- Original Article
- Open Access
A method for facilitating the seed germination of a mycoheterotrophic orchid, Gastrodia pubilabiata, using decomposed leaf litter harboring a basidiomycete fungus, Mycena sp.
© The Author(s) 2017
- Received: 5 September 2017
- Accepted: 1 December 2017
- Published: 8 December 2017
Mycoheterotrophic plants are one of the most difficult plant groups to conserve because they are entirely dependent on symbiotic fungi. Establishment of viable culture systems would greatly aid their conservation. We describe a simple culture system for the mycoheterotrophic orchid, Gastrodia pubilabiata, that does not require laboratory facilities. The orchid is symbiotic with leaf-litter-decomposing fungi.
Gastrodia pubilabiata seeds were incubated in plastic boxes or glass bottles filled with leaf litter collected from the natural habitat of the species. Seed germination was observed after 35 days and seedling development followed. Fungal isolates from seedlings were identified as Mycenaceae (Basidiomycota), a leaf-litter-decomposing mycorrhizal fungus of Gastrodia species.
Our method can be used to conserve endangered mycoheterotrophic plants associated with leaf litter-decomposing fungi efficiently, and can also serve as a model system for physiological and molecular studies of such plants.
- Leaf litter-decomposing fungi
- Mycoheterotrophic plants
Mycoheterotrophic plants depend on mycorrhizal fungi for their carbon supply throughout their life cycle (Leake 1994). Most such plants are rather rare and seriously endangered, principally because of habitat destruction (Merckx et al. 2013). However, they are also one of the most difficult plant groups to conserve both ex situ and in situ because they are strongly dependent on specific symbiotic fungi. Development of a viable culture system would be of great assistance in terms of efficient plant propagation and subsequent reintroduction into natural habitats. In addition, mycoheterotrophic plants have dust-like seeds with minimal nutrient reserves, and germination occurs mostly underground, rendering it difficult to investigate seed germination ecology in their habitat. Seedling growth in culture would greatly aid studies of seed dormancy, seedling germination, and development. As many biological characteristics of mycoheterotrophic plants remain unknown, a simple culture method would serve as a useful model system for elucidating the physiological and molecular aspects of mycoheterotrophy.
Successful flowering of mycoheterotrophic plants co-cultured with symbionts has been reported for several orchids associated with leaf-litter or wood-decomposing fungi, including Didymoplexis minor (Burgeff 1936), Gastrodia verrucosa (Tashima et al. 1978), Gastrodia elata, (Xu and Guo 2000), and Epipogium roseum (Yagame et al. 2007). However, these studies used fungal isolates from mycorrhizal tissues and required laboratory facilities. As a wide range of people, from professional scientists to amateur volunteers, are involved in orchid conservation programs, a versatile method without expensive culture facilities is required. An easy symbiotic culture system without fungal isolation has been successfully achieved for Australian green orchids by incubating orchid seeds and soil from their natural habitat (Brundrett and Ager 2011). Seedlings of Gastrodia nipponica, a mycoheterotrophic orchid that is associated with litter-decomposing fungi (Kinoshita et al. 2016), were successfully established by incubating the seeds in a plastic box filled with leaf litter from their habitat (Umata and Nishi 2010).
The mycoheterotrophic orchid Gastrodia pubilabiata is principally associated with litter-decomposing fungi in the families Mycenaceae and Marasmiaceae (Kinoshita et al. 2016); both the plant and the fungi can be cultured in vitro. In a previous study, seeds of G. pubilabiata were successfully germinated and developed into plantlets using in vitro symbiotic culture with a fungus from the mycorrhizal roots of G. verrucosa (Umata et al. 2000). Asymbiotic seed germination (Godo et al. 2007) and flowering under symbiotic cultivation (Shimaoka et al. 2017; S. Inagaki personal communication) have also been achieved. Seventeen prefectures in Japan list G. pubilabiata as an endangered plant (Association of Wildlife Research and EnVision 2013). Furthermore, this orchid is < 30 cm in height and easy to handle, making it a suitable model plant for physiological and molecular studies.
This paper describes a simple method for germinating seeds of G. pubilabiata using decomposing leaf letter, circumventing the need for laboratory facilities. We also describe the basidiomycete fungus implicated in the germination process. This paper presents a new model system that can be applied to other mycoheterotrophic orchids for conservation purposes.
Sample collection and seed germination
The culture vessels and leaf litter used in this study and the results of seed germination
Size of the vessels
Location of leaf litter collected
L260 × W190 × D100a
L411 × W337 × D139
Seedlings were washed three times with sterile water and pelotons in mycorrhizal tissue were teased with needles as described by Rasmussen (1995); these pelotons were cultured on potato dextrose agar (PDA) plates with 50 ppm of streptomycin and tetracycline each. Fungal colonies from single pelotons were transferred to fresh PDA plates for subculture. The fungal isolates obtained in this study were preserved in the Biological Resource Center of the National Institute of Technology and Evaluation (NBRC).
DNA was extracted from fungal isolates as described by Izumitsu et al. (2012). PCR and sequencing were performed as described by Ogura-Tsujita and Yukawa (2008). To identify the mycorrhizal fungi, fungal nuclear ribosomal internal transcribed spacer (ITS) regions were amplified using the ITS1F/ITS4 primer pair (White et al. 1990; Gardes and Bruns 1993). As the collected leaf litter might have been contaminated with seeds from other orchid species, the plant species of the seedlings were also identified molecularly using the 17SE/26SE primer pair (Sun et al. 1994), and we confirmed whether the germinated seedlings were consistent with the seeds used in this study. An entire seedling from Box 1 and a tuber larger than 1 cm from Bottle 3 were used for plant identification according to the above method.
The results of molecular identification of the fungal isolates from G. pubilabiata seedlings
Accession no. of ITS sequence
Closest mach in GenBank
Mycena abramsii voucher KA12-0434 (KR673481)
Mycorrhizal fungus of G. nipponica (LC013372)
Mycorrhizal fungus of G. pubilabiata (LC013346)
Mycena plumbea (JN198391)
Mycena sp. MKACC51882 (FJ785523)
Seeds of G. pubilabiata germinated in four of the five culture vessels used in this study (Table 1), and germination was achieved within 35 days after sowing. There were no differences in seedling establishment among the four types of culture vessel, although the culture in Box 1 resulted in the large number of protocorms (> 50 individuals), while the largest tuber was observed in Bottle 3. The leaf litter components also did not affect seed germination. Fungal rhizomorphs growing from dead leaves and tree branches were often connected to protocorms and roots (Fig. 2a, b, e), indicating that litter-decomposing fungal rhizomorphs associate with G. pubilabiata to transfer carbon from leaf litter to the seedlings. Long-term seedling culture was not achieved in this study. As the leaf litter in the box was broken down into small pieces and white vigorous rhizomorphs turned yellowish, it seems likely that the fungal growth resources became depleted over time. Continuous replenishment of leaf litter may be required for long-term seedling culture. Successful fungal isolation from seedlings indicates that the culture system established in this study could be useful as a seed baiting technique to detect symbiotic fungi from orchid habitats, as shown for terrestrial green orchids (Brundrett et al. 2003). In addition to the leaf litter culture, 70 packets containing orchid seeds were buried for 1 year in the same habitats, but only five seedlings (< 5 mm) were obtained from these packets (data not shown). It seems likely that the leaf litter culture may serve as a propagation method for G. pubilabiata, although additional replicates are required.
The molecular data imply that the Mycenaceae promote seed germination and seedling growth. The Mycenaceae family constitutes prominent fungal partners of G. pubilabiata and two other Gastrodia species, G. confusa and G. nipponica (Ogura-Tsujita et al. 2009; Kinoshita et al. 2016). Mycena osmundicola has been shown to induce germination of G. elata seeds (Xu and Guo 1989). In our study, the fungal isolates were highly homologous to the symbiotic fungi of G. nipponica and G. elata, indicating that the symbiotic Mycena species that we detected may be associated with several Gastrodia species. Furthermore, the F69 isolate was 99% homologous to a symbiotic fungus from adult G. pubilabiata plants, suggesting that a single Mycena species may be present throughout the entire life cycle of G. pubilabiata, in contrast to the situation in G. elata, in which the fungal partner shifts from Mycena to Armillaria as seedling development proceeds (Xu and Guo 2000).
We found that G. pubilabiata seeds germinated in culture vessels filled with leaf litter collected from the natural habitat; fungal isolation was unnecessary. Our system may assist ex situ conservation programs, allowing seedling culture, propagation, and reintroduction of Gastrodia and other mycoheterotrophic orchids associated with litter-decomposing fungi such as Didymoplexis (Burgeff 1932). Furthermore, our method may serve as a model system for observing seedling development and physiological and molecular studies of mycoheterotrophic plants.
KH performed the plant cultivation. KR performed the fungal isolation and identification. YY contributed with the sample collection and field survey. TY provides advice and guidance. YO designed the experiments, analyzed the data and wrote the paper. All authors read and approved the final manuscript.
The authors thank M. Goto, A. Kinoshita and T. Tanaka for collecting samples, and S. Inagaki for valuable advices for culture methods.
The authors declare that they have no competing interests.
Availability of data and materials
All fungal isolates were deposited to the Biological Resource Center of the National Institute of Technology and Evaluation (NBRC).
Consent for publication
Ethics approval and consent to participate
This study was supported by JSPS KAKENHI (Grant Nos. 17K07536 and 15H04417), Research Grant for Plant Science from The New Technology Development Foundation and a National Museum of Nature and Science research grant entitled “Integrated analysis of natural history collections for conservation of highly endangered species”.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Association of Wildlife Research and EnVision (2013) Search system of Japanese Red Data. http://www.jpnrdb.com/
- Brundrett M, Ager E (2011) Wheatbelt orchid rescue project final report 7. Seed collecting, soil baiting and propagation of orchids. In: Wheatbelt orchid rescue project, University of western Australia. Available via DIALOG. http://repository.uwa.edu.au/R/-?func=dbin-jump-full&object_id=29545&current_base=GEN01-INS01
- Brundrett M, Scade A, Batty AL, Dixon KW, Sivasithamparam K (2003) Development of in situ and ex situ seed baiting techniques to detect mycorrhizal fungi from terrestrial orchid habitats. Mycol Res 107:1210–1220View ArticlePubMedGoogle Scholar
- Burgeff H (1932) Saprophytismus und symbiose. Studien an tropischen orchideen. Gustav Fischer, JenaGoogle Scholar
- Burgeff H (1936) Samenkeimung der orchideen und entwicklung ihrer keimpflanzen. Gustav Fischer, JenaGoogle Scholar
- Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes-application to the identification of mycorrhizae and rusts. Mol Ecol 2:113–118View ArticlePubMedGoogle Scholar
- Godo T, Hashimoto T, Nakata S, Miyoshi K (2007) Aseptic seed germination of rare mycoheterotrophic orchid, Gastrodia pubilabiata. In: Abstracts of the symposium of Japanese society for plant cell and molecular biology, vol 25. pp 100 (In Japanese) Google Scholar
- Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A, Gafur A, Ohta A, Kawai M, Yamanaka T, Neda H, Ota Y, Tanaka C (2012) Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience 53:396–401View ArticleGoogle Scholar
- Kinoshita A, Ogura-Tsujita Y, Umata H, Sato H, Hashimoto T, Yukawa T (2016) How do fungal partners affect the evolution and habitat preferences of mycoheterotrophic plants? A case study in Gastrodia. Am J Bot 103:207–220View ArticlePubMedGoogle Scholar
- Leake JR (1994) The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol 127:171–216View ArticleGoogle Scholar
- Merckx VS, Smets EF, Specht CD (2013) Biogeography and conservation. In: Merckx VS (ed) Mycoheterotrophy. Springer, New YorkView ArticleGoogle Scholar
- Ogura-Tsujita Y, Yukawa T (2008) High mycorrhizal specificity in a widespread mycoheterotrophic plant, Eulophia zollingeri (Orchidaceae). Am J Bot 95:93–97View ArticlePubMedGoogle Scholar
- Ogura-Tsujita Y, Gebauer G, Hashimoto T, Umata H, Yukawa T (2009) Evidence for novel and specialized mycorrhizal parasitism: the orchid Gastrodia confusa gains carbon from saprotrophic Mycena. Proc R Soc Lond B Biol Sci 276:761–767View ArticleGoogle Scholar
- Rasmussen H (1995) Terrestrial orchids: from seed to mycotrophic plant. Cambridge University Press, CembridgeView ArticleGoogle Scholar
- Shimaoka C, Fukunaga Y, Inagaki S, Sawa S (2017) Artificial cultivation of Gastrodia species and identification of mycorrhizal fungi. In: Abstracts of the 16th annual meeting of the Japanese society for plant systematics, University of Kyoto, Kyoto, 9–12 March 2017 (In Japanese) Google Scholar
- Sun Y, Skinner DZ, Liang GH, Hulbert SH (1994) Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. TAG Theor Appl Genet 89:26–32View ArticlePubMedGoogle Scholar
- Tashima Y, Terashita T, Umata T, Matsumoto M (1978) In vitro development from seed to flower in Gastrodia verrucosa under fungal symbiosis. Trans Mycol Soc Jpn 19:449–453Google Scholar
- Umata H, Nishi M (2010) Ecological characteristics and water supply effect on in situ conservation in Gastrodia nipponica and G. pubilabiata, the endangered mycho-heterotrophic orchids. Res Bull Kagoshima Univ For 37:137–149 (In Japanese with English abstract) Google Scholar
- Umata H, Yamauchi H, Hashimoto T (2000) In vitro culture of a myco-heterotrphic orchid Gastrodia pubilabiata with a mycorrhizal symbiont of G. verrucosa. Res Bull Kagoshima Univ For 28:27–30 (In Japanese with English Abstract) Google Scholar
- White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic Press, California, pp 315–322Google Scholar
- Xu J, Guo S (1989) Fungus associated with nutrition of seed germination of Gastrodia elata–Mycena osmundicola Lange. Acta Mycol Sin 8:221–226Google Scholar
- Xu J, Guo S (2000) Retrospect on the research of the cultivation of Gastrodia elata Bl, a rare traditional Chinese medicine. Chin Med J 113:686–692PubMedGoogle Scholar
- Yagame T, Yamato M, Mii M, Suzuki A, Iwase K (2007) Developmental processes of achlorophyllous orchid, Epipogium roseum: from seed germination to flowering under symbiotic cultivation with mycorrhizal fungus. J Plant Res 120:229–236View ArticlePubMedGoogle Scholar