimum
and L. regale by applying an ovules-with-placental-tissue culture procedure. The
hybridity of ¯owering plants obtained was evaluated through karyotype, isozyme,
and random ampli®ed polymorphic DNA (RAPD) analyses, as well as by
morphological traits.
2. Materials and methods
2.1. Plant materials, pollination and observation of pollen tube growth
Lilium nobilissimum and L. regale were grown in pots under natural and/or
forced cultural conditions to adjust ¯owering time. Following castration 1 day
before anthesis, pistils were capped with aluminum foil to avoid contamination
and pollinated at anthesis with fresh pollen or stored pollen, as described
previously (Niimi and Shiokawa, 1992).
The growth of pollen tubes in the styles was observed as follows: styles were
collected for 144 h after pollination at intervals of 24 h, ®xed in FAA solution
(formalin: acetic acid; 70% ethanol 5:5:90), softened in 1 N NaOH solution at
608C for about 60 min, and then dissected according to a method described
previously (Niimi, 1991). After staining with a 0.1% cotton-blue solution, the 10
longest pollen tubes in each style were observed under a light microscope and the
ratio of tube length to entire style length was calculated based on the average
length of pollen tubes observed in each style (Niimi et al., 1997). At least ®ve
styles were used for each treatment.
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
193
2.2. Ovule-with-placental-tissue culture and establishment of hybrid seedlings
Swollen ovaries were collected on 30 and 40 days after cross-pollination
between Lilium nobilissimum and L. regale (Table 2). They were surfacedisinfected with 70% ethanol for 1 min and then with a commercial bleach
solution containing 2% active chlorine for 20 min, and rinsed three times with
sterilized distilled water. The basal part of the ovary was discarded, and only
middle or swollen parts of the ovary were used. Each carpel was longitudinally
separated from the ovary along the line of carpel union. Ovules with placental
tissue, considered as an explant, were excised from each carpel (Niimi et al.,
1995). Each explant (about 15 mm long) contained 24 ovules on average.
Each explant was placed in a test tube (18 180 mm) containing 15 ml of
basal medium. The basal medium was composed of major salts of B-5
macronutrient (Gamborg et al., 1968), micronutrient, Fe-EDTA and vitamins of
MS (Murashige and Skoog, 1962) medium, 5% sucrose and 0.2% gellan gum,
and adjusted to pH 5.7 before autoclaving at 1218C for 10 min at 1.2 kg cmÿ2.
Test tubes were covered with an aluminum foil cap.
The explants were maintained in the dark at 24 18C. After 8 weeks, swollen
and/or germinated ovules were isolated from placental tissue and cultured in a
50 ml Erlenmeyer ¯ask containing 20 ml of the fresh basal medium at 258C under
continuous illumination (1000±1200 lux) supplied by white ¯uorescent lamps.
Eight weeks later, the number of seedlings developing bulblets was recorded.
2.3. Growth of bulblets transplanted to soil and morphological characteristics
of hybrids
The bulbs obtained were cultured in the basal medium for several months.
Scales excised from each bulb were cultured on MS (1962) medium
supplemented with 1.0 mg lÿ1 NAA, 0.01 mg lÿ1 BA, 5% sucrose and 0.65%
agar, in the dark for 8±12 weeks at 24 18C. Bulblets which had developed on
the scales were isolated, washed with tap water, and cold-treated for 12 weeks at
48C after they were mixed with moist vermiculite (Niimi, 1995). They were then
transplanted into a plastic tray (50 35 7.5 cm) with mixed soil (red clay
lumps:peat moss:leaf mold 1:1:1, v/v/v) and cultivated in a greenhouse, where
the night (10 208C) and day (15 308C) temperatures ¯uctuated depending on
the weather, for 162 days from 1 April 1996.
The experiment was made with a split-plot-design in 10 random blocks, each
block consisting of 20 cells, with ®ve cells being used for the bulbs of each
combination. Each bulb was scored for sprouting date and the number of bulbs
with healthy and/or wilted leaves at intervals of 3 days. All bulbs were allowed to
grow in soil within 162 days after transplanting or until leaves were withered. In
the latter case, bulbs were harvested when leaf-tip scorch, blighting spot and/or
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
streaking had spread over more than half of one of the fully-expanded leaves.
After harvest, bulbs were individually weighed after removal of leaves and roots
and the percentage gain in fresh weight was calculated.
Morphological characteristics were investigated using parental and hybrid
plants. The fertility of mature pollen was tested at anthesis by staining with 2%
aceto-carmine and observing under a light microscope.
2.4. Karyotype analysis
Root tips were taken from ¯owering plants growing in pots, pretreated with
0.1% colchicine at 258C for 6 h, ®xed in a solution of ethanol±acetic acid (3:1,
v/v) at 58C for at least 20 h, then hydrolyzed in a 2:1 mixture of 45% acetic
acid:1 N HCl (2:1, v/v) at 608C for 20±40 s. Root tips rinsed with distilled water
were stained with aceto-orcein (1%) using a squash technique. Measurement of
the length of each chromosome was made on photographs magni®ed 2300 using
a digitizer-personal computer system (Uchiyama et al., 1988). Nomenclature for
centromere positions of chromosomes was made according to the methods
described by Levan et al. (1964).
2.5. Isozyme analysis
All ¯owering plants were subjected to isozyme analysis to con®rm their hybridity.
Esterase (EST), acid phosphatase (ACP), glucose 6-phosphatase dehydrogenase
(G6-PDH), malate dehydrogenase (MDH), and peroxidase (POX) isozymes were
analyzed according to the method of Wetter and Dyck (1983) with slight
modi®cations. Leaves from potted plants with a fresh weight of about 100 mg were
homogenized in a mortar with 1 ml of an extraction buffer of 10% glycerol, 0.14% 2mercaptoethanol, and 50 mM Tris±HCl buffer at pH 7.5. Crude extracts were
electrophoresed on 12.5% polyacrylamide slab gels with 50 mM Tris±glysine buffer,
pH 8.3, at a constant voltage of 150 V for 4 h, and stained for isozymes.
2.6. Random ampli®ed polymorphic DNA (RAPD) analysis
To verify the hybridity of ¯owering plants, RAPD analysis was also carried out
according to the method of Yamagishi (1995) with several modi®cations. To
extract DNA, about 100±200 mg of fresh leaves from parental and hybrid plants
in pots were homogenized by using the cetyltrimethylammonium bromide
(CTAB) method (Rogers and Bendich, 1985). Polymerase chain reaction (PCR)
was conducted on a 25 ml reaction mixture containing 30 ng template DNA,
1.5 mM MgCl2, 250 nM of primer, 0.25 units of Ampli Taq DNA polymerase
(Perkin-Elmer, USA), 200 mM each of dATP, dCTP, dGTP and dTTP (PerkinElmer, USA), and 1 reaction buffer (Perkin-Elmer, USA). One random primer
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
(OPB06 50 -TGC TCT GCC C-30 ) (Operon Technologies, USA) and two semirandom primers (Y35 50 -TGG TAT CAG AGC C-30 ; Y37 50 -GTC AAG CGC
G-30 ) (Yamagishi, 1995) were used. After an initial denaturation at 948C for
5 min, the reaction mixtures were subjected to ampli®cation in a program
temperature control system (2400-R, Perkin-Elmer, USA) for 40 cycles, each of
which consisted of 1 min at 948C, 1 min at 508C, and 2 min at 728C, followed by
5 min at 728C. Ampli®cation products were electrophoresed on 3% polyacrylamide gels with TBE buffer (2 mM EDTA in 89 mM Tris±borate buffer, pH 8.0) at
a constant voltage of 100 V for 1 h. Gels were then stained with 2.5 mg mlÿ1
ethidium bromide and photographed on an UV transilluminator.
3. Results
3.1. Growth of pollen tubes and ovule culture
The relative length of pollen tubes after pollination was 100% in both selfpollination, 67% in Lilium nobilissimum L. regale and 93% in L. regale Lilium
nobilissimum, after 96 h of pollination. In both cross-pollination, pollen tubes grew
slowly and almost reached the base of style after 144 h of pollination (Table 1).
Cross-pollinated ovaries gradually grew until 30±40 days, and enlarged only a little.
Enlarged ovaries turned brown and shrank by 60 days after pollination, the capsules
containing only sterile seeds having a trace of degraded embryo but no mature seeds
at all (data not presented). Thus, no mature seeds were obtained from either crosscombination, suggesting that a post-fertilization barrier existed in both combinations. To overcome the barrier, ovules-with-placental-tissue excised at 30 and 40
days after pollination (DAP) were cultured on a basal medium.
The effect of ovules-with-placental-tissue culture at 30 and 40 DAP on the
production of the hybrids between Lilium nobilissimum and L. regale is summarized
Table 1
Relative length of pollen tubes to style length in self- and cross-pollination of Lilium nobilissimum
and L. regalea
Combination
Relative pollen tube length (%), hours after pollination
48
96
120
144
Self-pollination
Lilium nobilissimum
L. regale
±b
56 4
100
100
100
±
100
±
Cross-pollination
Lilium nobilissimum L. regale
L. regale Lilium nobilissimum
±
80 5
67 15
93 8
82 10
±
94 6
±
a
b
Values represent the mean SE of at least ®ve replications.
Not determined.
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Table 2
Effect of combinations and number of DAP on the production of interspeci®c hybrids between
Lilium nobilissimum and L. regale by ovules-with-placental-tissue culture
Combinations
DAP
Total number of
ovules cultured
Number of
seedlings obtained
Lilium nobilissimum L. regale
30
40
30
40
145
168
305
277
0
6
9
25
L. regale Lilium nobilissimum
(0)
(3.6)
(3)
(9)
in Table 2. Ovules-with-placental-tissue showed browning within 5 weeks, and few
ovules germinated in situ (Fig. 1A). Enlarged and/or germinating ovules were
isolated from placental tissue after 8 weeks of culture and then subcultured on the
fresh basal medium (Fig. 1B). In L. regale Lilium nobilissimum, 3% of ovules
excised at 30 DAP and 9% excised at 40 DAP developed into seedlings. In Lilium
nobilissimum L. regale, only 3.6% of ovules excised 40 DAP developed into
seedlings, and none of the ovules excised 30 DAP produced any seedlings.
3.2. Growth of bulbs in the greenhouse
Fig. 2 shows the time course changes in percentage of bulblets with good
healthy leaves: sprouting began at around 24 days after transplant (DAT), and 90±
100% of bulblets sprouted until 60 DAT. Afterward, leaf-top scorch, which might
have been caused by physiological disorders, and browning spots and/or streaking
on leaves, which might have been caused by Botrytis, gradually appeared on scaly
Fig. 1. Ovules-with-placental-tissue culture and seedlings with developing bulblets of L.
regale Lilium nobilissimum: (A) Six-week-old cultures; ovules-with-placental-tissue were
excised at 40 DAP and cultured on a modi®ed B-5 medium in darkness. (B) Seedlings with
developing bulblets; developing ovules excised from placental tissue were cultured on fresh
medium under a light condition. Bars 1 cm.
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
197
Fig. 2. Time course changes in percentage of bulblets with good healthy leaves. Bulblets, (&)
Lilium nobilissimum Lilium nobilissimum, (*) L. regale L. regale, (&) Lilium
nobilissimum L. regale, and (*) L. regale Lilium nobilissimum, were cultivated in a glass
house for 162 days.
leaves. The percentage of bulblets with healthy leaves decreased rapidly after 120
DAT, in the beginning of August, particularly among Lilium nobilissimum
bulblets.
Percentage gains in the fresh weight of bulbs are summarized in Table 3. The
highest value was observed in bulbs of L. regal, the lowest in Lilium nobilissimum,
with the values of hybrid bulbs nearly intermediate between the two.
Table 3
Growth of bulbs transplanted to soil
Combinations
Lilium nobilissimum
Lilium nobilissimum
L. regale L. regale
Lilium nobilissimum L. regale
L. regale Lilium nobilissimum
a
At transplantation
At harvesta
Mean fresh
weight of
bulbs
Gain in
fresh
weight (%)
Number
of
bulbs
Mean fresh
weight of
bulbs
50
400 4
644 45
161 11
40
50
50
118 2
407 2
409 3
610 45
1540 118
1218 100
522 42
379 29
298 24
All bulbs were allowed to grow in soil for 162 days or until leaves were withered. In the latter
case, bulbs were harvested at the time when leaves were withered. After harvest, bulbs were
individually weighed after removal of leaves and roots and the percentage gain in fresh weight was
calculated.
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Table 4
Characteristics of Lilium nobilissimum, L. regale, and their hybrids at ¯owering time
Indices
Lilium
nobilissimum
L. regale
Lilium
nobilissimum
L. regale
L. regale
Lilium
nobilissimum
Sprouting time
Flowering time
Late March
Late July
Late April
Late June
Early April
Middle July
Early April
Middle July
Leaf
Shape
Ovate-oblong
Linear and
narrow lanceolate
Without petiole
Wide lanceolate
Wide lanceolate
Without petiole
Without petiole
Petiole
With petiole
Flower
Inflorescence
Attachment
Petals
Raceme
Vertical
Barely recurving
Umbel
Horizontal
Noticeably
recurving
Raceme or umbel
Horizontal
Recurving
Umbel
Horizontal
Recurving
Pollen
Color
Yellow
Yellow
Yellow
94.8 2.1
94 2.1
Reddish-brown
and yellow
0.6 0.3
Fertility
1.4 0.6
3.3. Characteristics of hybrids
The morphological and other characteristics of both parental and hybrid plants
are summarized in Table 4 and Fig. 3. The hybrids were nearly intermediate
between the two parent plants. The times of sprouting and ¯owering were near
those of Lilium nobilissimum, and hybrids ¯owered in mid-July. The leaves were
wide lanceolate (0.9±1.3 cm wide) without petioles. The in¯orescence of the
hybrid plants was umbel, except one Lilium nobilissimum L. regale plant which
had the same raceme in¯orescence as Lilium nobilissimum. The hybrid ¯owers
were white and tinged rose-purple, and squarely attached to the stem,
characteristics which were more similar to L. regale than to Lilium nobilissimum.
The pollen grains of the hybrids were reddish-brown and yellow, and their
fertility was quite low (Table 4).
3.4. Analyses of hybrid plants by karyotype, isozyme and RAPD
3.4.1. Karyotype analysis
Hybrids obtained were found to be 2n 2x 24. Karyotypes of Lilium
nobilissimum (Fig. 4A) and L. regale (Fig. 4B) were clearly distinguishable by
the existence and position of secondary constriction in the ®rst and second
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
199
Fig. 3. Plants of parental species and their hybrids: (A) Lilium nobilissimum, (B) L. regale, (C)
Lilium nobilissimum L. regale, and (D) L. regale Lilium nobilissimum.
longest chromosomes. The secondary constriction was observed only in the
®rst chromosome in Lilium nobilissimum (Fig. 4A) and in the ®rst and second
chromosomes in L. regale (Fig. 4B). All of the hybrids examined had
chromosomes characteristic of both parents (Fig. 4C and D).
3.4.2. Isozyme analysis
Among ®ve isozymes (EST, ACP, G6-PDH, MDH, and POX) tested, only EST
generated clear zymograms. The EST zymograms of Lilium nobilissimum and
L. regale were distinctly different, and the hybrid plants had common bands for
both parents (Fig. 5).
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Fig. 4. Individual chromosomes of parental species and their hybrids: (A) Lilium nobilissimum, (B) L. regale, (C) Lilium nobilissimum L. regale, (D)
L. regale Lilium nobilissimum. Chromosomes of n1 and n2 are speci®c to Lilium nobilissimum and those of r1 and r2 to L. regale. Each arrow
indicates a secondary constriction.
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
201
Fig. 5. Isozyme patterns for esterase (EST) in parental species and their hybrids. Arrows indicate
species-speci®c bands: (Lane 1) Lilium nobilissimum, (Lane 2) L. regale, (Lane 3) Lilium
nobilissimum L. regale, (Lane 4) L. regale Lilium nobilissimum.
3.4.3. RAPD analysis
RAPD analysis provided additional evidence for hybridization (Fig. 6). Among
three primers (OPB06, Y35, and Y37) tested, only Y35 generated polymorphic
patterns of PCR products between the parents. The clear and speci®c ampli®ed
DNA fragments of Lilium nobilissimum were 620, 950 and 1550 bp, whereas
those of L. regale were 470, 550, and 750 bp. In all hybrids analyzed, three
fragments from Lilium nobilissimum and two fragments from L. regale were
ampli®ed, although RAPD pro®les showed a slight difference between the hybrid
individuals.
4. Discussion
Recently, the importance of the lily as a horticultural crop has increased and
interspeci®c hybridization of Lilium has been attempted by many researchers
(Van Tuyl and Van Holsteijn, 1996). However, interspeci®c hybridization is often
limited by pre- and/or post-fertilization barriers, the latter of which has partly
been overcome by applying ovary culture, ovule culture, and/or embryo rescue
(Van Tuyl et al., 1991; Okazaki et al., 1992, 1994; Niimi et al., 1996; FernaÂndez
et al., 1996; Arzate-FernaÂndez et al., 1998). Results obtained in the present
research showed that the interspeci®c hybridization between Lilium nobilissimum
and L. regale was limited by a post-fertilization barrier, which could be overcome
202
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Fig. 6. PCR analysis showing ampli®cation products generated from DNA-templates of the
parental species and their hybrids with primer Y35. Arrows, from top to bottom, indicate 1550, 950,
750, 620, 550, and 470 bp fragments, respectively. (Lane 1) Lilium nobilissimum, (Lane 2) L.
regale, (Lane 3) Lilium nobilissimum L. regale, (Lane 4) L. regale Lilium nobilissimum. M
indicates the DNA size maker (1000 bp ladder and Hind III-digested l DNA).
by using an ovules-with-placental-tissue culture procedure. Furthermore, the
technique of ovules-with-placental-tissue culture appears to have the following
advantages: many ovules can be handled at once, and, in comparison with embryo
culture, a less complicated manipulation is required in particular with regard to
the composition of the medium, because the placental tissue may supply nutrients
or hormones to stimulate embryo growth and germination (FernaÂndez et al.,
1996).
The hybrids transplanted to soil showed some resistance to leaf-top scorch,
possibly caused by physiological disorders, basal rot due to Fusarium, and
browning spots and/or streaking due to Botrytis. These traits seem to be
introduced from L. regale, because bulbs of L. regale are highly resistant to these
af¯ictions but those of Lilium nobilissimum are easily affected. This suggests that
L. regale can be used as a parental plant to obtain interspeci®c hybrids resistant to
these diseases.
Previous reports showed that karyotype (Asano, 1978; Kanoh et al., 1988),
isozyme (Van Tuyl et al., 1986; Okazaki et al., 1992, 1994), RAPD (Yamagishi,
1995; Arzate-FernaÂndez et al., 1998), and nuclear rDNA (Niimi et al., 1996)
analyses may be used to ascertain the hybridity of lily plants. The present
karyotype analysis showed that the secondary constriction on the ®rst longest
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
203
chromosome of Lilium nobilissimum and on the ®rst and second longest
chromosomes of L. regale, which are characteristic of the respective species,
could be used as evidence for hybridization, as reported previously by Stewart
(1947) and Noda (1987), who mentioned that the ®rst and second longest
chromosomes are important in the karyotype analysis of Lilium. Isozyme and
PCR analyses also provided additional evidence for hybridization. The present
results suggest that these techniques could be a useful means by which to discover
true hybrids at the juvenile stage in lily breeding programs.
We conclude that the ovules-with-placental-tissue culture technique is one
effective method of producing interspeci®c hybrids between sexually incompatible lily species that are dif®cult to hybridize by conventional methods.
Acknowledgements
The authors are grateful to D.H. Goldstein, Professor of Keiwa Gakuen
University and Dr. G.J. De Klerk, Center for Plant Tissue Culture Research in the
Netherlands, for reading and correcting the English text, and to Dr. M. Nakata,
Botanical Garden of Toyama Prefecture, for his valuable advice on karyotype
analysis. This work was partly supported by a Grant-in-Aid for Scienti®c
Research (No. 09460017) from the Ministry of Education, Science and Culture.
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Lilium `Enchantment': In¯uence of pistil age on seed set. J. Japan. Soc. Hort. Sci. 65, 835±
842.
Noda, S., 1987. Lily chromosomes. In: Simizu, M. (Ed.), The Lilies of Japan, Species and Hybrids.
Seibundo Shinkou-sha, Tokyo, pp. 98±110 (in Japanese).
North, C., Wilis, A.B., 1969. Interspeci®c hybrids of Lilium lankogense Franchet produced by
embryo culture. Euphytica 18, 430±434.
Okazaki, K., Umeda, Y., Urashima, O., Kawada, J., Kunishige, M., Murakami, K., 1992.
Interspeci®c hybrids of Lilium longi¯orum and L. formolongi with L. rubellum and L.
japonicum through embryo culture (in Japanese with English summary). J. Japan. Soc. Hort. Sci.
60, 997±1002.
Okazaki, K., Asano, Y., Oosawa, K., 1994. Interspeci®c hybrids between Lilium `Oriental' hybrid
and L. `Asiatic' hybrid produced by embryo culture with revised media. Breed Sci. 44, 59±64.
Rogers, S.O., Bendich, A.J., 1985. Extraction of DNA from milligram amounts of fresh, herbarium,
and mummi®ed plant tissues. Plant Mol. Biol. 5, 77±84.
Stewart, R.N., 1947. The morphology of somatic chromosomes in Lilium. Amer. J. Bot. 34, 9±26.
Uchiyama, H., Nakata, M., Morihiro, S., Tanaka, R., 1988. Use of personal computer and digitizer
for karyotype analysis (in Japanese). La Kromosomo II 50, 1652±1658.
Van Creij, M.G.M., Van Raamsdonk, L.W.D., VanTuyl, J.M., 1992. Wide interspeci®c hybridization
of Lilium: Preliminary results of the application of pollination and embryo-rescue methods. The
Lily Yearbook of the NALS, vol. 45, pp. 28±37.
Van Tuyl, J.M., Franken, J., Jongerius, R.C., Lock, A.M., Kwakkenbos, T.P.M., 1986. Interspeci®c
hybridization in Lilium. Acta Hort. 177, 591±595.
Van Tuyl, J.M., Van DieÈn, M.P., Van Creij, M.G.M., Van Kleinwee, T.C.M., Franken, J., Bino, R.J.,
1991. Application of in vitro pollination, ovary culture, ovule culture, and embryo rescue for
overcoming incongruity barriers in interspeci®c Lilium crosses. Plant Sci. 74, 115±126.
Van Tuyl, J.M., Van Holsteijn, H.C.M., 1996. Lily breeding research in the Netherlands. Acta Hort.
414, 35±45.
Wetter, L., Dyck, J., 1983. Isoenzyme analysis of cultured cells and somatic hybrids. In: Evans,
D.A., Sharp, W.R., Ammirato, P.V., Yamada, Y. (Eds.), Handbook of Plant Cell Culture, vol. 1.
Macmillan, New York, pp. 607±628.
Yamagishi, M., 1995. Detection of section-speci®c random ampli®ed polymorphic DNA (RAPD)
markers in Lilium. Theor. Appl. Genet. 91, 830±835.
Interspeci®c hybrids between
Lilium nobilissimum and L. regale produced
via ovules-with-placental-tissue culture
Yumi Obata, Yoshiji Niimi*, Masaru Nakano,
Keiichi Okazaki, Ichiro Miyajima
Faculty of Agriculture, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan
Accepted 31 August 1999
Abstract
Reciprocal pollination was made between Lilium nobilissimum and L. regale. Pollen tubes
reached the base of style within 144 h after pollination, but no mature seeds were obtained in either
cross combination. Explants, or ovules-with-placental-tissue excised from each carpel 30 and 40
days after pollination (DAP), were cultured on a medium composed of major salts of B-5
macronutrient (Gamborg, O.L., Miller, R.A., Ojima, K., 1968. Exp. Cell Res. 50, 151±158),
micronutrient, Fe-EDTA and vitamins of MS (Murashige, T., Skoog, F., 1962. Physiol. Plant. 15,
473±497), 5% sucrose and 0.2% gellan gum. In L. regale Lilium nobilissimum, 3% of ovules
excised at 30 DAP and 9% of those excised at 40 DAP developed into seedlings. In Lilium
nobilissimum L. regale, only 3.6% of ovules excised 40 DAP developed into seedlings, and none
of the ovules excised 30 DAP produced any seedlings. Bulbs of L. regale and hybrids transplanted
to soil showed some resistance to bulb-rot, leaf-top scorch, browning spots and/or streaking on
leaves, but those of Lilium nobilissimum were sensitive to these diseases. Flowering individuals
were nearly intermediate between parents in their morphological characteristics. All ¯owering
individuals (2n 24 chromosomes) were identi®ed as hybrids based on karyotype, isozyme and
random ampli®ed polymorphic DNA analyses. # 2000 Elsevier Science B.V. All rights reserved.
Keywords: Lilium spp.; Interspeci®c hybrids; Isozyme; Karyotype; Ovules-with-placental-tissue
culture; RAPD
*
Corresponding author. Tel.: 81-25-262-6614; fax: 81-25-262-6614.
E-mail address: himesa@agr.niigata-u.ac.jp (Y. Niimi).
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 9 9 ) 0 0 1 1 5 - 6
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
1. Introduction
The issue of solving problems with diseases in horticultural plants is an
important one, because the future use of pesticides and fungicides in agriculture
will be restricted (Jaap et al., 1992). To breed hearty lilies, interspeci®c
hybridization has been used to incorporate disease resistance, using the methods
of embryo rescue (North and Wilis, 1969; Asano, 1978, 1982; Okazaki et al.,
1992, 1994), culture of ovules-with-placental-tissue (Niimi et al., 1995), and
ovary slice culture (Hayashi et al., 1986; Kanoh et al., 1988; Niimi et al., 1996;
Van Tuyl and Van Holsteijn, 1996; FernaÂndez et al., 1996; Arzate-FernaÂndez
et al., 1998). Lilium nobilissimum Makino, a species native to Japan, where they
no longer grow naturally in the ®eld, has potential as an ornamental plant because
of its erect, funnel-shaped white ¯owers and pleasant fragrance although it is very
sensitive to fungal diseases, while L. regale Wilson, a species native to China, has
economically and horticulturally desirable traits as a commercial plant. The
introduction of the variable traits of L. regale into other Lilium spp. has been
attempted (Van Creij et al., 1992; Niimi et al., 1996).
The purpose of this study was to develop hybrids between Lilium nobilissimum
and L. regale by applying an ovules-with-placental-tissue culture procedure. The
hybridity of ¯owering plants obtained was evaluated through karyotype, isozyme,
and random ampli®ed polymorphic DNA (RAPD) analyses, as well as by
morphological traits.
2. Materials and methods
2.1. Plant materials, pollination and observation of pollen tube growth
Lilium nobilissimum and L. regale were grown in pots under natural and/or
forced cultural conditions to adjust ¯owering time. Following castration 1 day
before anthesis, pistils were capped with aluminum foil to avoid contamination
and pollinated at anthesis with fresh pollen or stored pollen, as described
previously (Niimi and Shiokawa, 1992).
The growth of pollen tubes in the styles was observed as follows: styles were
collected for 144 h after pollination at intervals of 24 h, ®xed in FAA solution
(formalin: acetic acid; 70% ethanol 5:5:90), softened in 1 N NaOH solution at
608C for about 60 min, and then dissected according to a method described
previously (Niimi, 1991). After staining with a 0.1% cotton-blue solution, the 10
longest pollen tubes in each style were observed under a light microscope and the
ratio of tube length to entire style length was calculated based on the average
length of pollen tubes observed in each style (Niimi et al., 1997). At least ®ve
styles were used for each treatment.
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
193
2.2. Ovule-with-placental-tissue culture and establishment of hybrid seedlings
Swollen ovaries were collected on 30 and 40 days after cross-pollination
between Lilium nobilissimum and L. regale (Table 2). They were surfacedisinfected with 70% ethanol for 1 min and then with a commercial bleach
solution containing 2% active chlorine for 20 min, and rinsed three times with
sterilized distilled water. The basal part of the ovary was discarded, and only
middle or swollen parts of the ovary were used. Each carpel was longitudinally
separated from the ovary along the line of carpel union. Ovules with placental
tissue, considered as an explant, were excised from each carpel (Niimi et al.,
1995). Each explant (about 15 mm long) contained 24 ovules on average.
Each explant was placed in a test tube (18 180 mm) containing 15 ml of
basal medium. The basal medium was composed of major salts of B-5
macronutrient (Gamborg et al., 1968), micronutrient, Fe-EDTA and vitamins of
MS (Murashige and Skoog, 1962) medium, 5% sucrose and 0.2% gellan gum,
and adjusted to pH 5.7 before autoclaving at 1218C for 10 min at 1.2 kg cmÿ2.
Test tubes were covered with an aluminum foil cap.
The explants were maintained in the dark at 24 18C. After 8 weeks, swollen
and/or germinated ovules were isolated from placental tissue and cultured in a
50 ml Erlenmeyer ¯ask containing 20 ml of the fresh basal medium at 258C under
continuous illumination (1000±1200 lux) supplied by white ¯uorescent lamps.
Eight weeks later, the number of seedlings developing bulblets was recorded.
2.3. Growth of bulblets transplanted to soil and morphological characteristics
of hybrids
The bulbs obtained were cultured in the basal medium for several months.
Scales excised from each bulb were cultured on MS (1962) medium
supplemented with 1.0 mg lÿ1 NAA, 0.01 mg lÿ1 BA, 5% sucrose and 0.65%
agar, in the dark for 8±12 weeks at 24 18C. Bulblets which had developed on
the scales were isolated, washed with tap water, and cold-treated for 12 weeks at
48C after they were mixed with moist vermiculite (Niimi, 1995). They were then
transplanted into a plastic tray (50 35 7.5 cm) with mixed soil (red clay
lumps:peat moss:leaf mold 1:1:1, v/v/v) and cultivated in a greenhouse, where
the night (10 208C) and day (15 308C) temperatures ¯uctuated depending on
the weather, for 162 days from 1 April 1996.
The experiment was made with a split-plot-design in 10 random blocks, each
block consisting of 20 cells, with ®ve cells being used for the bulbs of each
combination. Each bulb was scored for sprouting date and the number of bulbs
with healthy and/or wilted leaves at intervals of 3 days. All bulbs were allowed to
grow in soil within 162 days after transplanting or until leaves were withered. In
the latter case, bulbs were harvested when leaf-tip scorch, blighting spot and/or
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
streaking had spread over more than half of one of the fully-expanded leaves.
After harvest, bulbs were individually weighed after removal of leaves and roots
and the percentage gain in fresh weight was calculated.
Morphological characteristics were investigated using parental and hybrid
plants. The fertility of mature pollen was tested at anthesis by staining with 2%
aceto-carmine and observing under a light microscope.
2.4. Karyotype analysis
Root tips were taken from ¯owering plants growing in pots, pretreated with
0.1% colchicine at 258C for 6 h, ®xed in a solution of ethanol±acetic acid (3:1,
v/v) at 58C for at least 20 h, then hydrolyzed in a 2:1 mixture of 45% acetic
acid:1 N HCl (2:1, v/v) at 608C for 20±40 s. Root tips rinsed with distilled water
were stained with aceto-orcein (1%) using a squash technique. Measurement of
the length of each chromosome was made on photographs magni®ed 2300 using
a digitizer-personal computer system (Uchiyama et al., 1988). Nomenclature for
centromere positions of chromosomes was made according to the methods
described by Levan et al. (1964).
2.5. Isozyme analysis
All ¯owering plants were subjected to isozyme analysis to con®rm their hybridity.
Esterase (EST), acid phosphatase (ACP), glucose 6-phosphatase dehydrogenase
(G6-PDH), malate dehydrogenase (MDH), and peroxidase (POX) isozymes were
analyzed according to the method of Wetter and Dyck (1983) with slight
modi®cations. Leaves from potted plants with a fresh weight of about 100 mg were
homogenized in a mortar with 1 ml of an extraction buffer of 10% glycerol, 0.14% 2mercaptoethanol, and 50 mM Tris±HCl buffer at pH 7.5. Crude extracts were
electrophoresed on 12.5% polyacrylamide slab gels with 50 mM Tris±glysine buffer,
pH 8.3, at a constant voltage of 150 V for 4 h, and stained for isozymes.
2.6. Random ampli®ed polymorphic DNA (RAPD) analysis
To verify the hybridity of ¯owering plants, RAPD analysis was also carried out
according to the method of Yamagishi (1995) with several modi®cations. To
extract DNA, about 100±200 mg of fresh leaves from parental and hybrid plants
in pots were homogenized by using the cetyltrimethylammonium bromide
(CTAB) method (Rogers and Bendich, 1985). Polymerase chain reaction (PCR)
was conducted on a 25 ml reaction mixture containing 30 ng template DNA,
1.5 mM MgCl2, 250 nM of primer, 0.25 units of Ampli Taq DNA polymerase
(Perkin-Elmer, USA), 200 mM each of dATP, dCTP, dGTP and dTTP (PerkinElmer, USA), and 1 reaction buffer (Perkin-Elmer, USA). One random primer
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
(OPB06 50 -TGC TCT GCC C-30 ) (Operon Technologies, USA) and two semirandom primers (Y35 50 -TGG TAT CAG AGC C-30 ; Y37 50 -GTC AAG CGC
G-30 ) (Yamagishi, 1995) were used. After an initial denaturation at 948C for
5 min, the reaction mixtures were subjected to ampli®cation in a program
temperature control system (2400-R, Perkin-Elmer, USA) for 40 cycles, each of
which consisted of 1 min at 948C, 1 min at 508C, and 2 min at 728C, followed by
5 min at 728C. Ampli®cation products were electrophoresed on 3% polyacrylamide gels with TBE buffer (2 mM EDTA in 89 mM Tris±borate buffer, pH 8.0) at
a constant voltage of 100 V for 1 h. Gels were then stained with 2.5 mg mlÿ1
ethidium bromide and photographed on an UV transilluminator.
3. Results
3.1. Growth of pollen tubes and ovule culture
The relative length of pollen tubes after pollination was 100% in both selfpollination, 67% in Lilium nobilissimum L. regale and 93% in L. regale Lilium
nobilissimum, after 96 h of pollination. In both cross-pollination, pollen tubes grew
slowly and almost reached the base of style after 144 h of pollination (Table 1).
Cross-pollinated ovaries gradually grew until 30±40 days, and enlarged only a little.
Enlarged ovaries turned brown and shrank by 60 days after pollination, the capsules
containing only sterile seeds having a trace of degraded embryo but no mature seeds
at all (data not presented). Thus, no mature seeds were obtained from either crosscombination, suggesting that a post-fertilization barrier existed in both combinations. To overcome the barrier, ovules-with-placental-tissue excised at 30 and 40
days after pollination (DAP) were cultured on a basal medium.
The effect of ovules-with-placental-tissue culture at 30 and 40 DAP on the
production of the hybrids between Lilium nobilissimum and L. regale is summarized
Table 1
Relative length of pollen tubes to style length in self- and cross-pollination of Lilium nobilissimum
and L. regalea
Combination
Relative pollen tube length (%), hours after pollination
48
96
120
144
Self-pollination
Lilium nobilissimum
L. regale
±b
56 4
100
100
100
±
100
±
Cross-pollination
Lilium nobilissimum L. regale
L. regale Lilium nobilissimum
±
80 5
67 15
93 8
82 10
±
94 6
±
a
b
Values represent the mean SE of at least ®ve replications.
Not determined.
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Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Table 2
Effect of combinations and number of DAP on the production of interspeci®c hybrids between
Lilium nobilissimum and L. regale by ovules-with-placental-tissue culture
Combinations
DAP
Total number of
ovules cultured
Number of
seedlings obtained
Lilium nobilissimum L. regale
30
40
30
40
145
168
305
277
0
6
9
25
L. regale Lilium nobilissimum
(0)
(3.6)
(3)
(9)
in Table 2. Ovules-with-placental-tissue showed browning within 5 weeks, and few
ovules germinated in situ (Fig. 1A). Enlarged and/or germinating ovules were
isolated from placental tissue after 8 weeks of culture and then subcultured on the
fresh basal medium (Fig. 1B). In L. regale Lilium nobilissimum, 3% of ovules
excised at 30 DAP and 9% excised at 40 DAP developed into seedlings. In Lilium
nobilissimum L. regale, only 3.6% of ovules excised 40 DAP developed into
seedlings, and none of the ovules excised 30 DAP produced any seedlings.
3.2. Growth of bulbs in the greenhouse
Fig. 2 shows the time course changes in percentage of bulblets with good
healthy leaves: sprouting began at around 24 days after transplant (DAT), and 90±
100% of bulblets sprouted until 60 DAT. Afterward, leaf-top scorch, which might
have been caused by physiological disorders, and browning spots and/or streaking
on leaves, which might have been caused by Botrytis, gradually appeared on scaly
Fig. 1. Ovules-with-placental-tissue culture and seedlings with developing bulblets of L.
regale Lilium nobilissimum: (A) Six-week-old cultures; ovules-with-placental-tissue were
excised at 40 DAP and cultured on a modi®ed B-5 medium in darkness. (B) Seedlings with
developing bulblets; developing ovules excised from placental tissue were cultured on fresh
medium under a light condition. Bars 1 cm.
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
197
Fig. 2. Time course changes in percentage of bulblets with good healthy leaves. Bulblets, (&)
Lilium nobilissimum Lilium nobilissimum, (*) L. regale L. regale, (&) Lilium
nobilissimum L. regale, and (*) L. regale Lilium nobilissimum, were cultivated in a glass
house for 162 days.
leaves. The percentage of bulblets with healthy leaves decreased rapidly after 120
DAT, in the beginning of August, particularly among Lilium nobilissimum
bulblets.
Percentage gains in the fresh weight of bulbs are summarized in Table 3. The
highest value was observed in bulbs of L. regal, the lowest in Lilium nobilissimum,
with the values of hybrid bulbs nearly intermediate between the two.
Table 3
Growth of bulbs transplanted to soil
Combinations
Lilium nobilissimum
Lilium nobilissimum
L. regale L. regale
Lilium nobilissimum L. regale
L. regale Lilium nobilissimum
a
At transplantation
At harvesta
Mean fresh
weight of
bulbs
Gain in
fresh
weight (%)
Number
of
bulbs
Mean fresh
weight of
bulbs
50
400 4
644 45
161 11
40
50
50
118 2
407 2
409 3
610 45
1540 118
1218 100
522 42
379 29
298 24
All bulbs were allowed to grow in soil for 162 days or until leaves were withered. In the latter
case, bulbs were harvested at the time when leaves were withered. After harvest, bulbs were
individually weighed after removal of leaves and roots and the percentage gain in fresh weight was
calculated.
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Table 4
Characteristics of Lilium nobilissimum, L. regale, and their hybrids at ¯owering time
Indices
Lilium
nobilissimum
L. regale
Lilium
nobilissimum
L. regale
L. regale
Lilium
nobilissimum
Sprouting time
Flowering time
Late March
Late July
Late April
Late June
Early April
Middle July
Early April
Middle July
Leaf
Shape
Ovate-oblong
Linear and
narrow lanceolate
Without petiole
Wide lanceolate
Wide lanceolate
Without petiole
Without petiole
Petiole
With petiole
Flower
Inflorescence
Attachment
Petals
Raceme
Vertical
Barely recurving
Umbel
Horizontal
Noticeably
recurving
Raceme or umbel
Horizontal
Recurving
Umbel
Horizontal
Recurving
Pollen
Color
Yellow
Yellow
Yellow
94.8 2.1
94 2.1
Reddish-brown
and yellow
0.6 0.3
Fertility
1.4 0.6
3.3. Characteristics of hybrids
The morphological and other characteristics of both parental and hybrid plants
are summarized in Table 4 and Fig. 3. The hybrids were nearly intermediate
between the two parent plants. The times of sprouting and ¯owering were near
those of Lilium nobilissimum, and hybrids ¯owered in mid-July. The leaves were
wide lanceolate (0.9±1.3 cm wide) without petioles. The in¯orescence of the
hybrid plants was umbel, except one Lilium nobilissimum L. regale plant which
had the same raceme in¯orescence as Lilium nobilissimum. The hybrid ¯owers
were white and tinged rose-purple, and squarely attached to the stem,
characteristics which were more similar to L. regale than to Lilium nobilissimum.
The pollen grains of the hybrids were reddish-brown and yellow, and their
fertility was quite low (Table 4).
3.4. Analyses of hybrid plants by karyotype, isozyme and RAPD
3.4.1. Karyotype analysis
Hybrids obtained were found to be 2n 2x 24. Karyotypes of Lilium
nobilissimum (Fig. 4A) and L. regale (Fig. 4B) were clearly distinguishable by
the existence and position of secondary constriction in the ®rst and second
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
199
Fig. 3. Plants of parental species and their hybrids: (A) Lilium nobilissimum, (B) L. regale, (C)
Lilium nobilissimum L. regale, and (D) L. regale Lilium nobilissimum.
longest chromosomes. The secondary constriction was observed only in the
®rst chromosome in Lilium nobilissimum (Fig. 4A) and in the ®rst and second
chromosomes in L. regale (Fig. 4B). All of the hybrids examined had
chromosomes characteristic of both parents (Fig. 4C and D).
3.4.2. Isozyme analysis
Among ®ve isozymes (EST, ACP, G6-PDH, MDH, and POX) tested, only EST
generated clear zymograms. The EST zymograms of Lilium nobilissimum and
L. regale were distinctly different, and the hybrid plants had common bands for
both parents (Fig. 5).
200
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Fig. 4. Individual chromosomes of parental species and their hybrids: (A) Lilium nobilissimum, (B) L. regale, (C) Lilium nobilissimum L. regale, (D)
L. regale Lilium nobilissimum. Chromosomes of n1 and n2 are speci®c to Lilium nobilissimum and those of r1 and r2 to L. regale. Each arrow
indicates a secondary constriction.
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
201
Fig. 5. Isozyme patterns for esterase (EST) in parental species and their hybrids. Arrows indicate
species-speci®c bands: (Lane 1) Lilium nobilissimum, (Lane 2) L. regale, (Lane 3) Lilium
nobilissimum L. regale, (Lane 4) L. regale Lilium nobilissimum.
3.4.3. RAPD analysis
RAPD analysis provided additional evidence for hybridization (Fig. 6). Among
three primers (OPB06, Y35, and Y37) tested, only Y35 generated polymorphic
patterns of PCR products between the parents. The clear and speci®c ampli®ed
DNA fragments of Lilium nobilissimum were 620, 950 and 1550 bp, whereas
those of L. regale were 470, 550, and 750 bp. In all hybrids analyzed, three
fragments from Lilium nobilissimum and two fragments from L. regale were
ampli®ed, although RAPD pro®les showed a slight difference between the hybrid
individuals.
4. Discussion
Recently, the importance of the lily as a horticultural crop has increased and
interspeci®c hybridization of Lilium has been attempted by many researchers
(Van Tuyl and Van Holsteijn, 1996). However, interspeci®c hybridization is often
limited by pre- and/or post-fertilization barriers, the latter of which has partly
been overcome by applying ovary culture, ovule culture, and/or embryo rescue
(Van Tuyl et al., 1991; Okazaki et al., 1992, 1994; Niimi et al., 1996; FernaÂndez
et al., 1996; Arzate-FernaÂndez et al., 1998). Results obtained in the present
research showed that the interspeci®c hybridization between Lilium nobilissimum
and L. regale was limited by a post-fertilization barrier, which could be overcome
202
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
Fig. 6. PCR analysis showing ampli®cation products generated from DNA-templates of the
parental species and their hybrids with primer Y35. Arrows, from top to bottom, indicate 1550, 950,
750, 620, 550, and 470 bp fragments, respectively. (Lane 1) Lilium nobilissimum, (Lane 2) L.
regale, (Lane 3) Lilium nobilissimum L. regale, (Lane 4) L. regale Lilium nobilissimum. M
indicates the DNA size maker (1000 bp ladder and Hind III-digested l DNA).
by using an ovules-with-placental-tissue culture procedure. Furthermore, the
technique of ovules-with-placental-tissue culture appears to have the following
advantages: many ovules can be handled at once, and, in comparison with embryo
culture, a less complicated manipulation is required in particular with regard to
the composition of the medium, because the placental tissue may supply nutrients
or hormones to stimulate embryo growth and germination (FernaÂndez et al.,
1996).
The hybrids transplanted to soil showed some resistance to leaf-top scorch,
possibly caused by physiological disorders, basal rot due to Fusarium, and
browning spots and/or streaking due to Botrytis. These traits seem to be
introduced from L. regale, because bulbs of L. regale are highly resistant to these
af¯ictions but those of Lilium nobilissimum are easily affected. This suggests that
L. regale can be used as a parental plant to obtain interspeci®c hybrids resistant to
these diseases.
Previous reports showed that karyotype (Asano, 1978; Kanoh et al., 1988),
isozyme (Van Tuyl et al., 1986; Okazaki et al., 1992, 1994), RAPD (Yamagishi,
1995; Arzate-FernaÂndez et al., 1998), and nuclear rDNA (Niimi et al., 1996)
analyses may be used to ascertain the hybridity of lily plants. The present
karyotype analysis showed that the secondary constriction on the ®rst longest
Y. Obata et al. / Scientia Horticulturae 84 (2000) 191±204
203
chromosome of Lilium nobilissimum and on the ®rst and second longest
chromosomes of L. regale, which are characteristic of the respective species,
could be used as evidence for hybridization, as reported previously by Stewart
(1947) and Noda (1987), who mentioned that the ®rst and second longest
chromosomes are important in the karyotype analysis of Lilium. Isozyme and
PCR analyses also provided additional evidence for hybridization. The present
results suggest that these techniques could be a useful means by which to discover
true hybrids at the juvenile stage in lily breeding programs.
We conclude that the ovules-with-placental-tissue culture technique is one
effective method of producing interspeci®c hybrids between sexually incompatible lily species that are dif®cult to hybridize by conventional methods.
Acknowledgements
The authors are grateful to D.H. Goldstein, Professor of Keiwa Gakuen
University and Dr. G.J. De Klerk, Center for Plant Tissue Culture Research in the
Netherlands, for reading and correcting the English text, and to Dr. M. Nakata,
Botanical Garden of Toyama Prefecture, for his valuable advice on karyotype
analysis. This work was partly supported by a Grant-in-Aid for Scienti®c
Research (No. 09460017) from the Ministry of Education, Science and Culture.
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