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© 2021 The Japan Mendel Society Cytologia 86(1): 61–65 New Cytogenetic Information of Wild Species and Cultivars in the Genus Epidendrum (Orchidaceae) Haruka Kondo, Shinji Kikuchi, Ayumi Deguchi and Kazumitsu Miyoshi* Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba 271–8510, Japan Received December 7, 2020; accepted December 17, 2020 Summary Chromosome numbers of wild species in the genus Epidendrum have previously been reported in the range of 2n=24–240. However, the ploidy levels of cultivars in the given genus originating from interspecific hybridization among several wild species have never been reported. To elucidate their ploidy levels, we analyzed the chromosome number of cultivars and related wild species. Four wild species showed a new record of chromo- some number in the present study, namely, E. radicans 2n=38, 80, E. secundum 2n=60, and E. cinnabarinum 2n=64. Six cultivars examined were revealed to have more chromosomes than the wild species, with a range of 2n=84–164, suggesting that the cultivars have high polyploid levels. A significant correlation between the nucle- ar DNA amount and the chromosome number in wild species as well as cultivars was observed. Those cultivars originated from hybridization. Thus, the results from the present study suggest that the polyploid cultivars in this genus could have resulted from pollination involving unreduced gametes during breeding. Keywords Epidendrum, Interspecific hybrid, Polyploidy, Chromosome, Flow cytometry. Orchidaceae is the largest family of flowering plants from the breeding of several wild species, i.e., E. radi- divided into five subfamilies, Apostasioideae, Cypripe- cans, E. secundum, E. cinnabarinum, and E. jamiesonis. dioideae, Orchidoideae, Vanilloideae, and Epidendroi- Those cultivars have larger flowers and thicker leaves deae (Chase 2005). The genus Epidendrum belongs to than the wild species. The most common consequence of Epidendroideae and is a neotropical genus consisting polyploidy is the increase in plant organs caused by the of ca.1,500 species, with a distribution ranging from augmented number of copied genes, referred to as the the southeastern United States to northern Argentina “gigas” effect (Sattler et al. 2016, Hlaing et al. 2020). We (Pridgeon et al. 2005). The chromosome numbers in the postulated that the Epidendrum cultivars, which have members of Epidendrum range from 2n=24 to 2n=240, larger flowers and thicker leaves, are polyploid. How- and some wild species are polyploid (Pinheiro et al. ever, to the best of our knowledge, there is no informa- 2009, Assis et al. 2013). Interspecific hybrids and in- tion on their number of chromosomes. The information terploidy hybrids have been detected in the sympatric regarding ploidy in Epidendrum is essential for future zones of some wild species in nature (Moraes et al. breeding programs because a reproduction barrier in in- 2013, Marques et al. 2014). terploid crosses has been reported (Behrend et al. 2015). In ornamental plants, inter- as well as intra-specific In this study, we first confirmed the variation of chro- hybridizations have commonly been conducted to obtain mosome numbers among interspecific cultivars. We also novel characteristics in flowers, such as color and size, analyzed the nuclear DNA amount in interspecific culti- as well as other important traits for commercial produc- vars and wild species in Epidendrum. We revealed a cor- tion, including altered flowering periods, environmental relation between them, enabling the estimation of chro- stress tolerance, and disease resistance (Nimura et al. mosome numbers and ploidy levels easily by measuring 2006, 2008, Vendrame et al. 2007, Laskowska et al. the amount of DNA through flow cytometry (FCM). We 2015, Zhang et al. 2017, Kondo et al. 2020). Especially postulated the ploidy levels of interspecific cultivars in in orchids, including epidendrums, interspecific hy- Epidendrum and compared them with the data obtained bridization is commonly conducted with less difficulty from these two analyses. and has extensively been employed in the breeding of novel cultivars; over 800 interspecific hybrid names in Materials and methods the genus Epidendrum were registered in the database Orchid Wiz version 12.3 (OrchidWiz, LLC. America). Plant materials Interspecific cultivars of Epidendrum mainly originated E. radicans ‘Col’ was collected in situ in Colombia, E. radicans ‘Lavender,’ E. secundum, and E. cinnabarinum * Corresponding author, e-mail: [email protected] were purchased from Matsumoto Orchid (Tokyo, Japan), DOI: 10.1508/cytologia.86.61 E. secundum var. purpureum was purchased from the 62 H. Kondo et al. Cytologia 86(1) Mochizuki Orchid Nursery (Ibaraki, Japan), and E. radi- a coefficient variation of <4% (data with a coefficient cans ‘Miura’ was purchased from Orchid Valley Miura variation of more than 4% was not included). Eustoma (Kanagawa, Japan). Six cultivars, i.e., E. Narrative Genji grandiflorum, which has a 2C DNA amount=3.26 pg ‘Fujitsubo,’ E. ‘White1,’ E. Sunny Girl ‘Toki,’ E. Venuspi- (Lindsay et al. 1994), was used as the internal standard ars ‘Akatsuki,’ E. Venuspiars ‘Towalemon,’ E. Venuspiars throughout the analysis. DNA amounts were calculated ‘Towaorange’ were gifts from the Floriculture Niyodo by comparing Eustoma grandiflorum and expressed in Nursery (Kochi, Japan). These plant materials were pot- ‘units’ instead of ‘picograms’ because the DNA amount ted in bark chips and cultivated in a greenhouse kept was analyzed only by DAPI staining (Nimura et al. lower than 38°C in the summer and higher than 14°C in 2008). Amount of DNA per chromosome number (DNA the winter. Sumitomo No.2 (Sumitomo Chemical Co., amount of chromosome) was calculated by dividing the Ltd.) was used as aqueous fertilizer, diluted to contain nuclear DNA amount by the number of chromosomes. 0.1% nitrogen, and regularly applied. Five fungicides and four pesticides were sprayed in rotation each month. Results The fungicides were Benlate® (Sumitomo Chemical Co., Ltd.), Daconil® (Sumitomo Chemical Co., Ltd.), Topsin Chromosome numbers of the six wild species plant M® (Nippon Soda Co., Ltd.), Bellkute® (Nippon Soda materials consisting of three taxa in E. radicans, two Co., Ltd.), and Ridomil Gold MZ® (Syngenta Japan Co., taxa in E. secundum, and a taxon of E. cinnabarinum Ltd.) and the four pesticides were Agri-Mek® (Syngenta were examined in this study and ranged from 2n=30–80 Japan Co., Ltd.), Actara® (Syngenta Japan Co., Ltd.), (Fig. 1, Table 1). E. radicans ‘Col’ had 2n=38, E. radi- Marathon® (Sumitomo Chemical Garden Products Inc.), cans ‘Lavender’ had 2n=60, E. radicans ‘Miura’ had and Hachi-Hachi® (Nihon Nohyaku Co., Ltd.). 2n=80, E. secundum var. secundum had 2n=30, E. secundum var. purpureum had 2n=60, and E. cinnabari- Counting the chromosome number num had 2n=64. The chromosome number of E. secun- The methods for chromosome counting followed those dum var. purpureum was two times that of E. secundum of Koehler et al. (2008) and Kondo et al. (2020) unless var. secundum. The resultant DNA amount of chromo- otherwise stated. Chromosome numbers were counted using a minimum of eight yellow root tips collected from three or more vegetatively propagated plants. The root tip sampling was conducted at least one week after spraying agricultural chemicals to avoid unexpected adverse effects. Root tips of 5–8 mm long were excised at around 10:30 a.m. and pretreated in 2 mM 8-hydroxy- quinoline for 48 h at 10°C. They were then fixed with a mixture of ethanol and acetic acid at a 3 : 1 (v/v) ratio for 3–5 h at room temperature. After fixation, the root tips were stored in 70% ethanol at 4°C. In preparation for chromosome observation, the stored root tips were soaked in distilled water for one hour to remove the ethanol. Then, the root segments were cut on a glass slide to isolate the distal 0.5 mm portion of the root tips and an addition of a 10 µL enzyme solution con- taining 2% (w/v) Cellulase Onozuka RS (Yakult Phar- maceutical Industry Co., Ltd.) and 2% (w/v) Pectolyase Y-23 (Kikkoman Corporation) was added to them on the slide. The slides were incubated for 20 min at 37°C in a sealed container humidified with water, and staining and observation were conducted identically to the method used by Kondo et al. (2020). The chromosome lengths of six wild taxa were measured using Image J (https:// imagej.nih.gov/ij/). Measurement of the nuclear DNA amount Methods were the same as mentioned in Kondo et al. Fig. 1. Metaphase chromosomes of Epidendrum wild species. a. E. (2020) unless stated otherwise. FCM was used to ana- radicans ‘Col’ (2n=38). b. E. radicans ‘Lavender’ (2n=60). c. E. radicans ‘Miura’ (2n=80). d. E. secundum (2n=30). e. lyze the latent plantlet buds. For each sample, approxi- E. secundum var. purpureum (2n=60) f. E. cinnabarinum mately 3,000 nuclei were analyzed for a mean value with (2n=64). Scale bar=10 µm. 2021 New Cytogenetic Information in the Genus Epidendrum 63 Table 1. Chromosome numbers and DNA amounts of the Genus Epidendrum. 2n DNA DNA amount/ Chromosome Plant materials chromosome amount chromosome Previous reports* length (µm) number (units) number (×100) E. radicans ‘Col’ wild species 38 2.01 5.30 1.99±0.10 n=19; ME70, ‘Lavender’ wild species 60 3.93 6.55 2.57±0.24 2n=40, 57, 60, 62, 64 TK1984, ‘Miura’ wild species 80 5.11 6.39 2.29±0.18 PI09 E. secundum var. secundum wild species 30 2.05 6.82 2.06±0.13 2n=28, 30, 40, 42, 48, 50, PI09, FG10; var. purpureum wild species 60 4.28 7.13 2.58±0.14 52, 54, 56, 58, 68, 80, 84 AS13 E. cinnabarinum wild species 64 3.73 5.83 2.01±0.09 n=108, 124; 2n=240 FG10, AS13 E.