Inheritance of Flower Pigment in Crosses between Cyclamen Cultivars and Cyclamen purpurascens

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1 Inheritance of Flower Pigment in Crosses between Cyclamen Cultivars and Cyclamen purpurascens T. Takamura, M. Aizawa and S.Y. Kim Faculty of Agriculture, Kagawa University Miki-cho, Kagawa M. Nakayama National Institute of Floricultural Science Tsukuba H. Ishizaka Horticultural Laboratory, Saitama Prefecture Agricultural and Forestry Research Center, Kuki Keywords: anthocyanin, Cyclamen persicum, 5-glucosyltransferase, interspecific hybrid, petal Abstract Although interspecific hybrids between cyclamen (Cyclamen persicum) cultivars and C. purpurascens were produced by using the ovule culture, mode of inheritance of flower pigment remains obscure. Therefore, inheritance of anthocyanin in the petals in the crosses between diploid cyclamen cultivars and C. purpurascens was investigated in the present study. In every cross combination, including the crosses by using acyanic cyclamen cultivars without the eye, all the F 1 progenies had a purple or reddish-purple eye in the petals, showing the eye expression was a dominant characteristic. All the F 1 progenies contained anthocyanins in the petals, whereas some progenies in the crosses by using a yellow-flowered cultivar contained very small amount of anthocyanin. The main flower pigment of C. purpurascens was malvidin 3,5-diglucoside. All the F 1 progenies also contained 3,5- diglucoside type anthocyanins, such as malvidin 3,5-diglucoside, peonidin 3,5-diglucoside and cyanidin 3,5-diglucoside, in the petals as the main pigments even in the crosses by using acyanic cultivars or cyanic cultivars containing malvidin 3-glucoside or peonidin 3-neohesperidoside in the petals as the main pigment. These results suggest that the expression of 5-glucosyltransferase in anthocyanin synthesis in the petals was dominant even in the crosses between cyclamen cultivars and C. purpurascens. INTRODUCTION Among the genus Cyclamen consisting of 22 species (Grey-Wilson, 2003), only C. persicum has been used as the major commercial plants. The other Cyclamen species, however, have desirable characteristics like sweet fragrance in C. purpurascens. Although intespecific hybridization between C. persicum and the other species had been difficult because of the strong cross-incompatibility (Legro, 1959; Grey-Wilson, 2003), Ishizaka and Uematsu (1992) overcame the difficulty with the ovule culture system. The system was also used successfully for obtaining interspecific hybrids between C. persicum cultivars and C. purpurascens (Ishizaka and Uematsu, 1995a; Ewald, 1996; Takamura et al., 2002), and the hybrids had sweet fragrance in their flower like C. purpurascens (Ishizaka and Uematsu, 1995a; Ewald, 1996). Flower color is another important characteristic for ornamental plants so that cultivars with desirable color and fragrance are demanded. While there are a few reports on the inheritance in crosses among cyclamen cultivars (Van Bragt, 1962; Takamura et al., 1995, 2000a,b,c), no report on the inheritance in interspecific crosses in the genus Cyclamen is available. Therefore, we investigated inheritance of anthocyanin in the petals in crosses between diploid cyclamen cultivars and C. purpurascens. Proc. IX th Intl. Symp. on Flower Bulbs Eds.: H. Okubo, W.B. Miller and G.A. Chastagner Acta Hort. 673, ISHS

2 MATERIALS AND METHODS Diploid cyclamen (C. persicum) cultivars were crossed with C. purpurascens and their F 1 progenies were obtained through the ovule culture following Ishizaka and Uematsu (1995a). In diploid cyclamen cultivars, yellow-flowered Golden Boy, white-flowered Mirabelle, red-flowered Largo and Strauss, reddish-purple-flowered Cochineal, and purple-flowered Wase Murasaki were used. The petals of F 1 progenies were collected, and the flower colors were recorded. Anthocyanins in petals of the F 1 progenies and some mother plants were investigated by using high performance liquid chromatography (HPLC). For the HPLC analysis, only slips (petals without their eye parts) were used. The slips were dried, and then soaked in 5% HCOOH methanolic solution at 5 C overnight. The extract was filled up to 5 ml. The filtered petal extract was injected to HPLC, in which chromatography systems were developed with LC-10AT pumps (Shimadzu Co. Ltd., ) equipped with Cosmosil 5C 18 AR-II columns (4.6 mm I.D. x 50 mm mm I.D. x 250 mm; Nakarai Tesque Co. Ltd., ) and an SPD-10AV detector (Shimadzu Co. Ltd.) set at 530 nm. The column temperature was maintained at 40 C. Liner gradient elution for 40 min from 25 to 40 or 41% solvent B (1.5% H 3 PO 4, 20% HCOOH and 25% CH 3 CN in H 2 O) in solvent A (1.5% H 3 PO 4 in H 2 O) were employed as the solvent system, and the flow rate of 0.8 ml was maintained. RESULTS AND DISCUSSION All the F 1 progenies in every cross combination, including the crosses by using acyanic cyclamen cultivars without cyanic eye, had a purple or reddish-purple eye in their petals (data not shown). The results suggest that the eye expression was dominant in the crosses as Takamura et al. (2000a) indicated that the eye expression in intraspecific cyclamen crosses was dominant. Van Bragt (1962) and Takamura et al. (2000b) reported that anthocyanin expression in slip parts of cyclamen petals was a dominant characteristic. All the F 1 progenies in the present study contained anthocyanins in their slips, suggesting that the anthocyanin expression was also dominant in the interspecific crosses (Table 1). All the F 1 progenies also contained 3,5-diglucoside type anthocyanins, such as malvidin 3,5-diglucoside (Mv3,5dG), peonidin 3,5-diglucoside (Pn3,5dG) and cyanidin 3,5-diglucoside (Cy3,5dG), in the slips as the main pigments even in the crosses by using acyanic cultivars or cyanic cultivars of which main anthocyanins in the slips were malvidin 3-glucoside or peonidin 3-neohesperidoside. Van Bragt (1962) and Takamura et al. (2001) suggested that synthesis of anthocyanidin 3,5-diglucoside was dominant in cyclamen cultivars. Results of the present study suggest that the expression of 5-glucosyltransferase in anthocyanin synthesis in the slips may be also dominant in the interspecific crosses. Since the main flower pigment of C. purpurascens was Mv3,5dG, genes in C. purpurascens should contribute to the expression. In Golden Boy x C. purpurascens crosses some F 1 progenies contained a little anthocyanin in their slips, whereas the other progenies accumulated relatively much anthocyanin. The differences in anthocyanin accumulation in the progenies caused the difference in flower color expression (Fig. 1): The slips of the progenies with a little anthocyanin were very pale pink. In Largo x C. purpurascens crosses all the F 1 progenies contained Cy3,5dG, Mv3,5dG and Cy3,5dG in their slips (Table 1). However, the primary anthocyanin was Mv3,5dG in some F 1 progenies, and the others contained Pn3,5dG in their slips as the primary anthocyanin (data not shown). A chimeric plant with both pink and purple flowers were found in progenies obtained by Strauss x C. purpurascens crosses. The main anthocyanin of the pink flowers differed from that of the purple flowers (Fig. 2). The factors that caused the segregation in anthocyanin accumulation and chimeric plant production in the cross combinations remains obscure, and further research on the factors is desirable Results of the present study should contribute to clarify inheritance of flower pigment in interspecific crosses between diploid cyclamen cultivars and C. purpurascens. The F 1 progenies in the crosses were, however, sterile (data not shown). Ishizaka and 438

3 Uematsu (1995b) bred amphidiploid plants between cyclamen cultivars and C. purpurascens, regarded as invaluable genetic resource for cyclamen breeding. Therefore, research on inheritance of flower colors and pigments in the breeding program by using the amphidiploids should be desirable, and we have already started the research. ACKNOWLEDGEMENT We wish to thank Ms. Mayumi Kawamura for her assistance. This research was partially supported by Research Project for Utilizing Advanced Technologies in Agriculture, Forestry and Fisheries of the Ministry of Agriculture, Forestry and Fisheries of. Literature Cited Ewald, A Interspecific hybridization between Cyclamen persicum Mill. and C. purpurascens Mill. Plant Breeding 115: Grey-Wilson, C Cyclamen. Timber Press, Portland. Ishizaka, H. and Uematsu, J Production of interspecific hybrids of Cyclamen persicum Mill. and C. hederifolium Aiton. by ovule culture.. J. Breed. 42: Ishizaka, H. and Uematsu, J. 1995a. Interspecific hybrids of Cyclamen persicum Mill. and C. purpurascens Mill. produced by ovule culture. Euphytica 82: Ishizaka, H. and Uematsu, J. 1995b. Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicines in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses. Euphytica 86: Legro, R.A.H The cytological background of Cyclamen breeding. Meded. Landbouwhogeschool, Wageningen 59:1-51. Takamura, T., Kim, S.Y. and Tanaka, M Inheritance of glycosylation of main anthocyanin in cyclamen petals and its effects on the petal color. (In ese). J.. Soc. Hort. Sci. 70(Suppl. 1):157. Takamura, T., Kitamura, H. and Tanaka, M. 2000a. Expression and inheritance of flower color and pigment in the eye of cyclamen petal. (In ese). J.. Soc. Hort. Sci. 69(Suppl. 1):350. Takamura, T., Sugimura, H. and Tanaka, M. 2000b. Inheritance of flower color and pigment in crosses between cyanic and acyanic cyclamen. (In ese). J.. Soc. Hort. Sci. 69(Suppl. 2):453. Takamura, T., Sugimura, T. and Tanaka, M. 2000c. Inheritance of yellow-flowered characteristic in crosses between diploid cyanic and yellow-flowered cyclamen cultivars. Acta Hort. 508: Takamura, T., Tomihama, T. and Miyajima, I Inheritance of yellow-flowered characteristic and yellow pigments in diploid cyclamen (Cyclamen persicum Mill.) cultivars. Scientia Hort. 64: Takamura, T., Yamada, R. and Tanaka, M Effects of genotypes of seed parents on the production of interspecific hybrids in Cyclamen persicum Mill. x C. purpurascens Mill. crosses. (In ese with English summary). Tech. Bull. Fac. Agr. Kagawa Univ. 54: Van Bragt, J Chemogenetical investigations of flower colours in Cyclamen. Meded. Landbouwhogeschool, Wageningen 62:

4 Tables Table 1. Main anthocyanin in slips in F 1 progenies between diploid cyclamen cultivars and C. purpurascens. 2 Cross combination or parents Anthocyanin expression 1 Main anthocyanin in slips No. of plants Cochineal C. purpurascens + Mv3,5dG 4 Golden Boy C. purpurascens + Mv3,5dG 5 ± Mv3,5dG 7 Largo C. purpurascens + Pn3,5dG+ Mv3,5dG+ Cy3,5dG 15 Mirabelle C. purpurascens + Mv3,5dG 11 Strauss C. purpurascens + Pn3,5dG+ Mv3,5dG+ Cy3,5dG 5 + Mv3,5dG 2 + Mv3,5dG (purple flowers) or 1 3 Pn3,5dG+ Mv3,5dG+ Cy3,5dG (pink flowers) Wase Murasaki C. purpurascens + Mv3,5dG 14 Cochineal + Mv3G - Golden Boy Largo + Pn3Nh - Mirabelle Strauss Pn3G, Pn3Nh - Wase Murasaki + Mv3,5dG - C. purpurascens + Mv3,5dG - 1 +, ± and - indicate much, a little and no anthocyanin was detected, respectively. 2 Cy3,5dG, cyanidin 3,5-diglucoside; Mv3,5dG, malvidin 3,5-diglucoside; Mv3G, malvidin 3-glucoside; Pn3,5dG, peonidin 3,5-diglucoside Pn3G peonidin 3-glucoside; Pn3Nh, peonidin 3-neohesperidoside. 3 A chimeric plant with both pink and purple flowers. Figures b* 10 A a* Fig. 1. Color of slips with a little (A) and relatively much (B) anthocyanin in F 1 progenies between Golden Boy and C. purpurascens. 440

5 Fig. 2. HPLC profiles of anthocyanins in purple (upper) and pink (lower) slips of a chimeric plant obtained by Strauss C. purpurascens crosses. 441