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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.55 No.4 pp.77-83
DOI : https://doi.org/10.14397/jals.2021.55.4.77

Comparisons of Morphological and Chromosomal Characteristics of Phalaenopsis Mini Type Cultivars

Vo Thi-Co1†, Hye-Min Lee1†, Deen Mohammad Deepo1, Yoon-Jung Hwang2, Hong-Yul Kim3, Ki-Byung Lim3*
1Department of Horticultural Science, Kyungpook National University, Daegu 41566, Korea
2Department of Life Science, Sahmyook University, Seoul 01795, Korea
3Institution of Agricultural Science and Technology, Kyungpook National University, Daegu, 41566, Korea

These authors contributed equally to this work.


* Corresponding author: Ki-Byung Lim Tel: +82-53-950-5726 Fax: +82-53-950-5722 Email: kblim@knu.ac.kr
July 21, 2021 ; August 19, 2021 ; August 20, 2021

Abstract


The purpose of the study is to assess morphological and chromosomal comparison of Mini type Phalanopsis ‘KS Little Gem’ and four domestic market available cultivars (‘Queen Beer’, ‘Tony Pink’, ‘Vaviche’ and ‘Rorens’). ‘KS Little Gem’ had the highest number of leaves (14.9), while the other four types had fewer than ten. The longest leaf length was 21.5㎝ for ‘Tony Pink’, followed by 16.2㎝, 18.0㎝ and 17.5㎝ for ‘Queen Beer’, ‘Rorens’ and ‘Vaviche’ respectively. The length and width of the petals of ‘KS Little Gem’ were 29.5㎜ and 25.6㎜ respectively indicating a round shape flower compared to other cultivars. When the flower lifespan of ‘KS Little Gem’ was compared to four cultivars of the domestic market, it was found that it had a 123-day shelf life, which was twice longer than that of the four cultivars. According to chromosome analysis ‘KS Little Gem’, ‘Rorens’, ‘Tony Pink,' and ‘Vaviche’ were tetraploid (2n = 4x = 76) while ‘Queen Beer’ was diploid (2n = 2x = 38). The estimated DNA content of ‘KS Little Gem’, ‘Rorens’, ‘Tony Pink’ and ‘Vaviche’ had 4918.4, 4794.2, 4705.2 and 4964.3 Mbp respectively, which were roughly double than that of P. cornu-cervi (control, 2n = 2x = 38). However, ‘Queen Beer’, had an estimated DNA content of 2802.2Mbp, similar to that of P. cornu-cervi. The morphological features, genome size and chromosomal data reported in these studies can be used by breeders to create more efficient Phalaenopsis breeding programs.



초록


    Introduction

    Phalaenopsis is one of the most popular potted plants in the horticulture industry due to its colorful and long-lasting blossoms. It is economically important pots and cut flowers in the horticultural market because of being easily programmed to flower throughout the year and flowers have a shelf life of 3–4months (Hwang et al., 2014;Jung et al., 2020). Furthermore, compared to other flowers, it has a longer blossoming life, making it particularly popular among orchids. (Hwang et al., 2014). The production of Phalaenopsis was 5.51 million pots in 2019 which was 39% of total orchid production and the value was 19,595 million won (KRW), which was 48% of the total orchid production (MAFRA, 2020).

    The genus Phalaenopsis consists of more than 70 species distributed from subtropical and tropical Asia, Korea, Japan and Taiwan to northern Australia (Kao et al., 2001;Hwang et al., 2014). Phalaenopsis amabilis, Phalaenopsis equestris and Phalaenopsis schilleriana are popular species (An et al., 2019). In Korea, Phalaenopsis breeding started in the early 1990s and new varieties were developed from 2002. As of 2021, 140 varieties (http://www.seed.go.kr) have been registered. Although the market value of domestically cultivated Phalaenopsis orchid varieties is increasing, most of the mini-sized popular Phalaenopsis orchids distributed in Korea are bred and imported from Taiwan and Japan. Korea are highly dependent on imports and thus paying royalties is a burden (Yea et al., 2014;Hwang et al., 2015).

    Phalaenopsis orchids distributed in Korea are mostly medium and large varieties which are mainly used for gifts purposes. However, since the uses of them as gifts have recently been decreased, the demand for mini sized Phalaenopsis orchids has risen for casual flowers and one-table one flower decoration purposes. As mini type varieties are not diverse, it is necessary to develop such kinds Phalaenopsis orchid (Vo et al., 2019;An et al., 2019). Plant shape and genetic features are important factors in parental selection for creating new varieties. (Vo et al., 2015;Jo et al., 2018;Jo et al., 2019;Mohammad et al., 2020;Tränkner et al., 2020). In case of medium and large Phalaenopsis orchids, various studies including morphological characteristics have been reported (Been et al., 2007;Been, 2010;Been et al., 2011;Lim et al., 2015;Hwang et al., 2015). However, the morphology and genetic characteristics of mini type Phalaenopsis orchid have not yet been reported and it is active research arena. Therefore, the purpose of the experiments is to compare morphological and chromosomal traits of Phalaenopsis ‘KS Little Gem’ with four cultivars which are available in domestic market (‘Queen Beer’, ‘Rorens’, ‘Tony Pink’, ‘Vaviche’).

    Materials and Methods

    1. Plant materials

    Mini type Phalanopsis ‘KS Little Gem’ which were raised in Gangsan Nanwon (Gangseo-gu, Busan) and four domestic market available cultivars (‘Queen Beer’, ‘Tony Pink’, ‘Vaviche’ and ‘Rorens’) were used in this experiment.

    2. Morphological characteristics analysis

    Phalaenopsis orchids of five cultivars were collected and they were cultivated in a glasshouse of Kyungpook National University. They were investigated according to the first revised UPOV survey criteria in 2014 (Yea et al., 2014). Leaf number, leaf length, leaf breadth and leaf thickness were analyzed for vegetative growth features, whereas inflorescence length, petals, calyxes, labellum, number of florets and flowering life were investigated for reproductive growth parameters. The blooming life was recorded from the first floret’s flowering date to the last floret’s closure date.

    3. Flow cytometry analysis

    Young leaves of P. cornu-cervi (Control), ‘KS Little Gem’, ‘Queen Beer’, ‘Tony Pink’, ‘Vaviche’, ‘Rorens’ were collected to determine the DNA content. The leaves were cut into (0.5㎠) pieces, put in a 5×12㎜ petri dish, chopped with a blade in 500㎕ of nuclei isolation buffer. Then, they were poured through a filter into a tube to remove the debris. Next, 2㎖ DAPI mixed staining buffer (Partech, GmbH, Munster, Germany) was poured into the tube. The nuclei suspension was then poured into a flow cytometry analyzer (Partec PA, Ploidy Analyzer, Germany). As a reference standard, samples of the young leaves from diploid P. aphrodite plantlets with a 2C nuclear DNA content of 2.8 picogram (pg) (Lin et al., 2001) were used.

    4. Chromosome observation

    Root tips of ‘KS Little Gem’, ‘Queen Beer’, ‘Tony Pink’, ‘Vaviche’ and ‘Rorens’ plants were collected early in the morning, treated with 2mM 8-hydroxyquinoline at 20℃ for 4 hours and fixed overnight in a solution (ethanol: acetic acid=3:1). The root tips were then put in a refrigerator with 70% ethanol. They were washed with distilled water before being treated with a 0.3 percent pectolyase, 0.3 percent cellulase and 0.3 percent cytohelicase enzyme solution in 150mM citrate buffer for 40 minutes at 37℃. The root-tips were then squashed in 17μl of 68% acetic acid on a glass slide and air-dried. Under a fluorescence microscope, the chromosomes were stained with 6-diamino-2-phynylindole (DAPI) in the VECTASHIELD solution and counted (BX 61, Olympus, Japan). Chromosome analysis was conducted according to Lim et al. (2001) with minor modifications.

    Results and Discussion

    The findings of a leaf attribute comparison between elegant ‘KS Little Gem’ and four domestic market available (‘Queen Beer’, ‘Rorens’, ‘Tony Pink’, ‘Vaviche’) cultivars are shown in Table 1. ‘KS Little Gem’ had the most leaves, with 14, followed by the remaining four varieties with fewer than 10 leaves. ‘Tony Pink’ had the longest leaves at 21.5㎝, followed by ‘Queen Beer,’ ‘Rorens’ and ‘Vaviche’, which were 16.2㎝, 18.0㎝ and 17.5㎝, respectively. Phalaenopsis ‘KS Little Gem’ was the shortest leaf length which was 13.9㎝ and considered as the most compact cultivar among the small and medium- sized orchids used in this experiment. (Table 1). Vo et al. (2015) stated that leaf characteristics follow dominant traits except for the number of leaves. In this experiment, it has been determined that hybridizing ‘KS Little Gem’ and ‘Queen Beer’ with small leaves can produce phalaenopsis orchids with compact leaves.

    Inflorescence and floret characteristics of mini type Phalaenopsis are compared in Table 2 and Fig. 1. ‘Tony Pink’ and ‘Vaviche’ had the longest florets at 54.1㎝ and 54.8㎝, respectively, while ‘Rorens’ had the smallest at 30.4㎝. ‘Rorens’ and ‘Vaviche’ had pedicel lengths of more than 36.0 ㎜, which was 4.0㎜ longer than ‘KS Little Gem' and ‘Queen Beer’. On contrary, there had no significant difference in the thickness of the pedicel (Table 2). The size of the floret varied each variety; however, the length and width were consistent and the overall form was spherical (Fig. 1). Hybrids usually have an intermediate type of inflorescence and floret between the parents (Tang et al., 2007). As a consequence of the findings of this experiment, it is concluded that Phalaenopsis with numerous florets can be produced by crossing ‘KS Little Gem’ with ‘Tony Pink’.

    The comparison findings of flower organ characteristics of mini type Phalaenopsis are shown in Table 3 and Fig. 1. The petals of ‘KS Little Gem’ measured 29.5㎜ in length and 25.6 ㎜ in width, indicating a more rounded shape than other types. (Table 3, Fig. 1A). In comparison to other types, ‘KS Little Gem’ has the longest labellum at the bottom of the bloom, measuring 25.3㎜ in length and 15.3㎜ in breadth.

    Shelf life of flower Phalaenopsis ‘KS Little Gem’ and four domestic market available cultivars were compared (Fig. 2). The longest was ‘KS Little Gem’, which lasted 123 days, followed by ‘Rorens’, ‘Vaviche’, ‘Tony Pink’ and ‘Queen Beer,’ which took 59, 58, 58 and 46 days, respectively. Flower longevity of four types was less than half that of 'KS Little Gem’. Through a forward crosses of multiple Phalaenopsis orchids, it was stated that a link between the flowering lifespan of the parental and F1 individuals, as well as the floral organ (Park et al., 2015). As a result of the analysis, if the width of the calyx became narrower than that of the length, the shelf life was long and the flower longevity of the female had an effect on that of the F1 individual. In addition, in this experiment, ‘KS Little Gem’ showed a longer and narrower calyx (Table 3) and a longer flower longevity (Fig. 2) than other small and medium-sized Phalaenopsis. In terms of morphological traits and bloom lifetime, ‘KS Little Gem’ is deemed to have extremely high utility value as a female for growing small and medium-sized Phalaenopsis. Therefore, if ‘KS Little Gem’ is used as the female and other small and medium-sized Phalaenopsis orchids are crossed as the male, it is expected that a new variety with a compact and long flower longevity can be developed.

    The findings of flow cytometry of five of mini type Phalaenopsis using diploid P. cornu-cervi as a control are shown Table 4. Flow cytometry enables easy identification of ploidy level which is responsive to the environment in term of evolutionary fitness (Galbraith, 2009). The DNA content of the control P. cornu-cervi was assessed to be 2680.7 Mbp. The estimated DNA content of ‘KS Little Gem’, ‘Rorens’, ‘Tony Pink’ and ‘Vaviche’ had 4918.4, 4794.2, 4705.2 and 4964.3 Mbp respectively (Table 4), which were roughly double than that of P. cornu-cervi. However, ‘Queen Beer’, had an estimated DNA content of 2802.2 Mbp, similar to that of P. cornu-cervi.

    The chromosome number determination is a primary cytogenetic tool. However, detecting the chromosome number of the Phalaenopsis species with a large number of small size chromosomes is often difficult. We performed photomicrographs of 50 well-spread cells from each species to confirm the chromosome number. The findings of chromosome observation of five mini type Phalaenopsis cultivars employing diploid P. cornu-cervi as a control, are shown in Fig. 3. As a result of chromosome observation, P. cornu-cervi was 2n = 38, which was diploid. ‘KS Little Gem’, ‘Rorens’, ‘Tony Pink’ and ‘Vaviche’ were confirmed as tetraploids with chromosome number 2n = 4x = 76, whereas, ‘Queen Beer’ was diploid with chromosome number 2n = 38 (Fig. 3). Griesbach (1985) reported that crossing between plants with different levels of ploidy, the success rate became very poor. Therefore, it is very important to know the ploidy level of parents prior to breeding. Wang et al. (2016) reported, triploid plants are partially or completely sterile. Because all the mini type of Phalaenopsis employed in this study were tetraploid and diploid which will be useful for the breeding. Manzoor et al. (2019) reported that ploidy level affects plant growth and tolerance to environmental stress. It will be feasible to establish a novel variety of Phalaenopsis with great growth and compactness utilizing the tetraploid lines studied in this experiment. As a result, it is believed that the findings of this study will be valuable in developing new kinds of Phalaenopsis orchids through hybridization in the future.

    Acknowledgement

    This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (iPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number. iPET318021-4). This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (iPET) through the Export Promotion Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number. iPET313009-4).

    Figures

    JALS-55-4-77_F1.gif

    Floret characteristics of ‘KS Little Gem’ and four domestic market available cultivars.

    A: ‘KS Little Gem’, B: ‘Queen Beer’, C: ‘Rorens’, D: ‘Tony Pink’, E: ‘Vaviche

    JALS-55-4-77_F2.gif

    Flower longevity of ‘KS Little Gem’ and four domestic market available cultivars.

    Bars mean ± standard error (n=5)

    JALS-55-4-77_F3.gif

    Chromosomes of P. cornu-cervi (A; 2n=2x=38), ‘KS Little Gem’ (B; 2n=4x=76), ‘Queen Beer’ (C; 2n=2x=38), ‘Rorens’ (D; 2n=4x=76), ‘Tony Pink’ (E; 2n=4x=76), ‘Vaviche’ (F; 2n=4x=76).

    Tables

    Leaf characteristics of ‘KS Little Gem’ and four domestic market available cultivars

    Inflorescence characteristics of ‘KS Little Gem’ and four domestic market available cultivars

    Flower characteristics of ‘KS Little Gem’ and four domestic market available cultivars

    Analysis of nuclear DNA content by flow cytometer

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