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Introduction

 The genus Cymbidium (Orchidaceae) comprises ~ 52 species that are distributed throughout South and East Asia from Northwest India to Japan, South through the Malay Archipelago to North and East Australia (Du Puy and Cribb 2007). The genus is represented by a wide-ranging geographical occurrence and reveals a great diversity in growth habits. Cymbidium first species was introduced to China and Europe in late 18^th century but in mid of 19^th century new species were explored. In recent years, it is considered as one of the most desirable and popular orchids in the world flower trade because of its attractive multicolor flowers that can be used in floral arrangements and corsages (Fukai et al. 2002). These plants can also be used as excellent flowering houseplants due their long lasting spectacular flowers. 

 In the last few decades, many plant scientists have been involved in the research related to ecological and genetic aspects of Cymbidium (Fukai et al. 2002; Nagl 1972; Sharma et al. 2010; Vij 1985). However, limited literature is available regarding karyological analysis of this species (Singh 1984; Vij and Shekhar 1987; Wimber 1956). Many plant scientists have been involved in cytogenetical studies of different Cymbidium species such as: C. kanran (Fukai et al. 2002), C. floribundum (Aoyama et al. 1986), C. quiubeiense (Yu-Ge et al. 2002), C. suavissimum (Yu-Ge et al. 2003), C. wenshanense (Yingxiang and Fangyuan 1990), C. quinquelobum (Zhong-Jian et al. 2006), and C. serratum (Li et al. 2002). Extensive studies revealed information about chromosome number in somatic as well as gamete cells, nuclear DNA contents, aneuploidy/ polyploidy and details of B-chromosomes in genus Cymbidium (Aoyama 2010; Qing-yun et al. 2009). Most of the Cymbidium hybrids are triploids or tetraploids having x=20 as basic chromosome number (Aoyama 1989; Bhattarai and Malla 1993). 

 Polyploidy analysis is considered as a valuable cytogenetical approach for elucidating phylogenetic and taxonomic relationships and also used in breeding programs (Stace 2000). The investigations on comparative ploidy levels and chromosome number have substantial role to understand the chromosome structure, genome organization and progression at interspecific and intraspecific levels within genus (Ehrendorfer 1980). The comparison in the polyploidy data is considered as base for genetic variations, as well as relatedness or distant among diverse genotypes (Sharma et al. 2010). Karyotype characterization of Asiatic cymbidiums: C. hookerianum, C. mastersii, and C. eburneum reveals that unequivocal species disparities have been hampered by almost same chromosome numbers (2n=40) and slight variations in chromosome morphology and low heteromorphism with no strong evidence for morphologically divergent satellite chromosomes (Sharma et al. 2010; Sharma et al. 2012). Chromosome observation and counting are important to elucidate the function, structure, and as well as organization of genes and genomes (Lv et al. 2009). To determine the ploidy level in plants may require some sophisticated and efficient tool. Flow cytometry has been used extensively for ploidy determination and this is proved as powerful and accurate method for ploidy analysis (Dolezel 1998; Gamiette et al. 1999). This method is non-destructive as works with a small portion of tissue, and this tool can even analyze precisely large populations of cells where aneuploidy or mixoploidy present (Dolezel 1997). In present study, ploidy analysis and DNA nuclear contents of nine Cymbidium genotypes were determined which will provide a baseline for further cytogenetic studies.

Materials and Methods

Plant material and Chromosome preparation

 Cymbidium germplasm were provided by Rural Development Administration (RDA), Suwon, Korea and cultured in MS medium at 25℃ in tissue culture laboratory, Kyungpook National University, Daegu, Korea.

 Root tips of actively growing cymbidium plants were pretreated with saturated α–bromonaphtalene for 5 hrs at 20℃. Fixation was carried out in acetic acid and ethanol solution (v/v, 1:3) for 24 hrs at room temperature. The material was kept at -20℃ in 70% ethanol solution prior to use. For chromosome study, the root tips were washed thoroughly with distilled water and then treated with a mixture of enzymes (0.3% cellulase, 0.3% cytohelicase, 0.3% pectolyase in 150 mM of citrate buffer) for 1 hr at 37℃. Then root tips were squashed in a drop of 60% acetic acid and followed by air drying.

 The chromosomes were counter-stained with 2 μL · mL^-1 of 4’, 6-diamidino-2-phenylindole (DAPI) in vecta-shield (Vector laboratories Inc., California, USA) and observed under the fluorescent microscope (Nikon BX 61, New York, USA). Images were taken through charge coupled device (CCD) and then images processing were done through the Genus imaging system (Applied Imaging Corporation, Genus version 3.8 program, USA).

Slide preparations and flow-cytometric analysis

 Slide preparation was done as adopted by Lim et al. (2001). 20 mg of leaf per plantlet were sliced with a sharp blade in a petri dish having 500 mL of nuclei extraction ice-cool buffer (Partech, GmbH, Münster, Germany) to get a fine suspension. The sample was strained through a nylon mesh (30 μm). Then, 2.5 mL of staining buffer (Partech, GmbH, Münster, Germany) was added and the suspension was added with 4’, 6-diamidino-2-phenylindole; subsequently, ploidy level from each sample was recorded using a flow cytometer (CyFlow, polidy analyzer, Partech, GmbH, Münster, Germany). Leaf tissue from the L. Longiflorum hybrid ‘White Tower’ (genome size 37,701 Mbp) plants grown in the greenhouse was used as a diploid external reference to adjust the flow cytometer (Note: This Lilium species was used to check the calibration of the flow cytometer as its ploidy level and nuclear contents were already known). All the experiments were replicated for three times. Data were presented as histograms to indicate the relative DNA contents of each sample. The peak area in the histogram is showing the number of nuclei for each ploidy level.

Results and Discussion

 Compared to the chromosome number (2n=40), the chromosome counts and ploidy level were estimated in nine genotypes of Cymbidium. This study results indicated that among nine genotypes of Cymbidium, six were tetraploid (2n=4x=80), two diploid (2n=2x=40) and one triploid (2n=3x=60). It was found that C. finlaysonianum as diploid (Fig. 1A), C. ‘Silk Road’ as a triploid (Fig. 1B) whereas, C. eburneum as a tetraploid (Fig. 1C). The 2C nuclear contents and genome size of six genotypes of Cymbidium are presented in Table 1. The 2C nuclear contents range from ~ 12 to ~ 17 pg in selected Cymbidium genotypes whereas, the genome size range from 6,140 to 8,683 Mbp (Table 1). DNA contents of nuclei isolated from the root tips of Cymbidium genotypes and analyzed with the standard genotype L. logiflorum hybrid ‘White Tower’ shows that most of the nuclei represent 2C DNA content (Fig. 2). For each genotype of Cymbidium, the histograms displayed one peak of 2C. Nuclear DNA contents in six species and 15 varieties of orchid were measured through flow cytometry tool (Uchiyama et al. 2003) and six-fold variation in 2C DNA contents ranged from 2.11 pg to 12.64 pg were observed. High correlation (R²=0.95) was recoded between chromosome numbers and nuclear DNA contents. This confirmed that, it is possible to estimate the chromosome number of Cymbidium by using the C value (Aoyama 2010). 

Table 1. The 2C nuclear contents and genome size of six genotypes of Cymbidium.

Fig. 1. Chromosomes in root tips’ cells of Cymbidium plants stained with DAPI. Somatic metaphase chromosome of (A) C. finlaysonianum (2n=2x=40), (B) C. ‘Silk Road’ (2n=3x=60), and (C) C. eburneum (2n=4x=80).

Fig. 2. Histograms of DNA content obtained after analyses of nuclei isolated from root tissue of A. C. finlaysonianum, B. C. ‘Toyo’ C. C. eburneum, D. C. ‘Toyo Tetra’, E. C. ‘Sundust’, F. C. ‘Silk Road’.

 In a previous study, while observing the pattern of chromosome morphology symmetrical formation of chromosomes was recorded in seven Cymbidium species and one variety from China (Li et al. 2002). Felix and Guerra (2000) have reported chromosomes and ploidy level details of orchids particularly focusing on members of Cymbidioid phylad. 44 species of genus Cymbidium were categorized and the pattern of polyploidy within the genera was reported. The variations in chromosome number was observed like in Psygmorchis pusilla (2n=10) and Oncidium Swartz (2n=168), while investigating chromosome counts of sub-tribes, variations were also observed within and between sub-tribes (Felix and Guerra 2000). It is also reported that at resting stage morphological characteristics of condensed chromatin were correlated with possibility of crossing among the different genera in family Orchidaceae (Tanaka 1971). Many intergeneric hybrids were developed after crossing with Cymbidium to introduce new genes into cymbidiums from other genera (Tanaka 1971). 

 It was found the Cymbidium roots were highly polysomatic as also reported in Dendrobium and Phalaenopsis (Mill et al. 1997; Jones and Kuehnle 1998). The present results strongly support the report that diversity in the Cymbidium species is due to polyploidy (Fukai et al. 2002). In Cymbidium the primary cultivars are basically diploid, although some cultivars showed tetraploidy. The cultivars with standard flower type are mostly tetraploid whereas some hybrid cultivars with excellent flower type are triploid. Similar trend was observed in Phalaenopsis polyploids (Aoyama 2010). Felix and Guerra (2000) reported that generally orchids and particularly cymbidioid phylad are widely benefited by the occurrence of inconstant basic number of chromosomes followed by attaining higher ploidy levels. From the literature about chromosome counts in Cymbidium as well as in allied species it was found that genus Cymbidium exhibited x=10 as the basic number of chromosome and therefore most of the species showed somatic chromosome number (2n=40) with few exceptions (Aoyama and Tanaka 1988; Felix and Guerra 2000; Li et al. 2002; Li et al. 2003). The present study likewise supports the previous findings with respect to x=10 as the basic number in genus Cymbidium. The genus Cymbidium is well-known for uniformity in the somatic chromosome counts (2n=40) as described by Sharma et al. (2010) but, some unusual chromosome numbers in different species like C. lancifolium, C. javanicum and C. macrorhizon (2n=38) were also reported by Aoyama (1989). The heteromorphic pair documented in C. devonianum, C. aloifolium, C. tigrinum, C. elegans, C. tracyanum, and C. lowianum, have been exhibited the heterogeneity and revealed less stability of their genome that leads to structural alterations in species due to the speciation process (Sharma et al. 2010).

 This study results can provide useful data on chromosome numbers including speciation based on heteromorphic chromosomes, basic chromosome number, and estimating ploidy level in genus Cymbidium. For cytogenetic studies and breeding programs, an accurate investigation of ploidy level of cultivars is prerequisite. The use of a reliable tool of flow cytometry for determination of ploidy in covenant with chromosome counts suggests the most appropriate method for this purpose (Gamiette et al. 1999).

Acknowledgment

 “This work was carried out with the support of Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ00929609), Rural Development Administration, Republic of Korea”.