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Interspecific Hybrids Production Between Lilium Brownii Var

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Interspecific Hybrids Production Between Lilium Brownii Var J. Japan. Soc. Hort. Sci. 81 (2): 191–197. 2012. Available online at www.jstage.jst.go.jp/browse/jjshs1 JSHS © 2012 Interspecific Hybrids Production between Lilium brownii var. colchesteri and Its Close Relatives by Ovary Slice Culture Nguyen Thi Lam Hai1*, Michikazu Hiramatsu2, Jong-Hwa Kim3, Jun-ichiro Masuda2 and Hiroshi Okubo2 1Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan 2Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan 3Department of Horticulture, Kangwon National University, Chuncheon 200-707, Korea Lilium brownii var. colchesteri (BRO) has a unique trait in its flower color. The color at anthesis is yellowish cream, and changes to white after one day. To introduce this trait and establish a sexually reproductive cultivation system for cut flower production in Lilium, interspecific crosses of L. × formolongi, L. formosanum, L. longiflorum (as seed parents) and BRO (as a pollen parent) were carried out. Capsules with unviable seeds were found in all crosses. To overcome the incongruity barrier, ovary slices excised from developing ovaries at 7 to 28 days after pollination (DAP) were cultured in Murashige and Skoog medium supplemented with 40 g·L−1 sucrose, 40 g·L−1 D-mannitol and 2.5 g·L−1 gellan gum under 24 h illumination. Hybrid plantlets were obtained from crosses of L. × formolongi × BRO and L. formosanum × BRO, but not from the cross of L. longiflorum × BRO. After acclimatization, 79 (44.1%) of 179 hybrid plants produced flowers within a year of cultivation. The hybrids developed flowers changing from yellowish cream to white during anthesis as does the pollen parent. Twenty- eight of 79 flowered plants sprouted two or more flower stalks, characteristics of L. formosanum. The hybrids exhibited relatively high pollen fertility and produced capsules, but no mature seed was obtained by self- pollination. Key Words: interspecific cross, L. brownii var. colchesteri, L. × formolongi, L. formosanum, L. longiflorum. been observed in other lilies. It was confirmed by Introduction molecular phylogenetic studies that the species is a close The genus Lilium spreads over the Northern relative of L. formosanum and L. longiflorum (Nishikawa Hemisphere and is centered mainly in Asia, North et al., 2001). To date, the lily has been grown naturally America, and Europe (Beattie and White, 1993), being in house gardens in Korea or has been cultivated as a composed of almost one hundred species (McRae, 1998). vegetable or as a component of traditional Chinese More than 7,000 cultivars have been bred until now medicine in China (Okubo, 2006; Sashida et al., 1992). (Leslie, 1982), mainly by intra- or inter-specific The species was, probably for the first time, used in hybridization, and more than 100 new cultivars have breeding to establish Centifolium hybrids, later renamed been released annually over the past two decades. Olympic hybrids, at Oregon Bulb Farms, USA by Jan Lilium brownii (F. E. Brown) Miellez var. colchesteri de Graaff and his collaborators, who initiated their lily E. H. Wilson is endemic to nearly all parts of central breeding program about 1940 (Benschop et al., 2010; China at elevations up to 1,500 meters (McRae, 1998). Jefferson-Brown and Howland, 1995), although McRae It seems to have been introduced and widely cultivated (1998) said that the species was not used for hybridiz- as an ornamental plant in Japan for about 400 years ing there. Interspecific crosses of L. brownii with (Okubo, 2006). It has unique ornamental floral traits L. longiflorum (Van Tuyl, 1980) and with L. formosanum with graceful harmony in flower and anther color (brown (by Zalivski) were also reported by McRae (1998), but color), elegant fragrance and petal color changing from it does not seem that cultivars with the species-specific yellowish cream to white during anthesis, which has not characters of L. brownii or var. colchesteri have ever been distributed. It is, however, difficult to cultivate the Received; September 1, 2011. Accepted; November 25, 2011. lily in our climate conditions, probably because it is * Corresponding author (Email: [email protected]). susceptive to virus infection. 191 192 N.T.L. Hai, M. Hiramatsu, J-H. Kim, J. Masuda and H. Okubo Lilium formosanum, natively distributed in mainland hybridization process in lily, the authors attempted ovary Taiwan from sea level to 3,500 meters (Yang, 2000), slice culture at different periods after stigmatic reaches anthesis within 12 months after seed sowing pollination. The precocious flowering ability enables the (Hiramatsu et al., 2002) unlike other Lilium species expected new cultivars to grow from seeds, and this that need several years to start flowering from reproduction process can eliminate virus transmission. seeds (Shimizu, 1971). The introduction of the early- The hybridity of developed progenies was evaluated by flowering ability of L. formosanum has been realized in isozyme analysis. Sexual reproductive ability and flower only one cultivar group, L. × formolongi, a hybrid of characteristics of F1 hybrids were also investigated. L. formosanum and L. longiflorum (Beattie and White, Materials and Methods 1993; Shimizu, 1971). The hybrid was used as parental materials to breed new lily cultivars with novel traits in Plant materials flower color (Van Creij et al., 1993; Van Tuyl et al., Lilium brownii var. colchesteri (BRO), collected in 1986), multiple stalks, early flowering (Saruwatari et al., South Korea and vegetatively established from one 2008), and fusarium resistance (Löffler et al., 1996). clone, was used in all crosses as a paternal genotype. Style manipulations and embryo techniques were applied L. formosanum naturalized in Fukuoka Prefecture, Japan, to conquer the pre- and post-fertilization barrier of L.×formolongi cvs. ‘F1 Augusta’, ‘F1 Julia’, ‘Raizan 1’, interspecific crosses between these Longiflorum lilies ‘Sakigake Raizan’, and ‘White Lancer’; L. longiflorum with other species in the genus Lilium (Fukai and Tsuji, ‘Hinomoto’ and seedlings established from the seeds of 2004; Kanoh et al., 1988; Van Creij et al., 1998; Van nine natural genotypes collected in the Ryukyu Tuyl et al., 1991). However, variation in floral charac- Archipelago and Taiwan (Table 1) were used as maternal teristics among these species is very poor; the flowers genotypes. The plants were grown in the field and in an show only white-colored petals, and funnel-shaped unheated greenhouse in Kyushu University, Fukuoka flowers with strong fragrance; therefore, introducing new Prefecture, Japan. floral traits into these species can enhance the variation of their flower color and enrich the lily world. Pollination and ovary slice culture The purpose of this study was to demonstrate the Normal pollination was carried out according to the possibility of interspecific breeding between L. brownii method described by Van Tuyl et al. (1986). The ovaries var. colchesteri and its close relatives including L. × at 7, 14, 18, 21, and 28 days after pollination (DAP) formolongi, L. formosanum, and L. longiflorum, with the were surface-sterilized by immersion in 70% ethanol for target of producing new lily cultivars endowed with the 1 min followed by 2% sodium hypochlorite solution interesting floral traits of both parents, such as precocious (NaClO) with a few drops of surfactant (Tween 20) for flowering and petal color changing. To improve the 15 min and washed three times in sterilized distilled Table 1. Capsule set in interspecific crosses using L. brownii var. colchesteri as the pollen parent. Number of capsules Number of Female parent Without embryo crosses Aborted With embryo (%) L. × formolongi ‘F1 Augusta’ 37 27 10 (27.0) 0 ‘F1 Julia’ 13 9 4 (30.8) 0 ‘Raizan 1’ 13 8 5 (38.5) 0 ‘Sakigake Raizan’ 27 21 6 (22.2) 0 ‘White Lancer’ 28 21 7 (25.0) 0 L. formosanum 51 51 0 0 L. longiflorumz ‘Hinomoto’ 41 41 0 0 LPI (Pitouchiao, Taiwan) 40 32 8 (20.0) 0 LFU (Fulung, Taiwan) 5 5 0 0 LYO (Yonaguni island, Ryukyu) 25 25 0 0 LIS (Ishigaki island, Ryukyu) 70 70 0 0 LMI (Miyako island, Ryukyu) 9 9 0 0 LKU (Kume island, Ryukyu) 64 64 0 0 LYR (Yoron island, Ryukyu) 33 33 0 0 LOE (Okinoerabu island, Ryukyu) 14 14 0 0 LKI (Kikai island, Ryukyu) 46 46 0 0 z Populations of L. longiflorum are arranged geographically. J. Japan. Soc. Hort. Sci. 81 (2): 191–197. 2012. 193 water. In vitro culture was carried out by the method agar (Niimi and Shiokawa, 1992) at 25°C for 4 h. At described by Kanoh et al. (1988) and Saruwatari et al. least 500 pollen grains were observed in each plant. (2008) with some modification. The swollen parts of the Results ovaries were cut into 2–3 mm thick slices; each sliced ovary disk with about 30 ovules was placed on culture A total of 524 interspecific pollinations were carried medium in a test tube (18 × 180 mm) and maintained at out in the flowering seasons of 2009 and 2010. Some ° 25 C in the dark for four weeks before exposure to cross combinations, such as with L. × formolongi ‘F1 continuous illumination with fluorescent light. Ovule Augusta’, ‘F1 Julia’, ‘Raizan 1’, ‘Sakigake Raizan’, and germination was recorded until ten months after culture. ‘White Lancer’ and with L. longiflorum ‘LPI’ (originated A modified MS medium (Murashige and Skoog, 1962) in Pitouchiao, mainland of Taiwan), produced capsules containing 40 g·L−1 sucrose, 40 g·L−1 D-mannitol, and but they all failed to set mature seed (Table 1). Pollinated 2.5 g·L−1 gellan gum was used as the test medium. The ovaries enlarged within one to six weeks after pollina- medium was adjusted to pH 5.8 and sterilized by tion; however, all the swollen capsules eventually withered autoclaving at 121°C and 1.1 kg·cm−2 for 15 min. and shrank before reaching the size of normal capsules.
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