Regular Article Stable Chromosome Number in Horticultural

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Chromosome Science 14: 53-62, 2011 Okuno et al. 53

Regular Article

Stable chromosome number in horticultural cultivars of Farfugium japonicum (Asteraceae), with descriptions of their morphological characteristics

Hajime Okuno, Masashi Nakata and Masahiro Mii

Abstract Introduction A total of 111 horticultural cultivars of Farfugium ja- Farfugium japonicum (L.) Kitam. (Asteraceae), Japanese ponicum were studied cytologically and described mor- name: ‘Tsuwabuki’, is a perennial herb native to Japan, phologically for the first time. All 111 cultivars with South Korea, Taiwan and the middle part of China various unusual morphologies, such as dwarf, rumpled (Kitamura 1981). In Japan it grows on rocky coastal cliffs in leaves, etc., had 2n=60 chromosomes, which was the Honshu (southwestern part), Shikoku, Kyushu, Ryukyu and same number as in wild plants. Only one individual ob- Ogasawara Islands (Horikawa 1972, Koyama 1995). Wild tained by tissue culture of the cultivar ‘Yukibeni-botan’ plants of F. japonicum have been used as a garden plant had exceptional chromosome number with 2n=59. In since the Edo period (Kaibara 1694), and some cultivars 14 cultivars with 2n=60 chromosomes examined, no ab- showing morphological variations or variegation in leaves normal chromosome behavior was observed through have been described in the literatures published more than the microsporogenesis, and at the meiotic metaphase 180 years ago (Iwasaki 1828, Mizuno 1829). After that, I, 30 bivalents were observed. Among the flowered 96 these variants have been preserved mainly by collectors. individuals in 95 cultivars, seven individuals had no pol- The number of cultivars has gradually increased and 73 len grains, and those having atrophied head, such as variants were listed in “Hana-ninki” (2001). Now more “SHISHI” and “KURUMA”, showed relatively low pollen than one hundred cultivars are known (Okuno et al. 2007). stainabilities, 48.3 and 23.4% in average, respectively, Chromosome numbers of wild F. japonicum have whereas the other cultivars had high stainability (91.3% been reported to be n=30 (Ishikawa 1916, as Ligularia in average). The 2n=59 somaclonal variant of the cultivar tussilaginea) and 2n=60 for the plants from Japan (Arano ‘Yukibeni-botan’ showed the lowest stainability (0.3%). 1962, Miyagi 1971, Okuno et al. 2005), Taiwan (Hsu These results indicate that horticultural cultivars of F. ja- 1970) and China (Su and Liu 1995, Liu 2001). Recently we ponicum are stable diploids from the cytogenetical view- examined chromosome number for 190 individuals of wild point, and that the morphological diversity in the culti- F. japonicum, including four taxonomic varieties, from 68 vars should not be ascribed to the numerical changes of localities in 31 prefectures of Japan and two localities in chromosomes but must have been caused by mutation Taiwan. The all individuals had the chromosome number of gene(s) responsible for morphogenesis. of 2n=2x=60 with high pollen stainability (93.9±8.8%) except for one spontaneous aneuploid (2n=61) (Okuno et al. 2009). However, no cytological studies have been Key words: Asteraceae, chromosome number, Farfugi- conducted yet on horticultural cultivars of this species. um japonicum, horticultural cultivar, pollen stainability Thus, in this paper, we describe the results of the studies on chromosome number and pollen fertility of 111 horticultural cultivars in F. japonicum with artificial classification and brief descriptions of the morphology of them. Hajime Okuno 3250-201 Fukaisawa-machi, Naka-ku, Sakai, Osaka 599-8236, Materials and Methods Japan Masashi Nakata (*) The cultivars of F. japonicum used in this study had Botanic Gardens of Toyama, 42 Kamikutsuwada, Fuchu-machi, been collected by the first author in the last 25 years from Toyama 939-2713, Japan nurseries or private collectors of the species throughout Tel: +81-76-466-4187, Fax: +81-76-465-5923 Japan. The 112 individuals of 111 horticultural cultivars e-mail: [email protected] as shown in Table 1 had been cultivated in pots at the Masahiro Mii first author’s private garden at least for five years under Laboratory of Plant Cell Technology, Graduate School of Horticul- continuous observations. Vouchers have been preserved at ture, Chiba University, 648 Matsudo, Matsudo, Chiba 271-8510, Ja- the Botanic Gardens of Toyama (TYM). pan For mitotic and meiotic chromosome observation, fresh 54 Chromosome number of horticultural cultivars of Farfugium japonicum

Table 1. Characteristics, chromosome numbers, pollen stainabilities, and vouchers of the horticultural cultivars of Farfugium japonicum examined. Chromo- some Pollen Cultivar Characteristics number stainability Voucher (2n) (%) Group Ⅰ . “SHISHI (BOTAN)” : Leaf blade rolled; margin crisped and waved. Head atrophied 1. ‘Midori-hime’ Leaf blade slightly rolled; margin rough crisped, low waved. Head at- 60 28.1 HO5084 rophied. 2. ‘Hira-jishi’ Leaf blade slightly (or no) rolled; margin rough crisped, medially 60 62.3 HO5023 waved. Head atrophied. 3. ‘Mai-jishi’ Leaf blade slightly rolled; margin rough crisped, highly waved. Head 60 44.7 HO5027 (Fig. 1-1) atrophied. 4. ‘Genkai-daruma-jishi’ Leaf blade slightly rolled; margin rough crisped, highly waved; peti- 60 78.1 HO5045 ole highly fasciated. Head atrophied. 5. ‘Sekka-botan-jishi’ Leaf blade medially rolled; margin rough crisped, highly waved; peti- 60 93.6 HO5015 ole highly fasciated. Head atrophied. 6. ‘Daruma-jishi’ Leaf blade medially rolled; margin rough crisped, highly waved; peti- 60 67.7 HO5014 (Fig. 1-2) ole extreamly fasciated. Head atrophied. 7. ‘Hotei-sama’ Leaf blade medially rolled; margin medially crisped, highly waved; 60 34.8 HO5049 petiole short, etreamly fasciated. Head atrophied. 8. ‘Kumano-jishi’ Leaf blade slightly rolled; upper surface bearing leaf-like prominence; 60 not flowered HO5086 margin medially crisped, low waved. 9. ‘Uzushio’ Leaf blade slightly rolled; margin medially crisped, medially waved. 60 54.0 HO5001 Head atrophied. 10. ‘Shirahama-jishi’ Leaf blade slightly rolled; margin medially crisped, highly waved. 60 48.6 HO5016 Head atrophied. 11. ‘Genkotsu’ Leaf blade medially rolled; margin medially crisped, highly waved; 60 13.4 HO5051 petiole highly fasciated. Head atrophied. 12. ‘Yukibeni-botan’ Leaf blade slightly rolled; margin fine crisped, medially waved. Head 60 46.1 HO5029 atrophied. 59 0.3 HO5024 13. ‘Satsuma-botan’ Leaf blade slightly rolled; margin fine crisped, medially waved. Head 60 7.7 HO5012 atrophied. 14. ‘Yukibeni-henge’ Leaf blade slightly rolled; margin fine crisped, medially waved; peti- 60 no pollen HO5064 ole subbranched. Head atrophied. 15. ‘Unryu-jishi’ Leaf blade slightly rolled, recurved; margin fine crisped, medially 60 no pollen HO5071 waved; petiole subbranched. Head atrophied. 16. ‘Kirin-ju’ Leaf blade slightly rolled; margin fine crisped, highly waved; petiole 60 no pollen HO5006 subbranched. Head atrophied. Group Ⅱ . “KURUMA (FUGIRE)”: Leaf blade incised 17. ‘Fukuju-botan’ Leaf blade medially rolled, medially and fine incised; lower surface 60, 30 II 23.4 HO5040 (Fig. 1-3)" bearing hook-like prominence; margin highly waved. Head atrophied. 18. ‘Temari’ Leaf blade medially rolled, deeply and fine incised; lower surface 60 no pollen HO5041 bearing hook-like prominence; margin highly waved. Head atrophied. Group Ⅲ .“NOKOGIRI (TSUNO-DASHI)”: Leaf margin dentate 19. ‘Ryukaku’ Leaf margin low- and rough dentate, slightly waved; petiole short, 60 93.4 HO5063 (Fig. 1-4) fasciated. 20. ‘Hakkaku-uryu’ Leaf margin rough dentate; lower surface bearing hook-like promi- 60 93.3 HO5077 (Fig. 1-5) nence. 21. ‘Ura-no-mai’ Leaf margin low- and rough dentate, low waved; blade slightly rolled; 60 92.2 HO5067 petiole short, fasciated. 22. ‘Oya-jishi’ Leaf margin low- and densely dentate, low waved; blade slightly 60 97.9 HO5002 rolled; petiole short, fasciated. 23. ‘Hime-daruma’ Leaf margin densely dentate, low waved; blade slightly rolled; petiole 60 93.3 HO5022 short, fasciated. 24. ‘Sakae-jishi’ Leaf margin medially dentate, slightly waved; blade slightly rolled; 60 97.3 HO5011 petiole short, fasciated. 25. ‘Ryujin’ Leaf margin medially dentate, medially waved; blade slightly rolled; 60, 30 II 95.8 HO5034 petiole short, fasciated. 26. ‘Genkai-jishi’ Leaf margin densely dentate, low waved; blade slightly rolled; petiole 60 97.2 HO5048 short, fasciated. 27. ‘Kushira-jishi’ Leaf margin densely dentate, medially waved; blade slightly rolled; 60 95.4 HO5007 petiole short, fasciated. 28. ‘Fuku-manryo’ Leaf margin densely dentate, highly waved; blade rolled; petiole 60 98.7 HO5025 short, highly fasciated. 29. ‘Hishakaku’ Leaf margin large- and rough dentate, highly waved; blade outward- 60 not flowered HO5020 bended at the margin. Okuno et al. 55

Table 1. (continued) Chromo- some Pollen Cultivar Characteristics number stainability Voucher (2n) (%) 30. ‘Ryuho’ Leaf margin large- and rough dentate, slightly waved; blade 60 92.7 HO5069 (Fig. 1-6) slightly rolled; petiole short, fasciated. 31. ‘Hime-shinryu’ Leaf margin large- and rough dentate, low waved; blade slightly 60 93.7 HO5021 rolled; petiole short, fasciated. 32. ‘Keikan’ Leaf margin large- and rough dentate, medially waved; blade 60, 30 II 99.2 HO5008 (Fig. 1-7) slightly rolled; petiole short, fasciated. 33. ‘Fuku-daruma’ Leaf margin large- and rough dentate, highly waved; blade slight- 60 95.5 HO5073 ly rolled; petiole short, fasciated. 34. ‘Kyoshiba’ Leaf margin large- and medially dentate. 60, 30 II 94.2 HO5037 35. ‘Kojin’ Leaf margin large- and densely dentate, highly waved; blade 60 91.9 HO5009 slightly rolled; petiole short, fasciated. 36. ‘Shinryu’ Leaf margin large- and very densely dentate, highly waved; blade 60, 30 II 75.6 HO5017 slightly rolled; petiole short, fasciated. Group Ⅳ . “RASHA”: Leaf blade rough-surfaced 37. ‘Itohime’ Extreamly dwarf. Leaf blade round in shape, small, rough-sur- 60 not flowered HO5044 (Fig. 1-8) faced. 38. ‘Raijin’ Leaf blade rough-surfaced; margin low waved. 60 80.6 HO5075 39. ‘Bonten’ Dwarf. Leaf blade rough-surfaced; margin low waved. 60 not flowered HO5076 40. ‘Rashomon’ Dwarf. Leaf blade irregular in shape, rough-surfaced. 60 not flowered HO5032 (Fig. 1-9)" 41. ‘Kazanho’ Leaf blade pitcher-shaped, rough-surfaced, bearing rod-like 60, 30 II 97.7 HO5004 prominence at base; margin outward-bended. Group Ⅴ . “CHIRIMEN”: Leaf blade rumpled, outward-bended at the margin 42. ‘Ryumen’ Leaf blade rumpled, slightly rolled; margin outward-bended. 60 not flowered HO5066 43. ‘Yozan’ Leaf blade irregular in shape, fine rumpled; margin exceptionally 60 not flowered HO5031 no bending. 44. ‘Aka-tombo’ Leaf blade rumpled; margin outward-bended; petiole brown-col- 60 93.9 HO5074 or. Ray floret fimbriate. 45. ‘Beni-koryu’ Leaf blade rumpled; margin outward-bended; upper surface 60 not flowered HO5026 (Fig. 1-10) bearing needle-like prominence. 46. ‘Kimen’ Dwarf. Leaf blade rumpled; margin outward-bended. Ray floret 60 not flowered HO5005 fimbriate. 47. ‘Beni-tatsugashira’ Leaf blade rumpled; margin outward-bended; petiole long. 60 not flowered HO9001 48. ‘Kaun’ Leaf blade rumpled; margin outward-bended. Head atrophied. 60 98.0 HO5043 (Fig. 1-11) Ray floret fimbriate. 49. ‘Yamakuno’ Leaf blade rumpled; margin outward-bended. Head atrophied. 60 97.2 HO5042 Ray floret fimbriate. 50. ‘Tatsugashira’ Leaf blade rumpled; margin outward-bended; upper surface 60 90.8 HO5019 bearing rod-like prominence. Head atrophied. Ray floret fimbriate. 51. ‘Hoo-ryu’ Leaf blade narrow, rumpled; margin outward-bended. 60 not flowered HO5079 52. ‘Ryusen-no-mai’ Leaf blade rumpled, slightly rolled; margin large- and densely 60 26.1 HO5035 (Fig. 1-12) dentate, highly weved; outward-bended; upper surface bearing rod-like prominence; petiole short, fasciated. Ray floret fimbriate. Group Ⅵ . “EDAWAKARE”: Composed leaf 53. ‘Clover’ Leaflets 2-4; margin low- and rough dentate, low waved. 60 97.0 HO5060 54. ‘Miwaku’ Leaflets 2-4, sometimes simple; margin low- and rough dentate, 60, 30 II 87.4 HO5028 low waved, outward-bended; blade slightly rolled; petiole long. 55. ‘Shishinden’ Leaflets 2-4, sometimes simple; margin low- and rough dentate, 60 95.7 HO5072 (Fig. 1-13) low waved, outward-bended; blade slightly rolled. 56. ‘Hime-clover’ Dwarf. Leaflets 2-4; margin low- and rough dentate, low waved. 60, 30 II 99.2 HO5036 57. ‘Goryu’ Leaflets 3-5; margin large- and rough dentate, low waved; lower 60 94.3 HO5082 surface of blade bearing hook-like prominence. 58. ‘Kara-kaede’ Leaflets 2-5; margin large- and rough dentate, low waved; lower 60 90.5 HO9022 surface of blade bearing hook-like prominence. Head double flowering “Anemone-flowered type”. 59. ‘Amadare’ Leaflets 2-4, sometimes simple; margin large- and rough dentate, 60 95.4 HO5083 low waved; blade slightly rolled; lower surface bearing hook-like prominence. 60. ‘Seiryukaku’ Leaflets 2-4, sometimes simple; margin large- and rough dentate, 60 97.2 HO5018 highly waved, outward bended; blade slightly rolled. Group Ⅶ . Other variation in leaves 61. ‘Kodakara’ Uppper surface of leaf blade bearing small leaf-like prominence. 60 91.2 HO5080 56 Chromosome number of horticultural cultivars of Farfugium japonicum

Table 1. (continued) Chromo- some Pollen Cultivar Characteristics number stainability Voucher (2n) (%) 62. ‘Higo-fukuju’ Leaf margin bearing small leaf-like prominences; petiole short. 60 94.1 HO5081 (Fig. 1-14) 63. ‘Odoriko’ "Dwarf. Leaf blade irregular in shape; lower surface bearing nee- 60 no pollen HO5003 (Fig. 1-15) dle-like prominence. 64. ‘Hime-ogi’ Dwarf. Leaf blade slightly incised; lower surface bearing needle- 60 not flowered HO5050 like prominence; margin slightly dentate. 65. ‘Ryugetsu’ Leaf blade irregular in shape, deeply incised; lower surface bear- 60 60.5 HO5033 ing hook-like prominence; margin large dentate. Head atrophied. 66. ‘Kamemaru’ Dwarf. Leaf blade depressed at veins. 60 not flowered HO5061 (Fig. 1-16) 67. ‘Ryokuho’ Leaf blade bearing wart-like prominence. 60 97.7 HO5070 (Fig. 1-17) Group Ⅷ . Leaf color variation 68. ‘Ukigumo-nishiki’ Leaf blade yellowish-white margined or splashed variegation. 60 95.8 HO6001 (Fig. 1-18) 69. ‘Kinkan’ Leaf blade yellow-margined variegation in spring leaves. 60, 30 II 75.9 HO6002 (Fig. 1-19) 70. ‘Inazuma’ Leaf blade yellow centered variegation. 60 98.2 HO6019 71. ‘Oda-shibori’ Leaf blade yellowish-white centered variegation. 60 97.3 HO6026 72. ‘Samidare’ Leaf blade yellowish-white splashed and mottled variegation in 60 97.7 HO6009 (Fig. 1-20) both spring and autumn leaves. 73. ‘Shichifuku’ Leaf blade whitish-yellow splashed variegation 60, 30 II 94.2 HO6004 74. ‘Basho-sen’ Leaf blade yellow spotted variegation; base rotundate. 60 98.9 HO9013 75. ‘Temboshi’ Leaf blade yellow spotted variegation; base cordate. 60 98.3 HO6005 (Fig. 1-21) 76. ‘Awayuki’ Leaf blade whitish-yellow mottled variegation. 60 93.6 HO6023 77. ‘Ryokukan’ Leaf blade yellowish-white mottled variegation in spring leaves. 60 92.9 HO6010 78. ‘Kaimon’ Leaf blade white margined and mottled variegation in spring 60 92.3 HO6016 leaves. 79. ‘Ryokuei’ Leaf blade yellow with green mottled variegation in both spring 60 97.5 HO6024 and autumn in early stage, and wholly green in final. 80. ‘Gion’ Leaf blade yellowish-white reticulate variegation in both spring 60 92.3 HO6018 and autumn in early stage, and wholly green in final. 81. ‘Kogane-zuki’ Leaf blade whitish-yellow in both spring and autumn in early 60 88.9 HO6011 (Fig. 1-22) stage, and wholly green in final. 82. ‘Aki-akebono’ Leaf blade yellowish-white in autumn in early stage, and wholly 60 93.1 HO6020 green in final. 83. ‘Hakucho’ Leaf blade yellowish-white in both spring and autumn in early 60 99.0 HO6017 stage, wholly green in final. 84. ‘Hengemo’ Leaf blade yellowish-white in both spring and autumn in early 60 97.7 HO6021 stage, reticulate or mottled variegation later, and wholly green in final. 85. ‘Okan’ Leaf blade yellow in both spring and autumn in early stage, and 60 92.4 HO6008 wholly green in final. Group Ⅸ . Leaf shape variation and variegation 86. ‘Beni-sango’ Leaf blade rolled, yellow-spotted variegation; margin medially 60 not flowered HO9010 crisped, highly waved. 87. ‘Hoshi-guruma’ Leaf blade slightly incised, yellow-spotted variegation; margin 60 48.2 HO9008 slightly waved. 88. ‘Kagami-jishi’ Leaf blade slightly incised, slightly rolled, yellow-spotted variega- 60, 30 II not flowered HO9014 tion; margin slightly waved. 89. ‘Hoshi-botan’ Leaf blade deeply incised, rolled, yellow-spotted variegation; 60 not flowered HO9017 margin highly waved. 90. ‘Kujaku-maru’ Leaf blade slightly rolled, yellowish-white veined variegation; 60 96.9 HO9016 margin low- and rough dentate, slightly waved; petiole short, fasciated. 91. ‘Tama-jishi’ Leaf blade slightly rolled, yellowish-white veined variegation; 60 94.7 HO5010 margin low- and rough dentate, low waved; petiole short, fasciated. 92. ‘Heisei-jishi’ Leaf blade slightly rolled, yellow-spotted variegation; margin 60 98.4 HO9007 (Fig. 1-23) large and rough dentate, highly waved; petiole short, fasciated. 93. ‘Ryuko’ Leaf blade rumpled, yellowish-white centered variegation. 60 52.3 HO9019 (Fig. 1-24) 94. ‘Gion-komachi’ Leaf blade irregular in shape, rumpled, yellowish-white irregular- 60 91.1 HO9004 shaped variegation. Okuno et al. 57

Table 1. (continued) Chromo- some Pollen Cultivar Characteristics number stainability Voucher (2n) (%) 95. ‘Tsukuda-jima’ Dwarf. Leaf blade spoon-shaped, rumpled, yellowish-white mot- 60 no pollen HO9006 (Fig. 1-25) tled variegation. Head atrophied. 96. ‘Hikoboshi’ Leaf blade rumpled, yellow-spotted variegation; margin slightly 60 93.3 HO9018 outward-bended, Ray floret fimbriated. 97. ‘Ama-no-gawa’ Leaf blade rumpled, outward-bended at the margin, yellow-spot- 60 94.7 HO9002 ted variegation. Ray floret fimbriated. 98. ‘Yuzen’ Leaf blade rumpled, yellowish-white mottled variegation; margin 60 96.1 HO9012 outward-bended. Ray floret fimbriated. 99. ‘Kiten-no-kawa’ Leaf blade rumpled, yellow-spotted variegation; margin outward- 60 92.8 HO9005 bended. Ray floret fimbriated. 100. ‘Hoshifu-koryu’ Leaf blade fold-rumpled, yellow spotted variegation. Head atro- 60 86.6 HO9009 phied. 101. ‘Hoshi-otome’ "Leaf blade yellow-spotted variegation. Pedicel long elon- 60 95.2 HO9024 (Fig. 1-26) gated. Head double flowering “Anemone-flowered type”. " Group X. Flower shape variation 102. ‘Manju’ Head double flowering “Informal decorative type”. 60 no pollen HO7005 (Fig. 1-27) 103. ‘Senju’ Head double flowering “Anemone-flowered type”. 60 95.6 HO7001 (Fig. 1-28) 104. ‘Yae-botaru’ Head double flowering “Anemone-flowered type”. Leaf blade yel- 60 82.4 HO9021 low-spotted variegation. 105. ‘Yae-kirin’ Head double flowering “Anemone-flowered type”. Petiole green. 60 95.1 HO9020 106. ‘Shikoku-shinryu’ Ray floret tubular. Leaf blade slightly rolled; margin large- and 60 95.2 HO9023 (Fig. 1-29) densely dentate, highly waved; petiole short, fasciated. 107. ‘Nami-chidori’ Ray floret irregular in shape. 60, 30 II 94.7 HO7002 Group XI. Flower color variation 108. ‘Hakuto’ Ray floret yellowish-white, revolute when aged. 60 96.7 HO8002 (Fig. 1-30) 109. ‘Shiranami’ Ray floret yellowish-white. 60, 30 II 84.0 HO8003 110. ‘Izumi-yamabuki’ Ray floret darkyellow. 60, 30 II 93.2 HO8001 111. ‘Yubae’ Ray floret orange. 60, 30 II 91.1 HO8004 (Fig. 1-31) root tips and pollen mother cells were used, respectively. appeared in spring, though the other characteristics The methods of chromosome preparations, staining, and were not season-specific in these cultivars. As a result of observation are the same as described in our previous morphological observation on both leaves and flowers, the paper (Okuno et al. 2009). Pollen fertility was estimated by 111 horticultural cultivars were classified into following 11 the stainability of pollen grains with lactophenol–cotton groups; 7 types of leaf shape variations (groups I to VII), blue solution (Okuno et al. 2009). Abnormal-shaped pollen “Leaf color variation” (group VIII), “Leaf shape variation grains were also judged as “sterile”. Generally, more than with leaf color variation” (group IX), “Flower shape 700 pollen grains (the minimum 552 and the maximum variation” (group X), and “Flower color variation” (group 2132) were surveyed for each individual. XI) (Table 1). Among the 7 types of leaf shape variations, 6 types were characterized by their morphological characteristics with traditional Japanese names (described Results in capitals hereafter), “SHISHI (or BOTAN)” (group I), “KURUMA (or FUGIRE)” (group II), “NOKOGIRI (or Artificial classification of cultivars TSUNO-DASHI)” (group III), “RASHA” (group IV), “CHIRIMEN” (group V) and “EDAWAKARE” (group VI), Some cultivars exhibited season-specific expression and the cultivars with other type of leaf shape variations not of characteristics in leaf morphology or variegation. For categorized in these traditional classifications were grouped example, “fasciated petiole” in cultivar ‘Daruma-jishi’ as “Other leaf shape variation” (group VII). Explanations (HO5014, Fig.1-2), “dentate and wavy margin of leaf blade for the Japanese group names and characteristics of the with short and fasciated petiole” in ‘Fuku-daruma’, “rod- cultivars are briefly described in Table 1, and more details like prominence on upper surface of leaf blade” in ‘Ryusen- are presented in our previous review written in Japanese no-mai’ (HO5035, Fig. 1-12) and “spoon-shaped leaf” (Okuno et al. 2007). Photographs of the representative in ‘Tsukuda-jima’ (HO9006, Fig. 1-25) were observed cultivars of the groups are presented in Fig. 1. It should mainly in autumn, whereas “hook-like prominence on be noted that with respect to flower morphology, the lower surface of leaf blade” in ‘Amadare’, “warty leaf blade” phenotype of “atrophied head” correlated closely with those in ‘Ryokuho’ (HO5070, Fig. 1-17) and “non-chimeric of “SHISHI” and “KURUMA”. variegated leaf” in ‘Kinkan’ (HO6002, Fig. 1-19) usually 58 Chromosome number of horticultural cultivars of Farfugium japonicum

Somatic chromosome number Discussion

All 112 individuals in 111 horticultural cultivars of Cytological features of the cultivars F. japonicum had the chromosome number of 2n=60 as exemplified in Fig. 2A (‘Mai-jishi’, HO5027, Fig. 1-1) As mentioned in our previous paper (Okuno et except for one aneuploid individual of 2n=59 (HO5024, al. 2009), chromosomal changes in number, such as Fig. 2B), which had been produced through leaf blade polyploidy (especially high-level polyploidy), aneuploidy, culture of ‘Yukibeni-botan’ (HO5029). Even in cultivars and mixoploidy can cause freak morphology in general of unusual morphology, such as ‘Itohime’ (HO5044, Figs. (Goodspeed and Avery 1939, Nishiyama 1952, Stebbins 1-8, 2C), ‘Ryusen-no-mai’ (HO5035, Figs. 1-12, 2D), and 1971, Griesbach and Kamo 1996, Kobayashi et al. 2008). ‘Tsukuda-jima’ (HO9006, Figs. 1-25, 2E), the chromosome Thus, we have expected that freak-form cultivars of F. numbers were normal with 2n=60. This is the first report japonicum, such as dwarf-form, rumpled leaves, irregular- of the chromosome number for horticultural cultivars of F. formed leaves, etc., are cytological variants. However, the japonicum. The result is comparable with that of wild plants results of our observation failed to meet our expectations. of F. japonicum, which showed 2n=60 in 176 individuals The chromosome number of 2n=60, which is common with one exceptional aneuploid of 2n=61 (Okuno et al. in the species, was observed in all the 111 horticultural 2009). cultivars examined. Moreover, normal bivalents of 2n=30II in meiosis were also detected in some selected cultivars. Meiotic chromosomes and pollen fertility These results indicate that those cultivars are stable diploid like as wild plants (Okuno et al. 2009). The wide range of Figure 2F shows meiotic metaphase I chromosomes in morphological variations in these cultivars must have been microsporogenesis in cultivar ‘Senju’ (HO7001, Fig. 1-28). caused by gene(s) responsible for morphogenesis. Cross Normal 30 bivalents were observed. In other 13 cultivars, experiments and molecular approaches are necessary to 2n=30II=60 configurations were also observed at the same clarify the cause of variations. stage or at diakinesis (Table 1). This confirmed previously It is curious that ‘Fukuju-botan’, which has quite low reported n=30 count by Ishikawa (1916). Any abnormal pollen stainability, showed normal 30 bivalents at meiotic chromosome behaviors, such as lagging chromosomes and metaphase I. Similar cases have been reported in some chromosome bridges were not observed in the subsequent interspecific hybrids such as tetraploid hybrids ofLeymus microsporogenesis. (Kishii et al. 2003, Tsujimoto pers. comm.), diploid hybrids Pollen stainability was examined in 112 individuals of Rubus (Iwatsubo et al. 1996, Iwatsubo & Naruhashi except for unflowered 16 cultivars (HO5005, 5020, 5026, 1996, 1998), and diploid hybrid of Potentilla (Iwatsubo 5031, 5032, 5044, 5050, 5061, 5076, 5079, 5066, 5086, & Naruhashi 1992). In these cases, interspecific hybrids 9001, 9010, 9014, 9017) and 7 cultivars with male sterile showed normal (or almost normal) bivalents at meiotic flower (HO5003, 5006, 5041, 5064, 5071, 7005, 9006). All metaphase I, but had complete sterile or fairly sterile pollen. “dwarf form” plants (HO5003, 5005, 5032, 5044, 5050, This could be explained that the parents are closely related 5061, 5076, 9006) except for ‘Hime-clover’ (HO5036) were species with similar genome in chromosomal level, but difficult to produce flowers or tended to produce pollen- differed each other in some gene(s) responsible for pollen less flowers. Figure 3A shows an example of normal pollen fertility. In contrast, wild F. japonicum is clarified as diploid grains having 98.0% stainability (‘Kaun’, HO5043, Fig. despite of its high chromosome number 2n=60 (Okuno et 1-11). Cultivars of the group I “SHISHI” excluding the al. 2009, r-DNA FISH unpublished data). Therefore, very aneuploid of ‘Yukibeni-botan’ and the group II “KURUMA” low pollen fertility in ‘Fukuju-botan’ might have resulted had relatively low values of stainability, 48.3±25.3% from mutation in gene(s) involved in microsporogenesis, (N=12) in the former and 23.4% (N=1) in the latter, or small structural change or loss in chromosome, which is respectively. Figures 3B and 3C show pollen grains (46.1 not responsible for chromosome pairing, in this cultivar. % stainability) and the abnormal tetrad with micronuclei, respectively, in the normal 2n=60 individual of ‘Yukibeni- Conservation and breeding of cultivars botan’ (HO5029), although normal tetrads were also observed in this individual. In ‘Fukuju-botan’, although In this study, characteristics of three somaclonal normal bivalents were observed at meiotic metaphase variants obtained through leaf blade culture of ‘Yukibeni- I, the pollen stainability was quite low (23.4%). Almost botan’ (2n=60, HO5029) were examined. Two of them are complete sterility (0.3% pollen stainability) was found in ‘Yukibeni-henge’ (HO5064) and ‘Unryu-jishi’ (HO5071), the 2n=59 aneuploid individual (HO5024) of ‘Yukibeni- both of which are 2n=60 diploid individuals showing botan’ (Fig. 3D). In contrast, the cultivars of the other different morphology from the original ‘Yukibeni-botan’ groups (groups III – XI) exhibited relatively high values and bearing pollen-less flowers, whereas remaining one is of stainability (91.3±12.0%, N=75, Table 1). The cultivars the 2n=59 aneuploid individual (HO5024) showing similar with “atrophied head” tended to either remain unflowered morphology to the original ‘Yukibeni-botan’ but having or produce pollen-less flowers. Figures 3E and 3F show the almost no pollen stainability, despite of the moderate part of anthers of the pollen-less double-flowered cultivar pollen stainability of the original cultivar. These results ‘Manju’ (HO7005, Fig. 1-27), and the normal flower with indicate that tissue culture includes a risk of producing 95.8% pollen stainability of ‘Ukigumo-nishiki’, a variegated cytological variants despite of its effectiveness for rapid cultivar (HO6001, Fig. 1-18), respectively. In the pollen-less micropropagation of cultivars. In order to maintain anthers, no sporoderm formation was observed (Fig. 3E) cultivars with minimized risk of somaclonal variations, although microspores were recognized. propagation by conventional division might be still a better choice. When we use tissue culture for propagating cultivars, we must at least check chromosome number Okuno et al. 59

Figure 1 Representatives of the horticultural cultivars of Farfugium japonicum cytologically investigated in this study. 1: ‘Mai-jishi’. 2: ‘Daruma-jishi’. 3: ‘Fukuju- botan’. 4: ‘Ryukaku’. 5: ‘Hakkaku-uryu’ 6: ‘Ryuho’. 7: ‘Keikan’. 8: ‘Itohime’. 9: ‘Rashomon’. 10: ‘Beni-koryu’. 11: ‘Kaun’. 12: ‘Ryusen-no-mai’. 13: ‘Shishinden’. 14: ‘Higo-fukuju’. 15: ‘Odoriko’ 16: ‘Kamemaru’. 17: ‘Ryokuho’. 18: ‘Ukigumo-nishiki’. 19: ‘Kinkan’. 20: ‘Samidare’ 21: ’Temboshi’. 22: ‘Kogane-zuki’. 23: ‘Heisei-jishi’ 24: ‘Ryuko’. 25: ‘Tsukuda-jima’. 26: ‘Hoshi-otome’. 27: ‘Manju’. 28: ‘Senju’. 29: ‘Shikoku-shinryu’. 30: ‘Hakuto’. 31: ‘Yubae’. Scale bars indicate 5 cm. 60 Chromosome number of horticultural cultivars of Farfugium japonicum

Figure 2 Chromosomes of horticultural cultivars of Farfugium japonicum. A: Mitotic metaphase chromosomes of ‘Mai-jishi’ (HO5027, Fig. 1-1), showing 2n=60. B: Mitotic metaphase chromosomes of the aneuploid (2n=59) (HO5024) individual of ‘Yukibeni-botan’. C: Mitotic metaphase chromosomes of the dwarf cultivar ‘Itohime’ (HO5044, Fig. 1-8), showing 2n=60. D: Mitotic metaphase chromosomes of the deformed leaf cultivar ‘Ryusen-no-mai’ (HO5035, Fig. 1-12), showing 2n=60. E: Mitotic metaphase chromosomes of the dwarf and rumpled leaf cultivar ‘Tsukuda-jima’ (HO9006, Fig. 1-25), showing 2n=60. F: Meiotic metaphase I chromosomes in microsporogenesis of ‘Senju’ (HO7001, Fig. 1-28), showing 2n=60=30 II. Scale bars indicate 10 μm (A-E) and 20 μm (F), respectively.

Figure 3 Pollen grains, tetrad in microsporogenesis, and anthers of horticultural cultivars of Farfugium japonicum. A: Normal pollen grains of ‘Kaun’ (HO5043, Fig. 1-11) having high pollen stainability (98.0%). B: Pollen grains of the 2n=60 individual of ‘Yukibeni-botan’ (HO5029) which shows stainability in almost half of the pollen (46.1%). Oil-drops attaching to the pollen grains were occasionally photographed. C: An abnormal tetrad having micronulei (arrows) observed in the 2n=60 individual of ‘Yukibeni-botan’ (HO5029). D: Pollen grains of the 2n=59 individual (HO5024) of ‘Yukibeni-botan’ showing the lowest stainability (0.3%). E: Part of anthers of ‘Manju’ (HO7005, Fig. 1-27), which has no pollen grains. F: Part of anthers of ‘Ukigumo-nishiki’ (HO6001, Fig. 1-18), which has high pollen stainability of 95.8%. Scale bars indicate 50 μm (A, B, and D), 30 μm (C), and 100 μm (E and F), respectively. for avoiding the unexpected propagation of cytological can be expected that cytological variants accompanying somaclonal variants. morphological variations will be eventually produced by Conversely, somaclonal variations could be used as changing culture conditions or by adding some mutagens. the source of novel characters for the breeding. In case of Since high stainability of pollen grains was observed the cultivar ‘Yukibeni-botan’, both an aneuploid without in most cultivars except for those in “SHISHI” and morphological change and morphological variants “KURUMA” groups, they could be used for the breeding by without changing in chromosome number have been hybridization among them. Actually we have started cross produced so far by prolonged tissue culture. Therefore, it breeding between some cultivars with successful results Okuno et al. 61

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