- Email: [email protected]
Ploidy estimation in Hemerocallis species and cultivars by flow cytometry
Scientia Horticulturae 97 (2003) 185–192
Short communication
Ploidy estimation in Hemerocallis species and cultivars by flow cytometry Hiroyuki Saitoa,b, Keiko Mizunashia, Shigefumi Tanakaa, Yukiko Adachia, Masaru Nakanoa,* a
Faculty of Agriculture, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan Plant Functions Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b
Accepted 10 August 2002
Abstract Ploidy level was estimated for nine species and 94 cultivars in the genus Hemerocallis by flow cytometry (FCM) analysis. By using parsley (Petroselinum crispum (Mill.) Nyman ex A.W. Hill) as an internal control, the ratio of the relative fluorescence intensity (RFI) of nuclei from each Hemerocallis genotype and parsley was calculated. The values (Hemerocallis/parsley ratio) for 103 Hemerocallis genotypes were clearly classified into three groups: 1.84–2.13 (group 1), 2.94–3.10 (group 2), and 3.74–4.26 (group 3). Since the diploid H. fulva var. littorea ð2n ¼ 22Þ and the triploid H. fulva var. kwanso ð2n ¼ 33Þ belonged to groups 1 and 2, respectively, the other Hemerocallis genotypes belonging to groups 1, 2 and 3 were considered to be di-, tri- and tetraploid, respectively. Chromosome counting in root tip cells revealed that Hemerocallis genotypes belonging to groups 1, 2 and 3 had 22, 33 and 44 chromosomes, respectively. No cytochimeras and polysomaty were observed. Thus, FCM analysis has proven useful for simple and rapid estimation of the ploidy level of Hemerocallis species and cultivars. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Chromosome counting; Daylily; Ploidy level
Hemerocallis spp., commonly called daylilies, are herbaceous perennials belonging to the family Liliaceae. They are very popular as garden plants for their attractive and showy flowers, high resistance to pests and diseases, and adaptability to a wide range of soils and climatic conditions. Hemerocallis spp. are also cultivated as a medicinal and vegetable in the Far East (Krikorian et al., 1990). Breeding of Hemerocallis spp. has so far been carried * Corresponding author. Present address: Plant Functions Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel./fax: þ81-25-262-6858. E-mail address: [email protected] (M. Nakano).
0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 1 5 0 - 4
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H. Saito et al. / Scientia Horticulturae 97 (2003) 185–192
out by inter- and intra-specific sexual hybridization (Baker, 1937; Krikorian et al., 1990; Tomkins et al., 2001) and polyploidization (Griesbach et al., 1963; Arisumi, 1964, 1972; Chen and Goeden-Kallemeyn, 1979), and numerous cultivars of varying ploidy levels have been produced (Krikorian et al., 1990; Baxter, 1999). The breeding efficiency of Hemerocallis spp. may strongly be enhanced by the knowledge of the ploidy level of breeding materials. Although microscopic chromosome counting in root tip cells has conventionally conducted to determine the ploidy level (Bennett and Smith, 1976; Dolezel, 1997), this process is laborious and time-consuming (De Laat et al., 1987; Dolezel, 1997). Recently flow cytometry (FCM) analysis has been alternatively applied for determining the ploidy level in a number of plant species such as Rhododendron spp. (De Schepper et al., 2001), Vaccinium spp. (Costich et al., 1993) and Actinidia spp. (Ollitrault-Sammarcelli et al., 1994). This method has the advantages of accuracy, convenience, simplicity, low cost and rapidly as compared with conventional chromosome counting (Galbraith et al., 1983; Arumuganathan and Earle, 1991; Dolezel, 1997), and thus a large number of samples can be analyzed in a short period. In the present study, we examined the applicability of FCM analysis for estimating the ploidy level in various Hemerocallis species and cultivars. Nine wild species and 94 cultivars of the genus Hemerocallis (Table 1) were analyzed in the present study. They were grown in the field of Faculty of Agriculture, Niigata University, Niigata, Japan. We also used parsley (Petroselinum crispum (Mill.) Nyman ex A.W. Hill) as an internal standard for FCM analysis. Basal tissues of young and healthy leaves were sampled from all Hemerocallis genotypes. Other tissues, i.e. middle and distal parts of leaves and roots, rhizomes, flower stalks and scapes, were also sampled from several genotypes. For parsley, mature and healthy leaves were sampled. Preparation of nuclei was carried out according to Saito and Nakano (2001) with several modifications. Sampled tissues (ca. 5 mm 5 mm) were chopped with a razor blade in 0.5 ml of solution A (Plant High Resolution DNA kit, Type P; Partec GmbH, Mu¨ nster, Germany) in a Petri dish. Following filtration with a 30 mm nylon mesh, 1.5 ml of a staining solution [10 mM 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), 50 mM sodium citrate, 2 mM MgCl2, 1% (v/w) polyvinylpyrolidone K-30 (PVP-K30), 0.1% (v/v) TRITON X-100 and 2 mg l1 4,6-diamidino-2-phenylindole (DAPI), pH 7.5] (Mishiba and Mii, 2000) was added to the nuclear suspensions. After staining for 5 min at room temperature, nuclear suspensions were subjected to measurement of the relative nuclear DNA content by using Partec PA (Partec GmbH) equipped with a mercury lamp and filter combinations of KG1, BG38, UG1, TK420 and GG435. Instrument gain was adjusted in order that the relative fluorescence intensity (RFI) of nuclei isolated from parsley leaves located at around channel 50. This calibration was carried out for every five samples in order to minimize variation due to runs and kept constant during analyses of samples prepared from Hemerocallis genotypes of unknown ploidy. At least 2000 nuclei were examined for each sample. A software package (Partec GmbH) was used for calculating the mean RFI of the peak and its coefficient of variation (CV). The ratio of RFI of Hemerocallis genotype and parsley was calculated for each measurement. Histograms obtained from basal tissues of young leaves of all Hemerocallis genotypes revealed a single peak of value with a CV of less than 3.0, which corresponded to nuclei in
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187
Table 1 Hemerocallis species and cultivars subjected to FCM analysis and the estimated ploidy levels 2 H. H. H. H. H. H.
dumortieri var. dumortieri dumortieri var. middendorffii fulva var. littorea fulva var. longituba fulva var. rosea hakuunensis
H. lilioasphodelus H. thunbergii Abbeville Sunset Bare Essence Beverly Hills Black Eyed Stella Brent Gabriel Carmen Marie Champagne Bauble Chorus Lime Clean Cut Coral Dawn Country Fellow Dot Le Blanc Eau La La Elsie Spalding Enchanting Blessing Far East Dream Glory In Ruffles Homeward Bound Howard Gliddem Memorial Ice Carnival Jade Tip Jason Salter John Yonski Jolyene Nichole Just For Laughs Keeno
2
3
4
Lalabel Lavender Tonic Lil Ledie Little Deeke Little Missy Little Strawberry Shortcake Midnight Splendor Minstrel Boy Monica Marie My Friend Nell Ono Optical Delight Oriental Ruby Pear Ornament Pixie parasol Pojo Pumpkin Kid Rainbow Connection Rosella Sheridan Sabie Sadie Lou Seductress Shaman Shibui Splendor Sir Galahad So Excited Solano Bulls Eye Spanish Masquerade Spinel Stella d’Oro Stop The Show Will Return
H. fulva var. kwanso Celtic Sunrise Forbidden City
Alvatine Taylor Amber Goddess Audacity Plus Blushing Lemon Bookmark Byzantine Mask Chinese Cloisonne Chinese Temple Flower Clemenceaux Comanche Drums Coral Rock Czarina Czars Treasure El Camino Gourmet Bouquet Hudson Valley Lucky Me Lunar Sea Mary Todd Matisse May Unger Mayan Poppy Nile Plum Palace Lantern Ramona Fay Red Inferno Scarlet Chalice Scarlet Orbit Seductor Serengeti Siamese Royalty Tamil Too Hot Venetian Crepe
the G0/G1 phase of the cell cycle. Little background noise was detected. Thus, the leaf basal tissues seem to be suitable for FCM analysis in Hemerocallis spp. All other tissues subjected to FCM analysis in the present study, i.e. middle and distal parts of leaves and roots, rhizomes, flower stalks and scapes, also showed a single peak of the same RFI as leaf basal tissues for each genotype (data not shown). Although cytochimeras (Va¨ ino¨ la¨ , 2000; Zonneveld and Iren, 2000; De Schepper et al., 2001) and polysomaty (Mishiba and Mii, 2000; Kudo and Kimura, 2001) have been detected by FCM analysis in several plant species, our results showed that neither cytochimeras nor polysomaty were found in Hemerocallis spp. Thus, all somatic tissues seem to consist of cells of the same ploidy level, and the single peak obtained by FCM analysis might reflect the given ploidy level in Hemerocallis spp.
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Typical histograms of RFI of nuclei isolated from leaf basal tissues of Hemerocallis genotypes as well as from leaf tissues of parsley are shown in Fig. 1. When the peak of RFI for parsley was set at around channel 50 (A), the peaks for Hemerocallis genotypes ranged from channel 92.6 to 213.4, but these were concentrated at around channel 100 (B), channel 150 (C) or channel 200 (D). When the peak for parsley was at channel 50.2, those for the diploid H. fulva var. littorea and the triploid H. fulva var. kwanso appeared at channel 102.0 and 152.4, respectively. Frequency distribution of the ratio of RFI of nuclei from each Hemerocallis genotype and parsley is shown in Fig. 2. The values (Hemerocallis/parsley ratio) for 103 Hemerocallis genotypes clearly classified into three groups: 1.84–2.13 (group 1), 2.94–3.10 (group 2), and 3.74–4.26 (group 3). Among 103 Hemerocallis genotypes analyzed, eight wild species and 58 cultivars belonged to group 1, one wild species and two cultivars did to group 2, and 34 cultivars did to group 3 (Table 1). H. fulva var. littorea and H. fulva var. kwanso are known as diploid and triploid, respectively (Kitamura and Tsukamoto, 1989), they belonged to groups 1 and 2, respectively. Therefore, the other Hemerocallis genotypes belonging to groups 1, 2 and 3 were considered to be di-, tri- and tetraploid, respectively (Table 1). For verifying the ploidy level, chromosome counting in root tip cells was performed for all Hemerocallis wild species and several cultivars randomly selected from each of group 1, 2 and 3. Root tips (ca. 1 cm) were incubated in water at 4 8C for 12 h and then fixed in ethanol:acetic acid (3:1, v/v) at 0 8C for over 24 h. After hydrolyzing with 1 N HCl:45% acetic acid (1:2, v/v) at 60 8C for 10–15 s, root tips were stained with 1% aceto-orcein and squashed under a cover glass. At least five root tips per plant were observed under a light microscope (LEICA DMLB; Leica, Wetzlar, Germany). Among nine wild species, H. fulva var. kwanso had 2n ¼ 33 chromosomes (Fig. 3B), and the other eight including H. fulva var. littorea had 2n ¼ 22 chromosomes (Fig. 3A). Cultivars belonging to groups 1, 2 and 3 had 2n ¼ 22, 33 and 44 (Fig. 3C) chromosomes, respectively. As the basic chromosome number of 11 has been demonstrated for Hemerocallis spp. (Stout, 1934; Brennan, 1992), Hemerocallis genotypes classified into groups 1, 2 and 3 were confirmed to be diploid (2), triploid (3) and tetraploid (4), respectively. Although H. hybrida cv. Gourmet Bouquet is registered as diploid and cvs. Monica Marie, Shaman, and Stop The Show as tetraploid in the Daylilies Online (http://www.assumption.edu/HTML/daylilies/about.html), cv. Gourmet Bouquet was verified to be tetraploid and cvs. Monica Marie, Shaman, and Stop The Show were verified to be diploid in the present study (Table 1). In addition, cvs. Celtic Sunrise and Forbidden City, both of which are registered as diploid in the Daylilies Online, were found to be triploid in the present study (Table 1). In the present study, the ploidy level of Hemerocallis spp. could be estimated by FCM analysis using parsley leaves as an internal control. The accuracy of FRI by FCM analyses can be improved by using an internal control (Arumuganathan and Earle, 1991). Parsley leaves used here seem to be a suitable control for FCM analysis of Hemerocallis spp., because its RFI was nearly the same as the half (one genome) of that of the diploid Hemerocallis genotypes. FCM analysis has proven useful for simple and rapid estimation of the ploidy level of a large number of Hemerocallis species and cultivars. This method might be used in
H. Saito et al. / Scientia Horticulturae 97 (2003) 185–192
189
Fig. 1. Histograms from FCM analyses of nuclear DNA content of Hemerocallis spp. DNA content is expressed as RFI on the horizontal axis, and the number of nuclei on the vertical axis: (A) parsley; (B) H. fulva var. littorea; (C) H. fulva var. kwanso; (D) H. hybrida cv. Scarlet Orbit.
190 H. Saito et al. / Scientia Horticulturae 97 (2003) 185–192 Fig. 2. Frequency distribution of the ratio of RFI of nuclei from each Hemerocallis genotype and parsley. The values (Hemerocallis/parsley ratio) were classified into three groups.
H. Saito et al. / Scientia Horticulturae 97 (2003) 185–192
191
Fig. 3. Microphotographs showing root tip cells of Hemerocallis genotypes at metaphase: (A) H. fulva var. littorea ð2n ¼ 22Þ; (B) H. fulva var. kwanso ð2n ¼ 33Þ; (C) H. hybrida cv. Scarlet Orbit ð2n ¼ 44Þ.
association with Hemerocallis breeding, especially as a very early and easy marker for ploidy manipulations such as polyploidization, haploidization and somatic hybridization. Also, FCM analysis might be suitable for the detection of mixoploidy as well as for the determination of the ploidy level in progenies of conventional intra- and inter-specific crosses.
Acknowledgements This work was supported in part by a grant-in-aid for Scientific Research (no. 13660024) from the Ministry of Education, Science and Culture, Japan, and by Sasakawa Scientific Research Grant from the Japan Science Society.
References Arisumi, T., 1964. Colchicine-induced tetraploid and cytochimeral daylilies. J. Hered. 55, 254–261. Arisumi, T., 1972. Stabilities of colchicine-induced tetraploid and cytochemical daylilies. J. Hered. 63, 15–18. Arumuganathan, K., Earle, E.D., 1991. Estimation of nuclear DNA content of plants by flow cytometry. Plant Mol. Biol. Rep. 9, 229–241. Baker, V.M.H., 1937. Hemerocallis: the day lily. J. Roy. Hortic. Soc. 62, 399–411. Baxter, G., 1999. Hemerocallis check list 1994–1998. Am. Hemerocallis Soc. Bennett, M.D., Smith, J.B., 1976. Nuclear DNA amounts in angiosperms. Phil. Trans. Roy. Soc. Lond. Ser. B 274, 227–274. Brennan, J.R., 1992. Chromosomes of Hemerocallis. Daylily J. 47, 73–77. Chen, C.H., Goeden-Kallemeyn, Y., 1979. In vitro induction of tetraploid plants from colchicine-treated diploid daylily callus. Euphytica 28, 705–709. Costich, D.E., Ortiz, R., Meagher, T.R., Bruederle, L.P., Vorsa, N., 1993. Determination of ploidy level and nuclear DNA content in blueberry by flow cytometry. Theor. Appl. Genet. 86, 1001–1006. De Laat, A.M.M., Go¨ hde, W., Vogelzang, M.J.D.C., 1987. Determination of ploidy of single plants and plant populations by flow cytometry. Plant Breed. 99, 303–307. De Schepper, S., Leus, L., Mertens, E., Van Bockstaele, E., De Loose, M., Debergh, P., Heursel, J., 2001. Flow cytometric analysis of ploidy in Rhododendron (subgenus Tsutsusi). HortScience 36, 125–127. Dolezel, J., 1997. Application of flow cytometry for the study of plant genomes. J. Appl. Genet. 38, 285–302.
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Galbraith, D.W., Harkins, K.R., Maddox, J.M., Ayres, N.M., Sharma, D.P., Firozabady, E., 1983. Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science 220, 1049–1051. Griesbach, R.A., Fay, O.W., Gorsfall, L., 1963. Induction of polyploidy in newly-germinated Hemerocallis seedling. Hemerocallis J. 17, 70–75. Kitamura, S., Tsukamoto, Y., 1989. Hemerocallis spp. In: Tsukamoto, Y. (Ed.), The Grand Dictionary of Horticulture, vol. 4. Shogakukan, Tokyo, pp. 358–361 (in Japanese). Krikorian, A.D., Kann, R.P., Corbin, M.S.F., 1990. Daylily. In: Ammirato, P.V., Evans, D.A., Sharp, W.P., Bajaj, Y.P.S. (Eds.), Handbook of Plant Cell Culture, vol. 5, Ornamental Species. Springer, New York, pp. 285–293. Kudo, N., Kimura, Y., 2001. Flow cytometric evidence for endopolyploidy in seedlings of some Brassica species. Theor. Appl. Genet. 102, 104–110. Mishiba, K., Mii, M., 2000. Polysomaty analysis in diploid and tetraploid Portulaca grandifolia. Plant Sci. 156, 213–219. Ollitrault-Sammarcelli, F., Legave, J.M., Michaux-Ferriere, N., Hirsch, A.M., 1994. Use of flow cytometry for rapid determination of ploidy level in the genus Actinidia. Sci. Hortic. 57, 303–313. Saito, H., Nakano, M., 2001. Flow cytometric analysis of partially synchronized suspension cultures of Hemerocallis hybrida. Plant Biotechnol. 18, 229–231. Stout, A.B., 1934. Daylilies: The Wild Species and Garden Clones, Both Old and New, of the Genus Hemerocallis. Macmillan, New York. Tomkins, J.P., Wood, T.C., Barnes, L.S., Westman, A., Wing, R.A., 2001. Evaluation of genetic variation in the daylily (Hemerocallis spp.) using AFLP marker. Theor. Appl. Genet. 102, 489–496. Va¨ ino¨ la¨ , A., 2000. Polyploidization and early screening of Rhododendron hybrids. Euphytica 112, 239–244. Zonneveld, B.J.M., Iren, F.V., 2000. Flow cytometric analysis of DNA content in Hosta reveals ploidy chimeras. Euphytica 111, 105–110.
Short communication
Ploidy estimation in Hemerocallis species and cultivars by flow cytometry Hiroyuki Saitoa,b, Keiko Mizunashia, Shigefumi Tanakaa, Yukiko Adachia, Masaru Nakanoa,* a
Faculty of Agriculture, Niigata University, 2-8050 Ikarashi, Niigata 950-2181, Japan Plant Functions Laboratory, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
b
Accepted 10 August 2002
Abstract Ploidy level was estimated for nine species and 94 cultivars in the genus Hemerocallis by flow cytometry (FCM) analysis. By using parsley (Petroselinum crispum (Mill.) Nyman ex A.W. Hill) as an internal control, the ratio of the relative fluorescence intensity (RFI) of nuclei from each Hemerocallis genotype and parsley was calculated. The values (Hemerocallis/parsley ratio) for 103 Hemerocallis genotypes were clearly classified into three groups: 1.84–2.13 (group 1), 2.94–3.10 (group 2), and 3.74–4.26 (group 3). Since the diploid H. fulva var. littorea ð2n ¼ 22Þ and the triploid H. fulva var. kwanso ð2n ¼ 33Þ belonged to groups 1 and 2, respectively, the other Hemerocallis genotypes belonging to groups 1, 2 and 3 were considered to be di-, tri- and tetraploid, respectively. Chromosome counting in root tip cells revealed that Hemerocallis genotypes belonging to groups 1, 2 and 3 had 22, 33 and 44 chromosomes, respectively. No cytochimeras and polysomaty were observed. Thus, FCM analysis has proven useful for simple and rapid estimation of the ploidy level of Hemerocallis species and cultivars. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Chromosome counting; Daylily; Ploidy level
Hemerocallis spp., commonly called daylilies, are herbaceous perennials belonging to the family Liliaceae. They are very popular as garden plants for their attractive and showy flowers, high resistance to pests and diseases, and adaptability to a wide range of soils and climatic conditions. Hemerocallis spp. are also cultivated as a medicinal and vegetable in the Far East (Krikorian et al., 1990). Breeding of Hemerocallis spp. has so far been carried * Corresponding author. Present address: Plant Functions Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Tel./fax: þ81-25-262-6858. E-mail address: [email protected] (M. Nakano).
0304-4238/02/$ – see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 1 5 0 - 4
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out by inter- and intra-specific sexual hybridization (Baker, 1937; Krikorian et al., 1990; Tomkins et al., 2001) and polyploidization (Griesbach et al., 1963; Arisumi, 1964, 1972; Chen and Goeden-Kallemeyn, 1979), and numerous cultivars of varying ploidy levels have been produced (Krikorian et al., 1990; Baxter, 1999). The breeding efficiency of Hemerocallis spp. may strongly be enhanced by the knowledge of the ploidy level of breeding materials. Although microscopic chromosome counting in root tip cells has conventionally conducted to determine the ploidy level (Bennett and Smith, 1976; Dolezel, 1997), this process is laborious and time-consuming (De Laat et al., 1987; Dolezel, 1997). Recently flow cytometry (FCM) analysis has been alternatively applied for determining the ploidy level in a number of plant species such as Rhododendron spp. (De Schepper et al., 2001), Vaccinium spp. (Costich et al., 1993) and Actinidia spp. (Ollitrault-Sammarcelli et al., 1994). This method has the advantages of accuracy, convenience, simplicity, low cost and rapidly as compared with conventional chromosome counting (Galbraith et al., 1983; Arumuganathan and Earle, 1991; Dolezel, 1997), and thus a large number of samples can be analyzed in a short period. In the present study, we examined the applicability of FCM analysis for estimating the ploidy level in various Hemerocallis species and cultivars. Nine wild species and 94 cultivars of the genus Hemerocallis (Table 1) were analyzed in the present study. They were grown in the field of Faculty of Agriculture, Niigata University, Niigata, Japan. We also used parsley (Petroselinum crispum (Mill.) Nyman ex A.W. Hill) as an internal standard for FCM analysis. Basal tissues of young and healthy leaves were sampled from all Hemerocallis genotypes. Other tissues, i.e. middle and distal parts of leaves and roots, rhizomes, flower stalks and scapes, were also sampled from several genotypes. For parsley, mature and healthy leaves were sampled. Preparation of nuclei was carried out according to Saito and Nakano (2001) with several modifications. Sampled tissues (ca. 5 mm 5 mm) were chopped with a razor blade in 0.5 ml of solution A (Plant High Resolution DNA kit, Type P; Partec GmbH, Mu¨ nster, Germany) in a Petri dish. Following filtration with a 30 mm nylon mesh, 1.5 ml of a staining solution [10 mM 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), 50 mM sodium citrate, 2 mM MgCl2, 1% (v/w) polyvinylpyrolidone K-30 (PVP-K30), 0.1% (v/v) TRITON X-100 and 2 mg l1 4,6-diamidino-2-phenylindole (DAPI), pH 7.5] (Mishiba and Mii, 2000) was added to the nuclear suspensions. After staining for 5 min at room temperature, nuclear suspensions were subjected to measurement of the relative nuclear DNA content by using Partec PA (Partec GmbH) equipped with a mercury lamp and filter combinations of KG1, BG38, UG1, TK420 and GG435. Instrument gain was adjusted in order that the relative fluorescence intensity (RFI) of nuclei isolated from parsley leaves located at around channel 50. This calibration was carried out for every five samples in order to minimize variation due to runs and kept constant during analyses of samples prepared from Hemerocallis genotypes of unknown ploidy. At least 2000 nuclei were examined for each sample. A software package (Partec GmbH) was used for calculating the mean RFI of the peak and its coefficient of variation (CV). The ratio of RFI of Hemerocallis genotype and parsley was calculated for each measurement. Histograms obtained from basal tissues of young leaves of all Hemerocallis genotypes revealed a single peak of value with a CV of less than 3.0, which corresponded to nuclei in
H. Saito et al. / Scientia Horticulturae 97 (2003) 185–192
187
Table 1 Hemerocallis species and cultivars subjected to FCM analysis and the estimated ploidy levels 2 H. H. H. H. H. H.
dumortieri var. dumortieri dumortieri var. middendorffii fulva var. littorea fulva var. longituba fulva var. rosea hakuunensis
H. lilioasphodelus H. thunbergii Abbeville Sunset Bare Essence Beverly Hills Black Eyed Stella Brent Gabriel Carmen Marie Champagne Bauble Chorus Lime Clean Cut Coral Dawn Country Fellow Dot Le Blanc Eau La La Elsie Spalding Enchanting Blessing Far East Dream Glory In Ruffles Homeward Bound Howard Gliddem Memorial Ice Carnival Jade Tip Jason Salter John Yonski Jolyene Nichole Just For Laughs Keeno
2
3
4
Lalabel Lavender Tonic Lil Ledie Little Deeke Little Missy Little Strawberry Shortcake Midnight Splendor Minstrel Boy Monica Marie My Friend Nell Ono Optical Delight Oriental Ruby Pear Ornament Pixie parasol Pojo Pumpkin Kid Rainbow Connection Rosella Sheridan Sabie Sadie Lou Seductress Shaman Shibui Splendor Sir Galahad So Excited Solano Bulls Eye Spanish Masquerade Spinel Stella d’Oro Stop The Show Will Return
H. fulva var. kwanso Celtic Sunrise Forbidden City
Alvatine Taylor Amber Goddess Audacity Plus Blushing Lemon Bookmark Byzantine Mask Chinese Cloisonne Chinese Temple Flower Clemenceaux Comanche Drums Coral Rock Czarina Czars Treasure El Camino Gourmet Bouquet Hudson Valley Lucky Me Lunar Sea Mary Todd Matisse May Unger Mayan Poppy Nile Plum Palace Lantern Ramona Fay Red Inferno Scarlet Chalice Scarlet Orbit Seductor Serengeti Siamese Royalty Tamil Too Hot Venetian Crepe
the G0/G1 phase of the cell cycle. Little background noise was detected. Thus, the leaf basal tissues seem to be suitable for FCM analysis in Hemerocallis spp. All other tissues subjected to FCM analysis in the present study, i.e. middle and distal parts of leaves and roots, rhizomes, flower stalks and scapes, also showed a single peak of the same RFI as leaf basal tissues for each genotype (data not shown). Although cytochimeras (Va¨ ino¨ la¨ , 2000; Zonneveld and Iren, 2000; De Schepper et al., 2001) and polysomaty (Mishiba and Mii, 2000; Kudo and Kimura, 2001) have been detected by FCM analysis in several plant species, our results showed that neither cytochimeras nor polysomaty were found in Hemerocallis spp. Thus, all somatic tissues seem to consist of cells of the same ploidy level, and the single peak obtained by FCM analysis might reflect the given ploidy level in Hemerocallis spp.
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Typical histograms of RFI of nuclei isolated from leaf basal tissues of Hemerocallis genotypes as well as from leaf tissues of parsley are shown in Fig. 1. When the peak of RFI for parsley was set at around channel 50 (A), the peaks for Hemerocallis genotypes ranged from channel 92.6 to 213.4, but these were concentrated at around channel 100 (B), channel 150 (C) or channel 200 (D). When the peak for parsley was at channel 50.2, those for the diploid H. fulva var. littorea and the triploid H. fulva var. kwanso appeared at channel 102.0 and 152.4, respectively. Frequency distribution of the ratio of RFI of nuclei from each Hemerocallis genotype and parsley is shown in Fig. 2. The values (Hemerocallis/parsley ratio) for 103 Hemerocallis genotypes clearly classified into three groups: 1.84–2.13 (group 1), 2.94–3.10 (group 2), and 3.74–4.26 (group 3). Among 103 Hemerocallis genotypes analyzed, eight wild species and 58 cultivars belonged to group 1, one wild species and two cultivars did to group 2, and 34 cultivars did to group 3 (Table 1). H. fulva var. littorea and H. fulva var. kwanso are known as diploid and triploid, respectively (Kitamura and Tsukamoto, 1989), they belonged to groups 1 and 2, respectively. Therefore, the other Hemerocallis genotypes belonging to groups 1, 2 and 3 were considered to be di-, tri- and tetraploid, respectively (Table 1). For verifying the ploidy level, chromosome counting in root tip cells was performed for all Hemerocallis wild species and several cultivars randomly selected from each of group 1, 2 and 3. Root tips (ca. 1 cm) were incubated in water at 4 8C for 12 h and then fixed in ethanol:acetic acid (3:1, v/v) at 0 8C for over 24 h. After hydrolyzing with 1 N HCl:45% acetic acid (1:2, v/v) at 60 8C for 10–15 s, root tips were stained with 1% aceto-orcein and squashed under a cover glass. At least five root tips per plant were observed under a light microscope (LEICA DMLB; Leica, Wetzlar, Germany). Among nine wild species, H. fulva var. kwanso had 2n ¼ 33 chromosomes (Fig. 3B), and the other eight including H. fulva var. littorea had 2n ¼ 22 chromosomes (Fig. 3A). Cultivars belonging to groups 1, 2 and 3 had 2n ¼ 22, 33 and 44 (Fig. 3C) chromosomes, respectively. As the basic chromosome number of 11 has been demonstrated for Hemerocallis spp. (Stout, 1934; Brennan, 1992), Hemerocallis genotypes classified into groups 1, 2 and 3 were confirmed to be diploid (2), triploid (3) and tetraploid (4), respectively. Although H. hybrida cv. Gourmet Bouquet is registered as diploid and cvs. Monica Marie, Shaman, and Stop The Show as tetraploid in the Daylilies Online (http://www.assumption.edu/HTML/daylilies/about.html), cv. Gourmet Bouquet was verified to be tetraploid and cvs. Monica Marie, Shaman, and Stop The Show were verified to be diploid in the present study (Table 1). In addition, cvs. Celtic Sunrise and Forbidden City, both of which are registered as diploid in the Daylilies Online, were found to be triploid in the present study (Table 1). In the present study, the ploidy level of Hemerocallis spp. could be estimated by FCM analysis using parsley leaves as an internal control. The accuracy of FRI by FCM analyses can be improved by using an internal control (Arumuganathan and Earle, 1991). Parsley leaves used here seem to be a suitable control for FCM analysis of Hemerocallis spp., because its RFI was nearly the same as the half (one genome) of that of the diploid Hemerocallis genotypes. FCM analysis has proven useful for simple and rapid estimation of the ploidy level of a large number of Hemerocallis species and cultivars. This method might be used in
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Fig. 1. Histograms from FCM analyses of nuclear DNA content of Hemerocallis spp. DNA content is expressed as RFI on the horizontal axis, and the number of nuclei on the vertical axis: (A) parsley; (B) H. fulva var. littorea; (C) H. fulva var. kwanso; (D) H. hybrida cv. Scarlet Orbit.
190 H. Saito et al. / Scientia Horticulturae 97 (2003) 185–192 Fig. 2. Frequency distribution of the ratio of RFI of nuclei from each Hemerocallis genotype and parsley. The values (Hemerocallis/parsley ratio) were classified into three groups.
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Fig. 3. Microphotographs showing root tip cells of Hemerocallis genotypes at metaphase: (A) H. fulva var. littorea ð2n ¼ 22Þ; (B) H. fulva var. kwanso ð2n ¼ 33Þ; (C) H. hybrida cv. Scarlet Orbit ð2n ¼ 44Þ.
association with Hemerocallis breeding, especially as a very early and easy marker for ploidy manipulations such as polyploidization, haploidization and somatic hybridization. Also, FCM analysis might be suitable for the detection of mixoploidy as well as for the determination of the ploidy level in progenies of conventional intra- and inter-specific crosses.
Acknowledgements This work was supported in part by a grant-in-aid for Scientific Research (no. 13660024) from the Ministry of Education, Science and Culture, Japan, and by Sasakawa Scientific Research Grant from the Japan Science Society.
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