Bull. Natl. Mus. Nat. Sci., Ser. B, 46(3), pp. 111–118, August. 21, 2020

In vitro Seed Propagation and Conservation of the Rediscovered Rare
Liparis hostifolia (Orchidaceae)
Chie Tsutsumi*, Tsuyoshi Hosoya and Tomohisa Yukawa
Department of Botany, National Museum of Nature and Science,
4–1–1 Amakubo, Tsukuba, Ibaraki 305–0005, Japan*E-mail: tsutsumi@kahaku.go.jp(Received 7 May 2020; accepted 24 June 2020)

Abstract Liparis hostifolia is a rare orchid species that was recently rediscovered, after anapproximately 80-year gap in the record, on Minami-iwo-to Island in the Bonin (Ogasawara)
Archipelago, Japan. We conducted in vitro propagation of L. hostifolia with asymbiotic and symbiotic seed germination techniques. Two strains of Tulasnella (Tulasnellaceae) were isolated from L.
hostifolia and used as fungal partners for symbiotic germination. While the germination rate was
low and the protocorm growth was slow in an asymbiotic culture, symbiotic cultures produced ahigh germination rate and rapid protocorm growth. Symbiotic in vitro culture will be useful for
propagation and conservation of this rare, endangered orchid.
Key words : Asymbiotic germination, conservation, Liparis, mycorrhiza, orchid, propagation,
symbiotic germination, Tulasnella.

Introduction
Liparis Rich. is a cosmopolitan orchid genus
consisting of about 320 species (Pridgeon et al.,2005). In Japan, there are 17 recognized speciesof Liparis (Yukawa, 2015). Liparis hostifolia(Koidz.) Koidz. ex Nakai was recorded on
Chichi-jima Island and Minami-iwo-to Island of
the Bonin (Ogasawara) Archipelago, Japan(Koidzumi, 1916; Tuyama, 1937). However,there had been no records from Minami-iwo-to
Island since 1936 or from Chichi-jima Island
since 1938. Recently, Takayama et al. (2019)
rediscovered the species in a 2017 ﬁeld survey of
Minami-iwo-to Island and used the living collection to examine its biological properties. Molecular phylogenetic analyses showed that L. hostifolia is likely a sister to the clade comprising L.
longiracemosa Tsutsumi, T.Yukawa, & M.Kato;
L. makinoana Schltr.; L. suzumushi Tsutsumi,© 2020 National Museum of Nature and Science

T.Yukawa, & M.Kato; and L. yongnoana N.S.
Lee, C.S.Lee & K.S.Lee. Analyses also suggested
that L. hostifolia had probably immigrated from
East Asia and had long been isolated in the Bonin
Archipelago. Liparis hostifolia may currently survive in the natural environment of Minami-iwo-to
Island, which has been well preserved because
the island is unpopulated. In contrast, the species
may be extinct from Chichi-jima Island, the vegetation of which is excessively exploited. This
species is ranked as Critically Endangered at thenational level (Ministry of the Environment,
Japan, 2020). The only ex-situ collection of L.
hostifolia consists of three individuals cultivatedat Tsukuba Botanical Garden, National Museumof Nature and Science in Tsukuba, Japan. Consequently, development of artiﬁcial propagation
methods should be prioritized for conservation ofthis species.
Under natural conditions, orchid seed germination requires an association with compatible
mycorrhizal fungi (Rasmussen, 1995). Germina-

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Chie Tsutsumi et al.

tion of the dust-sized seeds depends on thesemycorrhizal associations, which provide ﬁxed
carbon and mineral nutrients (e.g., Alexander etal., 1984; Hadley, 1984; Alexander and Hadley,
1985; Eriksson and Kainulainen, 2011). Most
mycorrhizal fungi associated with Orchidaceae
belong to a small number of taxa within the
Basidiomycota, such as Ceratobasidiaceae,
Tulasnellaceae, and Sebacinaceae, whereas
Glomeromycota, which are common as arbuscular mycorrhizae in most land plants, have notbeen recorded with the Orchidaceae (Yukawa etal., 2009; Leake and Cameron, 2012; Martos etal., 2012). Tulasnella (Tulasnellaceae) is associated with several Liparis species that are closely
related to Liparis hostifolia, including ʻL.
japonica (Miq.) Maxim.ʼ [the correct nameshould be L. makinoana or a relative (Tsutsumi
et al., 2019)]; L. kumokiri F.Maek.; L. liliifolia(L.) Rich. ex Lindl.; L. loeselii (L.) Rich.; and L.
purpureovittata Tsutsumi, T.Yukawa & M.Kato.
(McCormick et al., 2004; Illyés et al. 2005;
Shimura et al., 2009; Ding et al., 2014; Tsutsumi
et al., 2016).
In vitro asymbiotic germination of seeds is a
useful tool for propagation of both ornamental
and endangered orchid species. Protocols havebeen established for many orchid species (Ardittiand Ernst, 1984; Rasmussen, 1995; Arditti,2008). In uncultured species, however, the optimal culture condition should be determined byexamining factors such as sterilization, lightintensity and duration, temperature, and culture
medium. While asymbiotic germination is a popular technique for orchid seed germination, symbiotic seed germination has recently gained popularity in conservation and restoration projects(Kauth et al., 2008). Multiple studies havereported that symbiotic seed germination andseedling development methods hold advantages
over asymbiotic methods (see Results and Discussion). However, these advantages appear to be
dependent upon selection of a compatible mycobiont. For the Liparis species, in particular species allied to L. hostifolia, there have been studies on asymbiotic and symbiotic seed germination(L. fujisanensis, L. kumokiri, L. koreojaponica inTsutsumi et al., 2011; L. liliifolia in Rasmussen
and Whigham, 1998, Whigham et al., 2002,
McCormick et al., 2004; L. loeselii in Illyés etal., 2005). Here, we report on propagation of L.
hostifolia using asymbiotic and symbiotic invitro seed germination techniques.
Materials and Methods
Seed collection
Three Liparis hostifolia individuals collectedfrom Minami-iwo-to Island in 2017 (Takayama
et al., 2019) are cultivated in the greenhouse of
Tsukuba Botanical Garden, National Museum of
Nature and Science in Tsukuba, Japan. Artiﬁcial
self-pollinations were performed in 2017, andartiﬁcial cross-pollinations were carried out in2018. It took 10–16 months for fruits to ripen.
After self-pollination in 2017, a mature capsulewas harvested and seeds were collected from thecapsule on December 26, 2018 (hereafterreferred to as 2018-seeds). After cross-pollination in 2018, a mature capsule was harvested andseeds were collected on October 9, 2019 (2019seeds). The seeds were separated from the pericarp, dried at room temperature, and used for
germination analyses within 10 days of collection.
Fungal isolation
Fungal hyphae were isolated from two Liparis
hostifolia corms. After a corm was thoroughly
washed in running water and the outer tissues
were trimmed, the remaining inner tissue wassliced into three pieces of ca. 5–10 mm squareand ca. 1 mm thick using clean razor blades.
Each piece was washed three times in sterilized
distilled water and crushed using a sterilized
glass rod in a Petri dish with about 15 mL of cornmeal agar (CMA; Nissui Pharmaceutical Co.,Tokyo) containing 150 ppm streptomycin and
50 ppm tetracycline. After incubation at approximately 25°C in the dark for 3–5 days, fungal
hyphae were transferred to new CMA plates for
puriﬁcation. The isolates were transferred andConservation and propagation of Liparis hostifolia

cultured on potato dextrose agar slants (Nissui
Pharmaceutical Co.) at approximately 25°C in
the dark.
The hyphae were directly used for polymerase
chain reaction (PCR) ampliﬁcation. The internaltranscribed spacer (ITS) regions together withthe 5.8S region of nuclear ribosomal DNA wereused for fungal identiﬁcation. We used a pair ofprimers, ITS1F and ITS4 (White et al., 1990;
Gardens and Bruns, 1993). The procedures of
PCR and puriﬁcation followed Tsutsumi et al.
(2016). Sequences were analyzed using ABI
3500xl (Applied Biosystems) and assembled
using Seqman II (DNAstar Lasergene, WI).
The sequences were submitted to BLAST
searches (Altshul et al., 1997) against the NCBI
sequence database (GenBank) to detect closely
matched sequences. We assigned genus or familynames to our samples based on registeredsequences with ＞ 97% ITS similarity. The two
isolated Tulasnella strains, s1_1_3 and s2_1_3,
were used for symbiotic germination (see Resultsand Discussion). Their sequences were registeredat the DNA Data Bank of Japan (DDBJ) (Accession numbers: s1_1_3, LC552073 and s2_1_3,
LC552074).
Asymbiotic in vitro germination
We conducted asymbiotic germination tests
using both 2018-seeds and 2019-seeds. The procedures for sowing and culturing seeds followed
Miyoshi and Mii (1998). Seeds were sterilized ina solution of NaClO containing 0.25% active
chlorine for 30 min, and then rinsed three times
with sterilized distilled water. Approximately
150-500 seeds were sown in each of 90 × 20 mm
Petri dishes (SH-20S, Terumo Co., Tokyo for the
2018-seeds; SH90-20, AGC Techno Glass Co.,
Shizuoka for the 2019-seeds). Four replicate
plates were prepared. Each plate contained 80 mL
of ND medium solidiﬁed with 2 g of agar per
liter (Tokuhara and Mii, 1993). Immediately aftersowing, each plate was sealed with two layers of
Paraﬁlm™ (Pechiney Plastic Packaging, Menasha, WI). The plates were incubated in the dark
at 25°C. Seed germination and seedling develop-

113

ment were monitored for 40 weeks after sowing(WAS) for the 2018-seeds and 15 WAS for the
2019-seeds. After 15 WAS, the plates weremoved to incubation with weak light. The protocorms were subcultured after 52 WAS.
The rates of germination and protocorm formation were measured under a stereomicroscope(Olympus SZX-12; Olympus Optical, Tokyo,
Japan) at 5, 9, 14, 24, and 40 WAS for the 2018seeds, and at 5, 10, and 15 WAS for the 2019seeds. Germination was deﬁned as the stage
when an embryo emerged from the ruptured seed
coat, and protocorm formation was deﬁned as thestage when both a shoot primordium and rhizoids
were visible. The average rates of germinationand embryo formation were calculated fromthose of the four replicates.
Symbiotic in vitro germination
Symbiotic germination tests were performed
using the 2019-seeds. The seeds were sterilized
as described above. Approximately 150–500
seeds were sown in each of 90 × 20 mm Petridishes (AGC Techno Glass Co.). Each dish contained 80 mL of OMA medium [2.5 g oatmeal
agar (BD, NJ), 6.5 g agar (Wako Chemical,
Kanagawa), and 1 L distilled water, pH 5.8 (modiﬁed from Yagame et al., 2013)]. Two fungal isolates (s1_1_3 and s2_1_3) were used. At 3WAS,
the mycelium of each isolated fungus was inoculated onto three replicate plates on which the seeds
were sown. The dishes were sealed with Paraﬁlm
and incubated in the dark at 25°C. No germination
was observed prior to fungal inoculation. Therefore, monitoring of seed germination and seedlingdevelopment started after fungal inoculation. The
rates of germination and protocorm formation oneach plate were calculated at 5 and 10 weeks after
fungal inoculation (WAI). The averages of thethree replicate cultures were calculated.
Results and Discussion
Asymbiotic germination
Germination rates were quite low (＜ 1% on
average) at 5 weeks after sowing (WAS) for both

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Chie Tsutsumi et al.

2018- and 2019-seeds and gradually increased
with incubation time (Figs. 1, 2). Protocorms
were observed at 9 WAS, and protocorm rates
also gradually increased with time. The rates ofseed germination and protocorm formation in the
2018-seeds at 40 WAS were 23.6% and 19.6%,
respectively. The rate of protocorm formation
was about 25% at 52 WAS (data not shown),
before they were subcultured in the new medium,Fig. 1. Average rates of germination (white) andprotocorm formation (gray) of 2018-seeds inasymbiotic culture at 4, 9, 15, 24 and 40 WAS(weeks after sowing). Bars represent standard
deviations.

Fig. 2. Average rates of seed germination (white)and protocorm formation (gray) of 2019-seeds
in asymbiotic germination at 5, 10, and 15 WAS(weeks after sowing) and in symbiotic germination at 5 and 10 WAI (weeks after inoculation).
Two isolated Tulasnella stains, s1_1_3 ands2_1_3, were used in symbiotic germination.
Bars represent standard deviations.

although exact numbers could not be determinedbecause the enlarged protocorms overlappedeach other after 40 WAS. We obtained more than
300 protocorms from the 2018-seeds.
In the asymbiotic culture, seed germination
required more time and the rates of seed germination and protocorm formation were lower thanthose of allied species, L. fujisanensis, L. koreojaponica, and L. kumokiri, as demonstrated by
Tsutsumi et al. (2011). Germination rates of thethree species in our previous preliminary experiment were more than 80% under the same methods of sterilization, culture medium, temperature,and light conditions as this study (data notshown). The reason for the low rates and slow
germination of L. hostifolia in the asymbiotic
culture remains uncertain. The seeds of L. hostifolia have a high germination ability, as indicatedby the high rates of germination in the symbiotic
culture using seeds from the same capsule as in
the asymbiotic culture (Fig. 2). Further analyses
are needed to determine appropriate asymbiotic
culture conditions.
Fungal identiﬁcation
We obtained 19 fungal isolates from two L.
hostifolia corms. BLAST searches of the ITS
regions of the 19 isolates showed that 10 had
high aﬃnities with Tulasnella (Tulasnellaceae,
Basidiomycota), which are suggested to be
mycorrhizal partners with other allied Liparis
species (McCormick et al., 2004; Illyés et al.
2005; Shimura et al., 2009; Ding et al., 2014;
Tsutsumi et al., 2016). Nine of the ten isolates ofTulasnella were 100% identical to each other andthe remaining one was 99.2% identical (except
heteropeaks). One of the nine genetically identical strains (s1_1_3) and the other strain (s2_1_3)
were used for symbiotic germination.
Of the other nine isolates, three were assigned
to each Cladosporium, Mycoleptodiscus, andNeopestalotiopsis, and the remaining six weremembers of Pleosporales or Hypocreales. Cladosporium is common in many natural environments and considered to be a contaminant. Mycoleptodiscus and Neopestalotiopsis have beenConservation and propagation of Liparis hostifolia

reported as plant pathogens, non-pathogenic
endophytes, or saprobes (Jiang et al., 2018;
Hernández-Restrepo et al., 2019). Pelosporales
and Hypocreales have been regarded as either
endophytic fungi or contaminants (Dearnaley etal., 2012; Oliveira et al., 2014). Therefore, thosenine isolates were excluded as candidates of
mycorrhizal fungi for L. hostifolia.
Symbiotic germination
The germination rates in symbiotic culture
were remarkably high at 5 weeks after fungal
inoculation (WAI) (Fig. 2). The rate in cultureswith the s1_1_3 strain exceeded 90% at 5 WAI
and 10 WAI. The average rate in cultures withthe s2_1_3 strain was 63.5% at 5 WAI and 85.6%
at 10 WAI. In cultures with the s1_1_3 strain,
84.1% of seeds grew to protocorms at 10 WAI. In
cultures with the s2_1_3 strain, the rates of protocorm formation varied depending on the plates
and the rates were 0–83.6% at 5 WAI and 0.5–
88.5% at 10 WAI. These results suggest that
Tulasnella, particularly the 1_1_3 strain, is ahighly compatible fungal partner of L. hostifolia.
Compared to the asymbiotic culture, the seeds
germinated well and grew quickly in the symbiotic culture (Figs. 1–3). Thus, symbiotic culture
is a useful tool for the propagation and growthpromotion of L. hostifolia. Several studies haveshown that symbiotic seed germination has
advantages compared to asymbiotic germinationin terrestrial orchid species such as Dactylorhiza

115

majalis (Rchb.) P.F.Hunt & Summerh. (Rasmussen et al., 1990); Epipactis palustris (L.) Crantz(Rasmussen, 1992); Pterostylis arenicola
M.A.Clem. & J.L.Stewart (Jusaitis and Sorensen,
1993); Diuris longifolia R.Br. and Elythrantherabrunonis (Endl.) A.S.George (Oddie et al.,1994); Platanthera integrilabia (Correll) Luer
(Zettler and McInnis, 1994); Pecteilis radiata(Thunb.) Raf. (Takahashi et al., 2000); Eulophia
alta (L.) Fawc. & Rendle (Johnson et al., 2007);and Caladenia latifolia R.Br. (Bustam et al.,2014). However, direct comparisons between
asymbiotic and symbiotic techniques are questionable because the culture media are very different (Rasmussen et al., 1990). In this study, wewere unable to ﬁnd optimal asymbiotic conditions for L. hostifolia because the paucity of theseeds obtained limited the scope of the experiment. Nevertheless, propagation of L. hostifolia
was achieved under conditions based on previous
successful or standard methods used for allied
species.
We have also succeeded in in vitro ﬂowering
using asymbiotic culture in several Liparis species closely allied to L. hostifolia (C. Tsutsumi,
unpubl. observ.). The Liparis species are generally diﬃcult to cultivate in pots in the nursery forlong periods of time. Therefore, in vitro culture isuseful and eﬃcient for both propagation and exsitu conservation of Liparis and other plants thatare diﬃcult to maintain in living collections in
the nursery.

Fig. 3. Seedlings of Liparis hostifolia. A, at 40 WAS in asymbiotic culture of the 2018-seeds. B, at 10 WAI insymbiotic culture of the 2019-seeds. Scale bars ＝ 1 mm.

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Chie Tsutsumi et al.

Acknowledgments
We thank K. Suzuki, Y. Yamada, and M.
Nakajima (Tsukuba Botanical Garden, National
Museum of Nature and Science) for cultivating
the living plants and fungi, and K. Miyoshi(Graduate School of Horticulture, Chiba University) and J. P. Abe (Graduate School of Life andEnvironmental Sciences, University of Tsukuba)
for the help in the asymbiotic and symbiotic germination techniques. We are grateful to
K. Takayama (Department or Botany, Graduate
School of Science, Kyoto University), D.
Kawaguchi (Ogasawara Islands Branch Oﬃce,
Tokyo Metropolitan Government), and H. Kato
(Department of Biological Sciences, Tokyo
Metropolitan University) for providing the samples, and Y. Hirayama (Department of Botany,
National Museum of Nature and Science) for
help with the molecular experimental work. We
are also grateful to the research team of
Minami-iwo-to lsland 2017 for ﬁeld support,
which was carried out cooperatively by the Tokyo
Metropolitan Government, Japan Broadcasting
Corporation (NHK), and Tokyo Metropolitan University. Funding came from JSPS KAKENHI
Grant Number 19K06815 and research projectsof the National Museum of Nature and Science
entitled “Biological Properties of Biodiversity
Hotspots in Japan” and “Integrated Analysis of
Natural History Collections for Conservation ofHighly Endangered Species”.
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