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ISSN : 1225-5009(Print)
ISSN : 2287-772X(Online)
Flower Research Journal Vol.32 No.4 pp.336-345
DOI : https://doi.org/10.11623/frj.2024.32.4.14

In vitro Asymbiotic Germination of Immature Seed and Seedling Development of Wild White-egret Flower (Habenaria radiata (Thunb.) Spreng) in Korea

Jin-ho Kim1,3†, Ara Cho1, Seong Hyeon Yong1, Yerim Choi1, Yeon-ji Lee1, Sung won Son2, Mi jin Jeong1*
1Division of Forest Biodiversity, Korea National Arboretum, Pocheon 11186, Korea
2Research Planning and Coordination Division, Korea National Arboretum, Pocheon 11186, Korea
3Honam National Institute of Biological Resources, 99 Gohadoangil, Mokpo, 587262, Korea



†These authors contributed equally to this work.


Correspondence to Mi jin Jeong Tel: +82-31-540-8814 E-mail: mjjeong121@korea.kr ORCID: https://orcid.org/0000-0001-8774-2266
18/10/2024 17/12/2024

Abstract


Habenaria radiata, commonly known as the Egret Flower, is a critically endangered terrestrial orchid facing rapid population decline due to overharvesting in Korea. Asymbiotic seed propagation offers a promising conservation method for this rare species. This study investigates the effects of different nutrient media on the germination and plantlet development of immature seeds of H. radiata, with the goal of establishing optimal propagation conditions. Among four media types, with the highest germination rate (75.6%) observed on OBTSG medium, followed by 1/2 MS, OMM, and OSGM. Protocorm development varied across media, with OBTSG and OMM supporting the most robust growth. A two-step culture method involving OBTSG for germination followed by OMM for plantlet development proved most effective, achieving an 84.4% survival rate. These findings highlight the importance of media composition and transitioning for successful propagation. The results provide a practical protocol for in vitro propagation, contributing to both in situ and ex situ conservation efforts aimed at preserving H. radiata and other endangered terrestrial orchids.



endangered species , in vitro propagation , plant media , protocorm , terrestrial orchid

초록

    Introduction

    Habenaria radiata (Thunb.) Spreng, commonly known as the Egret Flower, is a rare terrestrial orchid found in Japan, China and Korea. This species is highly valued as an ornamental plant due to its distinctive flowers, which resemble the wings of an egret (Fig. 1). However, H. radiata has been rapidly declining in Korea due to illegal collection driven by its attractive appearance (Lee and Choi 2006). This species was once widely distributed in regions such as Gyeonggi, Gangwon, Gyeongbuk, and Gyeongnam, but unsustainable harvesting has led to its near disappearance from most habitats, except Gyeonggi Province (Lee 1984;National Institute of Biological Resources 2012). Some organizations have recently begun efforts to restore the species’ habitat. Consequently, H. radiata is classified as Critically Endangered (CR) on the IUCN Red List (Korea National Arboretum 2021).

    Deficiencies in data related to the phenology of seed development and maturation for the majority of wild species (Broadhurst et al. 2008;Mortlock 2000) often lead to asynchronous timing of seed collection (Merritt and Dixon 2011). This issue is particularly problematic in terrestrial orchids, where seed collection and propagation are further complicated by rapid seed maturation, germination issues related to dormancy, and sensitivity to desiccation (Kendon et al. 2017). For many orchid species, higher germination frequencies have been achieved by culturing immature seeds rather than mature ones (Arditti et al. 1982a, b;Ballard 1987;Light and MacConaill 1998;Lindén 1980;Mitchell 1989;DePauw and Remphrey 1993;Rasmussen 1995;Withner 1955). The germination potential of immature seeds varies depending on their developmental stage, and this technique is commonly referred to as ovule, embryo, green pod, or green fruit culture (Deb and Pongener 2011). Lo et al. (2004) demonstrated that the maximum number of Dendrobium tosaense seedlings was produced from the immature seeds of 12-week-old capsules. Similarly, Yamazaki and Miyoshi (2006) confirmed that germination rates of immature seeds at various developmental stages fluctuate based on seed maturity.

    Various studies have shown successful germination of Habenaria species using both asymbiotic and symbiotic methods. For example, Takahashi et al. (2000) achieved a 48.8% germination rate on Hyponex medium after 28 days with H. radiata, while nearly 80% germinated on oat meal agar co-cultured with Ceratobasidium fungi. Similarly, Stewart and Kane (2006b) reported germination rates of 65.7% for H. macroceratitis using symbiotic culture, further reinforcing the potential of both methods. Asymbiotic germination, on the other hand, simplifies this process as it bypasses the need for fungal isolation. Asymbiotic seed propagation is recognized as one of the most effective methods for the conservation of native terrestrial orchids, including H. radiata (Stewart and Kane 2006a). Despite some success, results have been reported asymbiotic in vitro seed germination using the mature capsule of H. radiata. In previous studies, the germination media used were Hyponex or Murashige and Skoog (MS) media, which showed low results of 29.6% or 48.8% (Kim et al. 2019;Lee 2006;Takahashi et al. 2000). Kim et al. (2019) reported that seed of H. radiata germination maximum (90.2%) on MS medium containing 2 μM thidiazuron (TDZ) and 3% (w/v) sucrose.

    Most terrestrial orchids have conspicuous storage organs (such as corms, rhizomes, or tuberoids) (Arditti 1992;Dressler 1981;Zimmerman 1990). Habenaria grows from a small underground tuber, supported by fleshy, branchless roots. As a deciduous species, the tuber provides energy early in its growth cycle for leaf and flower spike development. New bulbs form on short underground stems (stolons) while the old bulb dies by early autumn. Over the fall, the new bulbs are fully formed, leaf growth begins, and the roots from that season die, leaving the new bulbs as independent plants. The formation of bulb is crucial for the successful growth of H. radiata. However, the impact of various medium on asymbiotic seed germination, growth and tuber development of H. radiata is not well documented. Our study investigates the influence of nutrient medium composition on germination of immature seed and plantlet growth of H. radiata. The primary objective of this study is to establish optimal conditions for asymbiotic seed germination and plantlet propagation of H. radiata, aimed at supporting both in situ and ex situ conservation efforts.

    Materials and Methods

    Collection and in-vitro establishment

    Green capsules of Habenaria radiata (Thumb.) Spreng. were collected from growing naturally in Korea National Arboretum during 15th Sep. The capsule was cut in half and seed observed under Hirox digital microscope (KH-8700, Hirox, Japan). The sizes of the seeds, which were transparent white and light yellow, were measured separately in dissected fruits.

    For the viability test, 100 - 200 seeds from capsule were put in a sample tube and seeds was added to 2 mL of 1% sucrose solution and left a 25°C for 24 hours. The solution was removed and stained with 1% triphenyl tetrazolium chloride solution (TTC, Sigma-Aldrich, St Louis, MI, USA) for 24 hours at 25°C in the dark (Zhang et al. 2015). The seeds stained red were observed under the Hirox digital microscope (KH-8700, Hirox, Japan).

    Capsules washed with Tween 20 0.05% (v/v) for 5 minutes, rinses with distilled water and then sterilized with 70% (v/v) ethanol for 5 minutes, followed by treatment with NaClO 3% (v/v) for 6 minutes, and washed 5 times with sterile distilled water. After drying capsules were opened longitudinally using a sterile surgical blade and seeds (light yellow color) were placed on asymbiotic orchid seed germination medium.

    Asymbiotic media effects on seed germination

    Immature seeds were surfaced of four different asymbiotic media (Table 1), i.e. Phytamax Orchid Maintenance Medium (OMM; Sigma, USA), 1/2-strength Murashige & Skoog Medium (1/2 MS, Duchefa, The Netherlands), Orchid Seed Germination Medium (OSGM; MBcell, Korea), and Orchid BM1 Terrestrial Seed Germination & Stem Propagation Medium (OBTSG; MBcell, Korea). All media were commercially prepared and four of the media contained 2% sucrose, and 0.38% gelrite and pH was adjusted to 5.6 before autoclaving at 121°C for 15 minutes. Seeds were placed in grid Petri dished (100 x 20 mm) containing 25 ml of medium. All plates were kept in the dark for at 25 ± 1°C at 12 weeks. The experiment was repeated three times. The seed germination percentage was determined microscopically after 12 weeks of culture and calculated as the number of germinated seeds divided by total number of seeds with protocorms X 100. Germination and protocorm development were recorded on a scale of 0-5 stages, where 0 = No germination, viable embryo; 1 = Swelled embryo, production of rhizoid(s), germination; 2 = Continued embryo enlargement, rupture of testa; 3 = Appearance of protomeristem; 4 = Emergence of first leaf; 5 = Elongation of first leaf (Stewart and Zettle 2002).

    Development and growth of protocorm

    Induced protocorm were subcultured on basal medium after 16 weeks of culture. Plantlet developments and growth from protocorm was examined on four basic media, i.e. 1/2 MS, OMM, and Orchid Terrestrial Medium (OTM; MBcell, Korea) and Orchid Growth Medium (OGM; MBcell, Korea). The developed plantlet was investigated at the end of 8 and 16 weeks. They were cultured under a 12-h photoperiod illumination of approx. In vitro seedling development was scored after 30 weeks incubation. The survival rate was calculated as the percentage of plants that survived after being transferred to a new medium. Effects of photoperiods on tuber and leaf number were recorded.

    Statistical analysis

    The experiment was conducted using a completely randomized design with three replicates, if not specified otherwise. Significant differences were determined by Duncan’s Multiple Range Test (DMRT) using R software (Version 4.4.1; R Foundation for Statistical Computing, Austria).

    Results

    Immature seed and stainability of seeds with TTC

    When the green fruit, the seeds are very immature of H. radiata. The capsule was very moist and contains transparent white or light-yellow seeds. Size of clear white seeds were 0.63 ± 0.12 mm length and 0.22 ± 0.06 mm wide and light-yellow seeds were 0.77 ± 0.15 mm and length 0.25 ± 0.03 mm wide. Fig. 2 shows the results of stainability of seeds by TTC. Most seeds were stained, with means value of 82.3 - 90.2% (data not shown, Fig. 2). Seeds of slightly different conditions also showed differences in the seed coat. In TTC staining, the degree of staining of the pear is very visible in transparent white seeds (Fig. 2c), but in light yellow seeds, the seed coat is more solid and less visible than in transparent white seeds (Fig. 2d). There are also some differences in condition among the immature seeds at this collection time.

    Asymbiotic media effects on seed germination

    The highest germination rate and protocorm formation were observed on OBTSG medium with a germination rate of 75.6 ± 2.5%, followed by 1/2 MS (41.1 ± 0.3%), OMM (33.1 ± 0.8%), and OSGM (7.4 ± 1.8%) (Fig. 3). Seeds on OSGM began swelling, while seeds on OMM and OBTSG initiated protocorm development with rhizoids within 2 weeks. Protocorm formation also commenced on 1/2 MS within 2 weeks, although not consistently. In this study, protocorms developed on 1/2 MS and OBTSG were light yellow, while those on OMM appeared green. As shown in Fig. 4, protocorm development progressed through protocorm stages at 2, 8, and 12 weeks. The protocorms induced in the Orchid Seed Germination medium did not develop beyond stage 4 and started to exhibit browning and necrosis from week 12 onwards.

    Development and growth of protocorm

    Protocorms induced for 16 weeks were transferred to four different growth media (excluding OSGM) for further growth into plantlets, and the survival rate and development of the plants were monitored for an additional 16 weeks. In OMM, 1/2MS, and OTM, nearly all protocorms produced shoots and tubers (Fig. 5). Meanwhile, protocorms cultured in Orchid Growth Medium (OGM) after being transferred from 1/2MS and OMM grew until 8 weeks but all necrosed by 16 weeks. In this results, highest survival rate was obtained after culturing in OTM from developed protocorm in OBTSG (84.4 ± 1.2) followed by 1/2MS (80.4 ± 1.4), OMM (78.6 ± 2.2) and OTM from OMM (78.6 ± 1.0), OTM form 1/2 MS (78.2 ± 1.6) and OMM from OBTSG (78.0 ± 0.8) (Table 2). This indicates that plant growth during germination was influenced by the medium composition. Plantlet development and growth from protocorms differed depending on their nutritional requirements. There was a difference in the number of tubers that were enlarged compared to general roots during plant formation, and this was also related to the growth of the leaves (Fig. 6). Tubers measured in this experiment were the number of parts with a thickness of 1 mm or more. In the 1/2 MS and OMM, less than 1 tuber was measured per plant, while in the case of OTM, more than 1.5 tubers were measured. In addition, the number of roots was also the largest at 2.6. In contrast, OMM and 1/2 MS were measured to have 2.5 leaves, which is more than the 1 leaf for OTM.

    Discussion

    The present study demonstrated the importance of tailored medium composition for optimizing seed germination and protocorm development in Habenaria radiata using immature seed. Among the tested media, OBTSG exhibited the highest germination rate (75.6 ± 2.5%, Fig. 3) and protocorm formation efficiency, highlighting its suitability for the early developmental stages of terrestrial orchids. This result aligns with previous findings indicating that OBTSG, enriched with casein enzymatic hydrolysate, L-glutamine, and glycine, provides organic nitrogen sources critical for germination and protocorm formation in terrestrial orchid species (Malmgren 1996;Kauth et al. 2008). These components likely create an optimal biochemical environment for seed imbibition, metabolic activation, and early tissue differentiation. However, while OBTSG proved effective in early-stage development, its performance declined in subsequent stages, as protocorms cultured on this medium failed to progress beyond stage 4, with browning and necrosis observed by 16 week (not data). This limitation emphasizes the need for a two-step culture system, where a medium optimized for germination is followed by a medium tailored for plantlet development.

    In this study, the germination rate was not higher than that of other media, but OMM showed balanced performance in germination and protocorm development stages (Fig. 4). These were also observed in previous reports for terrestrial and epiphytic orchid including Encyclia adenocaula (Ruiz et al. 2008), Cypripedium macranthus (Huh 2016), Pelatantheria scolopendrifolia (Kim et al. 2021) and Dendrobium moniliforme (Hwang et al. 2024). This media closely resembles the widely used MS medium in terms of its composition, including both macro- and micronutrients (Table 1). Furthermore, the enhanced iron chelation and transport facilitated by disodium EDTA dihydrate and ferrous sulfate heptahydrate may have played a pivotal role in supporting these processes. This finding is consistent with previous studies showing that micronutrient bioavailability significantly impacts orchid seedling growth and survival (Curtis and Spoerl 1948).

    Interestingly, the plantlet development patterns varied significantly among the media. Protocorms transferred to OTM exhibited the highest number of tubers (≥ 1.5 per plant) and roots (2.6 per plant), suggesting that this medium supports the formation of storage and anchorage structures critical for the transition to autotrophic growth. Conversely, OMM and 1/2 MS favored leaf development (2.5 leaves per plant), suggesting differential allocation of resources based on nutrient composition. These results reflect the influence of ion concentration and nutrient balance on organogenesis during plantlet development. It was evident that seed maturity and appropriate media selection play critical roles in achieving successful germination and development.

    The observed effects of L-glutamine and glycine further highlight the stage-specific nutritional requirements of orchid species. While L-glutamine has been widely reported to enhance germination in species such as Cypripedium reginae and Dactylorhiza maculata (Harvais 1982;Van Waes and Debergh 1986), the impact of glycine appears to vary with developmental timing, as reported by Curtis and Spoerl (1948). These findings underscore the complexity of medium optimization, where both macroand micronutrients must be adjusted to accommodate dynamic developmental demands. Furthermore, the findings suggest that media composition, without reliance on exogenous hormone supplementation or photoperiod manipulation, can effectively induce the formation of storage organs such as tubers, which are essential for survival and establishment in natural habitats.

    In conclusion, the two-step culture protocol involving OBTSG for germination followed by OMM for plantlet development represents the most effective approach for the propagation of H. radiata. The ability to support in vitro growth of terrestrial orchids through optimized media formulations not only aids in their propagation but also offers a potential method for reintroducing these species into their natural habitats. This approach may serve as a key conservation tool, ensuring the survival and recovery of this rare species in the wild.

    Acknowledgments

    This study was supported by ‘Preparatory research based on the collection and quality certification system of Korean native plant seeds for forest restoration (KNA1-2-41-22-1)’ project funded by Korea National Arboretum.

    Figure

    FRJ-32-4-336_F1.gif

    Flowering plants of the egret flower (Habenaria radiata).

    FRJ-32-4-336_F2.gif

    Immature seed and TTC staining of H. radiata. A: Transparent white seeds, B: light-yellow seeds, C: stained by tetrazolium test of transparent white seeds and D: stained by tetrazolium test of light-yellow seed.

    FRJ-32-4-336_F3.gif

    Effect of culture media on percent seed germination of H. rediata within 12 weeks. The different letters indicate significantly differences at p < 0.05 (Duncan’s multiple range tests).

    FRJ-32-4-336_F4.gif

    Seed germination and protocorm stages of H. radiata cultured on OMM medium. A: seed test ruptured and rhizoids are formed on the surface of the protocorm within 2 weeks B: appearance of protomeristem within 8 weeks, C: emergence of first leaf within 12 weeks and D: elongation of first leaf within 16 weeks of culture.

    FRJ-32-4-336_F5.gif

    The seedling with developing shoots and roots of H. radiata after 32 weeks of culture. A: 1/2MS, B: OMM, and C: OTM (bar = 1 cm).

    FRJ-32-4-336_F6.gif

    Effects of growth media on number of tubers, root and leaf per in vitro cultivation of H. radiata. The different letters indicate significantly differences at p < 0.05 (Duncan’s multiple range tests).

    Table

    Components of culture media used in the asymbiotic germination and growth of Habenaria radiata. Orchid Seed Germination (OSGM), Orchid BM1 Terrestrial Seed Germination & Stem Propagation (OBTSG), 1/2 strength Murashige & Skoog (1/2 MS), Phytamax Orchid Maintenance (OMM), Orchid Terrestrial (OTM), Orchid Growth (OGM) and not present (empty).

    znot included.

    Survival of two step culture on germination to growth medium in H. rediata after 8 and 16 weeks of subculture.

    zMean separation within columns by Duncan’s multiple range test at p < 0.05.
    y1/2 strength Murashige & Skoog (1/2 MS), Phytamax Orchid Maintenance (OMM) and Orchid BM1 Terrestrial Seed Germination & Stem Propagation (OBTSG).
    x1/2 strength Murashige & Skoog (1/2 MS), Phytamax Orchid Maintenance (OMM), Orchid Terrestrial (OTM) and Orchid Growth (OGM).

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    2. Journal Abbreviation : 'Flower Res. J.'
      Frequency : Quarterly
      Doi Prefix : 10.11623/frj.
      ISSN : 1225-5009 (Print) / 2287-772X (Online)
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