Full paper

In vitro symbiotic seIndian endemic orch

*, Thgan

artmen

, Depa

Unive

ting mycorrhizal root

t as Epulorhiza sp., by

ribosomal RNA gene.

Epulorhiza sp. Uninoc-

n of germination, and

l hyphae entered the

ents, or through the

bryonic cells, the em-

erminated seeds were

n occurred only after

fungal association. Sixty-three percent of the protocorms developed their first leaf by 90

al inoculation. All the

pelotons. Our results

vitro production of C.

1. Introduction

Orchids have minute seeds with a very small embryo and

lack an endosperm (Arditti 1992). Mycorrhizal fungi are

therefore a prerequisite for orchid seed germination, as the

germinating seeds are incapable of obtaining nutrients suc-

cessfully without the interaction with mycorrhizal fungi

(Uetake et al. 1992). So, orchids obligately depend on

* Corresponding author. Tel.: 91 9965631914.(K. Sathiyadash).

Available online at www.sciencedirect.com

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 1 8 3e1 8 9E-mail addresses: ksd.bio@gmail.com, ksd.bio@rediffmail.com 2013 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.days and 62% of these produced a second leaf by 120 days after fung

seedlings in green leaf stage produced roots and contained fungal

suggest that the Epulorhiza sp. could be successfully used in the in

nervosa for their reintroduction into its natural environment.Accepted 11 August 2013

Available online 15 October 2013

Keywords:

Epulorhiza sp.

Mycorrhiza

Pelotons

Protocorm

Seedling development

nation was established for C. nervosa. We isolated a fungus by pla

discs of the terrestrial orchid Eulophia epidendreae and identified i

sequencing the internal transcribed spacer (ITS) regions of the

Germination of C. nervosa seeds was higher when inoculated with

ulated seeds of C. nervosa ceased to develop soon after the initiatio

the embryo failed to rupture the seed testa. The isolated funga

germinating seeds either through the pores in-between the integum

rhizoids. After the fungal establishment (peloton formation) in em

bryo transformed into a protocorm and after 45 days, 66% of the g

transformed into protocorms. Nevertheless, promeristem formatioReceived in revised form

10 August 2013

process. The epiphytic orchid Coelogyne nervosa endemic to south India is exploited in an

unsustainable manner for its therapeutic value. So a protocol for symbiotic seed germi-Kullaiyan Sathiyadash a,

Shanmugaraj Bala MuruRadha Raman Pandey c

a Root and Soil Biology Laboratory, Dep

Indiab Plant Genetic Engineering Laboratory

Tamil Nadu, Indiac Department of Life Sciences, Manipur

a r t i c l e i n f o

Article history:

Received 3 January 20131340-3540/$ e see front matter 2013 The Mhttp://dx.doi.org/10.1016/j.myc.2013.08.005ed germination of Southid Coelogyne nervosa

angavelu Muthukumar a,b, Ramalingam Sathishkumar b,

t of Botany, Bharathiar University, Coimbatore 641 046, Tamil Nadu,

rtment of Biotechnology, Bharathiar University, Coimbatore 641 046,

rsity, Canchipur, Imphal 795 003, India

a b s t r a c t

Study on the dependence of orchids on fungi for seed germination and seedling develop-

ment provides a mean for understanding the role of fungi in the orchid developmentjournal homepage: www.elsevier .com/locate/mycycological Society of Japan. Published by Elsevier B.V. All rights reserved.

for 1 min of extension (30 cycles) and a final extension of 72 C

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 1 8 3e1 8 9184mycorrhizal fungi for their growth and survival either

throughout or during part of their life-cycle. The mycorrhizal

fungi provide carbohydrates, nutrients and water to orchids

under natural conditions (Smith 1966). Low water and

nutrient availability in aerial habitats make epiphytic orchids

dependent on mycorrhizas (Suarez et al. 2009). The uptake of

nutrients from the digesting fungal pelotons by the immature

embryo stimulates seed germination, protocormdevelopment

and seedling growth in orchids (Clements 1988; Rasmussen

1995; Arditti 1996). An appropriate fungal mycobiont there-

fore is essential not only for seedling development and

establishment, but also for nutritional support for long term

survival of the orchids. Therefore, the presence ofmycorrhizal

association is essential for successful colonization and

establishment of an orchid in nature, as well as inmanaged or

restored habitats (Zettler 1997ab; Rasmussen 2002; Leake

2004).

The most commonly found fungal symbionts of orchid

mycorrhizae belong to the form-genus Rhizoctonia. This form-

genus includes fungi with perfect states belonging to Cerato-

basidiaceae, Sebacinaceae and Tulasnellaceae of Agar-

icomycotina of Basidiomycota (Wells 1994). All the binucleate

strains of Rhizoctonia with imperforate parenthesomes and

telomorphs in Tulasnella were placed in the genus Epulorhiza.

Earlier studies have shown the presence of Epulorhiza (ana-

morph of Tulasnella) in roots of both epiphytic and terrestrial

orchids (Zettler et al. 1998; Pereira et al. 2003, 2005;

Nontachaiyapoom et al. 2010). Nevertheless, the mycobionts

of orchids in India are largely unknown.

The epiphytic or lithophytic genus Coelogyne consists of

around 125 species distributed from India and China through

South East Asia and Indonesia, the Philippines to the Pacific

and Fiji islands (Abraham and Vatsala 1981). Eight species of

Coelogyne occur in South India, of which the epiphytic Coelo-

gyne nervosa A. Rich. is endemic to south India. This orchid is

frequently found growing on the tree trunks in sholas of

Southern Western Ghats of Kerala and Tamil Nadu

(Murugesan 2005). Major threats for C. nervosa growing in

Velliangiri hills include habitat disturbance, over harvesting

for trade as a medicinal herb and human interference.

Because of their use in traditional medicine, mature plants of

C. nervosa are collected in large scale and commercially sold in

local markets without any restriction (Murugesan and

Balasubramanian 2008). Balasubramanian and Murugesan

(2004) listed C. nervosa among the most commercially exploi-

ted medicinal plants that are harvested for their roots, rhi-

zomes or whole plants. To conserve this natural resource,

appropriate strategies and conservation plans need to be

developed, which will save the orchid biodiversity in this re-

gion (Balasubramanian and Murugesan 2004). Several orchids

like C. nervosa that have therapeutic value (e.g., species of

Cymbidium, Gastrodia, Habenaria and Vanda) are currently

propagated through tissue culture (Sharma et al. 2007; Kaur

and Bhutani 2009; Giri et al. 2012). Such type of in vitro prop-

agation of medicinal orchids could substantially reduce the

pressure on the wild populations of these orchids thereby

enabling their conservation. In vitro symbiotic seed germi-

nation is one of the popular tool for raising orchids forexperimental and conservational purposes (Stewart et al.

2003). As C. nervosa is over exploited in this particularfor 5 min. The amplified PCR products were analyzed by using

1% agarose gel electrophoresis and the bands were visualized

in gel documentation unit using the software Alpha Digidoc

(Alpha Innotech Corporation, San Leandro, USA). The ampli-

fied products were sequenced at Chromous Biotech, Banga-habitat, and no information exists on the symbiotic seed

germination of this orchid, a study was undertaken to inves-

tigate the in vitro symbiotic seed germination of this endemic

orchid whichwould enable its conservation and rehabilitation

in future.

2. Materials and methods

2.1. Study site

The orchid roots for fungal isolation and pods for seed

extraction were collected from Velliangiri Hills (11 040 N &76 930 E; altitude: 520e1840 m. a. s. l.; annual rainfall:500e7000 mm) located in the southern most part of the Nilgiri

Biosphere Reserve of Western Ghats in south India.

2.2. Isolation of the fungus

The mycorrhizal fungus was isolated from the roots of Eulo-

phia epidendreae (Retz.) Fischer, a terrestrial orchid growing in

the same habitat in Velliangiri hills from where pods of C.

nervosa were collected for seed extraction. Roots of E. epiden-

dreae were washed free of soil and adhering debris in running

tap water, surface sterilized in 1% mercuric chloride solution

followed by 70% ethanol for 1 min each and washed in several

changes of sterile distilled water. The roots were then cut into

1-mm discs using sterile razor blade. The discs were then

transferred to Petri plates containing 20 ml potato dextrose

agar medium (PDA: Potato starch 4 g/l, dextrose 20 g/l, agar

15 g/l) after the removal of the velamen tissues. The plates

were incubated at room temperature (25 3 C) upto twoweeks. The hyphae emerging from the root discs were sub-

cultured on PDA slant for further studies.

2.3. DNA extraction, PCR amplification and sequencing

Total genomic DNA was isolated from mycorrhizal fungal

hyphae using Cetyl trimethyl ammonium bromide (CTAB)

method (Weising et al. 1995) and Polymerase Chain Reaction

(PCR) amplification was carried out in an Eppendorf thermal

cycler (Eppendorf, Hamburg, Germany). The PCR was per-

formed using the specific primers ITS1 and ITS4 for amplifi-

cation of fungal ITS region (White et al. 1990). The

amplification of the ITS region was carried out in 20 ml PCR

reaction containing 2 ml of dNTPs (2 mM) (Fermentas, Baden-

Wurttemberg, Germany), 2 ml of Buffer (10) (Fermentas),0.5 ml of ITS1 primer (10 pM), 0.5 ml of ITS4 primer (10 pM), 1 ml

of template (10 ng), 0.1 ml of Taq Polymerase (5 U) (Fermentas)

and 12.3 ml of water. The PCR condition consisted of an initial

denaturation at 94 C for 5 min followed by 94 C at 1 min fordenaturation, 55 C for 1 min for primer annealing and 72 Clore, India. The sequence similarity to database records were

investigated through National Centre for Biotechnology

Information (NCBI) platform (http:www.ncbi.nlm.nih.gov)

using Basic Local Alignment Search Tool (BLAST) (Altschul

et al. 1997). The sequence was submitted to NCBIs GenBank,

accession JF499918.

2.4. Seed collection and sowing

Naturally pollinated, indehiscent mature capsules of C. nervosa

were collected on 26 September 2010 from a shola forest in

Velliangiri Hills, stored in an ice pack and brought to laboratory

for further processing. Capsules were surface sterilized with

0.1% sodium hypochloride solution for 1 min, rinsed in sterile

distilled water and transferred to 70% ethanol for 2 min. The

capsules were washed free of ethanol in five changes of sterile

distilled water and blotted dry by placing it over a sterile filter

failed to survive or develop further in contrast to theM seeds

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 1 8 3e1 8 9 185paper. The capsules were split open and ca. 100 seeds were

sown per Petri plate (90 mm dia.) containing 20 ml of oats

medium using an inoculation loop. The pH of the medium was

adjusted to 5.8 prior to autoclaving. Plates intended for

mycorrhizal treatment were inoculated with 1-cm3 block of

fungal mycelium (hereafter referred to as M) and plateswithout fungus served as control (hereafter referred to as M).All these operationswere performed under sterile conditions in

a laminar air flow chamber. Petri plates were sealed using

parafilm and incubated (18 h light and 6 h dark, 18e20 C andlight intensity 1200 lux) for seed germination. There were five

replicate plates each for M and M treatments.Incubated plates were observed at weekly intervals for

seed germination and contamination. Germination percent-

age for each developmental stage was calculated by dividing

the number of seeds in particular stage by the total number of

seeds sown 100. Seed germination and seedling develop-ment were scored on a scale of 0e5 (modification of Zettler

et al. 1998); stage 0, no germination; 1, production of rhi-

zoid(s) by the embryo (germination); 2, rupture of testa by

enlarging embryo; 3, appearance of shoot; 4, emergence of 1st

leaf and 5, emergence of 2nd leaf.

2.5. Mycorrhizal observation

Germinating seeds in M plates and root bits used for fungalisolation were cleared in 2.5% KOH at 100 C, for 30 min rinsed

Table 1 e Molecular identification of the fungal ITSrecovered from the fungal isolate used in this study. Theclosest Epulorhiza sequences found in GenBank by Blastanalysis are shown.

Origin Host plant Homology Accessionnumber

Singapore Spathoglottis plicata 98% AJ313453

Singapore Spathoglottis plicata 97% AJ313452

Singapore Oncidium varimyce Oncidium nona

95% AJ313448

China Cymbidium goeringii 95% EF393628

Thailand Phaius tankervilleae 94% HM145897

China Cymbidium faberi 92% FJ613267

China Cymbidium faberi 92% FJ613258

Singapore Oncidium varimyce 92% AJ313447

Oncidium nonawhich failed to develop beyond stage 2. Significant (P < 0.001)

differences were evident in the duration taken for the seeds

to germinate (F5, 12 3031.30), to produce rhizoids(F5, 12 3966.84) and for the rupture of testa (F5, 12 3171.85) inM and M treatments (Table 2).

3.3. Protocorm and seedling development

In M plates, protocorms failed to develop and seed devel-opment ceased at the rhizoidal stage (stage 2). But 70% of the

seeds in M plates transformed into protocorms by 45 DAI,and the rupture of testa was complete. The protocorms

developed a promeristem by 60 DAI and 63% of the proto-in water, acidified for 5 min in 5 N HCl and stained in 0.05%

trypan blue overnight (Koske and Gemma 1989). Squashes of

the stained seeds or roots were examined for the presence of

pelotons or fungal structures using an Olympus BX51 micro-

scope (400; Olympus, Tokyo, Japan). The proportion of totalnumber of cells to the number of cells containing pelotons

was also noted.

2.6. Statistical analysis

One-way analysis of variance (ANOVA) was used to test the

significance of variation among seed germination and devel-

opmental stages.

3. Results

3.1. Molecular identification of orchid endophytes

Molecular sequencing of the DNA isolated from the fungal

culture of E. epidendreae revealed a high similarity (92e98%)

with Epulorhiza sp. isolated from various orchid roots (Table 1).

The ITS sequences of Epulorhiza sp. (AJ313453) from Singapore

by Ma et al. (2003) showed the highest similarity (98%) to our

fungal sequence.

3.2. Initiation of seed germination

Seeds started to swell within a week after plating, and seed

germination commenced within weeks in M treatment. Sixpercent of the seeds in M treatment germinated and pro-duced rhizoids at 15 days after inoculation (DAI) compared to

11% in M treatment. By 30 DAI, 84% of the seeds in Mformed rhizoids (Table 2; Fig. 1A). However, in M treatmentonly 71% of the germinated seeds produced rhizoids. Though,

71% of the seeds in M treatment germinated, none of theseentered stage 2 (rupture of testa).

In M plates, the fungal hyphae first fully encased theimbibed seed and then penetrated the testa directly or entered

the rhizoids to colonize embryonic and suspensor cells

forming pelotons (Fig. 1B, C). Certain regions of the fungal

hyphae developed swelling to form beaded moniliform hy-

phae (Fig. 1D). By this time, the testa was completely ruptured

and transformed into a protocorm. Only 13% of the M seedscorms had their first leaf after 90 DAI and 62% of these pro-

tocorms had their second leaf after 120 DAI (Table 2). At this

stage, the protocorms started producing small roots which

penetrated the medium exhibiting a positive geotropism.

These roots had cells that contained darkly staining pelotons

(Fig. 1E). After shoot initiation, only 89% of theMprotocormsdeveloped their first leaf and 95% of these seedling with first

leaf developed a second leaf (Fig. 1F). Nearly 25% of the

germinated M seeds failed to reach the 5th stage of devel-opment. Significant (P < 0.001) differences were observed in

4. Discussion

The ITS sequence of the fungal isolate (JF499918) originating

from the roots of E. epidendreae showed high similarity with

that of Epulorhiza isolates from the roots of Spathoglottis plicata

from Singapore (AJ313453, AJ313452) and Cymbidium faberi

from China (FJ613267) (Table 1). This confirms that the fungus

isolated from the roots of E. epidendreae belonged to Epulorhiza.

Table 2 e Effect of Epulorhiza on seed germination and protocorm development of Coelogyne nervosa at different dates ofsymbiotic in vitro culture.

Stage Germination rate (%)

15th day 30th day 45th day 60th day 90th day 120th day

Ma M M M M M M M M M M MNo germination (stage 0) 89 2.8b 94 1.67 29 1.92 16 1.38 0 0 0 0 0 0 0 0Production of rhizoid(s) by

the embryo (stage 1)

11 1.05 6 0.63 71 1.33 84 1.17 0 30 1.65c 0 0 0 0 0 0

Rupture of testa by enlarging

embryo (stage 2)

0 0 0 0 0 70 1.22 0 0 0 0 0 0

Appearance of shoot (stage 3) 0 0 0 0 0 0 0 70 1.30 0 7 1.40 0 0Emergence of 1st leaf (stage 4) 0 0 0 0 0 0 0 0 0 63 1.92 0 8 2.36Emergence of 2nd leaf (stage 5) 0 0 0 0 0 0 0 0 0 0 0 62 2.01a M, seeds inoculated with mycorrhizal fungi; M, seeds without mycorrhizal fungi.b Mean S.E.c The germinated seeds turned brown and failed to survive beyond this day.

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 1 8 3e1 8 9186the duration of different seedling developmental stages like:

appearance of the shoot (F5, 12 52.53), and emergence of first(F5, 12 16196.40) and second leaves (F5, 12 896.74).Fig. 1 e Stages of symbiotic seed germination in Coelogyne nerv

B: Mycorrhizal hyphae (white arrow heads) encasing the seed.

(arrow head) in the hyphae encasing the germinating seed. E: I

cells. F: Seedlings (white arrow heads) in two leaf stage on oatTaxa in Epulorhiza have been frequently found in association

with tropical epiphytic and terrestrial orchids (Ma et al. 2003;osa. A: Rhizoids and protocorm formation (arrow heads).

C: Peloton (p) formation in embryonic cells. D: Septation

ntact (ip) and degenerating (dp) pelotons in the embryonic

meal agar. Bars: AeC 100 mm; D, E 50 mm; F 0.5 cm.

germinated seed further appears to be influenced by their rate

myc o s c i e n c e 5 5 ( 2 0 1 4 ) 1 8 3e1 8 9 187Pereira et al. 2003; Sharma et al. 2003; Athipunyakom et al.

2004; Bonnardeaux et al. 2007; Illyes et al. 2009; Tan et al. 2012).

In vitro seed germination is an effective method for orchid

seedlings production and their reintroduction into natural

habitats (Stewart and Kane 2006). Although, successful

asymbiotic seed germination for Coelogyne species was previ-

ously reported (Kathiyar et al. 1987; Ananthan et al. 2003;

Basker et al. 2004; Sebastinraj et al. 2006; Nongrum et al.

2007; Naing et al. 2011), this is the first study documenting

the symbiotic seed germination for the genus Coelogyne and

more specifically for C. nervosa. Although, the percentage of

seeds germinated under non-mycorrhizal conditions (M)was higher (90%) for C. nervosa, the germinated seeds failed to

develop further resembling the observations made for some

terrestrial orchids. For example, seeds of the terrestrial orchid

Goodyera repens could germinate in the soil even in the absence

of the fungal symbiont, nevertheless such type of seed

germination was followed by high seedling mortality

(Rasmussen 2002). As orchids use fungi as a source of carbon,

vitamins, hormones and amino acids for their growth and

development (Zettler et al. 2007), their absence could defer not

only the development, but also result in high seedling mor-

tality during early stages of plant development.

The commencement of seed germination in M plateswithin a week after fungal inoculation was quite rapid

compared to time reported for asymbiotic seed germination in

C. nervosa. Different studies have shown that the time taken

for asymbiotic seed germination in C. nervosa could range

between 44 and 100 days (Shibu et al. 2012; Sonia et al. 2012).

Similarly, the time taken for symbiotic seed germination of C.

nervosa in the present study is also less than those reported for

other species of Coelogyne (14e60 days) under asymbiotic seed

germination (Kathiyar et al. 1987; Ananthan et al. 2003; Basker

et al. 2004; Sebastinraj et al. 2006; Nongrum et al. 2007; Naing

et al. 2011) or for other epiphytic orchids under symbiotic seed

germination. In Epidendrum nocturnum, the germination of

seeds inoculated with Eulophia repens commenced within 3

weeks after the fungal inoculation (Zettler et al. 2001). Simi-

larly, visible changes associated with seed germination were

observed only 30 days after fungal inoculation in the epiphytic

orchid Grammatophyllum speciosum (Salifah et al. 2011).

In the present study, the Epulorhiza sp., originating from

the roots of the terrestrial orchid E. epidendreae was able to

promote seed germination and seedling development in the

epiphytic orchid C. nervosa. This shows that the fungus iso-

lated from an orchid species may assist seed germination in

other orchid species. Previous reports have also adequately

demonstrated the in vitro symbiotic seed germination of Spi-

ranthes brevilabris (Stewart and Kane 2007; Zettler et al. 2007),

Microtis media, Thelymitra crista, Disa bracteata (Bonnardeaux

et al. 2007) and Encyclia tampensis (Zettler et al. 2007) using

mycobionts isolated from a different orchid species. There-

fore, the results of the present study clearly suggest that the

most effective fungal isolate which promotes seed germina-

tion need not have its origin from the same host. This obser-

vation also implies that species of Epulorhizamay be non-host

specific, and can associate and function across a wide range of

orchid life-forms. Evidence also exists in the literature of thefailure of the mycobiont originating from the adult plants to

induce seed germination due to switching of the fungalof spread. An important observation made in the present

study was that the germinated seeds possessing 50% or more

of their cells colonized by the fungus failed to survive. Salifah

et al. (2011) also showed that seeds of Grammatophyllum spe-

ciosum in stage 1 or 2 of the Oteros growth index failed to

survive when the fungal isolate had outgrown the seeds.

However, the reasons for the failure of the intensely colonized

seeds to survive are yet to be elucidated.

Of further interest is that the protocorm development of C.

nervosa was dependent on the presence of the fungus similar

to the observations made by Stewart and Kane (2006) in

Habenaria macroceratitis. It has been suggested that the colo-

nization of the protocorm by the mycobiont might trigger a

series of metabolic process like the utilization of starch re-

serves, initiation of growth and meristem differentiation in

orchids (Hadley and Williamson 1971). Clements (1988) indi-

cated that the presence of pelotons was an important indi-

cator of the successful establishment of the symbiosis and

was necessary for the transformation of protocorms into

seedlings. Further, the changes associated with the develop-

ment of the seedlings from protocorms in C. nervosa like the

enlargement of the embryo and its differentiation as well as

the widening of the shoot apical meristems are similar to the

observations made by Ochara et al. (2001) for Kenyan orchids.

Nevertheless, the number of leaves and roots produced by the

mycorrhizal C. nervosa by the end of 120 DAI is less compared

to those reported for the Kenyan Polystachya for the same

period (Ochara et al. 2001). As different species of orchids

respond differently to the culture conditions, the varied

response observed in tenable.

5. Conclusion

The endemic nature and over exploitation of C. nervosa for

traditional medicinal practices necessitate the development

of an efficient plant production protocol. The symbiotic seed

germination data presented here are highly valuable for the

conservation of C. nervosa. This information will be critical for

the future plant production and reintroduction efforts aimed

at the conservation of C. nervosa into its natural habitats.

Earlier studies have shown the successful reintroduction of

in vitro raised orchids like Cypripedium calceolus (Ramsay and

Stewart 1998), Diuris fragrantissima (Dixon and Hopper 1996),partners by the orchids during their development from juve-

nile to adult phases (Rasmussen 2002). However, the promo-

tion of seed germination and establishment of a functional

association in the C. nervosa seedlings by the Epulorhiza sp.,

originating from the roots of an adult E. epidendreae in the

present study do indicate that this fungus could successfully

establish a functional relation with orchids during different

stages of their life-cycle.

In this study, the fungal hyphae encased the seeds wholly

unlike the observations of Ochara et al. (2001) where the

fungal symbionts formed dense hyphal concentrations only at

the micropylar end of the germinating seeds. Further, the

ability of the isolated fungus to sustain the development ofOrchis laxiflora (Muir 1989), Platanthera clavellata (Zettler and

Hofer 1998) and Platanthera ciliaris (Andersen 1996) into their

investigations. Botanical Journal of the Linnean Society 122:183e211.

Clements MA, 1988. Orchid mycorrhizae associations. Lindleyana3: 73e86.

my c o s c i e n c e 5 5 ( 2 0 1 4 ) 1 8 3e1 8 9188Dixon KW, Hopper SD, 1996. Australia. In: Hagsater EE,Dumont VE (eds), Orchids: status survey and conservation actionplan. International Union for the Conservation of Nature andAthipunyakom P, Manoch L, Piluek C, 2004. Isolation andidentification of mycorrhizal fungi from eleven terrestrialorchids. Natural Science 38: 216e228.

Balasubramanian V, Murugesan M, 2004. A note on thecommercially exploited medicinal plants of the VelliangiriHills, Coimbatore District, Tamil Nadu. Ancient Science of LifeJournal 23: 9e12.

Basker C, Deepa MA, Narmatha Bai V, 2004. Asymbiotic seedgermination and seedling development of Coelogyne stricta ean endemic orchid. Journal of Non-Timber Forest Products 11:120e124.

Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J,Sivasithamparam K, 2007. Diversity of mycorrhizal fungi ofterrestrial orchids: compatibility webs, brief encounters,lasting relationships and alien invasions. Mycological Research111: 51e61.natural habitats. To investigate such process for C. nervosa is

our next task.

Disclosure

The authors declare no conflict of interest. All the experi-

ments undertaken in this study comply with the current laws

of Japan.

Acknowledgments

KS thanks the Department of Science and Technology, New

Delhi, India for the financial assistance (Grant No. SR/ITS/

3856/2011-2012) to attend 4th international barcode of life

conference.

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In vitro symbiotic seed germination of South Indian endemic orchid Coelogyne nervosa1 Introduction2 Materials and methods2.1 Study site2.2 Isolation of the fungus2.3 DNA extraction, PCR amplification and sequencing2.4 Seed collection and sowing2.5 Mycorrhizal observation2.6 Statistical analysis

3 Results3.1 Molecular identification of orchid endophytes3.2 Initiation of seed germination3.3 Protocorm and seedling development

4 Discussion5 ConclusionDisclosureAcknowledgmentsReferences