Identification of Ploidy Level of Cyclamen Rohlfsianum Plants Through Flow Cytometric and Cytological Analysis of C
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© 2009 The Japan Mendel Society Cytologia 74(4): 457–465, 2009 Identification of Ploidy Level of Cyclamen rohlfsianum Plants through Flow Cytometric and Cytological Analysis of C. rohlfsianum, C. purpurascens and their Hybrid Hiroshi Ishizaka1,*, Takayoshi Oku2, Kei-ichiro Mishiba3 and Masahiro Mii2 1 Horticultural Laboratory, Saitama Prefecture Agriculture and Forestry Research Center, Rokumanbu 91, Kuki, Saitama 346-0037, Japan 2 Plant Cell Technology Laboratory, Faculty of Horticulture, Chiba University, Matsudo City, Chiba 271-8510, Japan 3 School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen, Naka-ku, Sakai, Osaka 599-8531, Japan Received September 18, 2009; accepted December 15, 2009 Summary The ploidy level of Cyclamen rohlfsianum was analyzed through the measurement of the relative fluorescent intensity (RFI) of nuclei stained with 6-diamidino-2-phenylindole (DAPI) using flow cytometry (FCM), and through the observation of mitotic and meiotic chromosomes, as compared with those of C. persicum (2nϭ2xϭ48, Cpe2x), C. rohlfsianum (Cro2, 2nϭ96), C. purpurascens (2nϭ2xϭ34, Cpu3) and a hybrid (2nϭ65) of C. rohlfsianum (Cro2, 2nϭ96)ϫC. purpurascens (2nϭ2xϭ34, Cpu3). The mean chromosome pairing at diakinesis and/or metaphase I ϩ ϩ ϩ ϩ of PMCs in Cro2, Cpu3 and their hybrid was 0.65I 47.07II 0.03III 0.28IV, 0.16I 16.92II and ϩ ϭ ϭ 17.00I 24.00II, respectively. These findings suggest that Cro2 is an autotetraploid (2n 4x 96) and the Cro2ϫCpu3 hybrid is an allotriploid (2nϭ3xϭ65), produced by crossing an autotetraploid and diploid. The chromosome number of Cro3 was 2nϭ144, equal to 1.5 times that of Cro2 (2nϭ 4xϭ96) and the DNA content of Cro3 estimated by the RFI value corresponded to approximately 1.5 times that of Cro2, suggesting that Cro3 is an autohexaploid (2nϭ6xϭ144). Twelve C. rohlfsianum plants of uncertain chromosome number were classified into 3 autotetraploids and 9 autohexaploids by FCM analysis, suggesting intraspecific differentiation concerning the ploidy level in C. rohlfsianum. Key words Cyclamen rohlfsianum, C. persicum, C. purpurascens, Chromosome, Flow cytometry, Ploidy level. In the genus Cyclamen containing 22 species, Cyclamen persicum Mill. is the major commercial plant. C. persicum plants growing in the wild are consistently diploid with a chromosome number of 2nϭ2xϭ48, although many cultivars that are diploid (2nϭ2xϭ48) and autotetraploid (2nϭ4xϭ96) have been produced in the species through the selection of natural mutants from wild plants and their varietal crossings (Legro 1959). Autotriploids (2nϭ3xϭ72) of C. persicum have rarely been obtained from the reciprocal crossing of a diploid cultivar an autotetraploid cultivar by the conventional method (Legro 1959). Although autotriploids have been obtained at a high frequency by ovule culture to rescue weak hybrid embryos in the cross of diploid cultivarϫautotetraploid cultivar (Takamura and Miyajima 1996), they have not been used as commercial plants, probably because of the high production costs. On the other hand, the remaining 21 species in the genus Cyclamen have only been grown by gardening devotees, and have not been * Corresponding author, e-mail: [email protected] 458 H. Ishizaka et al. Cytologia 74(4) developed as major commercial plants (Grey-Wilson 2002). The observation of mitotic and meiotic chromosomes is a reliable method for clarifying ploidy levels and genetic relationships among species in many plants. The species in the genus Cyclamen cover a wide range of chromosome numbers as follows: 2nϭ20, 22, 30, 34, 48, 51, 68, 72, 84 and 96. Polyploids as well as diploids have been discovered in C. hederifolium (2nϭ2xϭ34, 3xϭ51, 4xϭ68), C. africanum (2nϭ2xϭ34, 4xϭ68) (Bennett and Grimshaw 1991), C. persicum (2nϭ 2xϭ48, 3xϭ72, 4xϭ96) (Legro 1959, Takamura and Miyajima 1996) and C. graecum (2nϭ4xϭ84) (Ishizaka 2003). Estimation of nuclear DNA content by FCM has been used as an effective tool for the determination of taxa or ploidy levels, the identification of hybrids in wide cross, and the analysis of polysomaty in numerous plants, including cyclamen (Ewald 1996, Kato and Mii 2000, Mishiba and Mii 2000, Mishiba et al. 2000, Kato et al. 2001, Takamura et al. 2001, Amano et al. 2007, Hayashi et al. 2007a, b). Legro (1959) reported that C. rohlfsianum had chromosomes of 2nϭ96, the same as autotetraploid C. persicum (2nϭ4xϭ96), but he did not refer to the ploidy level of the examined plants. In our collection of C. rohlfsianum, we found that several plants had chromosomes more than 2nϭ96, although detailed studies of the chromosome numbers and ploidy levels have not yet been done. In the present study, we produced a hybrid of C. rohlfsianumϫC. purpurascens by ovule culture and explored the ploidy level of C. rohlfsianum through cytological observation and FCM using C. persicum, C. rohlfsianum, C. purpurascens and the C. rohlfsianumϫC. purpurascens hybrid. Materials and methods Plant materials Mature tubers of C. rohlfsianum (Cro2 and Cro3; each represents a different individual) and C. purpurascens (Cpu3), and seeds of C. rohlfsianum were introduced from Tile Burn Nursery and the Cyclamen Society in the UK, respectively. The tubers were maintained in a greenhouse using the method for native cyclamen, described by Grey-Wilson (2002). The seeds were sown after water absorption and kept in the dark for germination. The seedlings were then grown under the same culture conditions as plants grown from mature tubers and 12 flowering plants were obtained from the seedlings. Flowering plants of C. persicum ‘Schubert’ (Cpe2x) were introduced from a cyclamen grower in Saitama Prefecture in Japan. FCM Nuclear samples were prepared from mature leaves of Cpe2x, Cpu3, Cro2 and Cro3, and 12 seed-derived plants of C. rohlfsianum. Cpe2x and Cro2 were used as an internal standard for FCM. To release nuclei, leaf tissues (25 mm2) were chopped with a razor blade in 0.3 ml solution A of Plant High Resolution DNA kit type P (Partec, Münster, Germany) in a plastic Petri dish. The crude nuclear suspension was passed through a nylon filter of 30 mm mesh size, and added to 1.5 ml staining solution [10 mM Tris, 50 mM sodium citrate, 2 mM MgCl2, 1% (w/v) PVP, 0.1% (v/v) Triton X-100, and 2 mg/l DAPI]. After 5 min staining, the relative fluorescent intensity (RFI) of the nucleus was measured by FCM using a PA flow cytometer (Partec, Münster, Germany). Interspecific hybridization between C. rohlfsianum and C. purpurascens Cro2 was used as the seed parent and Cpu3 was used as the pollen parent. Crossed ovaries were collected 18 days after pollination and ovules with a placenta were removed aseptically and explanted to a test tube containing Murashige and Skoog (1962) medium supplemented with 30 glϪ1 sucrose, adjusted to pH 5.8 and solidified with 3 glϪ1 gellan gum, and maintained at 25°C in the dark. Plantlets germinated from the ovules were transferred to a jar containing the same medium 2010 Ploidy level of Cyclamen rohlfsianum 459 and maintained at 25°C under fluorescent illumination (34 m mol mϪ2 SϪ1) with a 16 h light period and 8 h dark period. Plantlets that developed normally through ovule culture were potted in vermiculite and kept in a moist environment for about 3 weeks. Thereafter, these plantlets were cultivated in a greenhouse using the standard method for cyclamen. Cytological analyses Root tips were collected from 2 plants of C. rohlfsianum (Cro2 and Cro3), 1 plant of C. purpurascens (Cpu3) and 1 hybrid of Cro2ϫCpu3, pretreated with 0.05% colchicine for 6 h at room temperature, and fixed in ethyl alcohol : acetic acid (3 : 1 v/v) for 24 h. Thereafter, root tips were stained with aceto-carmine for 30 min, heated over an alcohol flame for a few seconds and squashed with a coverslip in 45% (v/v) acetic acid. Flower buds at the appropriate stages were collected from Cro2, Cpu3 and the Cro2ϫCpu3 hybrid. The anthers were excised from these buds and squashed on a glass slide to liberate pollen mother cells (PMCs), which were macerated and stained with aceto-carmine for 5 min. PMCs were then warmed gently over an alcohol flame for a few seconds and squashed with a coverslip. Chromosome pairings at either diakinesis or metaphase were studied in 5 anthers per sample. Results Morphology, somatic chromosome number and relative fluorescent intensity (RFI) of C. rohlfsianum Cro2 and Cro3 of C. rohlfsianum originating from mature tubers had flowers with anthers and style projecting beyond the rim of the corolla, and ivy-shaped leaves with coarse serrate margin Fig. 1. Flower and leaf of C. rohlfsianum, C. purpurascens and their hybrid. A and B: C. purpurascens (Cpu3), C and D: hybrid of C. rohlfsianum (Cro2)ϫC. purpurascens (Cpu3), E and F: C. rohlfsianum (Cro2), G and H: C. rohlfsianum (Cro3). Arrow heads show stamen and style projecting beyond the rim of the corolla. Barsϭ2 cm (A, C, E, G). Barsϭ5 cm (B, D, F, H). 460 H. Ishizaka et al. Cytologia 74(4) (Fig. 1E, F, G, H). Cro2 and Cro3 had 96 and 144 chromosomes in the root tip cells, respectively, and the chromosome number of Cro3 was equal to 1.5 times of that of Cro2 (Fig. 2A, B). RFI of Cro2 and Cro3 was 1.78 and 2.54, respectively, provided that RFI of Cpe2x was 1.00, and RFI of Cro3 was 1.49, provided that RFI of Cro2 was 1.00 (Table 1, Fig. 4B, C). Consequently, Cro3 showed DNA content that was approx. 1.5 times higher than Cro2. Twelve plants of C. rohlfsianum raised from seeds introduced from the Cyclamen Society in the UK were classified into 2 groups with different RFI, that is, the RFI of 3 plants coincided with that of Cro2 (2nϭ96) and the RFI of 9 plants coincided with that of Cro3 (2nϭ144) (Table 1).