T. Oomiya Hokkaido Ornamental Plants and Vegetables Research Center Takikawa, Hokkaido,

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1 Cross-compatibility in Interspecific and Intergeneric Hybridization among the Colchicaceous Ornamentals, Gloriosa spp., Littonia modesta and Sandersonia aurantiaca S. Kuwayama, Y. Mizuta and M. Nakano T. Nakamura Faculty of Agriculture, Niigata University Graduate School of Life Science Niigata Tohoku University, Sendai Japan Japan T. Oomiya Hokkaido Ornamental Plants and Vegetables Research Center Takikawa, Hokkaido, Japan Keywords: cross-pollination, FCM analysis, ovule culture, rhizome-like structures, wide hybridization Abstract Interspecific and intergeneric cross-pollination and subsequent ovule culture were carried out among 6 genotypes of Gloriosa spp., 1 genotype of L. modesta and 2 genotypes of S. aurantiaca in order to widen their variations and to develop novel cultivars. Ovules with placental tissues prepared from ovaries 14 days after pollination were cultured at 25 C in the dark on a medium containing.1 mg l -1 α-naphthaleneacetic acid (NAA) and.1 mg l -1 benzyladenine (BA), on which rhizome-like structures were produced from the ovules in 49 out of 61 cross-combinations. Following transfer to a medium containing.25 mg l -1 NAA and 2.5 mg l -1 BA under a 16-h photoperiod, some of the rhizome-like structures produced shoots and/or root-like structures. These shoots developed into plantlets and produced tubers on a plant growth regulator-free medium. In some of the plantlets derived from intergeneric cross-pollination, early confirmation of the hybridity was accomplished by flow cytometry analysis of relative DNA content of nuclei. INTRODUCTION Gloriosa spp., Littonia modesta and Sandersonia aurantiaca, all belonging to the family Colchicaceae, are tuberous plants native to South Africa. Gloriosa spp. have red or yellow flowers, in which tepals are reflexed turning upwards and backwards. L. modesta has orange, bell-shaped flowers. S. aurantiaca, a monotypic species, has orange, lantern-shaped flowers. Although these plants have recently become popular as cut-flowers, they have few variations in plant form, flower color and shape. Interspecific and intergeneric hybridization may widen variations in horticultural traits of these plants. However, there have been only 2 reports on wide hybridization in the family Colchicaceae: S. aurantiaca x L. modesta and L. modesta x S. aurantiaca (Morgan et al., 21), and S. aurantiaca x G. rothschildiana (Nakamura et al., 22). In the present study, we examined the production of interspecific and intergeneric hybrid plants using 6 genotypes of Gloriosa spp., 1 genotype of L. modesta and 2 genotypes of S. aurantiaca. MATERIALS AND METHODS Plant Materials Six genotypes of Gloriosa spp., 1 genotype of L. modesta and 2 genotypes of S. aurantiaca were used in the present study (Table 1). G. rothschildiana, G. superba Lutea, Gloriosa Verschild and Gloriosa Marron Gold were kindly provided by Takii Seed Co., Japan. Gloriosa African Gold, S. aurantiaca and S. aurantiaca Phoenix were Proc. IX th Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS

2 purchased from Kokkaen Co., Japan, and Gloriosa Rothschildiana Pink and L. modesta were purchased from Komoriya Nursery Co., Japan. Tubers of all genotypes were planted to pots on March 26 and cultivated in the greenhouse without heating. Flowers of the seed parent were emasculated 2 days before anthesis. Self-pollination of each genotype and reciprocal cross-pollination between various combinations were carried out at anthesis using fresh pollen. Ovule Culture Ovule culture was performed according to Nakamura et al. (22). Ovaries were collected 14 days after pollination. They were surface-disinfected with 7% ethanol for 3 s and then with a sodium hypochlorite solution (2% active chlorine) for 2 min, followed by 3 rinses with sterile, distilled water. Ovules with placental tissues were prepared from the ovaries and cultured at 25 C in the dark on half-strength MS medium (Murashige and Skoog, 1962) containing.1 mg l -1 α-naphthaleneacetic acid (NAA) and.1 mg l -1 benzyladenine (BA). All media used in the present study were supplemented with 3% sucrose and.2% gellan gum, and adjusted to ph 5.7 before autoclaving. In the present study, only enlarged ovules were subjected to culture. Ovules that produced rhizome-like structures were transferred to half-strength MS medium containing.25 mg l -1 NAA and 2.5 mg l -1 BA under a 16-h photoperiod for shoot induction. Regenerated shoots were detached from the rhizome-like structures, transferred for rooting to half-strength MS medium without plant growth regulators (PGRs), and cultured under the same conditions. Flow Cytometry (FCM) Analysis Relative DNA content of nuclei isolated from leaf tissues was measured using a flow cytometer (Partec PA, GmbH, Münster, Germany) as previously described (Saito et al., 23). Briefly, leaves harvested from in vitro-grown plantlets were chopped with a razor blade in Solution A (Partec GmbH kit) to isolate nuclei, and then stained for 5 min with Solution B (Partec GmbH kit) containing 4,6-diamidino-2-phenylindole (DAPI) prior to FCM analysis. RESULTS AND DISCUSSION Pollination and Ovule Culture In the present study, totally 7 different combinations of pollination, including self-pollination of each of the 9 genotypes and 61 combinations of pollination, were carried out (Table 2). In self-pollination of each genotype and most interspecific cross-combinations, relatively large numbers of ovules enlarged 14 days after pollination. In intergeneric cross-pollination, many enlarged ovules were also obtained when S. aurantiaca and L. modesta were used as a seed parent, but only a few or no enlarged ovules were obtained when Gloriosa spp. was used as a seed parent except for G. superba Lutea. Two months after the initiation of ovule culture, rhizome-like structures (Fig. 1A) were formed from the ovules in self-pollination of each genotype and 49 out of 61 cross-combinations (Table 2). Shoots (Fig. 1B) and/or root-like structures were regenerated from the rhizome-like structures 1-6 months of culture on shoot induction media. Rhizome-like structures without shoot regeneration turned brown and subsequently died. Following transfer to PGR-free medium, most of the regenerated shoots produced roots within 2 weeks. Plantlets thus obtained produced small tubers at the shoot base (Fig. 1C). In intergeneric cross-pollination, plantlets were obtained in 19 out of 4 combinations, and the number of plantlets was higher when G. superba Lutea, L. modesta and S. aurantiaca were used as a seed parent (Table 3). In interspecific cross-pollination in Gloriosa, plantlets were obtained in 15 out of 19 combinations. In self-pollination of 2 S. aurantiaca genotypes and intraspecific cross-pollination between them, only a few plantlets were obtained although many rhizome-like structures were produced. Irrespective of the seed parent, no plantlets were obtained in intergeneric cross-pollination using L. modesta as a pollen parent. Morgan et al. (21) succeeded in 422

3 the production of intergeneric hybrids of S. aurantiaca x L. modesta. Although the same combination of intergeneric crosses was carried out, no plantlets were obtained from crosses in the present study. Most of the plantlets obtained in the present study proliferated well on PGR-free medium. Especially, plantlets derived from self-pollination of Gloriosa Marron Gold and cross-pollination using this genotype as one parent showed vigorous proliferation. Plantlets were successfully acclimatized in a growth chamber, transplanted to pots and cultivated in the greenhouse as the mother plants (Fig. 1D). Confirmation of the Hybridity Four Gloriosa genotypes, in which the ploidy level has not yet been determined, were initially subjected to FCM analysis, and Gloriosa Verschild, Marron Gold, African Gold and Rothschildiana Pink were considered to be heptaploid, tetraploid, diploid and hexaploid, respectively (Table 1). FCM analysis was then carried out for confirming the hybridity of ovule culture-derived plantlets. The effectiveness of FCM analysis for early verification of the hybridity has already been demonstrated for intergeneric hybridization between S. aurantiaca and G. rothschildiana (Nakamura et al., 22). In the present study, several plantlets derived from cross-pollination between genotypes with apparently different DNA contents have so far been confirmed to be hybrids by FCM analysis (Table 4). For example, the positions of the G/G1 peak of Gloriosa Marron Gold and S. aurantiaca were apparently different, and the peak of 5 independent plantlets derived from S. aurantiaca x Gloriosa Marron Gold appeared in an intermediate position between them, indicating that the 5 plantlets were hybrids (Fig. 2). On the other hand, the G/G1 peak of 2 independent plantlets derived from S. aurantiaca x G. rothschildiana, 2 independent plantlets derived from S. aurantiaca x G. superba Lutea and 1 plantlet derived from S. aurantiaca x Gloriosa Verschild appeared in the same position as S. aurantiaca, and these 5 plantlets seemed not to be hybrids but to be derived from self-pollination of S. aurantiaca. Similarly, 1 plantlet derived from G. superba Lutea x S. aurantiaca Phoenix, 3 independent plantlets derived from L. modesta x Gloriosa Marron Gold and 1 plantlet derived from L. modesta x S. aurantiaca Phoenix were considered to be derived from self-pollination of the seed parent by FCM analysis. In the present study, we succeeded in the production of several interspecific and intergeneric hybrid plants among the Colchicaceous ornamental plants via ovule culture. In addition, our results indicate that early confirmation of the hybridity of plantlets derived from cross-pollination between Colchicaceous genotypes with apparently different DNA contents can readily be accomplished by FCM analysis. Verification of the hybridity by randomly amplified polymorphic DNA (RAPD) analysis and chromosome observation is now in progress. Also, detailed characterization of hybrids obtained in the present study with respect to flower color and shape, leaf shape, plant height, and vase life is necessary for the development of novel cultivars in the family Colchicaceae. Literature Cited Morgan, E.R., Burge, G.K., Seelye, J.F., Hopping, M.E., Grant, J.E., Warren, A.G.F. and Brundell, D. 21. Wide crosses in the Colchicaceae: Sandersonia aurantiaca (Hook.) x Littonia modesta (Hook.). Euphytica 121: Murashige, T. and Skoog, F A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: Nakamura, T., Saito, H. and Nakano, M. 22, Production via ovule culture of intergeneric hybrid plants between Sandersonia aurantiaca and Gloriosa rothschildiana. Proc. XXVIth Intl. Hort. Congr., Toronto, Canada August. p.88. Saito, H., Mizunashi, K., Tanaka, S., Adachi, Y. and Nakano, M. 23. Ploidy estimation in Hemerocallis species and cultivars by flow cytometry. Scientia Hort. 97:

4 Tables Table 1. Colchicaceous plants used in the present study. Species and cultivar Abbreviation Chromosome number (2n) Flower color Gloriosa rothschildiana Gro 6x = 66 Red with yellow center Gloriosa superba Lutea Gsu 2x = 22 Yellow Gloriosa Verschild Gve 7x = 77 * Red with yellow center Gloriosa Marron Gold Gma 4x = 44 * Purple with yellow center Gloriosa African Gold Gaf 2x = 22 * Yellow Gloriosa Rothschildiana Pink Grp 6x = 66 * Pink with yellow center Littonia modesta Lit 2x = 22 Orange Sandersonia aurantiaca Sau 2x = 24 Orange Sandersonia aurantiaca Phoenix Sph 2x = 24 Light yellow * Ploidy level and chromosome number of 4 Gloriosa cultivars, Verschild, Marron Gold, African Gold and Rothschildiana Pink, were estimated by FCM analysis. Table 2. Number of independent rhizome-like structures produced via ovule culture after self- or cross-pollination in Colchicaceae. Data were recorded 2 months after the initiation of ovule culture. Parenthesized figures represent the number of cultured ovules. Seed Pollen parent parent Gro Gsu Gve Gma Gaf Grp Lit Sau Sph Gro 288 (713) nc 1 54 (291) 94 (94) 71 (71) 72 (264) nc 2 (13) (11) Gsu 29 (38) 143 (549) 132 (132) 159 (387) 49 (294) 149 (295) 4 (4) 25 (61) 9 (9) Gve np 2 np 25 (133) np np np 1 (1) 6 (6) (1) Gma 64 (181) 48 (48) np 3 (3) 19 (19) 31 (66) nc 19 (19) nc Gaf nc 17 (162) np 6 (186) 11 (19) 13 (7) nc 1 (1) nc Grp np np np np 26 (26) 57 (21) nc nc nc Lit 14 (245) 25 (124) 41 (122) 13 (136) 5 (221) 5 (211) 56 (393) 36 (163) 52 (381) Sau 484 (96) 113 (226) 86 (197) 49 (89) 19 (24) 25 (59) 134 (321) 627 (1292) 526 (117) Sph 4 (64) 4 (7) 8 (8) 39 (61) 46 (55) 7 (46) 1 (14) 81 (12) 15 (15) 1 Ovule culture was not carried out because no enlarged ovules were obtained 14 days after pollination. 2 Pollination was not carried out. 424

5 Table 3. Number of independent plantlets produced via ovule culture after self- or cross-pollination in Colchicaceae. Data were recorded 1-6 months after transfer of the ovules with rhizome-like structures to the shoot induction medium. Seed Pollen parent parent Gro Gsu Gve Gma Gaf Grp Lit Sau Sph Gro 6 nc nc Gsu Gve np 2 np 1 np np np 1 Gma 4 1 np nc 4 nc Gaf nc np nc nc Grp np np np np 4 5 nc nc nc Lit Sau Sph Ovule culture was not carried out because no enlarged ovules were obtained 14 days after pollination. 2 Pollination was not carried out. Table 4. Number of independent interspecific and intergeneric hybrid plantlets in Colchicaceae which have so far been confirmed by FCM analysis. Seed Pollen parent parent Gro Gsu Gve Gma Gaf Grp Lit Sau Sph Gro sp 1 nc * nc Gsu 1 sp 1 7 * 4 * 2 1 Gve np 3 np sp np np np 1 Gma 4 1 np sp 9 2 nc nc Gaf nc * np 3 sp 1 nc * nc Grp np * np np np 3 sp nc nc nc Lit * 2 * 1 sp 1 2 Sau Sph sp * * sp 1 Self-pollination. 2 Ovule culture was not carried out because no enlarged ovules were obtained 14 days after pollination. 3 Pollination was not carried out. * Cross-combinations in which the hybridity could not be confirmed by FCM analysis because of the similarity of the G/G1 peak position of the parents. 425

6 Figures A B C D Fig. 1. Production of intergeneric hybrid plants of Sandersonia aurantiaca x Gloriosa superba Lutea via ovule culture. (A) Development of a rhizome-like structure (arrow) from a cultured ovule (arrowhead). Bar = 1 mm. (B) Shoot formation from a rhizome-like structure. Bar = 1 cm. (C) Plantlet regeneration and tuber formation (arrowhead). Bar = 1 cm. (D) A hybrid plant established in the greenhouse. Bar = 1 cm. 426

7 2 Sa Number of nuclei 15 1 H 5 Gma Relative fluorescence 2 Fig. 2. Histogram from FCM analysis of nuclear DNA content of Sandersonia aurantiaca (Sau), Gloriosa Marron Gold (Gma) and their hybrid (S. aurantiaca x Gloriosa Marron Gold ) produced via ovule culture (H). 427