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. InL. regaleLilium nobilissimum, 3% of ovules excised at 30 DAP and 9% of those excised at 40 DAP developed into seedlings. In Lilium nobilissimumL. regale, only 3.6% of ovules excised 40 DAP developed into seedlings, and none of the ovules excised 30 DAP produced any seedlings. Bulbs ofL. regaleand hybrids transplanted to soil showed some resistance to bulb-rot, leaf-top scorch, browning spots and/or streaking on leaves, but those ofLilium nobilissimumwere sensitive to these diseases. Flowering individuals were nearly intermediate between parents in their morphological characteristics. All ¯owering individuals (2n24 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).
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, whileL.regaleWilson, 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 betweenLilium nobilissimum
andL. regaleby 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% ethanol5:5:90), softened in 1 N NaOH solution at
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 surface-disinfected 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 (18180 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 2418C. 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 2418C. 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 (50357.5 cm) with mixed soil (red clay
lumps:peat moss:leaf mold1:1:1, v/v/v) and cultivated in a greenhouse, where
the night (10208C) and day (15308C) temperatures ¯uctuated depending on the weather, for 162 days from 1 April 1996.
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®ed2300 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% 2-mercaptoethanol, 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 25ml reaction mixture containing 30 ng template DNA, 1.5 mM MgCl2, 250 nM of primer, 0.25 units of Ampli Taq DNA polymerase
(OPB06 50
-TGC TCT GCC C-30
) (Operon Technologies, USA) and two semi-random 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% polyacryla-mide 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 2.5mg 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 self-pollination, 67% inLilium nobilissimumL. regaleand 93% inL. regaleLilium 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 cross-combination, suggesting that a post-fertilization barrier existed in both combina-tions. 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 betweenLilium nobilissimumandL. regaleis summarized
Table 1
Relative length of pollen tubes to style length in self- and cross-pollination ofLilium nobilissimum
andL. regalea
Combination Relative pollen tube length (%), hours after pollination
48 96 120 144
Self-pollination
Lilium nobilissimum ±b 100 100 100
L. regale 564 100 ± ±
Cross-pollination
Lilium nobilissimumL. regale ± 6715 8210 946
L. regaleLilium nobilissimum 805 938 ± ±
a
Values represent the meanSE of at least ®ve replications.
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). InL. regaleLilium nobilissimum, 3% of ovules
excised at 30 DAP and 9% excised at 40 DAP developed into seedlings. InLilium nobilissimumL. 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 byBotrytis, gradually appeared on scaly Table 2
Effect of combinations and number of DAP on the production of interspeci®c hybrids between
Lilium nobilissimumandL. regaleby ovules-with-placental-tissue culture
Combinations DAP Total number of
ovules cultured
Number of seedlings obtained
Lilium nobilissimumL. regale 30 145 0 (0)
40 168 6 (3.6)
L. regaleLilium nobilissimum 30 305 9 (3)
40 277 25 (9)
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 ofL. regal, the lowest inLilium nobilissimum, with the values of hybrid bulbs nearly intermediate between the two.
Fig. 2. Time course changes in percentage of bulblets with good healthy leaves. Bulblets, (&)
Lilium nobilissimumLilium nobilissimum, (*) L. regaleL. regale, (&) Lilium nobilissimumL. regale, and (*) L. regaleLilium nobilissimum, were cultivated in a glass house for 162 days.
Table 3
Growth of bulbs transplanted to soil
Combinations At transplantation At harvesta
Number of bulbs
Mean fresh weight of bulbs
Mean fresh weight of bulbs
Gain in fresh weight (%)
Lilium nobilissimum
Lilium nobilissimum
50 4004 64445 16111
L. regaleL. regale 40 1182 61045 52242
Lilium nobilissimumL. regale 50 4072 1540118 37929
L. regaleLilium nobilissimum 50 4093 1218100 29824
a
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 ofLilium 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 oneLilium nobilissimumL. regaleplant 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 toL. regalethan toLilium 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 2n2x24. 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 Table 4
Characteristics ofLilium nobilissimum,L. regale, and their hybrids at ¯owering time
Indices Lilium
Sprouting time Late March Late April Early April Early April Flowering time Late July Late June Middle July Middle July
Leaf
Shape Ovate-oblong Linear and narrow lanceolate
Wide lanceolate Wide lanceolate
Petiole With petiole Without petiole Without petiole Without petiole
Flower
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. regalewere distinctly different, and the hybrid plants had common bands for both parents (Fig. 5).
Fig. 3. Plants of parental species and their hybrids: (A)Lilium nobilissimum, (B) L. regale, (C)
Fig. 4. Individual chromosomes of parental species and their hybrids: (A)Lilium nobilissimum, (B)L. regale, (C)Lilium nobilissimumL. regale, (D)
L. regaleLilium nobilissimum. Chromosomes of n1 and n2 are speci®c toLilium nobilissimumand those of r1 and r2 to L. regale. Each arrow indicates a secondary constriction.
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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 betweenLilium nobilissimum
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 fromL. regale, because bulbs ofL. regaleare highly resistant to these af¯ictions but those ofLilium nobilissimumare easily affected. This suggests that
L. regalecan be used as a parental plant to obtain interspeci®c hybrids resistant to these diseases.
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 incompa-tible 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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