ArticlePDF AvailableLiterature Review

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development

May 2015
Planta 242(1)

May 2015
242(1)

DOI:10.1007/s00425-015-2301-9

Source
PubMed

Authors:

Jaime A. Teixeira da Silva

Independent

Elena A Tsavkelova

Lomonosov Moscow State University

Songjun Zeng

South China Botanical Garden, Chinese Academy of Sciences

Tzi Ng

The Chinese University of Hong Kong

Show all 8 authorsHide

The genus Dendrobium is one of the largest genera of the Orchidaceae Juss. family, although some of its members are the most threatened today. The reason why many species face a vulnerable or endangered status is primarily because of anthropogenic interference in natural habitats and commercial overexploitation. The development and application of modern techniques and strategies directed towards in vitro propagation of orchids not only increases their number but also provides a viable means to conserve plants in an artificial environment, both in vitro and ex vitro, thus providing material for reintroduction. Dendrobium seed germination and propagation are challenging processes in vivo and in vitro, especially when the extreme specialization of these plants is considered: (1) their biotic relationships with pollinators and mycorrhizae; (2) adaptation to epiphytic or lithophytic life-styles; (3) fine-scale requirements for an optimal combination of nutrients, light, temperature, and pH. This review also aims to summarize the available data on symbiotic in vitro Dendrobium seed germination. The influence of abiotic factors as well as composition and amounts of different exogenous nutrient substances is examined. With a view to better understanding how to optimize and control in vitro symbiotic associations, a part of the review describes the strong biotic relations of Dendrobium with different associative microorganisms that form microbial communities with adult plants, and also influence symbiotic seed germination. The beneficial role of plant growth-promoting bacteria is also discussed.

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REVIEW

Symbiotic in vitro seed propagation of Dendrobium: fungal

and bacterial partners and their inﬂuence on plant growth

and development

Jaime A. Teixeira da Silva

•Elena A. Tsavkelova

•Songjun Zeng

•

Tzi Bun Ng

4,5

•S. Parthibhan

•Judit Dobra

´nszki

•Jean Carlos Cardoso

•

M. V. Rao

Received: 6 March 2015 / Accepted: 8 April 2015

ÓSpringer-Verlag Berlin Heidelberg 2015

Abstract The genus Dendrobium is one of the largest

genera of the Orchidaceae Juss. family, although some of

its members are the most threatened today. The reason why

many species face a vulnerable or endangered status is

primarily because of anthropogenic interference in natural

habitats and commercial overexploitation. The develop-

ment and application of modern techniques and strategies

directed towards in vitro propagation of orchids not only

increases their number but also provides a viable means to

conserve plants in an artiﬁcial environment, both in vitro

and ex vitro, thus providing material for reintroduction.

Dendrobium seed germination and propagation are chal-

lenging processes in vivo and in vitro, especially when the

extreme specialization of these plants is considered: (1)

their biotic relationships with pollinators and mycorrhizae;

(2) adaptation to epiphytic or lithophytic life-styles; (3)

ﬁne-scale requirements for an optimal combination of nu-

trients, light, temperature, and pH. This review also aims to

summarize the available data on symbiotic in vitro Den-

drobium seed germination. The inﬂuence of abiotic factors

as well as composition and amounts of different exogenous

nutrient substances is examined. With a view to better

understanding how to optimize and control in vitro sym-

biotic associations, a part of the review describes the strong

biotic relations of Dendrobium with different associative

microorganisms that form microbial communities with

adult plants, and also inﬂuence symbiotic seed germina-

tion. The beneﬁcial role of plant growth-promoting bacteria

is also discussed.

&Jaime A. Teixeira da Silva

jaimetex@yahoo.com

&Elena A. Tsavkelova

tsavkelova@mail.ru

&Songjun Zeng

zengsongjun@scib.ac.cn

&Tzi Bun Ng

b021770@mailserv.cuhk.edu.hk

&S. Parthibhan

thibhan@gmail.com

&Judit Dobra

´nszki

dobranszki@freemail.hu

&Jean Carlos Cardoso

jeancardosoctv@gmail.com

&M. V. Rao

mvrao_456@yahoo.co.in

P. O. Box 7, Miki-cho Post Ofﬁce, Ikenobe 3011-2,

Kagawa-Ken 761-0799, Japan

Department of Microbiology, Faculty of Biology,

Lomonosov Moscow State University, Leninskie gory 1-12,

119234 Moscow, Russia

Key Laboratory of South China Agricultural Plant Molecular

Analysis and Gene Improvement, South China Botanical

Garden, Chinese Academy of Sciences, Guangzhou 510650,

China

School of Biomedical Sciences, Faculty of Medicine, The

Chinese University of Hong Kong, Hong Kong, China

Shenzhen Research Institute, The Chinese University of

Hong Kong, Shenzhen, China

Department of Plant Science, Bharathidasan University,

Tiruchirappalli 620 024, Tamil Nadu, India

Research Institute of Nyı

´regyha

´za, University of Debrecen,

Nyı

´regyha

´za, P.O. Box 12, 4400, Hungary

Department of Rural Development, Centro de Cie

ˆncias

Agra

´rias, UFSCar, Via Anhanguera, km 174, CP 153,

Araras CEP 13.600-970, Brazil

123

Planta

DOI 10.1007/s00425-015-2301-9

Keywords Dendrobium germination and propagation 

Mycorrhiza Orchid-associated bacteria Orchidaceae 

Symbiotic seed germination

Abbreviations

ABA Abscisic acid

AMF Arbuscular mycorrhizal fungi

C Carbon

CMA Cornmeal agar

DW Dry weight

FW Fresh weight

GA Gibberellin

Gibberellic acid

IAA Indole-3-acetic acid

ITS Internal transcribed spacer

MH Mycoheterotrophy

N Nitrogen

OMA Oatmeal agar

OMF Orchid mycorrhizal fungi

PGPR Plant growth-promoting rhizobacteria

PGR Plant growth regulator

PLB Protocorm-like body

SEM Scanning electron microscopy

Trp Tryptophan

Introduction

Importance: ornamental and medicinal

Orchids form the largest family of angiosperms, the

Orchidaceae Juss., with more than 26,500 species dis-

tributed globally (Kew 2011), an increase from previous

estimates of 25,000 (Dressler 1993,2005; Fay and Chase

2009). In the wild, these plants are threatened not only by

phytopathogens and pests, but also mostly by the anthro-

pogenic impact on tropical rainforests, resulting in defor-

estation, illegal poaching, and commercial

overexploitation, primarily for their ornamental and med-

icinal properties (Roberts and Dixon 2008; Swarts and

Dixon 2009). The genus Dendrobium s.l. (Epidendroideae)

comprises over 1100 species of epiphytic orchids, dis-

tributed from the Himalayas through India and Southeast

Asia to Japan, Australia, New Zealand, Tasmania, and the

Paciﬁc Islands (Kamemoto et al. 1999; Kumar et al. 2011).

Species of the genus Dendrobium are highly valuable

for their vast diversity and their highly ornamental features.

One such example is D. aqueum Lindl. (Fig. 1a–c), a

threatened orchid endemic to India (Parthibhan et al. 2015).

Dendrobium hybrids are desirable for their large variety of

ﬂower colors and shapes, for the number of ﬂowers that

bloom at once and for their recurrent ﬂowering character-

istics (Vendrame et al. 2008). In addition, some Dendro-

bium species have traditional medicinal properties,

particularly in China and India (Zainuddin et al. 2011;

Mohanty et al. 2012).

Dendrobium species and the compounds isolated from

them display a variety of medicinally important activities

(Ng et al. 2012), with several properties described in more

detail next.

Anticancer activity

Moscatilin from D. loddigesii dramatically suppressed the

mutagenicity of 3-amino-1,4-dimethyl-5H-pyrido[4,3b]in-

dole but not furylfuramide (Miyazawa et al. 1999).

Moscatilin thwarted the growth of A549 lung cancer cells,

repressed growth factor-induced neovascularization, and

suppressed the ERK1/2, Akt, and eNOS signaling path-

ways in HUVECs (Tsai et al. 2010). Moscatilin arrested the

growth of human esophageal squamous cell carcinoma and

adenocarcinoma cells, and induced apoptosis and mitotic

catastrophe with heightened expressions of polo-like kinase

1 and cyclin B1 (Chen et al. 2013a). Moscatilin inhibited

migration and invasion of human non-small cell lung

cancer H23 cells. This action of moscatilin was accompa-

nied by a decline in endogenous reactive oxygen species

and a downregulation of activated focal adhesion kinase

and activated ATP-dependent tyrosine kinase (Kowitdam-

rong et al. 2013). Human lung cancer H292 cells exposed

to 4,5,40-trihydroxy-3,30-dimethoxybibenzyl from D. el-

lipsophyllum demonstrated an increase of E-cadherin and a

reduction of vimentin and transcription factor SNAIL,

signifying inhibition of epithelial-to-mesenchymal transi-

tion. At the same time, the activities of activated protein

kinase B and activated extracellular signal-regulated kinase

involved in pro-survival pathways were attenuated (Chao-

tham et al. 2014). Denbinobin, a phenanthrene from D.

nobile, triggered apoptosis in SNU-484 human gastric

cancer cells and suppressed invasion (Song et al. 2012).

Moniliformediquinone from D. moniliforme arrested cell

growth at the S-phase of the cell cycle and brought about

apoptosis in the hormone-refractory metastatic prostate

cancer cell lines DU-145 and PC-3. It elicited DNA dam-

age accompanied by Chk1, Chk2, c-Jun and JNK activa-

tion, mitochondrial membrane potential disruption,

cytochrome c liberation, and activation of caspases-3 and 9

(Hsu et al. 2014). Treatment of C57BL/6 mice bearing

colon cancer, induced by azoxymethane and dextran sulfate

sodium, with an ethanolic extract of D. candidum, brought

about a rise in body weight, diminished serum concentra-

tions of proinﬂammatory cytokines, a signiﬁcant increase

in the mRNA and elevated protein expression levels of

apoptosis-related genes (bax,caspase-3and caspase-9) and

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123

attenuated bcl-2expression levels versus the control group,

resulting in preventive effects against colon carcinogenesis

in mice (Wang et al. 2014).

Anti-inﬂammatory activity

Ephemeranthol A from D. nobile curbed the formation of

nitric oxide and pro-inﬂammatory cytokines by suppressing

NF-jB (nuclear factor kappa-light-chain-enhancer of acti-

vated B cells) activation and mitogen-activated protein

kinase (MAPK) phosphorylation in macrophages (Kim

et al. 2014). D. huoshanense polysaccharides induced IL-

1ra with anti-inﬂammatory activity in human and murine

monocytes. The pathways were shown to be involved in

extracellular signal-regulated kinase/ETS domain-contain-

ing protein ERK/ELK), p38MAPK, phosphatidylinositol

3-kinase (PI3K) and NFjB (Lin et al. 2014).

Anti-angiogenic activity

The ethanolic extract of D. chrysotoxum reduced retinal

angiogenesis during the process of diabetic retinopathy by

inhibiting the expression of vascular endothelial growth

factor (VEGF) and VEGF receptor 2, and some other pro-

angiogenic factors such as matrix metalloproteinase 2/9,

basic ﬁbroblast growth factor, platelet-derived growth

factorA/B, and insulin-like growth factor 1. Retinal in-

ﬂammation was alleviated by suppressing the NFjB sig-

naling pathway. The elevated phosphorylation of p65 and

expression of intercellular adhesion molecule-1 were at-

tenuated (Gong et al. 2014).

Immunoenhancing activity

D. ofﬁcinale and its polysaccharides stimulated cellular

immunity (delayed-type hypersensitivity and activity of

natural killer cells) and nonspeciﬁc immunity (phagocytic

activity of peritoneal macrophages) and splenocyte inter-

feron-gamma production in mice. D. ofﬁcinale, but not its

polysaccharides, upregulated humoral immunity (serum

hemolytic complement activity) (Liu et al. 2011). Water-

soluble polysaccharides from D. tosaense stems given

orally for 3 weeks elevated, in recipient mice, the number

and cytotoxicity of splenic natural killer cells, cytokine

production in spleen cells and phagocytic activity of

macrophages (Yang et al. 2014a,b).

Antidiabetic activity

A polysaccharide from D. huoshanense had higher hypo-

glycemic potency and protective activity against alloxan-

elicited oxidative injury than its counterparts from D. no-

bile and D. ofﬁcinale (Pan et al. 2014). Gigantol and a new

ﬂavonol glycoside from D. devonianum, 5-hydroxy-3-

methoxy-ﬂavone-7-O-[b-D-apiosyl-(1?6)]-b-D-glucoside,

displayed more pronounced a-glucosidase inhibitory ac-

tivity than the antidiabetic drug acarbose (Sun et al. 2014).

D. huoshanense polysaccharide manifested improved

antiglycation activity after sulfation (Qian et al. 2014).

Antioxidant activity

Isoamoenylin, a dihydrostilbene from D. amoenum,

manifested mild antioxidative and meager antibacterial

activities (Venkateswarlu et al. 2002). D. huoshanense

polysaccharide decreased hepatic formation of malondi-

aldehyde (an index of lipid peroxidation), and upregulated

the activities of hepatic antioxidative enzymes and glu-

tathione level in mice exposed to the hepatotoxin carbon

tetrachloride (Tian et al. 2013).

Hepatoprotective activity

Three antiﬁbrotic phenanthrenes from D. nobile, denbinobin,

ﬁmbriol B and 2,3,5-trihydroxy-4,9-dimethoxyphenan-

threne, undermined the proliferation of hepatic stellate cells

and brought about cell loss through autophagy-linked apop-

tosis (Yang et al. 2012). Chronic administration of D. of-

ﬁcinale granules alleviated alcohol-induced hypertension,

hepatic and renal damage and blood lipid abnormalities (Lv

a b c

Fig. 1 Dendrobium aqueum Lindley. aPlant in ﬂowering stage growing in natural wild stand. bImmature and mature undehisced pods. cViable

(v, red staining) and nonviable (nv, pale brown) seeds (960 magniﬁcation). Unpublished photos: M. V. Rao

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123

et al. 2013). D. candidum protected against carbon tetra-

chloride-induced hepatic damage in mice as evidenced by a

decline in serum levels of aminotransferases and proinﬂam-

matory cytokines and mRNA expression levels of NF-jB,

inducible nitric oxide synthase and cyclooxygenase-2 (Li

et al. 2014).

Neuroprotective activity

An alkaloid-enriched extract from D. nobile curtailed

lipopolysaccharide-induced tau protein hyperphosphoryla-

tion in rat hippocampus and apoptosis in the rat brain

(Yang et al. 2014b,c). (-)-Syringaresinol-4,40-bis-O-b-D-

glucopyranoside and (-)-(7S,8R,70E)-4-hydroxy-3,30,5,50-

tetramethoxy-8,40-oxyneolign-70-ene-7,9,90-triol 7,90-bis-

O-b-D-glucopyranoside from D. aurantiacum var. den-

neanum stems protected against glutamate-induced neuro-

toxicity in PC12 cells. Shashenoside I showed selective

cytotoxic activity with an IC

value of 4.17 lM against

the acute myeloid leukemia cell line MV4-11, while it was

inactive against 10 other human tumor cell lines (Xiong

et al. 2013).

Anti-fungal, antimalarial and anti-herpes simplex

virus activities

A mannose-binding lectin from D. ﬁndleyanum pseudob-

ulbs suppressed fungal growth (Sattayasai et al. 2009).

Phoyunnanin E and densiﬂorol B from D. venustum

demonstrated potent activity while gigantol, batatasin III

and phoyunnanin C exhibited mild antimalarial activity.

Batatasin III and gigantol also exhibited slight anti-herpes

simplex virus activity (Sukphan et al. 2014).

Hair growth promoting activity

D. candidum polysaccharides enhanced hair growth with a

mechanism associated with an increase in vascular en-

dothelial growth factor mRNA expression (Chen et al.

2014).

Activity on submandibular glands

D. ofﬁcinale polysaccharides mitigated an abnormality in-

volving aquaporin 5, and suppressed pro-inﬂammatory

cytokines, lymphocyte inﬁltration and apoptosis in sub-

mandibular glands of the murine model of Sjo

¨gren’s syn-

drome in which there is a deﬁciency of saliva secretion

(Lin et al. 2011). D. ofﬁcinale polysaccharides activated

M3 muscarinic receptors and stimulated the inﬂow of ex-

tracellular calcium ions, resulting in aquaporin 5 translo-

cation to the apical membrane of human submandibular

gland epithelial cells. This is probably the mechanism

involved in the saliva-promoting activity of D. ofﬁcinale

polysaccharides (Lin et al. 2015).

However, commercial and pharmaceutical overex-

ploitation has resulted in a signiﬁcant reduction of wild and

natural Dendrobium populations, aggravated by an in-

creasing number of highly endangered species (Zhang et al.

2012).

Conservation: needs and approaches

Only integrated approaches, focusing both on ecological

and genetic studies, as well as in situ research and ex

situ propagation, can serve as the most effective strate-

gies for orchid conservation (Swarts and Dixon 2009).

In situ conservation includes orchid habitat management,

taking into account biotic liaisons of the host-plant, the

pollinators and mycorrhizae. Regardless of the storage

protocol, seed germination remains one of the most

widely used techniques since numerous seeds are pro-

duced, a desirable feature for commercial, large-scale

production (Vendrame et al. 2008), as well as for sci-

entiﬁc purposes of orchid reproduction and conservation.

In contrast to terrestrial orchids, the seeds of tropical

epiphytic orchids do not contain inhibitors and lack

physiological barriers to germination, and thus can ger-

minate easily in vitro in the presence of moisture, nu-

trient media and suitable temperatures (Rasmussen 1995;

Swarts and Dixon 2009; Kolomeitseva et al. 2012). At

the same time, when cultivated ex situ under artiﬁcial

conditions, the interactions between the host orchid and

its associated partners are no less important, since plants

undergo different biotic and abiotic stresses that are

aggravated by the lack of natural consortive relations

(Kolomeitseva et al. 2002). Ex situ conservation implies

seed and germplasm banking, selection and storage of

genetically representative seeds and somatic tissues, re-

generation of plants from the stored material, as well as

in vitro propagation (Swarts and Dixon 2009; Teixeira da

Silva et al. 2014b).

The principles of an integrated approach, described by

Swarts and Dixon (2009), might be adopted for orchid

preservation, regardless of the genotype. The long-term

cryoconservation storage of seeds and orchid mycorrhizal

fungi (OMF) could facilitate seed banking and further

propagation of Dendrobium species, and there are numer-

ous studies on successful cryopreservation of pollinia,

seeds, and protocorm-like bodies (PLBs) of Dendrobium

species and hybrids (Wang et al. 1998; Vendrame et al.

2008; Galdiano et al. 2012; Kolomeitseva et al. 2012; re-

viewed in Teixeira da Silva et al. 2014b). Low moisture

content in freezing material is critical for proper cryop-

reservation (Pritchard 1984), and the use of cryoprotectants

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may increase the success of a cryopreservation protocol

(Vendrame et al. 2008).

Batty et al. (2001) reported the long-term ex situ seed

storage of rare terrestrial orchids (Caladenia,Diuris,

Pterostylis, and Thelymitra) as well as their fungal sym-

bionts. OMF, untreated and treated with different

cryoprotectants, were successfully preserved in liquid

nitrogen. The long-term cryopreservation of orchid germ-

plasm and mycorrhizae might be a key strategy for ex situ

orchid conservation (Touchell and Dixon 1993; Batty et al.

2001). Numerous methods are available for general fungal

storage and conservation, but the best techniques are

lyophilization and cryopreservation, which allow the fun-

gal cultures to be maintained genetically and phys-

iologically stable and viable for a long period of time.

Modiﬁed protocols using vitriﬁcation and encapsulation

have been used to preserve the most recalcitrant fungi

(Ryan and Smith 2007; Homolka 2013).

The balanced interactions between the host plant and its

associative microorganisms guarantee ecological stability

and advantageous growth for the partners. Under natural

and horticultural conditions, typical mutualistic asso-

ciations are established between orchid roots and certain

heterogeneous groups of bacteria and fungi. This review

aims to discuss in vitro Dendrobium seed germination,

focusing on the biotic relations with orchid-associated

fungi and bacteria, thus looking at the ex vitro milieu for

clues and strategies to improve symbiotic associations for

better germination and propagation of Dendrobium orchids

in the in vitro milieu.

Orchid mycorrhiza relations

The extreme specialization of orchids is displayed in their

biotic interactions, including pollination strategy, epi-

phytism, and symbiotic seed germination (Rasmussen and

Rasmussen 2014), which make the process of orchid

propagation quite challenging both in natural and artiﬁcial

environments. Like no other plants, orchids form asso-

ciations with a wide range of endophytic fungi. Orchid

mycorrhizae have long been studied, starting from in-

vestigation of their role in seed germination by Noe

¨l

Bernard (1900). Despite this, there are still questions and

controversies about plant-fungal speciﬁcity, nutrient sup-

ply among partners, taxonomic determination, mechanics

of stable symbiosis functioning, and other aspects of

orchid–fungus interactions. Several reviews have been

written on the subject where detailed information about

orchid mycorrhizae can be found (e.g., Knudson 1925;

Burgeff 1959; Otero et al. 2002,2013; Dearnaley 2007;

Smith and Read 2008; Liu et al. 2010; Hynson et al.

2013).

Successful germination in vivo is only possible for an

orchid seed if it meets an appropriate symbiotic mycor-

rhizal fungus, which will provide the energetic and nutri-

tional needs for the seedling (Burgeff 1959; Clements

1988; Smith and Read 1997; Waterman and Bidartondo

2008). The majority of adult orchids are autotrophic, while

achlorophyllous species obtain their complete carbon

process termed mycoheterotrophy (MH) (Merckx et al.

2012). Adult autotrophic plants usually lack strong liaisons

with mycorrhizae, although MH is not directly dependent

on the prevalence of phototrophic activity (Rasmussen and

Whigham 2002). Mycoheterotrophic, achlorophyllous

orchids maintain symbiotic relations with fungi throughout

their lifetime (Roberts and Dixon 2008; Vakhrameeva et al.

2008), whereas a number of green orchids remain partial

mycoheterotrophs and obtain their C and nitrogen (N) both

autotrophically and from mycorrhizae (Gebauer and Meyer

2003). These orchids often associated with arbuscular

mycorrhizal fungi (AMF) that simultaneously form ecto-

mycorrhizae with trees (Bidartondo et al. 2004; Liebel

et al. 2010; Selosse et al. 2010; Merckx et al. 2012).

At the primary stage of germination, orchid seeds are

capable of swelling without fungal inﬂuence due to mini-

mal water uptake and optimal abiotic conditions such as

light and temperature (Smith and Read 2008). As a subset

of orchids, Dendrobium seeds that come into contact with a

moist substrate begin to absorb water and swell

(Kolomeitseva et al. 2012). For many orchid species, initial

expansion of the embryo and cracking of the testa occur

in vitro without the involvement of a fungal symbiont

(Smith and Read 2008). However, in the absence of an

exogenous carbohydrate supply, mycorrhizal colonization

is needed for further development of the protocorm and

seedling growth (Smith and Read 2008; Hynson et al.

2013).

When mycorrhizal hyphae penetrate a parenchyma cell,

they form coil-like globular structures, the pelotons, which

may occupy an entire plant cell; the host orchid digests the

pelotons and retains the nutrients (Burgeff 1959; Clements

1988; Peterson and Currah 1990; Beyrle et al. 1995; Ras-

mussen 1995; Waterman and Bidartondo 2008). Most

orchid mycorrhizal partners, including those of epiphytic

orchids, belong to the form genus Rhizoctonia, a diverse

group of pathogenic, saprotrophic and symbiotic fungi,

which includes anamorphs of basidiomycetes Tulasnella,

Ceratobasidium,Thanatephorus, and Sebacina (Bon-

nardeaux et al. 2007; Otero et al. 2002; Selosse et al. 2011;

Merckx et al. 2012). Most MH plants associated with AMF

occur in the tropics, although, due to the bulk of data

emerging from temperate regions, information about MH

reﬂects mainly the terrestrial species from northern lati-

tudes (Selosse et al. 2010; Hynson et al. 2013). Some fully

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MH orchids, including mostly the tropical species of the

Epidendroideae, are associated with wood- or litter-de-

caying saprotrophic species of the Psathyrellaceae,

Mycenaceae or Gymnopus-related fungi (reviewed in

Selosse et al. 2010; Hynson et al. 2013). Martos et al.

(2012) reported on the differences between the terrestrial

and epiphytic mycorrhizal networks of tropical orchids,

revealing that epiphytic associations were more conserva-

tive and specialized than terrestrial ones. Such tight rela-

tions are explained by co-evolution of the host orchid and

its associated fungi in a more stressful abiotic environment

in epiphytic niches, namely a long period of low water

availability, limited amounts of substrate for optimal

rooting, low nutrient availability, and greater exposure to

evaporation. The authors suggest epiphytism as a major

factor that affects ecological assemblage and evolutionary

constraint in tropical mycorrhizal symbioses (Martos et al.

2012).

Although evidence proving the transfer of C, N, phos-

phorous and other nutrients from the fungus to the host

orchid are scarce and are based almost exclusively on ul-

trastructural studies (e.g., Peterson and Currah 1990;

Dearnaley 2007; Smith and Read 2008), the most recent

studies have employed stable isotope labeling (

C and

N), conﬁrming the direct transfer of C as well as N from

OMF to its achlorophyllous partner (e.g., Rhizanthella

gardneri) (Bougoure et al. 2014). Sto

¨ckel et al. (2014), by

measuring the C and N isotope composition of seedlings

and adult terrestrial European orchids, showed that the

seedlings of plants associated with ectomycorrhizal fungi

were enriched in C and N similarly to fully myco-

heterotrophic adults. Rhizoctonia (fungi) deliver less C and

N compounds to their associated orchid protocorms, and

potentially adults than the ectomycorrhizal fungi provide to

their associated orchid protocorms and adults (Sto

¨ckel et al.

2014). Unfortunately, to the best of our knowledge, isotope

labeling has not been applied to investigations of nutrient

uptake and ﬂow between OMF and epiphytic host orchids.

In mycorrhizal symbioses, the ﬂow of elements is con-

sidered to be bi-directional, from the fungus to the host,

and vice versa (e.g., Cameron et al. 2006; Kuga et al.

2014). The ability to utilize a wide variety of nutrients

(including inorganic and organic nitrogen and phosphorus)

by OMF broadens the habitat both for the fungus and for

the host orchid, particularly in suboptimal environments

(Nurfadilah et al. 2013). The most recent studies on sym-

biotic protocorms of Spiranthes sinensis and its mycor-

rhizal fungus, Ceratobasidium sp. showed that both live

and degraded fungal pelotones transfer C and N to the

protocorms (Kuga et al. 2014). Although Bougoure et al.

(2014) reported that the uptake of nutrients most likely

occurs in a necrotrophic manner, in the case of Rhizan-

thella gardneri and its mycorrhiza, this occurs after lysis of

the pelotones. Even though mycorrhiza are essential for the

formation of stable and beneﬁcial symbiotic relations with

the host orchid, in many instances, particularly ex situ

when growth conditions are not optimal, the fungus dom-

inates the plant, and incompatible interactions may result in

complete necrosis of the plant (e.g., Bonnardeaux et al.

2007; Bougoure et al. 2014). When relatively high con-

centrations of carbohydrates are used for in vitro germi-

nation, OMF are known to become parasitic and kill

protocorms, whereas cellulose favor the symbiotic inter-

actions, as was shown for interactions with Rhizoctonia

fungi and Orchis morio plants (Beyrle et al. 1995).

Thus, to better understand how the successful initiation

and further development of tight symbiotic associations is

triggered in tropical epiphytic orchids, including Dendro-

bium species, new knowledge and up-to-date techniques

are required, as have been applied to orchids from tem-

perate zones.

Dendrobium-associated fungi

In this section, we report on Dendrobium-associated fungi,

their isolation, and possible role in growth and develop-

ment of adult plants, as well as germinated seeds.

Isolation and identiﬁcation

To isolate fungi, several roots (leaves or tubers) are usually

sampled. After rinsing in sterile tap water or buffer, roots

are aseptically sliced into 2–5 mm segments, which can be

placed (slightly pressed), directly onto solid agar medium,

or homogenized to obtain a solution, which is spread on the

surface. However, to distinguish between mycorrhiza and

other associated fungi, histological analysis is required. To

isolate endophytes, the roots should be surface sterilized

with 70 % ethanol and 2 % sodium hypochlorite before

slicing (Nogueira et al. 2005; Pereira et al. 2009). A

mycorrhizal afﬁliation is established when the fungus has

been aseptically isolated directly from the pelotones, and

its symbiotic nature is conﬁrmed by further stimulation of

seed germination. Selection of mycorrhizal roots (i.e., roots

containing OMF) (Brundrett 1991; Zhu et al. 2008) and

detection of colonized zones is important for the isolation

of OMF.

For Dendrobium-associated fungi, root sections were

used to isolate endophytes from D. ofﬁcinale (Zhang et al.

2012) and D. moschatum (Tsavkelova et al. 2003a,2008),

while a single peloton or a clump of pelotons as well as the

mycorrhiza-colonized cells from the root cortex from D.

nobile and D. chrysanthum (Zhu et al. 2008; Chen et al.

2011,2012) were useful. Different media are used to cul-

ture fungi, including potato dextrose agar (PDA), cornmeal

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agar (CMA), oatmeal agar (OMA), malt extract and bran

agar, water agar, or Czapek-Dox mineral agar (Tsavkelova

et al. 2003a; Kang et al. 2007; Hossain et al. 2013). To

prevent bacterial growth, media are supplemented with

antibiotics such as streptomycin, tetracycline, chloram-

phenicol or penicillin (Hossain et al. 2013). Together with

morphological, cultural and sporulation characteristics,

sequencing of internal transcribed spacer (ITS) regions is a

common molecular technique for fungal identiﬁcation

(Table 1). Other speciﬁc marker loci have been used for

more accurate identiﬁcation within the genus Fusarium,

including IGS1 (intergenic spacer), TEF (partial DNA se-

quence of the translation elongation factor 1-a), and Tub

(partial DNA sequence of the beta-tubulin gene)

(Tsavkelova et al. 2008).

Mycorrhizal fungi and promotion of seed

germination

Seed germination, plant growth and nutrition of D.

brymerianum,D. candidum,D. chrysotoxum,D. densiﬂo-

rum,D. loddigesii,D. nobile, and D. primulinum were

promoted by co-inoculation with a range of OMF, includ-

ing Ceratobasidium sp., Ceratorhiza sp., Epulorhiza sp.,

Mycena anoectochila,M. dendrobii,M.orchidicola,

M.osmundicola,Rhizoctonia sp., and Tulasnella sp. (cited

from He et al. 2010). In vitro symbiotic seed germination is

an important tool not only for the study of orchid-fungus

speciﬁcity but also for the production of mycobiont-in-

fected healthy seedlings that could be valuable for both

horticultural and conservation purposes (Nontachaiyapoom

et al. 2011). There have only been few reports of successful

symbiotic germination in vitro for Dendrobium. Guo and

Xu (1990) germinated 20 % of D. hancockii seeds in co-

culture with a fungal isolate and 40–70 % when supple-

mented with fungal liquid extract, while no germination

was observed in the absence of the symbiosis. Non-

tachaiyapoom et al. (2011) reported that three fungal iso-

lates of different anamorphic species of Tulasnella most

effectively promoted seed germination and protocorm de-

velopment of D. draconis. Wu et al. (2012) reported that

four symbiotic fungi isolated from D. catenatum (Epu-

lorhiza sp.) and D. loddigessi (Alternaria sp. and Epu-

lorhiza sp.) promoted seed germination of D. catenatum,

but to various degrees. Two strains of Epulorhiza resulted

in signiﬁcantly higher germination (88.41 and 74.10 %) of

D.catenatum seeds than the control (61.90 %), although

the seedlings could not develop further. Other strains of

Epulorhiza and Alternaria did not signiﬁcantly improve

seed germination, but seedlings formed successfully. De-

tails of these and other studies are provided in Table 2.

Swangmaneecharern et al. (2012) reported that Epu-

lorhiza promoted seed germination and protocorm

development of four Dendrobium species: in D. pulchel-

lum,D. crepidatum and D. ﬁndlayanum, and D. crys-

tallinum seeds co-cultured with compatible fungi

developed more rapidly than seeds sown on OMA, and as

rapidly as seeds sown on Murashige and Skoog (1962; MS)

medium. For D. crystallinum, some seeds sown on MS and

those co-cultured with compatible fungi developed to a

more advanced stage than seeds sown on OMA, although

there were no signiﬁcant differences in growth character-

istics between seeds and protocorms. Tan et al. (2014) used

two mycorrhizal isolates of Tulasnella sp. (JC-02, JC-05)

in the symbiotic germination of D. ofﬁcinale seeds: ger-

mination in OMA was 98.47 and 99.05 %, respectively,

higher than in the control (81.05 %); treatment with these

mycorrhiza simultaneously improved the development of

resulting D. ofﬁcinale plantlets in vitro. Symbiotic seed

germination may be a more expensive and tedious tech-

nique than asymbiotic culture due to fungal isolation,

compatibility testing and in vitro mass production before

the fungus can be used. Nevertheless, symbiotic seed ger-

mination with OMF might be more effective. Zhao et al.

(2013) reported that almost all D. ofﬁcinale seeds culti-

vated on OMA medium (Warcup 1981) with fungi ger-

minated to the third stage, which is characterized by the

appearance of a protomeristem (Stewart and Zettler 2002);

in contrast, all seeds cultivated on OMA medium without

fungi remained ungerminated. OMA culture medium was

used for symbiotic germination in three out of seven papers

and in six Dendrobium species (Table 3).

A suppression subtractive hybridization (SSH) cDNA

library from symbiotically germinated D. ofﬁcinale seeds

revealed an increase in the expression levels of 15 selected

genes, including those encoding UDP-glucosyltransferase

(17.5-fold), b-glucosidase (57.20-fold), an immediate-early

fungal elicitor (9.28-fold), chitinase (33.94-fold), a leucine-

rich repeat receptor kinase (LRR-RK) (29.37-fold), b-1,3-

glucanase (23.67-fold), cysteine protease (23.67-fold), and

a NAC transcription factor (83.34-fold). Based on their

putative functions and the results of the SSH library, these

genes were found to be representative of symbiotic ger-

mination. After conﬁrming the expression by real-time

quantitative PCR, their functions in symbiotic germination

were predicted.

The ‘‘seed packet technique’’, ﬁrst described in Ras-

mussen and Whigham (1993), by which orchid seeds are

sown and retrieved in the ﬁeld under natural conditions,

considers both abiotic (light, shade, humidity, substrate)

and biotic (mycorrhizal and antagonistic fungi, seed

predators) factors for ex situ germination. Using a modiﬁed

method, Wang et al. (2011) reported the ex situ germina-

tion of D. ofﬁcinale,D. nobile, and D. chrysanthum seeds.

‘‘Orchid seed baiting’’ was accomplished by burying the

packets with 50–100 seeds either adjacent to or at varying

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Table 1 Dendrobium fungal endophytes (OMF) and their methods of identiﬁcation (listed chronologically, then alphabetically within each year)

Host plant Name of fungal isolate Methods used for identiﬁcation References

D. moschatum Fusarium solani,F. culmorum,F. proliferatum, and F.

chlamydosporum were isolated as endophytes.

Trichoderma virens,T. viride,Fusarium ventricosum,

Alternaria alternate,Phoma sp., Myrothecium

verrucaria, and unidentiﬁed basidiomycetes were

isolated from the root surface

Morphological, cultural and sporulation characteristics

were studied through light microscopy and SEM;

sequencing of ITS rDNA regions

Tsavkelova et al. (2003a,2008)

D.nobile Xylaria spp., Guignardia mangiferae,Clonostachys

rosea,Trichoderma chlorosporum,Fusarium solani

SEM, sequencing of ITS region of nuclear rDNA and

phylogenetic analysis

Yuan et al. (2009a,b)

D.loddigesii Fusarium,Acremonium,Chaetomella,Cladosporium,

Colletotrichum,Nigrospora,Pyrenochaeta,

Sirodesmium, and Thielavia, etc

Sequencing of 5.8S ITS regions rDNA and

phylogenetic analysis for fungi

Chen et al. (2010)

D.nobile,D.chrysanthum Rhizoctonia-like strains belonging to Tulasnellales,

Sebacinales and Cantharellales. In addition, species

of Xylaria,Fusarium,Trichoderma,Colletotrichum,

Pestalotiopsis, and Phomopsis were identiﬁed

Identiﬁed based on spore and culture characteristics

(colony shape, height and color of aerial hyphae, base

color, growth rate, margin, surface texture, and depth

of growth into medium). Sequence-based methods

were also conducted through sequencing of ITS

regions rDNA, the 5.8S and large subunit rRNA

(nrLSU) and phylogenetic analysis

Chen et al. (2011)

D.devonianum,D.thyrsiﬂorum 30 endophytic fungi in D.devonianum were

categorized into 11 taxa and 23 fungal endophytes in

D.thyrsiﬂorum were grouped into 11 genera,

respectively

ITS and ultrastructural analysis. Antibacterial and

antifungal activity were tested: 10 endophytic fungi

in D.devonianum and 11 in D.thyrsiﬂorum exhibited

antimicrobial activity against at least one pathogenic

bacterium or fungus among 6 pathogenic microbes

(Escherichia coli,Bacillus subtilis,Staphylococcus

aureus,Candida albicans,Cryptococcus neoformans,

and Aspergillus fumigatus)

Xing et al. (2011)

D.candidum,D.nobile,D.falconeri,D.loddigesii,

D.primulinum,D.gratiosissimum,D.chrysanthum,

D.hancockii,D.pendulum,D.moniliforme

80 species of fungi belonging to 37 genera identiﬁed.

Among them Acremonium,Alternaria,Ampelomyces,

Bionectria,Cladosporium,Colletotrichum,

Fusarium,Verticillium and Xylaria were the

dominant fungal endophytes

Identiﬁed the fungal isolates based on microscopic and

culture characters and sequence analysis of the ITS

nuclear rDNA region

Chen et al. (2012)

D.ofﬁcinale Mycena sp. Morphological, cultural and sporulation characteristics

were studied through light microscopy and SEM; and

sequence analysis of the ITS nuclear rDNA regions

Zhang et al. (2012)

D.ﬁmbriatum,D.nobile,D.falconeri,D.chrysotoxum,

D.aphyllum,D.crystalinum,D.chrysanthum

Among the 961 endophytes newly isolated, 217

xylariaceous fungi (morphotaxa) were identiﬁed

using morphological and molecular methods

A phylogenetic tree was constructed using nrITS, LSU,

and b-tubulin sequences. Anamorphic xylariaceous

isolates were divided into at least 18 operational

taxonomic units

Chen et al. (2013b)

D. nobile Tulasnella isolates JC-02 and JC-05 Morphological characteristics and phylogenetic

analysis of the ITS sequences

Tan et al. (2014)

ITS internal transcribed spacer, LSU large subunit of ribosomal DNA, nrDNA nuclear ribosomal DNA, OMF orchid mycorrhizal fungus, rDNA ribosomal DNA, SEM scanning electron

microscopy

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Table 2 Symbiotic seed germination in vitro of Dendrobium species (listed chronologically, and alphabetically within each year)

Species and/or

cultivar

Explant used Sterilization

procedure

Culture medium, PGRs and additives Culture

conditions

Remarks, experimental outcome,

acclimatization and variation

References

D. hancockii Symbiotic germination

of mature seeds in

undehisced wild

capsules (age

unclear)

Substrate mixture

(sawdust, wheat

bran and leaves)

as sowing

medium was

sterilized

Fungi isolate and fungi liquid extract 8-h PP; 25 °C Seed germination exceeded 20 % with

fungi, but was 0 % without incubation.

Seed germination from 40 % to 70 %

when incubated in liquid extract

containing cultured fungal products, but

growth of protocorms and seedlings was

low

Guo and Xu (1990)

D. leonis,D.

nobile,D.

crumenatum

Bacterization;

symbiotic seed

germination. Seeds

of different ages

12 % household

bleach

20 min ?3X

SDW

Bacillus pumilus culture was inoculated

onto surface of basal KC medium (no

vitamins or PGRs); pH 5.5–6.0; seeds

then spread on top of medium

16-h PP, 4000 lux,

25 °C

B. pumilus promoted germination of D.

crumenatum (40 %), D. nobile (56.5 %),

and D. leonis (83.3 %). Without

bacterization, \10 % of seeds

germinated

Kolomeitseva et al.

(2002)

D. moschatum Bacterization;

symbiotic

germination of

mature seeds

Gentle rub with

70 %

EtOH ?10 %

NaOCl

15 min ?3X

SDW

0.5 ml suspension (10

cfu/ml) of

Rhizobium sp., Sphingomonas sp. and

Mycobacterium sp. bacterial cultures

inoculated onto surface of basal KC (no

vitamins or PGRs), pH 6.0; seeds were

spread above

16-h PP, 3000 lux,

22 ±3°C

No germination in the control as well as

Rhizobium sp.-inoculated seeds. Two

other bacterial strains initiated

germination in 60 days. After 100 days

of incubation, 1.2 % of germinated seeds

(2 ±0.15 mm length seedlings) with

Mycobacterium sp., and 10.4 %

(5 ±0.2 mm) of germinated seeds with

Sphingomonas sp.

Tsavkelova et al.

(2007)

D.draconis Symbiotic seed

germination. Seeds

from a green capsule

(zones between

dehiscence zones

turned yellow)

95 %

EtOH ?ﬂamed

About 600 seeds containing a piece of

Whatman No.1 ﬁlter paper cultured on

MS medium, OMA, or OMA inoculated

with one of 8 fungal isolates (5 95mm

block of PDA containing the fungal

hyphae)

In dark 25 °C for

one week in a

plant growth

chamber ?16-h

PP at 25 °C for

15 weeks

Three fungal isolates of different

anamorphic species of Tulasnella,C1-

DT-TC-1, Pv-PC-1-1, and C3-DT-TC-2,

were the most effective fungi in

germination (in 2 weeks) promoting

protocorms (at 13th week) and further

development (after 13 weeks, 69, 91.9

and 95.2 % respectively)

Nontachaiyapoom

et al. (2011)

D.pulchellum,

D.crepidatum,

D.ﬁndlayanum,

D.crystallinum

Symbiotic seed

germination. Mature,

undehisced capsules

collected from

naturally pollinated

plants

Flamed Seeds were sown on a piece of ﬁlter paper

placed on MS, OMA or OMA inoculated

with a PDA block in the center

containing one of the ﬁve isolates of the

orchid mycorrhizal fungus Epulorhiza sp.

(containing Tulasnella calospora

sequences, i.e., Da-KP-0-1, Pch-SM-TC-

1, Pv-PC-1-1, Ps-KT-0-1, and Cs-QS-0-

In dark at 25 °C for

2 weeks and then

in a 16-h PP for

7 weeks

Some Epulorhiza isolates could promote

seed germination and protocorm

development of four Dendrobium

species. For D.pulchellum,

D.crepidatum, and D.ﬁndlayanum,

seeds co-cultured with compatible fungi

developed more rapidly (within 3 weeks)

than seeds sown on OMA, and as rapidly

as seeds sown on MS. Some

D.crystallinum seeds sown on MS and

those co-cultured with compatible fungi

germinated within 2 weeks and

developed to a more advanced stage

compared to seeds sown on OMA

although no signiﬁcant difference in

average developmental stages of seeds

and protocorms

Swangmaneecharern

et al. (2012)

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distances from adult plants. These packets were made of

nylon with holes large enough to allow fungal spores to

penetrate, but not to allow the seeds to escape. Wang et al.

(2011) described their method as being an effective strat-

egy to investigate orchid–mycorrhizal interactions, seed

mycorrhization and germination under natural ﬁeld condi-

tions, thereby providing a way to localize, collect and

identify orchid-speciﬁc fungi in the wild. Zi et al. (2014)

reported that Tulasnella sp. isolated from seed baiting of D.

aphyllum signiﬁcantly enhanced seed germination by

13.6 %, protocorm formation by 85.7 % and seedling de-

velopment by 45.2 % compared to the control. In the same

study, Epulorhiza, another seed germination-promoted

fungus isolated from Cymbidium mannii, also enhanced

seed germination by 6.5 % and protocorm formation by

20.3 %. However, Trichoderma suppressed seed germina-

tion by 26.4 %.

Symbiotic seed germination, which is essential for

orchid propagation and reintroduction, can be employed for

the conservation of Dendrobium species in their natural

habitats. Hardening of tissue culture-raised plants is an-

other important aspect of orchid mycorrhiza. Plantlets

produced in vitro can not develop natural resistance against

microbial pathogens (Liu et al. 2010). In addition, phys-

iological and anatomical deﬁciencies of in vitro propagated

plants can make them incapable of surviving under ﬁeld

conditions (Liu et al. 2010). Since orchid seedlings can be

infected with mycorrhizal endophytes during seed germi-

nation in nature and subsequently develop resistance to

fungal infection at maturity, biological hardening could

improve the growth and survival of asymbiotically raised

orchid seedlings using an appropriate OMF (Zhang et al.

2012). Associated fungi initiate the expression and pro-

duction of defense mechanism-related secondary metabo-

lites and other OMF-speciﬁc compounds, which provide

protection against biotic and abiotic stresses, resulting in

higher plant survival (Zhang et al. 2012). Zhang et al.

(2012) reported that plant height, number of new buds,

plant fresh weight (FW) and dry weight (DW) of D. of-

ﬁcinale seedlings inoculated with Mycena F-23 increased

179.3, 150, 67.7 and 91.7 % more than the control,

respectively.

OMF, including speciﬁc Rhizoctonia strains, effectively

promoted seed germination and seedling growth of D.

amethystoglossum,D. ‘Chao Praya Garnet’, D. ‘Snow

Flake’, D. tobaense, and D. loddigesii (Guo and Xu 1990;

Kang et al. 2007; Jin et al. 2009; Chen et al. 2010; Non-

tachaiyapoom et al. 2010; Zhang et al. 2012). Some authors

(cited from Liu et al. 2010) could successfully germinate

Dendrobium seeds inoculated with Mycena sp. fungi, ini-

tially isolated from Cymbidium sinense, thus suggesting

low speciﬁcity in plant-mycorrhizal relations since differ-

ent fungi promoted seed germination of the same orchid,

Table 2 continued

Species and/or

cultivar

Explant used Sterilization

procedure

Culture medium, PGRs and additives Culture conditions Remarks, experimental outcome,

acclimatization and variation

References

D. catenatum Mature seeds from

wild, undehisced

capsules

Seeds dipped in

5 % NaOCl

20 min ?5X

SDW

Four symbiotic fungi isolated from the

roots of D. catenatum (C20) and D.

loddigessi (L12, L24b and L28) with

OMA.

10-h PP Strains L12, L28 and L24b began to swell

in 2 weeks and germinate after 3 weeks.

Strains L24b (Epulorhiza) and L28

(Epulorhiza) resulted in higher

germination in 18 weeks (88.41 and

74.10 %, respectively)

Wu et al. (2012)

D. ofﬁcinale Symbiotic seed

germination. Seeds

from mature

capsules

70 % EtOH

1 min ?2.5 %

NaOCl

15 min ?3X

SDW

1- week-old PDA agar plugs with

mycelium of active Tulasnella isolates

inoculated with seeds at 25 °C in dark

12-h PP, 25 °C Two Tulasnella isolates (JC-02 and JC-05)

promoted higher seed germination

(98.47 % and 81.05 %, respectively)

than the control (81.05 %). Without any

symbiotic fungi, seed development was

arrested at stage 2

Tan et al. (2014)

EtOH ethanol (as a percentage, v/v), KC Knudson’s C medium (Knudson 1921), MS Murashige and Skoog (1962) medium, NaOCl sodium hypochlorite, NR not reported, OMA oatmeal agar,

PDA potato dextrose agar, PGR plant growth regulator, PP photoperiod, SDW sterile distilled water

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while the same fungal strains form associations with dif-

ferent orchids. Mycena sp., which was isolated from the

roots and identiﬁed as a mycorrhizal fungus of D. ofﬁcinale

(Zhang et al. 2012), was used to inoculate D. ofﬁcinale

seedlings. This resulted in enhanced growth, including

plant height, number of new buds, plant FW and DW of

seedlings, increasing 179, 150, 68 and 92 % more than the

controls, respectively.

Dan et al. (2012) investigated the effects of OMF on the

growth of D. candidum and D. nobile protocorms and

plantlets in which 40 fungal endophytes were isolated from

different orchid species. Six of them (Mycena dendrobii,

Mycena anoectochila,Epulorhiza sp., Gliocladium sp., and

Cephalosporium sp.) signiﬁcantly improved plantlet

growth and height.

Production of biologically active substances

OMF are known to secrete plant growth regulators (PGRs)

such as gibberellic acid (GA

), auxin (indole-3-acetic acid;

IAA), abscisic acid (ABA), zeatin and zeatin riboside (Wu

et al. 2002; Liu et al. 2010). Trichoderma sp., an OMF

(GN21) from Pleione bulbocodioides, increased seed ger-

mination up to 84.6 %, signiﬁcantly higher than other 8

OMFs (21.9–82.6 %) and the control (77.6 %) on OMA

medium (Yang et al. 2008). Production of PGRs by OMF

might be of great importance not only at the stage of seed

germination and seedling growth, but also for adult plants.

Different pathogenic and symbiotic fungal species are ac-

tive auxin (mainly, IAA) producers (Tudzynski and Sharon

2002; Maor et al. 2004; Tsavkelova et al. 2006). Endo-

phytic fungi of D. moschatum, represented by several

species of the genus Fusarium (F. solani,F. culmorum,

F.proliferatum, and F.chlamydosporum) (Fig. 2) were

capable of producing active IAA (Tsavkelova et al. 2003b,

2012), which biosynthesis enhanced by supplementation of

exogenous tryptophan (Trp), an IAA precursor. Fusarium

sp., isolated as an OMF from the roots of D. densiﬂorum,

secreted GA as well as vitamins B

and B

(folic acid)

(Wu et al. 2002).

The application of elicitors from pathogenic fungi is also

an effective strategy to improve the productivity of useful

secondary metabolites in medicinal D. nobile and D. of-

ﬁcinale (Song and Guo 2001; Chen et al. 2008). Song and

Guo (2001) reported that the fungus AR-18 had signiﬁcant

effect on fresh and DW of D. nobile inoculated in vermi-

culite, increasing FW by 16 and 21 %, respectively. Chen

et al. (2008) reported that the growth of protocorms could

be inhibited when the fungal elicitor was added at an early

stage of culture but was improved when the fungal elicitor

Table 3 Quantiﬁcation of

sterilization procedures and

culture media to establish

symbiotic seed germination in

Dendrobium

Sterilization procedure Culture medium Total

PDA Fungal isolate/extract KC OMA MS

EtOH ?NaOCl 1 1 2

EtOH ?ﬂaming 1

Flaming 1

NaOCl 1 1 2

NR 1 1

Total

11 23 2

EtOH, ethanol, KC Knudson’s C medium (Knudson 1921); MS Murashige and Skoog (1962) medium,

NaOCl sodium hypochlorite, NR not reported, OMA oatmeal agar, PDA Potato-dextrose agar medium

In two papers (b and c) authors used more than one culture media to test symbiotic germination

b, c

The same paper used two types of culture medium

Fig. 2 Endophytic fungal hyphae branching and penetrating the

velamen of the substrate root of Dendrobium moschatum.FH fungal

hyphae, Pnatural perforations of the velamen; scanning electron

microscopy (SEM). Bar 10 lm (originally published in Tsavkelova

et al. 2003a;Ó2003, NAUKA-SPIKF, St. Petersburg, Russia, with

kind permission). D. moschatum grown as a pot plant in a greenhouse.

Substrate roots were cut into 2–5 mm fragments, ﬁxed with a solution

of glutaraldehyde, dehydrated in ethanol solutions of increasing

concentration and 100 % acetone, freeze-dried, coated with Au–Pd in

an IB-3 device (Hitachi), and examined with an 1830I scanning

electron microscope (Amray, USA). After isolation of the endophytic

fungi colonizing the substrate roots of D. moschatum, they were

identiﬁed as belonging to the genus Fusarium

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was added at a later stage of culture. Compared with the

control, MF23 elicitor added on MS solid medium con-

taining 20 % potato extract, 3 % sucrose and 0.8 % agar

medium at weeks 11 and 13 could signiﬁcantly increase the

FW by 16.4 and 23.4 %, respectively, and the DW by 6.7

and 17.9 %, respectively. MF23 added into the medium for

the whole culture period signiﬁcantly decreased the DW by

9.9 %. Compared with the control, MF24 elicitor added at

weeks 11 and 13 could signiﬁcantly increase the FW by

17.7 and 12.0 %, respectively, added at week 11 could

signiﬁcantly increase the DW by 9.2 %, and MF24 added

into the medium for the whole culture period signiﬁcantly

decreased the DW by 13.9 %. Once the symbiotic rela-

tionship has become established, OMF release antagonistic

compounds that effectively prevent the invasion of other

pathogens and indirectly enhance the survival of orchid

seedlings and promote their growth (Chen et al. 2003).

It is known that non-pathogenic Fusarium species,

speciﬁcally F. oxysporum and F. solani strains, can reduce

disease severity and suppress the growth of pathogenic

strains of the same species on crop plants (Olivain and

Alabouvette 1997; Forsyth et al. 2006; Kavroulakis et al.

2007). The capacity of Fusarium species to promote orchid

seed germination was ﬁrst noticed by Bernard (1900) and

more recently by Vujanovic et al. (2000), reporting on

Fusarium isolates that stimulated seed germination and

protocorm formation of the terrestrial orchid, Cypripedium

reginae.

Endophytic F. proliferatum, isolated from the roots of

D. moschatum, is able to produce some gibberellins (GAs),

namely low amounts of GA

, and relatively high levels of

and GA

(Tsavkelova et al. 2008). GA

is known to

affect the development of orchid seedlings either by in-

hibiting symbiotic seed germination in Pterostylis vittata

(Wilkinson et al. 1994), or by promoting shoot and leaf

growth of Cymbidium protocorms (Fonnesbech 1972). F.

proliferatum also produce auxin (46.95 lg/ml) under Trp

supplementation; IAA biosynthesis occurs by the indole

acetamide (IAM) pathway, previously described in bacteria

(Spaepen et al. 2007). Only this strain has a functional

pathway, while in plant pathogenic strains of F. verticil-

lioides,F. fujikuroi and F. oxysporum, the route is inactive

(Tsavkelova et al. 2012). Thus, the production of PGRs

might be important at certain stages of plant colonization

by OMF, depending on the host plant, infection strategy,

and lifestyle of the fungus. In orchids, particularly those

grown ex situ, a crucial aspect is to select an appropriate

community of associative microorganisms in order to

establish a balanced consortium that can resist pathogenic

invasions.

The role of saprotrophic fungi of the genera Fusarium,

Trichoderma,Xylaria,Pestalotiopsis,Cylindrocarpon etc.

in orchid germination and growth is still unclear, although

they are frequently isolated from Dendrobium roots

(Table 1). Among the fungi that inhabit the surface of D.

moschatum’s substrate roots, Trichoderma and Fusarium

strains were isolated, whereas the surface of its aerial roots

was not populated by Fusarium, but rather by Trichoder-

ma,Phoma,Alternaria, and Myrothecium strains

(Tsavkelova et al. 2003a). Trichoderma, which is ubiqui-

tous and among the most commonly known biocontrol

agents, is able to protect plants from diverse plant patho-

gens, including Fusarium species (Vinale et al. 2008;

Alabouvette et al. 2009). This difference in composition of

the associative fungi of aerial and substrate roots of epi-

phytic Dendrobium conﬁrms the suggestion of Martos et al.

(2012) that epiphytic associations are more specialized

than terrestrial ones.

OMF can be applied both in vitro and ex vitro. In vitro

inoculation (Table 2) may be achieved by placing the

asymbiotically raised seedlings on moistened cotton pads

overlying the fungal growth on PDA or other agar culture

media such as MS basal medium for 1 week and then

transferring to pots ex vitro. For the ex vitro application, in

contrast, ﬁrst the mycorrhizal inoculum is prepared by

growing the fungi in PDA for 1 week, while the fresh

fungal mycelium needs to be homogenized in sterile dis-

tilled water or MS basal medium. The inoculum is ap-

plied at the root surface before or after transplantation to

pots containing sterilized potting mixture as support

medium and kept under controlled environmental condi-

tions for a few weeks before reintroduction to the natural

habitat. Therefore, it is highly recommended to apply OMF

for the following purposes: (a) to increase the survival rate

of micropropagated plantlets or seedlings ex vitro; (b) to

enhance both vegetative and reproductive growth of orchid

plants; (c) to stimulate in vitro ﬂowering (Teixeira da Silva

et al. 2014a), early ﬂowering and enhance ﬂower quality;

and (d) to reduce disease infection rates (Hossain et al.

2013).

In orchid–microbial associations, interactions between

partners are complex, involving diverse fungal and bacte-

rial strains, colonizing the surface and the inner tissues of

the plant. These relations point towards a mechanism that

selects the most beneﬁcial community that would guaran-

tee the supply of nutrients and better adaptation to envi-

ronmental conditions (Tsavkelova 2011). One of the

selective factors could be diverse antimicrobials, produced

by orchids, which inhibit the growth of pathogenic fungi

and bacteria (Stoessl and Arditti 1984). Production of ﬂa-

vonoids, alkaloids, phenolic substances and metabolites

explain the use of orchids as antitumor, anticancer, an-

timicrobial, antiviral, and antifungal medicine in traditional

pharmacology (Stoessl and Arditti 1984; Singh and Duggal

2009; Pant 2013). The phytoncides (volatile organic com-

pounds with antimicrobial activity) of Dendrobium

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kingianum Bidw. suppressed bacterial growth, speciﬁcally

of Pseudomonas aeruginosa,Staphylococcus aureus, and

Staphylococcus saprophyticus cultures (Kharitonova

1999). Despite this, orchids, including Dendrobium,

establish strong beneﬁcial associations with different fungi

and bacteria, suggesting the selective and advantageous

choice within microbial partners. On the other hand, when

the subtle balance between the host plant and associative

microorganisms is broken, the pathogenic strains are strong

enough to overcome plant resistance barriers, particularly

in artiﬁcial environments such as greenhouses and com-

mercial ﬁelds.

Dendrobium-associated bacteria

Cyanobacteria

Among symbiotic bacteria, the most prominent role of

cyanobacteria (prokaryotic phototrophs) is their capacity to

ﬁx N

, although in lichens, cyanobionts act both as a

diazotrophic (N

ﬁxation) and an autotrophic (CO

ﬁxa-

tion) component (Rai and Bergman 2002; Adams 2000;

Henskens et al. 2012; Rikkinen 2013). The combination of

assimilation and N

ﬁxation allows the partners of

cyanobacterial symbioses to ﬂourish in a nutrient-poor

environment (Rai and Bergman 2002). Under greenhouse

conditions, two epiphytic species D. moschatum and D.

bigibbum were shown to form strong associations with

cyanobacteria. Under constant warm and moist conditions,

D. bigibbum’s long and interlaced aerial roots were cov-

ered with a thick layer of cyanobacterial biomass, while D.

moschatum, cultivated under less humid conditions, lack

such a phototrophic sheath (Tsavkelova et al. 2003c;

Tsavkelova 2011). The composition and abundance of the

cyanobacterial communities also differ between both

orchids; Nostoc spp. were the only diazotrophic species

colonizing the aerial roots of D.moschatum, whereas the

roots of D.bigibbum were inhabited by Nostoc,Scytonema,

and strains of the LPP (Lyngbia,Phormidium, and Plec-

tonema) group (Tsavkelova et al. 2003c; Tsavkelova 2011).

Under N-limiting conditions, all ﬁlamentous diazotrophic

cyanobacteria formed specialized N

-ﬁxing cells, the

heterocysts. The N

-ﬁxing activity of a cyanobacterial

community of another epiphyte, Phalaenopsis amabilis,

was high (about 800 nmol C

/h/g biomass) (Tsavkelova

et al. 2003c). For known cyanobacterial symbioses, it is

obvious that a great part of the ﬁxed N

is usually provided

to the host plants (Rai and Bergman 2002; Adams and

Duggan 2008; Lindblad 2009). For adult orchids, high N

ﬁxation conﬁrms an important role of cyanobacteria. Ad-

ditionally, the cyanobacterial community provides nutri-

ents for the microbes embedded in it. This cyanobacterial

covering may also play an important role in providing

nutrients to the mycorrhizal fungus. It is believed that the

aerial roots, which are not in touch with the bark or other

substrates, do not have any endomycorrhizal associations

(Katiyar et al. 1986). However, the cyanobacterial coating,

which offers diverse nutrients, contributes to better and

advantageous fungal growth that enable the mycorrhizal

fungus to penetrate inside the aerial root and form a sym-

biosis with the host plant: fungal hyphae and half-digested

pelotones were detected in the cortical cells of Pha-

laenopsis amabilis aerial roots (Tsavkelova et al. 2003d;

Tsavkelova 2011).

The principle roles of the velamen, which is formed by

dead mature cells ﬁlled with air (Noel 1974), are me-

chanical protection, retaining mineral salt solutions, water

conservation and preventing excess loss of water from the

cortex; it also reduces transpiration, reﬂects solar radiation,

and is involved in O

and CO

exchange (Dycus and

Knudson 1957; Sanford and Adanlawo 1973; Noel 1974;

Cockburn et al. 1985; Pridgeon 1986; Zotz and Winkler

2013). Thus, the velamen may protect microbial cells from

different biotic and abiotic factors by harboring microor-

ganisms, while root exudates provide nutrient support

(Tsavkelova 2011).

Rhizobacteria

Yu et al. (2013) identiﬁed a wide diversity of endophytic

bacteria colonizing D. ofﬁcinale roots, most of which be-

longed to Proteobacteria, including Xanthomonadaceae,

Burkholderiaceae,Enterobacteriaceae,Alcaligenaceae,

and Pseudomonadaceae, where the genera Burkholderia,

Sphingomonas,Novosphingobium, and Pseudomonas (in

decreasing abundance). The roots of D. moschatum are also

abundantly colonized by various heterotrophic microbes

(Fig. 3a, b), and bacteria colonizing the aerial roots differ

from those isolated from the substrate roots: the most

commonly isolated species of culturable bacteria from the

substrate roots were Acinetobacter,Rhizobium,Bacillus,

Paenibacillus,Pseudomonas,Mycobacterium, and Strep-

tomyces, whereas the aerial roots were inhabited mainly by

Bacillus,Paenibacillus,Microbacterium,Sphingomonas,

Flavobacterium,Nocardia,Pseudomonas,Rhodococcus,

and Xanthomonas strains (Tsavkelova et al. 2004,2007;

Tsavkelova 2011). In contrast to bacteria colonizing the

substrate roots that form pale, slight yellowish or grayish

colonies, bacteria from the aerial roots of Dendrobium have

bright colors, with pink, purple, orange, yellow and red

pigments (Fig. 4). These pigments usually offer ultraviolet

radiation (UVR) protection, and UVR tolerance is a com-

mon phenotype among phyllosphere and epilithic bacteria

(Roy et al. 1998; Sundin and Jacobs 1999). Pigments such

as carotenoids from Erwinia herbicola, neurosporene and

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123

b-carotene from Escherichia coli, and mycosporine and

scytonemin from Nostoc commune, produced from bacteria

and cyanobacteria play an important role in cellular pro-

tection from UV-A and UV-B radiation (Becker-Hapak

et al. 1997; Ehling-Schulz et al. 1997; Sandmann et al.

1998).

Promotion of seed germination and plant growth has

been successfully applied to diverse agricultural, industrial,

and ornamental species. In contrast to pathogenic bacteria,

associative rhizobacteria are recognized as having a fa-

vorable impact on plant development due to nitrogen

ﬁxation, production of PGRs, improvement of water uptake

and mineral nutrition, and the biosynthesis of antimicrobial

substances reducing the number of phytopathogens

(Tsavkelova et al. 2006; Lugtenberg and Kamilova 2009;

Saharan and Nehra 2011). The advantages brought by such

interactions are often due to the strains of plant growth-

promoting rhizobacteria (PGPR) that inhabit the plant’s

rhizosphere, rhizoplane, phylloplane, as well as inner tis-

sues (endophytes). The beneﬁcial inﬂuence of bacteria on

the growth and development of orchids was ﬁrstly noticed

by Lewis Knudson in 1922 (Knudson 1922), who

inoculated the seeds of Epidendrum and Laeliocattleya

with a diazotrophic strain of Rhizobium leguminosarum

(initially, Bacillus radicicola) to improve germination.

Orchid-associated bacteria, capable of producing auxin

(IAA) were reported for endophytic Pseudomonas spp.,

Bacillus spp., and Xanthomonas spp. strains isolated from

terrestrial orchids of Western Australia (Wilkinson et al.

1989,1994), for a number of different rhizobacteria asso-

ciated with the wild-grown terrestrial Paphiopedilum ap-

pletonianum and epiphytic Pholidota articulata

(Tsavkelova 2011), endophytic Paenibacillus strains of

Cymbidium eburneum (Faria et al. 2013), endophytic

Burkholderia sp., Curtobacterium sp., Enterobacter sp. and

Bacillus sp., which were isolated from the roots of Cattleya

walkeriana (Galdiano Ju

´nior et al. 2011). Sphingomonas

paucimobilis ZJSH1, an endophyte of D. ofﬁcinale, apart

from its capacity to ﬁx nitrogen, was shown to produce

diverse phytohormones, such as IAA, salicylic acid, and

zeatin (Yang et al. 2014a).

Originally isolated from D. moschatum, strains of Sph-

ingomonas sp., Microbacterium sp., Mycobacterium sp.

and Rhizobium sp. were among the most active IAA pro-

ducers, and when supplemented with exogenous Trp, they

produced 50.2, 53.1, 92.9, and 60.4 lg IAA/ml, respec-

tively (Tsavkelova et al. 2007). Tryptophan is a usual auxin

precursor in the IAA biosynthetic pathway, and when

media are supplemented with Trp, it signiﬁcantly stimu-

lates auxin production by microorganisms (Spaepen et al.

2007). Root exudates of several crop plants are known to

contain Trp, which may undergo further conversion to IAA

by plant-associated bacteria (Kravchenko et al. 2004;

Kamilova et al. 2006; Mehdipour Moghaddam et al. 2012),

thus plant root exudates enhance auxin production by as-

sociated bacteria.

To stimulate seed germination and the growth of orchid

seedlings by bacterial cultures in vitro, different techniques

may be used. In vitro seed germination and the subsequent

growth of orchids, including Dendrobium, can be divided

into ﬁve developmental stages (Fig. 5), as was previously

deﬁned for Paphiopedilum (Zeng et al. 2012). Wilkinson

et al. (1989,1994) reported on the successful propagation

of Pterostylis vittata (a terrestrial orchid) seeds on OMA by

co-inoculating with a mycorrhizal fungus (unidentiﬁed)

and several orchid-associated bacteria belonging to Pseu-

domonas putida,Bacillus sphaericus,B. cereus, and

Arthrobacter sp.). Growth was promoted and biomass and

leaf number increased when micropropagated seedlings of

Cattleya loddigesii were treated with a bacterial suspension

of Paenibacillus lentimorbus and P. macerans strains

(Faria et al. 2013). Galdiano Ju

´nior et al. (2011) reported

that endophytic PGPR strains of Bacillus sp. and Enter-

obacter sp. improved growth during ex vitro aclimatization

Fig. 3 Fragment of the Dendrobium moschatum aerial root surface.

SEM. Bbacteria, BA bacterial agglomerates covered with an

exopolisaccharide matrix (originally published in Tsavkelova et al.

2001;Ó2001, Pleiades Publishing, Ltd., Moscow, Russia, with kind

permission). Bar 1lm. The aerial root surface is abundantly

populated by bacteria, which are located as individual bacterial cells,

as well as covered by extracellular polysacharide matrix that covers

microcolonies (bacteria agglomerates)

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123

12 3

Fig. 4 Bacterial colonies (colony-forming units), isolated from the

aerial (a) and substrate (b) roots of Dendrobium moschatum. The

nutrient solid (agarized) media contain: 1glycerol, 2tryptone soya; 3,

5glucose-yeast extract, and 4starch-ammonium. When grown on

carbohydrate-rich media (3,5), bacteria produce exopolysacharides,

forming a mucoid matrix. (Elena Tsavkelova, unpublished data).

Among bacteria colonizing the substrate roots of the plant, there were

strains of Acinetobacter,Bacillus,Mycobacterium,Pseudomonas,

Rhizobium,Rhodococcus; and Bacillus,Curtobacterium,Flavobac-

terium,Microbacterium,Nocardia,Pseudomonas,Rhodococcus,

Sphingomonas, and Xanthomonas isolated from the rhizoplane of its

aerial roots (Tsavkelova et al. 2001,2004,2007)

Fig. 5 Six seed germination and seedling developmental growth

stages of Dendrobium nobile.aStage 0, ungerminated seed with

embryo (unruptured testa). bStage 1, ruptured testa caused by

enlarging embryo (i.e., germination). cStage 2, appearance of the

shoot (i.e., the protomeristem) and/or rhizoids. dStage 3, emergence

and enlongation of the ﬁrst leaf; eStage 4, one leaf and one or more

roots present; eStage 5, presence of two or more leaves and roots

(i.e., seedling). Unpublished photos: Songjun Zeng

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123

of Cattleya walkeriana plantlets by increasing the number

and length of roots, leaf area and dry plant mass. Sphin-

gomonas paucimobilis ZJSH1 signiﬁcantly promoted the

growth of D. ofﬁcinale seedlings, increasing stem length by

8.6 % and fresh weight by 7.5 % (Yang et al. 2014b). To

achieve this, a 50-ll bacterial suspension (10

cfu/ml) was

inoculated on the base of 45-day-old tissue-cultured seed-

lings that were grown at 25 °C and a 12-h photoperiod.

The in vitro germination of seeds of four Dendrobium

species (D. moschatum,D. crumenatum,D. nobile and D.

leonis) was stimulated by the direct co-inoculation with

bacterial cultures (Kolomeitseva et al. 2002; Tsavkelova

et al. 2007). Surface-sterilized seeds were cultured on

Knudson-C (KC) medium (Knudson 1946) together with

Bacillus pumilus,Sphingomonas sp., Mycobacterium sp.,

and Rhizobium sp. (biomass taken from the colonies) was

smeared evenly onto the surface, and seeds were spread

above. Otherwise, 0.1–0.5 ml of a 10

cfu/ml suspension

was added to the surface of KC agar medium, and mature

seeds were spread afterwards (Tavkelova et al. 2007).

Flasks were maintained at 22 °C (day) and 18 °C (night),

with a 16-h photoperiod at 8000 lux (108 lmol m

-2

-1

The germination of D. crumenatum,D. nobile, and D.

leonis seeds, together with Bacillus sp. culture after

120 days of incubation resulted in 40, 56.5 and 83.3 %

germination, respectively, while in the uninoculated con-

trol, the germinated seeds did not exceed 10 %

(Kolomeitseva et al. 2002; Fig. 6). After 60 days of incu-

bation, D. crumenatum seedlings reached a length of

1.6 mm, 39 % were already in the phase of ﬁrst green leaf

emergence, and 35 % of seeds formed protocorms. In the

control, non-bacterial culture, 15 % of seeds formed only

protocorms while the remaining seeds stayed intact

(ungerminated). Dendrobium leonis formed protocorms

and juvenile plants earlier than D. crumenatum.D. nobile,

in contrast, formed protocorms after 1 month, ﬁrst leaves

emerged after 4 months and the ﬁrst root after 6.5 months

after the start of incubation.

In another experiment, Sphingomonas sp., Mycobac-

terium sp., and Rhizobium sp. cultures, previously isolated

from D. moschatum, were added to mature D. moschatum

surface-sterilized seeds, and cultivated on KC medium

without any supplementary PGRs (Tsavkelova et al. 2007;

Tsavkelova 2011). While no germination was observed in

the control (no bacterial inoculation), after 100 days of

incubation with Sphingomonas sp., 10 % of seedlings had

one to two well-developed ﬁrst leaves, additional roots, and

were 5 ±0.2 mm long. Treatment of the seeds with My-

cobacterium sp. resulted in less effective germination, and

only 1 % of germinated seeds formed 2 ±0.15 mm

seedlings. Surprisingly, no seeds germinated in the pres-

ence of a well-known PGPR strain, Rhizobium sp., which

produced abundant exopolysaccharides on sucrose-

Unswollen

seeds

Swollen seeds Protocorms Seedlings with

first leaves

Fig. 6 Dendrobium crumenatum seeds germinated on the 60th day of

incubation without (control) and with bacterial culture of Bacillus

pumilus (modiﬁed from Kolomeitseva et al. 2002). Dark grey non-

inoculated seeds (control); light grey co-culture with B. pumilus.

Numbers are percentages

1 week 2.5 weeks 6 weeks 14 weeks 53 weeks

rh p

ecst

Fig. 7 Stages of Dendrobium nobile Lindl. seed germination. After

sterilization (15 min with 10 % houshold bleach and three rinses with

sterile distilled water), seeds are placed on MS medium and incubated

in the dark for 12 weeks, then placed under a 12-h photoperiod at

constant temperature (25 °C). After 1 week, rhizoids emerge (arrow)

and the testa is clearly ruptured as the seed swells (a). By 2.5 weeks,

protocorms (p) have clearly developed rhizoids (rh) and a

distinguishable shoot tip (st); some seeds remain ungerminated (us)

(b). By 6 weeks, seeds form well-developed protocorms with an

extended chlorophyllated shoot tip (ecst), but others remain less

developed, without an ecst (white, encircled protocorm) (c). By

14 weeks, protocorms have formed two well-developed leaves (d).

Small but well-developed plantlets can be obtained within 6 months

to 1 year (e). All ﬁgures unpublished (Elena Tsavkelova)

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123

containing KC medium, and which fully covered the seeds,

overwhelming them.

Thus, selection of appropriate bacterial strains as well as

media composition, are important for successful bac-

terization and orchid seed germination. The ability of

PGPR strains to produce high amounts of IAA is not a pre-

requisite for the initiation of seed germination like Bacillus

and Paenibacullus strains, producing less than 10 lg/ml

(Kolomeitseva et al. 2002; Tsavkelova et al. 2007; Faria

et al. 2013), and there are strains that do not produce much

IAA but which signiﬁcantly stimulate germination, and

there are strains that produce sufﬁcient IAA (e.g.,

Burkholderia sp. with about 20 lg IAA/ml (Galdiano

´nior et al. 2011)orRhizobium sp. with about 40 lg IAA/

ml (Tsavkelova et al. 2007)) but have no positive impact on

seed germination and propagation. Nevertheless, Dendro-

bium seed can be symbiotically germinated in vitro and co-

cultivated with PGPR, conﬁrming the successful applica-

tion of bacteria and showing the important role of orchid-

associated bacteria in stimulating seed germination when

no mycorrhizal fungus or PGRs are added (Tsavkelova

et al. 2007). Such bacteria-assisted germination may also

take place in vivo, although this has to be proven by in situ

experiments.

Conclusions and future perspectives

Even though asymbiotic orchid propagation has been

studied for a long time, there is still ample interest in im-

proving Dendrobium seed germination. The application of

beneﬁcial microorganisms that can stimulate the develop-

ment of seedlings and promote plant growth, both by pure

cultures or in combination with mycorrhizal fungi, is still a

weakly examined technique, although a promising one.

This is particularly true for germplasm that is in high de-

mand or for increasing the number and biomass of

medicinally important, but rare, germplasm. There are still

many unsolved questions about the diversity of populations

of associative and endophytic bacteria and fungi that in-

habit aerial and substrate roots of Dendrobium, about their

characteristics, functional activity and production of plant

growth stimulators that may inﬂuence plant development in

natural and artiﬁcial conditions, and about the interaction

between OMF and associated bacteria. Stable biotic rela-

tions between the host plant and its symbiotic microor-

ganisms might not only help seed germination but also may

allow the adult plant to better adapt to environmental

stresses, thus making orchid preservation and reintroduc-

tion into the wild (Tsavkelova 2011; Zeng et al. 2014)

more effective. To achieve this objective, seed develop-

ment needs to be studied as a function of time, and the

application of an associative symbiont should be in step

with seed development. While OMF should be added to the

ungerminated seeds for establishing mycorrhizal liaisons,

bacterial cultures show their positive effect on seed ger-

mination and plant growth through all stages (Fig. 5)of

orchid development. Both the bacterization of seeds as well

as of grown up plantlets can lead to beneﬁcial plant growth

and better adaptation to ex vitro conditions. In the case of

D. nobile, seed bacterization allows plantlets to form with

more developed leaves and roots (Fig. 7), relative to seeds

that are germinated in the absence of PGRs. Nevertheless,

bacterial concentration (cfu) should be carefully titrated to

avoid bacterial overgrowth at the expense of seed and

seedling development and survival.

Greater attention should be paid to cryoconservation

techniques that enable the storage of Dendrobium seeds

and protocorms (Teixeira da Silva et al. 2014b) as well as

mycobionts. An understanding of the in vitro milieu

(Teixeira da Silva et al. 2015) and its dynamics would also

allow seed-derived Dendrobium plants to produce in vitro

ﬂowers (Teixeira da Silva et al. 2014a) and to also form

seeds within an artiﬁcial in vitro environment under aseptic

conditions, which has tremendous, and as-yet undiscov-

ered, beneﬁts (Teixeira da Silva et al. 2014c). Finally, the

use of seeds derived from in vitro pollination or from ex

vitro could serve as novel targets for genetic transformation

(Teixeira da Silva et al. unpublished), an experimental

concept that has not yet been tested in Dendrobium.

Author contribution J. A. Teixeira da Silva, E.

A. Tsavkelova, S. Zeng, T. B. Ng, S. Parthibhan, J. Do-

bra

´nszki, J. C. Cardoso and M. V. Rao contributed equally

to the review.

Acknowledgments The authors thank Dr. Meesawat Upatham

(Prince of Songkla University, Thailand) for comments and opinions

on an earlier version of the manuscript.

Conﬂict of interest The authors declare no conﬂicts of interest.

References

Adams DG (2000) Symbiotic interactions. In: Whitton BA, Potts M

(eds) The ecology of cyanobacteria: their diversity in time and

space. Kluwer Academic Publishers, Dordrecht, pp 523–561

Adams DG, Duggan PS (2008) Cyanobacteria-bryophyte symbioses.

J Exp Bot 59:1047–1058

Alabouvette C, Olivain C, Migheli Q, Steinberg C (2009) Micro-

biological control of soil-borne phytopathogenic fungi with

special emphasis on wilt-inducing Fusarium oxysporum. New

Phytol 184(3):529–544

Batty AL, Dixon KW, Brundrett M, Sivasithamparam K (2001) Long-

term storage of mycorrhizal fungi and seed as a tool for the

conservation of endangered Western Australian terrestrial

orchids. Aust J Bot 49:619–628

Becker-Hapak M, Troxtel E, Hoerter J, Eisenstark A (1997) RpoS-

dependent overexpression of carotenoids from Erwinia herbicola

Planta

123

in OxyR-deﬁcient Escherichia coli. Biochem Biophys Res

Commun 239:305–309

Bernard N (1900) Sur quelques germinations diﬁciles. Revue

´ne

´rale Bot 12:108–120

Beyrle HF, Smith SE, Peterson RL, Franco CMM (1995) Coloniza-

tion of Orchis morio protocorms by mycorrhizal fungus: effects

of nitrogen nutrition and glyphosate in modifying the responses.

Can J Bot 73:1128–1140

Bidartondo MI, Burghardt B, Gebauer G, Bruns TD, Read DJ (2004)

Changing partners in the dark: isotopic and molecular evidence

of ectomycorrhizal liaisons between forest orchids and trees.

Proc R Soc London B, Biol Sci 271:1799–1806

Bonnardeaux Y, Brundrett M, Batty A, Dixon K, Koch J, Sivasitham-

param K (2007) Diversity of mycorrhizal fungi of terrestrial

orchids: compatibility webs, brief encounters, lasting relation-

ships and alien invasions. Mycol Res 111:51–61

Bougoure J, Ludwig M, Brundrett M, Cliff J, Clode P, Kilburn M,

Grierson P (2014) High-resolution secondary ion mass spec-

trometry analysis of carbon dynamics in mycorrhizas formed by

an obligately myco-heterotrophic orchid. Plant, Cell Env

37:1223–1230

Brundrett MC (1991) Mycorrhizas in natural ecosystems. In: Mac-

fayden A, Begon M, Fitter AH (eds) Advances in ecological

research, Academic Press, London 21:171–313

Burgeff H (1959) Mycorrhiza of orchids. In: Withner C (ed) The

orchids: a scientiﬁc survey. The Roland Press, New York,

pp 361–395

Cameron DD, Leake JR, Read DJ (2006) Mutualistic mycorrhizas in

orchids: evidence from plant-fungus carbon and nitrogen trans-

fers in the green-leaved terrestrial orchid Goodyera repens. New

Phytol 171:405–416

Chaotham C, Pongrakhananon V, Sritularak B, Chanvorachote P

(2014) A bibenzyl from Dendrobium ellipsophyllum inhibits

epithelial-to-mesenchymal transition and sensitizes lung cancer

cells to anoikis. Anticancer Res 34(4):1931–1938

Chen RR, Lin XG, Shi YQ (2003) Research advances of orchid

mycorrhizae. Chin J Applied Env Biol 9:97–101 (in Chinese

with English abstract)

Chen XM, Guo SX, Meng ZX (2008) Effects of the fungal elicitors on

the growth of Dendrobium candidum protocorms. Chin Trad

Herb Drugs 39:423–426 (in Chinese with English abstract)

Chen XM, Dong HL, Hu KX, Sun ZR, Chen J, Guo SX (2010)

Diversity and antimicrobial and plant-growth-promoting ac-

tivities of endophytic fungi in Dendrobium loddigesii Rolfe.

Plant Growth Regul 29:328–337

Chen J, Hu KX, Hou XQ, Guo SX (2011) Endophytic fungi

assemblages from 10 Dendrobium medicinal plants (Orchi-

daceae). World J Microbiol Biotechnol 27:1009–1016

Chen J, Wang H, Guo SX (2012) Isolation and identiﬁcation of

endophytic and mycorrhizal fungi from seeds and roots of

Dendrobium (Orchidaceae). Mycorrhiza 22:297–307

Chen CA, Chen CC, Shen CC, Chang HH, Chen YJ (2013a)

Moscatilin induces apoptosis and mitotic catastrophe in human

esophageal cancer cells. J Med Food 16(10):869–877

Chen J, Zhang LC, Xing YM, Wang YQ, Xing XK, Zhang DW, Liang

HQ, Guo SX (2013b) Isolation and identiﬁcation of endophytic

and mycorrhizal fungi from seeds and roots of Dendrobium

(Orchidaceae). PLoS One 8:e58268

Chen J, Qi H, Li JB, Yi YQ, Chen D, Hu XH, Wang ML, Sun XL,

Wei XY (2014) Experimental study on Dendrobium candidum

polysaccharides on promotion of hair growth. Zhongguo Zhong

Yao Za Zhi 39(2):291–295 (in Chinese)

Clements MA (1988) Orchid mycorrhizal associations. Lindleyana

3:73–86

Cockburn W, Goh CJ, Avadhani PN (1985) Photosynthetic carbon

assimilation in a shootless orchid, Chiloschista usneoides (Don)

LDL: a variant on crassulacean acid metabolism. Plant Physiol

77:83–86

Dan Y, Meng ZX, Guo SX (2012) Effects of forty strains of

Orchidaceae mycorrhizal fungi on growth of protocorms and

plantlets of Dendrobium candidum and D. nobile. Afr J

Microbiol Res 6:34–39

Dearnaley J (2007) Further advances in orchid mycorrhizal research.

Mycorrhiza 17:475–486

Dressler RL (1993) Phylogeny and classiﬁcation of the orchid family.

Deoscorides Press, Oregon

Dressler RL (2005) How many orchid species? Selbyana 26:155–158

Dycus A, Knudson L (1957) The role of the velamen of the aerial

roots of orchids. Bot Gazette 119:78–87

Ehling-Schulz M, Bilger W, Scherer S (1997) UV-B-induced

synthesis of photoprotective pigments and extracellular polysac-

charides in the terrestrial cyanobacterium Nostoc commune.

J Bacteriol 179:1940–1945

Faria DC, Dias AC, Melo IS, de Carvalho Costa FE (2013)

Endophytic bacteria isolated from orchid and their potential to

promote plant growth. World J Microbiol Biotechnol

29(2):217–221

Fay MF, Chase MW (2009) Orchid biology: from Linnaeus via

Darwin to the 21st century. Ann Bot 104:359–364

Fonnesbech M (1972) Growth hormones and propagation of Cym-

bidium in vitro. Physiol Plant 27:310–316

Forsyth LM, Smith LJ, Aitken EA (2006) Identiﬁcation and

characterization of non-pathogenic Fusarium oxysporum capable

of increasing and decreasing Fusarium wilt severity. Mycol Res

110:929–935

Galdiano Ju

´nior RF, Pedrinho EAN, Castellane TCL, Lemos EGM

(2011) Auxin-producing bacteria isolated from the roots of

Cattleya walkeriana, an endangered Brazilian orchid, and their

role in acclimatization. Rev Bras Cie

ˆncia Solo 35:729–737

Galdiano Ju

´nior RF, Lemos EGM, Faria RT, Vendrame WA (2012)

Cryopreservation of Dendrobium hybrid seeds and protocorms as

affected by phloroglucinol and Supercool X1000. Sci Hortic

148:154–160

Gebauer G, Meyer M (2003)

N and

C natural abundance of

autotrophic and myco-heterotrophic orchids provides insights

into nitrogen and carbon gain from fungal association. New

Phytol 160:209–223

Gong CY, Yu ZY, Lu B, Yang L, Sheng YC, Fan YM, Ji LL, Wang

ZT (2014) Ethanol extract of Dendrobium chrysotoxum Lindl

ameliorates diabetic retinopathy and its mechanism. Vascul

Pharmacol 62(3):134–142

Guo SX, Xu JT (1990) Effects of fungi and its liquid extract on seed

germination of Dendrobium hancockii Rolf. J Chin Mat Med

15(7397–7399):445 (in Chinese with English abstract)

He XH, Duan YH, Chen YL, Xu MG (2010) A 60-year journey of

mycorrhizal research in China: past, present and future direc-

tions. Sci China Life Sci 53:1374–1398

Henskens FL, Green TG, Wilkins A (2012) Cyanolichens can have

both cyanobacteria and green algae in a common layer as major

contributors to photosynthesis. Ann Bot 110:555–563

Homolka L (2013) Methods of cryopreservation in fungi. In: Gupta

VK, Tuohy MG, Ayyachamy M, Turner KM, O’Donovan A

(eds) Laboratory protocols in fungal biology current methods in

fungal biology. Springer, NY, p 604

Hossain MM, Kant R, Van PT, Winarto B, Zeng SJ, Teixeira da Silva

JA (2013) The application of biotechnology to orchids. Critical

Rev Plant Sci 32(2):69–139

Hsu JL, Lee YJ, Leu WJ, Dong YS, Pan SL, Uang BJ, Guh JH (2014)

Moniliformediquinone induces in vitro and in vivo antitumor

activity through glutathione involved DNA damage response and

mitochondrial stress in human hormone refractory prostate

cancer. J Urol 191(5):1429–1438

Planta

123

Hynson NA, Madsen TP, Selosse MA, Adam IKU, Ogura-Tsujita Y,

Roy M, Gebauer G (2013) The physiological ecology of

mycoheterotrophy in mycoheterotrophy. In: Merckx VSFT (ed)

The biology of plants living on fungi. Springer Science ?Busi-

ness Media, New York, pp 297–343

Jin H, Xu ZX, Chen JH, Han SF, Ge S, Luo YB (2009) Interaction

between tissue-cultured seedlings of Dendrobium ofﬁcinale and

mycorrhizal fungus (Epulorhiza sp.) during symbiotic culture.

Chin J Plant Ecol 33:433–441 (in Chinese with English abstract)

Kamemoto H, Kuehnle AR, Amore TD (1999) Breeding Dendrobium

orchids in Hawaii. University of Hawai‘i Press, Honolulu, p 166

Kamilova F, Kravchenko LV, Shaposhnikov AI, Azarova T,

Makarova N, Lugtenberg B (2006) Organic acids, sugars, and

L-tryptophane in exudates of vegetables growing on stonewool

and their effects on activities of rhizosphere bacteria. Mol Plant-

Microbe Interact 19:250–256

Kang ZH, Han SF, Han ZM (2007) Effects of orchidaceous

Rhizoctonias on the growth of Dendrobium candidum. J Nanjing

For Univ (Nat Sci) 31:49–52

Katiyar RS, Sharma GD, Mishra RR (1986) Mycorrhizal infections of

epiphytic orchids in tropical forests of Meghalaya (India). J Ind

Bot Soc 65:329–334

Kavroulakis N, Ntougias S, Zervakis GI, Ehaliotis C, Haralampidis K,

Papadopoulou KK (2007) Role of ethylene in the protection of

tomato plants against soil-borne fungal pathogens conferred by

an endophytic Fusarium solani strain. J Exp Bot 58:3853–3864

Kew (Royal Botanic Gardens) (2011) World checklist of selected

plant families. http://apps.kew.org/wcsp/incfamilies.do. Ac-

cessed 26 April 2015

Kharitonova IP (1999) Phytoncidal activity of prchids. Okhorona i

kul’tivuvannya orkhidei (Preservation and Cultivation of Orch-

ids). Kiev: Nauk. Dumka, pp. 131–133 (in Russian)

Kim JH, Oh SY, Han SB, Uddin GM, Kim CY, Lee JK (2014) Anti-

inﬂammatory effects of Dendrobium nobile derived phenanthre-

nes in LPS-stimulated murine macrophages. DOI, Arch Pharm

Res. doi:10.1007/s12272-014-0511-5

Knudson L (1921) La germinacio

´n simbio

´tica de las semillas de

orquideas. Bol Real Soc Espan

˜ola Hist Nat 21:250–260

Knudson L (1922) Nonsymbiotic germination of orchid seeds. Bot

Gazette 73:1–25

Knudson L (1925) Physiological study of the symbiotic germination

of orchid seeds. Bot Gazette 79:345–379

Knudson L (1946) A new nutrient solution for germination of orchid

seed. Am Orch Soc Bull 15:214–217

Kolomeitseva GL, Tsavkelova EA, Gusev EM, Malina NE (2002) On

symbiosis of orchids and active isolate of the bacterium Bacillus

pumilus in culture in vitro. Bull GBS Russian Acad Sci

183:117–126 (in Russian with English abstract)

Kolomeitseva GL, Antipina VA, Shirokov AI, Khomutovskiy MI,

Babosha AV, Riabchenko AS (2012) The orchid seeds: develop-

ment, structure, germination. Geos, Moscow, p 352 (in Russian)

Kowitdamrong A, Chanvorachote P, Sritularak B, Pongrakhananon V

(2013) Moscatilin inhibits lung cancer cell motility and invasion

via suppression of endogenous reactive oxygen species. Biomed

Res Int 2013:765–894

Kravchenko LV, Azarova TS, Makarova NM, Tikhonovich IA (2004)

The effect of tryptophan of plant root metabolites on the

phytostimulating activity of rhizobacteria. Mikrobiologiia

73:156–158 (in English) and 195–198 (in Russian)

Kuga Y, Sakamoto N, Yurimoto H (2014) Stable isotope cellular

imaging reveals that both live and degenerating fungal pelotons

transfer carbon and nitrogen to orchid protocorms. New Phytol

202:594–605

Kumar P, Rawat GS, Wood HP (2011) Diversity and ecology of

Dendrobiums (Orchidaceae) in Chotanagpur plateau. India.

Taiwania 56(1):23–36

Li GJ, Sun P, Wang Q, Qian Y, Zhu K, Zhao X (2014) Dendrobium

candidum Wall. ex Lindl. attenuates CCl

-induced hepatic

damage in imprinting control region mice. Exp Ther Med

8(3):1015–1021

Liebel HT, Bidartondo MI, Preiss K, Segreto R, Sto

¨ckel M, Rodda M,

Gebauer G (2010) C and N stable isotope signatures reveal

constraints to nutritional modes in orchids from the Mediter-

ranean and Macaronesia. Am J Bot 97:903–912

Lin X, Shaw PC, Sze SC, Tong Y, Zhang Y (2011) Dendrobium

ofﬁcinale polysaccharides ameliorate the abnormality of aqua-

porin 5, pro-inﬂammatory cytokines and inhibit apoptosis in the

experimental Sjo

¨gren’s syndrome mice. Int Immunopharmacol

11(12):2025–2032

Lin J, Chang YJ, Yang WB, Yu AL, Wong CH (2014) The

multifaceted effects of polysaccharides isolated from Dendrobi-

um huoshanense on immune functions with the induction of

interleukin-1 receptor antagonist (IL-1ra) in monocytes. PLoS

One 9(4):e94040

Lin X, Liu J, Chung W, Sze SC, Li H, Lao L, Zhang Y (2015)

Polysaccharides of Dendrobium ofﬁcinale induce aquaporin 5

translocation by activating M3 muscarinic receptors. Planta Med

81(2):130–137

Lindblad P (2009) Cyanobacteria in symbiosis with cycads. In:

Pawlowski K (ed) Prokaryotic symbionts in plants: micro-

biological monographs, vol 8. Springer, Dordrecht, pp 225–233

Liu HX, Luo YB, Liu H (2010) Studies of mycorrhizal fungi of

Chinese orchids and their role in orchid conservation in China—

a review. Bot Rev 76:241–262

Liu XF, Zhu J, Ge SY, Xia LJ, Yang HY, Qian YT, Ren FZ (2011)

Orally administered Dendrobium ofﬁcinale and its polysaccha-

rides enhance immune functions in BALB/c mice. Nat Prod

Commun 6(6):867–870

Lugtenberg B, Kamilova F (2009) Plant-growth-promoting rhizobac-

teria. Annu Rev Microbiol 63:541–556

Lv GY, Xia CQ, Chen SH, Su J, Liu XP, Li B, Gao JL (2013) Effect

of Dendrobium ofﬁcinale granule on long-term-alcohol-induced

hypertension rats. Zhongguo Zhong Yao Za Zhi

38(20):3560–3565 (in Chinese)

Maor R, Haskin S, Levi-Kedmi H, Sharon A (2004) In planta

production of indole-3-acetic acid by Colletotrichum gloeospori-

oides f. sp. aeschynomene. Appl Environ Microbiol 70:852–854

Martos F, Munoz F, Pailler T, Kottke I, Gonneau C, Selosse MA

(2012) The role of epiphytism in architecture and evolutionary

constraint within mycorrhizal networks of tropical orchids. Mol

Ecol 21(20):5098–5109

Mehdipour Moghaddam MJ, Emtiazi G, Salehi Z (2012) Enchanced

auxin production by Azospirillum pure cultures from plant root

exudates. J Agr Sci Technol 14:985–994

Merckx VS, Janssens SB, Hynson NA, Specht CD, Bruns TD, Smets

EF (2012) Mycoheterotrophic interactions are not limited to a

narrow phylogenetic range of arbuscular mycorrhizal fungi. Mol

Ecol 21(6):1524–1532

Miyazawa M, Shimamura H, Nakamura S, Sugiura W, Kosaka H,

Kameoka H (1999) Moscatilin from Dendrobium nobile,a

naturally occurring bibenzyl compound with potential antimu-

tagenic activity. J Agric Food Chem 47:2163–2167

Mohanty P, Das MC, Kumaria S, Tandon P (2012) High-efﬁciency

cryopreservation of the medicinal orchid Dendrobium nobile

Lindl. Plant Cell Tissue Organ Cult 109:297–305

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bioassays with tobacco tissue cultures. Physiol Plant 15:473–497

Ng TB, Liu JY, Wong JH, Ye XJ, Sze SCW, Tong Y, Zhang KY

(2012) Review of research on Dendrobium, a prized folk

medicine. Appl Microbiol Biotechnol 93:1795–1803

Noel ARA (1974) Aspects of cell wall structure and development of

velamen in Ansellia gigantean Reichb F. Ann Bot 38:495–504

Planta

123

Nogueira RE, Pereira OL, Kasuya MCM, Lanna MCS, Mendonc¸a MP

(2005) Fungos micorrı

´zicos associados a orquı

´deas em campos

rupestres na regia

˜o do Quadrila

´tero Ferrı

´fero, MG, Brazil. Acta

Bot Bras 19(3):417–424 (in Portuguese)

Nontachaiyapoom S, Sasirat S, Manoch L (2010) Isolation and

identiﬁcation of Rhizoctonia-like fungi from roots of three

orchid genera, Paphiopedilum,Dendrobium and Cymbidium,

collected in Chiang Rai and Chiang Mai provinces of Thailand.

Mycorrhiza 20:459–471

Nontachaiyapoom S, Sasirat S, Manoch L (2011) Symbiotic seed

germination of Grammatophyllum speciosum Blume and Den-

drobium draconis Rchb. f., native orchids of Thailand. Sci Hortic

130:303–308

Nurfadilah S, Swarts ND, Dixon KW, Lambers H, Merritt DJ (2013)

Variation in nutrient-acquisition patterns by mycorrhizal fungi of

rare and common orchids explains diversiﬁcation in a global

biodiversity hotspot. Ann Bot 111:1233–1241

Olivain C, Alabouvette C (1997) Colonization of tomato by a non-

pathogenic strain of Fusarium oxysporum. New Phytol

137:481–494

Otero JT, Ackerman JD, Bayman P (2002) Diversity and host

speciﬁcity of endophytic Rhizoctonia-like fungi from tropical

orchids. Am J Bot 89(11):1852–1858

Otero JT, Mosquera TA, Flanagan NS (2013) Tropical orchid

mycorrhizae: potential applications in orchid conservation,

commercialization, and beyond. Lankesteriana 13(1–2):57–63

Pan LH, Li XF, Wang MN, Zha XQ, Yang XF, Liu ZJ, Luo YB, Luo

JP (2014) Comparison of hypoglycemic and antioxidative effects

of polysaccharides from four different Dendrobium species. Int J

Biol Macromol 64:420–427

Pant B (2013) Medicinal orchids and their uses: tissue culture a

potential alternative for conservation. African J Plant Sci

7(10):448–467

Parthibhan S, Senthil Kumar T, Rao MV (2015) Phenology and

reintroduction strategies for Dendrobium aqueum Lindley—an

endemic, near threatened orchid. J Nature Conserv 24:68–71

Pereira MC, Pereira OL, Costa MD, Rocha RB, Kasuya MCM (2009)

Diversidade de fungos micorrı

´zicos Epulorhiza spp. isolados de

Epidendrum secundum (Orchidaceae). Rev Bras Cie

ˆn Solo

33(5):1187–1197 (in Portuguese)

Peterson RL, Currah RS (1990) Synthesis of mycorrhizae between

protocorms of Goodyera repens (Orchidaceae) and Ceratoba-

sidium cereale. Can J Bot 68:1117–1125

Pridgeon AM (1986) Anatomical adaptations in Orchidaceae. Lind-

leyana 1:96–101

Pritchard HW (1984) Liquid nitrogen preservation of terrestrial and

epiphytic orchid seed. CryoLetters 5:295–300

Qian XP, Zha XQ, Xiao JJ, Zhang HL, Pan LH, Luo JP (2014)

Sulfated modiﬁcation can enhance antiglycation abilities of

polysaccharides from Dendrobium huoshanense. Carbohydr

Polym 101:982–989

Rai A, Bergman B (2002) Creation of new nitrogen-ﬁxing cyanobac-

terial associations. Biol Env Proc Royal Irish Acad 102:65–68

Rasmussen HN (1995) Terrestrial orchids: from seed to mycotrophic

plant. Cambridge University Press, Cambridge

Rasmussen HN, Rasmussen FN (2014) Seedling mycorrhiza: a

discussion of origin and evolution in Orchidaceae. Bot J Linn

Soc 175:313–327

Rasmussen HN, Whigham DF (1993) Seed ecology of dust seeds

in situ: a new study technique and its application in terrestrial

orchids. Amer J Bot 80:1374–1378

Rasmussen HN, Whigham DF (2002) Phenology of roots and

mycorrhiza in orchid species differing in phototrophic strategy.

New Phytol 154:797–807

Rikkinen J (2013) Molecular studies on cyanobacterial diversity in

lichen symbioses. MycoKeys 6:3–32

Roberts DL, Dixon KW (2008) Orchids. Curr Biol 18:R325–R329

Roy A, Tripathy P, Adhikary SP (1998) UV protecting pigment of

epilithic cyanobacteria occuring on the various regions of India.

In: Subramanian G, Kaushik BD, Venkataraman GS (eds)

Cyanobacterial Biotechnology, Proceedings of an International

Symposium, Sep 18-21. ISBN 81-204-1269-9, Oxford & IBH

publishing, New Delhi, pp 439–447

Ryan MJ, Smith D (2007) Cryopreservation and freeze-drying of

fungi employing centrifugal and shelf freeze-drying. Methods

Mol Biol 368:127–140

Saharan BS, Nehra V (2011) Plant growth promoting rhizobacteria: a

critical review. Life Sci Med Res 2011:1–21

Sandmann G, Kuhn S, Boger P (1998) Evaluation of structurally

different carotenoids in Escherichia coli transformants as

protectants against UV-B radiation. Appl Environ Microbiol

64:1972–1974

Sanford WW, Adanlawo I (1973) Velamen and exodermis characters

of West African epiphytic orchids in relation to taxonomic

grouping and habitat tolerance. Bot J Linn Soc 66:307–321

Sattayasai N, Sudmoon R, Nuchadomrong S, Chaveerach A, Kuehnle

AR, Mudalige-Jayawickrama RG, Bunyatratchata W (2009)

Dendrobium ﬁndleyanum agglutinin: production, localization,

anti-fungal activity and gene characterization. Plant Cell Rep

28:1243–1252

Selosse MA, Martos F, Perry BA, Padamsee M, Roy M, Pailler T

(2010) Saprotrophic fungal symbionts in tropical achlorophyl-

lous orchids: ﬁnding treasures among the ‘molecular scraps’?

Plant Signal Behav 5:349–353

Selosse MA, Boullard B, Richardson D (2011) Noe

¨l Bernard

(1874–1911): orchids to symbiosis in a dozen years, one century

ago. Symbiosis 54:61–68

Singh A, Duggal S (2009) Medicinal orchids: an overview. Ethnob-

otanical Leaﬂets 13:351–363

Smith SE, Read DJ (1997) Mycorrhizal symbiosis, 2nd edn.

Academic Press, New York

Smith SE, Read D (2008) Mycorrhizal symbiosis, 3rd edn. Academic

Press, New York

Song JY, Guo SX (2001) Effects of fungus on the growth of

Dendrobium candidum and D. nobile in vitro culture. Acta Acad

Med Sin 23(6):547–551 (in Chinese with English abstract)

Song JI, Kang YJ, Yong HY, Kim YC, Moon A (2012) Denbinobin, a

phenanthrene from Dendrobium nobile, inhibits invasion and

induces apoptosis in SNU-484 human gastric cancer cells. Oncol

Rep 27(3):813–818

Spaepen S, Vanderleyden J, Remans R (2007) Indole-3-acetic acid in

microbial and microorganism-plant signaling. Microbiol Rev

31:425–448

Stewart SL, Zettler LW (2002) Symbiotic germination of three

semiaquatic rein orchids (Habenaria repens,H. quinquiseta,H.

macroceratitis) from Florida. Aquat Bot 72:25–35

Sto

¨ckel M, Tesitelova T, Jersakova J, Bidartondo MI, Gebauer G

(2014) Carbon and nitrogen gain during the growth of orchid

seedlings in nature. New Phytol 202:606–615

Stoessl A, Arditti J (1984) Orchid phytoalexins. In: Arditti J (ed)

Orchid biology: reviews and perspectives. Cornell University

Press, New York, pp 151–175

Sukphan P, Sritularak B, Mekboonsonglarp W, Lipipun V, Likhitwi-

tayawuid K (2014) Chemical constituents of Dendrobium

venustum and their antimalarial and anti-herpetic properties.

Nat Prod Commun 9(6):825–827

Sun J, Zhang F, Yang M, Zhang J, Chen L, Zhan R, Li L, Chen Y

(2014) Isolation of a-glucosidase inhibitors including a new

ﬂavonol glycoside from Dendrobium devonianum. Nat Prod Res

28(21):1900–1905

Sundin GW, Jacobs JL (1999) Ultraviolet radiation (UVR) sensitivity

analysis and UVR survival strategies of a bacterial community

Planta

123

from the phyllosphere of ﬁeld-grown peanut (Arachis hypogeae

L.). Microb Ecol 38:27–38

Swangmaneecharern P, Serivichyaswat P, Nontachaiyapoom S (2012)

Promoting effect of orchid mycorrhizal fungi Epulorhiza isolates

on seed germination of Dendrobium orchids. Sci Hortic

148:55–58

Swarts ND, Dixon KW (2009) Terrestrial orchid conservation in the

age of extinction. Ann Bot 104:543–556

Tan XM, Wang CL, Chen XM, Zhou YQ, Wang YQ, Luo AX, Liu

ZH, Guo SX (2014) In vitro seed germination and seedling

growth of an endangered epiphytic orchid, Dendrobium of-

ﬁcinale, endemic to China using mycorrhizal fungi (Tulasnella

sp.). Sci Hortic 165:62–68

Teixeira da Silva JA, Kerbauy GB, Zeng SJ, Chen ZL, Duan J (2014a)

In vitro ﬂowering of orchids. Crit Rev Biotechnol 34:56–76

Teixeira da Silva JA, Zeng SJ, Dobra

´nszki J, Galdiano R Jr, Cardoso

JC, Vendrame WA (2014b) In vitro conservation of Dendrobium

germplasm. Plant Cell Rep 33(9):1413–1423

Teixeira da Silva JA, Zeng SJ, Dobra

´nszki J, Cardoso JC, Kerbauy

GB (2014c) In vitro ﬂowering of Dendrobium. Plant Cell Tiss

Org Cult 119:447–456

Teixeira da Silva JA, Dobra

´nszki J, Cardoso JC, Zeng SJ (2015)

Dendrobium micropropagation: a review. Plant Cell Rep

34:671–704

Tian CC, Zha XQ, Pan LH, Luo JP (2013) Structural characterization

and antioxidant activity of a low-molecular polysaccharide from

Dendrobium huoshanense. Fitoterapia 91:247–255

Touchell DH, Dixon KW (1993) Cryopreservation of seed of Western

Australian native species. Biodiversity Conserv 2:594–602

Tsai AC, Pan SL, Liao CH, Guh JH, Wang SW, Sun HL, Liu YN,

Chen CC, Shen CC, Chang YL, Teng CM (2010) Moscatilin, a

bibenzyl derivative from the India orchid Dendrobrium loddi-

gesii, suppresses tumor angiogenesis and growth in vitro and

in vivo. Cancer Lett 292(2):163–170

Tsavkelova EA (2011) Bacteria associated with orchid roots. In:

Maheshwari DK (ed) Bacteria in agrobiology: plant growth

responses. Springer, Berlin, pp 221–259

Tsavkelova EA, Cherdyntseva TA, Lobakova ES, Kolomeitseva GL,

Netrusov AI (2001) Microbiota of orchid rhizoplane. Microbi-

ology 70:492–497 (in English) and 567–573 (in Russian)

Tsavkelova EA, Alexandrova AV, Cherdyntseva TA, Kolomeitseva

GL, Netrusov AI (2003a) Fungi associated with orchid roots in

greenhouse conditions. Mycol Phytopathol 37:57–63 (in Russian

with English abstract)

Tsavkelova EA, Cherdyntseva TA, Netrusov AI (2003b) Phytohor-

mones production by the fungi associated with orchids. Mycol

Phytopathol 37:75–83 (in Russian with English abstract)

Tsavkelova EA, Lobakova ES, Kolomeitseva GL, Cherdyntseva TA,

Netrusov AI (2003c) Localization of associative cyanobacteria in

the roots of epiphytic orchids. Microbiology 86:91–104 (in

English) and 99–104 (in Russian)

Tsavkelova EA, Lobakova ES, Kolomeitseva GL, Cherdyntseva TA,

Netrusov AI (2003d) Associative cyanobacteria, isolated from

the roots of epiphytic orchids. Microbiology 72:92–97 (in

English) and 105–110 (in Russian)

Tsavkelova EA, Cherdyntseva TA, Netrusov AI (2004) Bacteria

associated with the roots of epiphytic orchids. Microbiology

73:710–715 (in English) and 825–831 (in Russian)

Tsavkelova EA, Klimova YS, Cherdyntseva TA, Netrusov AI (2006)

Microbial producers of plant growth stimulators and their

practical use: a review. Appl Biochem Microbiol 42:133–143

(in English) and 133–143 (in Russian)

Tsavkelova EA, Cherdyntseva TA, Klimova SYu, Shestakov AI,

Botina SG, Netrusov AI (2007) Orchid-associated bacteria

produce indole-3-acetic acid, promote seed germination, and

increase their microbial yield in response to exogenous auxin.

Arch Microbiol 188:655–664

Tsavkelova EA, Bo

¨mke C, Netrusov AI, Weiner J, Tudzynski B

(2008) Production of gibberellic acids by an orchid-associated

Fusarium proliferatum strain. Fung Gen Biol 45:1393–1403

Tsavkelova E, Oeser B, Oren-Young L, Israeli M, Sasson Y,

Tudzynski B, Sharon A (2012) Identiﬁcation and functional

characterization of the genes for indole-3-acetamide-mediated

IAA biosynthesis in plant-associated Fusarium species. Fung

Gen Biol 49:48–57

Tudzynski B, Sharon A (2002) Biosynthesis, biological role and

application of fungal hormones. In: Osiewacz HD (ed) The Mycota

X: industrial applications. Springer-Verlag, Berlin, pp 183–211

Vakhrameeva MG, Tatarenko IV, Varlygina TI, Torosyan GK,

Zagulskii MN (2008) Orchids of Russia and Adjacent Countries

(within the Borders of the Former USSR). A.R.G Ganter Verlag,

Ruggell 690 pp

Vendrame WA, Carvalho VS, Dias JMM, Maguire I (2008)

Pollination of Dendrobium hybrids using cryopreserved pollen.

HortScience 43(1):264–267

Venkateswarlu S, Raju MS, Subbaraju GV (2002) Synthesis and

biological activity of isoamoenylin, a metabolite of Dendrobium

amoenum. Biosci Biotechnol Biochem 66:2236–2238

Vinale F, Sivasithamparam K, Ghisalberti EL, Marra R, Woo SL,

Lorito M (2008) Trichoderma–plant–pathogen interactions. Soil

Biol Biochem 40:1–10

Vujanovic V, St-Arnaud M, Barabe

´D, Thibeault G (2000) Viability

testing of orchid seed and the promotion of coloration and

germination. Ann Bot 86:79–86

Wang J, Ge J, Liu F, Bian H, Huang C (1998) Cryopreservation of

seeds and protocorms of Dendrobium candidum. CryoLetters

19:123–128

Wang H, Fang H, Wang Y, Duan L, Guo S (2011) In situ seed baiting

techniques in Dendrobium ofﬁcinale Kimuraet Migo and Den-

drobium nobile Lindl.: the endangered Chinese endemic Den-

drobium (Orchidaceae). World J Microbiol Biotechnol

27:2051–2059

Wang Q, Sun P, Li G, Zhu K, Wang C, Zhao X (2014) Inhibitory

effects of Dendrobium candidum Wall ex Lindl. on azox-

ymethane- and dextran sulfate sodium-induced colon carcino-

genesis in C57BL/6 mice. Oncol Lett 7(2):493–498

Warcup J (1981) The mycorrhizal relationships of Australian orchids.

New Phytol 87:371–381

Waterman RJ, Bidartondo MI (2008) Deception above, deception

below: linking pollination and mycorrhizal biology of orchids.

J Exp Bot 59:1085–1096

Wilkinson KG, Dixon KW, Sivasithamparam K (1989) Interaction of

soil bacteria, mycorrhizal fungi and orchid seed in relation to

germination of Australian orchids. New Phytol 112:429–435

Wilkinson KG, Dixon KW, Sivasithamparam K, Ghisalberti EL

(1994) Effect of IAA on symbiotic germination of an Australian

orchid and its production by orchid-associated bacteria. Plant

Soil 159:291–295

Wu JP, Qian J, Zheng SZ (2002) A preliminary study on ingredient of

secretion from fungi of orchid mycorrhizal. Chin J Applied Ecol

13(7):845–848

Wu HF, Song XQ, Liu HX (2012) Ex-situ symbiotic seed germination

of Dendrobium catenatum. Acta Ecol Sin 32(8):2491–2497 (in

Chinese with English abstract)

Xing YM, Chen J, Cui JL, Chen XM, Guo SX (2011) Antimicrobial

activity and biodiversity of endophytic fungi in Dendrobium

devonianum and Dendrobium thyrsiﬂorum from Vietman. Curr

Microbiol 62:1218–1224

Xiong L, Cao ZX, Peng C, Li XH, Xie XF, Zhang TM, Zhou QM,

Yang L, Guo L (2013) Phenolic glucosides from Dendrobium

Planta

123

aurantiacum var. denneanum and their bioactivities. Molecules

18(6):6153–6160

Yang YL, Liu ZY, Zhu GS (2008) Study on symbiotic seed

germination of Pleione bulbocodioides (Franch) Rolfe. Micro-

biology 35(6):909–912 (in Chinese with English abstract)

Yang H, Lee PJ, Jeong EJ, Kim HP, Kim YC (2012) Selective

apoptosis in hepatic stellate cells mediates the antiﬁbrotic effect

of phenanthrenes from Dendrobium nobile. Phytother Res

26:974–980

Yang LC, Lu TJ, Hsieh CC, Lin WC (2014a) Characterization and

immunomodulatory activity of polysaccharides derived from

Dendrobium tosaense. Carbohydr Polym 111:856–863

Yang S, Gong Q, Wu Q, Li F, Lu Y, Shi J (2014b) Alkaloids enriched

extract from Dendrobium nobile Lindl. attenuates tau protein

hyperphosphorylation and apoptosis induced by lipopolysaccha-

ride in rat brain. Phytomedicine 21(5):712–716

Yang S, Zhang X, Cao Z, Zhao K, Wang S, Chen M, Hu X

(2014c) Growth-promoting Sphingomonas paucimobilis ZJSH1

associated with Dendrobium ofﬁcinale through phytohormone

production and nitrogen ﬁxation. Microb Biotechnol

7(6):611–620

Yu J, Zhou XF, Yang SJ, Liu WH, Hu XF (2013) Design and

application of speciﬁc 16S DNA-targeted primers for assessing

endophytic diversity in Dendrobium ofﬁcinale using nested PCR

DGGE. Appl Microbiol Biotechnol 97:9825–9836

Yuan ZL, Chen YC, Yang Y (2009a) Diverse non-mycorrhizal fungal

endophytes inhabiting an epiphytic, medicinal orchid (Dendro-

bium nobile): estimation and characterization. World J Microbiol

Biotechnol 25:295–303

Yuan ZQ, Zhang JY, Liu T (2009b) Phylogenetic relationship of

China Dendrobium species based on the sequence of the internal

transcribed spacer of ribosomal DNA. Biol Plant 53:155–158

Zainuddin M, Julkiﬂe AL, Pobathy R, Sinniah UR, Khoddamzadeh A,

Antony JJ, Pavallekoodi J, Subramaniam S (2011) Preliminary

analysis of cryopreservation of Dendrobium Bobby Messina

orchid using an encapsulation dehydration technique with Evans

blue assay. Afr J Biotechnol 10:11870–11878

Zeng SJ, Wu KL, Teixeira da Silva JA, Zhang JX, Chen ZL, Xia NH,

Duan J (2012) Asymbiotic seed germination, seedling develop-

ment and reintroduction of Paphiopedilum wardii Sumerh., an

endangered terrestrial orchid. Sci Hortic 138:198–209

Zeng SJ, Zhang Y, Teixeira da Silva JA, Wu KL, Zhang JX, Duan J

(2014) Seed biology and in vitro seed germination of Cypri-

pedium. Crit Rev Biotechnol 34:358–371

Zhang L, Chen J, Lv Y, Gao C, Guo S (2012) Mycena sp., a

mycorrhizal fungus of the orchid Dendrobium ofﬁcinale. Mycol

Progress 11(2):395–401

Zhao MM, Zhang G, Zhang DW, Hsiao YY, Guo SX (2013) ESTs

analysis reveals putative genes involved in symbiotic seed

germination in Dendrobium ofﬁcinale. PLoS One 8(8):e72705

Zhu GS, Yu ZN, Gui Y, Liu ZY (2008) A novel technique for

isolating orchid mycorrhizal fungi. Fungal Div 33:123–137

Zi XM, Sheng CL, Goodale UM, Shao SC, Gao JY (2014) In situ seed

baiting to isolate germination-enhancing fungi for an epiphytic orchid

Dendrobium aphyllum (Orchidaceae). Mycorrhiza 24:487–499

Zotz G, Winkler U (2013) Aerial roots of epiphytic orchids: the

velamen radicum and its role in water and nutrient uptake.

Oecologia 171:733–741

Planta

123

Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies

Article

Full-text available

Jun 2024

Dendrobium officinale Kimura & Migo is a perennial herbaceous plant of the genus Dendrobium in the family of Orchidaceae with high medicinal value. Melatonin (MT) is an indole-like tryptamine with functions such as regulating plant growth and development. This experiment investigated the effects of different concentrations of MT on the growth and development of protocorms of D. officinale protocorm-like bodies (PLBs). The results showed that the changes in morphological indicators such as color, cluster size, and surface changes were more significant under 75 µM MT than those of 0 µM (CK), and the appearance of white on the PLB surface was expedited, which was more conducive to the proliferation of PLBs. MT treatment of 100 µM inhibited the differentiation of adventitious buds, and the contents of photosynthetic pigments, polysaccharides, and flavonoids were significantly increased. Moreover, as compared with CK, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PLBs increased significantly, while the content of malondialdehyde (MDA) decreased gradually with 75µM or less. In conclusion, a concentration of 75 µM melatonin can enhance the rapid propagation rate of D. officinale in vitro, providing insights into the effects of melatonin on the growth of tissue-cultured D. officinale seedlings.

In vitro asymbiotic seed germination and micropropagation of Dendrobium heyneanum Lindl. – an endemic orchid of Western Ghats, India

Article

Full-text available

Apr 2024
PLANT CELL TISS ORG

Dendrobium heyneanum Lindl. or Heyne’s Dendrobium is an endemic epiphytic orchid of Western Ghats, with a height of approximately 3 - 4 inches. The present study aimed to establish a conservation strategy using in vitro regeneration methods for this endangered taxon. Mature pods of the D. heyneanum were collected from the field, and aseptically inoculated on various nutrient media. Asymbiotic seed germination was most successful on half-strength macro-MS media, yielding an 86.70% germination rate within 12 days. Different morphogenic stages (I-VI) were observed, with 20.84% of seeds producing young seedlings with roots on half-strength macro-MS media. The micropropagation protocol of D. heyneanum was established by using the protocorms (Stage IV) from the asymbiotic germinated seeds, with the highest frequency (90.20%) observed in 1.0 mg/L Kinetin (KN). In vitro flower buds were observed at 0.5 mg/L and 1.0 mg/L 6-Benzyl amino purine (BAP) and callus induction at 2.0 mg/L of BAP. The synergistic effect of KN combined with auxins - α- Naphthaleneacetic acid (NAA), Indole- 3- acetic acid (IAA), and Indole- 3- butyric acid (IBA) plantlets were assessed, with KN + IAA (1.0 mg/L) inducing pseudobulb elongation (0.92 cm) and rooting (0.74 cm). The plantlets were subsequently acclimatized and hardened on pots containing cocopeat and brick pieces resulting in a survival rate of 52.73%. This study presents a comprehensive protocol for in vitro propagation of Dendrobium heyneanum Lindl., offering a viable method for ex-situ conservation efforts.

Optimizing the potential utilization of bioreactors for the mass propagation of Indonesian Dendrobium varieties

Article

Full-text available

Feb 2024

The study focuses on two Indonesia Dendrobium varieties, D. Dian Agrihorti (DDA) and D. Syifa Agrihorti (DSA), which have potential to be commercialized, but face limitations on the availability of qualified seedlings sustainably. The research aimed to establish an in vitro propagation protocol using a Temporary Immersion System (TIS) to produce high-quality seedlings efficiently. Various factors, including varieties, media, plant growth regulators, anti-phenol compounds, organic additives, and TIS settings, were investigated using the basal part of the plantlet as the explant source. Key findings revealed that DDA outperformed DSA across all observed variables. In the initiation phase, basal plantlets cultured in Murashige and Skoog (MS) medium supplemented with 1.0 mg L-1 thidiazuron and 0.5 mg L-1 N-6 benzylaminopurine enhanced embryogenic callus (EC) formation, with a 13.5-day initiation period, 72% potential explant growth, 0.41 cm callus size, and a 3.45 rate of multiplication. During the proliferation stage, the addition of 150 mg L-1 ascorbic acid (AC) and the application of a TIS with a 30-minute dry period and a 15-minute wet period resulted in a 515.5% increase in EC fresh weight for DDA accompanied by a 6.16 multiplication rate. Regeneration of shoots was achieved using a Vacin and Went medium with 150 g L-1 banana extract, yielding 29.2 shoots per clump. Subsequent rooting of the shoots in 2 g L-1 Hyponex® medium with 20 g L-1 sugar and 2% AC proved successful. Acclimatization of plantlets with Cycas rumpii bulk demonstrated a 100% survivability rate. The established propagation protocol for DDA holds significant potential for application to other Dendrobium varieties, offering a sustainable and efficient method for meeting commercial demands in the Indonesian market.

Dendrobium officinale Flowers Flavonoids Enriched Extract Protects against Acute Ethanol-induced Gastric Ulcers via AMPK/PI3K Signaling Pathways

Article

Feb 2024

Culture age of Tulasnella affects symbiotic germination of the critically endangered Wyong sun orchid Thelymitra adorata (Orchidaceae)

Article

Full-text available

Nov 2023
MYCORRHIZA

Orchids (Orchidaceae) are dependent on mycorrhizal fungi for germination and to a varying extent as adult plants. We isolated fungi from wild plants of the critically endangered terrestrial orchid Thelymitra adorata and identified them using a multi-region barcoding approach as two undescribed Tulasnella species, one in each of phylogenetic group II and III (OTU1) of the Tulasnellaceae. Using symbiotic propagation methods, we investigated the role of Tulasnella identity (species and isolate) and age post isolation, on the fungus’s ability and efficacy in germinating T. adorata. The group II isolate did not support germination. Seed germination experiments were conducted using either (i) three different isolates of OTU1, (ii) 4- and 12-week-old fungal cultures (post isolation) of a single isolate of OTU1, and (iii) T. subasymmetrica which is widespread and known to associate with other species of Thelymitra. Culture age and fungal species significantly (P < 0.05) affected the time to germination and percentage of seed germination, with greater and faster germination with 4-week-old cultures. Tulasnella subasymmetrica was able to germinate T. adorata to leaf stage, although at slightly lower germination percentages than OTU1. The ability of T. adorata to germinate with T. subasymmetrica may allow for translocation sites to be considered outside of its native range. Our findings on the age of Tulasnella culture affecting germination may have applications for improving the symbiotic germination success of other orchids. Furthermore, storage of Tulasnella may need to take account of the culture age post-isolation, with storage at − 80 °C as soon as possible recommended, post isolation.

Molecular characterization of vermicompost-derived IAA-releasing bacterial isolates and assessment of their impact on the root improvement of banana during primary hardening

Article

Full-text available

Oct 2023
WORLD J MICROB BIOT

The hardening step of micropropagation is crucial to make the in vitro raised plants mature and further enhancing their survivability in the external environment. Auxin regulates various root physiological parameters in plant systems. Therefore, the present study aimed to assess the impact of three vermicompost-derived IAA-releasing microbial strains, designated S1, S2, and S3, as biofertilizers on in vitro raised banana plantlets during primary hardening. The High-Performance Thin-Layer Chromatography (HPTLC) analysis of these strains revealed a higher IAA content for S1 and S2 than that of S3 after 144 h of incubation. In total, seven different treatments were applied to banana plantlets, and significant variations were observed in all plant growth parameters for all treatments except autoclaved cocopeat (100%) mixed with autoclaved vermicompost (100%) at a 1:1 ratio. Among these treatments, the application of S3 biofertilizer: autoclaved cocopeat (1:1), followed by S2 biofertlizer: autoclaved cocopeat (1:1), was found to be better than other treatments for root numbers per plant, root length per plant, root volume, and chlorophyll content. These findings have confirmed the beneficial effects of microbial strains on plant systems and propose a link between root improvement and bacterial auxin. Further, these strains were identified at the molecular level as Bacillus sp. As per our knowledge, this is the first report of Bacillus strains isolated from vermicompost and applied as biofertilizer along with cocopeat for the primary hardening of banana. This unique approach may be adopted to improve the quality of plants during hardening, which increases their survival under abiotic stresses.

Complete Genome Sequence of Dyella sp. Strain GSA-30, a Predominant Endophytic Bacterium of Dendrobium Plants

Article

Full-text available

Mar 2023

We report a complete genome sequence of Dyella sp. strain GSA-30, a predominant endophytic bacterium of Dendrobium plants. The genome consists of a circular 5,501,810-bp chromosome with a G+C content of 61.4%. The genome was predicted to harbor 6 rRNA genes, 51 tRNA genes, and 4,713 coding sequences.

Anticipation and Verification of Dendrobium-Derived Nanovesicles for Skin Wound Healing Targets, Predicated Upon Immune Infiltration and Senescence

Article

Full-text available

Feb 2024
INT J NANOMED

Background Dendrobium, with profound botanical importance, reveals a rich composition of bioactive compounds, including polysaccharides, flavonoids, alkaloids, and diverse amino acids, holding promise for skin regeneration. However, the precise mechanism remains elusive. Seeking a potent natural remedy for wound healing, exocyst vesicles were successfully isolated from Dendrobium. Aims of the Study This investigation aimed to employ bioinformatics and in vivo experiments to elucidate target genes of Dendrobium-derived nanovesicles in skin wound healing, focusing on immune infiltration and senescence characteristics. Materials and Methods C57 mice experienced facilitated wound healing through Dendrobium-derived nanovesicles (DDNVs). Bioinformatics analysis and GEO database mining identified crucial genes by intersecting immune-related, senescence-related, and PANoptosis-associated genes. The identified genes underwent in vivo validation. Results DDNVs remarkably accelerated skin wound healing in C57 mice. Bioinformatics analysis revealed abnormal expression patterns of immune-related, senescence-related, and pan-apoptosis-related genes, highlighting an overexpressed IL-1β and downregulated IL-18 in the model group, Exploration of signaling pathways included IL-17, NF-kappa B, NOD-like receptor, and Toll-like receptor pathways. In vivo experiments confirmed DDNVs’ efficacy in suppressing IL-1β expression, enhancing wound healing. Conclusion Plant-derived nanovesicles (PDNV) emerged as a natural, reliable, and productive approach to wound healing. DDNVs uptake by mouse skin tissues, labeled with a fluorescent dye, led to enhanced wound healing in C57 mice. Notably, IL-1β overexpression in immune cells and genes played a key role. DDNVs intervention effectively suppressed IL-1β expression, accelerating skin wound tissue repair.

Acclimatization with endophytic fungi and reintroduction of Guarianthe skinneri (Bateman) Dressler & W.E. Higgins, a threatened, native orchid of cultural value in southern Mexico

Article

Feb 2024
J NAT CONSERV

The Dendrobium Orchid: Botany, horticulture, and utilization

Article

Full-text available

May 2023
CROP SCI

Dendrobium, one of the largest genera of tropical orchids, is grown commercially on a large scale with an important economic impact in several ASEAN countries. Dendrobium orchids can be grown in a wide range of climates, but flower production is best under tropical conditions. Due to being the most popular orchid for cut flower production, many hybrids of Dendrobium have been produced with colorful and attractive blooms. Cut flowers of Dendrobium are popular worldwide. In addition, many Dendrobium cultivars can be grown as flowering potted plants and for other purposes such as in medicinal applications and for use in decorative products. Postharvest physiology and technology are important in maintaining the quality of both cut flowers and potted plants from production sites to the marketplace. Plants of the Dendrobium genus also contain a wide diversity of bioactive compounds that can be developed into medicinal and cosmetic products. This review describes the botany, cultivation, postharvest physiology, handling and utilization of Dendrobium.

Phytohormone production by fungi associated with orchids

Article

Full-text available

Jan 2003

It was shown that all of the tested fungi, isolated from the terrestrial and epiphytic tropical orchids (aerial and substrate roots), produced auxin-like substances. Exogenous tryptophan induced its synthesis. The presence and activity of IAA (indole-3-acetic acid) were demonstrated with chromatography (TLC) and biological tests. Some of the fungi were able to produce cytokinin-like substances. None of the gibberellins were identified in the fungi filtrates. The ability of the fungi to produce phytohormone-like substances is considered to be necessary to adult orchids and in the relations between the plant and associated microorganisms.

Breeding Dendrobium Orchids in Hawaii

Book

Feb 2023

Seed ecology of dust seeds in situ: a new study technique and its application in terrestrial orchids

Article

Dec 1993

A method is described by which seeds of terrestrial orchids are sown and retrieved in the field under almost natural conditions. For the first time it is possible to conduct a quantitative study of orchid germination in situ and observe seasonal growth and mortality of seedlings. The technique has also enabled us to investigate the relation between the site where the seeds are sown, the availability of an appropriate fungus to infect the seeds, and seedling establishment in the soil. Five local species were studied. Corallorhiza odontorhiza, Goodyera pubescens, and Galearis spectabilis all began to germinate in May–June, after 23-30 weeks in the soil. These species differed in their dependency on infection at germination time, but none of the seedlings developed beyond the point of rupturing the testa except when infected. Seeds of Liparis lilifolia and Tipularia discolor did not germinate within the first 12 months of the experiment. The implications and potential uses of this field sowing technique for further studies and for other kinds of minute seeds are discussed.

Mycorrhizal Symbiosis

Book

Jan 2008

The roots of most plants are colonized by symbiotic fungi to form mycorrhiza, which play a critical role in the capture of nutrients from the soil and therefore in plant nutrition. Mycorrhizal Symbiosis is recognized as the definitive work in this area. Since the last edition was published there have been major advances in the field, particularly in the area of molecular biology, and the new edition has been fully revised and updated to incorporate these exciting new developments. . Over 50% new material . Includes expanded color plate section . Covers all aspects of mycorrhiza . Presents new taxonomy . Discusses the impact of proteomics and genomics on research in this area.

A revised medium for rapid growth and bioassays with tabacco tissue cultures

Article

Jan 1962

A new nutrient solution for the germination of orchid seed

Article

Jan 1946

L. Knudson

Aspects of Cell Wall Structure and the Development of the Velamen in Ansellia gigantea Reichb. f.

Article

Mar 1974

A.R.A. Noel

A transmission and scanning electron microscope study was made of cell wall development in the root velamen of Ansellia gigantea. The pattern of fibrillar deposition in the primary walls was established by means of shadowed surface preparations. The helical wall thickenings were shown to originate by the localised apposition of bundles of orientated 20-30 nm cellulose fibrils. This proceeds from near the tips of the radially elongated cells. Although microtubules were associated with later stages of helix deposition, there was no evidence for cytoplasmic pre-patteming. The large wall perforations, circumscribed by secondary wall depositions, develop at the sites of pit fields. The implications of the present observations are discussed, particularly as they appear to relate to general principle of wall development in tracheary elements.

Effects of fungal elicitors on growth of Dendrobium candidum protocorms

Article

Mar 2008

Objective: To study the effects of two fungal elicitors on the growth of Dendrobium candidum protocorms cultured on the solid medium. Methods: The medium for propagation of protocorms was selected and the growth curve on that medium was obtained. According to the curve, the two fungal elicitors were added at the different growth stages of protocorms. The fresh weight (FW) and dry weight (DW) of protocorms were measured. Results: The medium for propagation of protocorms was 1/2MS+20% potato extract +3% sucrose + 0.8% agar. Compared with the control MF23 elicitor added at weeks 11 and 13 could significantly increase the FW by 16.4% (P<0.01) and 23.4% (P<0.01), respectively, and the DW by 6.7% (P<0.05) and 17.9% (P<0.01), respectively, and MF23 added into the medium for the whole culture could significantly decrease the DW by 9.9% (P<0.01). Compared with the control, MF24 elicitor added at weeks 11 and 13 could significantly increase the FW by 17.7% (P<0.01) and 12.0% (P<0.05), respectively, added at week 11 could significantly increase the DW by 9.2% (P<0.05), and MF24 added into the medium for the whole culture could significantly decrease the DW by 13.9% (P<0.01). Conclusion: The growth of protocorms can be inhibited when the fungal elicitor is added at the early stage of culture and be improved when the fungal elicitor is added at the later stage of the culture.

Enhanced Auxin Production by Azospirillum Pure Cultures from Plant Root Exudates

Article

Sep 2012

Bacteria of the genus Azospirillum are well known as plant growth promoting rhizobacteria. The ability to synthesize phytohormones is considered one of the most important mechanisms to promote plant growth and is widely distributed among plantassociated rhizobacteria. The most important phytohormone produced by Azospirillum is the auxin indole-3-acetic acid, with the L-tryptophan as the precursor. In the present study, we evaluate the capacity of eight Azospirillum strains isolated from rice and wheat, to produce in vitro auxins using plant exudates. Our results show that isolates produced auxins in tryptophan free media, but, generally, the amount produced increased when the tryptophan concentration increased. Some plants root exudates had a similar effect to tryptophan for the auxin production. In this sense, bean, rice and canola root extracts produced, respectively, 93.3%, 96.2%, and 88.31% more auxin than L-tryptophan. Azospirillum sp. isolate A3 had the maximal capacity to produce auxin. Therefore, the effect of cell free supernatant was studied on rice root development. Statistical analysis did not show any significant difference between root number and dry weight of the treated and control seedlings. However, significant differences were observed in root length and wet weight at α=0.01 and α=0.05, respectively.

Preliminary analysis of cryopreservation of Dendrobium Bobby Messina orchid using an encapsulation-dehydration technique with evans blue assay

Article

Sep 2011
AFR J BIOTECHNOL

In vitro grown protocorm-like bodies (PLBs) of Dendrobium Bobby Messina hybrid were cryopreserved in liquid nitrogen (LN) at -196°C by an encapsulation-dehydration technique. PLBs (1 to 2 and 3 to 4 mm) were precultured in half strength semi-solid MS media supplemented with six different concentrations of sucrose (0.0, 0.2, 0.4, 0.6, 0.8 and 1.0 M). The PLBs were then encapsulated to form the beads in halfstrength liquid MS media supplemented with different concentrations of sodium alginate (2.5, 3.0 and 3.5%). The beads were placed in 2 ml cryovials and plunged into LN for 24 h. The beads were then thawed in a 40°C water bath for 90 s and were placed in recovery media composed of half strength semisolid MS media supplemented with 2% sucrose for four days under dark condition. After 12 days, the Evans blue dye assay was carried out to determine the viability of the PLBs. The highest viability was found in 1 to 2mm PLBs precultured in half strength semi-solid MS media supplemented with 1.0 M sucrose and encapsulated in 2.5% sodium alginate. Biochemical content analyses (chlorophyll, total soluble protein and peroxidase activities) were done to investigate the physiological responses of the PLBs after cryopreservation.

Symbiotic in vitro seed propagation of Dendrobium: fungal and bacterial partners and their influence on plant growth and development

Abstract

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