(PDF) Progress and Prospect of Orchid Breeding: An Overview

Pragya Tiwari

Jen-Tsung Chen Editors

Advances in

Orchid Biology,

Biotechnology

and Omics

Advances in Orchid Biology, Biotechnology

and Omics

Pragya Tiwari •Jen-Tsung Chen

Editors

Advances in Orchid Biology,

Biotechnology and Omics

Editors

Pragya Tiwari

Department of Biotechnology

Yeungnam University

Gyeongbuk, Korea (Republic of)

Jen-Tsung Chen

Department of Life Sciences

National University of Kaohsiung

Kaohsiung, Taiwan

ISBN 978-981-99-1078-6 ISBN 978-981-99-1079-3 (eBook)

https://doi.org/10.1007/978-981-99-1079-3

Pte Ltd. 2023

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Preface

Orchids comprise the most exotic and multi-colored group of ﬂowering plants,

classiﬁed in the family Orchidaceae. The multi-faceted attributes and promising

socio-economic applications in the present era have commercialized orchid cultiva-

tion and global trade, substantially improved via the advances in high-throughput

technologies, omics biology, and metabolic engineering approaches. Widely culti-

vated for ornamental purposes as the cut ﬂower and artiﬁcially propagated varieties,

the present decade has witnessed the popularity of orchids on a global level, with

researchers investigating the multi-faceted attributes and applications of orchids in

the food sector, healthcare, and industries. Novel and high-value varieties of orchids

are being developed via substantial contributions in advanced plant tissue culture

techniques, plant breeding, and more recently, the genetic manipulation studies in

orchids for plant trait improvement and value addition.

Orchids include approximately 30,000–35,000 species, which are found in

diverse habitats across the world. The very ﬁrst report suggested that the Chinese

were the pioneers in the cultivation and description of orchids, with the description

of Bletilla striata and Dendrobium species in the book, Materia Medica of the

twenty-eighth-century BC by a Chinese legend. In addition, traditional medicine

systems, like Ayurveda also reports the extensive usage of orchid species for

therapeutic purpos es. Some distinct characteristics of the orchid—adaptive mecha-

nisms, mycorrhiza-dependent germination, perennial nature, and absence of woody

structures and the ﬂowers include bilateral symmetry (zygomorphism), resupinate

ﬂowers, fused stamen and carpels, and highly modiﬁed petals (labellum). Further-

more, orchids exhibit monopodial (stem grows from a single bud, with the growth of

new leaves on the apex every season) and sympodial growth (adja cent shoots are

produced, grow to a certain size, bloom, and then replaced), growing laterally and

following the surface for support. The orchids usually ﬂower in the spring season

and some of the key species that are grown as ornamentals include Renanthera,

Paphiopedilum, Cattleya, Phragmipedium, Dendrobium, and Vanilla sp.

The cultivation and demand of exotic orchid varieties have witnessed a tremen-

dous upsurge, attributed to the improved understanding and knowledge in areas of

v

orchid biology, classiﬁcation, phytochemistry, and cultivation strategies, among

other areas. Plant tissue culture and traditional plant breeding approaches form the

basis of orchid cultivation, contributing immensely to the cultivation of exotic orchid

varieties, however, multiple challenges including slow growth, complex orchid

genomes, and poor efﬁciency of transformation are major limitations. The classical

plant breeding approaches comprising crossbreeding and mutational breeding,

molecular marker-assisted breeding, in vitro orchid propagation, and cryopreserva-

tion have addressed these challenges to a considerable extent. These traditional

approaches also provided a sound platform for introducing genetic manipulation of

novel orchid varieties for trait improvement. The last decade has witnessed the

extensive application of plant tissue culture techniques for the propagation and

conservation of orchids, via utilizing different approaches and explants, namely

shoot nodes, stems, ﬂower stalks, root tips, etc. facilitating the translational success

of several varieties. Conventional breeding approaches in orchid propagation and

conservation have witnessed key translational success in the development of novel

varieties as well as conservation of the species with novel attributes.

vi Preface

In this direction, efforts were also made to understand the molecular mechanisms

of orchid mycorrhizal symbiosis for elucidating genetic information. While the

plant–fungal interactions are key to orchid development, the association of fungal

endophytes and their prospects in the production of antimicrobials highlight key

prospects in the discovery and development of novel antimicrobials. Another inter-

esting contribution aims to discuss the societal impact of some medicinal orchids,

providing valuable insights into the history and ethnomedicinal uses and the pros-

pects of socio-economic applications in healthcare. Catasetum genus consists of

showy epiphytic orchids, deﬁnes novel attributes, and is highly prioritized in horti-

culture; however, most of the species are difﬁcult to cultivate without a greenhouse.

The conservation of members in the Catasetum genus, therefore, requires immediate

attention and conservation via biotechnological strategies, as discussed in a key

literature contribution.

In recent times, genetic engineering approaches have focused on trait improve-

ment by creating novel hybrids of genera, for example, Oncidium, Vanda, Phalae-

nopsis, Cymbidium, and Dendrobium, among others. Agrobacterium-mediated

transformation of orchids has been the most successful technique to date creating

novel transgenics in orchid genera like Oncidium, Vanda, Dendrobium, and Phal-

aenopsis. In addition, to overexpression of key genes in heterologous systems for

desired trai ts, gene silencing studies have also been attempted in orchids like

Oncidium and Dendrobium species. The biotechnological interventions in different

orchid varieties have focused on the alteration of ﬂower fragrance, color, disease

resistance, and shelf-life, aiming for improved plant traits and varieties. A few key

examples of transgenic orchid varieties include RNAi-based gene silencing in

Phalaenopsis equestris for ﬂower color, gene overexpression in Dendrobium

Sonia for altering orchid morphology, and organogenesis and in vitro development

via permanent magnetic ﬁelds in Phalaenopsis species, among others. The scientiﬁc

approaches have made remarkable contributions to the development of exotic

varieties displaying multi-faceted attributes, namely novel plant traits, different color

patterns, and disease resistance, among others.

Preface vii

In the present era, orchid cultivation has witnessed a tremendous upsurge attrib-

uted to their recognition as food ingredients, ﬂoriculture, and/in healthcare. More-

over, omics and computational approaches have signiﬁcantly improved our

understanding of different concepts in orchid biology via better insights into the

metabolic pathways and their roles in the biosynthesis of diverse metabolites and

physiological mechanisms in orchid biology. While proteome analysis of orchid

species focused on ﬂower development and micropropagation methods, while the

omics approaches have identiﬁed the developmental stages in orchid biology and

improved orchid breeding, conservation, and commercialization of novel varieties.

With the emerging importance and multi-faceted role of orchids in ﬂoricultur e, the

food sector, and healthcare, the respective book aims to discuss the recent advances /

developments in orchid biology, biotechnology, and omics approaches. The book

provides further insights into the progress and the prospects in orchid breeding, the

importance of key medicinal orchids and their societal impact, and how the associ-

ation of the fungal endophytes with members of Orchidaceae deﬁnes key prospects

as antimicrobials in drug discovery, an interesting yet less-explored area of investi-

gation in orchids. Some prospective chapters discuss speciﬁc examples in detail

including ethnomedicinal, phytochemistry, and biotechnological strategies for the

conservation of Orchids in the Catasetum genus, and some terrestrial orchids. The

book provides valuable insights and contributions from renowned experts in orchid

biology and biotechnology from all over the world, with 9 chapters discussing

different sub-themes of wider signiﬁcance and applications in orchid biology.

This book provides comprehensive insights into the existing and emerging trends

in orchid biology and discusses the advances/contribution of omics, plant breeding,

and biotechnological approaches in this interesting ﬁeld. In addition, it aims to

bridge the gaps in knowledge de ﬁciencies and provide a combined platform

discussing multi-faceted areas of orchid biology and biotechnology in a single

book. With the development of high-throughput approaches and omics interven-

tions, orchids have gained enormous popularity in socio-economic applications and

witnessed a global demand for exotic varieties. Therefore, the respective book will

play a key role in providing an excellent basis for graduate, and post-graduate

students, Ph.D. scholars, and researchers, to improve and widen their scientiﬁc

knowledge in the ﬁeld of orchid biology, updates on biotechnological/omi cs

approaches in orchid cultivation and how these developments project to remarkably

impact orchid industry and commercialization on a global platform. With this aim,

the book brings together high-quality chapters from eminent researchers/experts

across the world and hopes to serve as a platform of literature for future initiatives

in orchid biology. Finally, the editors would like to thank the effort of all authors for

organizing their chapters and the assistance and instructions from the editorial ofﬁce

of the publisher are much appreciated.

Gyeongbuk, Republic of Korea Pragya Tiwari

Kaohsiung, Taiwan Jen-Tsung Chen

Contents

Understanding the Molecular Mechanisms of Orchid Mycorrhi zal

Symbiosis from Genetic Information ...... ..................... 1

Chihiro Miura, Galih Chersy Pujasatria, and Hironori Kaminaka

Breeding of Orchids Using Conventional and Biotechnological Methods:

Advances and Future Prospects .... ..... ..... ...... ..... ..... . 27

Jean Carlos Cardoso, Joe Abdul Vilcherrez-Atoche,

Carla Midori Iiyama, Maria Antonieta Germanà, and Wagner A. Vendrame

Biotechnological Interventions and Societal Impacts of Some

Medicinal Orchids . ... ... ... ... ... ... ... ... ... ... ... ... ... . 59

Kalpataru Dutta Mudoi, Papori Borah, Dipti Gorh, Tanmita Gupta,

Prasanna Sarmah, Suparna Bhattacharjee, Priyanka Roy,

and Siddhartha Proteem Saikia

Gene Expression Pro ﬁling in Orchid Mycorrhizae to Decipher the

Molecular Mechanisms of Plant–Fungus Interactions ... ... ... .. ... . 145

Silvia De Rose, Silvia Perotto, Raffaella Balestrini, and Fabiano Sillo

Exploring the Potential of In Vitro Cultures as an Aid to the Production

of Secondary Metabolites in Medicinal Orchids . .... ..... .... ..... 163

Arshpreet Kaur, Jagdeep Verma, Vikramaditya G. Yadav,

Sandip V. Pawar, and Jaspreet K. Sembi

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential, and

Biotechnology Strategies for the Conservation of Orchids from the

Catasetum Genus ... .... .... .... ..... .... .... .... ..... .... . 187

Luis J. Castillo-Pérez, Daniel Torres-Rico, Angel Josabad Alonso-Castro,

Javier Fortanelli-Martínez, Hugo Magdaleno Ramírez-Tobias,

and Candy Carranza- Álvarez

ix

x Contents

Diversity and Antimicrobial Potential of Orchidaceae-Associated Fungal

Endophytes ...... ........ ........ ........ ........ ........ . 209

Muhammad Adil, Pragya Tiwari, Jen-Tsu ng Chen, Rabia Naeem Khan,

and Shamsa Kanwal

Asymbiotic Seed Germination in Terrestrial Orchids: Problems,

Progress, and Pro spects . .. ... ... ... .. ... ... ... .. ... ... ... .. . 221

Nora E. Anghelescu, Yavar Vafaee, Kolsum Ahmadzadeh,

and Jen-Tsung Chen

Progress and Prospect of Orchid Breeding: An Overview ... .. ... ... 261

Khosro Balilashaki, Zahra Dehghanian, Vahideh Gougerdchi,

Elaheh Kavusi, Fatemeh Feizi, Xiaoyun Tang, Maryam Vahedi,

and Mohammad Musharof Hossain

About the Editors

Editors and Contributors

Pragya Tiwari is working as Research Professor in the Department of Biotechnol-

ogy, Yeungnam University, Republic of Korea. She possesses research/teaching

experience of more than 8 years in national/international instit utions in the ﬁeld of

plant sciences, with resear ch experience in areas of phytomolecules from medicinal

plants, plant-associated endophytes, plant-microbe interactions, and computational

biology in drug designing. She has been a recipient of various research fellowships/

recognitions from CSIR and ICAR, Govt. of India, and gained scientiﬁc recognitions

for her work in inter-disciplinary areas of plant sciences. She has been an active

participant/presenter in more than 30 symposiums/conferences on international/

national platforms and holds life memberships of scientiﬁc societies including

Korean Society of Biotechnology and Bioengineering, Indian Science Congress

Association, and The Society of Biological Chemist, India. Her research work has

gained broad interest through highly cited publications, book chapters, and invited

lectures. Her area of research comprises plant-endophyte interactions in Panax

ginseng and the signiﬁcance of bioactive metabolite (ginsenoside) in promoting

plant growth and as bio-stimulants in sustainable agriculture.

Jen-Tsung Chen is currently a professor at the National University of Kaohsiung in

Taiwan. He teaches cell biology, genomics, proteomics, medicinal plant biotechnol-

ogy, and plant tis sue culture. His research interests include bioactive compounds,

chromatography techniques, in vitro culture, medicinal plants, phytochemicals, plant

physiology, and plant biotechnology. He has published over 100 scientiﬁc papers

and serves as an editorial board member for Plant Methods and Plant Nano Biolog y .

xi

Contributors

xii Editors and Contributors

Muhammad Adil Pharmacology and Toxicology Section, University of Veteri-

nary and Animal Sciences, Lahore, Jhang Campus, Jhang, Punjab, Pakistan

Kolsum Ahmadzadeh Department of Horticultural Sciences and Engineering,

Faculty of Agricul ture, University of Kurdistan, Sanandaj, Iran

Medicinal Plants Breeding and Development Research Institute, University of

Kurdistan, Sanandaj, Iran

Angel Josabad Alonso-Castro División de Ciencias Naturales y Exactas,

Departamento de Farmacia, Universidad de Guanajuato, Guanajuato, Mexico

Nora E. Anghelescu Faculty of Horticulture, University of Agronomic Sciences

and Veterinary Medicine of Bucharest, Bucharest, Romania

Raffaella Balestrini Consiglio Nazionale delle Ricerche-Istituto per la Protezione

Sostenibile delle Piante, Torino, Italy

Khosro Balilashaki Department of Horticultural Science, Faculty of Agricultural

Science, University of Guilan, Rasht, Iran

Suparna Bhattacharjee Agrotechnology and Rural Development Division, CSIR-

North East Institute of Science and Technology, Jorhat, Assam, India

Papori Borah Agrotechnology and Rural Development Division, CSIR-North East

Institute of Science and Technology, Jorhat, Assam, India

Jean Carlos Cardoso Department of Biotechnology, Plant and Animal Produc-

tion, Lab of Plant Physiology and Tissue Culture, Center of Agricultural Sciences,

Federal University of Sao Carlos (DBPVA, CCA/UFSCar), Araras, Sao Paulo,

Brazil

Graduate Program of Plant Product ion and Associated Bioprocesses, CCA/UFSCar,

Araras, Sao Paulo, Brazil

Candy Carranza-Álvarez Programa Multidisciplinario de Posgrado en Ciencias

Ambientales, Universidad Autónoma de San Luis Potosí, San Luis Potosí, San Luis

Potosí, Mexico

Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis

Potosí, San Luis Potosí, Mexico

Facultad de Estudios Profesionales Zona Huasteca, Universidad Autónoma de San

Luis Potosí, Ciudad Valles, San Luis Potosí, Mexico

Luis J. Castillo-Pérez Programa Multidisciplinario de Posgrado en Ciencias

Ambientales, Universidad Autónoma de San Luis Potosí, San Luis Potosí, San

Luis Potosí, Mexi co

Jen-Tsung Chen Department of Life Sciences, National University of Kaohsiung,

Kaohsiung, Taiwan

Editors and Contributors xiii

Zahra Dehghanian Department of Biotechnology, Faculty of Agriculture,

Azarbaijan Shahid Madani University, Tabriz, Iran

Silvia De Rose Dipartimento di Scienze della Vita e Biologia dei Sistemi,

Università di Torino, Torino, Italy

Fatemeh Feizi Department of Horticultural Science, Faculty of Agricultural Sci-

ence, University of Guilan, Rasht, Iran

Javier Fortanelli-Martínez Instituto de Investigación de Zonas Desérticas,

Universidad Autónoma de San Luis Potosí, San Luis Potosí, San Luis Potosí,

Mexico

Dipti Gorh Agrotechnology and Rural Development Division, CSIR-North East

Institute of Science and Technology, Jorhat, Assam, India

Vahideh Gougerdchi Department of Plant Breeding and Biotechnology, Faculty

of Agriculture, University of Tabriz, Tabriz, Iran

Tanmita Gupta Agrotechnology and Rural Development Division, CSIR-North

East Institute of Science and Technology, Jorhat, Assam, India

Mohammad Musharof Hossain Department of Botany, University of Chittagong,

Chittagong, Bangladesh

Carla Midori Iiyama Department of Biotechnology, Plant and Animal Production,

Lab of Plant Physiology and Tissue Culture, Center of Agricultural Sciences,

Federal University of Sao Carlos (DBPVA, CCA/UFSCar), Araras, Sao Paulo,

Brazil

Graduate Program of Plant Product ion and Associated Bioprocesses, CCA/UFSCar,

Araras, Sao Paulo, Brazil

Hironori Kaminaka Faculty of Agriculture, Tottori University, Tottori, Japan

Shamsa Kanwal Microbiology Section, University of Veterinary and Animal

Sciences, Lahore, Jhang Campus, Jhang, Punjab, Pakistan

Arshpreet Kaur Department of Botany, Panjab University, Chandigarh, India

Elaheh Kavusi Department of Plant Breeding and Biotechnology, Faculty of

Agriculture, University of Tabriz, Tabriz, Iran

Rabia Naeem Khan Microbiology Section, University of Veterinary and Animal

Sciences, Lahore, Jhang Campus, Jhang, Punjab, Pakistan

Chihiro Miura Faculty of Agriculture, Tottori University, Tottori, Japan

Kalpataru Dutta Mudoi Agrotechnology and Rural Deve lopment Division,

CSIR-North East Institute of Science and Technology, Jorhat, Assam, India

Sandip V. Pawar University Institute of Pharmaceutical Sciences, Panjab Univer-

sity, Chandigarh, India

Silvia Perotto Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università

di Torino, Torino, Italy

xiv Editors and Contributors

Galih Chersy Pujasatria The United Graduate School of Agricultural Science,

Tottori University, Tottori, Japan

Hugo Magdaleno Ramírez-Tobias Facultad de Agronomía y Veterinaria,

Universidad Autónoma de San Luis Potosí, San Luis Potosí, San Luis Potosí,

Mexico

Priyanka Roy Centre for Infectious Disease, CSIR-North East Institute of Science

and Technology, Jorhat, Assam, India

Siddhartha Proteem Saikia Agrotechnology and Rural Development Division,

CSIR-North East Institute of Science and Technology, Jorhat, Assam, India

Prasanna Sarmah Agrotechnology and Rural Development Division, CSIR-North

East Institute of Science and Technology, Jorhat, Assam, India

Jaspreet K. Semb i Department of Botany, Panjab University, Chandigarh, India

Fabiano Sillo Consiglio Nazionale delle Ricerche-Istituto per la Protezione

Sostenibile delle Piante, Torino, Italy

Xiaoyun Tang College of Art Colleges of Landscape Architecture, Fujian Agri-

culture and Forest ry University, Fuzhou, China

Pragya Tiwari Department of Biotechnology, Yeungnam University, Gyeongsan,

Gyeongsangbuk-do, Republic of Korea

Daniel Torres-Rico Facultad de Ciencias Químicas, Universidad Autónoma de

San Luis Potosí, San Luis Potosí, San Luis Potosí, Mexico

Yavar Vafaee Department of Horticultural Sciences and Engineering, Faculty of

Agriculture, University of Kurdistan, Sanandaj, Iran

Medicinal Plants Breeding and Development Research Institute, University of

Kurdistan, Sanandaj, Iran

Maryam Vahedi Department of Horticultural Science, Faculty of Agricultural

Sciences and Engineering, College of Agriculture and Natural Resources, University

of Tehran, Tehran, Iran

Jagdeep Verma Department of Botany, Sardar Patel University, Mandi, Himachal

Pradesh, India

Joe Abdul Vilcherrez-Atoche Graduate Program of Plant Production and Associ-

ated Bioprocesses, CCA/UFSCar, Araras, Sao Paulo, Brazil

Graduate Program of Plant Genetics and Breeding, Escola Superior de Agricultura

Luiz de Queiroz, University of São Paulo, Piracicaba, Sao Paulo, Brazil

Vikramaditya G. Yadav Department of Chemical and Biological Engineering,

University of British Columbia, Vancouver, BC, Canada

School of Biomedical Engineering, University of British Columbia, Vancouver, BC,

Canada

r

Understanding the Molecular Mechanisms

of Orchid Mycorrhizal Symbiosis from

Genetic Information

Chihiro Miura , Galih Chersy Pujasatria , and Hironori Kaminaka

1 Introduction

Mycorrhiza, the oldest plant–microbe holobiont ever described, is an intricate plant–

fungus relationship (Frank 2005; Selosse et al. 2017). The fungus enters the plant’s

root system and forms specialized structures depending on the mycorrhizal types.

The earliest-to-evolve type, arbuscular mycorrhiza, is found in almost all ﬂowering

plants (Delaux et al. 2013) and is mainly characterized by the formation of tree-like

hyphal structures (arbuscules), although other structures, such as vesicles, are also

formed. The second type is ectomycorrhiza (ECM), which is found in several tree

species, such as Pinaceae, Fagaceae, and Betulaceae (Smith and Read 2008). The

third type, which is the main topic of this chapter, is orchid mycorrhiza (OM). Orchid

mycorrhizal fungi penetrate orchid seeds or roots through the suspensor (Peterson

and Currah 1990; Richardson et al. 1992; Rasmussen and Rasmussen 2009)o

epidermal hairs (Williamson and Hadley 1970) and then form dense mycelium

coils known as pelotons. Although other mycorrhizal symbioses exhibit mutualism,

OM symbiosis is known as parasitism: Other mycorrhizal plants obtain minerals

from fungi instead of supplying photosynthetic products to the fungi, whereas

orchids depend on carbon, nitrogen, and phosphorus sources provided by OM

fungi (Cameron et al. 2006, 2007; Kuga et al. 2014), at least during their

germination—a characteristic classiﬁed as initial mycoheterotrophy (Merckx

2013). Most orchids indicate the dual (photosynthetic and mycoheterotrophic)

carbon acquisition strategy for growth and development—a phenomenon known

as partial mycoheterotrophy (Gebauer and Meyer 2003; Merckx 2013)—whereas

C. Miura · H. Kaminaka (✉)

Faculty of Agriculture, Tottori University, Tottori, Japan

e-mail: cmiura@tottori-u.ac.jp; kaminaka@tottori-u.ac.jp

G. C. Pujasatria

The United Graduate School of Agricultural Science, Tottori University, Tottori, Japan

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_1

1

The two main methods for genomic DNA extraction include solvent extrac-

tion, such as a modiﬁed cetyltrimethylammonium bromide protocol (Murray

and Thompson ; Inglis et al. ; Hu et al. ), or column extraction,

such as DNeasy Plant Mini Kit protocol (Qiagen) and DNAsecure Plant Kit

201920181980

others have even evolved to be fully mycoheterotrophic and rely completely on

mycorrhizal fungi. Such orchids are commonly leaﬂess or achlorophyllous

(Lallemand et al. 2019; Li et al. 2022).

2 C. Miura et al.

OM fungi are mainly represented by ﬁlamentous basal orders of

Agaricomycotina: Sebacinales and Cantharellales (Weiß et al. 2016; Miyauchi

et al. 2020). Some of the members of these orders resemble Rhizoctonia, a famous

plant pathogen, necessitating the name “Rhizoctonia-like fungi.” Regardless of the

taxonomical disputes, the members of this group are Ceratobasidium, Sebacina,

Serendipita, and Tulasnella. However, some orchids—especially fully

mycoheterotrophic ones—evolve to associate with ECM fungi or even ascomyce-

tous fungi (Taylor and Bruns 1997; Sisti et al. 2019). They can also indirectly obtain

carbon from dead wood, in which their mycorrhizal fungi grow, or simply form a

mycorrhizal network with nearby living trees (Suetsugu et al. 2020). Interestingly,

some orchids can even switch their mycorrhizal fungi across development stages

(Umata et al. 2013; Chen et al. 2019), and OM fungi may turn parasitic against

orchid seeds (Adamo et al. 2020). Thus, OM symbiosis indicates a remarkable

physiological diversity among all kinds of mycorrhiza to date.

Along with traditional studies, molecular studies of OM have been advancing in

recent decades, ranging from mycorrhizal diversity to physiological omics, such as

transcriptomics, proteomics, and genomics. Their use is advantageous because they

can reveal even the innermost physiological phenomena that are easily overlooked

when using in vivo assays. However, guidelines for OM symbiosis analysis using

these omics techniques are unavailable. In this chapter, the tentative methods of

orchids’ whole-genome sequencing (WGS) and transcriptome analysis will be

introduced as well as their applications and prospects.

2 Methodology of the Genomics and Transcriptomics

of Orchids

2.1 Sample Preparation, Sequencing, and Bioinformatics

for Orchid Genome Sequencing

Whole-genome sequencing generally involves ﬁve steps: DNA extraction and iso-

lation, genomic DNA library construction, sequencing, de novo assembly, and

annotation (Fig. 1). Because some choices or options exist in these steps, researchers

need to select suitable methods for their samples. Here, we introduce the methodol-

ogies used by researchers for orchid WGS.

(i) DNA extraction and isolation

(TIANGEN). In any case, high-purity genomic DNA above a certain amount is

necessary for obtaining high-quality sequence data. Leaves, shoots, and ﬂowers

tend to be used for DNA extraction, whereas roots, rhizomes, or bulbs are not

used because these parts potentially include symbionts, except for an aseptic

culture.

The two main ways to obtain WGS are short- and long-read sequencing

(Goodwin et al. ). Regarding orchid WGS performed to date, the ﬁrst

method is Illumina sequence technology, and the latter is the PacBio sequel

2016

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 3

Fig. 1 Schematic overview

of a whole-genome

sequence analysis. The

illustrations were modiﬁed

and/or created with images

DBCLS TogoTV/CC-

BY-4.0)

(ii) Genomic DNA library construction and sequencing

system or Oxford Nano pore Technologies. Orchid WGS is often assembled

using both short and long reads. Combining short- and long-read data improves

genome assemblies of orchids whose genomes reveal a high content of repet-

itive elements that encompass ~82% (Li et al. ). How is sequencing depth

achieved using these sequence technologies to produce high-quality assembled

genomes? Notably, some short and long reads frequently contain sequence

errors (Sims et al. ), which can be overcome by increasing the number of

sequencing reads. A high-quality assembly of a eukaryote genome can gener-

ally be achieved based on more than ×70 sequence depths from hybrid

approaches that combine short- and long-read sequencing technologies (Faino

and Thomma . For orchids, many studies have a coverage depth of

approximately 240-fold, with at least 54-fold sequence coverage generating

high-quality reference genomes (Table ). The sequence coverage is calculated

based on the estimated genome size. Although several methods exist for

measuring genome size, two have mainly been conducted in the orchid WGS:

ﬂow cytometric and k-mer analyses. In the former analysis, the content of

relative DNA extracted from leaves and stained with a ﬂuorescent dye is

compared between query and reference samples using ﬂow cytometr y

(Sliwinska . In the latter analysis, the genome size is estimated based

on sequence data using the k-mer method (Simpson In this method, the

read sequences are fragmented by approximately 17–31 base pieces in this

manner, and the same sequence fragments are counted. The genome size is

estimated based on the count distribution of these fragments (see details in

Simpson )). Genome size data are important for evaluating the assembled

sequence quality, ploidy, and heterozygosity levels.

(2014

2014).

2018)

1

2014)

2014

2022

4 C. Miura et al.

(iii) Assembly and annotation

De novo genome assembly tools include velvet (Zerbino and Birney 2008),

SOAPdenovo (Luo et al. 2012), Abyss (Simpson et al. 2009; Jackman et al.

2017), Platanus (Kajitani et al. 2014), ALLPATHS-LG (Gnerre et al. 2011),

and MaSuRCA (Zimin et al. 2013). Collected reads from orchids can be

assembled using three main software tools: velvet, SOAPdenovo, and Platanus.

Recently developed software, such as Canu, can enable long-read assembly,

contributing to WGS accuracy (Ko ren et al. 2017). Repetitive element accu-

mulation could make orchid genomic assembly challenging. Whole-genome

sequencing analysis showed that repetitive elements generally occupy approx-

imately 68% of orchid genomes or even 82% of the Platanthera

guangdongenesis genome (Li et al. 2022). Some software tools for the analysis

of repetitive elements, such as RepeatModeler/RepeatMasker (https://www.

repeatmasker.org/), RepeatScout (https://github.com/mmcco/RepeatScout),

and LTR_FINDER (Xu and Wang 2007), are beneﬁcial. To improve sequenc-

ing accuracy, researchers need to select better tools according to the sequencing

method and genome features.

(continued)

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 5

Table 1 Summary of the published whole-genome sequencing data of orchids

Subfamily Species DNA ext. protocol Sequencer

Genome

assembler

Chromosomes

(2n=2x)

Assembled

genome

size (Gb)

Sequence

coverage

(fold

change)

Protein-

coding

genes

Repetitive

elements Reference

Apostasioideae Apostasia

ramifera

CTAB Illumina

HiSeq2000

SOAPdenovo2 Draft 0.36559 156 22841 44.99% Zhang

et al.

(2021)

Apostasia

shenzhenica

modiﬁed CTAB Illumina

HiSeq2000,

PacBio, 10X Geno-

mics Linked-Reads

ALLPATHS-LG 68 (n=34) 0.349 229 21841 42.05% Zhang

et al.

(2017)

Epidendroidae Bletilla striata Genomic DNA

Kit (Qiagen)

PacBio Sequel II,

Illumina

LACHESIS 32 (n=16) 2.37/2.43

a

85.4 26673/

26891

a

Jiang

et al.

(2022)

Cymbidium

sinense

modiﬁed CTAB GridION, Illumina? NextDenovo 40 (n=20) 3.45 258 29638 77.78% Yang

et al.

(2021)

Dendrobium

catenatum Lindl.

modiﬁed CTAB Illumina

HiSeq2000

SOAPdenovo2,

Platanus

38 (n=19) 1.01 220 28910 78.10% Zhang

et al.

(2016)

Dendrobium

chrysotoxum

modiﬁed CTAB MGI-SEQ2000,

PacBio, NovaSeq

Canu 38 (n=19) 1.37 290 30044 62.81% Zhang

et al.

(2021)

Dendrobium

huoshanense

CTAB PacBio, Illumina

HiSeqX-Ten

SMARTdebovo,

Pilon

38 (n=19) 1.285 352 21070 79.38% Han et al.

(2020)

Dendrobium

nobile

modiﬁed CTAB MGISEQ-2000,

PacBio Sequel II,

MGISEQ-2000

Canu, Pilon 38 (n=19) 1.19 110 29476 61.07% Xu et al.

(2022)

Dendrobium

ofﬁcinale

modiﬁed CTAB,

DNeasy Plant

Mini Kit (Qiagen)

Illumina

HiSeq2000, PacBio

SOAPdenovo Draft 1.35 125 35567 63.33% Yan et al.

(2015)

Dendrobium

ofﬁcinale

modiﬁed CTAB PacBio, Illumina

Hiseq4000,

HiSeq2500

Mecat2 38 (n=19) 1.23 208 27631 76.77% Niu et al.

(2021)

Table 1 (continued)

Subfamily Species DNA ext. protocol Sequencer

Genome

assembler

Chromosomes

(2n=2x)

Assembled

genome

size (Gb)

Sequence

coverage

(fold

change)

Protein-

coding

genes

Repetitive

elements Reference

Gastrodia elata DNeasy Plant

Mini Kit (Qiagen)

Illumina

HiSeq2500

ALLPATHS-LG Draft 1.06 169 18969 66.18% Yuan

et al.

(2018)

Gastrodia elata VAHTS PacBio Sequel II,

MGI-SEQ2000

CANU 36 (n=18) 1.043 107 21115 66.36% Xu et al.

(2021)

Gastrodia elata modiﬁed CTAB

and DNeasy Plant

Mini Kit (Qiagen)

Illumina

NovaSeq6000,

PacBio

FALCON Unzip

assembler v0.4

36 (n=18) 1.045 242 18844 74.92% Bae et al.

(2022)

Gastrodia elata

(Achlorophyllous)

DNeasy Plant

Mini Kit (Qiagen)

Illumina

HiSeq2000

SOAPdenovo Draft 1.12 351 24484 68.34% Chen

et al.

(2020a, b)

Gastrodia

menghaiensis

DNAsequre Plant

Kit

Illumina HiSeqX-

Ten, Illumina

HiSeq2500, PacBio

FALCON 36 (n=18) 0.863 408 17948 62.57% Jiang

et al.

(2022)

Papilionanthe

Miss Joaquim

'Anges'

Nanobind Plant

Nuclei Big DNA

Kit (Circulomics

Inc)

Illumina NovaSeq

6000, GridION

Flye 38 (n=19) 2.5 35 31529 78.00% Lim et al.

(2022)

Phalaenopsis

aphrodite

CTAB, DNeasy

Plant Mini Kit

(Qiagen)

Illumina

HiSeq2000/2500

ALLPATHS-LG 38 (n=19) 1.025 469 28902 60.30% Chao

et al.

(2018)

Phalaenopsis

equestris

modiﬁed CTAB Illumina

HiSeq2000

SOAPdenovo Draft 1.086 110 29431 62% Cai et al.

(2015)

Phalaenopsis

KHM190 cultivar

CTAB Illumina

HiSeq2000

Velvet Draft 3.1 97 41153 59.74% Huang

et al.

(2016)

Orchidoideae Platanthera

guangdongensis

modiﬁed CTAB PacBio Canu, Pilon 42 (n=21) 4.2 99 22559 82.18% Li et al.

(2022)

Platanthera

zijinensis

modiﬁed CTAB PacBio Canu, Pilon 42 (n=21) 4.19 99 24513 77.38% Li et al.

(2022)

6 C. Miura et al.

Vanilloideae Vanilla planifolia modiﬁed CTAB Illumina

HiSeq4000

SOAPdenovo2,

Mnia

Draft 2.2 92.4 Hu et al.,

(2019)

Vanilla planifolia KeyGene Illumina

HiSeq4000,

GridION,

PromethION

Miniasm 28 (n=14) 736.8/

744.2

a

54 29167/

29180

a

44.30% Hasing

et al.

(2020)

a

Haplotype A/B

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 7

RNA is often extracted using a column method, such as the RNeasy Plant

Mini Kit (Qiagen), or an organic solvent method, such as TRIzol reagent

(Invitrogen). In any case, RNA-seq requires a sufﬁcient amount of high-quality

RNA. Because RNA is more unstable than DNA and environmental conditions

can easily affect expression patterns, sampling methods should effectively be

8 C. Miura et al.

2.2 Sample Preparation, Sequencing, and Bioinformatics

for Transcriptome Analysis of OM Symbiosis

RNA-seq-based transcriptome analyses generally involve four steps: RNA extrac-

tion and puriﬁcation, cDNA library preparation, RNA sequenc ing, and data analysis

(Fig. 2). In this section, we introduce the methodologies where some choices exist

for transcriptome analysis of OM associations.

(i) RNA extraction and puriﬁcation

Fig. 2 Schematic overview

of an RNA-sequencing

analysis. The illustrations

were modiﬁed and/or

created with images from

TogoTV/CC-BY-4.0)

considered when collecting samples in situ. For example, naturally collected

tissue samples should be soaked in an RNA preservation solution, such as

RNAlater (Qiagen), and processed for RNA extraction as soon as possible.

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 9

(ii) cDNA library preparation and sequencing

Transcriptome analyses of OM roots or protocorms have mainly been

performed using Illumina short-read sequencing platforms (Yeh et al. 2019).

The cDNA libraries are prepared using commercially available kits according

to the objectives of analysis: The various types of library prep kits are available,

for example, the kits for strand-speciﬁc RNA-seq, for removing ribosomal

RNA, and for small RNA-seq. Our primary concerns in RNA-seq experiments

are the number of biological replicates and the sequencing depth required for

each sample. Unfortunately, there is no clear answer to this issue (Sims et al.

2014). Lamarre et al. (2018) recommended at least four biological replicates per

condition and 20-M reads per sample to be almost sure of obtaining approxi-

mately 1000 differentially expressed genes (DEGs) if they exist, according to

the meta-analysis with 16 RNA-seq projects involving the tomato fruit model

(Solanum lycopersicum). One may reason that a higher number of biological

replications and sequence reads are more accurate and more sensitive to

detecting DEGs, but this is often difﬁcult to achieve, especially in the analysis

of orchids in nature. Although only a few RNA-seq studies exist for mycorrhi-

zal symbiosis using wild orchids, Suetsugu et al. (2017) and Valadares et al.

(2020) performed RNA-seq analysis with three biological replicates of

Epipactis helleborine and Oeceoclades maculata, respectively.

(iii) Data analysis

The bioinformatics pipelines vary depending on the available reference

genome sequence. When reference genome sequences are available, data

analysis is divided into the following parts: mapping and counting of reads

and downstream analyses, such as differential expression, clustering, and

pathway analyses. In addition to these steps, the pooled reads need to be aligned

themselves to generate a de novo reference assembly when reference genome

information is unavailable. The extracted RNA from symbiotic roots or

protocorms contains plants and fungal RNAs. How to analyze multispecies

transcriptome analysis remains controversial. Because most aligners are opti-

mized for a single organism rather than multispecies datasets (Chung et al.

2021), the de novo assembled sequences are preferably divided into single

species. Previous studies have often applied BLAST searches of the de novo

assembly data against the NCB I nonredundant protein (nr) database to predict

the origins of the contigs (Perotto et al. 2014; Suetsugu et al. 2017; Valadares

et al. 2020, 2021). Perotto et al. (2014) examined the transcriptome of Serapias

vomeracea protocorms inoculated with Tulasnella calospora. The de novo

assembled transcriptomes were either compared with the NCBI-nr database

using the BLSTX algorithm on the Blast2Go program (Conesa et al. 2005) with

a cutoff E value <1.0e-10 or analyzed with an EST3 classiﬁer, which deter-

mines the origin of sequences in mixed sequence sets by codon frequencies

(Emmersen et al. 2007). Although the T. calospora genome has been

sequenced (Kohler et al. 2015) as a part of a DOE JGI Community Sequencing

Program coordinated by F. Martin (INRA, Nancy, France), only 79 sequences

(0.84%) matched T. calospora genes with an E value <1.0e-10 in Perotto’s

study (2014). This result reﬂected an extremely high degree of variability in the

ribosomal DNA sequences of Tulasnella (Moncalvo et al. 2006;Suárez et al.

2006; Taylor and McCormick 2008; Cruz et al. 2011; Fuji et al. 2020). The

transcriptome study of symbiotic Bletilla striata protocorms by Miura et al.

(2018) utilized the assembled genome scaffolds provided from pure cultures of

Tulasnella sp. The plant-derived sequences were conﬁrmed by subtracting the

result of a BLAST search of the assembled Tulasnella genome from the de

novo reference assembly of the transcriptome of symbiotic protocorms. Several

issues are being discussed, such as how to deﬁne an E value threshold for the

BLAST search and how to handle unannotated sequences other than plant and

fungi.

10 C. Miura et al.

3 New Insights into the Molecular Mechanisms of OM

Symbiosis

3.1 Orchid Genome Summary

The whole-genome sequences of orchids have been deposited in the NCBI (https://

www.ncbi.nlm.nih.gov/data-hub/genome/?taxon=4747) or the Chinese National

Genomics Data Center Genome Sequence Archive (https://ngdc.cncb.ac.cn/gsa/)

for 12 species at the chromosome level and 11 species of draft genomes. These

analyses estimate that the haploid genomes are 0.35–4.3 Gb, which contain approx-

imately 25,000 protein-coding genes (Table 1). The assembled average genome size

of 1.7 Gb is 4.5 and 14.2 times larger than that of rice (Oryza sativa cv. Nipponbare)

and Arabidopsis (Arabidopsis thaliana col-0 ecotype), respectively, and approxi-

mately the same as that of tree cotton of 1.7 Gb (Gossypium arbore um) (Fig. 3). The

orchid genomes contain a large number of repet itive sequences; that of Platanthera

guangdongensis comprises 82% repetitive elements (Li et al. 2022), making it the

most signiﬁcant proportion of the orchid genome to date. The ratio is similar to Zea

mays (approximately 85%). Although the biological function of repetitive DNA

sequences remains largely unknown, these sequences are important in the regulation

of mammalian gene expression (Faulkner et al. 2009). In plant species, transposable

elements are important for epigenome alterations under stress (Ragupathy et al.

2013). According to RNA-seq analysis by Vangelisti et al. (2019), AM fungi induce

the expression of speciﬁc retrotransposons in sunﬂower roots (Helianthus annuus

L.), implying a function for retrotransposons during symbiotic interaction. Thus, a

large number of repetitive sequences in orchid genomes may be involved in regu-

lating symbiosis.

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 11

Fig. 3 Overview of plant genome sizes. The genome size ranges were estimated using the Kew

Garden C-value database (https://cvalues.science.kew.org/). Flow cytometry was selected as the

estimation method. The genome sizes of each plant species were based on the assembled genome

size by whole-genome sequencing

Most studies of orchid WGS detected at least two whole-genome duplications

(WGDs) events in Orchidaceae (Zhang et al. 2017; Xu et al. 2021, 2022; Jiang et al.

2022). Most monocots are likely to share older WGD, and younger WGD might

represent an independent event speciﬁc to the Orchidaceae lineage (Zhang et al.

2017). One may infer that WGD events have driven gene family extension, thereby

expanding the evolutionary potential for functional diversiﬁcation. For example,

a comparative genome analysis of the Venus ﬂytrap (Dionaea muscipula) and its

close relatives revealed that a common WGD is the source of gene recruitment to

carnivory-related functions of carnivorous plants (Palfalvi et al. 2020). Orchidaceae

is one of the most diverse groups of ﬂowering plants, comprising approximately

25,000 species (Dressler 1993; Cribb et al. 2003; Chase et al. 2015). Unlike most

plants, almost all orchid species are heterotrophic in their early life stages (Leake

1994). Future studies should determine whether WGD events contribute to the

evolution characterizing the orchid species, such as mycoheterotrophy. However,

orchids have lost some gene families, such as photosynthesis-related genes and a part

of the MADS-box genes from their genomes. According to WGS analysis of leaﬂess

orchid Gastrodia elata and P. guangdongensis, the number of missing gene families

was higher in the fully mycoheterotrophic orchids than in most photosynthetic

plants, and many of the lost genes were involved in photosynthesis, corroborating

their inability to perform photosynthesis (Yuan et al. 2018; Li et al. 2022). Most

orchids lack the type I M-beta MADS-box genes involved in endosperm develop-

ment initiation (Masiero et al. 2011). Almost all orchids are initially

mycoheterotrophic: They produce tiny, endosperm-free seeds dependent on

mycorrhizal fungi for nutrient uptake during seed germination. The absence of

M-beta genes is thought to be related to endosperm deﬁciency (Zhang et al. 2017).

However, some orchid species undergo double fertilization and form a rudimentary

endosperm (Pace 1907; Sood and Mohana Rao 1988), and the loss of M-beta may

not be directly related to the loss of endosperm formation in orchids (Qiu and Köhler

2022).

12 C. Miura et al.

3.2 Nutritional Mode or Nutrition Transport

Almost all orchids depend on carbon and other nutrients provided by mycorrhizal

fungi during seed germination and subsequent early growth, which is classiﬁed as

initial mycoheterotrophy. Some orchids completely depend on fungal carbon during

their entire life cycle (“full mycoheterotrophy”) or combine autotrophy and

mycoheterotrophy at maturity (“partial mycoheterotrophy” or “mixotrophy”). The

orchid genome architecture reﬂects their lifestyle. Fully mycoheterotrophic species,

such as P. guangdongensis, G. elata, and Gastrodia menghaiensis, lost some

photosynthesis-related genes from their nucleus genomes (Chen et al. 2020b; Jiang

et al. 2022). These genes might be under “relaxed selection,” where environmental

change often eliminates or weakens a selection source that was formerly important

for maintaining a particular trait (Lahti et al. 2009). A positive correlation may exist

between the degree of heterotrophy in plants and the frequency of nonsynonymous

mutations in the genes responsible for the photosynthetic process and plastid and

leave functions (Chen et al. 2020b).

How do orchids acquire nutrients from symbionts under the relaxed selection of

photosynthetic-related genes? On the genomic side, several studies have shown the

expansion of trehalase genes in Gastrodia orchids, Platanthera orchids,

Dendrobium catenatum, and Phalaenopsis aphrodite (Li et al. 2022; Jiang et al.

2022). The experiments using

14

C-labeled glucose by Smith (1967) suggested that

orchids synthesize sucrose from fungal-derived trehalose. Ponert et al. (2021)

reported that the trehalose analog validamycin A, which has a strong inhibitory

effect on trehalases, reduced the growth of symbiotically germinated Dactylorhiza

majalis (Ponert et al. 2021). Additionally, trehalase activity was increased in sym-

biotic protocorms (Ponert et al. 2021). They proposed that orchids metabolize and

utilize fungal-derived trehalose as a carbon source, corroborating Smith’s hypothe-

sis. In transcriptomic studies, high expression of the genes encoding sugar trans-

porters (SWEET) was detected in vitro symbiotic protocorms of S. vomeracea

inoculated T. calospora AL13 (Perotto et al. 2014) and B. striata inoculated

Tulasnella sp. HR1–1 (Miura et al. 2018) and in situ symbiotic roots of Epipactis

helleborine (Suetsugu et al. 2017) and Limodorum abortivum (Valadares et al.

2021). A Medicago truncatula SWEET1b transporter contributes to arbuscule

maintenance during arbuscular mycorrhizal (AM) symbiosis (An et al. 2019).

Additionally, the SWEET11 gene was highly expressed in M. truncatula root

nodules (Kryvoruchko et al. 2016). Thus, in addition to the role of nutrient transport

in mycoheterotrophic orchids, SWEET transporters might be involved in

maintaining OM symbiotic systems.

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 13

In addition to organic carbon, nitrogen is probably a major nutrient transferred to

the plant from fungi (Gebauer and Meyer 2003; Hynson et al. 2013; Stöckel et al.

2014; Fochi et al. 2016), but the mechanisms remain largely unknown. According to

Li et al. (2022), Platanthera zijinensis and G. elata lost a nitrate reductase (NIA)

gene and a nitrite reductase (NIR) gene and P. guangdongensis lacked the NIA gene

and exhibited low expression of the NIR gene. This suggests that these plants may

not directly utilize nitrate from soil. Considering the genome’s gene repertoire,

nitrate compounds acquired from fungi may be glutamine or ammonium (Li et al.

2022). Gene expression proﬁles supported the hypothesis that organic nitrogen ﬂows

between plants and fungi during symbiosis (Zhao et al. 2014; Valadares et al. 2020,

2021). The transcriptome analysis of S. vomeraceae protocorms infected with

T. calospora by Fochi et al. (2016) revealed that plant and fungal amino acids and

peptide transporters were highly expressed during symbiosis establishment. Addi-

tionally, the high expression of genes associated with plant and fungal ammonia

permeases and the glutamine synthetase-glutamate synthase assimilation pathway

were detected in the symbiotic protocorms. The authors suggest that organic nitro-

gen is mainly transferred to the plant and that ammonium might be taken up by the

intracellular fungus from the apoplastic symbiotic interface. Although the reason

why fungi infect seeds and protocorms or, in other words, whether there are any

merits for colonizing fungi is under debate, Dearnaley and Cameron (2017) pro-

posed a model for bidirectional nutrient transport in OM across intact membranes.

The transcriptome analysis of symbiotic protocorms of G. elata inoculated with

Mycena dendrobii revealed signiﬁcant expression of plant genes involved in

clathrin-mediated endocytosis during symbiotic seed germination (Zhang et al.

2017). Future studies should fully elucidate the mechanisms of nutrient transport

across interfaces in orchid mycorrhizae.

3.3 Defense System

A delicate balance between plants and fungi creates unstable OM symbiosis. The

lady’s slipper orchid Cypripedium macranthos var. rebunense produces antifungal

compounds in seedlings to restrict fungal growth (Shimura et al. 2007). Orchid

mycorrhizal fungi act as pathogens to the B. striata seeds from which the seed coat

had been removed (Miura et al. 2019). These ﬁndings have led to the hypothesis that

plant defense reactions occur during OM symbiosis and that the ﬁne-tuning of the

defense response is essential for maintaining the plant–fungus relationship. In G.

elata, Gastrodia antifungal protein (hereafter GAFP) or also known as gastrodianin

genes encoding the monocot mannose-binding lectin antifungal proteins are

expanded, and more than 80% of the GAFP genes are highly expressed in

protocorms and juvenile tubers harvested from Xiaocaoba in Yunnan Province

(Yuan et al. 2018). Additionally, G. elata is likely to reduce the number of genes

related to plant pathogen resistance, particularly in salicylic acid (SA) receptor

genes, such as NPR3 and NPR4, and SA signaling genes, such as EDS1, PAD4,

ALD1, and FMO1 (Yuan et al. 2018; Xu et al. 2021). Elevated SA-mediated defense

responses are generally effective against biotrophic pathogens (Pieterse et al. 2012).

Owing to the loss of these genes involved in SA biosynthesis and signaling from the

parasitic plant Cuscuta australis genome (Xu et al. 2021), a common life strategy

may exist for heterotrophic plants.

14 C. Miura et al.

Moreover, what defense mechanisms are involved in OM symbiosis? Many

transcriptome studies of OM symbioses have reported that protocorms and mature

roots highly express genes related to reactive oxygen species detoxiﬁcation during

symbiosis (Zhao et al. 2014; Chen et al. 2017; Suetsugu et al. 2017; Gao et al. 2022).

These genes play an important role in defense responses against biotic stresses and

may be linked to peloton digestion (Blakeman et al. 1976; Suetsugu et al. 2017). The

transcriptome analyses further supported the possibility of plant cell–wall

remodeling or modiﬁcation in OM fungal infections, as well as in AM and pathogen

colonization (Zhao et al. 2014; Valadares et al. 2021; Balestrini et al. 2022). Orchids,

in essence, control these defense responses to the extent that they do not eliminate

symbiotic fungi, which Perotto et al. (2014) referred to as “a friendly plant–fungus

relationship.”

3.4 Phytohormones

Phytohormones play a crucial role in almost every aspect of plant biology, including

growth, development, pathogen defense, and microbial symbiosis. For example,

exogenous gibberellins (GAs) reduce hyphal colonization and arbuscule formation

during AM symbiosis in Pisum sativum, rice (O. sativa), and Lotus japonicus roots,

which form typical Arum-type arbuscules (El Ghachtouli et al. 1996; Yu et al. 2014;

Takeda et al. 2015). However, GA promotes fungal entry and colonization during

Paris-type AM in Eustoma grandiﬂorum inoculated with Rhizophagus irregularis

(Tominaga et al. 2020). Interestingly, Paris-type colonization is typical of forest

ﬂoor herbaceous and long-liv ed, woody, and evergreen plants (Dickson et al. 2007),

and some of them are mycoheterotrophic plants (Hynson et al. 2013; Imhof et al.

2013; Giesemann et al. 2020). The symbiotic germination experiment of

Dendrobium ofﬁcinal e inoculated with Tulasnella sp. S6 showed that exogenous

GA

3

treatment inhibited fungal colonization in the protocorms and seed germination

but did not signiﬁcantly affect asymbiotic germination in the 4-week-old protocorms

(Chen et al. 2020a). Transcriptomic studies have reported high expression of genes

related to GA biosynthesis (GA 3-oxidase (ox) and GA20ox) and the GA-GID1-

DELLA signaling module in the protocorms of Cymbidium hybridum inoculated

with Epulorhiza repens ML01 and Anoectochilus roxburghii inoculated with

unknown fungal species, respectively (Zhao et al. 2014; Liu et al. 2015). These

ﬁndings suggest that GAs play a key role in OM symbiosis.

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 15

After recognizing symbiotic factors in each other, the symbiotic process between

plants and fungi begins. Strigolactone (SL) is one of the key phytohormones in AM

symbiosis initiation. In the rhizosphere, SLs released from plant roots stimulate the

hyphal branching of AM fungi, which increases the chances of an encounter with a

host plant (Kretzschmar et al. 2012). Yuan et al. (2018) conﬁrmed that SL had

similar branch-inducing effects in the OM fungus Armillaria mellea. The whole-

genome sequences of orchid species have demon strated the expansion of the genes

encoding SL synthesis enzymes and receptors in G. elata, G. menghaiensis, and D.

ofﬁcinale (Wang et al. 2018; Chen et al. 2020b; Jiang et al. 2022). Because the

ancestral function of SLs as rhizosphere signaling molecules was already present in

the bryophyte Marchantia paleacea (Kodama et al. 2022), further studies will

determine the role of orchid SLs in OM symbiosis.

Abscisic acid (ABA) is essential for seed dormancy and adaptation to environ-

mental stress (Seki et al. 2007; Miransari and Smith 2014). Herrera-Medina et al.

(2007) reported that tomato mutants with reduced ABA concentrations were less

susceptible to AM fungus than wild-type plants, suggesting that ABA contributes to

the development of the complete arbuscule and its functionality. During the seed

germination of D. ofﬁcinale, the ABA concent ration was lower in symbiotic

protocorms inoculated with Tulasnella sp. than in asymbiotic protocorms (Wang

et al. 2018), revealing ABA involvement in OM symbiosis. The transcriptome

analysis of Cymbidium hybridum inoculated with Epulorhiza repens ML01 revealed

lower expression of 9-cis-epoxycarotenoid dioxygenase (NCED) and zeaxanthin

epoxidase (ZEP) genes, which are related to ABA biosynthesis, in symbiotic roots

than in mock-inoculated controls (Zhao et al. 2014). Collaboratively, ZEP and

NCED signiﬁcantly decreased in the early germination stage of symbiotic Cremastra

appendiculata inoculated with Coprinellus disseminatus compared with those of the

C. appendiculata seeds at the start of the experiment (Gao et al. 2022). In contrast,

Gao et al. (2022) reported that the ABA receptor pyrabactin resistance 1-like genes

were upregulated within the same period. Given that three events, germination,

symbiotic process, and defense response, could happen simultaneously in symbiotic

germination, the network complexity of these events is expected.

3.5 Common Symbiosis Pathway

The ﬁrst land plants to colonize Earth, possibly cryptophytes, appeared in the

Ordovician (approximately 450 million years ago), as conﬁrmed using fossil records

(Kenrick and Crane 1997). Fossilized fungal hyphae and spores that resemble

modern AM fungi (Glomerales) were found in fossils of the same age (Redecker

et al. 2000). Although evidence that these Ordovician fossil fungi were associated

with plants is unavailable, the symbiotic association formed with AM-like fungi is

thought to support plant terrestrialization (Rensing 2018). Following this founding

event, alternative or additional symbioses emerged accompanied by plant diversiﬁ-

cation (van der Linde et al. 2018; Radhakrishnan et al. 2020). Because AM fungi

were detected in Borya mirabilis roots, which belongs to the same order as orchids,

Asparagales, (Reiter et al. 2013), and the mycorrhizal fungi of Apostasia species,

members of the earliest-diverging clade of Orchidaceae, belong to families

Botryobasidiaceae and Ceratobasidiaceae (Yukawa et al. 2009), symbiont switching

and trophic mode shifts are thought to correlate with the evolutionary success of

Orchidaceae (Wang et al. 2021). This section will focus on the common symbiotic

pathway (CSP), a putative signal transduction pathway shared by AM and the

rhizobium–legume symbiosis, to discuss the mechanisms of OM symbiosis and

how the symbiosis has evolved. The transcriptome analysis of symbiotic protocorms

of B. striata inoculated with Tulasnella sp. HR1–1 revealed that the expression

patterns of genes related to the signaling pathway of AM symbiosis are partially

conserved in B. striata (Miura et al. 2018). Additionally, the authors tested whether

one of the CSP genes calcium- and calmodulin-dependent protein kinase (CCaMK)

gene in B. striata is functional, by performing a cross-species gene complementation

assay using the Lotus japonicus ccamk-3 mutant (Tirichine et al. 2006). This analysis

showed that the B. striata CCaMK gene retains the functional characteristics of that

in AM-forming plants (Miura et al. 2018). These ﬁndings and other studies suggest

that orchids possess, at least partly, the molecular mechanisms common to

AM-forming plants (Perotto et al. 2014; Suetsugu et al. 2017; Miura et al. 2018).

Consistent with this suggestion, the CSP genes, such as symbiosis receptor-like

kinase SymRK, CCaMK, and calcium signal decoding protein CYCLOPS, are present

in orchid species (Radhakrishnan et al. 2020; Xu et al. 2021). However, the genes

encoding the GRAS transcription factor REQUIRED FOR ARBUSCULE DEVEL-

OPMENT 1 and the half-ATP-binding cassette transporters STUNTED

ARBUSCULE (STR) and STR2, which could be involved in the lipid transfer in

AM symbiosis, are missing from orchid genomes (Radhakrishnan et al. 2020;Xu

et al. 2021). Similarly, the three genes SymRK, CCaMK, and CYCLOPS were found

but RAD1, STR, and STR2 were not detected in the transcriptome of Ericaceae plants

that form ericoid mycorrhiza (Radhakrishnan et al. 2020). Molecular studies of

various types of mycorrhizae will help understand mycorrhizal symbiotic evolution.

16 C. Miura et al.

4 Prospects for Conserving Wild Orchids

Many orchid species are widely known to be endangered. Globally, biodiversity

hotspots are facing threats from land conversions, logging, and so on. These changes

affect both orchids and other plant species. However, orchids are most likely facing

greater threats than other plants if the other organisms they interact with (e.g.,

pollinators and mycorrhizal fungus) are also affected (Besi et al. 2019; Kolanowska

et al. 2021). At a glance, orchid conservation seems to simply preserve the existence

of a species, but in fact, orchid conservation requires extensive, complex approaches

that should meet their survival requireme nts, especially during reintroduction into

natural habitats. Conservationists and horticulturists worldwide are struggling with

this problem, looking for new strategies involving both conventional and modern

biotechnology. Although traditional methods, including symbiotic germination and

meristem culture, are commonly prefer red for mass seedling production (Knudson

1922; Arditti and Krikorian 1996), reintroduction of seedlings produced from these

methods directly into natural habitats could be even more challenging. The difﬁculty

is due to the nature of orchids: Establishing a symbiot ic association with appropriate

fungi is crucial for orchids, and plant robustness depends on the encounter with the

fungal partners.

Understanding the Molecular Mechanisms of Orchid Mycorrhizal... 17

Consequently, the transplantation of symbiotic seedlings seems to be better in situ

growth than asymbiotically grown seedlings. However, only a few orchid species

have been successfully cultured in symbiotic environments since Noel Bernard

discovered OM symbiosis in 1899. Rapidly developing next- and third-generation

sequencing techno logies have the potential to make a breakthrough in biodiversity

conservation because these sequencers overcome the technological hurdles of ana-

lyzing nonmodel plants at the molecular level. In AM symbiosis, the unculturability

of AM fungi without plant hosts has been an issue for a long time but is now allowed

for their asymbiotic cultures based on past ﬁndings and the latest fungal genome

information (Kameoka et al. 2019; Sugiura et al. 2020; Tanaka et al. 2022). A former

study reported that the cocultivation of the AM fungus R. irregularis with bacterial

strains of Paenibacillus validus induced secondary infective spores without host

plants (Hildebrandt et al. 2005). The genomes of AM fungi lack genes encoding type

I fatty acid synthases in their genomes but have enzymatic machinery for fatty acid

modiﬁcations (Tisserant et al. 2013; Tang et al. 2016; Maeda et al. 2018; Kobayashi

et al. 2018). Kameoka et al. (2019) corroborated these ﬁndings: AM fungi produce

spores on palmitoleic acid which is one of the fatty acids containing media.

According to Tanaka et al. (2022), the base media containing fatty acids were

available for another AM fungus Rhizophagus clarus, which lacks type I fatty acid

synthase as well as R. irregularis (Kobayashi et al. 2018). Tanaka et al. (2022) also

suggest that the comparative genome analysis of Rhizophagus species can provide

essential contributions to establishing custom-made culture methods and identifying

key genes involved in fungal diversity (Tanaka et al. 2022). Recent ﬁndings in OM

symbiosis, such as nitrogen transport, phytohormone signaling, and defense/symbi-

otic components, will contribute to efﬁcient symbiotic/asymbiotic seed germ ination

and plant growth handling.

Transcriptome and genome analyses provide large datasets and important impli-

cations but require additional conﬁrmation. In orchids, obtaining further evidence to

support omics data is often difﬁcult owing to the lack of methods for in vitro

propagation and gene transfer, a requirement of speciﬁc materials and technology

to analyze, such as radioisotope and stable-isotope measurements, and some han-

dling problems due to tiny seeds. Advanced technologies and novel ideas from

researchers in various ﬁelds are required to address these challenges. Orchid species

have a huge demand as horticultural and raw materials for Chinese herbal medicines.

In addition to the studies of ﬂower formation and asymbiotic mass production of

orchids, research on the molecular mechanisms of OM symbiosis is a fascinating

subject in that it reveals the symbiotic evolution process and develops a novel

in vitro/ex vivo culture system or even in situ transplantation. The application of

information obtained from omics analyses may be unlike untying the Gordian knot:

It cannot be directly and completely used to solve challenges in orchid conservation.

However, omics information can be used to determine which orchid–fungus pair

yields the best outcome for seedling vigor during reintroduction into natural habitats

by taking the role of phytohormone/metabolite production. Molecular studies on

OM fungi are expected to be implemented in a broader range of orchids, including

those of nonmodels.

18 C. Miura et al.

Acknowledgments The authors thank Masahide Yamato and Takaya Tominaga for critical

reading of the manuscript, and Enago (www.enago.jp) for the English language review. The

work was supported by the Research Fellowships of JSPS for Young Scientists (grant number

201801755) to C.M., and the Japanese government MEXT scholarship to G.C.P.

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Breeding of Orchids Using Conventional

and Biotechnological Methods: Advances

and Future Prospects

Jean Carlos Cardoso, Joe Abdul Vilcherrez-Atoche, Carla Midori Iiyama,

Maria Antonieta Germanà, and Wagner A. Vendrame

1 Introduction to the Family Orchidaceae and Main

Commercial Groups Used in the Flower Market

The family Orchidaceae is considered one of the largest groups among angiosperms

(along with Asteraceae) in a number of species, with more than 28,000 species

distributed in more than 850 genera according to data from World Flora Online and

Kew Botanical Garden (WFO 2022; Kew 2022). It is also one of the groups with the

widest geographic distribution, with representatives on almost all continents of

Planet Earth, including species with epiphytic, terrestrial, and lithophytic growth

habits, of which approximately 70% of all epiphytic ﬂora in the world are orchids

(Zotz and Winkler 2013).

J. C. Cardoso (✉) · C. M. Iiyama

Department of Biotechnology, Plant and Animal Production, Lab of Plant Physiology and

Tissue Culture, Center of Agricultural Sciences, Federal University of Sao Carlos (DBPVA,

CCA/UFSCar), Araras, Sao Paulo, Brazil

Graduate Program of Plant Production and Associated Bioprocesses, CCA/UFSCar, Araras, Sao

Paulo, Brazil

e-mail: jeancardoso@ufscar.br

J. A. Vilcherrez-Atoche

Graduate Program of Plant Production and Associated Bioprocesses, CCA/UFSCar, Araras, Sao

Paulo, Brazil

Graduate Program of Plant Genetics and Breeding, Escola Superior de Agricultura Luiz de

Queiroz, University of São Paulo, Piracicaba, Sao Paulo, Brazil

M. A. Germanà

Dipartimento Scienze Agrarie, Alimentari e Forestali (SAAF), Università degli Studi di

Palermo, Palermo, Italy

W. A. Vendrame

Environmental Horticulture Department, University of Florida, Gainesville, FL, USA

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_2

27

28 J. C. Cardoso et al.

In addition to its great ecological importan ce, diversity, and a high degree of

speciation in different regions of the world (Givnish et al. 2015; Pérez-Escobar et al.

2017), this group has been economically exploited worldwide, especially for the

purpose of cultivation of ornamental plants. This is mostly due to the high diversity

and number of species with inﬂorescences and ﬂowers with different architectures,

colors, and shapes that attract the consumer public in general and move billions of

dollars in the world ﬂower market.

A part of this trade can be considered illegal, especially for the exploitation of

native species taken from their natural habitat and placed for direct commercializa-

tion among collectors of rare species and even commercial cultivation nurseries,

reaching high unit economic values for the use of rare species and those at risk of

extinction, and which has taken on greater proportions with the online trade, which

still has limited capacity to trace the origin and destination of the trade in orchids and

other rare species of interest in botanical collecting (Hinsley et al. 2017; Cardoso and

Vendrame, 2022).

However, most of the use of orchids as ornamentals has been used legally, using

moderate to high technology, and based on the exploitation of native genetic

material to obtain hybrid cultivars, the latter including characteristics of horticultural

and ornamental interest, in a single plant. In this context of commercial use, it is

expected that a cultivar with high potential for use on a large scale will contain as its

main characteristics its suitability for large-scale production, which basically

requires: (1) rapid ﬂowering that can be controlled under artiﬁcial conditions in

order to accelerate and facilitate the production that requires ﬂowering plants at

different times of the year; (2) uniformity of vegetative and reproductive develop-

ment, allowing its commercialization to be programmed and delivered in lots

(3) compact size plants, a consumer market demand that also allows for an increase

in the number of plants per square meter of cultivated area, which is currently

expensive to implement and maintain; (4) resilient plants that need less inputs,

such as water, fertilizers, and pesticides, especially due to the incre ase in production

costs and the current demands associated with the concept of sustainability; (5) mar-

ket novelties, which attract the consumer and feed a market marked by innovations

and rapid changes in the end consumer’s desire. These requirements are of a general

order demanded by practically all commercial groups of orchids. However, more

speciﬁc objectives can be efﬁcient strategies in the development of new cultivars and

vary according to the commercial group cultivated.

Since the ﬁrst arti ﬁcial orchid hybrid, registered between Calanthe

masuca × C. furcata (De Chandra et al. 2014), there are currently at least a hundred

thousand hybrids generated worldwide by collectors and by commercial companies

specialized in the development of cultivars and trade of seedlings, also known as

“Breeders.” Based on this, there are commercial groups of orchids of greater

relevance for cultivation as ornamental plants, and from which the genetic improve-

ment for the market of ornamental ﬂowers and plants is quite advanced, being these

associated with the genera Phalaenopsis, Dendrobium, Cymbidium, Oncidium,

Vanda, and Cattleya (Fig. 1). The term associated is not by chance, because in the

case of Cattleya and Oncidium, most hybrids used as ornamentals are multigeneric,

therefore, originating from crosses containing multiple genera in a single plant. In

Breeding of Orchids Using Conventional and Biotechnological Methods:... 29

Fig. 1 Most important commercial orchids groups used in industrial and trade ﬂoriculture

the case of Cattleya, crosses compatible with other orchid genera are common, such

as Brassavola, Guarianthe, Hoffmanseggella, Ryncholaelia, Sophronitis, and more

recently with the genus Encyclia.In Oncidiinae subtribe, crosses between Oncidium

and species of the genera Brassia, Gomesa, Miltonia, Odontoglosssum, and more

recently with the inclusion of other genera, such as Ionopsis (Cardoso et al. 2016)

and Rodriguezia are more conventionally used. These intergeneric Oncidiinae

hybrids are commercially called Cambria orchids.

30 J. C. Cardoso et al.

In the other commercial groups, interspeciﬁc hybrids prevail, with some com-

mercially relevant intergeneric hybrids, such as Ascocenda (Ascocentrum × Vanda),

which allowed the miniaturization of commercial varieties of Vanda. Also, using the

genus Rynchonopsis (Rhynchostylis × Phalaenopsis) is currently used to achieve the

natural blue color in Phalaenopsis ﬂowers (Wu et al. 2022).

In contrast, in Phalaenopsis, the genus with the greatest commercial importance

as an ornamental plant in the world, the greatest consistency is from hybrids obtained

within the genus. Due to a large number of Phalaenopsis hybrids, the com-

mercial groups or types are divided by the characteristics of their ﬂowers or

inﬂorescences into: standard cultivars, containing in ﬂorescences with a good num-

ber of white ﬂowers of medium to large size; multi-ﬂora, characterized by small-

sized hybrids or also called mini-phalaenopsis and with multiple, compact and small

sized inﬂorescences; and market novelties, with yellow and red ﬂowers and also the

so-called spotted; and the biggest recent novelty called “Harlequins,” which present

coloration containing the fusi on of spots resembling intense and large red spots. In a

recent work developed by Lee et al. ( 2020), it is possible to see different types of

cultivars of each of the described Phalaenopsis commercial groups. Also, more

valued in the market are genotypes capabl e of synchronously producing two or three

inﬂorescences, which may belong to any commercial group described above.

Also, biotechnological methods have been used more frequently and more

effectively in the last decade, contributing to the development of cultivars with

speciﬁc characteristics, especially using transgenics (Hsieh et al. 2020; Liang et al.

2020). In this context, the advance in knowledge and increase in the efﬁciency of

in vitro regeneration systems, especially through the formation of Protocorm-Like

Bodies (PLBs), the increase in the number of sequenced orchid species, and

advances in molecular techniques, have resulted in the growing use of these tech-

niques in orchids and other species of ornamental use. Even so, in many countries,

the strict regulation related to the release of transgenic cultivars keeps the transgenic

cultivars in the ﬁeld of research by public and private companies and continues to be

the main obstacle for these cultivars to reach the ﬁnal consumer.

2 Basis of Reproductive Biology and Its Application

in Conventional Orchid Breeding

Despite the high diversity of species in Orchidaceae, some characteristics are

striking and deﬁnitive of this group of plants, such as its ﬂowers, which in general

consist of three sepals and three petals; one petal is modiﬁed and known as lip. In

addition, the reproductive structure is fused into a columnar structure, known as a

gynostemium, in which the stigmatic cavity and the pollinia are located, the latter

consisting of a mass with millions of pollen grains (Wu et al. 2009).

Breeding of Orchids Using Conventional and Biotechnological Methods:... 31

Most orchid species have hermaphroditic ﬂowers, that is, they contain the female

and male reproductive organs in the same ﬂower and fused in the column or

gynostemium.

However, there are monoecious species, which therefore produce female ﬂowers

separately from male ﬂowers (rarely hermaphroditic), which occur especially in the

subtribe Catasetinae (Machnicki-Reis et al. 2015). In this subtribe, there are impor-

tant genera of orchids used by collectors, such as the genus Catasetum, and from

which there are important advances in breeding and obtaining hybrid cultivars with

exotic colors and hardly found in other orchids subtribes. However, the greatest

difﬁculty in this genus for the expansion of the market aiming at large-scale

ﬂoriculture has been the long dormancy period of these plants, which lose their

leaves in fall-winter, keeping only their pseudobulbs, producing new shoots only in

the spring and blooming in spring-summer. In this case, the dormancy of

pseudobulbs can be broken by favorable climatic conditions of climatized green-

houses, which would make this group of plants good potential for innovation in the

market of ﬂowers and ornamental plants.

However, all the most commercially important groups mentioned have a column

containing functional pollinia and stigmatic cavities. Although these structures

contribute little to the ornamental aspect, they are essential in conventional breeding,

aiming to combine different genomes towards the development of new cultivars of

commercial interest.

The process of fertilization in orchids begins with pollination, a process by which

pollinia are positioned/inserted in the stigmatic cavity of the ﬂowers. From the

pollinia, millions of pollen tubes can emerge containing nuclei that will fertilize

the ovules, also in large numbers, and that will give rise to seeds. Embryo develop-

ment, a process known as embryogenesis, can take from 3 to 18 months depending

on the species and type of cross. Even within the same genus, there can be large

variations in the period of seed development.

As an example, in Dendrobium, one of the genera with the largest number of

species, there are two main commercial groups, mostly of hybrid origin, known as

Nobile and Den-phal. In the Nobile group, the main species with the greatest

genomic contribution to the development of cultivars is Dendrobium nobile, and

the main characteristic of this group of cultivars is the presence of long pseudobulbs

containing short inﬂorescences with one to four ﬂowers distributed along the

pseudobulb (Floricultura 2021). In this group, fruits have a very slow development,

and the physiological maturity of seeds, as well as the dehiscence of fruits, occurs

from 8 to 14 months after pollination. In the case of the Den-phal group (Fig. 1),

Den. phalaenopsis and Den. bigibbum seem to have the greatest contribution,

especially because they have large and round ﬂowers. Despite this, some orchids

are classiﬁed in the Den-phal group, but in some cases do not have the genome of

these two species in their origin. Unlike the previous group, Den-phal orchids are

characterized by one or more inﬂorescences, usually contai ning numerous ﬂowers,

which arise from the apical region of the pseudobulbs (Cardoso 2012; Fig. 1). In this

group of orchids, seed and fruit development is faster, with fruit dehiscence occur-

ring between 4 and 6 months after pollination.

32 J. C. Cardoso et al.

Cattleya and Vanda have fruit and seed development time from 8 to 12 months. In

the genus Phalaenopsis, fruit development, from pollination to natural dehiscence,

takes 6–12 months after pollination, like what occurs in Oncidium.

Orchid seeds also represent an exclusive charact eristic of this family of plants,

and the embryos develop in a limited way until the moment of fruit dehiscence and,

consequently, their dispersal. Embryos are also devoid of reserves, such as the

endosperm and cotyledons, and for effective natural germination, it is necessary

the symbiotic association of embryos with mycorrhizal fungi or other

microorganisms.

Most likely, partly, or entirely because seeds do not have nutritional reserves for

the embryo, this is considered one of the families with the greatest capacity for

interspeciﬁc hybridization, including multigeneric hybrids, that is, obtained from

multiple and successive crosses between different genera and which, in the end,

generate fertile hybrids capable of new hybridizations.

An example of this high cross ability is found in the subtribe Laellinae, whose

main commercial representative is the genus Cattleya and in which, however, most

of the cultivars produced and marketed as ornamentals come from interspeciﬁc and

intergeneric hybrids. In this way, it is possible to cross the genus Cattleya with

species of the genera Brassavola (Ex: Brassocattleya Binosa), Hoffmansegella

(ex Laelia) (e.g., Laeliocattleya Brazilian Girl, Cardoso 2010; Laeliocattleya

Nobiles Confetti, Fig. 1), Sophronitis (Sophrocattleya), Epidendrum (e.g.,

Epicattleya “Renne Marques”), Encyclia (Catyclia), Broughtonia (Cattleytonia),

Caularthron (Caulocattleya), Rhincholaelia (Rhincholaeliocattleya), among other

multiple combinations of these hybrids.

Thus, if, on the one hand, this high diversity of crosses allows great segregation of

traits for breeding, this is a highly complex family in genomic terms. Due to the

multiple possible combinations, it can result in a great complexity for molecular and

cytogenetic analysis aimed at the identiﬁcation and origin of chromosomes and

genes from these multiple possible combinations, which now difﬁculty programs

to use molecular assisted breeding.

Also, for this reason, and the easy crosses, with good fruit set and seed develop-

ment, conventional breeding has been used for decades aiming at the improvement

of orchids and until today it has been the main method for use in professional

programs for breeding and development of new orchid cultivars.

3 Main Methods Used in Conventional Orchid Breeding

Conventional orchid breeding methods are still today, in the era of omics and genetic

editing, the main method of orchid breeding aiming at the production of new

cultivars.

Breeding of Orchids Using Conventional and Biotechnological Methods:... 33

The prevalence of these methods is currently due to the numerous species

diversity and high capacity for interspeciﬁc and intergeneric combinations in

orchids. Thus, allow the breeder to seek, in a conventional way, genotypes that

add diff erent traits to be inserted in commercial cultivars, only using controlled hand

pollination to the development of fruit/seeds containing the hybrid progeny.

The other step of this process is the in vitro germination, which has been done

through in vitro cultivation techniques, in which seeds, after fruit and seed devel-

opment, undergo asepsis procedures to eliminate the present microbiota, being

placed to germinat e in a suitable culture medium containing a carbon source to

support the development of embryos into seedlings. After the period of cultivation

and in vitro development of the progeny, seedlings are acclimatized in a greenhouse

and later selected in a cultivation environment like the one in which they will be

grown. Genotypes with desired traits are selected, cloned using micropropagation

techniques, and tested on a commercial scale to evaluate clonal stability and cultivar

performance under cultivation conditions.

3.1 Creation of Germplasm Banks and Their Relationship

with the Objectives of the Breeding Program

Germplasm banks are the main source used to start orchid breeding programs and

consist of collections of species, hybrids, or even different genotypes of the same

species with characteristics of interest to be inserted and developed in future new

cultivars. Most private and public companies with programs for breeding orchids

and other ornamental plants have their germplasm bank, and are made up of species

from different geographic regions where they naturally occur; the ex situ conserva-

tion in protected cultivation is the main method used by breeding companies. That is,

species and genotypes of different species of interest are kept outside their natural

habitat, in cultivation conditions that simulate this environment and that may involve

the use of temperature control technologies (heating and cooling), increase in

relative humidity, irrigation, and artiﬁcial light. Undoubtedly, the largest orchid

breeding programs developed by private companies are in The Netherlands, Taiw an,

and Thailand, these are known as “Breeders,” and they are responsible for the

maintenance of germplasm banks, development, and commercialization of new

cultivars, in addition to the production and commercialization of seedlings of these

cultivars. Most companies are known as “Breeders” work speciﬁcally in the market

for cultivars and plantlets production, providing the genetic material for the world

ﬂower market. In this way, ﬂower growers who use the technology of these

companies, pay as costs the value of the production of plantlets, but also the

technology used and associated with the cultivar, also called as royalties. Currently,

the plantlets + royalties’ has been the most relevant cost among a ll costs associated

with the production of ﬂowers, exceeding in recent years, the cost of labor for

cultivation. Thus, to reduce the production costs of these plantlets, large companies

have developed production areas and plantlet cultivation systems (owned or

outsourced) within the country where the plantlets are marketed, reducing risks

associated with currency ﬂuctuations, high costs and bureaucracy of importing

plant material.

34 J. C. Cardoso et al.

In this way, germplasm banks are the main genetic source of traits in these

companies, and the collection of species, genotypes, and hybrids is the one that

maintains a frequency of production of new cultivars based on diverse and controlled

crosses to target-speciﬁc traits.

The main characteristics desired and placed as objectives in the current orchid

breeding programs can be divided according to the vegetative and reproductive

stages of the plants. At the vegeta tive stage, the main objectives, in general, are

compact, rapid, and vigorous vegetative development, good rooting in pot and

substrate conditions, and resistance to pests and diseases of the roots and shoots.

At the reproductive stage, the most general objectives covering most commercial

groups are high adaptability to already established cultivation systems that respond

uniformly to the ﬂowering control process; high ﬂowering uniformity and homoge-

neity of cultivation lots; reduction of the juvenile period and, consequently, faster

ﬂowering; natural ﬂowering at different times of the year, therefore, less dependent

on speciﬁc climatic conditions (Cardoso et al. 2016); the greater number of in ﬂo-

rescences at the same time, which has resulted in higher market value; compact and

ﬂexible inﬂorescence that allows adequate staking; large and round-shaped ﬂowers,

and when small, they should be numerous for greater visual ﬁlling; novelties about

colors and shapes of ﬂowers and inﬂorescences.

However, speciﬁc features must be highlighted, especially for the genus Phalae-

nopsis, which differs in growth habit (monopodial) from most other commercial

groups (sympodial). In this case, early ﬂowering is not desired for some reasons:

ﬂowering in Phalaenopsis occurs after 12–18 months from seedling or plantlet

acclimatization in a greenhouse; although it is possible to observe early ﬂowering

in some plants, this generally results in reduced inﬂorescence size and a number of

ﬂowers, not being marketed; the early emergence of this inﬂorescence results in the

need for additional management aiming at its elimination, as it delays vegetative

development and delays commercial ﬂowering. Regarding the presence of more

ﬂexible ﬂoral stems to support staking, in Phalaenopsis there is also a different

demand for inﬂorescences with high ligniﬁcation degree and that do not need

additional staking, as this would result in reduced plant management. Further,

inﬂorescence ligniﬁcation is a hereditary trait associated with the type of inﬂores-

cence architecture (Pramanik et al. 2022). Also, in Phalaenopsis, one of the fungi

with the greatest impact on cultivation is the genus Botrytis, which causes spots and

necrotic spots mainly on ﬂowers. Although this fungus is a problem in all orchids,

Phalaenopsis seem to have a greater susceptibility and, consequently, the genus in

which there is greater damage due to their symptoms reducing the quality and

durability of ﬂowers. Thus, the search for more resistant cultivars or sources of

resistance should be included in breeding programs, either by conventional crosses,

or even biotechnol ogical methods.

In Dendrobium, especially in the Nobile group, in addition to innovations in the

color of the ﬂowers, above all, plants that ﬂower in almost all nodes of the

pseudobulb are sought, as most cultivars have ﬂowering nodes only in the middle

and upper third, and no ﬂowers in the basal third of the elongated pseudobulb. In the

Den-phal group, among the objectives are: innovation in relation to colors (Cardoso

2012); increased ﬂo wering synchronization, as most cultivars available on the

market still have time-dispersed ﬂowering, with less than 60% clonal individuals

in a lot with synchronized ﬂowering; production of compact plants with multiple

terminal inﬂorescences or compact plants with 1–5 terminal ﬂowers of large diam-

eter and rounded shape. Due to a large number of species in Dendrobium, there is a

good potential for the release of new commercial groups, such as those with pendant

inﬂorescences, especially hybrids with Den. densiﬂorum, Den. thyrsiﬂorum, and

others from the same group (Teixeira da Silva et al. 2016).

Breeding of Orchids Using Conventional and Biotechnological Methods:... 35

In Oncidium and its multigeneric hybrids, the search has been for large plants

with multiple inﬂorescences containing medium- to large-sized ﬂowers and, in the

opposite direction, for compact plants with short inﬂorescences and medium-to-

large-sized ﬂowers. Color innovation is one of the central objectives, as most

cultivars are between yellow and brown, based on the two groups of great commer-

cial relevance worldwide, which are the groups called “Golden Rains,” yellow in

color and without fragrance, like Onc. Aloha and Onc. Sweet Sugar, and the Onc.

“Sharry Baby,” with brown tone ﬂowers with intense fragrance, which resembles the

smell of chocolate (Cardoso et al. 2016). Another group that has gained commercial

importance is commonly called Cambrias and is grouped by different intergeneric

hybrids, such as Colmanara, a multigeneric hybrid (Oncidium × Odontoglos-

sum × Miltonia), Vuylstekeara (Cochlioda × Miltonia × Odontoglossum), Beallara

(Cochlioda × Miltonia × Oncidium × Odontoglossum) and which results in multiple

inﬂorescences with a good number, size, and color of the ﬂowers. One of the

successful examples of this hybrid and cultivated worldwide is Colmanara “Wild

Cat,” with yellow ﬂowers and brown spots, and Beallara “Tahoma Glacier,” with

compact inﬂorescence, large and star-shape white ﬂowers with red spots (Fig. 1).

In Oncidium and Dendrobium, as well as their hybrids, high-impact rust has

emerged more recently, causing spots on pseudobulbs and leaves, these spots are

also called shotgun blasts, as they are characterized by several necrotic spots and

which together have a more or less circular shape. The causal agent of the disease is

not yet fully elucidated, but it is probably due to phytopathogenic fungi of the

Cercospora and Alternaria genera. However, for all genera, the main pathogenic

fungus actually is from the genus Botrytis, which causes numerous brown color

spots in the petals and sepals, which reduces the quality and impedes their

commercialization.

In the genu s Cattleya and its hybrids, the main problem associated with cultiva-

tion are the time from cultivation to the ﬁrst ﬂowering, which often exceeds

3–5 years, putting these plants at a disadvantage in relation to the other genera

mentioned above, which normally ﬂower at 18–24 months of cultivation; the low

shelf life of its ﬂowers, which rarely exceeds 20 days in the best hybrids, and; the

high sensitivity of ﬂower buds to stresses caused by handling, transport, and change

of environment. These characteristics put this plant at a disadvantage in relation to

other genera used as ornamentals, such as Phalaenopsis, Dendrobium, and

Oncidium, since in these plants, the time from cultivation to commercial ﬂowering is

18–24 months, with a shelf life of around 30 days or more, with Phalaenopsis

hybrids that can last longer than 60 days of shelﬂife, with good resistance to handling

and transport.

36 J. C. Cardoso et al.

3.2 Crosses by Controlled and Directed Pollination

After the creation of the germplasm bank based on the objectives of the breeding

program, the process of directed crosses begins, in which pollinia of one genotype

are taken to the stigmatic cavity of the other. In this process, in addition to the choice

of parents for the purpose of breeding, there is also a strong inﬂuence on orchids in

the choice of the plant to be used as a parent. In crosses carried out with different

genotypes by our research group and breeding program with Cattleya, we have

observed a vegetative development (e.g., type and intensity of rooting, type of leaves

and pseudobulbs of the progenies) with a greater genetic inheritance of traits from

the mother parent.

Preferably, pollinia taken from the paternal parent should be removed and

immediately brought to the stigma for pollination. Nevertheless, due to the difﬁcul-

ties of synchrony in ﬂowering or even obtaining plants with different ﬂowering times

from the parents, it is possible to store pollinia. Pollinia lose their viability very

quickly after they are removed from the ﬂowers at room temperature, but they can be

stored for a few weeks or even a few months at low temperatures, ranging from -

20 °Cto 8 °C (Yuan et al. 2018).

After pollination, the germination of pollen grains in the sti gma and the long way

to the inferior ovary and eggs for fertilization begins, which usually takes a few days

to occur. After fertilization, the process of zy gote development begins and culmi-

nates in embryogenesis, which, as previously mentioned, can take from 90 to more

than 360 days depending on the genus and species of orchids used in the crosses.

Problems related to incorrect pollination and/or non-occurrence of fertilization

can result in ﬂower abortion, early fruit abortion after a period of development, or

even in the formation of seeds without embryos. These anomalies related to repro-

duction may be associated with the non-viability of pollinia caused by different

factors, the incompatibility in the crossing, and as observed in our studies, the

genetic factors contained in different genotypes, which result in different degrees

of fruit and seed production in orchids.

Interestingly in the case of orchids, most commercial hybrids, even after succes-

sive generations of interspeciﬁc hybridization, mai ntain different levels of fertility.

Crosses, therefore, can be species × species, hybrid × hybrid, or hybrid × species,

mostly with success in obtaining progenies.

After 24–48 h of pollination, senescence of ﬂowers due to pollination can be

observed, with important changes such as wilting and forward bending of petals,

sepals, and lip (Fig. 2a, b), as if they were protecting the reproductive organ, until the

moment they dry completely and are detached from the gynostemium/ovary. At the

Breeding of Orchids Using Conventional and Biotechnological Methods:... 37

p-f

fsd

fruit

seeds

p-f

gf

Fig. 2 (a) Intergeneric cultivar from Cattleya hybrid group (Potinara Free Spirit) within one

pollinated ﬂower (p-f) showing the closing of petals and sepals and the green and swelling of the

inferior ovary. (b) Pollinated ﬂowers of Den-phal hybrid showing the swelling of its ovaries. (c)

Green fruit (g-f) and fruit starting dehiscence (fsd) and yellowish color, with 8 months after

pollination. (d) Fruits and seeds of Oncidium orchid 8 months after pollination. (e) In vitro culture

of F1 hybrid progeny of Cattleya orchids. (f) Greenhouse cultivation of different F1 progenies

seedlings of Cattle ya intergeneric orchid

same time, there is a clear increase in the green color and swelling of the ovary

(Fig. 2a, b). This process of fruit swelling continues throughout fruit development,

until the moment of dehiscence or natural opening of the fruit (Fig. 2c), at which time

the three infertile valves detach from the fertile valves of the capsule (Dirks-Mulder

et al. 2019) with seed dispersal by wind.

38 J. C. Cardoso et al.

Fruit from the directed crossing, also called capsules, can be harvested shortly

before (unripe fruits) or at the beginning of the dehiscence, also called ripe fruits

(Fig. 2c, d). Harvesting unripe fruits require care, especially regarding knowledge

about the seed maturation time, which is very variable in orchids and is subject to the

risk of an early harvest, which results in abortion and non-germination of most seeds

obtained from the cross . When ripe, part of the seeds is loose inside the fruits and this

coincides with the maturation and dehiscence of the capsule.

After being removed from the capsule, seeds are ready to be placed for germina-

tion. As a standard procedure performed at the Laboratory of Plant Physiology and

Tissue Culture of the Federal University of São Carlos, fruit from directed crosses

are harvested at the beginning of the dehiscence, when capsules change from green

to yellowish-green, or even when it is noticed the beginning of the dehiscence, which

starts in the distal region of the fruit, close to the column (Fig. 2c). After harvesting,

fruits are opened, and seeds are exposed and kept to dry for at least 24 h (Fig. 2d),

followed by removing all the seeds with the aid of a brush. Seeds are then stored in

plastic tubes under a low temperature (8 °C). In this way, it is possible to store the

seeds, with good viability for at least 6 months. This is extremely valid when

working with many crosses and there is a need for reseeding due to

non-germination or other problems that arise from the ﬁrst seeding attempt.

3.3 Asymbiotic Cultivation as the Main Means for Obtaining

Progenies

Germination of orchid seeds under natural conditions, due to the limited or

abscence of nutrient reserves associated with seeds, is dependent on relationships

with microorganisms that make a symbiotic association with orchids, especially

mycorrhizal fungi and rhizobacteria (Tsavkelova et al. 2016; Chen and Nargar

2020). Although it is possible to isolate, cultivate and, later, subculture these

microorganisms together with orchid seeds to promote germination, a technique

known as “seed baiting,” this is a tiring technique, of more difﬁcult implementation,

which requires care with the microorganism, with the seed and with the interaction of

the two organisms. These characteristics hardly meet the objectives of a breeding

program, in which the main objective is to germinate many progenies to select new

cultivars with superior characteristics. Symbiotic cultivation has shown good appli-

cability in projects to understand the interaction microbiota and orchid species, in

orchid species in which symbiotic germination does not seem to result in success as

with terrestrial species, and in conservation and restoration projects with orchid

species (Yang et al. 2020).

Breeding of Orchids Using Conventional and Biotechnological Methods:... 39

Thus, the studies that began with Knudson (1922) greatly helped the breeding

programs, by developing a technique for cultivating and germinating orchid seeds in

an asymbiotic way, that is, without the need for microorganisms. This technique uses

a culture medium containing a nutrient solution, a source of sugar and agar. Culture

media such as those of Knudson (1922) or Murashige and Skoog (1962) containing

half the concentration of macronutrients (MS1/2) have been the most used culture

media for germination of seeds of different orchid genera and meet the need to obtain

high germination rates (Teixeira da Silva et al. 2015; Chen and Nargar 2020). A

critical point for culture media to promote high germination rates is that the pH of the

media should be adjusted to values between 5.5 and 6.0.

Asymbiotic germination under in vitro conditions can allow germination above

90% a nd allows the germination of seedlings that would hardly germinate or survive

the stresses associated with the natural environment, in which less than 1% of the

seeds germinate. At the same time, in a breedi ng program looking for

high-performance plants, the objective is to obtain a large number of plants for

post-germination selection of plants with interesting horticultural and ornamental

characteristics, under conditions very different from those in nature, using technol-

ogies such as environmental thermal control, availability of water and fertilizers

based on balanced irrigation and nutrition programs available throughout the life of

the plant. That is, the cultivation conditions of a new cultivar, including the plant

selection process, take place in a very different environment from the natural one, in

which plants are evaluated and selected according to their performance under

artiﬁcial cultivation, similar to the conditions in the which large-scale ﬂowering

plants are produce d.

After in vitro germination, not infrequently, several hundred seedlings are ger-

minated in a single cultivation ﬂask, which requires a process of subcultures and

plant selection from the beginning. In hybrids of the genus Cattleya, Cardoso et al.

(2016) developed a methodology for the systematic selection of seedlings, which

starts from in vitro cultivation and ends at the time of the second ﬂowering. This

process consists of, at each subculture and from the ﬁrst in vitro subculture, initiating

the selection of seedlings with better vegetative performance. Soon after the germi-

nation of orchid seeds, the so-called protocorms are formed, globular structures from

the multiple cell divisions of the embryo placed for in vitro germination. It is notable

for most asymbiotically germinated progenies, the production of two types of

structures, one of which remains as protocorms and another group of progenies

directly originates or evolves to the formation of seedlings, containing leaves, roots

and sometimes, pseudobulbs. In this ﬁrst selection, which occurs after 90–120 days

of cultivation, only seedlings are selected. These seedlings are then transferred to

subculture 1, from which after 90–120 days of cultivation, there is a second selection

based on plants with a good shoot and root development. It is possible that some

genotypes still need a third subculture before reaching 3–8 cm in length (Fig. 2e),

when the seedlings selected in vitro are taken to the acclimatization process, with the

removal of the plants from the in vitro conditions with cultivation in culture medium

to ex vitro environment with cultivation in a substrate.

Acclimatization can last for 90 to 180 days and is carried out in trays with the

adequate substrate under greenhouse conditions. As the most common substrates,

sphagnum, peat moss, and coconut ﬁber are used, but different mixtures prepared by

specialized companies can be found. Here, fertigation programs also begin, in which

fertilization or plant nutrition is offered together with irrigation. After this period of

cultivation, a new round of selection of superior plants is carried out, with a selection

of plants with vigorous vegetative development and absence of symptoms of pests

and diseases, excluding those with inferior development. These selected plants are

transplanted into an intermediate pot size (Cattleya and Oncidium), with 6–9cm in

diameter (Fig. 2f), or even into the deﬁnitive and large pot size (Oncidium, Phalae-

nopsis, Dendrobium, Vanda), with 9, 12, or 15 cm in diameter, depending on plant

size and cultivation objective, and in which plants are ﬁnally selected for faster

ﬂowering characteristics and ornamental attributes (Cardoso et al. 2016). This

methodology has been used effectively in different commercial genera of orchids

and new cultivars have already been obtained in the commercial groups of Cattleya

(Cardoso 2009; Cardoso et al. 2016), Denphal, a Dendrobium-type orchid (Cardoso

2012) and Oncidium (Cardoso 2017).

40 J. C. Cardoso et al.

Importantly, in this case, the selection of plants occurs more for characteristics of

horticultural interest, to the detriment of the ornamental aspect, aiming at the

selection of plants with rapid development and early ﬂowering. Ornamental traits

are selected from only those plants that ﬂower the fastest. Although this seems to be

a limitation of the technique, the choice of parents is an important step towards the

selection of plants that include rapid development and abundant ﬂowering and with a

shape/color of interest to the market.

4 Biotechnological Approaches Used for Breeding Orchids

Although conventional breeding prevails over other methods in the development of

new orchid cultivars, in the last decade there has been an important growth in the

contribution of biotechnological techniques that resulted in the development of new

groups of orchid cultivars. The advancement of research in the areas of sequencing,

omics, and genetic engineering has currently allowed advances in the application of

biotechnological tools for breeding ornamental plants, including orchids. The most

relevant cases with the greatest commercial impact have occurred in genera used as

ornamental plants and of greater commercial importance, such as Cymbidium and

Phalaenopsis (Balilashaki et al. 2022; Cai et al. 2015 Yang et al. 2021a, b), and have

made possible the identiﬁcation of genes correlated to pathways of great relevance

for breeding orchids, such as the ﬂoral scent in Cymbidium goeringii (Ramya et al.

2019) and the color of the ﬂowers in Phal. equestris and Phal. aphrodite (Hsu et al.

2022).

The most used techniques for this purpose include the induction and selection of

somaclonal variants from in vitro culture, in vitro polyploidization using mutagens,

transgenics, and the isolation and fusion of protoplasts. The main biotechnological

tools used for orchi d breeding, as well as the target traits achieved for each tech-

nique, are resumed in Fig. 3.

Breeding of Orchids Using Conventional and Biotechnological Methods:... 41

Fig. 3 Target traits achieved using biotechnological tools. Rectangle forms represent the target traits achieved and elliptical forms showed the techniques used

to induce the traits achieved

42 J. C. Cardoso et al.

4.1 Induction of In Vitro Somaclonal Variations

In vitro somaclonal variation consists of genotypic and phenotypic variation that

occurs in in vitro tissues. Somaclonal variations are more frequently observed in

Protocorm-Like Bodies (PLBs), which are globular structures similar to protocorms

but of somatic-origin in orchids. The PLBs are used for large-scale clonal propaga-

tion of different orchids genera. However, this technique of propagation, also called

Induction, Proliferation, and Regeneration of Protocorm-Like Bodies (IPR-PLBs) is

a source of in vitro somaclonal variation in orchids, such as in Phalaenopsis,

Dendrobium, and Oncidium (Cardoso et al. 2020).

Otherwise, somaclonal variation can be a genetic variation source of new traits of

interest such as biotic and abiotic stress resistance, and morphological and physio-

logical variations in ﬂowers (Wang et al. 2019). The occurrence of punctual genetic

mutation derived from PLBs is interesting in ornamental breeding because the

cultivar maintains the main characteristics of originals with changes in one or a

few traits. Therefore, somaclonal variation can be strategically applied to meet the

demand for novelties in the orchid ﬂower market, not requiring a long period of

hybridization and progeny selection.

As an example, one of the most actual, interesting, and commercial novelty in

orchids are the Harlequin-type cultivars of Phalaenopsis, attractive for the color of

their ﬂowers with red or magenta-black fused spots, initially obtained and derived

from an in vitro SV from the clonal system for propagation of Phalaenopsis Golden

Peoker “Brother” (Hsu et al. 2019; Lee et al. 2020). Currently, the Harlequin-type

cultivars represent an innovative and attractive cultivar group used as a new source

of genes for the color of ﬂowers, due to the heredity of this characteristic in crossings

with other Phalaenopsis groups (Lee and Chung 2021).

However, somaclonal variation is random and spontaneous and can result in

morphological abnormalities of plants, for example, the occurrence of creased leaves

(Tokuhara and Mii 2001) or the occurrence of deformity in ﬂower structures, such as

the absence of the labellum (Cardoso et al. 2020), which are not desired for clonal

mass propagation or breeding purposes.

Some of the factors that can affect the frequency of somaclonal variation (SV) in

orchids are the species or genotype used, the type and concentration of

phytorregulators in the culture medium, the origin of explant and the system used

for regeneration, the age of the in vitro culture, and also the number and environ-

mental conditions of subcultures (Chin et al. 2019; Cardoso et al. 2020).

Long-term cultures are one of the main factors leading to somaclonal variation in

orchids. In vitro somaclonal variation was observed in Doritis pulcherrima derived

from PLBs after 2 years of in vitro culture. The main changes observed between the

original and SV-derived were the color of leaves, purple in the somaclones, and

green in the original-type plantlets, in addition to differences observed in the size and

content of chlorophylls, which were higher in the original-type ones (Thipwong et al.

2022). Long-term subculture also resulted in the presence of somaclonal variation in

Oncidium “Milliongolds,” which was detected by SLAF-seq in PLBs-derived clones

after 10 years of in vitro culture (Wang et al. 2019).

Breeding of Orchids Using Conventional and Biotechnological Methods:... 43

Among phytoregulators, most reports point to cytokinins as the main cause of

SVs in orchids. Somaclonal variation was reported in PLBs of Dendrobium “Sabin

Blue” cultured for 2 years in a medium containing kinetin as phytoregulator, and

detected by ISSR and DAMD molecular markers (Chin et al. 2019) and in PLBs of

Dendrobium nobile cultured in medium containing thidiazuron (Bhattacharyya et al.

2016), a cytokinin-like component.

Besides PLBs, the use of indirect organogenesis by callus proliferation resulted in

increased somaclonal variation frequency in Vanilla planifolia Jacks, producing

chlorophyll-variegated plantlets regenerated from callus (Ramírez-Mosqueda and

Iglesias-Andreu 2015).

The presence of SVs in orchids can be detected by molecular markers, such as

ISSR (Inter Simple Sequence Repeats), RAPD (Random Ampliﬁed Polymorphic

DNA), SCoT (Start Codon Targeted), DAMD (Direct Ampliﬁcation of Minisatellite

DNA region), SLAF-seq (Speciﬁc-Locus Ampliﬁed Fragment Sequencing)

(Cardoso et al. 2020; Li et al. 2021a), or using morphological of adult plants until

their ﬂowering (Zanello and Cardoso 2019).

4.2 Transgeny

Transgenic technology is an efﬁcient breeding technique that allows the transference

of foreign new genes into a plant genome (Belarmino and Mii 2000). For orchids,

two methods have been employed: the particle bombardment or biolistic consists of

a physical and direct approach to transfer exogenous genes into plant tissues

delivered by microparticles of gold or tungsten, which penetrate plant cell wall;

the Agrobacterium tumefaciens-mediated transformation, which is a biological

method based on the infection of plant tissue with speciﬁc strains of Agrobacterium

tumefaciens, a soil bacterium, that has the capacity of transferring genes into the host

plant (Mii and Chin 2018).

Although Agrobacterium-mediated transformation was considered a method only

for dicotyledonous plants, since monocots are not a natural host of Agrobacterium,

monocots as orchids have been successfully transformed (Mirzaee et al. 2022). The

particle bombardment has as its advantage the independence of hostage limitation.

However, Agrobacterium-mediated system is preferred for the ease and high-

repeatability of the technique.

In the family Orchidaceae, genetic transformation has been establishe d in all main

commercial orchid genera (Li et al. 2021a; Zhang et al. 2022), and some factors are

important to achieve a successful genetic transformation system. Agrobacterium-

mediated transformation requires bacteria strains with an efﬁcient infec tion of orchid

cells, followed by the later regener ation of cells and tissues under in vitro culture.

Thus, the use of super virulent strains, such as EHA101 and EHA105 (Subramaniam

and Rathinam 2010; Mirzaee et al. 2022) has contributed to improving the

transformation efﬁciency. Strain EHA101 is the most used in Phalaenopsis, Cat-

tleya, Cymbidium, Dendrobium, and Vanda orchids. Strain EHA101 is more fre-

quently reported in Phalaenopsis, b ut recent studies involving genetic

transformation in this genus only used strain EHA105. In addition, EHA105 was

also the most used strain in Oncidium and Erycina (Mii and Chin 2018). The target

explant used for Agrobacterium transformation is also high important, and most

studies focused o n PLBs or protocorms.

44 J. C. Cardoso et al.

After the infection of plants using Agrobacterium, it is important to eliminate

bacteria from plant cells and tissues by using antibiotics. The main antibiotics for this

purpose are hygromycin, kanamycin, cefotaxime, and meropenem. Some antibiotics

are also helpful to select the transformants, when a marker gene was used for

transformation. Usually, antibiotic-resistance genes are used to certify the occur-

rence of transgeny and to select transformants from the non-transformed tissues and

individuals. Thus, transformants containing the antibiotic-resistance gene will sur-

vive when exposed to antibiotics while the non-transformants will be eliminated

(Mii and Chin 2018; Ozyigit and Yucebilgili Kurtoglu 2020).

In the family Orchidaceae, the ﬁrst reports of genetic transformation focused on

testing and improving the efﬁciency of the method, using only marker or reporter

genes to demonstrate that explants were successfully transformed. However, g enetic

transformation has enabled the change in ﬂower color, the induction of early

ﬂowering, the resistance to pathogens, such as Cymbidium Mosaic Virus

(CymMV) and Odontoglossum Ring Spot Tobamovirus (ORSV), and more recently,

the resistance to Erwinia carotovora (Li et al. 2021a), the production of miniaturized

Phalaenopsis by overexpression of the OsGA2ox6 gene (Hsieh et al. 2020), and the

modiﬁcation of the color of ﬂowers, such as violet-blue in the white-ﬂower Phalae-

nopsis cultivar (Liang et al. 2020).

The main limitations of genetic transformation in orchids are the low efﬁciency of

transgeny; the limited results until now, especially with changes in the color of

ﬂowers of transgenic plants, and the difﬁculties with the release of transgenic

cultivars in the ﬂower market.

4.3 In Vitro Polyploidization and Self-Duplication

of Genomic DNA

Polyploidy is a biological event in which eukaryotic organisms have more than two

complete sets of chromosomes, thus generating changes from the genetic level to

their relationship and adaptation with the environment (Fox et al. 2020; Soltis et al.

2009) and which are observed from humid tropical forests, desert regions, and

extremely cold environments. This characteristic of increased vigor in polyploid

organisms is due to genetic redundancy, which also serves as a defense mechanism

against the negative effects of mutations and heterosis (Comai 2005). Genetic studies

have analyzed genome duplication events in angiosperms, revealing that they all

have a paleopolyploid ancestor, as their genome has undergone at least one dupli-

cation event during evolution (Jiao et al. 2011; Renny-Byﬁeld and Wendel 2014).

Breeding of Orchids Using Conventional and Biotechnological Methods:... 45

There are two mechanisms for natural polyploid formation in plants: the produc-

tion of unreduced gametes (2n) and somatic duplication (Sattler et al. 2016). Somatic

duplication in plants is called endoreduplication, which is modulated by hormonal,

environmental, and nutritional factors. Endoreduplication is caused by errors during

the endocycle of mitosis, where cells replicate their genome but do not undergo

cytokinesis, generating different levels of ploidy within them (Maluszynska et al.

2013). Diploid organisms can also produce unreduced gametes due to errors during

the ﬁrst or second division of the restitution phase of meiosis (Sattler et al. 2016).

From the establishment of in vitro plant cultivation by Haberlandt (1902) and

with the ﬁrst report of polyploidization using this cultivation system (Murashige and

Nakano 1966), it was possible to determine that plant tissue culture could be used not

only for mass propagation of plants but also as a new and efﬁcient tool for artiﬁcially

obtaining polyploid plants (Dhooghe et al. 2011). Currently, the use of in vitro

cultivation system using colchicine as an antimitotic agent has become the most

common and popular strategy for plant polyploidization (Eng and Ho 2019).

In orchids, the chromosome number of more than 90% of species is the result of at

least one polyploidy event (Mondin and Neto 2006). Thus, natural polyploidy

events, such as endopolyploidy and the formation of unreduced gametes have

been reported in many orchid genera, especially those used as ornamentals

(Vilcherrez-Atoche et al. 2022). Endopolyploid tissues have been reported in almost

all commercial orchid genera, with DNA content ranging from 2C to 16C in

Cymbidium, 2C to 32C in Dendrobium, and 2C to 64C in hybrids of Phalaenopsis

(e.g., Doritaenopsis) and Vanda (Vilcherrez-Atoche et al. 2022). Regarding the

frequency of occurrence of unreduced gametes in orchids, this is not naturally

high, being observed in some commercial cultivars of Cymbidium, between

0.15–4.03% (Zeng et al. 2020).

Although the natural production of polyploid plants from non-reduced gametes or

tissues with high rates of endopolyploidy is possible, polyploidization with the use

of antimitotics is the most used technique for the artiﬁcial induction and increase of

the frequency of polyploids for orchid breeding (Vilcherrez-Atoche et al. 2022).

The artiﬁcial induction of polyploidy in orchids has already been reported, at least

once, in the main genera used as ornamental plants, and in more than 80% of

polyploidization studies, the mutagen used was colchicine (Vilcherrez-Atoche

et al. 2022). Dendrobium, Cymbidium, and Phalaenopsis were the ﬁrst genera

used for artiﬁcial chromosome duplication in Orchidaceae. Menninger (1963) ﬁrst

performed the induction of a tetraploid of Cymbidium using colchicine, followed by

Griesbach (1981) who exposed protocorms of Phal. equestris, Phal. fasciata, Phal .

“Betty Hauaserman” to colchicine, resulting in the generation of polyploid plants

with an average frequency of 46%. Chaicharoen and Saejew 1981 also successfully

performed artiﬁcial autopolyploidy of Dendrobium phalaenopsis using colchicine.

The procedure for autopolyploidization in orchids using colchicine basically

consists of choosing the tissues to be treated; in orchids protocorms, and PLBs are

the most used, with the concentration (50–500 mg L

-1

colchicine) and explant

exposure time (1–7 days) to the antimitotic agent (Vilcherrez-Atoche et al. 2022).

After this process, tissues are immersed or exposed in a culture medium containing

the antimitotic agent, followed by washing the tissues with deionized water and

transferring the treated explants in a culture medi um without the antimitotic agent, in

order to reduce phytotoxic effects and regenerate polyploidized individuals. Subse-

quently, there is a need to select polyploidized individuals, which has been

performed more frequently using ﬂow cytometry, which is a more practical method

that allows the analysis of a large number of plants in a short time, being more

effective than chromosome counting using microscopy (Vilcherrez-Atoche et al.

2022).

46 J. C. Cardoso et al.

Plant polyploidization results in a change in the architecture of polyploidized

plants, including stem size and diameter, as well as leaf dimensions, shape, and color

(Eng et al. 2021). Polyploid plants of Phal. amabilis var. grandiﬂora showed a

reduction in plant size and an increase in the number of leaves (Mohammadi et al.

2021). Likewise, polyploid plants of Den. nobile showed a decrease in size,

pseudobulb diameter and leaf width/length (Vichiato et al. 2007) in relation to

diploids. In Cym. lowianum, artiﬁcial chromosome duplication generated plants

with slow growth, short stems, and darker and thicker leaves (Xuejiao et al. 2010).

In the reproductive part, such as leaves and inﬂorescences, changes such as increased

size, color intensity, aroma, and durability of ﬂowers are also observed (Sattler et al.

2016; Vichiato et al. 2007). Changes like those described have already been

observed in polyploid plants of Phal. Golden Sands “Canary” showed an increase

in the size of the ﬂowers, in addition to a darker and more intense color (Griesbach

1985). Likewise, polyploid Den. ofﬁcinale plants generated ﬂowers with increased

lip length and gynostemium width (Zhang and Gao 2020).

Most commercial cultivars of Dendrobium, Cymbidium, and Phalaenop sis orig-

inate from interspeciﬁc crosses, in which it has been observed that many of these

hybrids have different levels of infertility due to irregularities during meiosis

(Bolaños-Villegas and Chen 2007; Sattler et al. 2016). Triploid hybrids are those

with the greatest infertility problems (De et al. 2014a, b), limiting their use in orchid

breeding programs. Artiﬁcial polyploidization of these triploid genomes may result

in the restoration of fertility in these hybrids (Sattler et al. 2016). An example of

fertility restoration in orchids was observed in the triploid hybrid Phal. Golden

Sands “Canary”, in which colchicine-treated protocorms generated fertile hexaploid

plants that were later used as progenitor s for the development of new cultivars, such

as Phal. Meadowlark (Griesbach 1985). On the other hand, the development of

triploid plants (3×), from the crossing of polyploidized plants (4×) with diploid

plants (2×), could result in hybrids of interest to companies focused on breeding,

since the infertility of these hybrids could limit its use by other competing compa-

nies’ genetic imp rovement programs.

Breeding of Orchids Using Conventional and Biotechnological Methods:... 47

4.4 Isolation, Culture, Regeneration, and Fusion

of Protoplasts in Orchids

Protoplasts are plant cells free of the cell wall, which can be obtained from different

plant tissues and organs, and which have the biological mechanisms necessary for

the reconstruction of a new cell wall aiming at the regeneration of a complete plant

(Naing et al. 2021).

The ﬁrst stage in the protoplast culture system is the isolation of cells from the

tissue or organ of the donor plant. There are two methods for removing the cell wall

from plant cells, either by mechanical procedures used to obtain small amounts of

protoplasts from larger cells (Davey et al. 2005) and by enzymatic digestion treat-

ments (Davey et al. 2003). Currently, treatments using enzymes are the most used, in

which intrinsic factors speciﬁc to the explant and extrinsic factors are considered

important during the release and acquisition of protoplasts (Giles 2013; Sinha et al.

2003).

There are some efﬁcient protocols for the isolation of protoplasts in different

orchid genera, as observed by Teo and Neumann (1978), in which they used

enzymatic treatment with cellulase (2%), macerosyme (1%), pectinase (0.5%),

and 0.7 M sorbitol for the isolation of protoplasts from protocorms, leaves, plantlets,

and shoots of Renantanda “Rosalind Cheok,” Phalaenopsis, Cattleya, Dendrobium,

and Paphiopedilum, respectively. Price and Earle (1984) also used isolated enzy-

matic treatments with 2% cellulase or in combination with driselase (0.5%) and

macerosyme (1%) and 0.2 M and 0.5 M sorbitol for the isolation of protoplasts in

Angraecum, Brassia, Cattleya, Dendrobium, Odontonia, Paphiopedilum, and

Vanilla.

After the isolation of protoplasts, it is necessary to determine some important

parameters such as density, viability, and yield of the isolated material to increase the

chances of establishing the culture and achieving a high efﬁciency of fusion and

regeneration (Naing et al. 2021). In orchids, protoplast/cell viability and isolated

protoplast density were determined by the ﬂuorescein diacetate and hemocytometer

method, respectively (Yasugi et al. 1986; Shrestha et al. 2007; Ren et al. 2020a, b).

These two parameters analyzed during the isolation of protoplasts and their possible

regeneration can be inﬂuenced by the type and endogenous characteristics of the

explant (Naing et al. 2021). Ren et al. 2020a, b observed in Cymbidium that the

highest yield (~2.50 × 107/g FW), viability (~92.09%) and durability (>70% intact

protoplasts for up to 3 days) of protoplasts were obtained with the use of leaf base

tissues, compared to ﬂower pedicels and young root tips. Explant age can also

inﬂuence protoplast yield (Khentry et al. 2006), in which two-month-old

Dendrobium Sonia “Boom 17” leaves generated a greater number of protoplasts

per fresh weight (g) compared to 1-month-old leaves. In Dendrobium Pompadour, it

was determined that the protoplast size and yield of leaves from plantlets >2.5 cm in

length (31.12 × 10

5

/g FW) were higher compared to leaves smaller than 2.5 cm in

length (28.33 × 10

5

/g FW) (Kanchanapoom et al. 2001). In a Cymbidium hybrid, a

difference in protoplast isolation efﬁciency was found using in vitro (5.2 × 10

4

/

g FW) and ex vitro (4.4 × 10

4

/g FW) leaves (Pindel 2007). Similar results were

reported by Kang et al. (2020).

48 J. C. Cardoso et al.

In orchids, extrinsic factors such as low temperature (5 °C) caused a decrease in

the percentage of isolation of protoplasts/cells after enzymatic treatment of leaves

and petals of Dendrobium Yukidahura “Rainha” (Yasugi 1986).

Once the protoplasts are obtained, they are suspended to obtain an optimal

density that allows theirs in vitro cultivation. In orchids, proto plast density is an

important factor during its cultivation, as observed in protoplasts of Dendrobium

Sonia “Bom 17,” in which a density of 2 × 10

5

protoplasts/mL showed a higher

division rate (20.18%) for the highest density, 5 × 10

5

protoplasts/mL (6.45%)

(Khentry et al. 2006). The authors attributed this lower division rate to excess

protoplasts, which would cause a rapid decrease in nutrients, interfering with cell

wall regeneration and normal protoplast division.

In orchids, there are studies on the cultivation of protoplasts in some genera, such

as Aranda (Kanchanapoom and Tongseedam 1994), Cymbidium (Pindel 2007),

Dendrobium (Khentry et al. 2006; Yasugi 1986; Kunasakdakul and Smitamana

2003), Phalaenopsis (Shrestha et al. 2007; Kobayashi et al. 1993; Ichihashi and

Shigemura 2002), Rhyncholaelia (Mota-Narvaez et al. 2018), and Vanilla (Montero-

Carmona and Jiménez 2015).

Among the factors with the greatest effect on the cultivation and regeneration of

Phalaenopsis and Dendrobium protoplasts are the culture medium, phytoregulators

and the gelling agent. Shrestha et al. (2007) reported that the use of sodium alginate

beads allowed a greater production of compact colonies of cells and that they

presented a high capacity of callus induction and plant regeneration in Phalaenopsis

when compared to the method with gellan gum or standard with agar. Plant regen-

eration from protoplast culture can follow the organogenic or embryogenic pathway.

About 70% of ornamental species follow the organogenic regeneration pathway

(Tomiczak 2020), but in orchids, regeneration via embr yogenesis and the subsequent

formation of PLBs has been more frequently observed (Shrestha et al. 2007;

Kobayashi et al. 1993; Mota-Narvaez et al. 2018; Kunasakdakul and Smitamana

2003).

Isolation and cultivation of protoplasts have different purposes and applications

in modern agriculture. Among these applications, the fusion of protoplasts from

different species, also called somatic hybridization, is a biotechnological tool that

allows the formation of different types of somatic hybrids depending on the degree

of fusion between two protoplasts of different origins (Grosser et al. 2010). This

technique has been used to hybridize species that cannot transmit their genetic

characteristics throu gh conventional breeding techniques via sexual hybridization

(Grosser et al. 2010). The formation of somatic hybrids by protoplast fusion has been

applied in ornamental plants (Naing et al. 2021) and in some orchid genera, such as

Dendrobium (Kanchanapoom et al. 2001; Thomas et al. 2017; Yasugi 1989),

Phalaenopsis (Sumardi and Indrianto 1991), and Vanilla (Montero-Carmona and

Jiménez 2015; Divakaran et al. 2008; Macareno et al. 2016).

There are two mechanisms to carry out the fusion of protoplasts in plants by

physical means—via electrofusion, and by chemical means—via polyethylene

glycol (PEG). Electrofusion allows the fusion of organelles and maintains the

viability and integrity of the protoplast (Davey et al. 2005). Polyethylene glycol is

a high molecular weight reagent that dehydrates and alters cell membranes, increas-

ing their ﬂuidity and afﬁnity between membranes (Begna 2020) and has been the

most used compound for the fusion of orchid protoplasts (Divakaran et al. 2008;

Sumardi and Indrianto 1991; Yasugi 1989). Yasugi (1989) managed to form somatic

hybrids of Dendrobium with Epidendrum, Cattleya, and Paphiopedilum, and

Sumardi and Indrianto (1991) performed the fusion of Dendrobium and Phalaenop-

sis protoplasts using PEG. In the Vanilla genus of orchids, electrofusion was also

used for somatic hybridization, which generated a high number of fusion events

(8.9%) (Montero-Carmona and Jiménez 2015).

Breeding of Orchids Using Conventional and Biotechnological Methods:... 49

4.5 Production of Haploid and Double-Haploid Plants

The production of haploid and do uble-haploid plants is one of the most promising

techniques in breeding programs for allogamous plants, with a high heterozygosity

rate. Obtaining intervarietal hybrids from homozygous lines has supported world

agriculture since the end of the last century, serving as the basis for high yields of

crops such as corn, and more currently in the cultivation of vegetables, from the

cultivation of hybrids originating from the F1 generation of crosses between homo-

zygous lines.

Conventionally, obtaining homozygous lines from heterozygous plants can take

between 7–9 generations of self-fertilization, making the process slow and tedious.

On the other hand, the techniques of in vitro culture of gamete tissues, such as the

culture of anthers or isolated microspores or the culture of eggs or ovaries in vitro are

strategies of great value in different crops and currently support the production of

homozygous strains in different species of agronomic and horticultural interest

(Chaikam et al. 2019; Germanà 2011).

Basically, in a process of obtaining haploid and double-haploid plantlets, gamete

cells from microspores or eggs are induced to enter consecutive cell divisions,

without the need for fertilization. Thus, through the embryogenic pathway, there

would be the formation of embryos and haploid plantlets, from a change from the

gametophytic to the sporophytic pathway. In this regeneration process, there may be

maintenance (haploid) or natural duplication of the haploid genome (double-

haploid), resulting in completely homozygous plants, which can be used to obtain

intervarietal hybrids.

Despite the wide applicability, there are few reports of obtaining haploid and

double-haploid plants in orchids. Kato and Ichihashi (2018) observed the formation

of haploid and double-haploid plantlets in orchids of the genus Bletilla via parthe-

nogenesis, that is, by the use and regeneration of plantlets from unfertilized eggs.

Sporophytic cell division has also been report ed, due to the occurrence of symmet-

rical divisions and multicellular structures from microspores of Dendrobium hybrid

and Spathoglottis orchids (Indrianto et al. 2015).

50 J. C. Cardoso et al.

Despite the few studies in this area, the development of breeding programs based

on obtaining haploid and double-haploid plants, this technology can be of great

value both to expand the diversity of genotypes available for breeding programs and

to produce hybrid seeds from homozygous lines, which could change, simplify, and

cheapen the enti re propagation system for ornamental orchids. This is because using

this technology, seeds of the F1 generation of homozygous lines are used as the main

source of propagation material, instead of complex systems conventionally used on a

large scale and which involve the regeneration and proliferation of somatic tissues

in vitro for cloning orchids (Cardoso et al. 2020).

If, on the one hand, each orchid fruit generates hundreds of thousands or even a

million seeds, and directed pollination is a simple method to be used in orchids, on

the other hand, some difﬁculties can limit the use of this technique, such as high

polyploidy rate of current commercial cultivars, which would result in the regener-

ation of dihaploid (n = 2x, from 2n = 4x) and non-haploid (n = x, from 2n = 2x)

tissues. Thus, for this technology to be properly employed, it requi res the need to

identify cultivars with commercial potential that have not yet undergone

polyploidization, resulting in haploid and double-haploid plant s that can be evalu-

ated as homozygous lines in crosses aiming at obtaining elite hybrids.

5 Orchid Breeding in the Generation of Genome

Sequencing and Editing

5.1 The Transient Gene Expression System in Orchids

Advances in genetic sequencing in some orchid genera (Chao et al. 2017; Hsiao et al.

2021) have allowed for the beginning of work on the identiﬁcation and functional

characterization of genes; this information is essential to understand the complex

mechanisms of development, ﬂowering, adaptation, nutrition, and reproduction in

this plant family (Hsieh et al. 2013, 2020; Su and Hsu 2003; Tan et al. 2005).

The transient gene expression system using protoplasts is a technique that

combines plant tissue culture through the isolation of protoplasts with genetic

transformation mediated by the incorporation of genetic material through a vector

using polyethylene glycol (PEG) or electroporation for the study of biological

activity of different genes or proteins in plant cells (Ren et al. 2021; Davey et al.

2005).

It is a tool that allows for characterizing and studying the behavior, regulation,

interaction, and expression of genes of interest within the plant transcriptome (Lin et

al. 2018). There are studies in the literature on some orchid genera, such as

Cymbidium (Ren, Gao, et al. 2020; Ren et al. 2021; Yang et al. 2021a, b; Ren

et al. 2020a, b), Dendrobium (Li et al. 2021a, b, c) and Phalaenopsis (Li et al. 2018;

Lin et al. 2018), which use the transient gene expression system using protoplasts.

Breeding of Orchids Using Conventional and Biotechnological Methods:... 51

In Cymbidium, the polyethylene glycol (PEG)-mediated transient gene expres-

sion system using leaf-based protoplasts was used to analyze the CsDELLA gene

responsible for gibberellin (GA) regulation for ﬂowering-related genes. Among the

results of this work is the high efﬁciency of protoplast transfection (80%), which

allowed the subcellular localization of the CsDELLA-GFP protein. The analyses

showed that the overexpression of the CsDELLA gene caused a decrease in the

expression of some genes related to ﬂowering and the CsSOC1 and CsFT genes,

while its silencing generated an upregulated expression of these aforementioned

genes. The expression of genes related to ﬂowering promoted by gibberellic acid

(GA) also caused the suppression of the CsDELLA gene (Ren et al. 2021).

Yang et al. (2021a) used protoplasts from young leaves and petals of Cym.

ensifolium to generate overexpression of the Ce-miR396 gene through the transient

gene expression system using polyethylene glycol (PEG). The results showed that

the overexpression of the Ce-miR396 gene generated a decrease in the transcription

of the CeGRF gene in both types of protoplasts, indicating that the Ce-miR396 gene

plays a key role in the development of plant organs and that regulatory differences in

each CeGRF gene are due to different tissue-speciﬁc expression patterns.

In Cymbidium, the transient gene expression system by protoplasts allowed not

only studies related to plant development but also virus–plant interaction studies, as

observed in Ren et al. (2020b), where it was possible to observe in protoplasts that

Cymbidium mosaic virus (CymMV) infection increased the expression of three

proteins (CsNPR1–2, CsPR1–1, and CsPR1–2) and when salicylic acid (SA) was

present, it increased the expression of CsNPR1–2. This SA-dependent protein

CsNPR1–2 response is a defense mechanism that Cymbidium has against CymMV

infection.

The PEG-mediated transient gene expression system also allowed the subcellular

localization (cytoplasm and nucleus) of the DOTFL1 protein in protoplasts of the

leaf mesophyll of Dendrobium “Chao Praya Smile.” The DOTFL1 protein is an

ortholog of TFL1 in Arabidopsis thaliana and is involved in vegetative growth as

well as ﬂoral transition events necessary for reproductive success in Dendrobium

(Li et al. 2021a, b, c).

In Phalaenopsis “Ruili Beauty” and Phalaenopsis aphrot ide subsp. formosa,a

transient gene expression system mediated by PEG was developed using protoplasts

from young leaves and petals, respectively; in which this genetic tool allowed the

subcellular localization of ﬂuorescence-labeled proteins in the cell nucleus and

membrane. Furthermore, the modiﬁed protoplasts served to provide speciﬁc infor-

mation, such as protein–protein interactions, transcription factor activity, and

response mechanisms to plant growth regulators (Li et al. 2018; Lin et al. 2018).

5.2 CRISPR Gene Editing

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9) is a

gene editing technique that has been efﬁciently used in plant genetics (Semiarti

et al. 2020). One of its advantages in relation to genetic transform ation is the fact that

it allows the production of non-transgenic plants, which facilitates their regulation

regarding genetic modiﬁcation. CRISPR can perform gene editing without transfer-

ring exogenous DNA, but silencing endogenous genes to achieve a trait of interest

(Corte et al. 2019).

52 J. C. Cardoso et al.

Advances in genome sequencing in the family Orchidaceae have an impact on the

development of this technique. Although some species of the genera Phalaenopsis,

Dendrobium, Cymbidium, Gastrodia, Bletilla, Platanthera, Vanilla, and Apostasia

have their genome sequenced (Zhang et al. 2022), the CRISPR/Cas 9 technique was

reported only in Phalaenopsis (Nopitasari et al. 2020; Tong et al. 2020) and

Dendrobium (Kui et al. 2017).

In Phalaenopsis amabilis, a successful CRISPR/Cas9 KO system was developed

using protocorms and PDS3 as target genes, in which transformant plants showed

albino phenotype in leaf tissues (Semiarti et al. 2020). A gene editing system was

developed using Agrobacterium-delivered CRISPR/Cas9 carrying the VAR2 gene

into Phal. amabilis protocorms, resulting in variegate patterns in the leaves of

transformant plants (Nopitasari et al. 2020). In Phal. equestris, CRISPR was used

to produce mutants, combined with Agrobacterium-mediated transformation with

MADS genes, which are important for ﬂower development (Tong et al. 2020). In

Dendrobium ofﬁcinale, CRISPR/Cas9 was applied for editing endogenous genes

associated with the lignocellulose biosynthesis pathway (Kui et al. 2017). In

Dendrobium Chao Praya Smile, the DOTFL1 was knockout aiming at rapid

ﬂowering and bulb formation, studying the role of DOTFL1 in plant development

(Li et al. 2021a, b, c).

These results demonstrate that CRISPR gene editing systems are promising for

the molecular breeding of orchids, enabling the transference of exogenous genes and

the deletion of endogenous genes, as well as the development of new cultivars with

characteristics of interest in the family Orchidaceae, regardless of the disadvantages

of conventional breeding techniques.

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Biotechnological Interventions and Societal

Impacts of Some Medicinal Orchids

Kalpataru Dutta Mudoi, Papori Borah, Dipti Gorh, Tanmita Gupta,

Prasanna Sarmah, Suparna Bhattacharjee, Priyanka Roy,

and Siddhartha Proteem Saikia

1 Introduction

Orchids are extremely fascinating plants that surpass all the plant groups in the

“Plant kingdom.” It belongs to the Orchidaceae family, which is the second largest as

well as the highly advanced family among ﬂowering plants. It encompasses approx-

imately 850 genera and 35 thousand species (Stewart and Grifﬁth 1995; Gutierrez

2010). Orchids are better known for their alluring, enchanting attractive ﬂoweret,

which are extremely precious globally in ﬂoricultural trades. Orchids became the

second most top-selling cut ﬂower s as well as potted ﬂoricultural products due to

their increasing demand in the globe for trading. Their aristocratic, adorable, and

wonderful colors, sometimes-intricate forms, have enchanted men and women

through the ages. Orc hids lend a charming beauty with their extraordinary ﬂower

heterogeneity, in terms of size, shape, structure, number, density, color, and fra-

grance. Besides their adorning values, the orchids are also mentioned specially for

their therapeutic medicinal properties as well as economic importance especially in

the traditional pharmacopeias extensively since time immemorial (Withner 1959;

Kaushik 1983). Earlier in China and Japan orchids were used as herbal medicine for

different illnesses nearly 3000–4000 years ago, respectively (Reinikka 1995; Bulpitt

2005; Jalal et al. 2008).

Many species of Vanda, Dendrobium, Habenaria, Malaxis, Cymbidium,

Coelogyne, Cypripedium, Anoctochilus, Bletilla, Calanthe, and Cymbidium, etc.

are signiﬁcantly important for having medicinal importan ce. Medicinal orchid

K. D. Mudoi (✉) · P. Borah · D. Gorh · T. Gupta · P. Sarmah · S. Bhattacharjee · S. P. Saikia

Agrotechnology and Rural Development Division, CSIR-North East Institute of Science and

Technology, Jorhat, Assam, India

P. Roy

Centre for Infectious Disease, CSIR-North East Institute of Science and Technology, Jorhat,

Assam, India

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_3

59

plays an outstanding part in therapeutics with the presence of important phytochem-

icals such as alkaloids, ﬂavonoids, carotenoids, sterols, saponins, anthocyanins, and

polyphenols either in their pseudo bulb, tubers, leaves, stems, ﬂowers, roots, or in the

complete plant (Okamoto et al. 1966; Williams 1979; Majumder and Sen 1991;

Majumder et al. 1996; Zhao et al. 2003; Yang et al. 2006; Singh and Duggal 2009).

Several ailments like arthritis, tumors, fever, malaria, snakebite, scorpion bite,

depression, tuberculosis, cervical carcinoma, diabetes, and biliousness, etc. are

cured by medicinal orchids (Szlachetko 2001). These orchids were also employed

as food and fodder, and local medicine by rural communities for their livelihoods

and revenue generation. Moreover, uprooting the whole plant from its habitat for

sale to the trade rs as well as over-exploitation by rural communities causes the

extinction of many important orchid species (Kala 2004). Other than that native

environment of many orchids is rapidly declining due to hefty desertiﬁcation, habitat

loss, urban sprawl, and usage of land for farming and cultivation. Therefore in

medicinal orchids, it leads to a wide gap between booms and busts.

60 K. D. Mudoi et al.

In recent years, in Western countries, the growing use of herbal medicine and its

demand is increasing. Ultimately, this type of over-exploitation requisites an intense

protection measure. But in situ or ex situ of medicinal orchids conservation in their

natural habitat is not sufﬁcient for propagation a s their rate is low. Orchid seeds are

small, have no endosperm, and require fungal pathogens to germinate; therefore,

germination rates in nature are very low (Arditti 1992). It takes a long time to obtain

the desired number of orchids through asexual reproductio n by rhizo mes, bulbs, or

rooting branches. Hence, it needs proactive mass distribution and re-establishing

them in nature. To meet their growing pressure and to reduce collection pressure on

wild species, biotechnological approaches such as the plant tissue culture technique

has contributed immensely to plantlets production on large scale and developed

different protocols for rapid cloning of desired genotypes using various types of

explants. This technique has come up as a key drive in the production of planting

quality material for commercially and medicinally important orchids to fulﬁll the

increasing demand and to reduce the collection pressure on wild orchids.

Under the above circumstances, biotechnological approaches enhance the in vitro

propagation as well as conservation and mass multiplication of important medicinal

orchids has raised high hopes by adopting asymbiotic seed germination, vegetative

explants materials, artiﬁcial seed technology and secondary metabolites production,

in vitro acclimatization of raised plantlets and their establishment in nature, etc. This

chapter brieﬂy endows the state-of-the-art information mediated on tissue culture

with biotechnological interventions in some medicinal orchids through

micropropagation, along with its societal impacts such as ethnomedicinal properties,

phytochemistry, biological activities, and economics that being the need of the hour.

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 61

2 In Vitro Propagation

To establish a successful propagation of orchids explants type selection is the most

crucial factor. Among the various vegetative explants materials, the leaf has been

utilized as a potent and potential source of explants for the mass multiplication of

orchids. Leaf has the viability for producing a large number of uniform plantlets

from a single leaf or leaf segment through direct embryogenesis or organogenesis.

Knudson (1922) explored the asymbiot ic seed germination in orchids under the

aseptic condition, which was the ﬁrst feasible technique of in vitro propagation that

formed the base of modern biotechnology (Knudson 1922). Later on, Rotor (1949)

developed a method to culture Phalaenopsis using uni-nodal ﬂower stalk cuttings

but all credit goes to George Morel for developing a micropropagation technique for

orchids at a large scale (Rotor 1949). Virus-free Cymbidium clones were obtained

from in vitro shoot meristem culture (Morel 1960). Later on, Morel (1964) reported

that it was possible to produce million of plantlets within a year using a single bud by

frequent sub-culturing of protocorm-like bodies (PLBs) that motivated the orchid

growers (Morel 1964). The present-day micropropagation in both basic and practical

aspects is much more organized than it was in the beginning. Though shoot-tips have

remained the most commonly used explants for propagating orchids, the regenera-

tion potential of other explants like axillary buds, stem discs, inﬂorescence seg-

ments, ﬂoral stalks, leaves, leaf peels, perennating organs (pseudobulbs, rhizomes,

tubers), and roots has also been utilized successfully (Vij et al. 2004; Arditti 2008).

2.1 Seed Germination

To produce ﬁrm seeds and ﬂowers, it takes 5–10 years for an orchid plant. Orchid

seeds are one of the most distinctive features of the Orchidaceae family. They are

tiny, very small, and powdered, and are produced in large quantities, with

1300–4000,000 seeds per capsule (Harley 1951; Arditti 1961). Very fragile, rela-

tively undifferentiated, and without endosperms or cotyledons, seeds are produced

from the majority of orchid species (Mitra 1971).

Due to a lack of metabolic machinery and functional endosperm, the natural

germination rate of orchid seeds is very poor. Only 0.2–0.3% germinates in natural

conditions (Prasad and Mitra 1975). It is well known that the seeds of almost all

orchids are entrusted to mycorrhizal fungi for germination in natural conditions.

Symbiotic fungi have been extensively exhibited to induce seed germination in both

terrestrial and epiphytic orchids for seedling development. But, asymbiotic seed

germination has imparted a systematic way for the mass multiplication of orchids

(Chen et al. 2022).

62 K. D. Mudoi et al.

2.1.1 Asymbiotic Seed Germination

The ability of orchid seeds to germinate asymbiotically by in vitro means was

demonstrated for the ﬁrst time by Knudson in Cattleya species (Knudson 1922).

Asymbiotic in vitro seed germination of orchids occurred by culturing immature

ovules often known as either embryo, fruit, or pod (Fig. 1a–d). The germ ination

potential of immature embryos was much better than that of mature ones and varied

with their developmental stages. Due to pH, dormancy, and other metabolic factors,

very young orchid oocytes cannot germinate and thus cannot form suitable explants

(Withner 1953). During in vitro seed germination of orchids, the intermediate

protocorm stage is followed by subsequent seedling development (Fig. 1e–f). A

protocorm is a chlorophyll-like, hairy, and pear-like bulbous or oblong structure that

originates from the apical or lateral suture of the seed coat and provides nutrients like

cotyledons during embryonic development and subsequent seedling growth (Lee

1987). Protocorms have been inconsistently assessed as uniform callus structures or

distinct shoots (Kanase et al. 1993). The protocorm-like body speciﬁed the orchids

for the regeneration of multiple plantlets which is a blessing to the world ﬂoricultural

market (Fig. 1g–j).

Asymbiotic seed germination of orchids was exploited for in vitro mass produc-

tion of orchids with commercial and medicinal importance for conservation and

ecorestoration. It was reported by several investigators from time to time.

Half strength of Murashige and Skoog (MS) medium (Murashige and Skoog

1962) were used for seed germination of Bletia purpurea (Dutra et al. 2008),

Coelogyne stricta (Parmar and Pant 2016), Cymbidium giganteum (Hossain et al.

2010), Cymbidium goeringii (Gong et al. 2018), and Spathoglottis plicata (Aswathi

et al. 2017; Hossain and Dey 2013). Accordingly, Cymbidium aloifolium was

germinated in 1.0 mg/L 6-benzylaminopurine (BAP) and 0.5 mg/L

α-naphthaleneacetic acid (NAA) supplemented (Paul et al. 2019). However, a

modiﬁed half-strength MS medium was tested for in vitro germination of

Dendrobium ovatum (Shetty et al. 2015).

Six different media compositions for testing were examined for their effective-

ness towards the growth of Dactylorhiza hatagirea (Warghat et al. 2014) and Bletia

purpurea seeds in BM-1 (Van Waes and Debergh 1986); 1/2 MS, Vacin and Went

modiﬁed (VW) medium (Vacin and Went 1949); Malmgren modiﬁed terrestrial

orchid medium (MM) (Malmgren 1996) and Knudson C (KC) medium (Knudson

1946). Dendrobium macrostachyum seeds were accomplished on MS, VW, and KC

medium having different accumulation, amalgamation of growth hormones, and

other additives. Among them, VW basal medium tested with 0.5 mg/L BAP and

5 mg/L NAA was more acceptable for plantlet formation (Li et al. 2018).

Dactylorhiza hatagirea was cultured on Heller and Lindemann (LD) medium

(Warghat et al. 2014), MM, VW, MS, and KC media. Both MS and KC medium

were examined for asymbiotic seed germination of Eria bambusifolia (Basker and

Bai 2010). MS, KC, and KC-modiﬁed Morel medium were used for Satyrium

nepalense (Mahendran and Bai 2009) seed germination. Seeds from mature capsules

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 63

Fig. 1 In vitro micropropagation of Cymbidium aloifolium, (a) Mother plant, (b) Seed capsule, (c) In vitro seed germination, (d) Swelling of seeds, (e) PLBs

formation, (f) Enlargement of PLBs, (g) Shoot formation, (h) Formation of shoot and root, (i) Shoot elongation, (j) Shoot multiplication, (k) Hardening and

acclimatization, (l, m) Acclimatized plantlets ready for ecorestoration

of Dendrobium trigonopus were augmented in B

5

, MS, and 1/2 MS with NAA,

BAP, and bark powder for in vitro germination (Pan and Ao 2014). MS + 1.0 mg/L

BAP + Phytamax™ were provided for seed germination of Dendrobium aphyllum

(Hossain et al. 2013).

64 K. D. Mudoi et al.

Vacin and Went (1949) medium was alone tested for seed germination of

Dendrobium parishii (Vacin and Went 1949; Kaewduangta and Reamkatog 2011).

Likely, on VW medium mature seeds of Dendrobium lasianthera were enhanced

with the incorporation of different concentrations of peptone of 1, 2, and 3 gm/L

(Utami et al. 2017). Mature seeds of Cypripedium macranthos were sown on

hyponex-peptone (HP) medium that contained 1 μM NAA and BAP after steriliza-

tion (Shimura and Koda 2004). Mature capsules of Ansellia africana were tested on

Vasudevan and Van Staden (2010) medium for seed germination in vitro

(Vasudevan and Van Staden 2010; Bhattacharyya et al. 2017a). However, in vitro

germination of Dendrobium nobile Lindl. (Bhattacharyya et al. 2014),

D. thyrsiﬂorum (Bhattacharyya et al. 2015), D. heterocarpum (Longchar and Deb

2022), Cymbidium iridioides (Pant and Swar 2011), C. kanran (Shimasaki and

Uemoto 1990), Cypripedium debile (Hsu and Lee 2021), and C. macranthos

(Shimura and Koda 2004) was reported in MS medium of full strength. Cymbidium

iridioides young pods were cultured on MS medium containing 1 mg/L of NAA and

BAP (Longchar and Deb 2022). Immature seeds of Cymbidium kanran were inoc-

ulated on MS medium for shoot multiplication (Shimasaki and Uemoto 1990).

Young pods of Cymbidium iridioides were cultured on MS medium having NAA

(1 mg/L) + BAP (1 mg/L) for micropropagation (Pant and Swar 2011).

2.2 Micropropagation of Orchids Via Vegetative Explants

Materials

In orchids, as a result of out crossing, heterozygous offspring were produced from

seeds. Therefore, it is necessary to augment various vegetative parts of mature plants

to validate micropropagation protocols in orchids. Georges Morel was the pioneer

for culturing Cymbidium shoot tips and attained protocorm-like bodies (PLBs) from

contaminated plants to regenerate mosaic virus-free plants (Morel 1960). He intro-

duced the term “protobulb (PLB)” in his work published in the Bulletin of the

American Orchid Society (Arditti 2010). At the same time, a number of orchid

species have yielded fruitful results, including Lycaste, Cattleya, Ondontoglossum,

Dendrobium, Phaius, Miltonia, and Vanda (Arditti and Ernst 1993).

Large-scale propagation of medicinal orchids through in vitro method, different

vegetative explants sources such as shoot tip, axillary bud, leaves, nodal segments,

and inﬂorescence were augmented through callus formation or PLB mediation or

direct shoot bud formation as described below:

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 65

2.2.1 Shoot Tip Culture

To induce efﬁcient clonal propagation of medicinal orchids, shoot tips have been

efﬁciently cultured. It was ﬁrst implemented in Cymbidium by Morel (Morel 1960).

This technique enables the rapid propagation of Vanda coerulea (Seeni and Latha

2000). Response of bud formation is obtained from the shoot tips in vitro and mature

plants in a medium having 8.8 μM BAP and 4.1 μM NAA. For forming multiple

shoots in Vanda tessella te BAP and NAA combination was found to be more

effectual as compared to indole-3-acetic acid (IAA), NAA, and kinetin at single

action (Rahman et al. 2009). Shoot primordium of Doritis pulcherrima was cultured

for rapid propagation and regeneration of plantlets (Mondal et al. 2013). In VW

medium, Dendrobium shoot tip was cultured containing 15% coconut water plus

10 ppm NAA for a rapid proliferation of PLB and plantlet formation as well as the

growth of seedlings (Soediono 1983). Sixty days old Dendrobium chrysotoxum

shoot tips was inoculated on MS + 0.1 mg/L NAA + 3% sucrose + 0.5 mg/L BAP

for proliferation, shoot induction (Gantait et al. 2009).

2.2.2 Nodal/Internodal Culture

Dendrobium ﬁmbriatum segments were conferred for shoot induction, and prolifer-

ation in MS + 0.2–0.5 mg/L NAA + 1.0–4.0 mg/L BAP (Huang et al. 2008). But MS

medium with NAA and BAP at 17.76 μM recorded maximal regeneration

(14.0 ± 0.47) of shoots (Paul et al. 2017). Stem nodes of Dendrobium devonianum

cultured at MS + 0.01–0.5 mg/L NAA + 1.0–4.0 mg/L BAP for PLB and shoot

induction and proliferation in vitro (Li et al. 2011, 2013a). 0.5–1.0 cm nodal

segments excised with axillary buds from 4–5-month-old Dendrobium chrysanthum

seedlings grown in vitro, half strength MS + 0.1 mg/L NAA + 6 mg/L BAP + 3%

sucrose + 0.65% agar (Mohanty et al. 2013a).

Nodal explants of Malaxis acuminata were cultured on MS + sucrose (3%

w/v) + 3 μM NAA + 3 μM BAP and resulted in well-developed plantlets with

shoots and root growth (Arenmongla and Deb 2012). Young healthy nodal shoot

segments from the newly grown branches of wild Bulbophyllum odoratissimum

were taken and cultured on BAP (4.0 mg/L) and IBA (0.5 mg/L) fortiﬁed MS

medium for producing maximum shoot proliferation (Prasad et al. 2021). Nodal

cultures of Ansellia africana were tested in an MS medium supplemented with 5 μM

NAA and 10 μM of meta-topolin (mT) for multiple shoot induction (Bhattacharyya

et al. 2017a). Pseudo-stem segments of Dendrobium nobile with nodes (0.5–1 cm)

was used as explants for induction of PLBs with varied concentration of thidiazuron

(TDZ) for culture (Bhattacharyya et al. 2014). Malaxis acuminata internode cultures

responded to MS + 0.5 mg/L NAA + 3 mg/L TDZ; MS + 0.5 mg/L NAA + 3 mg/L

TDZ + 0.4 mM spermidine (spd); MS + 1.5 mg/L activated carbon (AC) + 4 mg/L

IBA was used for shoot induction (Cheruvathur et al. 2010).

66 K. D. Mudoi et al.

2.2.3 Leaf Culture

Leaves and leaf tips of young orchids were cultured in vitro for PLB initiation and

shoot proliferation. Wimber (1965) showed the potential of Cymbidium leaves

(Wimber 1965). Growth stimulation in the nutrient pool, donor axis location, and

physiological age of the mother plant strongly determine the regeneration potential

(Trunjaruen and Taratima 2018). Therefore, factors like growth hormones, medium

nutrients composition, leaf part, leaf source (in vivo/in vitro), explants preparation,

leaf maturity, etc. determine the efﬁciency of a leaf explants micropropagation

protocol (Chugh et al. 2009).

The leaf base of Vandaceous orchids evinced greater proliferative potential than

leaf tips (Na and Knodo 1995; Jena et al. 2013; Seeni and Latha 1992; Nayak et al.

1997). Younger leaves perform better than older leaves. Lea ves of mature Vanda

coerulea did not respond to bud formation or PLB in vitro (Seeni and Latha 2000).

Whereas, mature plants of V. spath ulata (L.) Spreng the regeneration potential of

leaf explants was noticed with 28.5 μM IAA + 66.6 μM BAP medium (Mitra et al.

1976).

2.2.4 Axillary Bud Culture

Axillary bud culture also played a very important role in medicinal orchid

micropropagation. Cymbidium elegans’s axillary buds were responsive to PLBs

formation (Pant and Pradhan 2010). Axillary bud culture of Dendrobium longicornu

was tested in MS medium with 0.8% agar + 3% sucrose + 5 μM NAA and 15 μM

BAP (Dohling et al. 2012). In Cypripedium formosanum a quarter concentration of

MS medium containing 22.2 or 44.4 mM BAP was sufﬁcient to propagate 6.3 and

7.1 shoots per explant with an average length of 10.6–11.7 mm to produce cultures

after 90 days (Lee 2010). Five species of Dendrobium (D. crumenatum,

D. ﬁmbriatum, D. moschatum, D. nobile, and D. parishii) induced multiple shoots

when axillary buds were cultured in vitro (Sobhana and Rajeevan 1993). Field-

grown axillary buds of Lycaste hybrids were grown in half-strength MS basal

medium supplemented with 0.5 mg/L BAP and 1.0 mg/L TDZ and 2% (w/v) sucrose

(Huang and Chung 2011). Six to seven millimeter long shoot tips of Aranda

Deborah hybrids grown in VW medium supplemented with coconut water (20%

v/v) produced an average of 2.7 PLB after 45 days (Lakshmanan et al. 1995).

2.2.5 Pseudobulb Culture

The pseudobulb of Coelogyne cristata was cultured with basal medium + BAP

(1–10 mg/L) + kinetin (1–10 mg/L) alone and in combination with NAA (1–10 mg/

L). In parallel sets of experiments, 0.2% AC was used in the medium for shoot

multiplication (Sharma 2021); 6-BAP (2.0 mg/L) + NAA (0.5 mg/L) induced shoot

proliferation in C. ﬂaccid (Parmar and Pant 2016). The pseudobulb of Malaxis

acuminata was cultured on MS + 1.0 mg/L BAP + 1.0 mg/L NAA + 2.0 g/L AC

for PLB formation (Suyal et al. 2020).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 67

2.2.6 Flower Bud Culture

Ascoﬁnetia, Neostylis, and Vascostylis were the ﬁrst species to culture the young

ﬂower buds or inﬂorescence for medicinal orchid micropropagation (Intuwong and

Sagawa 1973). Similarly, Phalaenopsis, Phragmipedium, and Cymbidium were also

cultured equivalently (Kim and Kako 1984). The ﬂoral buds were exposed to either

higher auxin levels or higher cytokinin levels and anti-auxin levels (Zimmer and

Pieper 1977; Tanaka and Sakanishi 1978; Reisinger et al. 1976). Younger ﬂoral buds

or inﬂorescence were more responsible than the matured ones in terms of shoot or

PLB proliferation in Oncidium Gower Ramsey, Phalaenopsis capitola, Dendrobium

Miss Hawaii, Ascoﬁnetia (Intuwong and Sagawa 1973; Mitsukuri et al. 2009;

Nuraini and Shaib 1992).

2.2.7 Root and Rhizome Segment Culture

The in vitro root culture was so far attempted with success in a few species of

medicinal orchids. The capacity of orchid root to induce shoot regeneration was very

low as reported earlier (Kerbauy 1984). Thereafter roots of Catasetum,

Cyrtopodium, and Rhyncostylis were utilized to regenerate plantlets a very high

proliferation rates (Kerbauy 1984; Sanchez 1988; Sood and Vij 1986). Root tips

excised from Vanda hybrids and Rhyncostylis were cultured in 1.0 mg/L IAA,

1.0 mg/L BAP and 200 mg/L of casein hydrolysate for a speedy shoot proliferation

rate (Chaturvedi and Sharma 1986). Rhizome of Cymbidium goeringii responded to

MS + 0.2% (w/v) AC, 3% (w/v) sucrose, 0.2% (v/v) coconut water, and 0.8% (w/v)

agar powder (Park et al. 2018). Moreover, auxin, particularly NAA was responsible

for stimulating rhizome formation of some medicinal orchids and ultimately new

shoots were developed from a rhizome in a cytokinin-enriched medium of C. kanran

Makino (Shimasaki and Uemoto 1990), C. forrestii (Paek and Yeung 1991), and

Geodorum densiﬂorum (Roy and Banerjee 2002).

Rhizome tips were also tested for PLB formation and shoot development (Udea

and Torikata 1972). In a few cases, cytokinins were inductive for stimulation of

shoots from rhizome segments of medicinal orchids such as Cymbidium forrestii

(Paek and Yeung 1991) and Geodorum densiﬂorum (Lam.) Schltr. (Roy and

Banerjee 2002; Sheelavantmath et al. 2000). Sometimes BAP was responsible for

the reduction of rhizome growth and branching but induced certain rhizome tips

gradually into shoot s (Paek and Yeung 1991).

68 K. D. Mudoi et al.

2.2.8 Thin Cell Layer Culture

Longitudinal or transverse sections of the thin cell layers are isolated from different

plant parts such as leaves, ﬂoral primordia, stems, or PLBs. The efﬁciency of normal

plant tissue culture and thin cell layer culture techniques is compared very method-

ically (Rout et al. 2006). In vitro raised seedlings of Dendrobium chryso toxum,

cross-section (2 mm thickness) of stem-nodes is grown in MS medium (semi-solid

and liquid) supplemented with BAP 4.44 μM and Kinetin 4.65 μM induced shoot

buds (Kaur 2017).

2.2.9 Protoplasts Culture

Different explants of orchids like stem, root, leaf disc, petal, and protocorm were

used for the isolation of protoplasts. Chris K. H. Teo (Malaysian scientist) and

K. Neumann (German botanist) ﬁrst introduced the induction and synthesis of orchid

protoplasts (Teo and Neumann 1978a, b). Since then studies were carried out in this

ﬁeld for the isolation of orchid protoplasts. However, during the screening of more

than 24 orchid species, from bases of juvenile leaves of medicinal orchid Cymbidium

aloifolium protoplast culture was achieved (Seeni and Abraham 1986).

2.3 Root Induction

Concentrations of different auxins were incorporated into basal media either singu-

larly or in combination for testing their root-promoting efﬁciency in medicinal

orchids. For root induction of Dendrobium ﬁmbriatum with 100% rooting fre-

quency, MS + 0.5 mg/L NAA or 0.3–1.0 mg/L IBA and a combination of 0.5 mg/

L IBA and NAA were used (Huang et al. 2008). IBA, IAA, and phenolic elicitor PG

containing MS medium were responsible for root induction of Ansellia africana

within 6 weeks interval (Bhattacharyya et al. 2017a). IBA was responsible for root

promotion of medicinal orchids viz., 1.0 mg L/1 IBA in Acampe praemorsa (Nayak

et al. 1997) and Cymbidium iridioides (Pant and Swar 2011), and 1.5 mg L/1 IBA in

Dendrobium densiﬂorum (Pradhan et al. 2013).

A decline in root number and length was reported with increased concentration of

IBA. In Dendrobium nobile, IBA was better than NAA in maximizing root numbers

(Asghar et al. 2011). MS + 3% sucrose + 2 g/L AC + 0.2 mg/L IBA was used in

Dendrobium chrysotoxum (Gantait et al. 2009). Whereas, in the root formation of

Vanilla planifolia and Geodorum densiﬂorum, NAA exhibited a conducive effect

(Sheelavantmath et al. 2000; Tan et al. 2011).

In Dendrobium transparens (Sunitibala and Kishor 2009) and Dendrobium

primulinum (Pant and Thapa 2012) supplementation of IAA increased the rate of

root proliferation whereas its affectivity was poor during root formation. However,

rooting of Vanda spathulata shoots was observed within 3–9 weeks in a medium

containing 75 g/L banana pulp and 5.7 μm IAA. In vitro shoots of 2–5 cm in length

developed two to ﬁve roots easily in pots at 80–90% survival rates instead of

hardening (Decruse et al. 2003).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 69

2.4 Photoperiodic Condition

In vitro seed culture and micropropagation of medicinal orchids were inﬂuenced by

ambiance conditions, like photoperiod (P P) for efﬁcient early culture development.

Cool white light, 16/8-h PP, 1000 lux light intensity, 25 ± 2 ° C, and pH 5.2 have

been reported for Dendrobiu m moschatum (Kanjilal et al. 1999). Fluorescent light,

12/12-h PP, 60 μL mol m

-2

s

-1

,25 ± 2 °C was provided in D. parishii

(Kaewduangta and Reamkatog 2011). D. trigonopus was probably supplemented

with 14/12-h PP, 25 ± 2 °C, 50 μL mol m

-2

s

-1

(Pan and Ao 2014). In D. aphyllum

provide 14/12-h PP, 60 μL mol m

-2

s

-1

, cool white ﬂuorescent, 25 ± 2 °C (Hossain

et al. 2013). 1000–1500 lux, 12/12-h PP, white ﬂuorescent tube, 25 ± 1 °C extended

to D. candidum (Zhao et al. 2008). 50 μL mol m

-2

s

-1

, 12/12-h PP, 25 ± 2 °C was

furnished in D. chrysanthum (Mohanty et al. 2013a). In D. chrysotoxum 16/8-h PP,

30 μL mol m

-2

s

-1

, white ﬂuorescent tube, 60% RH, 25 ± 2 °C was supplied

(Gantait et al. 2009). Originally, 25 ± 2 °C in the dark for 2 weeks, 23 μL mol m

-

2

s

-1

25 ± 2 ° C, 16/8-h PP, (callus + PLB) was described in D. crumenatum

(Kaewubon et al. 2015). 350–500 lux 16/8-h PP, 25 ± 2 °C was supplied in

D. densiﬂorum (Pradhan et al. 2013). 1500–2000 lux, 12/12-h PP, 25 ± 2 °C and

pH 6.0 was suitable for D. devonianum (Li et al. 2011, 2013a). Cool white ﬂuores-

cent tubes, 12/12-h PP, 40 μL mol m

-2

s

-1

,25 ± 2 °C were used in D. draconis

(Rangsayatorn 2009). 2000 lux, 12/12-h PP, 25 °C and pH 5.4–5.6 was reported in

D. ﬁmbriatum (Huang et al. 2008). Cultures of Ansellia africana were maintained in

cool white ﬂuorescent tubes in a culture room with a light intensity of 40 μ mol m

-

2

s

-1

at 25 ± 2 °C under a dark and light cycle of 12 h (Bhattacharyya et al. 2017a).

D. ﬁmbriatum was cultured under a photoperiod of 14 h with a light intensity of

50 μ mol m

-2

s

-1

using cool-ﬂuorescent tube lights, at 25 ± 2 °C (Paul et al. 2017).

2.5 Hardening and Acclimatization

Hardening and acclimation of in vitro cultured plantlets are important steps of

micropropagation for better survival and successful plant establishment under ex

vitro conditions. The percentage of plant loss or damage is higher during the transfer

of in vitro growing plants to ex vitro conditions. Regenerates have to adapt to many

abnormal conditions such as high irradiance, low humidity, and water hydraulic

conductivity of the root and root- stem connections in an ex vitro environment (Fila

et al. 1998). Acclimatization of regenerates with gradually reducing humidity will

overcome this threat (Bolar et al. 1998).

70 K. D. Mudoi et al.

Well-rooted micropropagated orchid plantlets were ready for acclimatization after

attaining sufﬁcient growth in terms of root or shoot length. After removal from

ﬂasks, the well-rooted plants were cleaned thoroughly to remove the remnant of

artiﬁcial media such as sucrose and nutrient agar. Thereafter, clean plantlets were

soaked in an effective fungicide solution before shifting them into pots or poly

sleeves having a potting mixture. The blending of various potting mixtures plays an

important part in the survivability of orchid plantlets raised in vitro. A combination

of the potting mixture was pounded of dried coconut husk or coco peat, tiny pieces of

tree cortex, peat moss or sphagnum moss, and pieces of broken bricks or charcoals in

various ratios. The ideal potting mixture should have water retaining capacity along

with draining out of extra water and aeration for proper hardening and acclimatiza-

tion of plants (Diaz et al. 2010; Kang et al. 2020) (Fig. 1k–m).

Brick pieces and charcoal chunks (1:1) mixture were fruitful for acclimatization

of Dendrobium chrysanthum with a topmost cover of moss (Mohanty et al. 2013a).

Plantlets of Dendrobium moschatum were shifted for hardening to a blending of

charcoal, brick, coal, sand, and soil (1:1:1:1:1) with 48% survivability (Kanji lal et al.

1999). Rooted shoots of Dendrobium macrostachyum were provided with a perlite

and peat moss mixture and kept in the green house for acclimatization (Li et al.

2018). In the mixture of coco peat, litter, and clay in the ratio of 2:1:1 with a covering

of sphagnum moss Cymbidium aloifolium plantlets were acclimatized with an 85%

survival rate (Pradhan et al. 2013). Acclimatization was carried out for hardening

plantlets of Dendrobium draconis and shifted to cocopeat and perlite (1:1) compo-

sition with 92% achievement (Rangsayatorn 2009). In Coelogyne cristata, the

composition of pine bark, brick, moss, and charcoal pieces (1:1:1:1) was used for

transplanting (Sharma 2021). In Coelogyne ﬁnlaysonianum, brick, charcoal, coco

peat and litter (1:1:1:1); brick, charcoal, litter, and saw dust (1:1:1:1); brick, char-

coal, and litter (1:1:1); and brick and charcoal (1:1) were utilized for survival (Islam

et al. 2015). A mixture of humus and sand (1:1) was tested in Changnienia amoena

(Jiang et al. 2011). A composition of brick, charcoal, coconut husk, and sand (1:1:1:

1) was provided for acclimatization of Spathoglottis plicata (Grell et al. 1988). In

Cymbidium iridioides, plantlets were acclimatized by using cocopeat, peat moss, and

brick (Pant and Swar 2011). In the ratio of 1:1:1 substrate of brick, charcoal,

shredded bark, and a moss cover were imparted for the survivability of Dendrobium

longicornu in a greenhouse (Jaime et al. 2015). Eria bambusifolia was tested on

coconut husk, charcoal, brick pieces, broken tiles, and perlite (Basker and Bai 2010).

Hardening plantlets of Satyrium nepalense were transferred to a 1:1:1 ratio of a

mixture containing vermicompost, sand, and coconut husk in plastic pots

(Mahendran and Bai 2009). Rhynchostylis retusa was adapted in small plastic pots

containing (2:1) moss and bark (Naing et al. 2010). Cypripedium macranthos was

hardened in a plastic bag that contain wet vermiculite and acclimatized in a soil

mixture of coarse volcano ash and clay granules (Shimura and Koda 2004).

Dactylorhiza hatagirea was survived in a potting mixture consisting of (1:1:1)

cocopeat, vermiculite, and perlite (Warghat et al. 2014). Rooted plantlets of

Dendrobium lasianthera were planted in a composition of coconut husk and sphag-

num moss (3:1) and achieved a 90% survivability rate (Utami et al. 2017). In vitro

rooted Ansellia africana plantlets were tested with a mixture of vermiculite, sand,

and decaying litter (1:1:1) and found 87% survivability after 60 days (Bhattacharyya

et al. 2017a). Dendrobium nobile plantlets were acclimatized with various compo-

sitions of mixture viz., (1) charcoal and bricks in the ratio 1:1; (2) in the ratio 1:1 of

decaying litter and brick; (3) in the ratio 1:1:1 of brick chips, leaf litter, and charcoal;

and (4) brick chips, leaf litter, and charcoal in the ratio 1:1:1 in addition to the

topmost coating of moss. Among various compositions brick, charcoal, and

decaying litter treatment as well as moss covering received the highest 84.3%

survivability (Bhattacharyya et al. 2014). Composition of (a) brick and charcoal

(1:1) (b) brick and coco peat in the ratio 1:1 (c) coco peat, brick, charcoal pieces in

the ratio 1:1:1; and (d) leaf mold, brick chips, and cocopeat in the ratio 1:1:1 were

supplied for transplantation of Bulbophyllum odoratissimum in Green house condi-

tion with 90% relative humidity (RH) and 91.66% survival rate. Among the different

treatments, brick chips, charcoal, and coco peat (1:1:1) containing the mixture was

best for high water retention as well as good aeration capacity (Prasad et al. 2021).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 71

3 Ecorestoration

Ecosphere restoration is the “task reconstructing of an ecosystem that has been

damaged due to manmade catastrophe” (Libini et al. 2008). The main objective of

restoration is to re-establish the environmental system that is disturbed by various

factors with respect to its structure and functional properties.

After successful acclimatization, in vitro-raised Vanda coerulea plantlets were

transferred to tree trunks of forest segments, for successful ex situ harbor by using

the binding medium like moss and coconut husk with 70–80% survivability rate for

ecorestoration. Such a study commencing in India for restoring the natural habitat is

of great interest from a horticultural and conservation point of view (Seeni and Latha

2000). Similarly, Epidendrum ilense and Bletia urbana were also shifted to the forest

ecosystem or typical natural habitat for ecorestoration (Christenson 1989; Rublo

et al. 1989). During the lab to land transfer strategy, it was observed that host trees

with rough bark were selected and the in vitro-raised orchids were ﬁxed either to the

tree trunks with the roots or tree bark for ecorestoration efforts (Decruse et al. 2003;

Aggarwal and Zettler 2010; Aggarwal et al. 2012; Gangaprasad et al. 1999; Grell

et al. 1988; Kaur et al. 2017). Micropropagated plantlets of Smithsonia maculate

showed 48% survival after one year reinforced at Karamana river of Peppara

Wildlife Sanctuary, Kerala, India. The pilot trial on restoration through

micropropagation was useful for further reintroduction and population enhancement

for the practical conservation of this orchid (Decruse and Gangaprasad 2018). In

vitro rooted plantlets of Vanda spathulata were observed with a 50–70% survival

rate, which were introduced into forest segments at Ponmudi and Palo de in the

Southern Western Ghats of India (Decruse et al. 2003).

72 K. D. Mudoi et al.

The reintroduction trials of orchid plantlets should be conducted with well-

established in vitro-rooted plantlets during the monsoon period to corroborate the

maximum survival rate of the plantlets for ecorestoration or eco-rehabilitation study.

4 Artiﬁcial Seed Technology

The concept of artiﬁcial or synthetic seed was ﬁrst coined by Murashige and at

present it is well known by some different names such as manufactured seed,

synthetic seed, or synseed (Murashige 1977). Artiﬁcial seeds were originally deﬁned

as “encapsulated single somatic embryos” by Murashige (1978), i.e., a clonal

product that can grow into plantlets at in vitro or ex vivo conditions if used as real

seeds for sowing, storage, and transport (Murashige 1978). Gray and Purohit (1991)

also deﬁne somatic embryos with practical usage in commercial plant production

(Gray et al. 1991). Therefore, the production of synthetic seeds has previously been

restricted to those plants where somatic embryogenesis has been reported. Although

somatic embryogenesis is restricted to selective plant species, to overcome this

limitation, exploration of a suitable alternative to somatic embryos, i.e.,

non-embryogenic vegetative propagules like shoot tips, segmental/axillary buds,

protocorm-like bodies (PLBs), organs or embryogenic callus is practiced (Ahmad

and Anis 2010; Ara et al. 2000; Danso and Ford-Llyod 2003).

However, artiﬁcial/synthetic seeds or beads production was reported ﬁrst time by

Kitto and Janick (Kitto and Janick 1985). Since then, several ﬂowering plant species

have extensively utilized this technique including orchids. Production of synthetic

seeds opens a new vista in plant tissue culture technology by adding many fruitful

improvements on a commercial scale. Artiﬁcial seeds were utilized for transforma-

tion into plantlets under in vitro and in vivo circumstances. It was applied for the

multiplication of rare, threatened, and endangered plant species which are hard to

propagate by normal propagation process and by natural seeds.

Synthetic seed production in orchids is especially important as they produce

minute non-endosperm seeds. Corrie and Tandon (1993) have used protocorms to

produce synthetic seeds of Cymbidium giganteum which are transferred to a nutrient

medium or sterile sand and soil medium developed healthy seedlings (Corrie and

Tandon 1993). Comparable conversion frequencies of 100%, 88%, and 64% were

obtained on in vitro, sand, and sand-soil mixture condition, respectively. These

observations enable the direct transplantation of aseptically grown protocorms into

the soil as well as reduce the cost of growing plantlets in vitro and subsequent

acclimatization. As orchids produce tiny and non-endospermic seeds, the production

of artiﬁcial seeds was beneﬁcial.

Several reports on encapsulation using somatic embryos have been carried out

(Ara et al. 2000; Danso and Ford-Llyod 2003; Castillo et al. 1998; Ganapati et al.

1992). For synthetic seed production, meristematic shoot tips or axillary buds were

also utilized in orchids along with somatic embryos or PLBs (Ganapati et al. 1992;

Bapat et al. 1987; Piccioni and Standardi 1995). Encapsulation of PLBs is well

reported in many orchids such as Cymbidium giganteum, Dendrobium wardianum,

Dendrobium densiﬂorum, Phaius tonkervillae, and Spathoglottis plicata (Danso and

Ford-Llyod 2003; Sa iprasad and Polisetty 2003; Vij et al. 2001).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 73

In Dendrobium orchid, Saiprasad and Polisetty found that fractionated PLB was

best suited for encapsulation at leaf primordia stage 13–15 days after culture

(Saiprasad and Polisetty 2003). Encapsulation matrices prepared with MS medium

(3/4 strength) + 0.44 μMB BAP + 0.54 μM NAA result in 100% conversion of

encapsulated PLBs when cultured on MS medium + 0.44 μMB BAP + 0.54 μM

NAA (Dendrobium). Sarmah et al. (Sarmah et al. 2010) production of synthetic

seeds in an endangered monopod orchi d, i.e., Vanda coerulea by leaf-based encap-

sulating PLBs with 94.9% conversion frequency on immediate inoculation in

Ichihashi and Yamashita (IY) medium (Ichihashi and Yamashita 1977). 95% con-

version was achieved on encapsulating PLB of Flickingeria nodosa in Burgeff

medium (Withner 1955) + 2% sucrose + 2 mg/L Adenine sulfate + 1 mg/L IAA at

4 °C for 3 months (Nagananda et al. 2011). Alginate encapsulation of Aranda ×

Vanda PLB was also reported (Gantait et al. 2012). Three percent sodium alginate

and 75 mM calcium chloride support better encapsulation of individual PLBs (4 mm

long). Plant growth regulator (PGR)-free MS medium (1/2 strength) reported 96.4%

of conversion. Likely, short-term storage of PLBs of Dendrobium shavin (Bustam

et al. 2012); 60-day-old PLBs in Dendrobium nobile (Moha nty et al. 2013b) and

Coelogyne breviscapa (Mohanraj et al. 2009); 30-day-old PLBs in Geodorum

densiﬂorum (Datta et al. 1999); PLB of Spathoglottis plicata Blume (Haque and

Ghosh 2017); somatic embryos in Dendrobium candidum (Guo et al. 1994) were

used for encapsulation with varied binding solution, polymerization time, and

conversion percentage. During the sowing of artiﬁcial seeds contam ination is one

of the main barriers to the commercialization of encapsulation technology. However,

Chitosan was used as a fungal growth retardant.

5 Genetic Stability

The somaclonal variations are a phenomenon of plant tissue culture that is dependent

on medium composition, multiplication, explants type, adventitious shoots forma-

tion, culture period, and plant genotype (Côte et al. 2001). Despite several experi-

ences of in vitro regeneration, either genetic uniformity or variability was observed

in micropropagated plantlets (Larkin and Scowcroft 1981). Micropropagation pro-

vides a feasible substitute to seed propagation as it entitles rapid propagation of elite

stock cultivars in a fairly short duration of time. For the raising of quality plant

material, the genetic consistency of micropropagated plants is a prerequisite factor.

In contrast, genetic instability occurs in the in vitro-regenerated plants (somaclonal

variation) due to the use of hyper-optimum potency of growth regulators and

continuous sub-culturing. Orchid micropropagation was inte rrupted with an inter-

vening callus phase, which interfered with the integrity of the regenerated clonal

plantlets (Nookaraju and Agrawal 2012); on the other hand, micropropagation via

meristem culture was considered as uniform culture (Rani and Raina 2000).

74 K. D. Mudoi et al.

To examine the in vitro protocols, whether propagation was either true-to-type or

not clonal ﬁdelity was tested with various Single Primer Ampliﬁcation Reaction

(SPAR)-based methods such as Inter Simple Sequence Repeats (ISSR), Random

Ampliﬁed Polymorphic DNA (RAPD), and Direct Ampliﬁcation of Minisatellite

DNA (DAMD) markers (Zietkiewicz et al. 1994; Williams et al. 1990; Heath et al.

1993). In addition, a recently invented molecular marker, the Start Codon-Targeted

(SCoT) polymorphism (Collard and Mackill 2009) has gained popularity as a

powerful tool for the evaluation of clonal ﬁdelity or genetic diversity in regenerated

orchid plants (Bhattacharya et al. 2005; Ranade et al. 2009) (Table 1).

Very few studies were endured for testing of clonal ﬁdelity of micropropagated

orchids. Among them, the genetic stability of micropropagated Dendrobium plant-

lets was screened by Random Ampliﬁed Polymorphic DNA (RAPD) marker

(Ferreira et al. 2006). Likely, in Habenaria edgeworthii (Giri et al. 2012a); Aerides

crispa (Srivastava et al. 2018); Anoectochilus elatus (Sherif et al. 2017);

Changnienia amoena (Li and Ge 2006); Cymbedium ﬁnlaysonianum

(Worrachottiyanon and Bunnag 2018); Cymbidium giganteum (Roy 2012); Cym-

bidium aloifolium (Sharma et al. 2011; Choi et al. 2006); Dendrobium densiﬂorum

(Mohanty and Das 2013); Dendrobium chrysotoxum (Tikendra et al. 2019a);

Dendrobium ﬁmbriatum (Tikendra et al. 2021); Dendrobium heterocarpum

(Longchar and Deb 2022); Dendrobium moschatum (Tikendra et al. 2019b);

Dendrobium nobile (Bhattacharyya et al. 2014); Eulophia dabia (Panwar et al.

2022); Rhynchostylis retusa (Oliya et al. 2021); Spathoglottis plicata (Auvira et al.

2021); Vanda coerulea (Manners et al. 2013) and in Vanilla planifolia (Sreedhar

et al. 2007) genetic uniformity was tested by RAPD marker.

Moreover, Inter Simple Sequence Repeats (ISSR) marker was tested in

Anoectochilus elatus (Sherif et al. 2017, 2018); Anoectochilus formosanus (Lin

et al. 2007; Zhang et al. 2010); Bletilla striata (Wang and Tian 2014); Bulbophyllum

odoratissimum (Prasad et al. 2021); Cymbidium aloifolium (Sharma et al. 2011,

2013; Choi et al. 2006); Dendrobium aphyllum (Bhattacharyya et al. 2018);

Dendrobium chrysotoxum (Tikendra et al. 2019a); Dendrobium crepidatum

(Bhattacharyya et al. 2016a); Dendrobium ﬁmbriatum (Tikendra et al. 2021); and

in Dendrobium nobile (Bhattacharyya et al. 2014); Dendrobium thyrsiﬂorum

(Bhattacharyya et al. 2015); Habenaria edgeworthii (Giri et al. 2012a); Platanus

acerifolia (Huang et al. 2009); Vanda coerulea (Manners et al. 2013; Gantait and

Sinniah 2013); and Vanilla planifolia (Gantait et al. 2009; Sreedhar et al. 2007;

Bautista-Aguilar et al. 2021) for studying the effectiveness of in vitro protocol.

Simple Sequence Repeats (SSR) marker was tested in Vanilla planifolia (Bautista-

Aguilar et al. 2021). Ampliﬁed Fragment Length Polymorphism (AFLP) marker was

tested in Anoectochilus formosanus (Zhang et al. 2010) and Dendrobium

thyrsiﬂorum (Bhattacharyya et al. 2017b). Inter-Retrotransposon Ampliﬁed Poly-

morphism (IRAP) marker was tested in Bletilla striata (Guo et al. 2018) and

Dendrobium aphyllum (Huang et al. 2009). Directed Ampliﬁcation of

Minisatellite-region DNA (DAMD) marker was tested on Cymbidium aloifolium

At the inter-speciﬁc level, 90% of polymorphism was observed. Among

the species, the average cumulative genetic similarity was 66%. The

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 75

Table 1 Genetic stability analysis of some medicinal orchids with various markers

Sl

no Plant species Markers Findings References

1Aerides crispa RAPD RAPD was used to conﬁrm the genetic variations among 52 in vitro

morphological variants. Among these, only 15 mutant lines were

established based on genetic diversity

Srivastava et al. (2018)

2Anoectochilus elatus ISSR 2.38% polymorphism and 97.61% monomorphism with genomic uni-

formity that of the mother plant was revealed with band patterns using

ISSR

Sherif et al. (2017)

ISSR Using ISSR, homogeneity in direct somatic embryo regenerated plants

was found to be 94.22% whereas 93.05% from plants elevated from an

indirect somatic embryo

Sherif et al. (2018)

3Anoectochilus

formosanus

ISSR and AFLP Among the regenerated shoots, the range of genetic variation was from

0.00% to 5.43%

Lin et al. (2007)

ISSR Among the total 1810 scorable bands, 94% were genetically similar

whereas only 2.76% polymorphism was observed

Zhang et al. (2010)

4Ansellia africana SCoT Using SCoT in micropropagated plants, an increment in clonal variabil-

ity with a higher gene ﬂow value (Nm = 1.596) was recorded

Bhattacharyya et al. (2017a)

5Bletilla striata SCoT and IRAP 96.17% polymorphic bands were recorded using the SCoT marker and

94% polymorphic bands were recorded using the IRAP marker

Guo et al. (2018)

ISSR Clonal ﬁdelity assessment by ISSR markers revealed 99.8 –100.0 %

similarity between the regenerants and their mother plants and

99.5–100.0 % similarity among the regenerants

Wang and Tian (2014)

6Bulbophyllum

odoratissimum

ISSR The genetic homogeneity degree using ISSR markers was high among

the clones

Prasad et al. (2021)

7Changnienia amoena RAPD Percentage of polymorphic bands at the species level was 76.5% and at

the population level it was 37.2%

Li and Ge (2006)

8Cymbidium

aloifolium

ISSR Sharma et al. (2013)

Table 1 (continued)

Sl

no Plant species Markers Findings References

range of average polymorphism at the intra-speciﬁc level was 29.8–69.9

% within ﬁve Cymbidium species

RAPD, ISSR, and

DAMD

Polymorphism in ﬁve species of Cymbidium viz., C. aloifolium,

C. mastersii, C. elegans, C. eburneum, and C. tigrinum was found to be

96.6% at an inter-speciﬁc level and 51.2–77.1% at an intra-speciﬁc level

Sharma et al. (2011)

RAPD Similarity values for total bands score analysis ranged from 0.501 for

Cymbidium aloifoilum and C. kanran to 0.935 for Cymbidium ensifolium

and Cymbidium marginatum

Choi et al. (2006)

9Cymbedium

ﬁnlaysonianum

RAPD The genetic stability of the cryopreserved synthetic seeds was conﬁrmed

with a similar index value of 0.998

Worrachottiyanon and

Bunnag (2018)

10 Cymbidium

giganteum

RAPD 5.81% molecular variation was detected in the regenerants Roy (2012)

11 Dendrobium

aphyllum

IRAP and ISSR Among the regenerants, the pooled data revealed 5.26% clonal variabil-

ity whereas individually 7.69% (IRAP) and 4% (ISSR) variability was

detected

Bhattacharyya et al. (2018)

12 Dendrobium

chrysotoxum

RAPD and ISSR Among the in vitro clones and mother plants, 96.30% of monomorphism,

and 3.6% of polymorphism was detected

Tikendra et al. (2019a)

13 Dendrobium

crepidatum

SCoT and ISSR Cumulative ISSR and SCoT data revealed high genetic ﬁdelity among

the regenerates with 6.25% clonal variability. Whereas within the

micropropagated plants SCoT data revealed a 10% total variability

Bhattacharyya et al. (2016a)

14 Dendrobium

densiﬂorum

RAPD No genetic variation was observed Mohanty and Das (2013)

15 Dendrobium

ﬁmbriatum

RAPD, ISSR &

SCoT

Among the regenerants, 100% monomorphism was observed, while low

genetic polymorphism of 1.52%, 1.19%, and 3.97% with RAPD, ISSR,

and SCoT markers, respectively, was exhibited

Tikendra et al. (2021)

16 Dendrobium

heterocarpum

RAPD, DAMD,

and SCoT

Genetic homogeneity of the regenerates was conﬁrmed with 96.89%

monomorphism and 3.11% polymorphism

Longchar and Deb (2022)

76 K. D. Mudoi et al.

17 Dendrobium nobile RAPD and SCoT 94.04% monomorphism and 5.95% polymorphism conﬁrmed the high

degree of genetic stability within the in vitro propagated plants

Bhattacharyya et al. (2014)

SCoT The very high degree of clonal ﬁdelity within the propagated plantlets

was conﬁrmed

Bhattacharyya et al.

(2016b)

18 Dendrobium

thyrsiﬂorum

ISSR and SCoT In detecting clonal variability, SCoT is more ef ﬁcient compared to ISSR Bhattacharyya et al. (2015)

AFLP High genetic diversity with 98.50% polymorphism was observed Bhattacharyya et al.

(2017b)

19 Eulophia dabia RAPD Genetic stability was evaluated which proved true to typesets of the

in vitro-raised plants

Panwar et al. (2022)

20 Habenaria

edgeworthii

RAPD Genetic stability was conﬁrmed among regenerates Giri et al. (2012a)

21 Platanus acerifolia ISSR A genetically stable micropropagated line of P. acerifolia was conﬁrmed

with 2.88% polymorphism

Huang et al. (2009)

22 Rhynchostylis retusa RAPD Genetic uniformity among all the analyzed in vitro samples and with the

mother plant was conﬁrmed

Oliya et al. (2021)

23 Spathoglottis plicata RAPD 53.28% polymorphism was reported in the orchid variants Auvira et al. (2021)

SCoT Genetic uniformity of the regenerates with the mother plant was

conﬁrmed

Manokari et al. (2022)

24 Vanda coerulea ISSR Genetic stability was conﬁrmed in plantlets from converted capsules

stored in 4 and 25 °C

Gantait and Sinniah (2013)

RAPD and ISSR Natural genetic diversity with 58.88% polymorphism was shown at the

intra-speciﬁc level

Manners et al. (2013)

25 Vanilla plantifolia RAPD & ISSR No genetic diversity was recorded among the micropropagated plants Sreedhar et al. (2007)

SSR & ISSR High genetic stability with low polymorphism percentages was detected Bautista-Aguilar et al.

(2021)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 77

(Sharma et al. 2011) and Dendrobium heterocarpum (L ongchar and Deb 2022). Start

Codon-Targeted Polymorphism (SCoT) was performed in micropropagated plantlets

of Anseilla africana (Vasudevan and Van Staden 2010); Bletilla striata (Guo et al.

2018); Dendrobium crepidatum (Bhattacharyya et al. 2016a); Dendrobium

ﬁmbriatum (Tikendra et al. 2021); Dendrobium heterocarpum (Longchar and Deb

2022); Dendrobium nobile (Bhattacharyya et al. 2014, 2016b); Dendrobium

thyrsiﬂorum (Bhattacharyya et al. 2015), and Spathoglottis plicata (Manokari et al.

2022) for homogeneity demonstration.

78 K. D. Mudoi et al.

Genetic variation or polymorphism was analyzed in Bulbophyllum

odoratissimum as 3.94% (Prasad et al. 2021); 2.76% in Anoectochilus formosanus

(Zhang et al. 2010); 2.53% in Dendrobium chrysotoxum;2% in Dendrobium

moschatum (Tikendra et al. 2019a, b); 2.38% in Anoectochilus elatus (Sherif et al.

2018); and 2.88% in Platanus acerifolia (Huang et al. 2009). The results of the ISSR

analysis conﬁrmed the feasibility of the micropropagation protocol of orchids

although tiny dissimilarity in genomic constituents was noticed. Such negligible

variation may be due to the maintenance of in vitro culture for a longer duration,

concentration of growth regulators, and in vitro stress conditions that lead to clonal

variations (Tikendra et al. 2019a; Razaq et al. 2013; Devarumath et al. 2002).

6 Ethno-Medicinal Properties

Orchids are the backbone of traditional herbal medicines and have been extensively

studied because of their pharmacological importance. From ancient times orchids are

being used in traditional systems of medicine like Ayurveda, Siddha, Yunani,

Homeopathy, Traditional Chinese Medicine (TCM), etc. Chinese described a

Dendrobium species and Bletilla stri ata in Materia Medica of Shen-Nung (twenty-

eighth century B.C.) and in many other Chinese writings orchids symbolize friend-

ship, perfection, numerous progeny, noble, and elegant (Reinikka 1995). In India,

there are nearly 1600 species that constitute about 9% of the total ﬂora (Medhi and

Chakrabarti 2009). The therapeutic importance of Indian orchids in treating ailments

is well documented in the literature (Lawler 1984; Handa 1986) (Table 2).

Several orchid species have important ingredients in various traditional medicinal

formulations. Whole plants or their parts are used as a paste or in boiled form, single

or mixed with other food stuffs as therapeutics in several ailments (Pant 2013;

Gopalakrishnan and Seeni 1987).

The roots of Acampe papillosa are used in rheumatism, burning, boils, expecto-

rant, biliousness, asthma, bronchitis, eyes, and blood, and help in curing infections,

curing secondary syphilis, uterine diseases, tuberculosis, fever, and throat troubles

(Hossain 2009; Zhan et al. 2016; Chopra et al. 1969). The root of Acampe praemorsa

is used as a tonic for rheumatism and treats neuralgia, sciatica, syphilis, and uterine

disorders. Various parts of this orchid are used for the treatment of cough, stomach-

ache, ear-ache, and eyes diseases, reduce body temperature, antibiotic for wounds,

traumatic pain, backache, menstruation pain, burning sensation, asthma, bronchitis,

Epiphytic

Karnataka: Districts of

Hassan, Mysuru, Ballari,

Chikkamagaluru,

Chitradurga, Kodagu

(Coorg), Shivamogga,

-

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 79

Table 2 Distribution and therapeutic importance of some medicinal orchids

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

1Acampe

papillosa

Small Warty Acampe Epiphytic

Bangladesh, Bhutan, India

(North West Himalaya,

Sikkim, West Bengal);

Laos, Myanmar, Nepal,

Thailand, and Vietnam

Root Asthma, bronchitis, eyes,

and blood

Helps to cure syphilis and

uterine diseases, tuberculo-

sis, poisonous infections,

throat troubles, and fever.

Also used as a cooling

agent, astringent, and

expectorant

Crusted roots are used as a

tonic; pasted roots are used

for rheumatic pains, sciat-

ica, and neuralgia

Piri et al. (2013), Hossain

(2009), Chopra et al.

(1969)

2Acampe

praemorsa

Wight’s Acampe, Brittle

Orchid

Kannada: Seete hoo, Seete

dande; Konkani:

Kanphoden

Epiphytic

Tropical Africa, India,

eastwards to China and

southwards to Malaya,

Indonesia, The Philippines,

and New Guinea

Root Used as a tonic for arthritis,

rheumatism, sciatica, neu-

ralgia, syphilis, and uterine

disorders. Pulverized plant

mixed with egg white and

calcium heal fractured

limbs. Freshly prepared

paste of its roots along with

Asparagus recemosus root

paste cures arthritis

Suja and Williams (2016),

Perfume workshop (n.d.-

a), Hossain (2009), Lean-

der and Lüning (1967),

Shanavaskhan et al.

(2012), Devi et al. (2015),

Panda and Mandal (2013),

Nongdam (2014), Mishra

et al. (2008)

3Aerides crispa Curled aerides

Marathi: Pan Shing

2–3 drops of boiled pul-

verized plant with neem is

used to treat earache

Jayashankar and Darsha

(2021), Perfume work-

shop (n.d.-a)

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

Uttara Kannada, Dakshina

Kannada

Terrestrial

Throughout the UK, many

European countries

4Aerides

multiﬂorum

The Multi-Flowered

Aerides—In Thailand—

Aiyaret—Phuang Malai,

Fox brush orchid, Maana

Terrestrial, epiphytic, sap-

rophytic

Found in Bangladesh,

eastern Himalayas, India,

Nepal, western Himalayas,

Andaman Islands, Myan-

mar, Thailand, Laos, Cam-

bodia, and Vietnam

Elevation: 1100 m

Whole plant Leaf paste is applied on

wounds and earaches. The

powdered leaf is used as a

tonic. In vitro tubers and

leaves have an antibacterial

effect and antimicrobial

effects, respectively

Lal et al. (2020), Perfume

workshop (n.d.-a), Baral

and Kurmi (2006), Basu

et al. (1971), Behera et al.

(2013), Bhattacharjee

(1998)

5Aerides

odorata

Fragrant Fox Brush

Orchid, Fragrant Aerides,

Fragrant Cat's-tail Orchid

Mizo: Nau-ban

Epiphyte

Native to South-Central

and South-East China,

Bangladesh, East

Himalaya, West Himalaya,

Nepal, India, Cambodia,

Laos, Myanmar, Thailand,

Vietnam, Borneo, Jawa,

Lesser Sunda Islands,

Malaya, Philippines, Sula-

wesi, and Sumatera

Roots,

leaves, fruits

Leaf paste and Fruits are

used to heal wounds and

cure tuberculosis. Leave

juice and seeds are used for

treating boils in the ear,

nose and other skin disor-

ders. Combination of the

fresh root of A. odorata,

root powder from Saraca

asoca, bark from

Azadirachta indica and

common salt used as an

oral medicine for painful

swollen joints

Hongthongkham and

Bunnag (2014), Devi et al.

(2013), Perfume work-

shop (n.d.-a), Leander and

Lüning (1967), Hossain

(2009), Baral and Kurmi

(2006), Behera et al.

(2013)

6Anacamptis

pyramidalis

Pyramidal Orchid - For skin whitening;

exhibits antioxidant and

scavenging capacities

Parker (2016), Perfume

workshop (n.d.-a)

80 K. D. Mudoi et al.

including Slovenia, in

North Africa and the Near

East

Elevation: 0–1600 m

(continued)

7Anocetochilus

elatus

South Indian Jewel Orchid

Malayalam: Nagathali

Assamese: Boga-kopou-

phul

Terrestrial

Distributed along Southern

Western Ghats of India

Whole Plant Used in the chest and

abdominal pain and to treat

snake bites

Sherif et al. (2012, 2018)

8Anocetochilus

formosanus

Jewel orchid Terrestrial

Widely distributed in Tai-

wan and Fujian Province of

China, and Japan

Whole plant The whole plant is used as

a cooling agent, an antipy-

retic, for relieving pain in

the waist and knee, and for

treating tuberculosis, dia-

betes, bronchitis, renal

infections, snake bites, and

stomach aches. The plant

also possesses anti-

cancerous properties

Jiang et al. (2015), Per-

fume workshop (n.d.-a),

Aswandi and Kholibrina

(2021), Nandkarni (1976)

9Ansellia

africana

Leopard orchid Perennial, and epiphyte,or

sometimes terrestrial

Tropical and subtropical

areas of southern Africa

Whole plant Stem infusion is used as an

antidote to bad dreams.

Leaves and stems are used

for treating madness

Bhattacharyya and Staden

(2016), Saleh-E-In et al.

(2021)

10 Arundina

graminifolia

Bamboo orchid, Bird

Orchid, Kinta Weed

Manipuri: Kongyamba

lei; Mizo: Le-len

Terrestrial

Myanmar, India, Sri

Lanka, Nepal, Thailand,

Vietnam, the Ryukyu

Islands, Malaysia, Singa

pore, China to Indonesia,

the Philippines and New

Guinea

Whole plant It possesses anti-bacterial

activity. The root is used as

a pain reliever. The

scrapped bulbous stem is

applied on the foot heels to

treat the cracks

Hu et al. (2013), Aswandi

and Kholibrina (2021),

Hossain (2009), Kumar

(2002), Dakpa (2007)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 81

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

The entire plant is used to

improve blood circulation,

heal abscesses,

Suetsugu and Fukushima

( ), Perfume2014

11 Bletilla striata Hyacinth orchid or Chi-

nese ground orchid

Terrestrial

Japan, Korea, Myanmar

(Burma), and China

(Anhui, Fujian, Gansu,

Guangdong, Guangxi, Gui

zhou, Hubei, Hunan,

Jiangsu, Jiangxi, Shaanxi,

Sichuan, Zhejiang)

Tuber, Root Tubers are used in treating

hemorrhage, tuberculosis,

and bleeding. It promotes

the regeneration of muscle

and other tissues. They are

used to treat sores, chapped

skin, dysentery, fever,

malignant ulcers, gastroin-

testinal disorders, hemor-

rhoids, anthrax, malaria,

eye diseases, etc. The

powdered roots mixed with

oil are applied to burns and

skin diseases. Effective

against leucorrhea. Leaves

are used to cure lung

disease

He et al. (2017), Perfume

workshop (n.d.-a), Kong

et al. (2003), Bulpitt et al.

(2007)

12 Bulbophyllum

odoratissimum

Lithophytic

China, India

Native to:

Andaman Is., Assam,

Cambodia, China South-

Central, China Southeast,

East Himalaya, India,

Laos, Myanmar, Nepal,

Thailand, Tibet, Vietnam

Whole plant Fractures, pulmonary

tuberculosis, hernia pain

Perfume workshop (n.d.-

a), Bhattacharjee (1998)

13 Calanthe

discolor

Japanese Hardy Orchid Terrestrial

Korea, Japan, and China

Whole plant

82 K. D. Mudoi et al.

rheumatism, bone pain,

and traumatic injuries as

well as treat skin ulcers and

hemorrhoids

workshop ( ),

Yoshikawa et al. ( ) 1998

n.d.-a

Malanga, aloe-leafed cym-

bidium

Boat Orchid

Epiphytic herb

Global Distribution

India, Sri Lanka, Thailand,

(continued)

14 Changnienia

amoena

Whole

plant, roots

Teoh (2019)

15 Coelogyne

cristata

Swarna Jibanti; Jibanti

India: Hadjojen (bone

joiner)

Nepal: ban maiser,

jhyanpate

Found in moss forests

associated with tree bark

and rocks, often exposed to

sun

India, Bhutan, Nepal, Tibet

and mountainous regions

of Northern Thailand

Elevation: 1500–2600 m

Pseudobulbs Pseudo bulbs are used for

constipation and aphrodi-

siac. The juice is used for

healing wounds, boils, and

sores

Sharmaa et al. (2014),

Mitra et al. (2018), Per-

fume workshop (n.d.-a),

Pant and Raskoti (2013),

Subedi et al. (2011),

Pamarthi et al. (2019)

16 Coelogyne

ﬂaccida

Bearded Coelogyne, loose

Coelogyne

China: Lilinbeimu Lan,

Guishangye

Epiphyte or lithophyte

Himalayas, Nepal, North

India, Bhutan, China, and

Myanmar

Elevations: 900–1400 m

Pseudo bulb Used to treat headache,

fever, and indigestion

Kaur and Bhutani (2013),

Pant and Raskoti (2013),

Teoh (2016), Pamarthi

et al. (2019), Perfume

workshop (n.d.-a)

17 Coelogyne

nervosa

Veined coelogyne Epiphytic

Southern Western Ghats of

Kerala and Tamil Nadu

Whole plant Has potential antimicro-

bial, antioxidant, and anti-

cancer properties

Sathiyadash et al. (2014),

Ranjitha et al. (2016)

18 Coelogyne

stricta

The Rigid Coelogyne

Pseudobulb

India: Harjojan

Found on tree trunks or

lithophytes on mossy rocks

Elevations: 1400–2000 m

North-East India, Sikkim,

Bhutan, Myanmar, and

Nepal

Pseudobulbs The paste is used to cure

headaches and fever

Perfume workshop (n.d.-

a), Basker and Bai (2006),

Yonzone et al. (2012),

Pamarthi et al. (2019)

19 Cymbidium

aloifolium

Rhizome,

root, pseudo

bulbs

The paste is used to treat

fractured and dislocated

bones

Behera et al. (2013), Per-

fume workshop (n.d.-a),

Pamarthi et al. (2019)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 83

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

Tamil Nadu:

panaipulluruvi

Assam: Kopou-Phul

Indonesia, Java, Indo-

Malaysia

20 Cymbidium

ensifolium

Golden-thread orchid,

burned-apex orchid, spring

orchid, and rock orchid

Epiphytic

Global Distribution: India

and Sri Lanka

Native to:

Assam, Cambodia, China

South-Central, China

Southeast, Hainan, Japan,

Korea, Laos, Myanmar,

Philippines, Taiwan,

Thailand, Tibet, Vietnam

Root, ﬂower Root decoction is used to

treat gonorrhea. Flower

decoction used in eye sore

disorders

Chang and Chang (1998),

Tsering et al. (2017)

21 Cymbidium

ﬁnlaysonianum

Finlayson’s Cymbidium

Malay: Sepuleh

Thai: Ka Re ka Ron Pak

Pet

Terrestrial (Primary

Rainforest, Secondary

Rainforest, Coastal Forest)

Thailand, Vietnam, Cam-

bodia, Peninsular Malay-

sia, Java, Borneo and the

Philippines

Elevation: 0–1200 m

- Restore health Islam et al. (2015), Per-

fume workshop (n.d.-a)

22 Cymbidium

giganteum

Iris-like Cymbidium Epiphytic

Chinese Himalayas, India,

eastern Himalayas, Nepal,

western Himalayas, Myan-

mar, and Vietnam

Elevation: 0–1200 m

Leaves Wounds Hossain et al. (2010),

Bulpitt (2005), Fonge

et al. (2019),

Linthoingambi et al.

(2013)

84 K. D. Mudoi et al.

(continued)

23 Cymbidium

goeringii

Noble orchid

Japan: Chun Lan (spring

orchid)

Terrestrial

East Asia including Japan,

China, Taiwan, and South

Korea

Elevation: 300–3000 m

Seed, whole

plant

Seeds are used to cure

wounds and injuries and

also in curing fractures, and

traumatic soft tissue

injuries

Perfume workshop (n.d.-

a), Teoh (2016)

24 Cymbidium

iridioides

Iris Cymbidium

Chinese: Huang chan Lan

Epiphytic

China, India, Bhutan,

Nepal, Myanmar; and

Vietnam

Elevation: 900–2,800 m

Leaves,

pseudo

bulbs, roots

Fresh juice of this plant is

used to stop bleeding. The

powder is used as a tonic.

During diarrhea, pseudo

bulbs and roots are

consumed

Perfume workshop (n.d.-

a), Aggarwal and Zettler

(2010), Arditti et al.

(1982), Arditti and Ernst

(1984), Medhi and

Chakrabarti (2009)

25 Cymbidium

kanran

The Cold Growing

Cymbidium

Terrestrial

Exclusively distributed in

Northeast Asia including

China, Japan, and Korea

Whole plant Cures coughs and asthma.

Roots are used to cure

ascariasis and

gastroenteritis

Perfume workshop (n.d.-

a), Jeong et al. (2017)

26 Cymbidium

lancifolium

Lance leafed Cymbidium Grows in broad-leaved

forests where the soil is

rich in humus and also

plenty of leaf litter

In the Himalayas, India,

Nepal, Bhutan, China, Tai-

wan, Japan

Elevation: 300–2300 m

Whole plant Used to cure rheumatism,

improve blood circulation

and treat traumatic injuries

Perfume workshop (n.d.-

a)

27 Cymbidium

longifolium

Red-Spotted Lip Cymbid-

ium; In China Chang Ye

Lan

Epiphytic, lithophytic, or

terrestrial

Found in China, Eastern

Himalayas, Nepal, Bhutan,

Burma, and India

Elevation: 1000–2500 m

Pseudo bulb The fresh shoot is used for

nervous disorders, mad-

ness, epilepsy, hysteria,

rheumatism, and spasms.

Salep used as demulcent.

An aqueous solution of

powdered pseudo bulbs is

taken orally on an empty

stomach

Nongdam (2014), Sood

et al. (2006), Yonzone

et al. (2013), Zhan et al.

(2016)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 85

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

Rhizome,

ﬂower,

Used to treat skin disease,

roots and stem promote

Shimura and Koda ( ),

Shimura et al. ( ),2007

2004

28 Cymbidium

sinense

Japan—Hosai-Ran—Tai-

wan-Ran—In China Mo

Lan

Terrestrial

Found in India, Myanmar,

northern Thailand, Viet-

nam and east China, Japan

Whole plant Used in purifying heart,

lungs, treating cough and

asthma

Perfume workshop (n.d.-

a)

29 Cypripedium

calceolus

Lady’s-slipper orchid

Japanese: Ko-atsumori-

sou

Shady, deciduous and

mixed woodland, predomi-

nantly on calcareous soils

Spain, Europe, China,

Siberia, Sakhalin Island,

and Japan

Elevation: 2000 m

Root,

rhizome

It acts as a sedative, pro-

motes sleep, and reduces

pain when powdered roots

are mixed with sugar water.

A tea prepared from roots

is used to treat jangling

nerves and headaches

Kull (1999), Kolanowska

and Busse (2020), Singh

and Dey (2005)

30 Cypripedium

debile

Frail lady’s slipper

Lan (two leaf spoon

orchid)

Japan, Korea, Taiwan, and

China

Whole plant Used to improve blood cir-

culation, reduce swelling,

relieves pain, and act as a

diuretic

Perfume workshop (n.d.-

a)

31 Cypripedium

formosanum

Formosa lady’s slipper Terrestrial

Found on sandy ﬂoor of the

forest and in open areas in

Taiwan

Elevation: 2000–3000 m

Whole plant Improves blood circula-

tion, regulates menses, and

relieves pain and itching.

Roots and stems are used to

treat malaria, snake bites,

traumatic injury, and

rheumatism

Perfume workshop (n.d.-

a)

32 Cypripedium

guttatum

Spotted lady’s slipper Hardy terrestrial

European Russia to Korea,

Alaska to Yukon

Elevation: 1000–4100 m

Roots and

leaves

Used to treat epilepsy Zhang et al. (2007), Per-

fume workshop (n.d.-a)

33 Cypripedium

macranthos

Large ﬂowered lady’s

slipper

86 K. D. Mudoi et al.

Terrestrial

East Belarus to temperate

East Asia

stem, and

root

dieresis, reduce swelling,

expel gas, relieve pain and

improve blood ﬂow. Dried

ﬂowers are used to stop in

wound bleeding

Perfume workshop (n.d.-

a)

Epiphytic

Western Himalayas, India,

(continued)

34 Cypripedium

parviﬂora

Yellow lady’s slipper or

moccasin ﬂower

Terrestrial

Native to:

Delaware, Nebraska, North

Dakota, Québec, Rhode I.,

Elevation: 1400 m

Rhizome Cures insomnia, anxiety,

headache, emotional ten-

sion, fever, palpitations,

tumors, irritable bowel

syndrome, neuralgia, and

reduces menstrual and

labor pain

Meier et al. (2018),

Moerman (1986), Grieve

(1998), Kumar et al.

(2005)

35 Cypripedium

pubescens

Yellow lady’s slipper Deciduous and coniferous

forest, meadows, fens

Newfoundland to British-

Columbia, south to Geor-

gia, Arizona, Washington,

and Europe

Elevation: 5750–11,000 ft.

Root The plant is diaphoretic,

hypnotic, nervine, anti-

spasmodic, sedative, and

tonic. Used in diabetes,

diarrhea, dysentery, paral-

ysis, joint pain, convales-

cence, impotence, and

malnutrition

Pant and Rinchen (2012),

Wani et al. (2020),

Shrestha et al. (2021),

Perfume workshop (n.d.-

a), Singh and Duggal

(2009), Khory (1982)

36 Dactylorhiza

hatagirea

Himalayan Marsh Orchid

India: Munjataka in

Ayurveda

Terrestrial

India, Pakistan, Afghani-

stan, Nepal, Tibet, and

Bhutan. Elevation:

2500–5000 ft.

Tubers Used as a tonic, heals

wound, fever, and control

burns and bleeding. Also

used as food due to the

presence of starch

Pant and Rinchen (2012),

Wani et al. (2020),

Shrestha et al. (2021),

Perfume workshop (n.d.-

a), Aggarwal and Zettler

(2010), Arditti (1967,

1968, 1992), Arditti et al.

(1982), Arditti and Ernst

(1984)

37 Dendrobium

amoeneum

The Lovely Dendrobium Venkateswarlu et al.

(2002)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 87

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

eastern Himalayas, Nepal,

Bhutan, Sikkim,

Bangladesh, Myanmar

Elevation: 600–2000 m

Pseudo

bulbs,

leaves

The freshly prepared paste

is used to cure skin dis-

eases and dislocated bones

38 Dendrobium

aphyllum

Thai names: Uean sai,

Ueang sai long laeng, etc.

Assamese: Haliki-thutia-

phul

Epiphytic

Continental Southeast

Asia, Southwest China,

Sikkim, and Nepal

Pseudo

bulbs

Leaf paste is applied on the

abnormal and deformed

head parts of the newly

born baby to get a normal

shape

Liu et al. (2018), Perfume

workshop (n.d.-b), Pant

(2013)

39 Dendrobium

candidum

Shihu in Chinese and

Sekkoku in Japanese

Epiphytic

Southern China, Taiwan,

Nepal, Thailand, Vietnam,

India, Myanmar

Elevation: 2000–3000 m

Leaves Used to treat diabetes Nongdam (2014), Wu

et al. (2004)

40 Dendrobium

chrysanthum

Golden yellow-ﬂowered

dendrobium

Epiphytic and Lithophytic

India, Nepal, Bhutan,

Burma, China, Thailand,

Laos, and Vietnam

Elevation of 450–2000 m

Stem, leaf The stem is used as a tonic

to enhance the immune

system, promote body ﬂuid

production, and reduce

fever. The leaf is used as an

antipyretic and mild skin

disease as well as beneﬁts

the eyes

Nongdam (2014), Bulpitt

(2005), Jalal et al. (2008,

2010), Li et al. (2016)

41 Dendrobium

chrysotoxum

Golden Orchid

Thai: Uang Khan

Vietnam: Kim diep

Epiphytic

North-East India, Nepal,

Bhutan, Burma, China,

Thailand, Laos, and

Vietnam

Whole plant The whole plant possesses

antitumoral and

anticancerous properties.

Stem and ﬂower extract is

used as tonic and leaf

extract as antipyretic

Nongdam (2014), Per-

fume workshop (n.d.-b),

Sood et al. (2006), Bulpitt

et al. (2007), Joshi et al.

(2009)

88 K. D. Mudoi et al.

Fringe Lipped

Dendrobium

China: Liusushihu

(tasseled stone orchid)

Epiphytic, lithophytic and

terrestrial

China, Western Himalayas,

Bangladesh, Eastern

Himalayas, India, Nepal,

(continued)

42 Dendrobium

crepidatum

Shoe-Lip Dendrobium

China: Meigui Shihu (rose

Dendrobium)

Epiphytic Pseudo

bulbs, stem

Pseudo bulb paste is used

to treat the fracture and

dislocated bones. Stems are

used as a tonic for treating

arthritis and rheumatism

Perfume workshop (n.d.-

b), Joshi et al. (2009),

Joshi and Joshi (2001), Hu

et al. (2016)

43 Dendrobium

crumenatum

Pigeon orchid, Dove

orchid

India: Jivanti

Malay: bunga angin (wind

orchid)

Malaysia, Singapore Leaf Leaves are used to treat

boils and pimples

Perfume workshop (n.d.-

b), Joshi and Joshi (2001),

Topriyani (2013)

44 Dendrobium

densiﬂorum

Pineapple Orchid

Thai: Ueang Mon Kai

Liam

Vietnam: Thy-tien

Epiphytic

China, Bhutan,NE India,

Myanmar, Nepal, Thailand

Elevation: 400–1000 m

Pseudo

bulbs, leaf

Pulps of the pseudo bulbs

are used to treat boils,

pimples, and other skin

eruptions. Leaf paste is

used on fractured bones, to

relieve sprains and

inﬂammations

Perfume workshop (n.d.-

b), Arditti (1992), Arditti

et al. (1982), Arditti and

Ernst (1984), Keerthiga

and Anand (2014), Pant

et al. (2022)

45 Dendrobium

devonianum

Devon's Dendrobium

China: Chiban Shihu

(teeth pedal Dendrobium)

Epiphytic

Native to south China, the

eastern Himalayas (Bhu-

tan, Assam), Myanmar,

Thailand, Laos, Vietnam

Stem Dried stems are used as an

immune system enhancer

Li et al. (2011, 2013a),

Perfume workshop (n.d.-

b), Cakova et al. (2017)

46 Dendrobium

draconis

Thai names: Ueang ngoen,

ueang ngum

Myanmar Name: Kein na

ri

Terrestrial

India, Cambodia, Laos,

Myanmar, Thailand, and

Vietnam

Stem Used in antipyretic and

hematinic

Rangsayatorn (2009),

Perfume workshop (n.d.-

b)

47 Dendrobium

ﬁmbriatum

Whole plant Used in upset of liver and

severe anxiety. Leaves are

used for treating fractured

bone, the pseudo bulbs are

used in fever

Huang et al. (2008),

Nongdam (2014), Per-

fume workshop (n.d.-b),

Arditti et al. (1982)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 89

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

India: Fringed lip

Dendrobium

Bhutan, Laos, and Vietnam

Elevation: 800–2400 m

Epiphytic

Northeast India, Bhutan

and Nepal across Myanmar

48 Dendrobium

heterocarpum

Golden-Lip Dendrobium

Thailand: Ueang Si Tan in

Epiphyte

Native to:

China, Nepal, Bhutan, the

Indian subcontinent and

Southeast Asia

Pseudo bulb The paste is used to treat

fractured and dislocated

bones

Arditti and Ernst (1984),

Warinhomhoun et al.

(2022)

49 Dendrobium

lasianthera

Sepik Blue Orchid Epiphyte

New Guinea, Papuasia,

Asia Tropical

Roots, stem,

leaves

Anticancer Utami et al. (2017)

50 Dendrobium

longicornu

Long-horned dendrobium Epiphyte or terrestro-litho-

phyte

Native to southern China,

the Himalayas (Nepal,

northeastern India, Bhutan,

Bangladesh) and northern

Indo-China region

Elevation : 1200–3000 m

Whole plant The plant juice mixed with

lukewarm water is used for

treating children with

fever. The boiled root is

used to feed the livestock

to remove cough

Dohling et al. (2012),

Perfume workshop (n.d.-

b)

51 Dendrobium

macrostachyum

Fringed Tree Dendrobium Epiphytic

India, Myanmar, Sri Lanka

and on the Cape York

Peninsula

Native to Australia, tropi

cal Asia, and eastern

Malaysia

Tender

shoot tip

Tender shoot tip juice is

used for earaches

Pyati et al. (2002), Per-

fume workshop (n.d.-b),

Reddy et al. (2001)

52 Dendrobium

moschatum

Musk Dendrobium

Thai: Ueang Champa

Pseudo bulb Pseudo bulb paste is used

to treat dislocated and

fractured bones

Kanjilal et al. (1999), Per-

fume workshop (n.d.-b)

90 K. D. Mudoi et al.

and Thailand to Laos,

Vietnam, and China

(continued)

53 Dendrobium

nobile

Noble Dendrobium

China: Jinchashihu (gold

hairpin Dendrobium)

Japanese name: Koki

Epiphytes or lithophytes

Himalayas and China

Pseudo

bulb, seed,

Stem

The pseudo bulb extracts

cure eye infections and

burns; the plant is used to

treat pulmonary tuberculo-

sis, ﬂatulence, and dyspep-

sia, and reduce salivation,

night sweats, fever, and

anorexia. Also used as an

antiphlogistic, tonic. Seeds

are used to heal wounds;

stems to cure fever and

tongue dryness; stems are

used for longevity

Bhattacharyya et al.

(2014), Asghar et al.

(2011), Luo et al. (2010),

Singh and Duggal (2009),

Perfume workshop (n.d.-

b), Arditti (1967), Arditti

et al. (1982)

54 Dendrobium

ovatum

Green Lipped Dendrobium

India: Anantali Maravara

Epiphytic

Global Distribution: West-

ern Ghats of India

Whole plant Fresh plant juice cures

stomach ache, excites bile,

also acts as a laxative to the

intestines, and cures

constipation

Pujari et al. (2021), Shetty

et al. (2015), Perfume

workshop (n.d.-b),

Kirtikar and Basu (1981),

Caius (1986)

55 Dendrobium

parishii

Parish’s Dendrobium

Thai: Ueang Khrang Sai

San

Epiphyte.

Native to the Eastern

Himalayas, China,

Thailand, Myanmar, Laos,

Cambodia, and Vietnam

Pseudo

bulbs

Antipyretic encourages the

secretion of body ﬂuids

Kongkatitham et al.

(2018), Perfume work-

shop (n.d.-b)

56 Dendrobium

primulinum

Primrose Yellow

Dendrobium

Epiphyte

Assam, Himalayas, Nepal,

Andaman Islands, Myan-

mar, Thailand, China, and

Vietnam

Dried stems Immune system enhancer Pant and Thapa (2012)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 91

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

57 Dendrobium

thyrsiﬂorum

Pinecone-like raceme

dendrobium

Epiphytic, lithophytic, or

terrestrial

Native to the Eastern

Himalayas, China,

Thailand, Myanmar, Laos,

Cambodia, and Vietnam

Elevation: 1200–2000 m

Stem Used to resist heat, beneﬁts

the stomach, and promotes

the production of body

ﬂuid

Wrigley (1960), Ruixuan

et al. (2015), Perfume

workshop (n.d.-b)

58 Dendrobium

transparens

Translucent Dendrobium Epiphytic

Western Himalayas,

Bangladesh, eastern

Himalayas, India, Nepal,

Bhutan, Sikkim, Myanmar,

China, and Vietnam

Elevation: 500–2100 m

Pseudo bulb The paste is used to treat

fractures and dislocated

bones

Sunitibala and Kishor

(2009), Arditti and Ernst

(1984)

59 Dendrobium

trigonopus

Thailand: Triangular Col-

umn Foot Dendrobium

Epiphyte

The plant grows in the for-

est of Burma, Thailand,

SW China, Laos and Viet-

nam

Elevations: 300–1500 m

Stem Used to cure fever and

anemia

Hu et al. (2008a), Perfume

workshop (n.d.-b)

60 Doritis

pulcherrima

Beautiful Moth Orchid Terrestrial, epiphytic

Myanmar, Thailand,

China, Laos, and Vietnam

Elevation: 1000–4900 ft.

Leaves Used to treat ear infections Perfume workshop (n.d.-

c)

61 Eria

bambusifolia

Bamboo-Leaf Eria Epiphytic

World distribution: India,

Thailand

Elevation: 1000–1300 m

Whole plant

parts

Treating hyperacidity and

stomach disorders

Basker and Bai (2010),

Zhan et al. (2016)

92 K. D. Mudoi et al.

Saprophytic

Nepal, Bhutan, India,

Japan, North Korea,

Used in stroke, tetanus,

migraine, malaise, general-

ized dermatitis dizziness,

(continued)

62 Eulophia dabia Dubious Eulophia

Salibmisri, Sung Misrie

Terrestrial

Afghanistan, Baluchistan,

Uzbekistan, Southern

Himalayas, South China

Tubers Stimulate appetite, cures

stomach ache, and stimu-

lates blood ﬂow

Pant (2013), Perfume

workshop (n.d.-b),

Panwar et al. (2022)

63 Eulophia

epidendraea

Epidendrum Eulophia

Katou kaida maravara

Terrestrial

South India, Sri Lanka,

Bangladesh

Tubers Cure tumor, and diarrhea;

acts as an appetizer,

anthelmintic, aphrodisiac,

stomachic, and worm

infestation, stimulate appe-

tite, and puriﬁes blood

during heart troubles

Perfume workshop (n.d.-

d), Narkhede et al. (2016)

64 Eulophia

graminea

Grass Eulophia

Kattuvegaya

Terrestrial

India, Sri Lanka, Southeast

Asia, China, and Japan

Whole plant Juice to treat earache Perfume workshop (n.d.-

d)

65 Eulophia nuda Terrestrial

Found in the Western

Ghats of India, tropical

Himalayas, Myanmar and

South China, Indochina,

Malaysia, Indonesia, Phil-

ippines and the Paciﬁc

Islands

Whole plant A thick paste of tubers is

applied on the stomach to

kill intestinal worms, cure

rheumatoid arthritis, bron-

chitis, scrofulous glands,

and tumors, purify the

blood, and used as a tonic,

acts as an anti-aphrodisiac,

demulcent and anthelmin-

tic. The leaf is used as a

vermifuge, the whole plant

is used in stomachache and

snake bites, and the stem is

used to stop bleeding and

pain from trauma

Hada et al. (2020)

66 Gastrodia elata Tianma China: Ming

Tianma, Japan: Tenma,

Korean name: Cheon ma

Tuber Perfume workshop (n.d.-

d), Chen et al. (2014)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 93

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

Siberia, Taiwan, and China

Elevations: 400–3200 m

sleepiness, insomnia, high

blood pressure, blood cir-

culation, rheumatism,

numbness, paralysis, back-

ache, skin boils, ulcers, and

body pain

Terrestrial

Shandong, Tibet, Dongbei,

The root is beneﬁcial for

the lungs and kidney,

strengthen muscles and

67 Geodorum

densiﬂorum

Nodding Swamp Orchid

Bangladesh: Kukurmuria

China: Dibao Lan

India: Kukurmuria

Terrestrial

Japan, China, Taiwan, Sri

Lanka, Myanmar, Philip-

pines, Indochina, Thailand,

Malaysia, Ryukyu Islands,

Indonesia, Nepal, India

Pseudo

bulbs, roots

Used as a disinfectant.

Root paste mix with ghee

and honey in menstrual

disorders and root paste is

applied on insect bites and

wounds

Nongdam (2014), Per-

fume workshop (n.d.-d),

Sheelavantmath et al.

(2000)

68 Gymnadenia

conopsea

China: shou shen,

Shouzhangshen Japan:

Tegata-chidori

Lithophytes

Russia, Europe, Japan,

Korea

Stem Treat kidney disorders,

cough, dysfunction, dis-

charge, traumatic injuries,

thrombosis, chronic hepa-

titis, lactation failure stops

bleeding, and fever

Perfume workshop (n.d.-

d), Gustafsson (2000)

69 Habenaria

edgeworthii

Terrestrial Leaves and

roots

Cooling and spermophytic Singh and Duggal (2009)

70 Habenaria

pectinata

Comb Habenaria Terrestrial

Assam, China South Cen-

tral, East Himalaya, Myan-

mar, Nepal, Pakistan, West

Himalaya

Bulb Bleeding diathesis, burning

sensation, fever, and

phthisis

Singh and Duggal (2009)

71 Herminium

lanceum

Lanceleaf Herminum

China:

Shuangchunjiaopan Lan

Roots Perfume workshop (n.d.-

d)

94 K. D. Mudoi et al.

Guangxi, Taiwan

Elevation: 1100–3500 m

bones, stops bleeding, and

treats tuberculosis

Cylindrical Vanda, Parrot

Flower

China: Banghua Lan,

India: Chaitek Lei in

Epiphytic

India, Andaman Island.,

Bangladesh, China South-

Central, East Himalaya,

India, Laos, Myanmar,

Stem and leaves are used to

improve blood ﬂow and

reduce swelling. The paste

is used to treat dislocated

bone. Leaf paste is applied

(continued)

72 Liparis odorata Fragrant Liparis Terrestrial

Global distribution: Wide-

spread

Native to: Japan,

Bangladesh, Cambodia,

China South-Central,

China Southeast, East

Himalaya, India, Laos,

Myanmar, Nansei-shoto,

Nepal, Sri Lanka,

Thailand, Tibet, West

Himalaya

Whole part The whole plant is used for

external use, tubers are

used to treat stomach dis-

orders and its paste is for

chronic ulcers

Perfume workshop (n.d.-

e)

73 Malaxis

acuminata

Jeevak Terrestrial

Bangladesh, India, Nepal,

Myanmar, Thailand, Laos,

Cambodia, Vietnam,

Malaysia, and Philippines

Elevation: 1500–2100 m

Pseudo bulb Used as tonic, Aphrodisiac,

styptic, antidysentery and

febrifuge. The paste is

applied on insect bites, and

treats rheumatism, bleed-

ing, burning sensation, and

lungs disease

Pushpa et al. (2011)

74 Oberonia

ensiformis

Word-Leaf Oberonia

China: Jian Ye Yuan Wei

Lan

Lithophytic, epiphytic

Nepal, India, China,

Myanmar, Thailand, Laos,

and Vietnam

Elevation: 600–1000 m

Used to encourage diuresis,

treat cystitis, urethritis,

injuries, and fractures and

improve blood circulation

Perfume workshop (n.d.-

c)

75 Papilionanthe

teres

Stem and

leaves

Perfume workshop (n.d.-

c)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 95

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

Manipuri, cylindrical

Vanda

Nepal, Thailand, Vietnam

Elevation: 600 m

to reduce fever. Stem juice

protects from coughs and

colds

France, Germany, Great

Britain, Albania, Austria,

Baltic States, Belarus, Bel-

gium, Bulgaria, Denmark,

Finland, Greece, Hungary,

Iran, Iraq, Ireland, Italy,

76 Pholidota

articulata

Rattlesnake orchids

India: Harjojan; Jivanti

Myanmar: Kwyet mee pan

myo kywe

Nepal: Thurjo, Pathakera

Epiphytic

Montane to submontane

zones, Uttarakhand

Himalayas, Arunachal

Pradesh, and Indo-China to

Malaysia

India, Nepal, Bhutan

Myanmar, Thailand, Cam-

bodia, Vietnam, Malaysia,

and Indonesia

Whole plant Enriched in remove gas

and reduce swelling, treat

coughs, headache, dizzi-

ness, traumatic injuries,

sores and ulcers, irregular

menses and uterine prob-

lems, and fractures, used as

a stimulant, demulcent, and

tonic. Pseudo bulbs paste is

applied on dislocated

bones. Powdered root treat

cancer and capsule juice

are used to treat skin erup-

tions and ulcers

Perfume workshop (n.d.-

c)

77 Pholidota

pallida

China: Eumaishixiantao Epiphytic

Bhutan, Central Nepal,

Northeast India

Root and

pseudo bulb

Its powder induces sleep

and juice to remove

abdominal pain. Root and

pseudo bulb paste is used

to cure fever

Perfume workshop (n.d.-

c)

78 Platanthera

chlorantha

Greater butterﬂy-orchid Whole Plant The whole plant is used in

strengthening the kidneys

and lungs, and cures sexual

dysfunction, hernia, and

enuresis affecting children

Perfume workshop (n.d.-

c)

96 K. D. Mudoi et al.

Krym, Netherlands, North

Caucasus, Norway,

Poland, Romania, Sicilia,

Spain, Sweden, Switzer-

land, Turkey, Ukraine,

Yugoslavia

(continued)

79 Rhynchostylis

retusa

Foxtail orchid

Blunt Rhynchostylis

India: Kopou phool,

draupadi mala, panas keli

Nepal: ghoge gava

Epiphytic

Global Distribution: Indo-

Malaysia, India

Leaf, root,

ﬂower

Leaves and roots paste are

used in rheumatism. Leaf

juice is used in constipa-

tion, gastritis, acidity, and

as an emollient. Root juice

is used to heal cuts and

wounds, and root is used in

menstrual pain and arthri-

tis. Dry ﬂowers are used as

an emetic

Basu et al. (1971),

Bhattacharjee (1998),

Bulpitt et al. (2007),

Dakpa (2007) Dash et al.

(2008)

80 Satyrium

nepalense

Nepal Satyrium Terrestrial

Sri Lanka, India, Bhutan,

and Myanmar

Elevation: 2400–5000 m

Tubers Treats diarrhea, dysentery,

and malaria. Tubers are

consumed as an aphrodi-

siac and used as children’s

growth supplements. Juice

is used in cuts and wounds.

The powder is used as a

tonic and to treat colds,

coughs, and fever

Baral and Kurmi (2006);

Behera et al. (2013),

Bulpitt et al. (2007),

Gutierrez (2010)

81 Spathoglottis

plicata

Philippine ground orchid,

Large purple orchid

Terrestrial

Taiwan, Southern India,

Indonesia, Japan, Malay-

sia, New Guinea, Philip-

pines, Sri Lanka, Thailand,

Vietnam, Australia, Tonga

and Samoa

Pseudo bulb Treat rheumatic swelling,

relieve pain, and uplift

blood circulation

Teng et al. (1997), Friesen

and Friesen (2012)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 97

Table 2 (continued)

Sl.

No. Species

Common Name and Local

Name Habitat and Distribution Part Used Therapeutic Importance References

82 Thunia alba White Thunia Epiphytic

India, China, and Southeast

Asia

Elevation: 2000 m

Whole plant Cough pneumonia, bron-

chitis, bone break treat-

ment, and injury

Xu et al. (2019a)

83 Vanda coerulea Blue Orchid, blue vanda,

autumn lady’s tresses

India: Kwaklei

Lawhlei

Vandara

Epiphytic

Native to: North East India

Elevation: 2500–4000 ft.

Flower Flower juice is used in

treating glaucoma, cataract,

and blindness

Roy et al. (2011)

84 Vanda

roxburghii

Rasna Epiphytic

Widely distributed

throughout Bangladesh

Root Treat fever, nervous sys-

tem disease dyspepsia,

snake bites bronchitis, hic-

cough, piles, rheumatism,

allied disorders

Uddin et al. (2015),

Uprety et al. (2010)

85 Vanda

spathulata

Spoon-Leaf Vanda

India: Ponnampumaravara

Terrestrial

South India and Sri Lanka

India: Karnataka, Kerala,

and Tamil Nadu

Dried

ﬂowers

Dried ﬂower powdered

juice is used to treat

asthma, and depression,

enhance memory, and

antioxidant activity, and

alleviate chronic disease,

and degenerative ailments

such as cancer, autoim-

mune disorders, hyperten-

sion, delay the aging

process, and

atherosclerosis

Decruse et al. (2003),

Jeline et al. (2021), Gupta

and Katewa (2012)

86 Vanda

tessellata

Grey orchid or Checkered

Vanda

Leaves Chowdhury et al. (2014)

98 K. D. Mudoi et al.

Epiphytic

India, Myanmar, China,

and Sri Lanka

Inﬂammations, rheuma-

tism, dysentery, bronchitis,

dyspepsia, and fever

87 Vanda testacea Small ﬂowered Vanda Epiphytic

India, Myanmar, and Sri

Lanka

Roots,

leaves, and

ﬂowers

The powdered extract is

used in nervous disorders,

piles, inﬂammations, rheu-

matism, bronchitis, and

anticancerous drugs

Kaur and Bhutani (2009)

88 Vanilla

planifolia

Flat-leaved vanilla Terrestrial or epiphytic

South America

Native to: Mexico and

Central America

Fruits Treats intestinal gas and

fever, increases sexual

desire, used as ﬂavoring

syrup and perfume

fragrance

Rxlist (n.d.)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 99

and mild uterine diseases (Pant 2013; Perfume workshop n.d.-a; Leander and Lüning

1967; Shanavaskhan et al. 2012; Devi et al. 2015; Panda and Mandal 2013;

Nongdam 2014; Mishra et al. 2008). The paste of leaves of Aerides multiﬂorum is

used for wounds, cuts, earaches, and consumed as a tonic (Perfume workshop n.d.-a;

Baral and Kurmi 2006; Basu et al. 1971; Behera et al. 2013; Raja 2017). The leaf of

Aerides odorata is applied in cuts, wounds, and tuberculosis, the fruit is used to heal

the wound. Leave juice and seeds are used in treating boils in ear, nose, and skin

disorders (Pant 2013; Perfume wor kshop n.d.-a; Leander and Lüning 1967; Baral

and Kurmi 2006; Basu et al. 1971; Behera et al. 2013). The whole plant of

Anocetochilus elatus is used to relief chest and abdominal pain and treats snake

bites (Raja 2017; Sherif et al. 2012; Jiang et al. 2015).

100 K. D. Mudoi et al.

The whole plant of Anocetochilus formosanus is used as an antipyretic, in

detoxiﬁcation, and treats tuberculosis, diabetes, bronchitis, infections in the kidney,

bladder, cramps, snake bites, stomach ache, inﬂammation, hematemesis, nocturnal

emission, nephritis, vaginal discharge, hepatitis, hypertension, and convulsions The

plant possesses antioxidant, anti-hyperglycemic, hepatoprotective, anticancerous

properties, and pharmacological effects, such as antiosteoporosis, antihyperliposis,

and antifatigue (Perfume workshop n.d.-a; Aswandi and Kholibrina 2021;

Nandkarni 1976). The leaf and stem of Ansellia africana are used for treating

madness. Besides it also possesses anti-acetylcholinesterase activity in treating

Alzheimer’s disease (Saleh-E-In et al. 2021; Bhattacharyya and Staden 2016). The

whole plant of Arundin a graminifolia is used for curing rheumatic, trauma, bleeding,

and snake bites. To relieve body aches root is used. In cracks scrapped bulbous stem

is applied on the foot-heels (Pant 2013; Aswandi and Kholibrina 2021; Kumar 2002;

Dakpa 2007).

Bletilla striata is used for tonic, against leucorrhea; leaves are used in treating

lung disease; tubers are used for regeneration of musc le and other tissues, in

hemorrhage dyspepsia, dysentery, fever, malignant ulcers, gastrointestinal disorders,

anthrax, malaria, eye diseases, ringworm, tumors, necrosis, silicosis, traumatic

injuries, coughs, chest pain, cures tuberculosis, sores, scaling, chapped skin, blood

puriﬁcation, strengthening, and lungs consolidation, malignant swellings, breast

cancer, pustules ulcers, demulcent, and expectorant (Perfume workshop n.d.-a;

Kong et al. 2003; Bulpitt et al. 2007). The Bulbophyllum odoratissimum plant is

used to cure fractures, pulmonary tuberculosis, hernia pain, infusion, or decoction is

used to treat tuberculosis and chronic inﬂammation (Perfume workshop n.d.-a; Chen

et al. 2008; Bhattacharjee 1998). The entire plant of Calanthe discolor is used for

improving blood ﬂow, circulation, abscesses, scrofula, rheumatism, bone pain, and

traumatic injuries, treating skin ulcers and hemorrhoids (Perfume workshop n.d.-a;

Yoshikawa et al. 1998). Changnienia amoena plant cools the blood, acts as anti-heat

and antitoxic, cures coughs, blood-streaked sputum, sores, and furuncles (Teoh

2016). The pseudo bulbs of Coelogyne cristata are used in constipation and aphro-

disiac (Pant and Raskoti 2013; Subedi et al. 2011; Pamarthi et al. 2019). Coelogyne

stricta pseudo bulb paste cures headaches and fever (Pamarthi et al. 2019; Yonzone

et al. 2012). Coelogyne ﬂaccida pseudo bulb paste cures headache and fever, juice

helps in indigestion (Teoh 2016; Pant and Raskoti 2013; Pamar thi et al. 2019).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 101

The rhizome paste of Cymbidium aloifolium is applied on fractured and

dislocated bones. Bulbs are used as demulcent agents (Pamarthi et al. 2019). The

root of Cymbidium ensifolium decoction used to treat gonorrhea and ﬂower decoc-

tion used in eye sore disorders (Tsering et al. 2017). The leaves of Cymbidium

giganteum are applied over wounds (Bulpitt 2005; Fonge et al. 2019; Linthoingambi

et al. 2013). The seed of Cymbidium goeringii is used to treat cuts and injuries; entire

plant parts are used in curing fractures (Teoh 2016). The leaf juice of Cymbidium

iridioides is used to cease blood; its powder as a tonic; pseudo bulbs and roots are

consumed in diarrhea (Aggarwal and Zettler 2010; Medhi and Chakrabarti 2009;

Arditti et al. 1982; Arditti and Ernst 1984). The whole plant of Cymbidium kanran is

used in heart puriﬁcation, cures cough and asthmatic problems, and its roots are used

to cure ascariasis and gastroenteritis. The whole plant of Cymbidium lancifolium is

used in the treatment of rheumatism, improves blood ﬂow, and traumatic injuries.

The whole plant of Cymbidium sinense is used in purifying the heart, lungs; treat

cough and asthma (Perfume workshop n.d.-a). The dried powdered pseudo bulb of

Cymbidium longifolium is consumed on an empty stomach and fresh shoot is used

for nervous disorders, madness, epilepsy, hysteria, rheumatism, and spasms. Salep

used as demulcent (Zhan et al. 2016; Teo h 2016; Yonzone et al. 2013).

The powdered roots of Cypripedium calceolus promote sleep and reduce pain and

tea prepared by the roots cures nerves and headaches (Singh and Dey 2005). The

whole plant of Cypripedium debile is used for improving blood ﬂow, swellings,

pain, and diuretic. Likely, Cypripedium formosanum is used to improve blood ﬂow,

menses, expels gas, pain and itching whereas roots along with stems are used in

treating malaria, snake bites, traumatic injury, and rheumatism. The roots and leaves

of Cypripedium guttatum are used in treating epilepsy (Perfume workshop n.d.-a).

The rhizomes, roots, and stems of Cypripedium macranthos are used to treat skin

disease, promote dieresis, swelling, and pain and improve the ﬂowing of blood; dried

ﬂowers are used to stop blood (Shimura et al. 2007). The rhizome of Cypripedium

parviﬂora helps to treat insomnia, fever, headache, neuralgia, emotional tension,

tumors, delirium, convulsions, anxiety, menstruate pain, and child birth (Moerman

1986; Grieve 1998; Kumar et al. 2005). The whole plant of Cypripedium pubescens

is used as antispasmodic, diaphoretic, hypnotic, sedative, tonic, diabetes, diarrhea,

dysentery, paralysis, and malnutrition, also in cases of nervous irritability, functions

of the brain and promotes sleep. The dry powder roots are used as drugs for joint

pains and treating stomach worms (Singh and Duggal 2009; Khory 1982).

The tubers of Dactylorhiza hatagirea are used as food and tonic and help in

healing wound and fever and control burns and bleeding (Arditti 1992, 1967, 1968;

Aggarwal and Zettler 2010; Arditti et al. 1982; Arditti and Ernst 1984). The leaves

and pseudo bulb paste of Dendrobium amoenum are applied on skin diseases, burnt

skin, and dislocated bones (Venkateswarlu et al. 2002). The leaf paste of

Dendrobium aphyllum is applied on deformed abnormal head of a new born baby

in order to form a normal shape (Pant 2013). The leaves of Dendrobium candidum

are used to treat diabetes (Wu et al. 2004). The stem of Dendrobium chrysanthum is

used as a tonic, enhances the immune system, and reduces fever. Leaves are used as

antipyretic and mild skin diseases, which beneﬁt the eyes (Bulpitt 2005; Jalal et al.

2008, 2010; Li et al. 2016). The whole plant of Dendrobium chrysotoxum possesses

antitumorous and anticancerous properties, stem and ﬂower extract is used as tonic

and leaf extract as antipyretic (Bulpitt et al. 2007; Sood et al. 2006; Joshi et al. 2009).

The pseudo bulb paste of Dendrobium crepidatum is used in fractured and dislocated

bones. Stems are used as a tonic, in arthritis and rheumatism (Joshi et al. 2009;

Reddy et al. 2001; Joshi and Joshi 2001). The leaves of Dendrobium crumenatum

are used to cure boils and pimples (Joshi and Joshi 2001). The pseudo bulb pulps of

Dendrobium densiﬂorum are used to cure boils, pimples, and various skin eruptions,

leaf paste is applied upon fractures bones, sprains, and inﬂammations (Arditti 1992;

Arditti et al. 1982; Arditti and Ernst 1984). The dried stems of Dendrobium

devonianum is used as an enhancer for the immune system (Cakova et al. 2017).

The stem of Dendrobium draconis are used in antipyretic and hematinic (Perfume

workshop n.d.-b). The whole plant of Dendrobium ﬁmbriatum is used during upset

of the liver and severe anxiety; leaves are used in bone fracture and as a tonic, the

pseudo bulbs are used in fever (Aggarwal and Zettler 2010; Arditti et al. 1982). The

pseudo bulb paste of Dendrobium heterocarpum is used in treating fractured and

bone dislocate (Arditti and Ernst 1984). The root, stem, and leaf of Dendrobium

lasianthera act as anticancer (Utami et al. 2017).

102 K. D. Mudoi et al.

The whole plant juice of Dendrobium longicornu is added to lukewarm water to

bath for fever; roots are boiled to feed the livestock, to remove cough; stem juice is

used to treat fever (Perfume workshop n.d.-b). The tender shoot tip juice of

Dendrobium macrostachyum is used for earaches (Zhan et al. 2016). The pseudo

bulb paste of Dendrobium moschatum is used for dislocated and fractured bone

(Reddy et al. 2001). The pseudo bulb extracts of Dendrobium nobile are used in

treating burns, and eye infections; the plant is used to cure pulmonary tuberculosis,

fever, general debility, ﬂatulence, dyspepsia, reduce salivation, parched, thirsty

mouth, night sweats, antiphlogistic, and tonic. Seeds are used to heal wounds;

stems to cure fever and tongue dryness; stems are used in longevity, aphrodisiac,

stomachic, and analgesic (Aggarwal and Zettler 2010; Arditti et al. 1982; Arditti

1967). Whole plant juice of Dendrobium ovatum cures stomach aches, excites bile,

and is a laxative for the intestines, curing constipation (Kirtikar and Basu 1981;

Caius 1986). The dried stem of Dendrobium primulinum acts as an enhancer for the

immune system (Pant and Thapa 2012). The pseudo bulb paste of Dendrobium

transparens is used in treating fractures and dislocated bones (Arditti and Ernst

1984). The stem of Dendrobium trigonopus is used to cure fever and anemia

(Perfume workshop n.d.-b). Doritis pulcherrima leaf is used to treat ear infections

(Perfume workshop n.d.-c).

The whole plant of Eria bambusifolia is used in treating hyper acidity and various

stomach aches (Zhan et al. 2016). The tubers of Eulophia dabia tubers are used as a

tonic and aphrodisiac help to cure stomach aches, and stimulate blood ﬂow, also

used for consumption mixed with milk, sugar, and ﬂavored species (Panwar et al.

2022). The tuber of Eulophia epidendraea is applied upon boils; controls pain in

breast feeding mother; cures tumor and diarrhea; acts as an appetizer, anthelmintic,

aphrodisiac, stomachic, worm infestation, stimulate appetite and puriﬁes blood

during heart troubles (Narkhede et al. 2016). The whole plant of Eulophia nuda is

used in stomachache and snake bites; the stems are used to stop bleeding and trauma

pain; a thick paste of tuber is applied on the stomach to kill intestinal worms, cures

rheumatoid arthritis, bronchitis, scrofulous glands, tumors, puriﬁes blood, used as a

tonic, acts as anti-aphrodisiac, demulcent, and anthelmintic. The leaf is used as a

vermifuge (Hada et al. 2020). The tuber of Gastrodia elata is used to cure stroke,

tetanus, migraine, headaches, backache, skin boils, ulcers, and pain in the lower

extremities; for generalized dermatitis dizziness, sleepiness, insomnia, high blood

pressure, blood circulation, rheumatism, numbness, and paralysis (Chen et al. 2014).

The root paste of Geodorum densiﬂorum is applied on insect bites and wounds; the

root paste by mixing with ghee and honey to correct menstrual disorders and the

poultice made from pseudo bulbs is used as a disinfectant (Sheelavantmath et al.

2000). The stem of Gymnadenia conopsea helps the kidney, treats cough, lactation

failure, sexual dysfunction, traumatic injuries, thrombosis, and chronic hepatitis

(Gustafsson 2000).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 103

The leaves and roots of Habenaria edgeworthii act as cooling and spermopiotic;

the pseudo bulb of Habenaria pectinata is used during diathesis bleeding, burning

sensation, fever, and phthisis (Singh and Duggal 2009). The root of Herminium

lanceum is beneﬁcial for the lungs and kidneys, strengthens muscles, bones, stops

bleeding, and treats tuberculosis (Perfume workshop n.d.-d). The whole plant of

Liparis odorata is soaked in wine for external use; tubers are used during stomach

disorders (Perfume workshop n.d.-e). The pseudo bulb of Malaxis acuminata is used

as a tonic, aphrodisiac, styptic, antidysentery, and febrifuge (Pushpa et al. 2011). The

stem and leaves of Papilionanthe teres are used for improving blood ﬂow and

reducing swellings. The whole plant of Pholidota articulata is used to remove gas

and reduce swelling, treat coughs, headaches, dizziness, ulcers, sores, traumatic

injuries, uterine, and menses problems. The roots and pseudo bulb paste of Pholidota

pallida are used to cure fever and induce sleep and juice to remove abdomen pain.

The whole plant of Platanthera chlorantha is used to strengthen the kidneys and

lungs, hernia, and sexual dysfunction (Perfume workshop n.d.-c).

The leaves and roots paste of Rhynchostylis retusa are used in rheumatism, leaf

juice is used in constipation, gastritis, acidity, and as emollient; root juice is used to

heal cuts and wounds; root is used to treat menstrual pain and arthritis; dry ﬂower is

used as emetic (Basu et al. 1971; Dakpa 2007; Bulpitt et al. 2007; Bhattacharjee

1998; Dash et al. 2008). Tubers of Satyrium nepalense are used to treat diarrhea,

dysentery, and malaria, consumed as an aphrodisiac, and used as a children’s growth

supplement. Juice is used in cuts and wounds (Gutierrez 2010; Baral and Kurmi

2006; Basu et al. 1971; Behera et al. 2013; Bulpitt et al. 2007). The pseudo bulb of

Spathoglottis plicata is used in rheumatic swelling; the hot fomentation is pressed on

to draw out pus from the infected part, helps in proper blood ﬂow and reduces pain

(Friesen and Friesen 2012). The whole plant of Thunia alba is used in treating

cough, pneumonia, bronchitis, bone break treatment, and injury (Mathew 2013).

The ﬂower juice of Vanda coerulea is used in treating glaucoma, cataract, and

blindness. The root of Vanda roxburghii is used to treat fever, dyspepsia, bronchitis,

cough, piles, snake bites, rheumatism, allied disorders, and nervous system disease

(Uprety et al. 2010). The dried ﬂower powdered juice of Vanda spathulata are used

to treat asthma, depression, enhance memory, antioxidant activity, and alleviate

chronic disease, and degenerative ailments such as cancer, autoimmune disorders,

hypertension, delay in aging process, and atherosclerosis (Jeline et al. 2021). The

leaf of Vanda tessellata is used in inﬂammation, rheumatism, dysentery, bronchitis,

dyspepsia, and fever (Chowdhury et al. 2014). The leaf, root, and ﬂower powdered

extract of Vanda testacea is used in nervous disorders, piles, inﬂammations, rheu-

matism, bronchitis, and anti-cancerous drugs (Kaur and Bhutani 2009). The fruit of

Vanilla planifolia is used to treat intestinal gas and fever, increase sexual desire, used

as ﬂavoring syrup and perfume fragrance (Rxlist n.d.).

104 K. D. Mudoi et al.

The phytochemicals such as alkaloids, ﬂavonoids, and glycosides made the

orchids therapeutically important (Hossain 2011); they are, however, mainly used

as nutraceuticals because the active principles responsible for their medicinal prop-

erties are yet to be identiﬁed with further accuracy.

7 Phytochemistry

Gas Chromatography and Mass Spectrometry (GC/MS) analyzed the essential oil

and the oleoresins for various medicinal orchids. In our present study, we accessed

and summarized the phytochemicals of 45 orchid species (Table 3).

Major phytochemicals reported in Ansellia africana namely n-Hexanal, Mesityl

oxide, 4-Heptenoic acid, 3,3-dimethyl-6-oxo-methyl ester, Pentadecanoic acid,

Succinic acid, 3,7-dimethyloct-6-en-1-yl pentyl ester, Linoleic acid, Linolenic

acid, l-Ascorbyl 2,6-Dipalmitate, Toluene, Ethylbenzene, Mesitylene, Erythro-1-

Phenylpropane-1,2-diol, Styrene, Hyacinthin, 2-Ethylbutyric acid,

3-methylbenzylester which possess cytotoxic effect against cancerous cell line

(Saleh-E-In et al. 2021). Gramniphenol, a potent marker reported in Arundina

graminifolia showed anti-tobacco mosaic virus activity (Gao et al. 2012). Phyto-

chemicals of B. striata showed major biological activity in aiding hemostasis,

cytotoxicity, antimicrobial, anti-inﬂammation, anti-oxidation, immunomodulation,

anti-ﬁbrosis, antiaging, and anti-allergy (He et al. 2017). Densiﬂorol B, the most

active compound reported from Bulbophyllum odoratissimum exhibit cytotoxic

activity against the ﬁve tested cell line s (Chen et al. 2008). Major stilbenoids,

ﬂaccidin, oxo ﬂaccidin and isoﬂaccidin were reported in Agrostophyllum callosum,

Coelogyne ﬂaccida (Majumder and Maiti 1988, 1989, 1991; Majumder et al. 1995).

5-hydroxy-3-methoxy-ﬂavone-7-O-[β-D-apiosyl-(1 → 6)]-β-D-glucoside, an alpha-

glucosidase inhibitor reported from Dendrobium devonianum (Sun et al. 2014).

Sesquiterpene such as alloaromadendrene, emmotin, an d picrotoxane from

Dendrobium nobile possesses immunomodulatory potential (Ye et al. 2002).

Dendroparishiol a marker reported from Dendrobium parishii exhibited antioxidant

and anti-inﬂammatory activity against RAW264.7 cells (Kongkatitham et al. 2018).

9, 10-dihydrophenanthrene, a novel marker reported from Eria bambusifolia showed

anticancer activity against the human cell line (Rui et al. 2016). Major aromatic

phytochemicals were reported in Platanthera chlorantha namely β-Ocimene, Lilac

Species Phytochemicals References

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 105

Table 3 Screening of phytochemicals in some medicinal orchids

Sl.

No.

1Anacamptis

pyramidalis

Disaccharide, Citric acid, Parishin

G isomer-1, Parishin G isomer-2,

Gastrodin derivative, Parishin B,

Gastrodin derivative, Parishin C,

Dihydroxybenzoic acid derivative,

Caffeic acid derivative, Acacetin

derivative, Oxo-dihydroxy-

octadecenoic acid, Trihyd roxy-

octadecenoic acid

Fawzi Mahomoodally et al.

(2020)

2Ansellia

africana

2,4,4-Trimethyl-1-hexene,

2-Hexene, 2,5,5-trimethyl,

2,3-Dimethyl-2-heptene,

Cyclopentane, 1,2,3,4,5-

pentamethyl, pentane, 1,2,3,4,5-

pen, Nonane 4,5 dimethyl, Octane

5-ethyl-2-methyl, n-Decane,

1-Undecane, 4-methyl, Dodecane,

Cyclohexane, (1,2,2-

trimethylbutyl), tetradecane,

pentadecane, Hexadecane

4-methyl, heptadecane,

Nonadecanol, Lignoceric alcohol,

cis-4-Hexen-1-ol, n-Hexanal,

Mesityl oxide, 4-Heptenoic acid,

3,3-dimethyl-6-oxo-methyl ester,

Pentadecanoic acid, Succinic acid,

3,7-dimethyloct-6-en-1-yl pentyl

ester, Linoleic acid, Linolenic acid,

l-Ascorbyl 2,6-Dipalmitate, Tolu-

ene, Ethylbenzene, Mesitylene,

Erythro-1-Phenylpropane-1,2-diol,

Styrene, Hyacinthin, 2-Ethylbutyric

acid, 3-methylbenzylester

Saleh-E-In et al. (2021)

3Arundina

graminifolia

graminibiben-zyls A, 5,12-

dihydroxy-3-methoxybibenzyl-6-

carboxylic acid, dihydropinosylvin,

2,5,2′,5′-tetrahydroxy-3-

methoxybibenzyl, rhapontigen,

pinosylvin, bauhiniastatin D,

arundinaol, coelonin, cucapitoside,

blestriarene A, isoshancidin,

obovatin, kaempferol-β-3-O-

glycos, dihydropinosylvin, 4′-

-methylpinosylvin, 3-(γ,-

γ-dimethylallyl)resveratrol, 5-(γ,γ

dimethylallyl)oxyresveratrol,

3-hydroxy-4,3′,5′-trimethoxy-

trans-stilbene, gramniphenol, 9'-

dehydroxy-vladinol, vladinol F,

Gao et al. (2012), Hu et al. (2013),

Zhang et al. (2021)

Species Phytochemicals

9-O-β-D-xylopyranoside-

vladinol F, 4,9-dihydroxy-4',7-

epoxy-8',9'-dinor-8,5'-neolignan-7'-

oic acid

(continued)

106 K. D. Mudoi et al.

Table 3 (continued)

Sl.

No. References

4Bletilla striata 3,3′-dihydroxy-5-methoxybibenzy,

gigantol, 5,4′-dimethoxybibenzyl-

3,3′-diol, 3 ′-hydroxy-5-

methoxybibenzyl-3-O-β-D-

glucopyranoside, 5-hydroxy-4-

(p-hydroxybenzyl)-3′,3-

dimethoxybibenzyl, bulbocol,

gymconopin D, bulbocodin D,

blestritin B, 4,7-dihydroxy-2-

methoxy-9,10-

dihydrophenanthrene, 9,10-

dihydro-4,7-

dimethoxyphenanthrene-2,8-diol,

blestriarene A, 2,4,7-trimethoxy-

phenanthrene, 7-hydroxy-2-

methoxyphenanthrene-3,4-dione,

3′,7′,7-trihydroxy-2,2′,4′-

-trimethoxy-[1,8′-biphenanthrene]-

3,4-dione, cyclomargenone,

β-sitosterol, stigmasterol,

protocatechuic acid, cinnamic acid,

p-hydroxybenzaldehyde,

3,7-dihydroxy-2,4,8-

trimethoxyphenanthrene, 9,10-

dihydro-4,7-

dimethoxyphenanthrene-2,8-diol,

9,10-dihydro-1-(4'-

hydroxybenzyl)-4,7-

dimethoxyphenanthrene-2,8-diol,

3',4"-dihydroxy-5',3",5"-

trimethoxybibenzyl, batatasin III

He et al. (2017), Woo et al. (2014)

5Bulbophyllum

odoratissimum

Moscatin, 7-hydroxy-2,3,4-

trimethoxy-9,10-

dihydrophenanthrene, coelonin,

densiﬂorol B, gigantol, batatasin

III, Tristin, vanillic acid,

syringaldehyde, 3,7-Dihydroxy-

2,4,6-trimethoxyphenanthrene,

Bulbophyllanthrone

Chen et al. (2008), Shariﬁ-Rad

et al. (2022)

6Coelogyne

cristata

Coelogin, coeloginin, 3,5,7-trihy-

droxy-1,2-dimethoxy-9,10-

dihydrophenanthrene, 3,5,7-trihy-

droxy-1,2-dimethoxyphenanthrene

Majumder et al. (2001)

Species Phytochemicals References

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 107

Table 3 (continued)

Sl.

No.

7Coelogyne

ﬂaccida

Callosin, ﬂaccidin, oxoﬂaccidin,

2,7-dihydroxy-6-methoxy-5H-

phenanthro [4,5-bcd] pyran-5-one

Majumder and Sen (1991),

Majumder and Maiti (1988,

1989), Majumder et al. (1995)

8Cymbidium

aloifolium

1,2 diarylethanes, 9,10

dihydrophenanthrene, 6-0-

methylcoelonin, batatasin III,

coelonin, gigantol, 5-hydroxy-3-

methoxy-1,4-phenanthraquinone,

Friedelin, sitosterol,

n-hexadecanoic acid, 9,12-

octadecadienoic acid, 9,12,15-

octadecatrienoic acid, octadecanoic

acid, phytol; 2-butyne;

2-cyclopenten-1-one; and

1,4-benzenedicarboxylic acid

Juneja et al. (1987), Barua et al.

(1990), Rampilla and Khasim

(2020)

9Cymbidium

ensifolium

Cymensiﬁns, cypripedin, and

gigantol

Jimoh et al. (2022)

10 Cymbidium

ﬁnlaysonianum

1-(4-Hydroxybenzyl)-4,6-

dimethoxy-9,10-

dihydrophenanthrene-2,7-diol,

Cymbinodin-A

Lertnitikul et al. (2018)

11 Cymbidium

giganteum

1,2-diarylethane, gigantol, 4ξ-(β-d-

glucopyranosyloxymethyl)-14-

α-methyl-22ξ,24ξ, 25,28-

tetrahydroxy-9,19-cyclo-5α,9-

β-ergostan-3-one

Juneja et al. (1985), Dahmén and

Leander (1978a)

12 Cymbidium

goeringii

Gigantol Won et al. (2006)

13 Cymbidium

kanran

Vicenin-2, Schaftoside isomer,

Schaftoside, Vicenin-3, Vitexin,

Isovitexin

Jeong et al. (2017)

14 Dendrobium

amoenum

3,4′-dihydroxy-5-methoxybibenzyl

and 4,4′-dihydroxy-3,3′,5-

trimethoxybibenzyl, 3,4,5-

trimethoxybenzaldehyde,

picrotoxinin, aduncin, 9,10-

dihydro-5H-phenanthro-(4,5-b,c,

d)-pyran, amoenumin, (E)-13-

docosenoic acid; oleic acid;

11-octadecenoic acid, methyl ester;

and hexadecanoic acid,

2,3-dihydroxypropyl ester,

aphyllone B, (R)-3,4-dihydroxy-

5,4′,α-trimethoxybibenzyl,

4-[2-[(2S,3S)-3-(4-hydroxy-3,5-

dimethoxyphenyl)-2-

hydroxymethyl-8-methoxy-2,3-

dihydrobenzo (Stewart and Grifﬁth

Venkateswarlu et al. (2002),

Majumder et al. (1999), Dahmén

and Leander (1978b), Veerraju

et al. (1989), Paudel and Pant

(2017)

Species Phytochemicals

1995; Kaushik ) dioxin-6-yl]

ethyl]-1-methoxyl benzene,

dendrocandin B, 4,4′-dihydroxy-

3,5-dimethoxybibenzyl,

3,4-dihydroxy-5,4′-

-dimethoxybibenzyl, 3-O-

methylgigantol, dendrophenol,

gigantol, dendrocandin C,

dendrocandin D, and 3,3′,4,4′-

-tetrahydroxy-5-methoxybibenzyl

1983

(continued)

108 K. D. Mudoi et al.

Table 3 (continued)

Sl.

No. References

15 Dendrobium

candidum

3,4′-dihydroxy-5-

methoxybibenzyl, uridine, sucrose,

adenosine

Li et al. (2008, 2009)

16 Dendrobium

chrysanthum

Denchrysan B, dengibsin,

moscatin, dendroﬂorin,

denchrysan A, moscatilin, gigantol,

batatasin III, Tristin,

4,9-dimethoxy-2,5-

dihydroxyphenanthrene,

3,4-dihydroxybenzoic acid, dibutyl

phthalate, stigmasterol, β-sitosterol,

daucosterol

Li et al. (2016)

17 Dendrobium

chrysotoxum

Chrysotoxols A and B, bibenzyls,

phenanthrenes, ﬂuorenones, cou-

marin, ﬂavonoid, gigantol, 3-O-

methylgigantol, moscatilin,

4-[2-(3-hydroxy-4-methoxyphenyl)

ethyl]-2,6-dimethoxyphenol,

crepidatin, chrysotoxine, erianin,

isoamoenylin, batatasin III, tristin,

nobilin C, moscatin, 2,5-dihydroxy-

4,9-dimethoxyphenanthrene,

confusarin, nudol, ﬁmbriatone,

1,5,6,7-tetramethoxy-2-

hydroxyphenanthrenol, 7-hydroxy-

2,3,4-trimethoxyphenanthrene,

1,2,6,7-tetrahydroxy-4-

methoxyphenanthrene,

2,4-dihydroxy-7-methoxy-9,10-

dihydrophenanthrene, erianthridin,

2,5-dihydroxy-4-methoxy-9,10-

dihydrophenanthrene, 1,4,7-trihy-

droxy-5-methoxy-9H-ﬂuoren-9-

one, nobilone, 6-methylesculetin,

and homoeriodictyol

Hu et al. (2012), Liu et al. (2022)

18 Dendrobium

crepidatum

Crepidatuols A, (±)-

homocrepidine A, Crepidatin,

crepidatumines A and B,

Li et al. (2013), Hu et al. (2016),

Xu et al. (2020, 2019b), Ding

et al. (2021)

Species Phytochemicals

dendrocrepidine B, crepidatumines

C and D, crepidine, isocrepidamine,

crepidamine, octahydroindolizine

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 109

Table 3 (continued)

Sl.

No. References

19 Dendrobium

densiﬂorum

Densiﬂorol, Dendroﬂorin Fan et al. (2001)

20 Dendrobium

devonianum

Quercetin, Taxifolin, Rutin,

Luteolin, Kaempferol, Myricetin,

(-)-Epiafzelechin,

5-Hydroxyauranetin, 6-C-Hexosyl-

hesperetin O-hexoside, 8-C-

Hexosyl-apigenin

O-feruloylhexoside, 8-C-Hexosyl-

apigenin O-hexosyl-O-hexoside,

8-C-Hexosyl-chrysoeriol

O-feruloylhexoside, Isorhamnetin

hexose-malonate, Isorhamnetin

O-acetyl-hexoside, Isorhamnetin-3-

O-rutinoside, Isoschaftoside,

Isovitexin, Isovitexin 7-O-gluco-

side, Jaceosidin, Kaempferide

3-O-β-D-glucuronide, Ladanein,

Naringenin, Nepetin, Peonidin 3-O-

glucoside chloride, Pinobanksin,

Quercitrin, Rhoifolin, Schaftoside,

Tamarixetin, Tangeretin, Tricin

7-O-hexoside, Tricin 7-O-hexosyl-

O-hexoside, Tricin

O-malonylhexoside, Tricin

O-saccharic acid, Tricin

O-sinapoylhexoside, Violanthin,

Vitexin, Vitexin 2''-O-β-L-

rhamnoside, Vitexin-2-O-D-

glucopyranoside, 5-hydroxy-3-

methoxy-ﬂavone-7-O-[β-d-apiosyl-

(1 → 6)]-β-d-glucoside

Zhao et al. (2021), Sun et al.

(2014)

21 Dendrobium

draconis

5-methoxy-7-hydroxy-9,

10-dihydro-1,4-

phenanthrenequinone, hircinol,

gigantol, batatasin, 7-methoxy-

9,10-dihydrophenanthrene-2,4,5-

triol

Sritularak et al. (2011)

22 Dendrobium

ﬁmbriatum

Plicatol B, hircinol, plicatol A, and

plicatol C, 1 bibenzyl (3′,4-

dihydroxy-3,5′-

-dimethoxybibenzyl), furostanol,

protodioscin, Denﬁgenin, gigantol-

5-O-β-d-glucopyranoside, 9,10-

dihydro-aphyllone A-5-O-β-d-

Talapatra et al. (1992), Xu et al.

(2017), Favre-Godal et al. (2022)

Species Phytochemicals

glucopyranoside, ﬁcusal-4-O-β-d-

glucopyranoside, botrydiol-15-

O-β-d-glucopyranoside

(continued)

110 K. D. Mudoi et al.

Table 3 (continued)

Sl.

No. References

23 Dendrobium

heterocarpum

Methyl 3-(4-hydroxyphenyl) propi-

onate, 3,4-dihydroxy-5,4’-

-dimethoxybibenzyl,

dendrocandin B,

dendrofalconerol A, syringaresinol,

batatasin III, 3-O-methylgigantol,

gigantol, moscatilin,

dendrocandin A, (S)-3,4,-

α-trihydroxy-4′,5-

dimethoxybibenzyl, densiﬂorol A,

dendrocandin I, dendrocandin F,

coelonin, carthamidin, 4-hydroxy-

2-methoxy-3,6-dimethylbenzoic

acid

Warinhomhoun et al. (2022),

Xiao-bei et al. (2019)

24 Dendrobium

longicornu

Longicornuol A,

4-[2-(3-hdroxyphenol)-1-

methoxyethyl]-2,6-

dimethoxyphenol, 5-hydroxy-7-

methoxy-9,10-

dihydrophenanthrene-1,4-dione,

7-methoxy-9,10-

dihydrophenanthrene-2,4,5-triol,

erythro-1-(4-O-β-D-

glucopyranosyl-3-methoxyphenyl)-

2-[4-(3-hydroxypropyl)-2,6-

dimethoxyphenoxy]-1,3-

propanediol, Longicornuol B

Hu et al. (2008b, 2010)

25 Dendrobium

nobile

Vitamin A Aldehyde; Longifolene;

1-Heptatriacotanol; Z,Z6,28-

Heptatriactontadien-2-One and

Dendroban-12-One,

alloaromadendrane, emmotin,

picrotoxane, dendronobilate, 4-O-

demethyl-nobilone, dendronobilate,

4-O-demethyl-nobilone

Ye et al. (2002), Cao et al. (2021),

Meitei et al. (2019)

26 Dendrobium

ovatum

Stilbenoid Pujari et al. (2021)

27 Dendrobium

parishii

(-)-Dendroparishiol Kongkatitham et al. (2018)

28 Dendrobium

primulinum

2,4,7-trihydroxy-9,10-

dihydrophenanthrene, denthyrsinol,

moscatin, moscatilin, gigantol,

batatasin III, tristin, 3,4,5-

trihydroxybibenzyl, 3,6,9-

Ye et al. (2016)

Species Phytochemicals

trihydroxy-3,4-dihydroanthracen-1

(2H)-one, -sitosterol, -daucosterol

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 111

Table 3 (continued)

Sl.

No. References

29 Dendrobium

thyrsiﬂorum

Denthyrsin, denthyrsinol,

denthyrsinone, 2,3,5-Trihydroxy-4-

methoxyphenanthrene,

3,7-Dihydroxy-2,4-

dimethoxyphenanthrene,

2,7-Dihydroxy-1,5,6-

trimethoxyphenanthrene,

Syringaresinol, Pinoresinol,

Ayapin, Scopoletin, and

6,7-Dimethoxycoumarin,

4, 7-dihydroxy-2-methoxy-9,

10-dihydrophenan-threne,

syringaldehyde, moscatin, gigantol,

batatasin Ill, tristin, stigmasterol

Zhang et al. (2005), Wrigley

(1960), Ruixuan et al. (2015)

30 Dendrobium

trigonopus

Trigonopols A and B, gigantol,

tristin, moscatin, hircinol,

naringenin, 3-(4-hydroxy-3-

methoxyphenyl)-2-propen-1-ol, (-

)-syringaresinol

Hu et al. (2008a)

31 Eria

bambusifolia

Erathrins A and B, bambusifolia,

batatasin III, tristin, 3-hydroxy-5-

methoxy bibenzyl, gigantol, 3′,5-

dimethoxy-9,9′-diacetyl-

4,7′-epoxy-3,8′-bilign-7-ene-4′-

-methol, and balanophonin

Rui et al. (2016)

32 Eulophia

epidendraea

β-sitosterol, β-sitosterol glucoside,

β-amyrin, lupeol

Maridass and Ramesh (2010)

33 Eulophia nuda Eulophiol, Nudol, 2,3,4,7-

tetramethoxyphenanthrene, 9,10-

dihydro-4-methoxyphenanthrene-

2,7-diol,

l,5-dimethoxyphenanthrene-2,7-

diol, 1,5,7-

trimethoxyphenanthrene-2,6-diol,

5,7-dimethoxyphenanthrene-2,6-

diol, 4,4,8,8-tetramethoxy-

[1,1-biphenanthrene]-2,2,7,7-

tetraol, 2,2,4,4,7,7,8,8-

octamethoxy-1,1-biphenanthrene,

Lupeol, 9,10-dihydro-2,5-

dimethoxyphenanthrene-1,7-diol,

9,10-dihydro-4-

methoxyphenanthrene-2,7-diol,

1,5-dimethoxyphenanthrene-2,7-

diol, 1,5,7,-

trimethoxyphenanthrene-2,6-diol,

Hada et al. (2020), Bhandari et al.

(1985), Tuchinda et al. (1988)

Species Phytochemicals

5,7-dimethoxyphenanthrene-2,6-

diol, and 4,4′,8,8′-tetramethoxy

[1,1′-biphenanthrene]-2,2′,7,7′-

-tetrol. 4-Hydroxybenzaldehyde,

4-hydroxybenzyl alcohol,

2,7-dihydroxy-3,4-

dimethoxyphenanthrene

(continued)

112 K. D. Mudoi et al.

Table 3 (continued)

Sl.

No. References

34 Gastrodia

elata

Parishins B and C, gastrodin A,

gastrol A

Lin et al. (1996), Li et al. (2007)

35 Gymnadenia

conopsea

Gymnoside, loroglossin,

dactylorhin, daucosterol, dioscin,

gymconopin, blestriarene,

2,6-dimethoxy phenol, eugenol,

4-hydroxybenzene, 4-methoxy

phenylpropanol, 4-ethoxy

phenylpropanol, contra-

hydroxybenzyl, dithioether,

syringol, syringaldehyde, gastrodin,

arabinose, xylose, lupenone,

4,4-dimethyl-5α-cholesta-8,14,24-

trien-3β-ol, lupeol, cirsimarin,

astragalin, kaempferol-7-O-gluco-

side, desmethylxanthohumol,

isorhamnetin, naringenin chalcone,

equol, galangin,

1-((4-hydroxyphenyl)methyl)-4-

methoxy-2,7-phenanthrenediol,

gymconopin A,9,10-dihydro-2-

methoxy-4,5-phenanthrenediol,

blestriarene A, gymconopin,

blestriarene B

Gustafsson (2000), Shang et al.

(2017)

36 Liparis

odorata

Anodendrosin A, Liparisglycoside,

Liparis alkaloid, 4-(O- β-D-

Glucopyranosyl)-3,5-bis(3-methyl-

2-butenyl) benzoic acid, Adeno-

sine, D-α-2-Alanin,

p-Hydroxybenzoic acid

Liang et al. (2019)

37 Malaxis

acuminata

Catechin, phloridzin, rutin, Caffeic

acid, chlorogenic acid, ellagic acid,

3-hydroxy benzoic, 4-hydroxy

benzoic, protocatechuic acid,

3-hydroxy cinnamic acid,

p-coumaric acid, Stigmasterol and

β-sitosterol, Sibutramine, limonene,

diethylene glycol, p-cymene, euge-

nol, benzene, piperitone, glycerol,

ribitol, and myo-inositol,

6-octadecenoic acid,

Suyal et al. (2020)

Species Phytochemicals

8-octadecenoic acid, 9-octadecenal,

batatasin III, bulbophythrin A, butyl

oleate, cerasynt, cis-oleic acid,

cyclopentadecanolide, diethyl

phthalate, cyclopentanetridecanoic

acid

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 113

Table 3 (continued)

Sl.

No. References

38 Phalaenopsis

cornucervi

1,2-saturated pyrrolizidine mono-

esters, T-phalaenopsine

Frölich et al. (2006)

39 Pholidota

pallida

Oelonin, lusianthridin, ﬂavanthrin,

batatasin-III, 3′,5-dihydroxy-2-

(4-hydroxybenzyl)-3-

methoxybibenzyl, gigantol,

3-[2-(3-hydroxyphenyl) ethyl]-2,4-

bis[(4-hydroxyphenyl) methyl]-5-

methoxyphenol, hydroxytyrosyl

butyrate, (24R)-ethylcholest-5-en-

3-ol-7-one, taraxerone, friedelin,

hydroxytyrosyl

Yu et al. (2021)

40 Platanthera

chlorantha

β-Ocimene, Lilac aldehyde,

β-Elemene, α-Bergamotene,

Cedrene, Germacrene D,

Pentadecane, b-Bisabolene,

b-Sesquiphellandrene, 1,2,3-

Trimethoxy-5-(2-propenyl) ben-

zene, Tetradecanal, Benzophenone,

Galaxolide, Docosane, Tetradecyl

benzoate

D'Auria et al. (2020)

41 Platanus

acerifolia

5,7,40-trihydroxy-8-

(1, 1-dimethylallyl)-30-

methoxyﬂavonol, 5,7,40-trihy-

droxy-60-prenyl-30-

methoxyﬂavonol, Kaempferol-3-O-

a-L-(300-E-p-coumaroyl)-

rhamnoside, Quercetin-3-O-α-l-

(2″-E-p-coumaroyl-3″-Z-p-

coumaroyl)-rhamnopyranoside (E,

Z-3′-hydroxyplatanoside, and

quercetin-3-O-α-l-(2″-Z-p-

coumaroyl-3″-E-p-coumaroyl)-

rhamnopyranoside (Z,E-3′-

-hydroxyplatanoside, 8-methoxy-6-

C-methyl-5,7-dihydroxyﬂavonol,

8-C-(1,1-dimethyl-2-propen-1-yl)-

5,7-dihydroxyﬂavonol, and 8-C-

(1,1-dimethyl-2-propen-1-yl)-4′-

-methoxy-5,7-dihydroxyﬂavonol

Wu et al. (2022), Kaouadji

(1989), Thai et al. (2016)

42 Thunia alba Batatasin-III, lusianthridin,

3,7-dihydroxy-2,4-

Majumder et al. (1998), Ya-ping

et al. (2019), Yan et al. (2016)

Species Phytochemicals

dimethoxyphenanthrene,

3,7-dihydroxy-2,4,8-

trimethoxyphenanthrene,

cirrhopetalanthrin and ﬂavanthrin,

hircinol, scoparone, β-sitosterol,

3,7-dihydroxy-2,4-

dimethoxyphenanthrene,

lusianthridin, coelonin, thunalbene

aldehyde, β-Elemene, α-Bergamotene, Cedrene, Germacrene D, Pentadecane,

b-Bisabolene, b-Sesquiphellandrene, 1,2,3-Trimethoxy-5-(2-propenyl) benzene,

Tetradecanal, Benzophenone, Galaxolide, Docosane, Tetradecyl benzoate (D'Auria

et al. 2020). Quercetin-3-O-α-L-(2″-E-p-coumaroyl-3″-Z-p-coumaroyl)-

rhamnopyranoside (E, Z-3′-hydroxyplatanoside and quercetin-3-O-α-L-(2″-Z-p-

coumaroyl-3″-E-p-coumaroyl)-rhamnopyranoside (Z, E-3′-hydroxyplatanoside)

markers reported from Platanus acerifolia. The leaves exhibit antimicrobial activity

against Staphylococcus aureus (Wu et al. 2022). Phytochemicals reported in genus

Vanda possess major pharmacological activities, markers such as stigmasterol,

γ-sitosterol, β-sitosterol, β-sitosterol-D-glucoside, tetracosylferulate possess anti-

aging, antimicrobial, anti-in ﬂammatory, antioxidant, neuroprotective, membrane

stabilizing, and hepato-protective activities (Khan et al. 2019).

114 K. D. Mudoi et al.

Table 3 (continued)

Sl.

No. References

43 Vanda

coerulea

Imbricatin, methoxycoelonin,

gigantol, phenanthropyrans,

bibenzyl, dihydrophenanthrenes

Simmler et al. (2009)

44 Vanda

tessellate

Tessalatin, Oxo-tessallatin,

2,5-Dimethoxy-6,8-dihydroxy iso-

ﬂavone, Gallic acid, 2.7.7-

Trimethyl bicycle () heptanes,

Octacosanol, Heptacosane

Khan et al. (2019)

45 Vanda

roxburghii

Stigmasterol, γ-sitosterol,

β-sitosterol, β-sitosterol-D-gluco-

side, tetracosylferulate

Khan et al. (2019)

46 Vanilla

planifolia

Vanillin Podstolski et al. (2002)

7.1 Secondary Metabolites

A wide range of secondary metabolites is present in Orchids, of which only a very

slight portion was analyzed. Normally several phytochemicals viz., alkaloids, sapo-

nins, ﬂavonoids, anthocyanins, carotenoids, polyphenols, sterols, etc. were produced

and integrated into in vitro culture of orchids (Mulabagal and Tsay 2004; Yesil-

Celiktas et al. 2007; Shinde et al. 2010). Among them, polyphenols were responsible

for their crucial role in curing many degenerative and age-linked ailments (Brewer

2011; Procházková et al. 2011). Likely, other bioactive compounds like ﬂavonoids,

tannins, and alkaloids were bestowed for the medication of several chronic diseases

(Lu et al. 2004; Zhang et al. 2005; Harris and Brannan 2009).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 115

7.1.1 Bioactive Compounds

Various plant parts (leaf, root, and pseudobulb) of orchids possess a group of

important phenolic acids such as gentisic acid, gallic acid, salicylic acid,

protocatechuic acid, syringic acid, caffeic acid, sinapic acid, ferulic acid as well as

ﬂavonoids viz., catechin, apigenin, myricetin, naringin, rutin, quercetin, kaempferol,

and alkaloids viz., chysine, drobine, dendronine, grandifolin, crepidine, and vanilin

in higher concentration. In in vitro rais ed plants, bioactive compounds were more

dominant than in wild plants of medicinal orchids (Fig. 2).

The majority of bioactive compounds viz., ayapin, n-octastylferulate, crepidatin,

confusarin, physcion, scopolin, rhein, ﬁmbriatone, and β-sitosterol were reported in

Dendrobium ﬁmbriatum which were important for pharmacological point of view

(Paul et al. 2017; Bi et al. 2003; Shailajan et al. 2015). However, studies on the

phytochemical analysis of medicinal orchids raised in vitro are very few

(Bhattacharyya et al. 2014, 2015, 2018, 2016a,b; Bhattacharyya and Staden 2016;

Giri et al. 2012b; Bose et al. 2017). A bioactive compound such as bisbenzyl erianin

was isolated from the callus culture of Dendrobium chrysotoxum which was the

potential as an antioxidant, antitumor, and antiangiogenic agent (Zhan et al. 2016).

The presence of polyphenols was reported in Habenaria edgeworthii culture (Giri

et al. 2012a). Different biochemical constituents like total phenolic, ﬂavonoid,

alkaloids, and tannins contents were analyzed and comparisons were reported

between the various parts of mother plants and micropropagated plants of

Dendrobium nobile (Bhatt acharyya et al. 2014). Compounds with higher concen-

trations are reported in micropropagated plants of Herminium lanceum (Singh and

Babbar 2016) and Habenaria edgeworthii (Giri et al. 2012a) than in wild plants. The

phytochemical evaluation of various parts of the mother plant and in vitro propa-

gated plants of Bulbophyllum odoratissimum was performed by using HPLC (Prasad

et al. 2021). Extracts of Dendrobium crepidatum contained bioactive compounds

like tetracosane, hexadecanoic acid, triacontane, phenol derivatives, and

tetradecanoic acids are responsible for antioxidant and cytotoxic activities (Paudel

et al. 2019).

7.1.2 Biological Activity

Antioxidant Activity

Bioactive components exhibited vigorous antioxidant properties in divergent in vitro

methods which showed high scavenging potentiality to various Reactive Oxygen

116 K. D. Mudoi et al.

Fig. 2 Chemical structure

of bioactive molecules of

medicinal orchids (Drawn in

Chemdraw 8.0)

Species (ROS) viz. hydroxyl radical, peroxynitrite, superoxide anion, and

hypochlorous acid (Halliwell 2008). Unlike synthetic antioxidants, vigorous studies

were conducted on antioxidants present in natural fruits, vegetables and medicinal

plants, which are considered less toxic due to their effective free radical scavenging

activity.

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 117

1,1- diphenyl-2-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power

(FRAP) assay were used for the analysis of the antioxidant activity of the plant

extracts of mother and micropropagated Dendrobium nobile plants (Cao et al. 2021).

Both the assays describe the antioxidant response of Dendrobium nobile determining

the high antioxidant potential in samples of leaf due to its high content of poly-

phenols, alkaloids, and ﬂavonoids. Among the different solvents and plant parts of

the tested species, the DPPH activity of the methanolic leaf extraction was the

highest (89.8 ± 2.9%), but the activity of radical scavenging of the chloroform

leaf extraction was the lowest (28 ± 2.9%) of the micropropagated plant. D. nobile

plantlets grown through tissue culture reported higher levels of free radical scav-

enging activity than mother plants (Bhattacharyya et al. 2014). Total phenol content

(TPC), radical scavenging activity DPPH and ABTS (2,2′-azino-bis

(3-ethylbenzothiazoline-6-sulfonic acid), Total Flavonoid Content (TFC) as well

as total reducing power ability is being reported from all plant material extracts of

mother plants and in vitro-cultured plants of Bulbophyllum odoratissimum (Prasad

et al. 2021). DPPH radical scavenging activity was studied in some of the following

orchid species viz. Acampe papillosa, Aer ides odorata, Bulbophyllum lilacinum,

Arundina graminifolia, Cymbidium aloifolium, Dendrobium aphyllum,

Papilionanthe teres, Luisia zeylanica, Dendrobium tortile, Rhynchostylis retusa

(Rahman and Huda 2021); Rhynchostele rossii (Gutiérrez-Sánchez et al. 2020);

Dendrobium candidum (Wang et al. 2016); Dendrobium chrysanthum (Aswandi

and Kholibrina 2021); Dendrobium draconis (Sritularak et al. 2011); Pholidota

articulata (Singh et al. 2016a); Papilionanthe teres (Mazumder et al. 2010);

Geodorum densiﬂorum (Keerthiga and Anand 2014). DPPH assay measures the

total phenolic, alkaloid and ﬂavonoid content by using Folin-Ciocalteu, spectropho-

tometry and modiﬁed acid-alkalimetry methods in Dendrobium crumenatum

(Topriyani 2013). DPPH radical, column chromatography Diaion HP-20 or

reverse-phase silica gel column chromatography was studied in Gymnadenia

conopsea (Shang et al. 2017). A DPPH radical, spectrophotometric method, Liquid

Chromatography Mass Spectrometry (LC-MS) was studied in Paphiopedilum

villosum (Khamchatra et al. 2016). DPPH and ABTS assay were studied in Cym-

bidium kanran (Axiot is et al. 2022); Dactylorhiza hatagirea (Kumari et al. 2022);

Dendrobium moschatum (Robustelli della Cuna et al. 2018); Geodorum densiﬂorum

(Keerthiga and Anand 2014); Gastrodia elata (Song et al. 2016). DPPH, ABTS

radical scavenging assays and reducing capaci ty assays have been studied in

Dendrobium aphyllum (Liu et al. 2017) and Dendrobium macrostachyum

(Sukumaran and Yadav 2016). DPPH, ABTS, and metal chelating in Malaxis

acuminata (Bose et al. 2017) and in Dendrobium nobile hydroxyl radicals scaveng-

ing assay was also studied (Luo et al. 2010). MTT (3-(4, 5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide) assay in Dendrobium aphyllum (Liu et al. 2018) and

DPPH assay in Dendrobium densiﬂorum (Pant et al. 2022), and in Dendrobium

crepidatum by using GC–MS (Gas Chromatography and Mass Spectrometry) was

used to identify the compounds (Paudel et al. 2019). DPPH, ORAC, and deoxyri-

bose assays in Dendrobium parishii (Raja 2017); DPPH scavenging activity, reduc-

ing power and chelating activity against iron ions (Fe

2+

)in Dendrobium candidum

(Ng et al. 2012). DPPH and FRAP assay were studied in Dendrobium devonianum

(Wang et al. 2018) and Dendrobium ﬁmbriatum (Paul and Kumaria 2020). Deoxy-

ribose assays, non-site-speciﬁc scavenging assays, or antioxidants and iron ions, also

known as site-speciﬁc scavenging assays have been studied in Dendrobium

chrysotoxum (Zhao et al. 2007) (Table 4).

118 K. D. Mudoi et al.

Antimicrobial Activity

Five different multidrug resistance (MDR) bacterial clinical isolates were used for

testing the antibacterial activity of the epiphytic orchid Pleione maculata which

includes Escherichia coli (2461), Enterococcus sp. (2449), Staphylococcus aureus

(2413), Serratia sp. (2442), and Acinetobacter sp. (2457) along with

antimycobacterial activity with Mycobacterium tuberculosis strain (H37Rv)

(Bhatnagar and Ghosal 2018). Likely methanolic extracts of tubers of Satyrium

nepalense were studied against both Gram-negative and -positive food pathogenic

bacteria namely Staphylococcus mutans, Pseudomonas aeruginosa, Staphylococcus

aureus, and Klebsiella pneumonia and 6 mg/100 μL concentration was responsible

for the minimal effect against all the tested microorganisms (Mishra and Saklani

2012).

Ethanolic and hexane extracts of Coelogyne cristata and Coelogyne ﬁmbriata,

leaves and pseudobulbs were explored against human pathogens like Gram-positive

Bacillus cereus (ATCC 14579), Staphylococcus aureus (ATCC 12600), and Gram-

negative Escherichia coli (ATCC 10798), Yersinia enterocolitica (ATCC 9610), and

Klebsiella pneumonia (ATCC BAA-3079) bacteria. Only 70% of ethanolic leaf

extracts inhibited the growth of the investigated human pathogens (Pyakurel and

Gurung 2008; Subedi 2002; Wati et al. 2021; Subedi et al. 2013). Methanolic and

water extract of Peristylus densus showed better antimicrobial activity against

bacterial and fungal strains with an inhibition zone of 8–10 mm when tested against

S. typhi, P. aeruginosa, S. aureus, E.coli, and Aspergillus niger (Jagtap 2015).

Methanolic and ethanolic extract of Malaxis acuminata revealed strong antimicro-

bial activity against P. aeruginosa and S. aureus strain in Minimum Inhibitory

Concentration (MIC) assay and Butanol extract showed a strong inhibition zone of

32 mm compared to control 28 mm against Candida albicans (Suyal et al. 2020).

Ethyl acetate extract showed signiﬁcant antimicrobial activity against bacterial

strains K. pneumoniae, S. enteric and E. coli with an inhibition zone of 14–18 mm

in Pholidota articulata (Singh et al. 2016b). Whereas ethanolic extract of the species

showed antimicrobial activity against microbial strains S. aureus, Vibrio cholerae,

B. subtilis, E. coli, and K. pneumoniae with inhibition zone ranges from 9 to 12 mm.

No activity was observed in V. cholerae (Marasini and Joshi 2012). Ethanolic extract

Species Antioxidant activity References

(continued)

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 119

Table 4 Testing of antioxidant activity of some medicinal orchids

Sl

No.

1Cymbidium

kanran

DPPH and ABTS assays Axiotis et al.

(2022)

2Dactylorhiza

hatagirea

DPPH and ABTS assays. Further, UPLC-DAD

analysis

Kumari et al.

(2022)

3Dendrobium

aphyllum

DPPH and ABTS-free radical scavenging assays

and the reducing power assay. MTT assay

Liu et al. (2017)

4Dendrobium

candidum

DPPH scavenging activity, 2,2-diphenyl-1-

picrylhydrazyl (DPPH) scavenging activity,

reducing power, and ferrous ion (Fe

2+

) chelating

activity

Wang et al.

(2016), Ng et al.

(2012)

5Dendrobium

chrysanthum

DPPH radical scavenging activity Xiao-Ling et al.

(2014)

6Dendrobium

chrysotoxum

Deoxyribose assay, non-site-speciﬁc scavenging

assay) or antioxidants and iron ions (referred as a

site-speciﬁc scavenging assay)

Zhao et al. (2007)

7Dendrobium

crepidatum

DPPH (2, 2-diphenyl-1-picrylhydrazyl) and MTT

(3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide) assays

Paudel et al.

(2019)

8Dendrobium

crumenatum

1-1 Diphenyl-2-picrylhydrazyl (DPPH) method,

measurement of total phenol, ﬂavonoid, and alka-

loid content using Folin-Ciocalteu method, spec-

trophotometry method, and modiﬁed acid-

alkalimeter method

Topriyani (2013)

9Dendrobium

draconis

DPPH-free radical assay Sritularak et al.

(2011)

10 Dendrobium

densiﬂorum

DPPH and MTT assays Pant et al. (2022)

11 Dendrobium

devonianum

DPPH Radical-Scavenging Assay, Ferric Reduc-

ing Antioxidant Power (FRAP) Assay

Wang et al.

(2018)

12 Dendrobium

ﬁmbriatum

DPPH and FRAP assay Paul and Kumaria

(2020)

13 Dendrobium

macrostachyum

DPPH, ABTS radical scavenging, and reducing

power activity

Sukumaran and

Yadav (2016)

14 Dendrobium

moschatum

DPPH assay and ABTS assay Robustelli della

Cuna et al. (2018)

15 Dendrobium

nobile

Free radical scavenging activity assay; ABTS

assay; DPPH assay; hydroxyl radicals scavenging

assay

Luo et al. (2010)

16 Dendrobium

parishii

DPPH, ORAC, and deoxyribose assays Kongkatitham

et al. (2018)

17 Gastrodia elata The DPPH and ABTS radical scavenging activities Song et al. (2016)

18 Geodorum

densiﬂorum

DPPH method (1,1-diphenyl-2-picrylhydrazine) Keerthiga and

Anand (2014)

19 Gymnadenia

conopsea

Diaion HP-20 column chromatography (reverse-

phase silica gel column chromatography, DPPH

radical

Shang et al.

(2017)

Species Antioxidant activity References

of Pholidota imbricata showed effectiveness against S. aureus, V. cholerae,

B. subtilis, E. coli, and K. pneumonia microbia l strains with inhibition zone ranges

from 8 to 14 mm (Marasini and Joshi 2012). Rhyzopus stolonifer, Candida albicans,

and Mucor sp. were tested with the different orchid species. No activity against

fungal organisms reported in Coelogyne stricta (leaf), Coelogyne stricta

(Pseudobulb), and Dendrobium amoenum. Whereas Pholidota imbricata and

P. articulata extracts showed ﬁne activity. Dendrobium nobile, Eria spicata,

Rynchostylis retusa, Bulbophyllum afﬁne, and Vanda cristata showed very weak

to moderate activity against selected fungal pathogens (Marasini and Joshi 2012).

120 K. D. Mudoi et al.

Table 4 (continued)

Sl

No.

20 Malaxis

acuminata

DPPH, metal chelating, and ABTS Bose et al. (2017)

21 Paphiopedilum

villosum

Anti-free radical activity (DPPH), spectrophoto-

metric methods, liquid chromatography coupled to

mass spectrometry (LC-MS)

Khamchatra et al.

(2016)

22 Papilionanthe

teres

DPPH assay Mazumder et al.

(2010)

23 Pholidota

articulata

DPPH radical scavenging Singh et al.

(2016a,b)

Cytotoxic Activity

The cytotoxic activity of crude extracts from Dendrobium longiﬂorum plants was

determined by the Mean Transit Time (MTT) assay (Mosmann 1983; Sargent and

Taylor 1989). This study tested tumor cells of the human brain (U251) and cervical

cancer cells (HeLa). The cytotoxicity results of D. longicornu acetonic extract

showed a signiﬁcant cell growth inhibitory effect on the U251 cell line which may

be due to high levels of ﬂavonoids, while ethanolic extract had no signiﬁcant

cytotoxic activity on U251 cells. Similarly, the higher ﬂavonoid levels in the

ethanolic extract of D. longicornu showed signiﬁcant results on the cytotoxic

activity of the HeLa cell line. The cytotoxic activity of ﬂavonoids has been described

by previous researchers (Patel and Patel 2011; Awah et al. 2012; Jeune et al. 2005).

Methanolic extract of the whole plant of Pleione maculata was tested for cell

cytotoxicity and found to be within permissible limit, i.e., 7% at MIC assay. This

supports scientiﬁc evidence in favor of folk medicinal utilization of Pleione

maculata for various ailment treatments (Bhatnagar and Ghosal 2018). However,

no cytotoxic effect was observed at an extract dosage of 50–100 μg/mL in the

methanolic extract of Pholidota articulata, whereas 200–400 μg/mL of the extract

showed better activity in HeLa cells (IC50 673.04) compared to U251 cells (IC50

3170.55). The control showed a better cytotoxic effect (Joshi et al. 2020). Similarly

in Papilionanthe uniﬂora no cytotoxic effect was observed at a methanolic extract

dosage of 50–100 μg/mL, whereas 200–400 μg/mL of the extract showed better

activity in HeLa cells (IC50 781.85) compared to U251 cells (IC50 2585.88) and

control showed better cytotoxic effect (Joshi et al. 2020).

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 121

The cytotoxic activity of Dendrobium crepidatum was determined against HeLa

(Human Cervical Cancer) and U251 (Human Glioblastoma) cell lines. The extract

contains bioactive compounds like tetracosane, tetradecanoic acid, triacontane,

phenol derivatives, and hexadecanoic acid which cause cytotoxic activity. The

percentage of growth inhibition of HeLa cells for extraction of hexane (DCH) at

100 g/mL and chloroform extract (DCC) at 800 g/mL was found to vary from 19.84

to 4.31% and 81.49–0.43%, respectively. Whereas higher growth inhibition pe rcent-

age was recorded in DCC at 800 μg/mL and in the extraction of acetone (DCA) at

400 μg/mL (74.35–0.59%) of HeLa cell, which was signiﬁcantly different compared

to other extracts. Likewise, ethanol extracts (DCE) at 100 μg/mL and methanol

extracts (DCM) at 200 μg/mL showed signiﬁcantly higher growth inhibition per-

centages of HeLa cells (Paudel et al. 2019).

8 Economics

Orchids are popular due to their attractive and long-lasting ﬂowers, with unique

shapes and forms. This is a ﬂowering plant consisting of diverse genera and species.

Nowadays using the micropropagation technique it has become easy to multiply

some of the rare medicinal orchids. Flowers have bagged a signiﬁcant position in

present-day contemporary society. Therefore, a potential pressure for ﬂowers was

created especially in terms of the orchid ﬂower as they have a plethora of ﬂower

forms and colors. As Orchid reproduction is in a very germinal stage in India,

different medicinal orchid varieties can be reproduced by adopting a well-planned

Orchid augmentation strategy for the cut-ﬂower trade.

Orchids were the ﬁrst horticultural crop mass multiplied successfully through the

micropropagation technique and the commercial aspects of this group were being

increasing day by day for their medicinal importance. Commercial Tissue Culture

laboratories around the globe have aided the orchid’s mass multiplication and helped

the orchid industry revolutionize in the form of cut ﬂower business in several

countries. The Indian ﬂower market is expected to grow to INR 661 billion by

2026. North East India, along with Sikkim, Arunachal Pradesh, and Himachal

Pradesh, is the orchid-rich state in the country. In southern India, Kerala and

Tamil Nadu have high humidity, low temperatures, abundant rainfall, and a pleasant

climate suitable for commercial orchid cultivation. The orchid industry in India is in

its infancy in terms of in vitro micropropagation or commercial cult ivation. This is

due to inappropriate or unsuitable planting material for large-scale cultivation, a

deﬁcit of technology for commercial mass propagation techniques, a lack of post-

harvest commercial techniques for the cut-ﬂower market for international trade,

export policies, inappropriate commercial planting methods, etc. However, in

India, it can be possible to grow commercially viable orchid varieties such as

Cattleya, Cymbidium, Dendrobium, Oncidiums, Phalaenopsis, Paphiopedilum, and

Vandaceous for cut ﬂower production. Presently, the inward demand for orchid

cut-ﬂower is mainly reﬁlled through imports from outside India. However, wi th the

installation of in vitro propagation technology, cost-effective greenhouses, and

post-harvest and storage technology, the orchid cut-ﬂower industry can commence

in other parts of India also.

122 K. D. Mudoi et al.

According to the National Horticultural Database released by the National Hor-

ticultural Administration, in 2020–2021, the ﬂower planting area in India is 322,000

hectares, producing 2152 thousand tones of scattered ﬂowers and 828,000 tones of

cut ﬂowers. Growing orchids is more than just a pleasure these days. This is an

international trade that accounts for about 8% of the world’s horticultural business

and has the potential to change a country’s economic outlook.

According to Biotech Consortium India Limited (Biotechnology Division) and

Agri-Business of Small Farmers’ Consortium, Indian Tissue Culture Market

Research, 2005, Dendrobium sp. as cut ﬂowers and Vanilla as spice are the most

important plants in India which are suitable for micropropagation. Growing orchids

in India, different agro-climatic regions, low labor costs, and accelerating high-end

customer markets create a successful impact on society (Singh et al. 2008). But, the

orchid cut ﬂower business is consistently retarded by the unruly condition in

airports; large numbers of infected and deserted cut ﬂowers; moreover chemically

processed ﬂowers are rejected in Indian cities for violation of bio-safety norms

(De 2008).

Presently, worth millions of dollars industry of orchid cut ﬂower are ﬂourishing in

different countries such as Malaysia, Australia, Thailand, and Singapore among the

top ten cut ﬂowers of the world, the cut ﬂower grasps sixth position and 3%

Cymbidium orchid alone contributes in this list (De and Debnath 2011).

9 Conclusion

Biotechnological interventions and plant tissue culture techniques are accelerating

the large-scale reproduction of the delicate and rare medicinal orchid for its potential

uses as therapeutics. Since orchids are exotic breeders, they propagate by seed to

produce hybrid plants. Therefore, protocols that allow regeneration from different

vegetative parts of the plant are needed to achieve suitable types of micropropagation

of medicinal orchids, which have shown amazing developments in germplasm

conservation in recent years. Hardening and acclimatization of in vitro-propagated

orchids have maintained in different ratios of the organic medi um before ex vitro

survivability. In recent years, as a research tool addition to being used, plant tissue

culture techniques have also been of great industrial signiﬁcance in the plant

propagation ﬁeld, plant improvement, and secondary metabolites production.

Furthermore, testing of clonal ﬁdelity of micropropagated medicinal orchids by

using markers like RAPD, ISSR, and SCOT can be adequately utilized in the

sustainable implementation of plant genetic resources by identifying and eliminating

the difﬁculties of somaclonal variations. However, from various parts of the in vitro-

raised medicinal orchid many compounds have been isolated which are a good

source of bioactive molecules as well as phytochemicals. Antioxidant activities

and ethnomedicinal properties have been offering better possibilities for the occur-

rence of value-added products, for the treatment of diseases with herbal medicines to

boost health beneﬁts.

Biotechnological Interventions and Societal Impacts of Some Medicinal Orchids 123

Similar to micropropagation technology, synthetic seed technology has attracted

much attention in recent years due to its broader application of germplasm conser-

vation in natural habitats. Although little progress has been made in proving the

feasibility of synseeds, their successful implementation in the conservation of orchid

ornamental/medicinal genetic resources is achievable.

Emphasis on eco-rehabilitation study provides a new gateway for ex situ conser-

vation of in vitro-raised medicinal orchids in their natural habitats. The host tree and

orchid species symbiosis still maintains a proper balance for further reintroduction

and population enhancement for the practical conservation of important orchids.

Orchids have both ﬂower value and medicinal value and are more demanding in the

international market. Endemic and rare orchids have a plethora of ﬂower shapes and

colors that require scientiﬁc attention for their use in the cut ﬂower industry.

Comprehensive research is still necessary to extensively study the different

orchid species for various ailments. However, due to limited understanding and

knowledge about the therapeutic values of these locally available plants, the use of

orchids in the traditional healing process is restricted. For commercial scale, very

less effort has been made for medicinal orchid cultivation due to its small size

population and restriction in distribution. Different p recious orchid species that

have reached either the threatened or extinct category can survive with biotechno-

logical interventions and human support for their mass propagation. Therefore, to

meet the current need for medicinal orchids and to reduce the pressure on its natural

population, plant tissue culture can be an acceptable alternative for its sustainable

utilization which is the need of the hour.

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Gene Expression Proﬁling in Orchid

Mycorrhizae to Decipher the Molecular

Mechanisms of Plant–Fungus Interactions

Silvia De Rose, Silvia Perotto, Raffaella Balestrini, and Fabiano Sillo

1 Introduction

The Orchidaceae fami ly comprises over 27,000 plant species (WFO 2022 http://

www.worldﬂoraonline.org/) adapted to live in diverse environments, ranging from

soil (terrestrial orchids), rock surfaces (lithophytic orchids) and on other plant

species (epiphytic orchids) (Zhao et al. 2013).

Like 90% of plant species (Bonfante and Genre 2010), orchids form symbiotic

associations with mycorrhizal fungi. From an ecological point of view, the study of

orchid mycorrhizal (OM) symbiosis is pivotal since many orchid species are rare or

at risk of extinction due to habitat destruction and over-harvesting. The presence of

compatible OM fungi, necessary for seed development and plant growth, is therefore

extremely crucial for the survival of plants in nature, as well as for ex situ horticul-

tural growth. Furthermore, symbiotic germination from seeds may favor genetic

variability compared to monocultures created by asexual propagation (Fig. 1)

(Dearnaley et al. 2016).

In most mycorrhizal symbioses, the fungus provides inorganic nutrients in

exchange for ﬁxed carbon (C) from the photosynthetic host plant, which achieves

several beneﬁcial effects from this association (Genre et al. 2020). However , the OM

symbiosis appears to be an unusual association because the usual mechanism of

mycorrhizal nutrient exchange is reversed, at least during early development

(Dearnaley and Cameron 2016); under natural conditions, orchids are entirely

dependent on their associated symbiotic fungi for the supply of carbon and other

S. De Rose · S. Perotto

Dipartimento di Scienze della Vita e Biologia dei Sistemi, Università di Torino, Torino, Italy

R. Balestrini ( *) · F. Sillo

Consiglio Nazionale delle Ricerche-Istituto per la Protezione Sostenibile delle Piante, Torino,

Italy

e-mail: raffaella.balestrini@ipsp.cnr.it

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_4

145

nutrients, particularly during seed germination and early plant development, appar-

ently without getting anything in return (Selosse and Roy 2009). The plant depen-

dency on the associated fungus is maintained throughout the orchid life cycle in

achlorophyllous species. This peculiar trophic strategy, whereby orchids obtain

carbon from their mycorrhizal fungi, instead of supplying carbon to their partner,

is called mycoheterotrophy (Dearnaley et al. 2016).

146 S. De Rose et al.

Fig. 1 In vitro development stages from seeds into the adult stage of Serapias vomeracea.(a, b)

seeds seen under a stereomicroscope; (c) swollen seed; (d, e) protocorms; (f) protocorm with

pre-leaf; (g) seedling; (h) adult plant

The advent of -omics approaches, particularly transcriptomics, has contributed to

elucidating the molecular mechanisms of this intriguing symbiosis and to providing

insights into the nutrient exchanges between orchids and their associated fungi, thus

helping to dissect this paradigm. By reﬂecting the gene expression changes during

the development of organisms, as well as under the effects of biotic and abiotic

factors, the transcriptome allows ﬁlling the gap between the genome and its pheno-

type at a particular time (Stark et al. 2019). Next-generation sequencing (NGS)

approaches have now become widely available, thus providing the opportunity to

explore differential gene expression at great resolution by the sequencing of whole

transcriptomes, i.e., RNA-sequencing (RNA-seq; Marconi et al. 2014). RNA-seq

approaches make it possible to investigate the extreme complexity of cellular life in

the round, redeﬁning the ﬁelds of investigation that can be further explored by

integrating with other -omics techniques (Lowe et al. 2017) or with target tools

such as the use of laser microdissection (LM) technology (Balestrini et al. 2009).

Gene Expression Proﬁling in Orchid Mycorrhizae to Decipher the... 147

In the next sections, the contributions that transcriptomics has made to the broad

understanding of the mechanisms that drive this intriguing type of symbiosis, with

reference to nutrient exchange, fungal genes and plant responses involved in the

establishment of this association, are introduced and discussed.

2 Transcriptomics to Understand Nutrient Exchanges

between Orchids and their Symbionts

As mentioned above, OM fungi play a key role during orchid seed germination and

plant development during the early stages. Orchid seeds are often small (0.3–14 μg)

and the embryos have limited nutrient reserves, which mainly consist of protein and

lipids (Arditti and Ghan i 2013; Zhao et al. 2013; Dearnaley et al. 2016). Germination

of these tiny seeds requires the interaction with a compatible OM fungal species/

isolate, which forms elaborate intracellular hyphal coils (Smith and Read 2008),

called “pelotons,” responsible for nutrient exchanges between symbionts. The sub-

sequent orchid develo pmental stages include the formation of tuber-like heterotro-

phic structures lacking chlorophyll, deﬁned as protocorms (Smith and Read 2008).

In the adult stage, orchids can develop different trophic strategies: most species

become autotrophic, with green leaves and photosynthetic capacity (Dearnaley

2007), but around 100 species are reported as achlorophyllous and therefore

completely dependent on the fungal symbiont for organic carbon (C), i.e.,

mycoheterotrophic (Selosse and Roy 2009; Hynson et al. 2013). Mixotrophy is an

interesting evolutionary intermediate between the ﬁrst two strategies, where the plant

takes advantage of the fungal supply of organic C while retaining photosynthetic

capacity (Julou et al. 2005; Gebauer et al. 2016). Mixotrophy allows strong adapta-

tion under shady environments or in situations of reduced photosynthetic capacity

(Girlanda et al. 2006; Lallemand et al. 2019).

Early studies on nutrient exchange based on experiments with isotope tracing

showed that the OM fungus is responsible for the supply of C, phosphorus (P), and

nitrogen (N) to protocorms and that the supply of P and N continues in seedlings and

adulthood plants (Cameron et al. 2006, 2007, 2008). By performing a high-

resolution secondary ion mass spectrometry, nutrient transfer through peloton lysis

in the obligate mycoheterotrophic orchid Rhizanthella gardneri has been observed

(Bougoure et al. 2014). In mycorrhizal protocorms of Spiranthes sinensis, the use of

imaging of stable isotope tracers at the cellular level also demonstrated that C and N

are translocated from the mycorrhizal fungus to the orchid cell either through intact

pelotons or through the release of hyphal cytoplasm during peloton degradation

(Kuga et al. 2014).

148 S. De Rose et al.

In the last years, several nutrient transporter genes have been detected and

characterized in OM by transcriptomics (Perotto et al. 2014; Zhao et al. 2014;

Fochi et al. 2017a, b), thus supporting the hypothesis that an active nutrient

exchange takes place at the plant–fungal interface. Investigations have been mainly

focused on symbiotic protocorms obtained in vitro, but recent work also considered

the symbiosis in the roots of adult plants (Valadares et al. 2020, 2021). Before the

advent of RNA-seq, methods relying on PCR-based ampliﬁcation of cDNA frag-

ments that differ from the control, like suppression subtractive hybridization (SSH),

led to the identiﬁcation of various genes involved in transport processes in the orchid

Dendrobium ofﬁcinale colonized by a Sebacina sp. fungus, including a cation

transporter of the plant and an inorganic phosphate transporter of the fungus (Zhao

et al. 2013).

By using RNA-seq technology, the transcriptomic responses of Cymbidium

hybridum plantlets co-cultivated with two different beneﬁcial fungi, one of them

non-mycorrhizal, were investigated (Zhao et al. 2014). Among the different genes

involved in nutrient transport, two plant phosphate transporters, co-regulated during

interactions with both fungal species, were identiﬁed (Zhao et al. 2014). Two genes

coding for phosphate transporters expressed in mycorrhizal roots of the adult green

orchid Oececlades maculata collected in natural conditions were also recently

identiﬁed (Valadares et al. 2020). These results provide evidence that the acquisition

of inorganic phosphorus in adult plants is mediated by the associated fungus, as

suggested for the terrestrial orchid Goodyera repens based on isotope studies

(Cameron et al. 2007). In soils with limited P availability, naturally occurring

orchids have been found to acquire signiﬁcant amounts of inorganic P from the

symbiotic fungal partners; plant–fungus combinations, which may be more or less

efﬁcient, strongly inﬂuence P acquisition, with plant-mediated niche differentiation

(Davis et al. 2022).

Bidirectional transfer of C between a green adult orchid and its fungal symbiont

has been demonstrated by isotope tracing experiments, thus allowing a more com-

plete view of C ﬂuxes in OM symb iosis (Cameron et al. 2006). Interestingly, an

up-regulated putative bidirectional sugar plant transporter belong ing to the SWEET

family has been identiﬁed by high-throughput transcriptomics of a normalized

cDNA library by 454 GS-FLX Titanium pyrosequenci ng in Serapias vomeracea

protocorms colonized in vitro by the fungus Tulasnella calospora (Perotto et al.

2014). Pathogenic microorganisms and beneﬁcial symbionts are both known to

target plant SWEET transporters for nutritional gain (Chen et al. 2010). SWEET

transporters were also identiﬁed in mycorrhizal roots of albino variants of Epipactis

helleborine, a mixotrophic orchid (Suetsugu et al. 2017); in symbiotic protocorms of

Bletilla striata (Miura et al. 2018) and mycorrhizal roots of adult orchids

O. maculata and Limodorum abortivum (Valadares et al. 2020, 2021) both collected

in nature. Intriguingly, a sucrose transporter that may allow sucros e import at the

symbiotic interface in mycoheterotrophic Gastrodia elata associated with the fungus

Armillaria has been found (Ho et al. 2021). Moreover, invertase, an enzyme cleaving

sucrose into glucose and fructose, has been identiﬁed, through proteomic

approaches, in mycorrhizal protocorms of the orchid Oncidium sphacelatum

(Valadares et al. 2014).

Gene Expression Proﬁling in Orchid Mycorrhizae to Decipher the... 149

Most orchid tissues are highly N-enriched (Hynson et al. 2013,) and the fungus

has been demonstrated to provide N to protocorms and adult green orchids by

exploiting inorganic and organic N sources in the substrate (Kuga et al. 2014;

Cameron et al. 2006). The exchange of N in OM has been clariﬁed thanks to

transcriptomics approaches. RNA-seq analysis of the plant and fungal N uptake

pathways in the model system S. vomeracea-T. calospora identiﬁed several

up-regulated plant and fungal genes associated with N metabolism (Fochi et al.

2017a). To understand the preferential N form taken up by the fungus and transferred

to the orchid protocorm, the fungal mycelium was grown on two different N sources.

Based on transcriptomic and genomic data, it has been hypothesized that the fungus

can obtain N from organic and inorganic sources, excluding nitrate, and two

ammonium fungal transporters were identiﬁed, one of which was up-regulated in

symbiosis. Plant transporters for N compounds resulted to be up-regulated in

symbiosis, such as ammonium and oligopeptide transporters, as well as amino

acid transporters, including a plant lysine histidine transporter (LHT1). A homolo-

gous LHT1 gene was previously reported in mycorrhizal roots of Cymbidium

hybridum (Zhao et al. 2014) and recently detected in symbiotic protocorm s of

B. striata (Miura et al. 2018) and symbiotic roots of adult orchid plants (Valadares

et al. 2021). This repertoire of fungal and plant genes was further investigated

through the use of laser microdissection (Balestrini et al. 2018; Fochi et al.

2017b). By combining a microscope and a computer-assisted laser beam to separate

various cellular components from sections placed under a microscope slide, laser

microdissection enables the quick separation of speciﬁc cells from a piece of

heterogeneous tissue making it possible to extract a variety of cellular compo unds,

including RNA (Balestrini et al. 2009, Balestrini and Fiorilli 2020). The analysis of

gene expression in RNA samples originating from orchid cells harboring fungal coils

at diverse developmental stages, as well as cells non-colonized by the fungus,

demonstrated that plant genes coding for transporters of N compounds are differen-

tially expressed in symbiosis (Fochi et al. 2017b). Based on these ﬁndings, it has

been hypothesized that N-rich amino acids may be transferred from the fungus to the

host plant, also contributing to the C requirement. Moreover, in addition to active

transport, recovery of organic N forms from peloton lysis may occur (Kuga et al.

2014). Recent transcriptomic and proteomic studies focused on orchid species

collected in nature support this hypothesis (Valadares et al. 2021). For example, in

the mycorrhizal roots of the orchid L. abortivum, genes encoding a lysine histidine

transporter 1 (LHT1) and an amino acid permease, as well as several NRT1/PTR

family members putatively associated with N transfer from the fungus to the host

plant, were found to be up-regulated (Valadares et al. 2021). Similarly, it has been

discovered that the mycorrhizal roots of the terrestrial orchid O. maculata regulates

genes for LHT1 and NRT1/PTR family members, which are amino acid transporters

(Valadares et al. 2021). Interestingly, members of this protein group have also been

discovered to transport peptides, but also chloride, nitrite, glucosinolates, and several

phytohormones such as auxin, abscisic acid, gibberellins, and jasmonate

(Corratgé-Faillie and Lacombe 2017). However, their roles in mycorrhizal symbiosis

are still under debate (Corratgé-Faillie and Lacombe 2017).

150 S. De Rose et al.

A recent metabolomic study using the model system S. vomeracea —T. calospora

integrated previous transcriptomic data (Fochi et al. 2017a, b) and showed that the

external mycelium of the mycorrhizal fungus freely growing close to the host

protocorms affected several metabolic pathways. The interaction between plant

and fungus increased compounds associated with structural, signaling, and energy,

mostly lipids, particularly glycerolipids (GP) and sphingolipids (SP) (Ghirardo et al.

2020). Lipids are known to be the main structural components of cell membranes but

also provide other important biological functions, ranging from signaling, C storage,

plant–microbe interactions, and even response to environmental stresses (Ghirardo

et al. 2020). Notably, a percentage decrement of N- and S-containing compounds in

the mycorrhizal fungus growing close to the host protocorms, led the authors to

hypothesize that this depletion may mirror a transfer of N compounds to the host

plant. However, among the identiﬁed S-containing compounds, the amount of

S-adenosylmethionine (SAM) increased in the mycelium surrounding protocorms

(Ghirardo et al. 2020). This molecule is used by methyltransferases as a methyl

group donor for a variety of target substrates (Mato et al. 1997; Ghirardo et al. 2020).

Currently, scarce information on S transfer from fungi to the host plant in OM is

available. This nutrient is essential for plant growth and develo pment, as a constit-

uent of amino acids such as cysteine and methionine and sulfated peptides (i.e.,

glutathione or phytosulfokines) (Kopriva et al. 2019). Very recently, experiments

with labeled S, N, and C showed that these elements could be translocated from the

substrate to the protocorm cells via the fungal hyphae (De Rose et al., submitted),

corroborated by target transcriptomic data which showed up-regulation of several

plants and fungal transporter genes, as well as genes related to S assimilation

enzymes involved in movement and redistribution of S in the cell. Overall, these

ﬁndings support the hypothesis of transfer during symbiosis of S in a reduced

organic form that also contains N, such as S-amino acids or small peptides, including

glutathione (De Rose et al., submitted).

Based on the reports so far available, the model originally proposed for nutrient

transport in OM (Dearnaley and Cameron 2016) can be strengthened and integrated

with transcriptomic data. In non-photosynthetic stages, orchids may receive

inorganic P, C, N, and S from the symbiotic fungal partner in the form of amino

acids, and export ammonium in exchange (Cameron et al. 2006, 2007; Kuga et al.

2014; Fochi et al. 2017a, b). This exchange takes place across intact membranes

(Kuga et al. 2014; Fochi et al. 2017b), but the lysis of senescent fungal pelotons may

also release C, N, and P (Bougoure et al. 2014). Signiﬁcant metabolic (particularly

lipid-related) changes in the mycelium outside the plant could also participate in the

nutrient supply to the host plant (Ghirardo et al. 2020). In photosynthetic orchids, C

as sugars could be exported from the plant to the fungus, while inorganic P continues

to be received by the mycorrhizal partner (Cameron et al. 2006; Valadares et al.

2020, 2021) in amounts that can vary, even greater, depending on plant-fungus

compatibility (Davis et al. 2022). The ﬂow of nutrients in the orchid mycorrhizal

symbiosis may be even more complex than presented when considering the hyphal

interconnections that may exist in natural ecosystems, well investigated for

mycoheterotrophic orchids, and the fungal diversity in orchid roots (extensively

reviewed by Yeh et al. 2019). An example is the ﬂow of C between the tree species

Salix repens and Betula pendula with the orchid Corallorhiza triﬁda by hyphal

network shared by the plants (McKendrick et al. 2000), or the coexistence of

different endosymbionts in Cymbidium hybridum (Zhao et al. 2014).

Gene Expression Proﬁling in Orchid Mycorrhizae to Decipher the... 151

3 Transcriptomics to Decipher the Mechanisms Involved

in the Establishment of Symbiosis

Fungi that form mycorrhizal associations with orchids exhibit great phylogenetical

and ecological diversity (McCormick et al. 2018). Orchids are known to associate

with a plethora of fungi, including ectomycorrhizal basidiomycetes and ascomycetes

wood degraders and other saprotrophic basidiomycetes (Bidartondo et al. 2004;

Selosse et al. 2004; Dearnaley 2007; Ogura-Tsujita and Yukawa 2008; Martos

et al. 2009; Kottke et al. 2010; Lee et al. 2015; Kinoshita et al. 2016). The most

common taxa in photosynthetic orchid species include Tulasnella, Ceratobasidium,

and Serendipita (Dearnaley et al. 2012).

The diversity of OM fungi in the soil may be a crucial element in determining the

distribution and future of orchids (McCormick et al. 2018) because orchids in nature

are entirely dependent on OM fungi at least for seed germination and early stages of

development. After initial contact and seed germination, protocorms are colonized

by OM fungi; the hyphae penetrate parenchyma cells, branch and fold to create thick

hyphal coils (the pelotons) that at last degrade (Miura et al. 2018). The symbiotic

germination of orchid seeds requires the coordinated expression of numerous func-

tional genes as well as a crosstalk between genes associated with the mycorrhizal

establishment and the germination process (Liu et al. 2015; Chen et al. 2020).

According to Evangelisti et al. (2014), plant hormones may act as a node in the

crosstalk between plant development and plant–microbe interactions. Strigolactones

(SLs), for instance, are signaling molecules produce d by plant roots either constitu-

tively (Akiyama et al. 2005) or in response to low phosphorus levels (Kretzschmar

et al. 2012) capable of recruiting arbuscular mycorrhizal (AM) fungi and promoting

hyphal branching. In orchids, a carotenoid cleavage dioxygenase, involved in the

strigolactones biosynthetic pathway, has been identiﬁed in a proteomic study in

O. sphacelatum mycorrhizal protocorms by Valadares et al. (2014). Since the

enzyme was more expressed in earlier stages of mycorrhizal protocorm develop-

ment, it has been hypothesized that SLs may have essential early functions in luring

compatible fungal symbionts to aid orchid seeds germination (Valadares et al. 2014).

The crosstalk of jasmonic acid (JA), abscisic acid (ABA), and SLs were inves-

tigated in D. ofﬁcinale seeds colonized by Tulasnella sp. during the germination

phase (Wang et al. 2018). The transcriptomic and RT-qPCR data, combined with the

quantiﬁcation of endogenous phytohormones, suggested that the OM fungus had a

role in hormone production (Wang et al. 2018). Additionally, endogenous JA, ABA,

or SLs levels were maintained low to promote the formation of the D. ofﬁcinale-

Tulasnella protocorm-like structures (Wang et al. 2018). However, the great phylo-

genetic and ecological diversity of OM fungi might suggest that other signals are

involved. A comparative analysis of gene expression in asymbiotic and symbiotic

Anoectochilus roxburghii seeds through Illumina HiSeq 4000 transcriptome

sequencing allowed focus on the regulatory module GA-GID1-DELLA (Liu et al.

2015). Gibberellins (GAs) are plant hormones that play key roles in growth and

development (Wang and Deng 2014). The GID1 receptor and the DELLA repressor

were found to be critical in the regulation of seed germination (Wang and Deng

2014). ABA, another important phytohormone, was found instead to inhibit the

process through a ﬁnely tuned crosstalk (Liu et al. 2015). Among the differentially

expressed plant transcripts identiﬁed in symbiotic and asymbiotic seeds, two tran-

scripts coding for gibberellin 20 oxidase (GA20ox), two transcripts coding for

gibberellin 2-oxidase (GA2ox) involved in GAs biosynthesis, and two transcripts

coding for DELLA proteins, members of GRAS superfamily (Hernández-García

et al. 2021), were established as common elements of the mycorrhizal signaling

pathway (Jin et al. 2016). This study suggested that OM fungi could modulate the

expression of these plant genes, possibly affecting the entire GA-GID1-DELLA

regulatory module.

152 S. De Rose et al.

Investigations focused on the impact of GAs on symbiotic seed germination in the

model system represented by D. ofﬁcinale and Tulasnella sp. were also performed

(Chen et al. 2020). Levels of endogenous gibberellic acid (GA3) using liquid

chromatography-mass spectrometry (LC-MS/MS) were determined during symbi-

otic and asymbiotic germination of orchid seeds, and a signiﬁcantly higher ratio

between GA3 and ABA was found in symbiosis (Chen et al. 2020). Phenotypic and

target gene expression investigations were conducted on the germination of seeds

treated with various concentrations of exogenous GA3, showing a negative effect of

high concentrations of GA3 on fungal colonization. These ﬁndings were combined

with data obtained from RNA-seq and proteomic analyses that highlighted a signif-

icantly higher expression of an ABA receptor protein, PYR1, during the early stages

of symbiotic germination in D. ofﬁcinale (Chen et al. 2017). The expression proﬁle

of genes involved in GAs and ABA biosynthesis, identiﬁed in the transcriptomic

data, and of genes reported to be part of the recognized common symbiotic pathway

(including a calcium-binding protein and a calcium-dependent protein kinase),

showed a ﬁne-tuned regulation under the different GA treatments (Chen et al.

2020). These results suggest that an interplay between GAs metabolism and the

establishment of symbiosis may occur in orchids (Chen et al. 2017, 2020).

A group of common symbiosis genes (CSG) have been identiﬁed in angiosperms

forming arbuscular mycorrhizal (AM) symbiosis, reported to act in the signaling

pathway for recognizing and transducing microbial signals that are diffuse through-

out root colonization and nutrient exchange (Stougaard 2001; Kistner et al. 2005;

Genre and Russo 2016). In contrast, angiosperms unable to form mycorrhizal

associations with AM fungi, such as members of Brassicaceae, showed no functional

or only a few members of CSG (Delaux et al. 2014). Members of the Pinaceae in the

gymnosperms, forming ectomycorrhiza, were also lacking CSG in their genomes

(Garcia et al. 2015).

Gene Expression Proﬁling in Orchid Mycorrhizae to Decipher the... 153

To explore the possible presence of CSG in orchids, protocorms of B. striata

colonized by the mycorrhizal fungal taxa Tulasnella sp. were analyzed at different

stages by transcriptomics (Miura et al. 2018). Notably, all of the CSG characterized

in other plant species were found in the B. striata transcriptome (Miura et al. 2018).

In vivo assay by functional complementation of L. japonicus CCaMK mutant

through B. striata CCaMK gene showed that this gene complemented the function

of LjCCaMK (Miura et al. 2018). Moreover, eight genes were strongly induced

during symbiosis (Miura et al. 2018). The high similarity of these genes to the AM

marker genes in rice (Gutjahr et al. 2008), as well as their strong induction during the

plant–fungus interaction, allow considering this set of eight genes as marker genes

also for OM (Miura et al. 2018).

A large-scale analysis of more than 250 transcriptomes and about 100 plant

genomes, encompassing the whole land-plant diversity, demonstrated that a shared

symbiosis signaling pathway occurred in all plants forming intracellular endosym-

bioses (Radhakrishnan et al. 2020). It is worth noting that co-evolution between

plants and fungi began approximately 400 million years ago and that four mycor-

rhizal types evolved at different times: arbuscular mycorrhiza, orchid mycorrhiza,

ericoid mycorrhiza, and ectomycorrhiza (Genre et al. 2020). The ﬁrst three types are

endosymbioses, i.e., fungal symbionts are harbored intracellularly inside plant cells.

In particular, AM symbiosis is probably the most ancient plant–fungus symbiosis

(Delaux et al. 2013) from which OM symbiosis appeared to be derived after plant

diversiﬁcation (Radhakrishnan et al. 2020). Comparative transcriptomic studies

allowed us to identify six genes lost in non-mutualistic plant taxa: CcaMK, calcium-

and calmodulin-dependent protein kinase, SymRK, a receptor-like kinase,

CYCLOPS and RAD1, two transcription factors, and two transporters STR and

STR2 showing a half-ATP-binding cassette (ABC). In particular, the ﬁrst three

genes were conserved in all plants forming intracellular endo symbiosis, while

STR, STR2, and RAD1 were supposed to be speciﬁc to AM (Radhakrishnan et al.

2020).

Starting from the earliest observations (Burgeff 1932; Burges 1939), OM has

been often argued to represent a balanced antagonism between plant and fungus,

since it has been documented that fungi occasionally might destroy the protocorm

(Adamo et al. 2020) and because plant receives C from the fungus lacking a clear

reward. However, large-scal e transcriptomic data integrated with target gene expres-

sion analysis with RT-qPCR allowed to demonstrate the absence of a defense

activation in S. vomeracea mycorrhizal protocorms (Perotto et al. 2014), as previ-

ously observed (Zhao et al. 2013). The OM symbiosis was suggested as “a friendly

plant–fungus relationship” and the lack of a strong defense response was succes-

sively conﬁrmed by other transcrip tomic studies in other orchid species (Suetsugu

et al. 2017; Miur a et al. 2018). Using 454 pyrosequencing, Perotto et al. (2014)

proposed a nodulin-like gene called SVNod1 as a marker of OM symbiosis (Perotto

et al. 2014). In this transcriptomic study, a set of plant and fungal genes expressed in

S. vomeracea protocorms colonized by OM fungus T. calospora was identiﬁed

(Perotto et al. 2014). In-depth gene expression analysis using an extremely efﬁcient

target approach, laser microdissection, tested the expression of several genes iden-

tiﬁed in the work by Perotto et al. (2014), conﬁrming the results and suggesting

SvNod1 as a marker gene of orchid symbiosis, since its transcript was detected in the

fungal colonized cells only (Perotto et al. 2014; Balestrini and Bonfante 2014).

154 S. De Rose et al.

An important process for endosymbiosis is clathrin-mediated endocytosis

(Leborgne-Castel et al. 2010). The plant plasma membrane plays a pivotal role in

the management of microbial interactions, as it senses and possibly allows the entry

of endocellular symbionts or microbial substances, and endocytosis can regulate the

entry of extracellular particles or cargoes into the cell (Zeng et al. 2017). The

molecular components of this process have been investigated by transcriptomics in

the peculiar OM system represented by the orchid Gastrodia elata, a fully

mycoheterotrophic orchid able to establish a symbiosis with two genera of fungal

partners, i.e., Mycena and Armillaria. Mycena species are known to interact with

Gastrodia as symbionts during the early stages of plant development, including

protocorm formation (Kim et al. 2006; Zeng et al. 2017). The transcriptomes of

G. elata symbiotic seeds and protocorms were analyzed by RNA-seq and among the

differentially expressed genes identiﬁed in the study, genes putatively linked to

energy met abolism, plant defense, molecular signaling, and secondary metabolism

were detected (Zeng et al. 2017). Genes coding for clathrin, an adaptor protein,

dynamin, and HSC70, were found to be constitutively expressed in seeds but

strongly expressed in protocorms, indicating that endocytosis mediated by clathrin

may be crucial in G. elata during interactions with Mycena fungi (Zeng et al. 2017).

Comparative transcriptome analysis was also used to unravel molecular mechanisms

underlying gastrodin biosynthesis since this compound has been reported to have

several positive effects on human health (Tsai et al. 2016). Two putative

monooxygenases and one glycosyltransferase key enzymes involved in gastrodin

biosynthesis were identiﬁed, which could be a target of genetic editing to improve

gastrodin production (Tsai et al. 2016).

In OM symbiosis, the fungal hyphae must enter the cell walls of the epidermal

cells to reach the internal parenchyma cells during the colonization of orchid seeds,

protocorms, or roots (Chen et al. 2014; Favre-Godal et al. 2020). The plant cell wall

is therefore the ﬁrst physical structure where interactions between the host plants and

the associated symbiotic partners take place (Balestrini and Bonfante 2014).

Recently, Chen et al. (2022) compared transcriptomic proﬁles during seed germina-

tion, protocorm formation, and seedling development under symbiotic and

asymbiotic conditions to further investigate changes in the expression of plant

genes related to plant cell wall biosynthesis, structure modiﬁcation, and the expres-

sion pattern of fungal genes related to plant/fungal cell wall degradation (i.e.,

CAZymes) (Chen et al. 2022). After being inoculated with two symbiotic fungi

(Tulasnella sp. and Serendipita sp.), D. ofﬁcinale seeds showed signiﬁcantly

increased expression of genes coding for secreted glycoproteins speciﬁcally associ-

ated with the epidermis, proline-rich receptor-like proteins, leucine-rich repeat

(LRR) extensin-like proteins, and extensin-like proteins during the symbiot ic

stage. Extensins are a large class of hydroxyproline-rich glycoproteins that play a

variety of roles in plant defense, such as reinforcing the cell wall to preclude invasion

by pathogens or to facilitate the interaction with symbiotic organisms (Chen et al.

2022). They were also probably essential for preventing fungal colonization of basal

cells and spreading inside the whole protocorms (Li et al. 2018). The observed

up-regulation of a microtubule-associated protein gene during the symbiotic stage

suggested that cytoskeletal remodeling took place during the fungal colonization of

orchid seeds (Chen et al. 2022). In addition, some genes involved in plant cell wall

biosynthesis, including genes coding for cellulose synthase and pectin esterase, were

signiﬁcantly up-regulated in seeds of D. ofﬁcinale inoculated with both fungal

species, suggesting that the interaction between seeds and mycorrhizal fungi was

particularly active in terms of modiﬁcation in plant cell wall biosynthesis pathways

(Chen et al. 2022). In the symbiotic fungal mycelium, several differentially

expressed CAZymes were found, representing approximately 24.8% and 36.7% of

the total number of CAZymes identiﬁed in Serendipita sp. and Tulasnella

sp. genomes, respectively (Chen et al. 2022). These genes were hypothesized to

play a role in the suppression of the plant defense responses, the identiﬁcation of

mycorrhizal fungi during the germination of orchid seeds, and also in the successful

fungal colonization (Chen et al. 2022).

Gene Expression Proﬁling in Orchid Mycorrhizae to Decipher the... 155

As mentioned earlier, OM fungi sometimes display a saprotrophic behavior that

leads to disruption of the mycorrhizal association, and recent research by Adamo

et al. (2020) focused on possible changes, during mycorrhizal symbiosis and

saprotrophic growth, in the expression of fungal genes encoding CAZymes capable

of breaking down the plant cell wall (PCW). They found that PCW-degrading

enzymes are ﬁnely regulated in T. calospora during mycorrhizal and saprotrophic

growth in the host and hypothesized that the expression of several important

CAZymes was connected to OM fungal transitions from symbiotic to saprotrophic

growth (Adamo et al. 2020).

Although most studies on OM have been focused on germinating seeds and

protocorms, omics approaches are also revealing molecular mechanisms and actors

involved in the adult stages of orchids. In comparison to protocorms, the interaction

in adult plants is expected to be different because of it involves the root, an

anatomically and metabolically complex plant organ (Valadares et al. 2020). Two

recent studies have shed light on the mechanisms driving plant–fungus interactions

in adult orchids in natural conditions (Valadares et al. 2020, 2021). The interaction

between adult plants of the terrestrial orchid O. maculata and its mycorrhizal fungus

Psathyrella candolleana (Basidiomycota) was analyzed using transcriptomic and

proteomic analyses (Valadares et al. 2020).

In nature, the same individual of O. maculata can form older roots compl etely

colonized by OM fungi as well as younger non-colonized roots (Valadares et al.

2020). For this reason, O. maculata is an excellent experimental system allowing the

study of the molecular changes related to OM formation and functioning since it can

be used to analyze both mycorrhizal and non-mycorrhizal roots from the same plant.

Integration of transcriptomic and proteomic data showed that a chitinase and a

mannose-binding lectin, two proteins involved in plant defense responses, decreased

in mycorrhizal roots, suggesting that the proximity of the fungal symbiont might

cause a local decrease of plant defense responses in the orchid tissues (Valadares

et al. 2020). Results also highlighted that allene oxide synthase transcripts, pre-

cursors involved in the biosynthesis of JA, were only found in non-mycorrhizal roots

of O. maculata, while genes annotated as 9-lipoxygenase and allene oxide synthase

were negatively regulated in mycorrhizal roots (Valadares et al. 2020). Based on

these ﬁndings, the authors suggested that inhibition of JA production is probably

needed to promote fungal colonization and OM formation (Valadares et al. 2020). In

addition, the up-regulation of three ethylene-induced calmodulin genes and 15 eth-

ylene-responsive transcription factors in the transcriptome of O. maculata mycor-

rhizal roots suggested that the activation of the ethylene pathways plays a role in OM

(Valadares et al. 2020). Similar outcomes were also reported in C. hybridum mycor-

rhizal roots (Zhao et al. 2014).

156 S. De Rose et al.

To further explore the mycorrhizal interactions in adult orchids in natural condi-

tions at the molecular level, a transcriptomic approach has been used to examine

gene expression in roots of the mixotrophic orchid Limodorum abortivum, able to

associate with ectomycorrhizal fungi of the taxon Russula (Valadares et al. 2021).

This study, which focused on how the plant responds to the mycorrhizal symbiont

(s) and used non-sterile non-mycorrhizal roots collected in nature as references,

addressed for the ﬁrst time OM interactions in an orchid species inte racting with an

ectomycorrhizal fungus. The comparison between non-sterile mycorrhizal and

non-mycorrhizal roots made it simpler to distinguish between the general orchid

responses to microbes and the mycorrhiza-speciﬁc plant responses. A shared core of

plant genes engaged in endomycorrhizal symbioses already identiﬁed in arbuscular

mycorrhiza was identiﬁed in L. abortivum and mirrored by the overexpression of

several molecular marker genes for symbiosis in mycorrhizal roots. Further studies

and gene characterization are needed to determine whether the unique characteristics

of OM depend on the precise regulation of these elements, or if additional genes are

involved in the process (Valadares et al. 2021). Among the genes differentially

expressed in planta, pectin methyl esterase (PME) genes were detected to be

signiﬁcantly down-regulated in L. abortivum, while PME inhibitor genes were

up-regulated in mycorrhizal roots, demonstrating that the main proportion of pectin

is in a highly methylated state in the outer cell wall and/or symbiotic interface

(Valadares et al. 2021). The high expression of two expansin coding genes in

mycorrhizal roots further supports the hypothesis that a loosening of the cell wall

during symbiosis may occur (Valadares et al. 2021).

The transcriptome of L. abortivum mycorrhizal roots also showed a signiﬁcant

up-regulation of subtilisin-like serine protease-coding genes. The major part of

subtilisin-like serine prote ases is primarily directed to the plant cell wall, in which

they can play a role in the regulation of the structural remodeling of the cell wall

(Schaller et al. 2018). Additionally, two genes in mycorrhizal L. abortivum roots

coding for syntaxin-132 (SYP132) proteins were found to be up-regulated, and thus

it has also been documented in mycorrhizal roots of other orchid species (Zhao et al.

2014; Valadares et al. 2020). Syntaxins have been characterized in the AM symbi-

osis and the SYP132α isoform has been demonstrated by knockdown mutant

experiments to be needed for arbuscule formation in the model plant Medicago

truncatula (Huisman et al. 2020). It has been hypothesized that SYP132, which is

localized on the perisymbiotic plant membrane surrounding functional arbuscules, is

essential for the development of a functional plant-fungus interface (Huisman et al.

2020). Even though it is currently unknown where the SYP132 proteins are located

in OM roots, it is intriguing to hypothesize that the AM and OM symbioses share a

similar exocytotic route (Valadares et al. 2021).

Gene Expression Proﬁling in Orchid Mycorrhizae to Decipher the... 157

4 Conclusion

In the last decades, thanks to advances in technology applied to transcriptomics, the

understanding of how orchids interact with their symbiotic partners has been

strongly improved. The application of RNA-seq in several orchid species has

allowed in-depth analyses of the molecular bases of nutrient transfer between OM

fungi and their orchid hosts, as well as the identiﬁcation of fungal and orchid genes

involved in the establishment of the symbiotic association. The current availability

of annotated orchid and fungal transcriptomes will help to ﬁll the gap between the

genomic data and the phenotypic observations, also in natural conditions. From the

plant side, a broad transcriptomics resource for orchid species has been developed,

named Orchidstra 2.0 database (http://orchidstra2.abrc.sinica.edu.tw), including data

from EST libraries and RNA-seq of 18 species from the ﬁve major subfamilies of the

Orchidaceae (Chao et al. 2017). This tool has already proven to be useful for the

comparison of whole transcriptomes across different orchid species. In addition,

international sequencing efforts, including the “1000 Fungal Genomes” project of

the U.S. Department of Energy (DOE) Joint Genome Institute (JGI) (Grigoriev et al.

2014), boosted knowledge on the fungal genomes and transcriptomes of several

fungal taxa, including OM fungi. The integration of this data with outcomes of other

-omics approaches, such as metabolomics, will improve our knowledge of the

orchids and their fungal “friends,” and allow a better understanding of this fascinat-

ing and complex symbiosis.

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Exploring the Potential of In Vitro Cultures

as an Aid to the Production of Secondary

Metabolites in Medicinal Orchids

Arshpreet Kaur, Jagdeep Verma, Vikramaditya G. Yadav,

Sandip V. Pawar, and Jaspreet K. Sembi

Abbreviations

BA 6-benzyladenine

CA Caffeic acid

CW Coconut water

CITES Convention on international trade in endangered species of wild fauna

and ﬂora

CHI Chalcone isomerase

FA Ferulic acid

KC Knudson C

Kn Kinetin

MeJA Methyl jasmonate

MemTR 6, 3-methoxybenzylamino-9-b-D-ribofuranosylpurine

M Mitra

MS Murashige and Skoog

mTR Meta-topolin riboside

NAA α-Naphthaleneacetic acid

A. Kaur · J. K. Sembi (*)

Department of Botany, Panjab University, Chandigarh, India

e-mail: jaspreet.sembi@pu.ac.in

J. Verma

Department of Botany, Sardar Patel University, Mandi, Himachal Pradesh, India

V. G. Yadav

Department of Chemical and Biological Engineering, University of British Columbia,

Vancouver, BC, Canada

School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada

S. V. Pawar

University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, India

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_5

163

164 A. Kaur et al.

pCA p-Coumaric acid

PAL Phenylalanine ammonia lyase

PLBs Protocorm-like bodies

SA Salicylic acid

STS Stilbene synthase

TDZ Thidiazuron

VW Vacin and Went

1 Introduction

Orchids belong to one of the largest and most diverse plant families in ﬂowering

plants (Christenhusz and Byng 2016). This diversity is due to their ability to

acclimatize to almost every type of habitat. Their exquisitely beautiful ﬂowers confer

these plants high ornamental and economic value in the global commercial market.

Apart from the immense ornamental signiﬁcance of orchids, these plants are also

medicinally important and have been utilized as therapeutics as acknowledged in the

traditional pharmacopeias worldwide (Hossain 2011; Sut et al. 2017). Habenaria

edgeworthii, Habenaria intermedia, Malaxis acuminata, and Malaxis muscifera are

components of Astavarga, which is a popular rejuvenating herbal formulation in

Ayurveda (Dhyani et al. 2010). In the traditional Chinese medicine system,

Anoectochilus roxburghii and A. formosanus have been used for preventing cancer,

protecting the liver, and treating diabetes and cardiovascular diseases (Han et al.

2008; Zhang et al. 2013). Shi-Hu, an orchid-based Chinese therapeutic formulation

derived from Dendrobium nobile, has been effectively used in treating lung, kidney,

and stomach diseases (Teoh 2016). Over the time, there have been numerous reports

on the wide usage of different plant parts of orchids in the treatment of a myriad of

diseases and ailments. The tubers of Bletilla striata are used in the treatment of

tuberculosis and gastric and duodenal ulcers (Ming et al. 2003). Dendrobium

candidum extracts maintain the tonicity of the stomach and have a body ﬂuid-

promoting effect (Ng et al. 2012). Pseudobulbs of Mala xis acuminata are used as

a curative for burning sensations, fever, and tuberculosis and as a nutritive tonic

(Hossain 2011). Whole plants of Ansellia africana are used for their aphrodisiac

properties (Chinsamy et al. 2011). Direct application of seeds of Acampe praemorsa

on wounds serves as a substitute for antibiotics (Shanavaskhan et al. 2012). Dried

powder of whole plants of Bulbophyllum odoratissimum is used to treat fractures,

chronic inﬂammations, and tuberculosis (Mohanty et al. 2015). There have been

wide ethnomedicinal evidence on the use of leaves of numerous species of

Dendrobium for treating musculoskeletal and nervous system problems (Wang

2021). The roots, rhizomes, pseudobulbs, stems, ﬂowers, and whole plant of species

belonging to the genus Calanthe are used in curing toothaches, rheumatism, jaun-

dice, typhoid, stomach-ache, ulcers, asthma, sore throat, etc. (Nanjala et al. 2022).

Additionally, there are a plethora of reports on the traditional medicinal usage of

Bulbophyllum species in various countries such as Nepal, India, China, Japan,

Bangladesh, Thailand, and Malaysia (Shariﬁ-Rad et al. 2022). It can therefore be

concluded that orchids have an immense therapeutic potential which is indicated by

the ethnobotanical reports and the presence of the wide variety of secondary

metabolites (Gantait et al. 2021).

Exploring the Potential of In Vitro Cultures as an Aid to the... 165

Plant seconda ry metabolites are a rich source of compounds having potent

biological activity. These metabolites are classiﬁed into numerous categories such

as alkaloids, ﬂavonoids, anthocyanins, and terpenoids (Sut et al. 2017; Ghai et al.

2021). The biosynthesis of these metabolites is regulated by pathways such as

phenylpropanoid pathway, mevalonate (MVA) pathway, and methyl-d-erythritol

4-phosphate (MEP) pathway. (Ghai et al. 2022). Some of the genes involved in

these pathways like Phenylalanine Ammonia Lyase (PAL), Chalcone synthase

(CHS), Chalcone Isomerase (CHI), Flavonol Synthase (FLS), and Stilbene Synthase

(STS) encode the key rate-limiting enzymes in speciﬁc secondary metabolites bio-

synthetic pathways, and hence regulate their biosynthesis (Ghai et al. 2022; Halder

et al. 2019; Kaur et al. 2022).

In orchids, pharmaceutically important biomolecules such as polysaccharides,

bibenzyl derivatives, phenylpropanoids, phenanthrene derivatives, alkaloids, and

ﬂavonoids are widely present (Hossain 2011; Sut et al. 2017). Numerous studies

on the evaluation of the biological activity of the phytochemicals extracted from

orchids have been reported (Table 1). There have been reports on disease ameliora-

tion using orchid phytochemicals. Antidiabetic properties of extracts of Aphyllorchis

montana and Anoectochilus roxburghii have been reported (Thalla et al. 2013; Cui

et al. 2013). Immunomodulatory effects of polysaccharides derived from orchids

have been evaluated in Bletilla striata where the polysaccharides derived from it

improved the spleen and thymus indices (Chen et al. 2020). Additionally, a Type II

arabinogalactan polysaccharide extracted from Anoectochilus formosanus stimu-

lated the maturation of dendritic cells to induce immune responses against patho-

gens, thus attributing to their immune-enhancing potential (Lai et al. 2015). The

compounds extracted from orchids also exhibit antimicrobial properties. For

instance, bibenzyl derivatives of Dendrobium nobile displayed broad-spectrum

antifungal activity against several phytopathogenic fungi (Zhou et al. 2016).

Retusiusines B, a phenylpropanoid compound extracted from Bulbophyllum

retusiusculum showed effective antifungal activity against Candida albicans (Fang

et al. 2018). The signiﬁcant antioxidant potential has also been evaluated through

DPPH (2, 2-Diphenyl-1-picrylhydrazyl) radical scavenging activity, for a ﬂavonoid

compound, rutin, isolated from Dendrobium ofﬁcinale (Zhang et al. 2017). The

compounds derived from orchids have also been reported to possess anticancer

properties. Bulbophythrins, the phenanthrene derivatives isolated from

Bulbophyllum odoratissimum displayed cytotoxic potential against human hepa-

toma, leukemia, adenocarcinoma, and stomach cancer cell lines (Xu et al. 2009).

Similarly, in Dendrobium nobile, ‘nudol’, a phenanthrene derivative, inhibited the

osteosarcoma cell growth (Zhang et al. 2019). High cytotoxicity against the growth

of Hela human cervical cancer cell line has been observed for the bibenzyl com-

pounds derived from Dendrobium ofﬁcinale (Ren et al. 2020). Different researches

Plant name Compound Reference

(continued)

166 A. Kaur et al.

Table 1 Compounds isolated from orchid species and their biological activity

Biological

activity

Antibacterial

activity

Bletilla ochracea Blestriarene A, Blestriarene B,

Blestriarene C

Yang et al. (2012)

Bletilla striata Bletistrin F, Bletistrin G,

Bletistrin J, Bulbocol, Shanciguol,

and Shancigusin B

Jiang et al. (2019b)

Bulbophyllum

retusiusculum

Retusiusines B Fang et al. (2018)

Liparis regnieri Erianthridin, Gigantol, Hircinol,

Nudol, Coelonin, Moscatin

Ren et al. (2016)

Antidiabetic

property

Aerides multiﬂora Aerimultin C Thant et al. (2021)

Dendrobium

crepidatum

Dendrocrepine Xu et al. (2020)

Dendrobium

formosum

Confusarin Inthongkaew et al.

(2017)

Dendrobium

loddigesii

Loddigesiinols G–J Lu et al. (2014)

Dendrobium

scabrilingue

Dendroscabrol B Sarakulwattana et al.

(2020)

Antioxidant

activity

Cremastra

appendiculata

Coelonin, Orchinol Tu et al. (2018)

Dendrobium

ofﬁcinale

Rutin Zhang et al. (2017)

Dendrobium

palpebre

Dendroﬂorin Kyokong et al.

(2019)

Dendrobium

parishii

Dendroparishiol Kongkatitham et al.

(2018)

Gastrodia elata Gastrodin Jiang et al. (2020)

Anticancer

activity

Bulbophyllum

odoratissimum

Bulbophythrins A and B Xu et al. (2009)

Cattleya tigrina Triterpene

24-methylenecycloartanol,

gigantol, phocantone

Ferreira et al. (2021)

Dendrobium

brymerianum

Moscatilin, gigantol, lusianthridin,

and dendroﬂorin

Klongkumnuankarn

et al. (2015)

Dendrobium

draconis

Gigantol Bhummaphan and

Chanvorachote

(2015)

Dendrobium

falconeri

Dendrofalconerol A Pengpaeng et al.

(2015)

Dendrobium

nobile

Nudol Zhang et al. (2019)

Dendrobium

williamsonii

Aloifol I, moscatilin,

moniliformine, balanophonin

Yang et al. (2018)

Goodyera

schlechtendaliana

Goodyschle A Dai et al. (2021)

Plant name Compound Reference

have been conducted to test the anti-inﬂammatory effects of the compounds derived

from different orchid species. The ethanolic extract of Bletilla striata yielded a

dihydrophenanthrene, coelonin, which possessed the potential to decrease inﬂam-

mation (Jiang et al. 2019a). The alkaloids and phenanthrene isolated from

Dendrobium crepidatum and Dendrobium chrysanthum, respectively, exhibited

anti-inﬂammatory activity (Hu et al. 2020; Yang et al. 2006). To summarise, it can

be inferred that the secondary metabolites of orchids have the potential to be used as

leads for therapeutic cures in pharmaceutical industries after systematised preclinical

and clinical studies.

Exploring the Potential of In Vitro Cultures as an Aid to the... 167

Table 1 (continued)

Biological

activity

Spiranthes

sinensis

Spiranthes phenanthrene A Liu et al. (2019)

Anti-inﬂam-

matory

activity

Dendrobium

chrysanthum

Dendrochrysanene Yang et al. (2006)

Dendrobium

crepidatum

(+)-Dendrocrepidamine A,

Dendrocrepidamine B, (+)-

Homocrepidine A

Hu et al. (2020)

The therapeutic properties of orchi ds have aroused curiosity amongst people all

over the world and have in turn led to unscrupulous collections from their natural

habitats for trade and consumption. As a result, these plants face threats due to their

habitat destruction and indiscriminate exploitation. Resultantly, the family

Orchidaceae is included in the Appendix II of the Convention on International

Trade in Endangered Species of Wild Fauna and Flora (CITES), and the interna-

tional trade in orchids is austerely governed (Hinsley et al. 2018). Hence, there is a

dire need to develop approaches for developing alternative methods for propagation

and protection of these high-value plants. In vitro culture methods play a pivotal role

in ameliorating the pressures and restoring the decimated natural populations.

Besides this, it facilitates the production of biomass and amassing of metabolites

in plant tissues and culture media. In vitro cultures also serve as a tool for extensive

investigation of the controls and mechanisms of metabolic pathways.

2 In Vitro Propagation and Production of Secondary

Metabolites

There are several limitations associated with the extraction of secondary metabolites

from the wild plants cultivated in the ﬁeld such as ﬂuctuations in the yield due to

variability in the geographic location, seasonal variation, and environment of the

plant, therefore, in vitro propagation has emerged as a better substitute (Murthy et al.

2014). In vitro cultures are grown on deﬁned media under controlled condition s. In

orchids, there are several nutrient media which are generally employed for tissue

culture such as media proposed by as Murashige and Skoog (MS) (Murashige and

Skoog 1962), Knudson C (KC) (Knudson 1946), Vacin and Went (VW) (Vacin and

Went 1949), Mitra (M) (Mitra et al. 1976), etc.

168 A. Kaur et al.

A general trend of decline in secondary metabolite production under normal

in vitro growing circumstances in comparison to wild plants has been observed.

However, culture media conditions, concentrations of plant growth regulators,

nitrogen source, carbon source, and other organic/inorganic additives have been

optimised for enhanced production of secondary metabolites (Chandran et al. 2020).

In Habenaria edgeworthii, three times more phenolic content was found in callus

grown on half strength Murashige and Skoog (MS) and 3 μM 6-benzyladenine (BA),

as compared to the wild tubers. These also showed enhanced antioxidant activity

evaluated by the standard in vitro assays (Giri et al. 2012). In Dendrobium

candidum, the supplementation of the MS basal medium with 0.5 mg L

1

NAA,

ratio 5:25 (mM) of NH

4

:NO

3

, 2.5% (w/v) sucrose, and 1% (v/v) banana homogenate

were favourable for the production of polysaccharides, polyphenolics, and ﬂavo-

noids (Cui et al. 2015). In D. huoshanense, phosphate at 0.312 mmol L

1

concen-

tration was optimum in the medium for maximum accumulation and production of

polysaccharides (Jiang et al. 2006). Similarly, the supplementation with 50 g L

1

sucrose in protocorm-like bodies (PLBs) cultures of D. huoshanense resulted in a

109-fold increase in the polysaccharide content compared to the media which lacked

sucrose feeding (Zha et al. 2007).

In vitro cultures with speciﬁc additives and growth conditions have been reported

to favour secondary metabolite content and resultant antioxidant potential in com-

parison to the mother plant as in Dendrobium nobile (Bhattacharyya et al. 2014),

Dendrobium thyrsiﬂorum (Bhattacharyya et al. 2015), Aphyllorchis montana

(Mahendran and Bai 2016), Dendrobium crepidatum (Bhattacharyya et al. 2016),

Malaxis acuminata (Bose et al. 2017), Coelogyne ovalis (Singh and Kumaria 2020),

Cymbidium aloifolium (Kumar et al. 2022). Additionally, antimicrobial activity was

also found to be higher in in vitro propagated plants of Aphyllorchis montana as

compared to the wild plants (Mahendran and Bai 2016). In Dendrobium longicornu,

the in vitro protocorms have higher anticancer, antioxidant, and antimicrobial

potential (Paudel et al. 2020). Similarly, the protocorms of Dendrobium chryseum

were reported to signiﬁcantly inhibit the growth of human cervical carcinoma cell

lines (Pant et al. 2021). Thus, besides mass multiplication for the production of

greater biomass, in vitro propagation protocols can also be used for the heightened

phytochemical production and signiﬁcant biological activities. Additionally, the use

of in vitro cultures offers a sustainable strategy for conser vation and utilization of

these endangered medicinal plants.

Exploring the Potential of In Vitro Cultures as an Aid to the... 169

3 Elicitation stimulates Secondary Metabolism

Several reports suggest the use of speciﬁc conditions/compounds as elicitors to

enhance secondary metabolism. Elicitor-speciﬁc receptors are present on the cell

membrane of plants which get activated in the presence of an elicitor which

subsequently induces a cascade of downstream signalling events in plant cells

involving changes in the expression of genes encoding rate-limiting enzymes of

the secondary metabolites biosynthetic pathways (Halder et al. 2019). Thus, stimu-

lation by an elicitor can increase the content of metabolites and/or also generate new

compounds, mimicking the inherent strategy of the plant to protect and adapt itself to

the abiotic and biotic stresses such as drought, salinity, UV-irradiation, and patho-

genesis (Khare et al. 2020). There are various types of elicitors that affect secondary

metabolite production in orchids. Abiotic elicitors are derived from non-living

sources and include light and plant hormones, etc., while biotic elicitors have a

biological origin like chitosan and microorganisms (Table 2). This biotechnological

technique of elicitation offers a beneﬁcial approach to exploit the therapeutic

potential of medicinal plants and has unfolded a hot topic for research which has

tremendous potential for the therapeutic industry (Fig. 1).

3.1 Variable Light Exposure alters Phytochemical Proﬁle

The growth and metabolism of the plants is inﬂuenced by light. A number of studies

have highlighted the effect of intensity and quality of light on the phytochemical

proﬁle in orchids. In Anoectochilus roxburghii, the plants grown under light ﬁltered

through blue and red ﬁlm for 8 months displayed enhancement in the content of

active compounds such as polysaccharides and ﬂavones and greater antioxidant

enzyme activities; the highest phenolic content was observed in the plants with red

ﬁlm treatment (Ye et al. 2017). In another report on the same plant, a noteworthy

increase in the total polyphenols and total ﬂavonoids upon supplementation of blue

light has been reported while yellow light treatment produced higher soluble sugar

and polysaccharide content (Wang et al. 2018). Some research studies have also

focussed on using combinations of lights of different wavelengths to promote the

production of active compounds. A treatment using a combination of blue-red light

in the ratio 1:4 in Anoectochilus roxburghii plants enhanced the total ﬂavonoid

content. An increase in the expression of genes such as CHI (Chalcone Isomerase)

and FLS (Flavonol Synthase) involved in the ﬂavonoid biosynthetic pathway was

also observed (Gam et al. 2020). In Dendrobium Enopi x Dendrobium Pink Lady

hybrid orchid, the in vitro PLB cultures showed the highest ﬂavonoid content upon

treating the PLBs pre-illuminated with cool-white LED with blue-red (1:1) LED

irradiation. In addition, the PLBs precultured with red ﬂuorescent light for two

subculture cycles upon exposure to blue LED light displayed the highest antioxidant

activity (Yeow et al. 2020). Thus, the technique of altering the light source during

170 A. Kaur et al.

Table 2 Use of elicitors in in vitro cultures to boost the production of secondary metabolites

Plant species Elicitor Type of culture Secondary metabolite Reference

Anoectochilus

roxburghii

Methyl jasmonate (MeJA) and salicylic

acid (SA)

Rhizome suspension cultures Kinsenoside and polysaccharide content Luo et al.

(2018)

Mycorrhizal fungi Symbiotic cultures of tissue

cultured plantlets

Flavonol-glycosides (narcissin, rutin,

isorhamnetin-3-O-β-d-glucoside, querce-

tin-7-O-glucoside, and kaempferol-3-O-

glucoside), two ﬂavonols (quercetin and

isorhamnetin), and two ﬂavones (nobiletin

and tangeretin)

Zhang et al.

(2020a)

Ansellia

africana

Meta-topolin riboside (mTR) and

6, 3-methoxybenzylamino-9-b-D-

ribofuranosylpurine (MemTR)

Protocorm-like bodies (PLBs)

culture

Phenolic compounds (benzoates and

cinnamates)

Bhattacharyya

et al. (2019)

Dendrobium

candidum

Methyl jasmonate (MeJA) Protocorm-like bodies (PLBs)

culture

Alkaloids, polysaccharides, and ﬂavonoid

and phenolic content

Wang et al.

(2016)

Dendrobium

ﬁmbriatum

Caffeic acid (CA), p-coumaric acid (pCA),

and ferulic acid (FA)

Cell suspension cultures

established from Protocorm-

like bodies (PLBs)

Flavonoid, phenolic, alkaloid, and tannin

content

Paul and

Kumaria

(2020)

Dendrobium

ovatum

L-phenylalanine Callus-derived plantlets Bibenzyl derivative (moscatilin) Pujari et al.

(2021)

Dendrobium

Sabin blue

Thidiazuron (TDZ) Protocorm-like bodies (PLBs)

culture

Anthocyanin content Chin et al.

(2021)

Habenaria

edgeworthii

6-Benzyladenine (BA) Callus suspension culture Phenolic content Giri et al.

(2012)

Exploring the Potential of In Vitro Cultures as an Aid to the... 171

Fig. 1 A schematic ﬂowchart depicting the use of plant tissue culture and pharmacological studies in medicinal orchids

the propagation of orchids is an effective choice to increase the production of

bioactive compounds.

172 A. Kaur et al.

3.2 Chemical Abiotic Elicitors trigger Stress Responses

There are various plant growth regulators which act as elicitors and play a key role in

modifying the secondary metabolism in plants. The exogenous applications of these

chemicals have frequently been used in cell or organ culture to accentuate secondary

metabolite biosynthesis (Thakur et al. 2019).

3.2.1 Methyl jasmonate

Methyl jasmonate (MeJA), a derivative of jasmonic acid is one such hormone that

functions as a signalling molecule and strongly activates secondary metabolism in

medicinal orchids as it induces defence response against pathogens and wounding

(Nabi et al. 2021). Also, it triggers the expression of pivotal genes involved in

ﬂavonoid and anthocyanins biosynthesis such as Phenylalanine Ammonia Lyase

(PAL), Stilbene Synthase (STS), and Chalcone Isomerase (CHI) which play a pivotal

role in the production of ﬂavonoids and anthocyanins (Nabi et al. 2021). There have

been reports on the role of MeJA in the accumulation of alkaloids in Dendrobium

ofﬁcinale (Chen et al. 2019). Besides alkaloids, elicitation with MeJA in the root

tissue of D. ofﬁcinale has been observed to induce the production of bibenzyl

compounds such as erianin and gigantol (Adejobi et al. 2021). The treatment of

75 μM MeJA to the protocorm-like bodies (PLBs) of Dendrobium candidum showed

augmentation in the production of alkaloids, polysaccharides, and ﬂavonoids while

the increase in phenolic content was observed under 100 μM MeJA treatment (Wang

et al. 2016). Thus, optimization of elicitation is required for the production of a

particular group of active compounds. Besides chemical concentration, the duration

of exposure also matters. The rhizome suspension cultures of Anoectochilus

roxburghii upon treatment with 550 μM MeJA for a period of 14 and 16 days,

resulted in the maximum production of kinsenoside and polysaccharide, respectively

(Luo et al. 2018). In addition, the increase in the concentration of MeJa beyond the

optimum concentration may result in the reduction of the metabolite content. For

instance, in Habenaria edgeworthii, the total phenolic content decreased beyond

10 μM MeJA (Giri et al. 2012). Similarly, using MeJA beyond 50 μM in the

protocorm-like bodies (PLBs) of Dendrobium Sabin Blue (a hybrid species between

Dendrobium Blue Angel and Dendrobium Sanan Blue) orchid resulted in the

reduction in anthocyanin content (Abd Malik et al. 2021).

Exploring the Potential of In Vitro Cultures as an Aid to the... 173

3.2.2 Salicylic acid

Salicylic acid (SA), a phenylpropanoid compound, is another common elicitor

involved in signalling in plants. Besides its signiﬁcant role in the physiological

processes of plants such as seed germination, photosynthesis, uptake of nutrients,

nodulation in legumes, and induction of ﬂowering, it also regulates the expression of

genes associated with the enzymes of secondary metabolism (Ali 2021). The effect

of this important signal molecule may differ in different plant tissues. In Coelogyne

ovalis, the leaf tissues treated with SA yielded the highest content of ﬂavonoids and

anthocyanins while the SA-treated pseudobulbs showed the highest phenolic con-

tent. In addition, the SA-treated plantlets exhibited signiﬁcantly higher antioxidant

activity (Singh and Kumaria 2021). SA, like MeJA, regulates the metabolite content

in a concentration and time-dependent manner. Alkaloids and polysaccharides

accumulated in high amounts in the protocorm-like bodies (PLBs) of Dendrobium

candidum upon elicitation with 75 μM SA while 100 μM SA led to high production

of ﬂavonoids (Wang et al. 2016). Anoectochilus roxburghii rhizomes exhibited

maximum kinsenoside and polysaccharide contents upon treatment with 500 μM

SA for 12 days (Luo et al. 2018). However, some sporadic studies also report the

inhibitory role of SA (Chin et al. 2021).

3.2.3 Cytokinins

Apart from playing a vital role in the growth and development of plants, cytokinins,

and their derivatives also inﬂuence the production of active compounds in plants. In

Habenaria edgeworthii, the callus grown on 3 μM BA showed a signiﬁcant

improvement in phenolic content and antioxidant activity (Giri et al. 2012). Simi-

larly, another cytokinin, topolin, and its derivatives like meta-topolin riboside (mTR)

and 6, 3-methoxybenzylamino-9-b-D-ribofuranosylpurine (MemTR) showed a pos-

itive impact in Ansellia africana. The PLBs showed an increase in the production of

phenolic compounds like benzoates and cinnamates along with an increase in

antioxidant activity (Bhattacharyya et al. 2019). The PLBs of Dendrobium Sabin

Blue supplemented with 4 mgL

1

thidiazuron (TDZ) depicted an increase in the

anthocyanin content (Chin et al. 2021).

Thus, chemical or hormonal elicitors are promising for increasing the production

of the metabolites and their utilization is considered an advantageous strategy.

However, it is important to formulate the optimum concentration and duration of

exposure to the elicitor being used.

174 A. Kaur et al.

3.3 Biotic Elicitors alter Secondary Mechanism as a Defence

Mechanism

3.3.1 Fungal elicitors

In orchids, mycorrhizal fungi play a key role in the germination of seeds and

development as the fungi supplement organic and inorganic nutrients for the grow-

ing entity (Dearnaley et al. 2012). Thus, orchid-mycorrhizal symbiosis constitutes a

pivotal part in the life cycle of orchids. Fungal elicitation also leads to the activation

of speciﬁc genes related to secondary metabolite biosynthetic pathways. Moreover,

fungal elicitors have been reported to be more promising in the biosynthesis of

metabolites in comparison to the chemical elicitors in a plethora of studies (Favre-

Godal et al. 2020). A signiﬁcant increase in the production of active metabolites in

the host plant upon inoculation with different types of mycorrhizal fungi has been

demonstrated in a few orchids. F-23 fungus (Mycena sp.) improved the product ion of

kinsenosides and ﬂavonoids of Anoectochilus formosanus (Zhang et al. 2013).

Similarly, Dendrobium nobile upon inoculation with the same fungus (F-23) showed

an increase in dendrobine level in the stem thus suggesting the role of mycorrhizal

fungi in dendrobine synthesis (Li et al. 2017). Another fungus, Ceratobasidium

sp. AR2 stimulated the accumulation of ﬂavonol-glycosides (narcissin, rutin,

isorhamnetin-3-O-β-d-glucoside, quercetin-7-O-gluco side, and kaempferol-3-O-

glucoside), ﬂavonols (quercetin and isorhamnetin), and ﬂavones (nobiletin and

tangeretin) in Anoectochilus roxburghii (Zhang et al. 2020a). Similar results show-

ing the promoting role of AR2 on ﬂavonoid production in Anoectochilus roxburghii

have been reported in another study (Zhang et al. 2020b). An enhancement in the

antioxidant and the hepatoprotective activity upon inoculation of Rhizoctonia

mycorrhizal fungi had also been observed in Anoectochilus formosanus (Cheng

and Chang 2011).

3.3.2 Chitosan

Another biotic elicitor, chitosan, a polysaccharide, derived from the exoskeletons of

insects and fungi showed a promoting role in the accretion of secondary metabolites

by augmenting the production of the enzymes involved in the biosynthetic pathways

of secondary metabolites (Zhao et al. 2005). Chitosan is a non-toxic natural bio-

polymer consisting of glucosamine and N-acetylglucosamine subunits (Sanford and

Hutchings 1987). It basically mimics the fungal pathogen and gets recognised at the

plant membrane through the mechanism of cell surface recognition which induces a

series of downstream events activating the defence response in plants (Singh and

Kumaria 2021). The micropropagated plantlets of Vanda coerulea when treated with

chitosan displayed an improvement in the phytoc hemical contents and antioxidant

potential. A positive correlation between the phytochemical content and Phenylal-

anine ammonia lyase (PAL) enzyme activity in Vanda coerulea upon treatment with

chitosan was also observed (Nag and Kumaria 2018), thus suggesting that chitosan

triggered the genes involved in secondary metabolism.

Exploring the Potential of In Vitro Cultures as an Aid to the... 175

4 Precursors Feeding in Cultures accentuates Secondary

Metabolite Production

The use of precursor molecules as elicitors has also been elucidated in some orchids.

Precursors are intermediates in the pathway of secondary metabolite biosynthesis

which upon adding to the culture medi a tend to increase the amount of the related

secondary metabolites. This strategy of precursor feeding is quite useful when the

precursor compound is available at a low cost compared to the ﬁnal desired product

(Namdeo et al. 2007).The highest phenolic and ﬂavonoid content in the cultures of

Dendrobium ﬁmbriatum was observed upon application of caffeic acid (CA) while

2 mM ferulic acid (FA) and 4 mM p-coumaric acid led to the highest alkaloid and

tannin content, respectively. Also, the cultures treated with caffeic acid exhibited the

highest antioxidant activity (Paul and Kumaria 2020). In a similar manner, in

Dendrobium ovatum, the use of L-Phenylalanine as a precursor ensured high content

of moscatilin, a bibenzyl derivative compound that possesses anticancer properties

(Pujari et al. 2021).

5 Bioreactors as Mini Factories for Scale-up

A bioreactor is an instrument for large scale in vitro propagation. It consists of a

closed and sterile culture vessel in which the internal environmental conditions can

be monitored and controlled (Mamun et al. 2015). The application of bioreactor

systems offers an alternative strategy for the production of bioactive compounds at

the industrial level. The method of bioreactor systems consumes less time and is

cost-effective compared to the use of gelled or semi-solid medium which requires the

transfer of the plant material into a fresh expensive media at periodic intervals of

time (Murthy et al. 2018). Moreover, in solid media all the plant parts are not in

direct contact with the medium and resultantly, growth occurs slowly (Zhang et al.

2018). Thus, to overcome these problems, different plant parts and culture media

under bioreactor systems have been utilised in orchids (Table 3). Several factors

affect the plant biomass and phytochemical production in a bioreactor that needs to

be optimised for desired results. The selection of a suitable type of bioreactor is vital

for the growth and metabolism of plant cultures. Temporary and continuous immer-

sion bioreactor systems are usually used for plant cultures. In the continuous

immersion system, the plant cultures are continuously immersed in the liquid

medium whereas the temporary immersion system works on the principle of tem-

porarily submerging the cultures in the medium at speciﬁc intervals of time (De Carlo

176 A. Kaur et al.

Table 3 Use of bioreactor cultures for mass production of active ingredients in orchids

Plant name

Plant part

used Culture media and PGRs Type of bioreactor Active ingredient Reference

Anoectochilus

roxburghii

Rhizomes 3/4 MS + 2 mg L

1

BA + 0.2 mg L

1

Kn + 0.5 mg L

1

NAA

CIB (continuous

immersion bioreactor)

Polysaccharide and Kinsenoside Jin et al.

(2017)

Dendrobium

candidum

Protocorms MS + 0.5 mg L

1

NAA CIB (continuous

immersion bioreactor)

Polysaccharides, coumarins, polypheno-

lics, ﬂavonoids, vitamin C and vitamin E

Cui et al.

(2014)

Dendrobium

nobile

Seedlings 1/2 MS + 0.5 mg L

1

+ NAA + 80 g L

1

CW

TIB (temporary immer-

sion bioreactor)

Alkaloids Zhang

et al.

(2022)

Bletilla striata Pseudobulbs 1/2 MS + 60 g L

1

potato

lixivium + 0.5 mg L

1

NAA

TIB (temporary immer-

sion bioreactor) system

Polysaccharides Zhang

et al.

(2018)

et al. 2021). In Anoectochilus roxburghii, the continuous immersion bioreactor

having a net at the bottom of the sphere of the bioreactor was found apt for the

mass production of rhizomes; nearly 2980.5 mg L

1

of kinsenoside and

5672.9 mg L

1

of polysaccharides were produced (Jin et al. 2017). In 2014, a

research group cultured the protocorms of Dendrobium candidum in different

bioreactor systems and found that the continuous immersion bioreactor system

was the most appropriate for the production of polyphenolics, ﬂavonoids,

vitamin C, a nd vitamin E, coumarins, and polysaccharides (Cui et al. 2014). In

Epipactis ﬂava, maximum in vitro micropropagation efﬁciency was obtained by

using the temporary immersion system in comparison to the continuous immersion

bioreactor system (Kunakhonnuruk et al. 2019). RITA

®

bioreactor based on a

temporary immersion system has been employed to cater to the demand of Cattleya

forbesii in the commercial market (Ekmekçigil et al. 2019). Similarly, in Vanda

tricolor, a temporary immersion bioreactor system has been established to be

efﬁcient for its commercial production (Esyanti et al. 2016).

Exploring the Potential of In Vitro Cultures as an Aid to the... 177

For optimisation of the bioreactor system, different aspects associated with bio-

reactors such as inoculation density, air volume, immersion frequency, and light

intensity hold signiﬁcant importance. Inoculation density affects the number of

nutrients that are available for each explant and aeration volume affects the mixing

of the constituents and oxygenation. Inoculation density of 50 g L

1

and an aeration

volume of 0.1 vvm (air volume per culture volume per minute) were found beneﬁcial

in protocorm immersion culture of Dendrobium candidum (Cui et al. 2014). In

Anoectochilus roxburghii, an inoculation density of 12.5 g L

1

, air volume lower

than 500 mL L

1

and 45 μmol m

2

s

1

light intensity was favourable (Jin et al.

2017). The temporary immersion frequency of 5 min every 6 h maximised the

biomass and total alkaloid content in the plantlets of Dendrobium nobile (Zhang

et al. 2022).

The secondary metabolite content reaches its peak value after a speciﬁc number

of days of initiation of bioreactor culture. For instance, for the rhizome immersion

bioreactor culture in Anoectochilus roxburghii, 30 days period was the optimum for

maximum polysaccharide (4251.2 mg L

1

) and kinsenoside (1724.0 mg L

1

) pro-

duction (Jin et al. 2017). However, for Dendrobium nobile, 20 days culture period

was most advantageous for alkaloids production (Zhang et al. 2022). Further, the

concentration of carbon source used in the culture medium also has a role in PLB

bioreactor cultures of Dendrobium candidum, the optimal concentration of sucrose

in the culture medium was found to be 30 g L

1

for improved polysaccharide and

alkaloid yields (Yang et al. 2015). Additionally, the elicitors have also been tested in

bioreactor cultures in a few orchids. In Dendrobium candidum, the MeJA treatment

to 30 days bioreactor cultured PLBs for 4 and 10 days has been observed to induce

mass production of alkaloids or polysaccharides and phenolics or ﬂavonoids,

respectively (Wang et al. 2016). The content of polysaccharide and kinsenoside

and antioxidant activity showed improvement upon elicitation with MeJA or

salicylic acid in rhizome immersion bioreactor cultures of Anoectochilus roxburghii

in comparison to the plants grown in the ﬁeld (Luo et al. 2018). In Dendrobium

nobile, 10 μM MeJA improved the accumulation of alkaloids in the bioreactor

Declarations: Conﬂicts of Interest/Competing Interests

culture of plantlets (Zhang et al. 2022). Similarly, elicitation with 0.25 mmole L

1

of

MeJA enhanced the biosynthesis of polysaccharides and enlarged the pseudobulbs

of Bletilla striata (Zhang et al. 2018). Hence, the use of elicitors in bioreactor

cultures is considered suitable for orchids to serve as mini-factories of important

metabolites by scaling up the secondary metabolites production.

178 A. Kaur et al.

6 Conclusions

In vitro cultures serve as a consistent source of valuable plant-speciﬁc metabolites in

orchids. Thus, this technique could be utilised for the scale-up process resulting in

the mass production of orchid-speciﬁc bioactive compounds to cater to the demands

of the pharmaceutical, cosmetic, and nutraceutical industries. Additionally, it

reduces overexploitation and unscrupulous collections pressures on the natural

populations of orchids. Hence, resulting in sustainable utilization, commercial

propagation, and conservation of high-value therapeutically important orchid spe-

cies. This review offers insights into the strategies for improvement of phytochem-

ical production in orchids and provides a baseline data for future research.

Acknowledgements AK is thankful to the Department of Science and Technology (DST) for

INSPIRE Fellowship for Research Students. (File No. DST/INSPIRE/03/2021/002638). JKS is

grateful to the Department of Science and Technology, Government of India, for partial ﬁnancial

support under Promotion of University Research and Scientiﬁc Excellence (PURSE) grant scheme.

The authors have no conﬂicts of interest to declare that are relevant to the content of this article.

Author’s Contributions JKS conceived the original idea and outline. AK and JV prepared the

original draft. SVP and VGY gave signiﬁcant inputs. All the authors critically revised the work and

approved the ﬁnal manuscript.

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Ethnomedicinal Uses, Phytochemistry,

Medicinal Potential, and Biotechnology

Strategies for the Conservation of Orchids

from the Catasetum Genus

Luis J. Castillo-Pérez, Daniel Torres-Rico, Angel Josabad Alonso-Castro,

Javier Fortanelli-Martínez, Hugo Magdaleno Ramírez-Tobias,

and Candy Carranza-Álvarez

L. J. Castillo-Pérez

Programa Multidisciplinario de Posgrado en Ciencias Ambientales, Universidad Autónoma de

San Luis Potosí, San Luis Potosí, San Luis Potosí, Mexico

e-mail: jesus.perez@uaslp.mx

D. Torres-Rico

Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí,

San Luis Potosí, Mexico

A. J. Alonso-Castro

División de Ciencias Naturales y Exactas, Departamento de Farmacia, Universidad de

Guanajuato, Guanajuato, Mexico

J. Fortanelli-Martínez

Instituto de Investigación de Zonas Desérticas, Universidad Autónoma de San Luis Potosí, San

Luis Potosí, San Luis Potosí, Mexico

e-mail: fortanel@uaslp.mx

H. M. Ramírez-Tobias

Facultad de Agronomía y Veterinaria, Universidad Autónoma de San Luis Potosí, San Luis

Potosí, San Luis Potosí, Mexico

e-mail: hugo.ramirez@uaslp.mx

C. Carranza-Álvarez (✉)

Programa Multidisciplinario de Posgrado en Ciencias Ambientales, Universidad Autónoma de

San Luis Potosí, San Luis Potosí, San Luis Potosí, Mexico

Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí,

San Luis Potosí, Mexico

Facultad de Estudios Profesionales Zona Huasteca, Universidad Autónoma de San Luis Potosí,

Ciudad Valles, San Luis Potosí, Mexico

e-mail: candy.carranza@uaslp.mx

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_6

187

188 L. J. Castillo-Pérez et al.

1 Introduction

The obtention of new drugs from plant secondary metabolites plays an important

interest in the pharmaceutical industry (Radice et al. 2020). Many medicinal plants

from the Solanaceae, Asteraceae, and Fabaceae families have been studied, whereas

other plant families like Apiaceae, Ranunculaceae, and Orchidaceae lack scientiﬁc

information validating their medicinal properties (Marrelli 2021).

The Orchidaceae family is the most diverse in the plant kingdom and represents

an important part of the biodiversity in the Neotropics. This family has a wide

distribution in this area. Some of the genera, including Laelia, Stanhopea,

Cyrtopodium, and Epidendrum belonging to this family have reports of medicinal

properties (Castillo-Pérez et al. 2019). These orchid genera have shown biological

activities as antihypertensive, antipyretic, anti-inﬂammatory, antinociceptive, and

antidysentery, among others (Vergara-Galicia et al. 2013; Morales-Sánchez et al.

2014; Emeterio-Lara et al. 2016; Arora et al. 2017).

A particular and low-studied genus within the Orchidaceae family is the

Catasetum genus, which possesses approximately 170 species and is widely distrib-

uted in the neotropical region of America (Milet-Pinheiro and Gerlach 2017).

However, some Catasetum hybrids have been successfully cultivated and adapted

to other regions, being grown in Europe, Asia, and America (Cantuaria et al. 2021).

They have diverse growth habits, most species are epiphytic (Fig. 1a), but some

species present terrestrial, lithophyte, or saprophyte development (Milet-Pinheiro

and Gerlach 2017). Moreover, they are sexually dimorphic and exhibit male and

female ﬂowers (Fig. 1b, c) (Gerlach 2013). Another important piece of data is these

orchids present mycorrhizal and myrmecophile ecological interactions for their

Fig. 1 Catasetum integerrimum (Orchidaceae) is a common example of the genus Catasetum. (a)

Whole plant in situ with epiphytic growth; (b) Male ﬂowers; (c) Female ﬂowers; (d) Interaction

with the bee of the Euglossini tribe, the main pollinator of the genus Catasetum

growth and defense, and the male bees of the Euglossini tribe are the main pollinat-

ing agent (Fig. 1d) (Gerlach 2013; Bonilla-Morales et al. 2016).

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 189

Currently, species of the Catasetum genus are used mainly as ornamental plants

in several parts of the world. Some species of this genus have medicinal properties

attributed to different population groups around the world. The objective of this

chapter is to summarize all the research ﬁndings available on various aspects, such as

botanical description and distribution, ethnopharmacology, phytochemistry, and

conservation of the Catasetum genus.

The information search was based on the following groups of keywords:

Catasetum orchids, Medicinal Catasetum, Phytochemical Catasetum, Biotechnol-

ogy Catasetum, and Ecology of Catasetum. We search the most relevant data in

“PubMed”, “ScienceDirect”, “Scopus”, “Web of Science”, and “Google Scholar”,in

addition, physical and digital books were consulted. The current taxonomy of the

species was validated using the website of The World Flora Online (http://www.

worldﬂoraonline.org/). The article search was carried out from 15 March 2022 to

15 August 2022. Based on all the compiled information, the research gap has also

been discussed. This chapter provides the basis for further studies on the conserva-

tion and development of identifying better therapeutic agents and health products

from the Catasetum orchids.

2 Botanical Description of the Species

This section describes the general characteristics shared by Catasetum orchids. We

suggest consulting the taxonomic keys provided in the botanical bibliography for the

speciﬁc description of any species from this genus. Most Catasetum orchids are

epiphytic, perennial, medium-sized with a height of 30–70 cm, composed of

pseudobulbs ovoid to fusiform and ﬁbrous roots at the base. The plants have leaves

that can be oblong-lanceolate to elliptic and deciduous. The ﬂowers are terminal

racemes, and some species develop non-resupinate, unisexual, dimorphic and fra-

grant ﬂowers. The column in the Catasetum species is short and truncated and has a

pollinial vestigial. After pollination, these species develop ellipsoid and glaucous

capsules with seeds minute and powdery. Most of the pseudobulbs are ﬂeshy,

smooth, shining, greenish, covered with membranous sheath, and slightly mucilag-

inous (Salazar et al. 1990). The ﬂowering of these species varies throughout the year

and some species can develop ﬂowers more than once a year. Anatomical and

histochemical studies revealed the presence of endophytic mycorrhizal fungus in

the root and protocorm (Silva et al. 2015). The anatomical similarity between

rhizomes and pseudobulbs indicates that species can be propagated from its rhi-

zomes as well as pseudobulbs.

190 L. J. Castillo-Pérez et al.

3 Habitat, Distribution, and Ecology

Catasetum orchids have different development forms, some species are epiphytic,

others are terrestrial or lithophyte and some species have even been described with

saprophyte growth. The species of the Catasetum genus usually develop mycorrhizal

and myrmecophile interactions for their growth, plant development, and defense.

Another interesting ecological aspect is that the wide majority of Catasetum species

share their main pollinating agent, the male bees of the Euglossini tribe, also known

as orchid bees (Milet-Pinheiro and Gerlach 2017; Gerlach 2013; Bonilla-Morales

et al. 2016).

The Catasetum genus is present only in the neotropical region of the American

continent and has approximately 170 species. Brazil encompasses the largest number

of Catasetum orchids (Romero-Gonzales 2012; Ramos et al. 2012; Chase et al.

2015). In the case of Mexico, two of the most important orchids of the Catasetum

genus, C. integerrimum, and C. laminatum are distributed in the states of Tamauli-

pas, San Luis Potosí, Hidalgo, Veracruz, Puebla, Querétaro, Oaxaca, Chiapas,

Tabasco, Campeche, Yucatan, and Quintana Roo (Salazar et al. 1990).

Table 1 shows the few available studies published about the habitat and ecology

of the Catasetum orchids. Most of these species are distributed in tropical forests,

which is not surprising since many of these orchids are epiphytes. Interestingly,

C. discolor grows in more arid ecosystems in countries like Bolivia, Brazil, and

Venezuela (Milet-Pinheiro and Gerlach 2017; Dodson 1978).

Catasetum orchids have their pollinating species. Nevertheless, there are few

records about the pollinating organisms of these orchids, including insects of the

Hymenoptera order, Apidae family, and Euglossini tribe, speciﬁcally two genera,

Eufriesea, Euglossa, and Eulaema (Table 1). However, this work found records of

16 Catasetum species, which represents a gap in the ecological knowledge of these

species.

Another ecological aspect with limited information is the time of ﬂowering in

these orchids. For example, of the 16 species presented in this work, this data is only

known in eight of the 16 species. Interestingly, some species such as

C. integerrimum and C. viridiﬂavum ﬂower for most of the year (Table 1) (Milet-

Pinheiro and Ger lach 2017; Hernández-Ramírez 2021).

4 Ethnomedicinal Uses

The medicinal uses conferred on the Catasetum orchids have been documented in

several reports (Table 2). Firstly, it was recorded in 1958 that the ashes of

pseudobulbs from C. maculatum were used for the treatment of inﬂammations,

abscesses, sores, and warts (Kunow 1958). Afterward, Arenas and Moreno-Azorero

(1977) documented using C. gardneri pseudobulbs as a sterilant. The application of

this orchid was recommended in conjunction with the rhizomes of another plant

(continued)

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 191

Table 1 Some ecological aspects of Catasetum orchids

Species Habitat

Main

growth

type Pollinator species

Flowering

season References

Catasetum arietinum

F.E.L. Miranda and

K.G. Lacerda

Neotropical cloud forests Epiphyte Euglossa nanomelanotricha,

Euglossa securigera

February–

July

Brandt et al. (2020)

Catasetum

integerrimum Hook

Tropical deciduous and semi-

deciduous forests, warm oak and

palm forests, neotropical cloud for-

ests, and montane forests

Epiphyte Eulaema cingulata, Eulaema

polichroma, Eulaema cingulate,

Eulaema meriana, Exaerete

frontalis

April–

November

Salazar et al. (1990),

Hernández-Ramírez

(2021)

Catasetum uncatum

Rolfe

The short palm stems in dry forest Epiphyte Euglossa nanomelanotricha,

Euglossa carolina

March–

May

Milet-Pinheiro et al.

(2015)

Catasetum pusillum

C. Schweinf

Semi-humid forests Lithophyte

and

terrestrial

Euglossa sp. February–

May

Huatangare-Córdova

(2000)

Catasetum saccatum

Lindl.

Primary forests Epiphyte Eufriesea violacens, Euglossa

augaspis, Euglossa chalybeata,

Euglossa cordata, Euglossa ignita,

Euglossa imperialis, Eulaema

cingulata

NM

a

Milet-Pinheiro and

Gerlach (2017),

Huatangare-Córdova

(2000)

Catasetum

peruvianum Dodson

and D.E. Benn

Primary and secondary forests Epiphyte NM NM Huatangare-Córdova

(2000)

Catasetum cernuum

(Lindl.) Rchb.f.

Tropical and riparian forests Epiphyte Eufriesea violacea NM Nunes et al. (2017)

Catasetum

ochraceum Lindl.

Tropical dry forests Terrestrial Euglossa modestior, Euglossa

gaiani, Euglossa deceptrix,

Euglossia liopoda

NM Romero and Nelson

(1986), Zapata-Hoyos

et al. (2021)

Table 1 (continued)

Species Habitat

Main

growth

type Pollinator species

Flowering

season References

Catasetum

macrocarpum Rich.

ex Kunth

Tropical forests bordering rivers Epiphyte

and

terrestrial

Eulaema bombiformis, Eulaema

nigrita

Starts in

January

Dodson (1978), Carvalho

and Machado (2002),

Ferreira et al. (2018)

Catasetum discolor

(Lindl.) Lindl

Savannah, in sand Terrestrial Eulaema bombiformis, Eulaema

bomboides Eulaema cingulata,

Eulaema nigrita, Eulaema meriana,

Euglossa ignita

May–

November

Milet-Pinheiro and

Gerlach (2017), Dodson

(1978)

Catasetum

longifolium Lindl.

NM Epiphyte Eulaema bombiformis

Eulaema meriana

NM Dodson (1978)

Catasetum

maculatum Kunth

NM Epiphyte Eulaema polychroma, Eulaema

meriana, Eulaema cingulata

Twice a

year,

March and

July

Janzen (1981)

Catasetum

viridiﬂavum Hook

Tropical cloud forest Epiphyte Eulaema cingulata, Eulaema

nigrita, Eulaema marcii, Exaerete

frontalis

April–

December

Milet-Pinheiro and

Gerlach (2017),

Zimerman (1991)

Catasetum galeritum

Rchb. f.

Tropical cloud forest Epiphyte Eufriesea superba NM Milet-Pinheiro et al.

(2018)

Catasetum gardneri

Schltr.

NM Epiphyte Eufriesea auriceps, Eufriesea

violacens, Eufriesea combinata,

Eulaema cingulata, Euglossa sp.

NM Milet-Pinheiro et al.

(2018), Coelho-Ferreira

(2005)

Catasetum barbatum

(Lindl.) Lindl

NM Epiphyte Euglossa augaspis, Euglossa

cognata, Euglossa cordata,

Euglossa mixta, Eulaema cingulata

NM Milet-Pinheiro et al.

(2018)

Catasetum

macroglossum

Rchb. f.

NM Epiphyte Eulaema cingulata, Eulaema

tropica, Eulaema bomboides,

Eulaema speciosa, Eulaema

polychroma

NM Milet-Pinheiro et al.

(2018), Vogel (1963)

a

NM Not mentioned

192 L. J. Castillo-Pérez et al.

(continued)

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 193

Table 2 Ethnopharmacological uses of the genus Catasetum

Species

Plant section

used Preparation way Ethnopharmacological uses

Country

where it is

used References

Catasetum

maculatum

Kunth

Pseudobulb Plaster Treatment of inﬂammations, abscesses,

sores, and warts

Mexico Kunow (1958), Cervantes-Reyes

(2008)

Catasetum

gardneri Schltr.

Pseudobulb Infusion with rhi-

zome of Typha

latifolia

Sterilization Paraguay Arenas and Moreno-Azorero (1977)

Infusion Contraceptive Paraguay

and Brazil

Teoh (2019)

Catasetum

barbatum

(Lindl.) Lindl.

Aerial parts NM Asthma and lumbago Paraguay Shimizu et al. (1988)

Catasetum

integerrimum

Hook

Leaf NM Treatment of pimples Mexico Ankli et al. (1999)

All plant NM Dermatological diseases Alonso-Castro et al. (2011)

Pseudobulb Liqueﬁed with

water

Supplement against kidney and urinary

infections

Castillo-Pérez et al. (2021)

NM Infusion with leaf

of Laelia

autumnalis

Cough treatment Cervantes-Reyes (2008)

NM Snake bite Téllez-Valdés et al. (1989)

Cure of tumors and in the treatment of

abscesses and wounds

Cox-Tamay (2013)

Burns and wounds Cruz-Garcia et al. (2014)

Antidiarrheal Teoh (2019)

Pseudobulb,

leaf, root,

capsule

Infusion or lique-

ﬁed with water

Treatment of colitis, diabetes, high blood

pressure, kidney conditions, and cancer

Galicia-Mendieta (2017),

Hernández-Bautista and Martínez-

Espinoza (2019)

Table 2 (continued)

Species

Plant section

used Preparation way Ethnopharmacological uses

Country

where it is

used References

Catasetum

expansum

Rchb. f.

Stem Plaster or poultice -Treatment of broken bones and bone

fractures

Ecuador Zambrano-Intriago et al. (2015)

Catasetum

macroglossum

Rchb. f.

Pseudobulb Plaster -Treatment of broken bones and bone

fractures—anti-inﬂammatory and anti-

rheumatic

Ecuador Ramos et al. (2012), Ramos-Corrales

et al. (2011)

NM Not mentioned

194 L. J. Castillo-Pérez et al.

denominate Typha latifolia. To obtain the sterilizing effect, both parts of the plants

should be boiled in water and consumed at the morning. The consumption of the

pseudobulbs of C. gardneri as an infusion is registered as a contraceptive method by

residents of indigenous regions from Paraguay and Brazil.

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 195

Fig. 2 Pseudobulbs on sale of C. integerrimum in a local market in the municipality of Matlapa,

Huasteca Potosina, Mexico

C. barbatum is another species of the Catasetum genus documented as medicinal.

This species is used in traditional medicine from Paraguay for the treatment of

asthma and lumbago. However, there is no information on the preparation of this

plant for the treatment of these diseases (Shimizu et al. 1988).

One of the Catasetum species with the most records of medicinal properties is

C. integerrimum (Table 2). In the late 1980s, this species was reported to be useful

for treating viper bites (Téllez-Valdés et al. 1989). Subsequently, it was reported that

the leaves of this species were used by Mayan communities in the state of Yucatan,

Mexico for the treatment of “large grains”, which possibly may allude to tumors

(Ankli et al. 1999). Another investigation carried out by Alonso-Castro et al. (2011)

mentioned the entire use of the orchid for the treatment of dermatological conditions,

and Cox-Tamay (2013), documented its application in the treatment of tumors,

abscesses, and wounds by communities of Yucatan, Mexico. Another use that has

been conferred to C. integerrimum is in the treatment of burns and wounds (Cruz-

Garcia et al. 2014), and recently in the state of Veracruz, Mexico, its application is

used for treating diarrhea (Teoh 2019). However, the information on which plant

part should be used for the medicinal purpose, the way of administration, and the

way of preparation are frequently omitted in the scientiﬁc literature.

In the Huasteca Potosina region, pseudobulbs of C. integerrimum are traded in

local markets with other medicinal plants and fruits (Fig. 2). The inhabitants of this

region comment that the pseudobulbs should be prepared as an infusion with water

and orally consumed for treating kidney, gastrointestinal, urinary tract infections,

and against diabetes mellitus (Castillo-Pérez et al. 2021).

196 L. J. Castillo-Pérez et al.

Other Catasetum species documented with medicinal properties are C. expansum

and C. macroglossum, used in communities in Provincias del Rio, Ecuador.

According to Zambrano-Intriago et al. (2015), C. expansum is used in the treatment

of broken bones and bone fractures, through the preparation of plaster or poultice,

made from the scape ﬂoral, which is then applied to the affected area. On the other

hand, Ramos-Corrales et al. (2011) mention that C. macroglossum is used in the

treatment of inﬂammation, pain and broken bones, also applied by making a poultice

from the pseudobulbs. Likewise, Ram os et al. (2012) reported the topical use of the

pseudobulbs of C. macroglossum as anti-inﬂammatory and anti-rheumatic in the

middle lands and forests of Ecuador.

Finally, some reports documented the consumption of the stem ﬂoral wand of

some Catasetum species for the reduction of headaches in Shuar communities

(Ecuador). However, the Catasetum species was not reported (Bennett 1992).

Likewise, Kunow (1958) and Teoh (2019) reported the use of Catasetum maculatum

used in traditional Mayan medicine for treating external tumors and abscesses. The

Catasetum genus has a wide variety of medicinal applications. However, the studies

that support these properties are scarce.

5 Phytochemicals Isolated and Pharmacological Activities

As shown in Table 3, the studies on the secondary metabolites isolated Catasetum

species and their pharmacological actions are limited to three species. Four com-

pounds, including the phenanthrene 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene

were isolated from an ethanolic extract of the aerial parts of C. barbatum and tested

on their anti-inﬂammatory and antinociceptive activity through the carrageenan-

induced plantar edema test and the histamine-induced contortion tests in rats

(Shimizu et al. 1988).

Currently, C. integerrimum is one of the orchids most studied under different

approaches. There are two studies carried out to verify its pharmacological activities.

First, 25 μg/mL ethyl acetate extract of leaves, roots, and pseudobulbs of

C. integerrimum showed cytotoxic activity by 79.47% and 97.79% on breast cancer

cell lines MCF-7 and MDA-MB231, respectively. The compounds identiﬁed

included phenolic acids (ferulic, gallic, p-coumaric, p-hydroxybenzoic, syringic,

and vanillin) and ﬂavonoids (phloretin, galangin, naringenin, quercetin, and rutin)

(Cruz-Garcia et al. 2014).

The antioxidant activity of a root extract from C. integerrimum and its metabo-

lites showed antioxidant activity in the ABTS and DPPH assays. The phytochemical

qualitative test revealed the presence of sterols, unsaturations, ﬂavonoids, and

coumarins in wild plants and vitroplants (Table 3). This is one of the ﬁrst studies

reporting the phytochemical proﬁle of Catasetum vitroplants (Torres-Rico 2021).

(continued)

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 197

Table 3 Biological activities and phytochemicals isolated from Catasetum

Species

Plant part

analyzed Biological activity studies

Tested

extracts Isolated chemical compounds References

Catasetum

barbatum

(Lindl.) Lindl

Aerial parts Anti-inﬂammatory activity by

carrageenan-induced plantar edema

Ethanolic

extract

1. 2,7-dihydroxy-3,4,8-

trimethoxyphenanthrene

2. 2,7-dihydroxy-3,4-

dimethoxyphenanthrene

3. 2,7-dihydroxy-3,4-

dimethoxy-9,10-

dihydrophenanthrene

4. 2,7-diacetoxy-3,4-

dimethoxy-9,10-

dihydrophenanthrene

Shimizu et al. (1988)

Catasetum

integerrimum

Hook

Leaf, root,

and

pseudobulb

Cytotoxic activity by breast cancer

cell lines (MCF-7 and

MDA-MB231)

Ethyl ace-

tate

extract

1. Ferulic acid

2. Gallic acid

3. p-coumaric acid

4. p-hydroxybenzoic acid

5. Syringic acid

6. Vanillin

7. Phloretin

8. Galangin

9. Naringenin

10. Quercetin

11. Rutin

Cruz-Garcia et al. (2014)

Root Antioxidant activity by DPPH and

ABTS assays

Ethanolic

extract

1. Flavonoids

2. Phenols

3. Reducing sugars

4. Alkaloids

5. Tannins

6. Saponins

7. Sterols

8. Terpenes

Torres-Rico (2021)

Table 3 (continued)

Species

Plant part

analyzed Biological activity studies

Tested

extracts Isolated chemical compounds References

Catasetum

macroglossum

Rchb. f.

Pseudobulb Anti-inﬂammatory activity by

carrageenan-induced plantar edema

Aqueous

extract

1. Reducing sugars

2. Flavonoids

3. Glucomannan

4. Phenanthrene

5. Stilbene

Ramos et al. (2012)

Antioxidant activity by DPPH assay Ethanolic

extract

1. Phenols

2. Flavonoids

3. 1,5-anhydro-D-sorbitol

4. Xylitol

5. Octanedioic acid

6. Fructose

7. Linoleic acid

Molina-Sandoval (2020), Buenaño-

Morales and Santillán-Chávez

(2021)

198 L. J. Castillo-Pérez et al.

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 199

Aqueous extract prepared from C. macroglossum pseudob ulbs showed anti-

inﬂammatory activity on the carrageenan-induced plantar edema test in Wistar

rats. These properties were attributed to the presence of ﬂavonoids. An HPLC-

DAD analysis determined the presence of phenanthrenic and stilbenic

dihydroderivatives (Ramos et al. 2012). Recent current works on

C. macroglossum suggested the presence of phenols, ﬂavonoids, various sugars,

and some fatty acids in this plant species (Table 3). Some of these compounds have

antioxidant effects, which can confer add value to many of these edible orchids. The

presence of biological activity in Catasetum species conﬁrms the traditional use of

these orchids, demonstrating the need for more ethnobotanical studies.

6 Propagation and Cultivation Effort

Current biotechnological efforts in plants are an integral part of the works associated

with in vitro and ex vitro conservation and propagation, genetic transformation,

acclimatization, and product development from several plant genera and species

(López-Puc and Herrera-Cool 2022). Several works were published on biotechno-

logical studies about the Catasetum genus (Table 4), focusing on the propagation

and in vitro conservation of these species from various types of explants, denoting a

preference for the conservation of the genus, but with little research focused on the

acclimatization and development of products from species with phytochemicals of

pharmacological potential.

The conservation protocols of six different Catasetum species were published.

Seeds and protocorms are the most widely used explants, although pseudobulbs,

roots, and in vitro plants have also been used. Seed germination and

micropropagation for mass propagation studies are available for these species.

Fernandes et al. (2015) used seeds from an immature capsule of Catasetum boyi

and obtained up to 90% germination. The percentage of seed germination is low,

approximately 5% of all seeds, under natural conditions (Arditti 1967).

Micropropagation work was also carried out for C. gardneri (Silva-Maia and

Pedroso-deMoraes 2017), C. macrocarpum (Ferreira et al. 2018), and

C. schmidtianum (Leles-Gaudêncio et al. 2014) using seeds as explants.

There are micropropagation protocols using roots as explants in two species of

Catasetum orchids (C. gardneri and C. integerrimum) have worked

micropropagation protocols using roots as an explant. In the case of C. gardneri,

in vitro plants were obtained with a developed of 3.75 cm root growth per explant

(Peres et al. 2009). Indirect organogenesis was tested and observed in the production

of C. integerrimum in vitro plants by adding kinetin as a plant growth regulator

(López-Puc and Herrera-Cool 2022). No Catasetum orchid micropropagation pro-

tocol has reported leaves as an efﬁcient explant to generate in vitro plants. Castillo-

Pérez et al. (2021) tested this type of explant in C. integerrimum, obtaining a null

response to regenerate seedlings (Fig. 3).

Species Response obtained References

(continued)

200 L. J. Castillo-Pérez et al.

Table 4 Propagation effort by plant tissue culture techniques in Catasetum species

Explant

type used

Composition of the

culture media

Catasetum boyi

Mansf.

Seed 30 mg L

-1

sucrose

2g L

-1

fertilizer B and

G

100 mg L

-1

coconut

water

2g L

-1

activated carbon

4g L

-1

agar

90% seed germination

was obtained

Fernandes

et al.

(2015)

Catasetum

gardneri Schltr.

Protocorm MS basal medium mod-

iﬁed with 1/2

macronutrients

Vitroplants were

obtained by direct organ-

ogenesis way with

growth of 6 cm per

explant, 3 roots devel-

oped per explant and

pseudobulbs with 3 cm in

diameter

Rego-

Oliveira

and de

Faira

(2005)

Commercial formulation

N.P.K (10–5-5)

2mL L

-1

Vitroplants were

obtained by direct

organogenesis way with

a growth of 8.04 cm per

explant

Seed MS basal medium

1g L

-1

activated carbon

30 g L

-1

sucrose

7g L

-1

agar

Jasmonic acid (concen-

tration not mentioned)

Vitroplants were

obtained by direct organ-

ogenesis way with the

development of 2.4 roots

per explant, 1 leaf per

explant and approxi-

mately 1.75 cm leaf and

root growth per explant

Silva-Maia

and

Pedroso-

deMoraes

(2017)

Roots Vacin and Went

medium modiﬁed by

substituting

Fe

2

(C

4

H

4

O

6

)

3

by

27.8 mg dm

-3

Fe-EDTA

MS micronutrients

Sucrose

Vitroplants were

obtained with a devel-

oped of 3.75 cm root

growth per explant

Peres et al.

(2009)

Vitroplants Vacin and Went

medium

Micronutrients of MS

0.01% thiamine

0.1% soy peptone

2% sucrose

0.2% phytagel

In this work ethylene

production showed a

decreasing trend in the

ﬁrst 4 months, presenting

an initial and a ﬁnal con-

centration of 66.11

± 10.68 and 21.92

± 6.67 μLg

-1

FW h

-1

,

respectively. Likewise,

an increase in ethylene

production was observed

at the end of the 8 months

Rodrigues

et al.

(2013)

Species Response obtained

(198.64 ±5.17), coin-

ciding with the termina-

tion of a growth cycle

(continued)

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 201

Table 4 (continued)

Explant

type used

Composition of the

culture media References

Nodal

explants

Vacin and Went

medium

Micronutrients of MS

0.1% activated charcoal

2% sucrose

0.7% agar

Ethylene

1-MCP

The chronic exposure to

exogenous ethylene-

induced severe growth

deterioration in young

plants during the

5 weeks of treatment, on

the contrary, the supply

of 1-MPC, induced

morphological effects

opposite to those

induced by ethylene

Catasetum

integerrimum

hook

Vitroplants 4.46 g L

-1

MS medium

8g L

-1

agar plant

30 g L

-1

sucrose

3g L

-1

activated carbon

IAA

BAP

Vitroplants were

obtained with 5.73

± 0.45 shoots per explant

and 5.84 ± 0.48 leaves

per shoot. Moreover,

vitroplants developed

11.20 ± 0.28 roots per

explant and 13.20

± 0.28 cm root growth

Castillo-

Pérez et al.

(2021)

Pseudobulb 4.46 g L

-1

MS basal

medium

8g L

-1

agar plant

30 g L

-1

sucrose

3g L

-1

activated carbon

1mg L

-1

IAA

1mg L

-1

BAP

By direct organogenesis

in vitro plants were

obtained with 1.00 ± 00

shoots per explant, 5.50

± 0.18 leaves per shoot,

4.37 ± 0.37 roots per

explant with a growth

rate of 4.88 ± 0.20 cm

and a plant growth of

7.96 ± 0.12 cm

Plantlet MS basal medium (half-

strength)

Sorbitol

Carbon

The treatment added with

3% carbon, and 2% sor-

bitol presented the lowest

value of growth in plant-

let length (17.70 ± 5.8).

In the same way, showed

the lowest shoot forma-

tion (1 ± 00)

López-Puc

and

Herrera-

Cool

(2022)

Root and

node

MS basal medium

3% sucrose

2.2 g L

-1

Gelrite

2g L

-1

activated carbon

BAP

Kinetin

Direct shoot organogen-

esis was observed in

node explant in

BAP-supplemented MS

and kinetin-

supplemented MS at all

concentrations tested.

Species Response obtained

Indirect shoot organo-

genesis was observed in

root explant in MS

supplemented with 4.64

or 9.29 μM kinetin

(continued)

202 L. J. Castillo-Pérez et al.

Table 4 (continued)

Explant

type used

Composition of the

culture media References

Catasetum

macrocarpum

Rich. ex Kunth

Seed ½ MS basal medium

0.4 mg L

-1

tiamin

100 mg L

-1

myo-inosi-

tol

2% sucrose

BA

NAA

Vitroplants were

obtained with 4.1 shoots

per explant and 6.1 roots

per explant

Ferreira

et al.

(2018)

Vitroplant First phase: Bioplant

Prata® with sphagnum

(1:1)

Second phase: Bioplant

Prata with Ouro Negro

substrate (1:2)

The survival rates

observed in the acclima-

tization process were

93.3% for the ﬁrst phase

and 96.6% for the sec-

ond phase

Catasetum

pileatum

Rchb. f.

Protocorm MS basal medium

3% sucrose

0.8% agar-agar

Kinetin

IBA

8.63 regenerated PLB

were obtained per

explant with 12.70 leaves

and 7.40 average roots

Zakizadeh

et al.

(2019)

Protocorm MS basal medium

3% sucrose

0.8% agar

1.00 mg L

-1

BA

0.50 mg L

-1

NAA

Colchicine

For the polyploid induc-

tion, treatment with

4.00 mg l

-1

colchicine

for 72 h was the only

treatment to result in a

mixoploid seedling.

Moreover, developed

4.16 and 4.12 cm root

growth per explant, 7.00

roots per explant,

4.58 cm leaf growth per

explant, and 6.66 cm leaf

per explant

Kazemi

and

Kaviani

(2020)

Catasetum

schmidtianum

F.E.L. Miranda

and

K.G. Lacerda

Protocorm 30 mg L

-1

sucrose

2g L

-1

fertilizer B and

G

200 mg L

-1

coconut

water

2g L

-1

activated carbon

4g L

-1

agar

1mg L

-1

extract

pyroligneous

By direct organogenesis

in vitro plants were

obtained with 27.6 cm

leaf growth per explant

and 4.1 roots per explant

Florestino-

Silva

(2021)

Species Response obtained References

Ethnomedicinal Uses, Phytochemistry, Medicinal Potential,... 203

Table 4 (continued)

Explant

type used

Composition of the

culture media

Seed 10 mL L

-1

Kudson C

medium

30 g L

-1

sucrose

24 g L

-1

natural gelatin

By direct organogenesis

in vitro plants were

obtained with 3 mm

Protocorm growth per

explant

Leles-

Gaudêncio

et al.

(2014)

Vitroplants Fertilizers B and G

Coconut water

Activated carbon

Agar

Sucrose

Water

Sphagnum Moss

Vermiculite

Carbonized rice straw

Charcoal

The acclimatization

treatment consists of

Chile Moss + vermiculite

+ carbonized Rice straw

+ charcoal (1:1:1:1 v/v)

presented the most suit-

able conditions for the

development of the

species

Arenas-

deSouza

and Vera-

Karsburg

(2016)

Fig. 3 Null in vitro response of leaves after 16 weeks of culture in an experiment with different

concentrations and types of plant growth regulators for induction of direct organogenesis in

C. integerrimum

Finally, the most used culture media for the micropropagation of Catasetum

orchids are the MS medium and the Vacin and Went medium. Furthermore, acti-

vated carbon is commonly used for the micropropagation of Catasetum orchids and

the most frequently used carbon source is sucrose. Plant growth regulators and

additives (vitamins or natural extracts) vary depending on the objective of each

study (Table 4).

204 L. J. Castillo-Pérez et al.

7 Future Prospective and Conclusions

Some Catasetum species showed in vitro anti-inﬂammatory, cytotoxic, and antiox-

idant activities. The ethnomedicinal information of these plant species was validated.

However, in vivo assays and their molecular mechanism of action remains to be

elucidated. Most of the secondary metabolites isolated from the Catasetum orchids

correspond to polyphenols, and many of these compounds have previously reported

anti-inﬂammatory and antioxidant actions. Nevertheless, some Catasetum orchids

lack of chemical composition of their metabolites. The isolation and elucidation of

the structure of new compounds obtained from the Catasetum genus should be

carried out.

There is limited information about the obtention of new compounds from the

Orchidaceae family. It is also necessary to work on the biological and ecological

aspects of the Catasetum orchids, such as growth and climatic conditions, seasons,

exposure to sunlight, altitude, and genetic composition, due to these abiotic factors

inﬂuence the chemical composition and the pharmacological effects of these plant

species.

Biotechnological plant tissue culture techniques, including symbiotic and

asymbiotic germination, clonal propagation, and direct organogenesis are available

in this orchid genus. The use of biotechnological techniques can prevent and control

the reduction of the pressure on wild species of this genus. In our laboratory

(Environmental Science Research Laboratory—Autonomous University of San

Luis Potosí, Mexico), we have worked with an efﬁcient propagation protocol for

C. integerrimum from pseudobulb sections and using the direct organogenesis

technique (Castillo-Pérez et al. 2021). Moreover, we have begun to study the

production of phytochemicals produced by in vitro orchids, inducing different

types of stress in vitro, and comparing them with the homologous produced by

wild plants for establishing a biotechnological technique for the production of

bioactive compounds. Overall, Catasetum orchids remain to be studied for their

pharmacological, ecological, botanical, chemical, and toxicological aspects.

Acknowledgments This work was partially supported by Consejo Nacional de Ciencia y

Tecnología (CONACYT, Mexico), grant number 321352 provided to HMRT and CCA. LJCP is

a student of doctors in science (773045) and DTR is a student of master’s in science (1181114),

both students supported by CONACYT fellowship.

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Diversity and Antimicrobial Potential

of Orchidaceae-Associated Fungal

Endophytes

Muhammad Adil, Pragya Tiwari, Jen-Tsung Chen, Rabia Naeem Khan,

and Shamsa Kanwal

1 Orchidaceae-Associated Fungal Endophytes:

Introduction and Signiﬁcance

As a major and diverse family of ﬂowering plants, Orchidaceae represents almost

750–850 genera and 25,000–35,000 species (Hossain 2011; Sarsaiya et al. 2019).

Subtropical and tropical regions are blessed with the highest diversity of these

ubiquitous plants, whereas, orchids are not found in hot deserts and Antarctica

(Hossain 2011). Orchids are capable of occupying a wide range of habitats including

forest ﬂoors, sandy dunes, and tree barks as epiphytes, lithophytes, saprophytes, and

terrestrial plants (Ma et al. 2015). Apart from the photosynthesis process,

mycoheterotrophism is also employed by the adult orchid plants for carbon acqui-

sition (Zhang et al. 2018). Orchids are characterized by their remarkable capability

of deceiving pollinators using several mechanisms such as rendezvous attraction,

shelter imitation, generalized food deception, sexual deception, brood-site imitation,

food-deceptive ﬂoral mimicry, and pseudo antagonism (Jersáková et al. 2006;

M. Adil (*)

Pharmacology and Toxicology Section, University of Veterinary and Animal Sciences, Lahore,

Jhang Campus, Jhang, Punjab, Pakistan

e-mail: muhammad.adil@uvas.edu.pk

P. Tiwari

Department of Biotechnology, Yeungnam University, Gyeongsan, Gyeongsangbuk-do,

Republic of Korea

e-mail: pragyatiwari@ynu.ac.kr

J.-T. Chen

Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan

R. N. Khan · S. Kanwal

Microbiology Section, University of Veterinary and Animal Sciences, Lahore, Jhang Campus,

Jhang, Punjab, Pakistan

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_7

209

Shrestha et al. 2020). Several orchid species have been associated with the produc-

tion of storage organs in terms of pseudo-bulbs or bulbs (Śliwiński et al. 2022).

Monopodial as well as sympodial growth patterns have been documented in orchids.

210 M. Adil et al.

Although, predominantly grown for ornamental purpose, orchids also exhibit

culinary and medicinal values, on account of different bioactive compounds includ-

ing ﬂavonoids, carotenoids, alkaloids, xanthones, and saponins (Hossain 2011;

Cheamuangphan et al. 2013). The ethnomedicinal signiﬁcance of orchids is consid-

erably exploited in Ayurvedic and Chinese medicines (Bulpitt et al. 2008; Kobayashi

2020). Calanthe, Ephemerantha, Coelogyne, Dendrobium, Galeola, Cymbidium,

Eria, Ludisia, Gastrodia, Cypipedium, Habenaria, Nevilia, Thunia, Luisia, and

Gymnadenia represent the maj or genera of medicinal orchids (Bungtongdee et al.

2019).

Fungal microorganisms, known for internally colonizing and inhabiting the

leaves, stems, roots, seeds, and ﬂowers of plants, without inﬂicting any damage or

infection, are referred to as endophytic fungi (Dhayanithy et al. 2019; Zhang et al.

2019). Therefore, the association of fungal endophytes with host plants is primarily

meant for mutual beneﬁts and described as mutualism or symbiosis (Khare et al.

2018). Endophytic fungi are mainly harbored by ﬂowering plants, ferns, and grasses

(Sudheep et al. 2017). Plants may be invaded by a single or multiple species of

endophytes. These beneﬁcial and non-pathogenic fungi are dependent upon their

host for shelter and nourishment, and improve the uptake of nutrients, growth as well

as tolerance of plants to abiotic and biotic stress in exchange (Velma et al. 2018;

Rana et al. 2019; Devi et al. 2020). Orchids are completely dependent on endophytic

fungi for the germination of seed and subsequent growth, due to lack of endosperm

(Shah et al. 2019). Certain secondary metabolites are secreted by the endophytes for

counteracting the plant defense mechanisms and thereby enhancing their viability

within the host tissues (Tiwari and Bae 2022). Besides, endophytes may potentially

modify or enhance the synthesis of phytometabolites (Ludwig-Müller 2015).

Orchidaceae-associated fungal endophytes can be cultured to harvest their bio-

active metabolites for agricultural, industrial, and pharmaceutical applications

(Bungtongdee et al. 2019). Several industrially-important extracellular enzymes

including cellulase, lipase, laccase, pectinase, and amylase have been isolated

from Orchidaceae-associated fungal endophytes (Paramanantham et al. 2019).

Orchidaceae-associated Penicillium isolates have shown tolerance to copper and

lead and can be potentially used for bioremediation purpose (Khan and Lee 2013;

Idris et al. 2019). Some beneﬁcial metabolites obtained from fungal endophytes have

been linked with the conferral of plant protection against pathogenic fungi and pests

(Duan et al. 2019; Yadav et al. 2020). The causal role of fungal endophytes in

orchid–endophyte inte raction has been explicated in terms of bioprotection,

bioregulation, and biofertilization (Pant et al. 2017).

Diversity and Antimicrobial Potential of Orchidaceae-Associated... 211

2 Diversity of Orchidaceae-Associated Fungal Endophytes

Despite the one milli on globally recorded species of fungal endophytes, less than

30% of the entire orchids genera have been screened for the isolation and identiﬁ-

cation of fungal endophytes (Sarsaiya et al. 2019). Since the conventional fungal

classiﬁcation is based on their spore-bearing structures and spores, the identiﬁcation

of some fungi becomes difﬁcult due to failure of in vitro sporulation. Endophytic

fungi belonging to Fusarium, Aspergillus, Trichoderma, Verticillium,

Colletotrichum, Xylaria, and Phomopsis genera have been frequently recovered

from orchids (Chen et al. 2013a; Ma et al. 2015). Fusarium, Penicillium, and

Aspergillus are most common, whereas Nigrospora, Guignardia, and Gliocladium

are relatively infrequent endophytes of Bulbophyllum orchids (Sudheep and Sridhar

2012; Sawmya et al. 2013). Cattleya orchids predominantly harbor Epulorhiza and

Colletotrichum genera, while, Tetracladium, Monilliopsis and Botrytis are their

minor endophytic fungi (Ovando et al. 2005; Da Silva et al. 2018). Apart from

Fusarium and Tulasnella as the most frequent endophytic fungi, Cylindrocarpon,

Cryptosporiopsis, and Cyperus have also been sporadically associated with Cym-

bidium orchids (Yu et al. 2015; Shubha and Srinivas 2017). Table 1 enlists the

important Orchidaceae-associated fungal endophytes.

Aspergillus, Fusarium, Trichoderma, Acremonium, Rhizoctonia, Xylaria,

Alternaria, Colletotrichum, and Phomopsis are major endophytes of Dendrobium

orchids, whereas, Aureobasidium, Curvularia, Thielavia, Westerdykella,

Chaetomium, and Scolecobasidium have been rarely isolated (Yuan et al. 2009;

Mangunwardoyo et al. 2012; Sour et al. 2015; Jin et al. 2017; Shrestha et al. 2018).

In addition to Rhizoct onia as the most frequent endophyt e, Oncidium orchids are less

commonly invaded by Pestalotia and Aspergillus (Otero et al. 2002; Mohamed and

Joseph 2016). Tulasnella is the most widespread endophyte of Paphiopedilum

orchids along with Valsa, Penicillifer, Lasiodiplodia, and Rigidoporus as the

uncommon inhabitants (Khamchatra et al. 2016; Rajulu et al. 2016; Parthibhan

and Ramasubbu 2020). Rhizoctonia and Tulasnella have been widely isolated

from Phalaenopsis orchids than Cochliobolus and Trichoderma (Saha and Rao

2006; Rachanarin et al. 2018). Vanda orchids have been commonly linked with

Ceratobasidium, Alternaria,and Fusarium, while, Agaricus, Mycena, Armillaria,

Russulaceae, and Moniliopsis are their infrequent endophytes (Sudheep et al. 2012;

Chand et al. 2020).

3 Antimicrobial Screening of Orchidaceae-Fungal

Endophytes

The association of orchid plant species with endophytes is attributed to the plant-

endophyte dynamics and microbial development via plant host association (Chutulo

and Chalannavar 2018). The screening of endophytic fungi for antimicrobial

Endophytic fungi References

(continued)

212 M. Adil et al.

Table 1 Diversity of Orchidaceae-associated fungal endophytes

Host orchid

plants

Bulbophyllum

kaitiense

Aspergillus, Penicillium Kasmir et al. (2011)

Bulbophyllum

neilgherrense

Aspergillus, Colletotrichum, Fusarium,

Gliocladium, Guignardia, Nigrospora, Penicil-

lium, Pestalotiopsis, Trichoderma, Xylaria

species

Sudheep et al. (2012);

Sawmya et al. (2013)

Cattleya

jongheana

Colletotrichum Da Silva et al. (2018)

Cattleya

skinneri

Epulorhiza, Penicillifer, Trichoderma, Fusarium,

Aspergillus, Tetracladium, Verticillium,

Pestalotiopsis, Monilliopsis, Botrytis

Ovando et al. (2005)

Cymbidium

aloifolium

Cyperus, Fusarium, Trichoderma, Alternaria,

Penicillium, Colletotrichum, Aspergillus

Shubha and Srinivas

(2017)

Cymbidium

dayanum

Corynascus, Fusarium, Xylaria, Phoma,

Pestalotiopsis, Chaetomium, Colletotrichum

Sour et al. (2015)

Dendrobium

friedericksianum

Fusarium, Pestalotiopsis, Xylaria

Dendrobium

hercoglossum

Chaetomium cochliodes, Xylaria Colletotrichum,

Nigrospora species

Cymbidium

faberi

Umbelopsis, Tulasnella, Fusarium, Trichoderma Yu et al. (2015)

Cymbidium

goeringii

Cylindrocarpon, Cryptosporiopsis, Nigrospora,

Fusarium, Exophiala, Tulasnella

Dendrobium

crumenatum

Cladosporium, Scolecobasidium, Colletotrichum,

Guignardia, Curvularia, Fusarium,

Westerdykella, Xylohypha, Pestalotiopsis, Xylaria

species

Mangunwardoyo et al.

(2012); Sour et al.

(2015)

Dendrobium

loddigesii

Acremonium, Cladosporium, Fusarium,

Colletotrichum, Sirodesmium, Chaetomella,

Pyrenochaeta, Nigrospora, Thielavia

Chen et al. (2010)

Dendrobium

nobile

Colletotrichum, Hypoxylon, Clonostachys,

Guignardia, Penicillium, Trichoderma,

Phomopsis, Fusarium, Pestalotiopsis, Rhizocto-

nia, Xylaria

Yuan et al. (2009)

Dendrobium

ofﬁcinale

Alternaria, Aspergillus, Aureobasidium,

Cochliobolus, Colletotrichum, Cystobasidium,

Epicoccum, Fusarium, Pestalotiopsis,

Trichoderma, Xylaria

Jin et al. (2017)

Dendrobium

speciosum

Epicoccum nigrum, Fusarium, Trichoderma,

Nigrospora, Phialophora, Tulasnella

Boddington and

Dearnaley (2008)

Dendrobium

aphyllum

Colletotrichum, Fusarium, Phomopsis,

Xylariaceae species

Chen et al. (2013a, b)

Dendrobium

chrysanthum

Dendrobium

chrysotoxum

Endophytic fungi References

potential is deﬁned by literature, suggesting that endophyt es modulate the host

defense mechanisms in accordance with the spectrum of pathogens. Orchid species

are being widely explored for endophytic associations and their potential to produce

promising antimicrobia l compounds. With recent advances in scientiﬁc technolo-

gies/assay systems, research initiatives are undertaken to screen the potential endo-

phytic fungi from various species of orchids. Different fermentation conditions and

types are employed for the synthesis of bioactive products using the endophytic

fungi (Tiwari et al. 2021a) and include potato dextrose medium (liquid culture),

Diversity and Antimicrobial Potential of Orchidaceae-Associated... 213

Table 1 (continued)

Host orchid

plants

Dendrobium

crystallinum

Dendrobium

falconeri

Dendrobium

ﬁmbriatum

Dendrobium

monoliforme

Aspergillus, Fusarium, Cladosporium,

Hypoxylon, Colletotrichum, Trichoderma,

Helminthosporium, Leptosphaerulina

Shrestha et al. (2018)

Dendrobium

transparens

Oncidium

altissimum

Rhizoctonia, Colletotrichum, Pestalotia, Xylaria Otero et al. (2002)

Oncidium

species

Rhizoctonia, Cercospora, Aspergillus Mohamed and Joseph

(2016)

Paphiopedilum

druryi

Colletotrichum, Penicillifer, Tulasnella Parthibhan and

Ramasubbu (2020)

Paphiopedilum

fairrieanum

Xylaria, Penicillium, Lasiodiplodia, Fusarium,

Cladosporium

Rajulu et al. (2016)

Paphiopedilum

villosum

Valsa, Coriolopsis, Nigroporus, Flavodon,

Ceratobasidium, Rigidoporus, Tulasnella

Khamchatra et al. (2016)

Phalaenopsis

manni

Cochliobolus, Trichoderma, Rhizoctonia Saha and Rao (2006)

Phalaenopsis

pulcherrima

Rhizoctonia, Epulorhiza, Tulasnella Rachanarin et al. (2018)

Vanda cristata Mycoleptodiscus, Agaricus, Fusarium,

Paraconiothyrium, Alternaria,

Pseudochaetospaeronema

Chand et al. (2020)

Cymbidium

sinense

Epulorhiza, Tulasnella Nontachaiyapoom et al.

(2010)

Paphiopedilum

sukhakulii

Vanda testacea Ceratobasidium, Fusarium, Xylaria, Rhizoctonia,

Tulasnella, Thanatephorus, Serendipita,

Russulaceae, Mycena, Moniliopsis,

Erythromyces, Ceratobasidium, Armillaria

Behera et al. (2013)

23 days culture, 25 C for mullein production from Penicillium janczewskii (Patil

et al. 2016), fermentation medium, for 8 days at 30 C for Taxol production from

Aspergillus aculeatinus (Qiao et al. 2017) mineral medium (liquid), 3 days at 25 C

for Vincristine production from Fusarium oxysporum (Patil et al. 2016), submerged

culture, 30 days at 25 C for pyrrocidine A and B production from Acremonium zeae

(Patil et al. 2016), grain–bran–yeast medium for 40 days at 28 C for rhizoctonic acid

production from Rhizoctonia species (Patil et al. 2016), among other techniques.

214 M. Adil et al.

The fungal endophytes from orchids are cultured via solid-state fermentation or

submerged fermentation and the conditions, namely temperature, pH, media com-

position, partial pressures of carbon dioxide and oxygen (pCO

2

and pO

2

), aeration,

etc. are optimized for maximum product recovery. These media parameters are

crucial to metabolite production and differ accordingly. The different fungal endo-

phyte strains are screened via antimicrobial (antibacterial, antifungal,

antiviral) assays for validation of their antimicrobial properties. Moreover, the

culture broth of endophytes is screened for bioactive properties via common

methods, namely mycelial radial growth test, disk diffusion technique, and agar

dilution assay (Songrong et al. 2005; Aly et al. 2008; Hoffman et al. 2008;

Pongcharoen et al. 2008). The increased bio-prospection of endophytes colonizing

different plants has demonstrated signiﬁcant antimicrobial potential, parti cularly

from medicinal plants including Paris polyph ylla var. yunnanensis, fungal endo-

phytes from Garcinia, Ophiopogon, and Cyrtomium species (Jiang et al. 2006;

Phongpaichit et al. 2006; Li et al. 2008; Zhao et al. 2010).

4 Antimicrobial Potential of Orchidaceae-Associated

Fungal Endophytes

Antimicrobial compounds are gaining popularity on account of their therapeutic

potential in combating the pathogenic microorganisms. Alternative biological

resources are extensively screened and employed to produce novel antimicrobials

(Tiwari et al. 2021a, 2022b). The endophyte species are documented to be proliﬁc

producers of bioactive metabolites (Tiwari et al. 2022a), exhibiting potent pharma-

cological activities and are commercialized as marketed drugs. For instance, taxol, a

multi-billion-dollar drug is synthesized by endophytic fungus, Taxomyces

andreanae that was isolated from Taxus brevifolia (Tiwari et al. 2021b, 2022a).

Orchid-associated endophytes have been implicated in the synthesis of diverse

antimicrobial metabolites. Fungal endophyte species, namely Xylaria, Phoma, and

Fusarium, isolated from Dendrobium devonianum, Dendrobium ofﬁcinale,

Acianthera teres, and Acianthera setaceus have been investigated for their antimi-

crobial potential. Fusarium oxysporum has been extensively screened for antimicro-

bial effects against various pathogenic microorganisms including Saccharomyces

cerevisiae, Staphylococcus aureus, Pseudomonas aeruginosa, Candida albicans,

Candida krusei, Sarcina lutea, and Escherichia coli (Vaz et al. 2009; Jin et al. 2017;

Bungtongdee et al. 2019). Table 2 enlists the antimicrobial activities of endophytic

fungi recovered from diverse orchid species.

Diversity and Antimicrobial Potential of Orchidaceae-Associated... 215

Table 2 The antimicrobial potential of fungal endophytes from diverse orchid species

Fungal species Orchid species Test microorganisms Reference

Fusarium nivale Dendrobium

crumenatum

Candida tropicalis, Candida

albicans

Mangunwardoyo

et al. (2012)

Streptomyces

strains DR5–1,

DR7–3, DR8–5,

DR8–8

Dendrobium

species

Alternaria alternate, Fusarium

oxysporium, Curvularia oryzae,

Colletotrichum gloeosporioides

Tedsree et al.

(2022)

Xylaria species Anoectochilus

setaceus

Methicillin-resistant, Staphylo-

coccus aureus, Bacillus subtilis

Ratnaweera et al.

(2014)

Aureobasidium

pullulan, fusarium

oxysporum

Dendrobium

ofﬁcinale

Staphylococcus aureus,

Escherichia coli, Pseudomonas

aeruginoa, Candida albicans

Jin et al. (2017)

Fusarium

oxysporum

Acianthera teres Staphylococcus aureus,

Escherichia coli, Candida

albicans, Candida krusei

Vaz et al. (2009)

Fungal endophyte

DO14

Dendrobium

ofﬁcinale

Candida albicans, Cryptococcus

neoformans, Trichophyton

rubrum, Aspergillus fumigatus

Wu et al. (2015)

Alternaria species Oncidium

warmingii

Staphylococcus aureus, Bacillus

cereus

Vaz et al. (2009)

Phoma species Dendrobium

devonianum,

Dendrobium

thyrsiﬂorum

Escherichia coli, Staphylococcus

aureus, Bacillus subtilis

Xing et al. (2011)

Bioactive products with promising antimicrobial activity have been derived from

different endophyte speci es. A triterpenoid, helvolic acid, isolated from the organic

endophyte extract of a Sri-Lankan orchid (Anoectochilus setaceus), displayed potent

antimicrobial effect against Bacillus subtili s and methicillin-resistant Staphylococ-

cus aureus (Ratnaweera et al. 2014). The an timicrobial properties of metabolites

from fungal endophy tes, found in Thai orchids were examined and out of the

97 isolates, 13 endophyte strains demonstrated antifungal activity against

Colletotrichum species, Fusarium species and Curvularia species. In addition,

endophyte CK F05–5 showed potent antifungal activity against Fusarium species

(Bungtongdee et al. 2019). A fungal endophyte was isolated, characterized from

Dendrobium moniliforme, and the presence of phenolics in the organic extract

contributed to the antimicrobial properties of the host plant (Shah et al. 2019).

Endophytic Pyrenochaeta species, recovered from Dendrobium loddigesii, revealed

antimicrobial activity agains t Bacillus subtilis and Aspergillus fumigatus (Chen et al.

2010). Phoma species of endophytic fungi also demonstrated signiﬁcant antimicro-

bial effects against Staphylococcus aureus, Bacillus, and Escherichia coli (Xing

et al. 2011). Surprisingly, the antibacterial efﬁcacy of Orchidaceae-derived fungal

endophytes was superior to that of some existing antimicrobial drugs such as

ampicillin (Xing et al. 2011).

216 M. Adil et al.

5 Conclusion and Future Perspectives

In addition to their ornamental and culinary values, orchids also possess a wide range

of phytochemical ingredients including terpenoids, phenanthrenes, steroids, and

ﬂavonoids (Zhang et al. 2015). Accordingly, the antimicrobial, anticancer,

neuroprotective, antioxidant, hypoglycemic, hepatoprotective, and immuno-

modulatory actions of these valuable plants are traditionally being exploited in

several forms of ethnomedicine for the treatment of various diseases (Kong et al.

2003;Pant 2013; Biswas et al. 2016). Besides, Orchidaceae-associated fungal

endophytes also synthesize diverse bioactive metabolites such as alkaloids, peptides,

quinones and phenolics, exhibiting anti-inﬂammatory, antineoplastic and antimicro-

bial properties (Jin et al. 2017; Pant et al. 2017).

Several species of Orchidaceae-fungal endophytes have been linked with antimi-

crobial effects (Singh et al. 2012). So far, the antimicrobial potential of Phoma,

Xylaria, and Fusarium species of fungal endophytes associated with different

orchids including Anoectochilus setaceus, Acianthera teres, Dendrobium

thyrsiforum, Dendrobium Ofﬁcinale, Dendrobium lindleyi, Dendrobium

devonianum, and Dendrobium crumenatum have been analyzed. Consequently,

the metabolic products of Orchidaceae-associated fungal endophytes can serve as

lead compounds for potential development of new antimicrobial agents against the

drug-resistant microbial pathogens (Cui et al. 2012). Recent advances in omics,

medicinal chemistry and computer-aided drug development are projected to expedite

the translation of complicated orchid–endophyte interaction into more proliﬁc and

ecofriendly therapeutic products. Nevertheless, challenges in terms of scarce taxo-

nomic data, lack of biotechnologically based in vitro reproduction and rapid deteri-

oration of orchid diversity necessitate adequate and long-term solutions. Moreover,

bioactive metabolites of Orchidaceae-associated fungal endophytes with proven

antimicrobial efﬁcacy during in vitro assays should be further evaluated through

appropriate in sil ico and in vivo studies.

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Asymbiotic Seed Germination in Terrestrial

Orchids: Problems, Progress, and Prospects

Nora E. Anghelescu, Yavar Vafaee, Kolsum Ahmadzadeh,

and Jen-Tsung Chen

1 Introduction

The species within the Orchidace ae family are among the largest and most diverse

groups of ﬂowering plants (Gaskett and Gallagher 2018). Scientiﬁc evidence indi-

cates that the most recent common ancestor of extant orchids lived about 76–84

million years ago (the Late Cretaceous) (Ramirez et al. 2007). Throughout history,

orchids have fascinated mankind, dating back thousands of years. Based on a Greek

myth, Orkhis was a prince who fell in love with a priestess of Bacchus, but the

creatures guarding her, tore him apart. The ﬂowers that grew from his bloodshed

were named after him (Ramirez et al. 2007). Therefore, this explains the origin of the

name of one temperate genus, genus Orchis L., which later gave the name of the

entire Orchidaceae family. The word “orchid” can also be traced back to the works

of Theophrastus between 370 and 285 BC (Yam and Arditti 2017a). Due to the

shape of the tuberoids of some orchids, they were considered to be aphrodisiacs, and

another myth suggests that the tuberoids were the favorite food of satyrs (Gaskett

and Gal lagher 2018). With 899 genera, 27,801 species, and about 70,000 to 100,000

N. E. Anghelescu

Faculty of Horticulture, University of Agronomic Sciences and Veterinary Medicine of

Bucharest, Bucharest, Romania

Y. Vafaee (✉) · K. Ahmadzadeh

Department of Horticultural Sciences and Engineering, Faculty of Agriculture, University of

Kurdistan, Sanandaj, Iran

Medicinal Plants Breeding and Development Research Institute, University of Kurdistan,

Sanandaj, Iran

e-mail: y.vafaee@uok.ac.ir

J.-T. Chen

Department of Life Sciences, National University of Kaohsiung, Kaohsiung, Taiwan

e-mail: jentsung@nuk.edu.tw

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_8

221

interspeciﬁc, cultivated hybrids, Orchidaceae is the second most species-rich family

among the ﬂowering plants, after Asteraceae, comprising 10% of all systematically

veriﬁed angiosperms and 40% of all monocotyledon species (Gaskett and Gallagher

2018; The Plant List 2020). From an evolutionary and phylogenetic point of view,

orchids are among the most evolved plant species with a broad range of inter- and

intra-speciﬁc variation, reﬂected as a wide morphological diversity including plant

architecture and ﬂower size, shape, color and smell, and variations that can be rarely

seen in other plant families (Zhang et al. 2018; Otero and Flanagan 2006). All

characteristics of the species undergoing active speciation are present among

orchids, which live in a delicately balanced equilibrium with their ecosystem

(Dressler 1982). Orchids represent the main interest in many scientiﬁc studies due

to their amazing ﬂower beauty, small and dust-like seed, unique pollination strate-

gies and reproduction system, as well as due to their complex symbiosis association

with mycor rhizal fungi (Zhang et al. 2018; Schluter et al. 2011). It is important to

note that these outstanding features are considered evolutionary forces to retain or

improve orchids’ diversity and survival (Gaskett and Gallagher 2018; Rasmussen

1995; Shefferson et al. 2020). Orchidaceae is one of the most adaptive plant families,

which has provided their species with the possibility of long-term survival

(Shefferson et al. 2020). A typical adaptation mechanism among Orchidaceae

species is the formation of a multilayer epidermis of dead cells, called velamen,

present in the roots of many orchids, especially tropical orchids, protecting the root

cortex from excessive drying and helping the water absorption (Zotz and Winkler

2013; Gravendeel et al. 2004). Another adaptation mechanism of orchids is their

extraordinary ﬂowers, which have a close and special relationship with pollinating

insects (Schluter et al. 2011; Waterman and Bidartondo 2008). The third mechanism

is the symbiotic relationship with mycorrhizal fungi, which makes orchids more

tolerant of non-suitable habitats, thus helping their global spreading (Gao et al. 2020;

Selosse et al. 2022). Therefore, members of the Orchidaceae family comprise a

substantial variety of life forms including epiphyte, lithophyte, aquatic, and terres-

trial, which are compatible with diverse niches from tropical forests to high alpine

regions, except Antarctica, with the greatest species diversity in the tropical and

subtropical region (Zhang et al. 2018; Tsiftsis et al. 2018; Acharya et al. 2011). They

are found all over the world, from deserts and semi-scrubs to rainforests and tundra

ecosystems (Acharya et al. 2011; Renz 1978). Renz (Renz 1978) indicated two

distinct border lines of Mediterranean orchids grown in the Iranian plateau and

Himalayan range (Fig. 1).

222 N. E. Anghelescu et al.

Among different life types of orchids, terrestrial species are typically grown in

soil and produce ﬂeshy underground round or palmate-shaped tubers. Many terres-

trial species within the Orchidaceae family are on the red list of rare and endangered

species of wild plants and animals (CITES) (Valletta et al. 2008; Hinsley et al. 2017)

and some of them are at risk of extinction because of climate change, deforesting,

land manipulation, tuber overexploitation, and illegal trade (Vafaee et al. 2021;

Ghorbani et al. 2014a, 2014b). The harvest of underground tubers of terrestrial

orchid species in the Anatolia and Middle East, the two main hotspots of terrestrial

orchid species, has intensiﬁed due to increased global demand. For instance,

approximately 7–11 million orchid plants are annually harvested from the main

terrestrial orchid diversity regions in Iran, and this pressure has put these medicinally

valuable species in danger of extinction (Ghorbani et al. 2014a). In the presence of

special mycorrhizal fungi, which provide essential nutrients for orchids, only a little

ratio of orchid seeds can germinate in nature (Acharya et al. 2011; Renz 1978).

Exploiting the large number of seeds produced within each capsule (about 0.2–2

million seeds (Valletta et al. 2008)), the asymbiotic seed germination procedure can

be employed for large-scale propagation of terrestrial orchids through the generation

of a high number of in vitro raised plantlets over a short period (Jolman et al. 2022).

The present book chapter introduces terrestrial orchid species, describes their biol-

ogy and conservational status, and focuses on in vitro conservation efforts performed

on terrestrial orchids, with major emphasis on asymbiotic seed germination barriers.

Moreover, it summarizes the performed research on asymbiotic seed germination of

terrestrial orchid species highlighting the important variables including media com-

ponents in particular organic supplements and plant growth regulators (PGRs).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 223

Fig. 1 The supposed distribution of distinct border lines for Europe-Mediterranean and Himalayan

orchids (Renz 1978)

224 N. E. Anghelescu et al.

2 Conservational Status of Orchids

Based on the state of the World’s Plants and Fungi report released by Royal

Botanical Gardens Kew, it has been highlighted that about 40% (two-ﬁfths) of all

plant species are at risk of extinction on a world scale which shows a double increase

in the number of threatened species from 2016 to 2020 (Nic Lughadha et al. 2020).

With the highest documented species number after Asteraceae, the Orchidaceae

family is on the front line of extinction. In this regard, ﬁve orchid species have

already been extinct, 87 are near threatening, 195 species are classiﬁed as vulnerable,

197 species are identiﬁed as critically endangered, and a total of 747 species are

classiﬁed as threatened based on the IUCN Global Red List 2020 (Wraith et al.

2020). An individual tropical tree can harbor hundreds of epiphytic orchid species,

so a small loss in habitat will impose profound negative impacts on orchid diversity

and survival (Go et al. 2020). Several major and minor factors can, directly and

indirectly, lead to the destruction of natural orchid habitats and declining their

diversity and survival, as summarized by Hágsater and Dumont (Hágsater and

Dumont 1996). Geographic distribution, habitat speciﬁcity, and population size all

affect the efﬁciency of these factors on a given orchid species. The main causes are

habitat destruction, modiﬁcation, and fragmentation due to lodging, agriculture,

artiﬁcial plantations, and overexploitation for ornamental, medicinal, and food

purposes (Hágsater and Dumont 1996; Sezik 2002). Rare species are generally

thought to have more speciﬁc habitat priorities than non-threatened species. A

further factor responsible for orchid decline is environmental destruction, which

can increase extinction risk through intensiﬁed climate change, soil erosion, and

drought, among other factors (Gale et al. 2018; Swarts and Dixon 2009). Intense

ﬁres, ﬂoods, or severe environmental ﬂuctuations are among the natural catastrophes

threatening rare orchid species (Wraith et al. 2020; Phillips et al. 2020). Small and

spatially isolated fragments of natural habitat destabilize populations and impede

pollen and seed exchange (Kropf and Renner 2008; Cozzolino et al. 2005). Genetic

diversity can be lost in fragmented populations, leading to decreasing the attraction

of a diverse range of pollinators. For example, it has been shown that some terrestrial

orchid species grown in Iran including Himantoglossum afﬁne (Boiss.) Schltr.,

Orchis simia Lam. and Anacamptis collina (Banks & Sol. ex Russell) R. M.

Bateman, Pridgeon & M. W. Chase are at risk of extinction due to environmental

and anthropogenic impacts (Gholami et al. 2021a, 2021b; Kaki et al. 2020; Vafaee

et al. 2017; Nosrati et al. 2011). Orchid conservation traditionally is based on three

procedures including developing action plans, determining the population or con-

servation status at the species or genus level, and propagating and reintroducing

cultivated individual plants of the threatened species into nature/the wild (Gale et al.

2018).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 225

3 Terrestrial Orchid

Genealogically and phenologically, temperate, terrestrial orchids are similar to

tropical orchids, the main differences being the underground ﬂeshy tubers formed

in soil and their rather smaller ﬂowers (Djordjević and Tsiftsis 2022). The signiﬁcant

feature of terrestrial orchid species includes their complicated ecology, rareness, and

capability to survive in almost all habitats (Rasmussen 1995; Swarts and Dixon

2017). Beyond their reproductive structures and pollination mechanisms, terrestrial

orchids are unusual in many ways (Shefferson et al. 2020). It is important to study

the terrestrial orchids, mostly temperate species, from a mycotrophic viewpoint if we

are going to understand their biology. It is well documented that terrestrial orchids

are more associated with mycorrhizal fungi than epiphytic counterparts. This is

because the seedlings of these species stay underground and rema in dependent on

mycorrhizal fungi for a long time. In contrast, orchid seedlings growing epiphyti-

cally access light at their early-life stages and can start photosynthesis once the

seedlings are established (Rasmussen 1995). Based on the World Conservation

Union, terrestrial species account for one-third of all taxonomically veriﬁed orchids,

while more than half of extinct, vulnerable, and critically endangered species belong

to this life type of orchids (Swarts and Dixon 2009, 2017). Due to the multiplicity of

threatening factors, terrestrial orchids have already experienced more injuries and are

also more vulnerable to experience extinction in the future (Swarts and Dixon 2009).

The terrestrial orchid genera which have attracted more attention in terms of

conservational activities are Cypripedium L. (Bernhardt and Edens-Meier 2010),

Orchis Tourn. ex L. (Fay et al. 2007), Ophrys L. (Devey et al. 2008), Platanthera

Rich. (Knudson et al. 2015), Dactylorhiza Neck. ex Nevski (Hedrén 2001),

Himantoglossum Spreng. (Dulić et al. 2019), Goodyera R.Br. (Wong and Sun

1999), Cephalanthera Rich. (Hasegawa et al. 2017), Epipactis Zinn (Squirrell

et al. 2002), and Serapias L. (Bellusci et al. 2009). In Fig. 2, the ﬂower and

inﬂorescence morphology of some endangered terrestrial species have been shown.

4 The Life Cycle of Terrestrial Orchids

Terrestrial orchids have a long-life cycle in nature where they need 2–5 years to enter

the reproductive phase and to produce mature seeds (Balilashaki et al. 2020;

Delforge 2006). Seeds, protocorms, juveniles, dormant adults, vegetative adults,

and ﬂowering individuals account for the six primary stages of the terrestrial orchid

life cy cle (Shefferson et al. 2020; Harrap and Harrap 2009) (Fig. 3). The life cycle of

terrestrial orchids starts with symbiotic seed germination, which is a complex

process requiring special microclimate and micro-edaphic conditions besides the

relationship with mycorrhizal fungi (Rasmussen et al. 2015; Fatahi et al. 2022a). Cell

division of the embryo within the dust-like seed leads to the formation of the

protocorm, a special structure containing leaf and shoot primordia (Cardoso et al.

2020). The protocorm is the primordial stage of terrestrial orchids’ life cycle, which

develops underground, and is found in the Orchidaceae and Pyroloideae families

(Shefferson et al. 2020; Yeung 2017). Upon the development of protocorm, a high

density of rhizoids will be generated, which will help the absorption of the essential

nutrients from the surrounding medium (Piria et al. 2008). The length of the ﬁrst

post-germination winter determines the seedling’s ability to transition from the

protocorm to the young plantlet stage. In this term, some species even require

more than one winter season (Rasmussen 1995). In the next stage, the protocorms

develop root-tuber structures (also known as mycorrhizae), from which small plant-

lets will start developing, after spending the dormant phase (Harrap and Harrap

2009). The seedlings can live underground for months or even years, where they

226 N. E. Anghelescu et al.

Fig. 2 The ﬂower and inﬂorescence morphology of some most endangered terrestrial orchid

species. (a) Orchis mascula (L.) L.; (b) Ophrys reinholdii subsp. straussii (H.Fleischm.) E.Nelson;

(c) Ophrys schulzei Bornm. & Fleischm.; (d) Dactylorhiza umbrosa (Kar. & Kir.) Nevski; (e)

Himantoglossum afﬁne (Boiss.) Schltr.; (f) Nigritella nigra subsp. bucegiana Hedrén, Anghel. &

R. Lorenz, subsp. nov.; (g) Anacamptis coriophora (L.) R. M. Bateman, Pridgeon & M. W. Chase;

(h) Orchis simia Lam.; (i) Steveniella satyrioides (Spreng.) Schltr.; (j) Orchis purpurea Huds., (k)

Dactylorhiza fuchsii (Druce) Soó subsp. carpatica (Batoušek & Kreutz) Kreutz var. albiﬂora; (l)

Anacamptis palustris (L.) R.M.Bateman, Pridgeon & M.W.Chase subsp. elegans (Heuff.) R. M.

exclusively depend on mycorrhizal fungi to obtain their required nutrients, being

called mycotrophs (Harrap and Harrap 2009). It is important to consider various

factors such as the depth of the germination, the porosity of the soil, concentration of

humus, climate and genetic variation that may affect how long the underground

phase lasts (Rasmussen 1995).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 227

Fig. 3 The life cycle of terrestrial orchids. After Shefferson, Jacquemyn (Shefferson et al. 2020).

(The diagram created with Biorender.com)

Like many other higher plants, orchids exploit asexual propagation means besides

sexual reproduction (Yam and Arditti 2017a). This includes vegetative reproduction

through the root-tuber structure which results in the generation of genetically

identical individuals. Vegetative reproduction can be seen in almost all types of

terrestrial orchid root systems as classiﬁed by Tsiftsis, Štípková (Tsiftsis et al. 2018)

including rhizomatous (Cephalanthera Rich., Corallorhiza Gagnebin, Epipactis

Zinn, and Epipogium Sw.), intermediate (Dactylorhiza Neck. ex Nevski,

Gymnadenia R.Br., and Platanthera Rich.) and tuberous orchids (Anacamptis,

Himantoglossum Spreng., Ophrys L. and Orchis Tourn. ex L.). In tuberous orchids,

during the autumn of the second year, root and shoot meristems started to activate,

producing a young plantlet that remains dormant during the winter (Malmgren

1996). During the spring of the third year, the axillary bud of the mother tuber

produces a new tuberoid that survives in the next dormant season and generates a

new shoot in the growing season (Figs. 4 and 5). In Iran and Turkey, the collectors

pick the daughter young tuber and leave the mother tuber for the next growing

season, a traditional conservational activity mitigating the overharvesting pressure

on terrestrial orchid species (Ghorbani et al. 2014a, 2014b).

228 N. E. Anghelescu et al.

Fig. 4 The growth phases and stages of terrestrial orchid species. The brown and gray parts are the

protocorm and mother tuber, respectively. The pink part is new formed daughter tuber. After

Rasmussen (1995) and Harrap and Harrap (Harrap and Harrap 2009). (The picture created with

Biorender.com)

Fig. 5 The development of the daughter tuber on the mother tuber as a vegetative reproduction

system in (a). Ophrys reinholdii subsp. straussii (H. Fleischm.) E. Nelson and (b). Gymnadenia

At the time of harvest, each plant has an old tuber, which has a stem and ﬂower,

and a fresh, ﬂeshy tuber, which is for the next year’s plant growth (Fig. 5). Old tubers

are rough and wrinkled. In most cases, collectors collect the plant before the seeds

are formed, which is a limitation of reproduction in wild populations (Kreziou et al.

2016).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 229

5 Salep and Tuber-Derived Products

Besides medicine, orchid products are widely used in the food industry to make

traditional ice creams and beverages with special rheological properties (Kurt 2021;

Şen et al. 2018; Kurt and Kahyaoglu 2015). The tubers of terrestrial orchids are rich

sources of glucomannan (GM), which consists of linear chains of glucose and

mannose connecting with 1–4 beta glycosidic bonds (Kurt 2021) (Fig. 6). In

Mediterranean and Middle East countries, the underground tubers usually harvest

and boil in water or milk, and then dry to prepare salep powder (Şen et al. 2018; Sen

et al. 2019). During salep preparation, no cleaning and puriﬁcation process is

performed and the resulting powder is used directly in various formulations (Ece

Tamer et al. 2006; Jagdale et al. 2009). However, salep has other constituents

including starch, protein, and ash, which usually consider factors reducing the

quality of the salep powder. A typical salep sample can include 8–48%

glucomannan, 5–44% starch, 2.7–12% protein, and 1.5–6.8% ash (Şen et al.

2018). As an anti-constipation constituent, GM generally causes bowel movements

for 12–24 h (Kurt 2021; Kurt and Kahyaoglu 2015). On the other hand, GM is a

natural water-soluble ﬁber that can regulate blood sugar, help hypoglycemia allevi-

ation, and reduce stress (Tekinşen and Güner 2010). It can also act as a preventive

agent for chronic diseases and obesity (Jagdale et al. 2009).

Fig. 6 Chemical structure of glucomannan, a polysaccharide found in a high ratio in terrestrial

orchid species. (Source: Kim, S., et al., PubChem 2023 update. Nucleic Acids Research, 2023. 51

(D1): p. D1373–D1380)

230 N. E. Anghelescu et al.

Fig. 7 Tuber morphology of tuber in selected terrestrial orchid species. Scale: 1 cm

In recent years, the high demand for salep-based beverages and ice creams and

also for its food and medicinal products has attracted the attention of collectors to

supply tuber material from nature (Ghorbani et al. 2014b; Sezik 2006). Salep is a

white ﬂour obtained by grinding the dry tubers from terrestrial orchid species (Kurt

2021). About 24 genera and 90 species of terrestrial orchids within the Orchidaceae

family are used to produce salep powder (Sen et al. 2019). The genera Anacamptis

Rich., Himantoglossum Spreng., Orchis Tourn. ex L., Ophrys L., and Serapis L. are

orchids with round or oval tubers and the genus Dactylorhiza Neck. ex Nevski with

palmate and ﬁnger-shaped tubers are among the most used taxa to produce salep

powder (Ece Tamer et al. 2006; Ghorbani et al. 2017). Approximately 30 tons of

orchids are annually harvested in Turkey (Kurt 2021) which accounts for 30–120

million individual terrestrial orchid plants. The shortage of natural populations of

terrestrial orchids in Turkey has shifted the harvesting pressure to neighboring

countries. In this regard, a volume of 7–13 million terrestrial orchid plants is

harvested in Iran belonging to 30 species and sub-species mainly growing in the

Alborz and the Zagros Mountain basins (Vafaee et al. 2021; Ghorbani et al. 2014a,

2014b). Figure 7 shows the tuber morphology of some terrestrial orchid species

grown on the Iranian plateau.

Hot salep is a viscous milky drink with unique rheological features that is widely

consumed in Turkey during the winter season (Karaman et al. 2012). It is prepared

by boiling salep powder and milk with sugar and then sprinkling cinnamon on top

(Ece Tamer et al. 2006; Dogan and Kayacier 2004). In Greece, it is also widely used

in local markets as a traditional warm beverage in winter. It is interesting that before

the introduction of coffee, the salep drink was common in Europe (Kreziou et al.

2016). Although there are alternatives such as carboxymethyl cellulose (CMC) due

to the unique and special organoleptic and rheological features of salep and also

because of CMC side effects, there is still an incre asing demand for original salep

powder (Kurt 2021; Sen et al. 2019; Kargar Jahromi et al. 2018).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 231

6 Seed and Embryo in Terrestrial orchids

Despite their microscopic size, orchid seeds are produced in large numbers where an

individual orchid capsule may contain about 0.2–2 million seeds (surprisingly about

four million seeds per capsule in Cycnoches ventricosum Bateman) (Sonkoly et al.

2016; Arditti and Ghani 2000). As a result, orchid seeds are among the smallest

known seeds in the plant kingdom. All orchids including terrestrial species have tiny

and dust-like seeds which makes the tracing of seed dispersal and monitoring of

germination and plantlet growth challenging (Rasmussen and Whigham 1993; Ren

et al. 2017). These stages could comprise seed releasing from dehiscent capsules to

the symbiotic establishment and seedling development (Rasmussen 1995;

Rasmussen et al. 2015; Süngü Şeker et al. 2021). However, with the advent of

current domestication platforms for terrestrial orchids, it is possible to trace and

monitor seed dispersal and establishment at pilot levels (Rasmussen 1995). On the

other hand, considering the geographical distrib ution of orchids and the physical

properties of seeds, some reasonable assumptions can be made about seed dispersal

(Rasmussen 1995). Aside from their size (from 0.05 in Anoectochilus imitans Schltr.

to 6 mm in length in Epidendrum secundum Jacq., showing 120-fold size differ-

ences) and shape, orchid seeds particularly in terrestrial species represent a remark-

able diversity in their testa architecture and sculpture (Vafaee et al. 2021; Gholami

et al. 2021b; Arditti and Ghani 2000; Barthlott et al. 2014). Having a large number of

air spaces, seeds are ideally adapted to being dispersed by wind (Hedrén et al. 2021).

This allows orchids to disperse their seed kilometers far from their main niches

(100 and 250 km for Orchis militaris L. and Orchis simia Lam., respectively

(Rasmussen 1995)) leading to higher dispersal rates, maintenance, and extension

of genetic diversity throughout geographical and ecolog ical boundaries and in the

same time reducing parental investment per seed (Hedrén et al. 2021). The seed

morphometric characteristics play an important role in the systematic and taxonomic

analyses of terrestrial orchid species, and species-speciﬁc patterns have been found

for various epiphytic and terrestrial orchid species (Vafaee et al. 2021; Barthlott et al.

2014). The morphometric variation of seed testa in orchids could be attributed to the

ways of dispersion and dormancy status (Barthlott et al. 2014). The color of orchid

seeds varies greatly from whitish to dark brown which is determined by the seed coat

and especially by the embryo. Figure 8 shows the seed morphology and structure of

some threatened terrestrial orchid species collected from the Iranian plateau.

Compared to other ﬂowering plants, orchids exhibit a unique seed development

pattern (Fang et al. 2016). The pattern of orchid embryo development is unique

among ﬂowering plants for several features including lack of cotyledon and endo-

sperm, the various morphology of suspensor, and the simple seed coat (Lee et al.

2007). The seeds of most ﬂowering plants are known for having an embryo that

differentiates into cotyledon(s), radicle, plumule, and hypocotyl, but in orchids,

embryo development is not as advanced as other ﬂowering plants (Yeung 2017;

Lee et al. 2007; Kauth et al. 2006). In orchids, the embryo is poorly differentiated

and the meristems and cotyledons are usually absent at the time of seed maturity

(Balilashaki et al. 2020; Yeung 2017). Although in some orchid species, there may

be more than one embryo (polyembryony), for example, the presence of more than

12 embr yos in the seed of Thecostele alata (Roxb.) E.C.Parish & Rch b. f. species

(Barthlott et al. 2014). An embryo gradually expands and ﬁlls the endosperm cavity,

as the polar-chalazal complex degenerates at the beginning of seed development

(Yeung 2017; Lee et al. 2007). An increase in embryo volume occurs during the

232 N. E. Anghelescu et al.

Fig. 8 The seed morphology of some threatened orchid species. (a) Anacamptis coriophora (L.)

R. M. Bateman, Pridgeon & M. W. Chase; (b) Dactylorhiza umbrosa (Kar. & Kir.) Nevski; (c)

Ophrys reinholdii subsp. straussii (H.Fleischm.) E. Nelson; (d) Orchis mascula (L.) L.; (e)

Himantoglossum afﬁne (Boiss.) Schltr.; (f) Orchis simia Lam.; (g) Ophrys cilicica Schltr. (prev.

Ophrys kurdistanica Renz); (h) Himantoglossum comperianum (Steven) P.Delforge (part of

research studies performed at the Research Center for Terrestrial Orchid, RCTO, university of

Kurdistan)

generation of globular embryos due to cell divisions in the outermost as well as the

inner layers of the embryo proper (Lee et al. 2007). The seed coat develops from the

integuments (maternal tissues) into a thin layer with varied surface characteristics. In

Fig. 9, the SEM and light microscopic images of seed-containing embryos have been

shown in some typical terrestrial orchid species.

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 233

SE

A

SE

ST

B

SE

Fig. 9 The testa and embryo as revealed by SEM in (a). Himantoglossum comperianum (Steven)

P.Delforge and (b). O. simia Lam. and using light microscopy in (c). Ophrys reinholdii subsp.

straussii (H.Fleischm.) E.Nelson. SE seed embryo; ST seed testa

7 Symbiosis with Mycorrhizal Fungi

Recent investigations have revealed that an increasingly large number of green

orchids, in the genera Cypripedium L., Cephalanthera Rich., Corallorhiza

Gagnebin, Epipactis Zinn, Epipogium Sw., Limodorum Boehm., Gymnadenia R.

Br., Neottia Guett., Orchis Tourn. ex L. and Platanthera Rich (Shefferson et al.

2020; Abadie et al. 2006), obtains large amounts of their carbon from associations

with ectomycorrhizal fungi. Current research using molecular techniques has begun

to elucidate the type of fungi found in association with orchids. All of the fungi

identiﬁed thus far that form orchid mycorrhiza typically belong to the division

Basidiomycota Moore, R.T. Rhizoctonia-forming fungi or higher fungi, which

occurred in the most ancestral orchid lineages, and today are most widespread in

the family. More speciﬁcally, the mycorrhizal fungi mainly come from four families,

Ceratobasidiaceae G.W. Martin (genus Rhizoctonia D.C., genus Ceratobasidium

D.P. Rogers), Sebacinaceae K.Wells & Oberw. (genus Sebacina Tul. & C.Tul.),

Tulasnellaceae Juel (genus Tulasnella J.Schröt., genus Epulorhiza R. T. Moore) and

Russulaceae Lotsy (genus Russula Pers.). They are usually saprotrophs, which feed

on decaying wood, leaf litter or dung, ectomycorrhizal fungi attached to tree roots,

and parasites on other plants (Rasmussen et al. 2015; Favre-Godal et al. 2020). The

ectomycorrhizal fungi are generally symbiotic with the roots of neighboring photo-

synthetic trees. They obtain simple carbohydrates from the photosynthetic leaves of

the trees and, in return, they provide minerals, amino acids, water, etc., to them

(Selosse et al. 2022). Studies showed that the orchids managed to hitch-hike the

hyphae of the ectomycorrhizal fungi and thus gain direct access to the ﬂow of readily

synthesized nutrients that come from the photosynthetic leaves. By associating with

these tree-symbiotic fungi (the ectomycorrhizal fungi), the orchids ultimately

became parasites on the trees, directing the abundant ﬂow of nutrients straight into

their roots (Selosse et al. 2022). This mutualistic association provides the fungus

with relatively constant and direct access to carbohydrates, such as glucose and

sucrose (Selosse and Cameron 2010). The carbohydrates are translocated from the

tree source, usually the leaves, to root tissue and onto the plant’s fungal partners. In

return, the plant gains the beneﬁts of the mycelium’s higher absorptive capacity for

water and mineral nutrients due to the large surface area of fungal hyphae, which are

much ﬁner than plant roots, thus improving the plant’s mineral absorption capabil-

ities. The mycelium can send extremely ﬁne ﬁlaments far out into the soil, which acts

as root extensions (Dearnaley et al. 2016). These ﬁlaments are far more effective in

nutrient and water absorption than the plant roots themselves. The mycorrhizae

enable them to grow much more quickly than they would otherwise. It has been

estimated that the mycorrhizae increase the nutrient absorption of the plant by a

factor of 100–1000 times (Li et al. 2021). This phenomenon used to be termed

epi-parasitism or hyper-parasitism (to be a parasite on another parasite). Yet orchids

are not alone in beneﬁtting from such a relationship with ectomycorrhizal fungi. It is

now known that 90% of plant species interconnect and have mutually beneﬁcial

relationships with mycorrhizae, but for these to exist, the soil must be undisturbed.

These fungi have been fundamental to plant growth for the last 460–400 million

years (Wang 2009; Kanchan et al. 2022).

234 N. E. Anghelescu et al.

7.1 The Damaging Effect of Phytoalexins

Nevertheless, at certain moments in time, the friendly, mycorrhizal fungus has the

potential to grow excessively and turn the tables on the orchid, becom ing parasitic on

the roots. If the infection would not be stopped on time, the fungus may extend its

pelotons to the entire rhizome, the base of the stem, and leaves. In protocorms, this

phenomenon may be lethal, the fungus being able to infect the whole protocorm

body, ultimately destroying it. Having said that, this sudden and seemingly

uncontrolled invasion generally does not take the orchid by surprise because these

plants have adapted to produce highly effective, "home-made," speciﬁc fungicides,

known as phytoalexins. These poisonous substances allow the orchid to keep control

of the expansion of the hyphae, without destroying or killing them. In most cases, the

fungi remain alive, for at least a certain period, and beneﬁt the orchid by releasing the

needed nutrients. Phytoalexins also limits the penetration of hyphae to speciﬁc areas,

such as the aboveground organs (leaves, stem, ﬂowers). They are synthesized

locally, before the initial fungal infection, initially by the protocorms’ rhizoids,

and later, by the entire root system. The production increases signiﬁcantly in

response to fungal infection or wounding (Pavarino 1909; Bernard 1911). Several

phytoalexins were isolated from orchids. To mention a few, orchinol, discovered in

1957 and isolated from Orchis militaris L., was shown to be widespread in many

European terrestrial orchids, as well as loroglossol and hircinol that were isolated

from Loroglossum hircinum (L.) Rich. [today Himantoglossum hircinum (L.)

Spreng.], both discovered by Bernard in 1910 (Bernard 1921; Bernard and Costantin

1916). Thus, the orchid can control and regulate the timing and degree of fungal

association, presumably providing a sufﬁcient reason for the fungi to colonize and

re-associate with it. The degree of colonization changes over the season, indicating

that the orchid is controlling the uptake of nutrients while preventing parasitism by

the fungus. While fungal food sources have become a life condition for orchids, one

might ask how strong is the impact of orchid predation on fungal survival and

evolution (Jones and Smith 2004). Up until now, it has not been demonstrated that

orchid poisonous effect substantially affects fungal health and vigor. However, in

some areas, it has been reported that the aboveground production of mushrooms (the

fruiting bodies of fungi) was much lower in mycelia that supported orchids, as

compared to mycelia of related fungal species, which do not associate with them.

This might indicate that fungal ﬁtness was reduced by the poisonous effect of

orchids on their fungal partner. Moreover, the damaging effect was, subsequently,

affecting other exosystemic interactions, damaging entire hyphal networks, and

reducing plant species’ resistance and development. It remains to be fully demon-

strated if the reduction in fungal diversity in speciﬁc habitats was solely due to the

poisonous effect of a sudden increase in the production of phytoalexins or if it was

also due to the simultaneous, combined effect of other factors such as changes in the

substrates pH, temperature, humidity, etc. Despite these observations, it is well

known that orchid mycorrhiza, as well as all the other complex mycorrhizal net-

works, can heal and recover rapidly. This would diminish, at least partially, the

effects orchids have on their speciﬁc fungi. At the same time, during their evolution,

the fungi have not developed any signiﬁcant avoidance or defense mechanisms

against orchid poisonous effects. This leads to the conclusion that fungi are generally

able to gradually recover and, in time, re-establish the ectomycorrhizal associations

speciﬁc to certain, particular ecosystems (Merckx and Bidartondo 2008). However,

in case of temporary loss/disappearance of speciﬁc symbiotic fungal partners,

germination of seeds may be signiﬁcantly affected, even if the loss of fungal parents

lasts for 1–6 months to 1 year. This generally would be sufﬁcient to affect entire

generations of germinating seeds or protocorms in their ﬁrst stages of development

when they are entirely dependent on healthy, strong fungal associations.

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 235

236 N. E. Anghelescu et al.

7.2 Destruction of Ecosystems/Natural Habitats

As mentioned in the previous section, orchids are highly dependent on the activities

of both the speciﬁc fungi and the trees that sustain them, from the initial, early stages

of development and, in many cases, throughout their adult life. This explains why

particular orchids are only found in woodlands that contain speciﬁc types of trees

(Yeh et al. 2019; Jacquemyn et al. 2017). For instance, the chlorophyll-deﬁcient

Corallorhiza triﬁda Châtel. is associated with the ectomycorrhizal fungi of the genus

Tomentella (Thelephoraceae family), which populate the roots of birch and willow

in some areas and pine trees in others (De Angelli and Anghelescu 2020). Recent

studies showed that Corallorhiza triﬁda Châtel. derives about 52% of its nitrogen

and 77% of its carbon from the associated fungi and therefore it is particularly

sensitive to the type of trees and fungi associated with. Another example is

Limodorum abortivum (L.) Sw., which in some areas has a particular association

with pine trees, while in others with beech or oak trees (Bellino et al. 2014; Wang

et al. 2021a). It seems that most orchid species grow in woods not because of the

shade and moisture they provide, but because of the presence of the speciﬁc fungi

that are dependent on certain trees. This makes the orchids particularly sensitive to

any environmental change or destruction. Dr. Kenji Suetsugu (Kobe University), in

2016, discovered the elusive Japanese orchid, Lecanorchis tabugawaensis Suetsugu

& Fukunaga, explains the signiﬁcance and importance of fungi-dependent plants for

the ecosystems in which they live: “Due to the sensitivity of mycoheterotrophic

plants, it has long been suggested that their species richness provides a useful

indicator of the overall ﬂoral diversity of forest habitats. A detailed record of the

distribution of these vulnerable plants, therefore, provides crucial data for the

conservation of p rimary forests.”

Wherever the natural habitats/ecosystems remain unaltered by human presence

and activity, a perfect balance is established between the varied plant and animal

species living together in the same natural habitat. As long as there is no human

disruptive intervention, each ecosystem self-regulates. But any interference by

humans can have unpredictable consequences, which can lead to the destruction of

the existing balance in that ecosystem. Due to their complex way of life, the

associations with a mycorrhizal fungus, and the sophisticated yet low-efﬁciency

reproductive cycle, orchids need stable ecosystems (Rasmussen et al. 2015). For

these sensitive plants, any change or alteration to their environment can lead to a

rapid reduction in numbers and eventually to their disappearance (Hágsater and

Dumont 1996). Where there is the correct blend/mixture of fungi present within the

soil, orchids, as well as other plant species can ﬂourish. When this equilibrium is

disturbed, for instance, by deep cultivation, land drainage, or slash-and-burn a gri-

culture, the composition of the soil and the mycorrhizal growth mat changes. As a

consequence, the symbiotic fungi could disappear and the subsequent uptake and

sharing of nutrients from the environment to the orchid can be severely inhibited.

Habitat destruction and habitat change are the major reasons for this, but the

collectors illegally removing plants, photographers, botanists, and visitors trying to

get a closer look, all contribute to their decline (Gale et al. 2018). All ecosystems are

complex, resilient systems that connect thousands of species of plants, allowing

them to intercommunicate and adapt. But they are also vulnerable, not only to

natural disturbances but also to a myriad of anthropic factors. Instead of hurting

and destroying them, we could reinforce and help them recover. The great thing

about natural environments is that they have an enormous capacity to regenerate

(Dukes 2007). Nevertheless, we should not be surprised if new research into the

social networks of plants will reveal the surprising beneﬁts that orchids provide to

their partners—the fungi and, ultimately, the whole ecosystems in which they live

(Anghelescu 2021).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 237

8 Barriers of Seed Germination in Terrestrial orchids

8.1 Seed Testa

The physical features of seeds in terrestrial orchid species and their biological roles

have been studied and reviewed by many researchers (Vafaee et al. 2021; Süngü

Şeker et al. 2021; Aybeke 2013, 2007; Calevo et al. 2017; Chase and Pippen 1988;

Gamarra et al. 2015a, 2015b, 2012; Ortúñez et al. 2006). Seeds in terrestrial orchids

are species-speciﬁc as they have unique features like embryo/airspace size ratio,

unique testa sculpture, and the presence of plant hormones and other regulators

which impact both symbiotic and asymbiotic seed germination (Arditti and Ghani

2000; Barthlott et al. 2014; Yang and Lee 2014). The seed testa phenotypic diversity

in orchids could be attributed to the dispersion strategy and seed dormancy (Ren

et al. 2017; Yang and Lee 2014; Prutsch et al. 2000). In this regard, air space within

the seed testa surrounding the embryo increases its air travel and ﬂoatability on the

water surface. However, the ligniﬁed, pectin layer can act as a barrier to wat er uptake

and embryo enlargement, thus preventing seed germination in terrestrial orchids

(Vafaee et al. 2021; Şeker and Şenel 2017).

Therefore, the ligniﬁed testa should be removed or softened during the seed

germination process to facilitate protocorm-like bodies and rhizoid formation,

which are the perquisites for the successful development of the in vitro raised

plantlets (Fatahi et al. 2022a, 2022b). Therefore, the hard seed testa is one of the

causes of extended dormancy observed in orchids, particularly in terrestrial species

occurring in seasonal climates (Arditti and Ghani 2000). This is because, unlike

tropical and epiphytic species with a seed testa composed of one layer, terrestrial,

mature seed testa has 2–3 layers of dead cells (Yang and Lee 2014). Seed testa

structure, cuticle thickness, seed cell number, and presence or absence of a distinct

cell size gradient also provide information on how easy mature seeds are to germi-

nate symbiotically and asymbiotically. One of the strategies to soften and eliminate

the strong and impenetrable testa is the treatment with sodium hypochlorite

(NaOCl), which simultaneously disinfects and scariﬁes the seeds. Depending on

the species, the concentration and treatment time differ for many terrestrial species.

In this regard, Malmgren has proposed the optimal NaOCl concentrations and

disinfection times for Euro-Mediterranean orchid species (Malmgren 1996). Ponert

Vosolsobě (Ponert et al. 2011), by studying different European temperate orchid

species, found that higher concentrations of NaOCl and longer disinfection times

have a negative effect on the germination of Dactylorhiza fuchsii (Druce) Soó and

Dactylorhiza majalis (Rchb.) P.F.Hunt & Summerh., while other species like

Dactylorhiza baltica (Kling e) Nevski, showed high germination rates. As ethanol

eliminates suberin, cutin, and other wax derivatives off the orchid seed surfaces, its

implementation could also improve seed germination (Jolman et al. 2022). Experi-

mentally, the important point is the color change of disinfected seeds from brown to

a milky, transparent/translucent color, which indicates successful sterilization. After

the color change, the seeds should be immediately sown on a culture medium

surface, as over-disinfection can lead to seed death. Fig. 10 shows how the color

change occurs in the seed of several terrestrial orchid species and what exactly

happens on the seed testa surface during the disinfection processes.

238 N. E. Anghelescu et al.

Fig 10 Test color change of seed testa in (a). Anacamptis coriophora (L.) R. M. Bateman,

Pridgeon & M. W. Chase; (b) Dactylorhiza umbrosa (Kar. & Kir.) Nevski; (c) Ophrys reinholdii

subsp. straussii (H. Fleischm.) E. Nelson; (d) Himantoglossum afﬁne (Boiss.) Schltr.; (e) Ophrys

schulzei Bornm. & Fleischm.; (f) Seed testa rupturing in Paphiopedilum armeniacum S. C. Chen &

8.2 Toxicity of Inorganic Nitrogen Sources

During orchid seed germination, nitrogen plays a crucial role in the synthesis of

macromolecules including proteins, nucleic acids, and enzymes. In this connection,

the form of nitrogen used in media is one of the most important factors affecting seed

germination. Living cells are stimulated to synthesize proteins by exogenous ammo-

nium, which activates glutamate dehydrogenase. Nitrate reductase enzymes also

respond to the reduced forms of nitrogen, making the nitrates absorbed more

efﬁciently (Rasmussen et al. 2015; Rasmussen and Whigham 1993). However,

inorganic forms of nitrogen have been included in some well-known media for

orchid micropropagation, the inhibition effect of organic nitrogen has been described

in both tropical and temperate orchids where both orchid groups prefer organic

nitrogen in the form of amino acids rather than ammonium (NH4

+

) or nitrates

(NO3

-

) (Rasmussen 1995; Rasmussen et al. 2015; Dijk and Eck 1995; Figura

et al. 2020; Nadarajan et al. 2011; Van Waes and Debergh 1986a). Terrestrial

orchids are growing in natural habitats with low strength of available nutrients and

therefore the reported nitrate sensitivity in asymbiotic seed germination of terrestrial

orchids may be part of their adaptive strategy (Figura et al. 2020, 2021; Ponert et al.

2013). It seems that each terrestrial orchid species shows a unique response to the

presence of nitrate and ammonium, therefore for the selection of an appropriate

medium, we should consider the ability of target species in metabolizing nitrogen

sources (Jolman et al. 2022). As an alternative conclusion, it has been stated that

some terrestrial orchid species possess low nitrate reductase activity or delayed

activation (Fatahi et al. 2022b; Johnson and Kane 2007; Van Waes and Debergh

1986b; Bektaş et al. 2013). The presence of both nitrate and ammonium not only can

have an inhibitory impact on asymbiotic seed germination but also negatively affect

the association of mycorrhizal fungi with orchids during in vitro symbiotic seed

germination. According to Cuenca and Azcón (Cuenca and Azcón 1994), arbuscular

mycorrhizal plants can enhance the nitrate absorption of fungi through nitrogen

metabolism (a symbiotic relationship). By increasing nitrate concentrations above

optimal levels, symbiosis loses its beneﬁcial effect on plant growth, and colonized

plants exhibit varying behaviors depending on the fungal species (Azcón et al.

2001). Further investigation is required to understand the details of nitrate and

ammonium metabolism during symbiotic and asymbiotic seed germination at phys-

iological and molecular scales. Our studies performed on terrestrial orchid species

from the Iranian plateau show that even common media like Murashige and Skoog

(MS) (Murashige and Skoog 1962) with low strengths (1/4 or 1/8 strength) can

inhibit seed germination. As is represented in Fig. 11, even the protocorms of rarely

germinated seed turned black and died.

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 239

8.3 Seed and Plantlet Dormancy

Terrestrial orchids have an annual cycle, whereby a period of growth is followed by

the loss of leaves, stems, and adventitious roots (Schiebold 2018). In many species,

the duration of the protocorm stage can extend from a few months, up to several

years, until leaves are produced. Thus, the absence of a compatible mycorrhiza may

last several years (Rasmussen et al. 2015; Dearnaley 2007). The period needed from

seed germination to reaching the adult stages (when the ﬁrst ﬂower is produced)

varies considerably and depends on the species. Consequently, the maturation time

for Cypripedium calceolus L. is 9 to 11 years, for Corallorhiza triﬁda Châtel. is 5 to

9 years, for Epipogium aphyllum Sw. is 10 years, for Ophrys apifera Huds. is about

6 to 8 years, for Neottia nidus-avis (L.) Rich and Cephalanthera damasonium (Mill.)

Druce is 9 to 11 years, for Neottia ovata (L.) Bluff & Fingerh. is 15 to 20 years, for

Dactylorhiza sambucina (L.) Soó is 12 years, for Dactylorhiza majalis (Rchb.) P.F.

Hunt & Summerh. and Dactylorhiza incarnata (L.) Soó is 16 years, for Orchis

mascula (L.) L. is 8 years, for Spiranthes spiralis (L.) Chevall. is 3 to 10 years, for

Neotinea ustulata (L.) R. M. Bateman, Pridgeon & M. W. Chase is 10 to 15 years,

etc. (Rasmussen 1995). The orchid remains below ground until conditions become

suitable for further growth. In the absence of a suitable fungus , the orchid

protocorms may remain viable in the soil, postponing their germination (Allen

1992; Allen et al. 1995). They survive by utilizing their minimal reserves very

slowly, waiting for a food source of simple nutrients to save them. When these are

provided (by fungal association), the development continues (Selosse and Cameron

2010; Selosse et al. 2022). Nevertheless, it is commonly known that only a very

small percentage of germinating seeds succeed and became adult plants. This is

usually due to the absence of the mycorrhizal partner that lacks from those particular

habitats. Despite the high survival potential of dormant protocorms, the prolonged

absence of the fungal symbiont usually leads to protocorm starvation and ultimately

to its death. Less than 5%, or even 1% in some temperate species, manage to survive

and successfully reach the age of reproduction when they are able to produce fruits

and viable seeds. The rest (seeds or protocorms), even perfectly viable, in the

absence of the fungus, remain dormant for good. The absence of fungi, as stressed

previously, may be due mainly to detrimental human intervention (anthropic fac-

tors), which usually led to major habitat and climatic changes (substrate pH,

temperature changes, ﬂooding, soil desiccation, deforestation, agriculture, tourism,

estate expansion, etc.), all ultimately leading to mycorrhizal network destruction and

plant species interaction disruption.

240 N. E. Anghelescu et al.

Fig. 11 The negative effect of high concentration of nitrate and ammonium present in MS medium

on asymbiotic seed germination of Ophrys reinholdii subsp. straussii (H. Fleischm.) E. Nelson. (a)

Ahmadzadeh.

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 241

9 Asymbiotic Seed Germination

However, almost all terrestrial orchid species needs a symbiosis relationship with

mycorrhizal fungi to germinate seed, develop protocorm, and establish plantlets in

nature, these events can also be proceeded both symbiotically (in the presence of

fungal symbiont) or asymbiotically (without fungal symbiont) (Ponert et al. 2013).

The establishment of a reciprocal relationship with mycorrhizal fungi could be

obligatory in some terrestrial orchid species for successful germination and

protocorm formation as they supply a big part of water, mineral nutrients, and

vitamins (Jolman et al. 2022). On the other hand, it has been represented that several

fungal species are joining the symbiotic relationship in terrestrial orchid roots

continuously or seasonally (Rasmussen 1995). One of the reasons for endangering

and threatening some terrestrial orchid species is the absence of mycorrhizal fungi

symbiosis due to climate change or habitat destruction (Li et al. 2021). In this regard,

one of the main problems to start a symbiotic seed germination experiment is the

need for a diverse range of ectomycorrhizal fungus species which usually have

unfavorable features like slow growth, difﬁcult cultivation, and high host speciﬁcity

(Rasmussen et al. 2015; Fatahi et al. 2022b; Kömpe 2022). Moreover, during the

symbiotic seed germination of terrestrial orchids, the nutritional and cultivation

condition requirements of both orchids and mycor rhizae should be provided (Ponert

et al. 2011). It is, moreover, not suitable for a wide range of physiological studies on

orchids because it is almost impossible to separate any effect of the fungus from the

direct effect of the factor under study. Unlike symbiotic germination, during

asymbiotic seed germination, the required nutrients are obtained by orchid seeds

through an artiﬁcial medium (Knudson 1922). Considering the obstacles of symbi-

otic culture establishment, asymbiotic germination procedures possess advantages

including an easier cultivation process, fast and large scale in vitro plantlet produc-

tion, and direct investigation of important variables affecting different biological

aspects of orchids’ life (Jolman et al. 2022; Swarts and Dixon 2009, 2017). To date,

the asymbiotic seed germination of different terrestrial orchid species has been

optimized as each terrestrial orchid taxon needs a speciﬁc and accurate combination

of organic and inorganic medium ingredients. Depending on the genus, species, and

even sub-species, there are drastically different developmental requirements, in

particular, based on the climate origin like tropical and temperate that necessitate

the exploitation of technically different germination procedures (Jolman et al. 2022;

Diantina et al. 2020). An extensive list of the performed research works on

asymbiotic seed germination of terrestrial orchid species highlighting the exploited

basal media, organic components, and PGRs, the highest reported seed germination

rate, and the country origin of the studied species has been shown in Table 1.

-

242 N. E. Anghelescu et al.

Table 1 The performed research studies on the asymbiotic seed germination of endangered terrestrial orchid species

Species Basal medium/media

Organic

supplements PGRs

Max.

germination

(%) Seed origin

Accl.

±Reference

Anacamptis longicornu MS, OM -95.5 Italy + Arcidiacono

et al. (2021)

Anacamptis morio KC, OM, MM Pep, PJ -88.91 Turkey + Hurkan et al.

(2018)

Anacamptis pyramidalis KC, MM Pep, CW,

PJ, CH, Gl

BA, Kin, 2-iP 73.79 Serbia -Ostojić et al.

(2022)

Anacamptis pyramidalis KC, OM, MM Pep, PJ -74.42 Turkey + Hurkan et al.

(2018)

Chloraea crispa MS, BM2 CH, Gl BAP, IBA 30.50 Chile -Quiroz et al.

(2017)

Cypripedium macranthos MS CW -68.10 South Korea -Huh et al. (2016)

Cypripedium macranthos MS, BM1, HP Pep BA, NAA 8.90 Japan + Shimura and

Koda (2004)

Cyrtopodium punctatum PT, MM, KC, MS,

P723

CH, Pep, Gl -27.30 USA + Dutra et al.

(2008)

Dactylorhiza hatagirea MS, MM, BM2, VW,

PT139, KC, LD

CH, Gl IBA, Kin 37.12 India + Warghat et al.

(2014)

Dactylorhiza romana KC, OM, MM Pep, PJ -66.30 Turkey + Hurkan et al.

(2018)

Dactylorhiza urvilleana OM Gy, TR Zeatin 62.39 Turkey -Bektaş (2016)

Epipactis ﬂava VW, MS, BM1, MM,

KC

CW, PE, CH -70.40 Thailand + Kunakhonnuruk

et al. (2018)

Epipactis veratrifolia FT Pep -79.60 Iran -Dianati Daylami

et al. (2017)

Eulophia ﬂava MS CW BA, NAA 26.39 Thailand -Vasupen et al.

(2022)

-

(continued)

Eulophia spectabilis MS, KC, BM1 CW, CH, Gl,

Ar

BAP, Kin 91.30 India + Nanekar et al.

(2014)

Eulophia promensis MS, P723 Pep BAP, NAA 100 Bangladesh + Hossain (2015)

Gastrodia cunninghamii MS, NG, WA, BM1 CH, Gl -~23 New Zealand -Diantina et al.

(2020)

Gymnadenia conopsea KC, MM Pep, CW,

PJ, CH, Gl

BA, Kin, 2-iP 69.88 Serbia -Ostojić et al.

(2022)

Habenaria macroceratitis MM BP -100 USA -Stewart and

Kane (2010)

Himantoglossum

adriaticum

MM Pep BA 5.10 Italy -Del Vecchio

et al. (2019)

Himantoglossum afﬁne MM Pep, CW,

AV, PJ, CH

-99.46 Iran + Fatahi et al.

(2022a)

Himantoglossum

calacaratum subsp.

jankae

KC, MM Pep, CW,

PJ, CH, Gl

BA, Kin, 2-iP 82.56 Serbia + Dulić et al.

(2019)

Liparis koreojaponica ND -15 Japan + Tsutsumi et al.

(2011)

Liparis kumokiri ND -30 Japan + Tsutsumi et al.

(2011)

Microtis arenaria BM1, MS, P723, Pa5,

W3

CW, BP,

CH, Gl

BA 99.20 Australia + Dowling and

Jusaitis (2012)

Neotinea tridentata KC, OM, MM Pep, PJ -55.03 Turkey + Hurkan et al.

(2018)

Ophrys apifera MM PSE, CW,

BP, YE

Zeatin, BA, GA, Kin,

TDZ

9.10 Italy + Pierce et al.

(2013)

Ophrys benacensis MM CM, PB, PJ -39.80 Italy + Pierce et al.

(2010)

Ophrys sphegodes MS, OM -12.0 Italy + Arcidiacono

et al. (2021)

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 243

Table 1 (continued)

Species Basal medium/media

Organic

supplements PGRs

Max.

germination

(%) Seed origin

Accl.

±Reference

-

Ophrys sphegodes KC, MM Pep, Gl, CH -62 Serbia -Dulić et al.

(2018)

Ophrys spp. MM CW, PJ -96 Greece -Kitsaki et al.

(2004)

Anacamptis coriophora OM, KC, LM, PM Pep, Tr IAA, IBA, 2,4-D, NAA,

BA, 2-iP, Kin, TDZ

44.20 Turkey -Bektaş et al.

(2013)

Orchis mascula MM, OM YE, CM BA 5.12 Italy -Valletta et al.

(2008)

Orchis militaris MM, KC, Ha CW, BS BA, 2-iP, IBA 82.60 Russia -Nabieva (2021)

Orchis simia MM Pep, CW,

AV, PJ, CH

-94.51 Iran + Fatahi et al.

(2022b)

Paphiopedilum

armeniacum

MS CaH, PE,

BH

NAA 96.20 China + Wang et al.

(2021b)

Paphiopedilum

spicerianum

MS, RE, CW BAP, NAA 21.65 China + Chen et al.

(2015)

Paphiopedilum tigrinum mHa, 1/2 MS CW BA, Kin 90.17 China + Yao et al. (2021)

Paphiopedilum venustum BM, BM1, KC, MM Pep, CW,

PJ, CH, Gl

-82.75 India + Kaur and

Bhutani (2016)

Paphiopedilum wardii MS CW NAA 65.33 China + Zeng et al.

(2012)

Pelatantheria

scolopendrifolia

1/2 MS, PM, BM1,

BM2

CH, Gl -94.10 South Korea -Kim et al. (2021)

Phragmipedium

warscewiczii

KC, MS -2.90 Costa Rica + Muñoz and

Jiménez (2008)

Phragmipedium

longifolium

KC, MS -41.30 Costa Rica + Muñoz and

Jiménez (2008)

244 N. E. Anghelescu et al.

-Phragmipedium pearcei KC, MS -38.70 Costa Rica + Muñoz and

Jiménez (2008)

Platanthera chapmanii P723 Pep -15.50 USA -Poff et al. (2016)

Pleione bulbocodioides 1/2 MS CW, PE, BE 2,4-D, BA, Kin, TDZ 73.32 China -Zhou et al.

(2021)

Prasophyllum pruinosum BM1, MS, P723, Pa5,

W3

CW, BP,

CH, Gl

BA 65.50 Australia + Dowling and

Jusaitis (2012)

Pseudorchis albida MM CW, BP,

ME, YE

BA, GA3, TDZ 50.50 Italy -Pierce and

Cerabolini

(2011)

Pterostylis banksii MS, NG, WA, BM1 CH, Gl -~34 New Zealand -Diantina et al.

(2020)

Pterostylis nutans BM1, MS, P723, Pa5,

W3

CW, BP,

CH, Gl

BA 91.20 Australia + Dowling and

Jusaitis (2012)

Satyrium nepalense MS, KC -BAP, Kin, TDZ 86.70 India + Mahendran and

Bai (2009)

Spiranthes spiralis KC, MM Pep, CW,

PJ, CH, Gl

BA, Kin, 2-iP 36.65 Serbia + Dulić et al.

(2019)

Thelymitra nervosa MS, NG, WA, BM1 CH, Gl -~35 New Zealand -Diantina et al.

(2020)

Thelymitra pauciﬂora BM1, MS, P723, Pa5,

W3

CW, BP,

CH, Gl

BA 60.90 Australia + Dowling and

Jusaitis (2012)

BM1 Basal medium/media, Ha harvais, HP hyponex-peptone, KC Knudson C, LD Lindemann, MM Malmgren, MS Murashige and Skoog, ND New Dogashima,

NG Norstog, Pa5 burgeffs N3f, PT139 Mitra, PT Phyto Technology orchid medium, RE Robert Ernst, PM Phytamax, OM Orchimax, W3 Western 3, WA water-

agar Organic supplements: Ar arginine, BH banana homogenate, BP banana powder, BS birch sap, CaH carrot homogenate, CM coconut milk, Gy glycine, ME

malt extract, PE potato extract, PJ pineapple juice, PSE pea seed extract, Tr tryptone, PGRs plant growth regulators, 2,4-D 2,4-dichlorophenoxyacetic acid; N6-

(2-Isopentenyl) adenine, BA benzyl adenine, BAP 6-benzylaminopurine, IAA indole-3-acetic acid, IBA indole-3-butyric acid, GA3 gibberellic acid, Kin kinetin,

NAA naphthalene acetic acid, TDZ thidiazuron

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 245

246 N. E. Anghelescu et al.

9.1 Asymbiotic Seed Germination Stages

There are different processes and stages between symbiotic and asymbiotic germi-

nation in terrestrial orchids as symbiotic germination requires an extra stage for

mycorrhizal association and symbiont development. In terrestrial orchid species,

during asymbiotic germination process, the embryos enlarge and produce small

structures called protocorm s, which have root and shoot meristematic centers. The

protocorm can develop completely only in the presence of adequate storage

resources for the shoot a nd root formation. By activation of root and shoot meristem,

the plantlets start to grow under in vitro conditions. Based on the description in the

literature, asymbiotic seed germination in terrestrial orchids can be divided into ﬁve

stages (Bektaş 2016; Nabieva 2021):

Stage I: “no-germination” stage. Unimbibed seed with intact testa.

Stage II: “swelling” stage. Embryo swelling and enlargemen t followed by testa

rupturing.

Stage III: the “pre-germination” stage. Complete rising of the embryo from

ruptured seed testa and formation of ﬁrst rhizoids.

Stage IV: “Rhizoid” stage. The formation of rhizoids on the surface of the

protocorm.

Stage V: “Protocorm or germination” stage. Enlargement of protocorm and

formation of proto meristem.

Stage VI: “Shoot” stage. Further enlargemen t and development of the ﬁrst

green leaf.

As a part of studies performed in the Research Center for Terrestrial Orchid,

RCTO, university of Kurdistan), the asymbiotic seed germination of Ophrys

reinholdii subsp. straussii (H.Fleischm.) E. Nelson (a threatened Euro-

Mediterranean terrestrial orchid species) has been shown in Fig. 12 highlighting

the main stages of germination.

10 Organic Supplements and Asymbiotic Seed Germination

As artiﬁcial media give different results depending on the target species, screening

media and supplements would be helpful to determine the best nutrient formulation

that maximizes seed germination in terrestrial orchids (Swarts and Dixon 2009,

2017; Cardoso et al. 2020). In this regard, terrestrial orchid speci es seeds are

sensitive to the inorganic form of nutrients in particular nitrogen and therefore a

variety of organic additives and compounds have been used for asymbiotic seed

germination of orchids (Utami and Hariyanto 2020; de Menezes Gonçalves et al.

2016; Kaur 2021). There are several such compounds, including peptone, coconut

water, pineapple juice, casein hydrolysate, yeast extract, and amino acid mixtures. It

is very important to use a suitable organic compound such as pineapple juice,

coconut milk, boiled potatoes, or other similar compounds. These compounds

contain vitamins and plant hormones, which are often the most suitable compounds

for orchid propagation. The use of organi c compounds is important in the in vitro

culture of various orchids as they provide vitamins and plant growth regulators. In

many cases, they have a positive effect on seed germination and plantlet growth

regardless of whether their components are known or unknown. Here, we discuss the

nature and the application of some organic supplements used for in vitro germination

and propagation of terrestrial orchids. The use of inexpensive organic complex

supplements can reduce the costs helping the large-scale in vitro micropropagation

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 247

Fig. 12 Seed germination stages and plantlet growth and development in Ophrys reinholdii subsp.

straussii (H.Fleischm.) E.Nelson. (a) SEM micrograph of seed testa micromorphology (Stage I); (b)

Embryo enlargement (stage II) and its excise from seed testa (stage III) 18–22 days after seed sown;

(c) Rhizoid formation (stage IV) 24–30 days after seed sown; (d, e) Enlargement of protocorm and

formation of protomeristem (stage V); (f) Developing green leaves (stage VI); ready for acclima-

tization 3–4 months after seed sown; (g) A clump of in vitro raised plantlets with healthy and green

leaves and small tuber; (h) The stages of in vitro protocorm development, rooting, and plantlet

growth. SE swelling embryo; RT rupturing testa; DP developing protocorm; RZ rhizoids; LP leaf

Ahmadzadeh

of endangered terrestrial orchi d species and their reintroduction to nature (Fatahi

et al. 2022a, 2022b).

248 N. E. Anghelescu et al.

10.1 Peptone

Different raw materials can be digested by acids or enzymes to produce a protein

hydrolysate named peptone (Nhut et al. 2008). Peptone is a product of animal tissue

and products digestion and is composed of low molecular weight const ituents (23%

glycine, 16.16% total nitrogen, 15.38% peptone nitrogen, 11% glutamic acid, 9.42%

monoamine nitrogen, 8% arginine, 5.9% aspartic acid) (Yam and Arditti 2017b).

Peptone not only is autoclavable and dialyzable but also stable under acidotic and

alkaline conditions (Jan et al. 1994). There are numerous reports on the exploitation

of peptone as one of the main organic media constituents not only in plant tissue

culture but also in animal and insect cell culture to supply carbon and nitrogen. In

orchids, peptone is used to improve symbiotic and asymbiotic seed germination. In

this regard, in Himantoglossum afﬁne (Boiss.) Schltr. as a Euro-Mediterranean

terrestrial orchid species, the highest germination rate (98.77 ± 0.37%) was obtained

with media containing pineapple juice plus peptone. Besides its positive impact on

seed germination and protocorm development and growth, peptone also positively

affects terrestrial orchid plantlet growth as it is a rich source of amino acids and

vitamins such as thiamin, biotin, pyridoxine (Utami and Hariyanto 2020). In this

regard, individual use of organic nitrogen compounds resulted in higher germination

efﬁciencies, and plantlets grown on media supplemented with peptone had the

highest plantlet length and weight compared to other organic nitrogenous com-

pounds (Fatahi et al. 2022a). Evaluation of different levels of peptone and banana

homogenate on in vitro micropropagation of terrestrial orchid Paphiopedilum

venustum (Wall. ex Sims) Pﬁzer revealed that BM-1 medium (Van Waes and

Debergh 1986b) containing 1 g/L peptone resulted in the highest shoot efﬁciency

of shoot multiplication (Kaur and Bhutani 2016). The adding peptone to seed

germination medium for Paphiopedilum hirsutissimum (Lindl. ex Hook.) Stein

and Paphiopedilum insigne (Wall. ex Lindl.) Pﬁzer resulted in a 30% higher

germination percentage (Zeng et al. 2016). Orchis simia Lam. is a threatened

terrestrial orchid growing in central and southern Europe with fragmented

populations due to climate change and overexploitation. Fatahi, Vafaee (Fatahi

et al. 2022b) by studying different organic compounds supplying nitrogen found

that commercially available amino acid mixture (Vamine) and casein hydrolysate

were more efﬁcient than peptone on seed germination and plantlet growth. They

stated that altogether the using of organic nitrogenous supplements can replace the

need for mycorrhizal fungi regardless of the nature of used organic nitrogenous

compounds. Malmgren medium containing peptone, coconut water, and glutamine

resulted in the highest germination rate in lizard orchid, Himantoglossum

calcaratum (Beck) Schltr. subsp. jankae (Somlyay, Kreutz & Óvári) R.M.Bateman,

Molnar & Sramkó (Dulić et al. 2019) showing the important role of amino acids in

the successful asymbiotic seed germination of terrestrial orchids. With lower per-

centages, the seed of Himantoglossum adriaticum H.Baumann was successfully

germinated on the same medium supplemented with 0.5 g/L peptone (Del Vecchio

et al. 2019).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 249

10.2 Coconut Water

Coconut water is the liquid obtained from the center part of the endosperm, while

coconut milk is the liquid obtained from the solid and ﬂeshy part (George et al. 2008;

Yong et al. 2009). Compared to coconut water, coconut milk which is also the source

of coconut oil has not been commonly used in terrestrial orchid tissue culture. There

are a variety of compounds found in coconut water, including amino acids, organic

acids, plant growth regulators, vitamins, sugars, sugar alcohols, minerals, nucleic

acids, and unknown growth substances, all of which can support and trigger in vitro

plant growth and development (George et al. 2008; Yong et al. 2009). Plant growth

regulators (PGRs) including auxins (mainly indole-3-acetic acid IAA), cytokinins

(trans-zeatin, trans-zeatin O-glucoside, N

6

-isopentenyladenine, and dihydrozeatin),

gibberellins (GA1 and GA3), and abscisic acid are among most prevalent PGRs in

coconut water (Yong et al. 2009; Shekarriz et al. 2014). Various mineral ions

including Ca, Fe, Mg, P, and K can be found in coconut water (Vasupen et al.

2022). Additionally, it contains B1, B2, B3, B5, B6, B7, and B9 vitamins (George

et al. 2008; Yong et al. 2009). Due to the presence of PGRs in particular cytokinins,

coconut water is extensively has been exploited in the propagation of terrestrial

orchid species through asymbiotic seed germination (George et al. 2008). By

providing faster energy to the cells and by triggering cell division through its

cytokinin content, CW implementation in culture media results in better responses

(Jolman et al. 2022). Coconut water could promote seed germination and develop-

ment of seed to post protocorm stage in Eul ophia ﬂava (Lindl.) Hook. f (Vasupen

et al. 2022). However, the supplementation of Malmgren medium with coconut

water resulted in lower asymbiotic germination rates in Himantoglossum afﬁne

(Boiss.) Schltr. (an endangered Euro-Mediterranean tuberous orchid), causing the

shortest time to germination compared to other used organic supplements (Fatahi

et al. 2022a). Orchis militaris L. is a cold-hardy terrestrial Euro-Siberian species

considered recalcitrant to in vitro seed germination response. Adding 5% coconut

water to Malmgren medium led to a higher number of protocorms and seedlings,

producing ﬁrst secondary roots and true leaves in Orchis militaris L. (Nabieva

2021). The perennial tuberous and rhizomatic orchid species, Pleione

bulbocodioides (Franch.) Rolfe, also known as Cremastra Pleione, is an endangered

species due to tuber overharvesting and natural low-rate propagation. It has been

reported that the rate of protocorm formation is higher in Pleione bulbocodioides

(Franch.) Rolfe uses coconut water than other organic compounds like peptone and

banana extract. Among three studied concentrations of coconut water (50, 100, and

150 mg/L), the protocorm induction percentage was at the highest value

(50.35 ± 0.60%) using 100 mg/L coconut water (Zhou et al. 2021). The Spiranthes

spiralis (L.) Chevall. seed sown on Knudson C medium supplemented with coconut

water had a high germination rate which has been attributed to a high content of

cytokinins supporting cell division and thus growth promotion (Duli ć et al. 2019).

Similar ﬁndings have been obtained with asymbiotic seed germination in threatened

terrestrial orchid Cypripedium macranthos Sw. on the MS medium nourished with

coconut water (Hu h et al. 2016).

250 N. E. Anghelescu et al.

10.3 Pineapple Juice

Sucrose, glucose, and fructose are among the most abundant ingredients of pineap-

ple, which comprise about 81–86% of its total soluble solids. On the other hand,

there is 2–3% ﬁber in pineapple which is high value within fruit crops (Malmgren

1996). Ascorbic acid is the most prevalent organi c acid in pineapple while there is

also a low level of citric acid present in pineapple juice (Kitsaki et al. 2004; George

et al. 2008). In this term, bromelain is a protease present in pineapple contributing

80% of total proteolytic activity that can break down other proteins. Minerals found

in pineap ples include calcium, chlorine, potassium, phosphorus, sodium, and copper

(Utami and Hariyanto 2020; George et al. 2008). Besides nutritional roles in

providing macro- and micronutrients as well as PGRs, pineapple juice can also

reduce phenolic compound production in the environment (Rasmussen 1995), which

has improved the propagation efﬁciency of European orchids through asymbiotic

seed germination (Malmgren 1996). A number of temperate terrestrial species have

also been found to beneﬁt from pineapple juice in terms of root differentiation and

growth (Rännbäck 2007). Malmgren stated that adding 15–125 mL pineapple juice

supplies about 30–40 mg/L potassium and also enough concentrations of microel-

ements (Malmgren 1996). Pineapple juice is a permanent organic supplement added

to the asymbiotic seed germination medium in different species members of terres-

trial orchid genera including Cypripedium L., Dactylorhiza Neck. ex Nevski,

Nigritella Rich., Gymnadenia R.Br., Orchis Tourn. ex L., Platanthera Rich., etc.

Pineapple juice could be successfully applied for asymbiotic seed germination in

endangered tuberous orchid species incl uding Orchis sim ia Lam. (Fatahi et al.

2022b), Himantoglossum afﬁne (Boiss.) Schltr. (Fatahi et al. 2022a), Cypripedium

spp. (Rännbäck 2007), Ophrys benacensis (Reisigl) O. Danesch, E. Danesch &

Ehrend, (Pierce et al. 2010), Himantoglossum calcaratum (Beck) Schltr. subsp.

jankae (Somlyay, Kreutz & Óvári) R. M. Bateman, Molnar & Sramkó and

Spiranthes spiralis (L.) Chevall. (Dulić et al. 2019), Anacamptis pyramidalis (L.)

Rich., and Gymnadenia conopsea (L.) R. Br (Ostojić et al. 2022). Moreover, a high

ratio of seed germination and plant development have been obtained with Malmgren

medium containing pineapple juice, however, the best results were obtained with

BM1 medium supplemented with casein hydrolysate. On the other hand, Kitsaki and

Zygouraki (Kitsaki et al. 2004) studied during the germination of different terrestrial

orchid species belonging to the Ophrys L. genus including Ophrys umbilicata Desf.,

Ophrys sphegodes subsp. spruneri (Nyman) E.Nelson, Ophrys speculum Link,

Ophrys tenthredinifera Willd., Ophrys sphegodes subsp. mammosa (Desf.) Soó ex

E.Nelson, Ophrys lutea Cav., Ophrys fusca subsp. iricolor (Desf.) K.Richt., Ophrys

ferrum-equinum Desf., Ophrys × delphinensis O. Danesch & E. Danesch, Ophrys

scolopax subsp. cornuta (Steven) E. G. Camus, Ophrys argolica H. Fleischm. and

Ophrys apifera Huds., the medium containing pineapple juice as the inorganic

supplement resulted in the best plantlet development (Kitsaki et al. 2004).

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 251

10.4 Casein Hydrolysate

The enzymatic or acidic hydrolysis of different natural products such as milk, plant

and animal tissues, and microbial cultures can result in the production of hydroly-

sates. There are mixed recommendations for using hydrolysates in the in vitro orchid

culture (Lee and Yeung 2018). There are several commercially available hydroly-

sates. Casein hydrolysate is a fraction product obtained from the enzymatic digestion

of mammalian milk. Casein hydrolysate is an important compo nent in various media

formulations since it provides a mixture of proteins, amino acids, and peptides as

reliable natural sources (George et al. 2008). Some micronutrients and vitamins are

also present (Dulić et al. 2019). Due to its high concentration of essential and

non-essential amino acids, vitamins, and phosphates, casein hydrolysate is known

as a germination and growth-inducing factor in the orchid tissue culture (Fatahi et al.

2022a; Kaur 2021). BM media are typical media used for asymbiotic seed germi-

nation of some terrestrial orchids, developed by Van Waes and Debergh (Van Waes

and Debergh 1986b), which contains 500 mg/L casein hydrolysate. However, other

commercial media used for multiplication, maintenance, and sub-culturing terrestrial

orchids usually contain 1–2 g/L of casein hydrolysate depending on the species

(Utami and Hariyanto 2020). In this term, among different combinations of organic

additives, 500 mg/L casein hydrolysate plus 15% coconut water (CW) represented

the best seed germination results in Eulophia spectabilis (Dennst.) Suresh is a

therapeutically important endangered orchid species from India (Nanekar et al.

2014). Using MS and Mitra media supplemented with 500 mg/L casein hydrolysate

and 1 mg/L N

6

-benzyladenine (BA), a high asymbiotic seed germination

(75 ± 2.5%) was obtained Crepidium khasianum (Hook.f.) Szlach (Deb 2006).

Similar results were obtained in Ophrys sphegodes Mill. using Knudson C and

Malmgren media containing peptone, L-glutamine, folic acid, and casein hydroly-

sate (Dulić et al. 2018). Chloraea crispa Lindl. (Quiroz et al. 2017), Anacamptis

pyramidalis (L.) Rich. and Gymnadenia conopsea (L.) R.Br. (Ostojić et al. 2022),

Orchis simia Lam. (Fatahi et al. 2022b), Himantoglossum afﬁne (Boiss.) Schltr.

(Fatahi et al. 2022a), Himantoglossum calcaratum (Beck) Schltr. subsp. jankae

(Somlyay, Kreutz & Óvári) R. M. Bateman, Molnar & Sramkó (Dulić et al. 2019),

and Eulophia spectabilis (Dennst.) Suresh (Nanekar et al. 2014) are among other

species whose asymbiotic seed germination has bene ﬁted from the improving

impacts of casein hydrolysate.

252 N. E. Anghelescu et al.

10.5 Amino Acid Mixtures

Based on the fact that the enzymatic systems of amino acid metabolism and

biosynthesis of the developing embryo change and evolve (Lee et al. 2007), they

can be exploited as easy-to-metabolize alte rnative nitrogen sources (Rasmussen et al.

2015; Utami and Hariyanto 2020; Kaur 2021). However , they may not be available

and considered primary nitrogen sources, they can be metabolized and used to

synthesize new essential structural and enzymatic proteins (Rasmussen et al.

2015). The metabolism of ready mixtures of amino acids (which are commercially

available) can be performed by orchids’ embryos and PLBs more efﬁciently com-

pared to other inorganic nitrogen sources (Valadares et al. 2014). This is because

under in vitro conditions, they can redirect or skip some nitrogen assimilation

pathways (Rasmussen 1995; Rasmussen et al. 2015). The response of asymbiotic

seed germination to amino acids supplementation is different depending on the

terrestrial orchid species (Fatahi et al. 2022b). Researchers believe that nitrogen in

the amino acid form may facilitate seed germination or growth of proto corms than

available inorganic nitrogen sources (Malmgren 1996; Kauth et al. 2008; Stewart

and Kane 2007). This fact has been also shown in asymbiotic seed germination of

Himantoglossum afﬁne (Boiss.) Schltr. where a ready amino acid mixture with the

commercial name Vamine was more effective in the induction of seed germination

than peptone and casein hydrolysate (Fatahi et al. 2022a). The slow growth of

orchids was attributed to the sluggish nitrogen metabolism using inorganic nitrogen

forms like NH

4 +

for seed germination, in the ﬁrst step ammonium is converted to

amino acids (Wu et al. 2013). Since all amino acids are not required during seed

germination, a combination of selected important amino acids can be used more

effectively to achieve high seed germination ratios. It has been shown in Orchis

simia Lam. that bigger protocorms (4.5-fold bigger) were obtained on media

supplemented with pineapple juice (PJ) in combination with Aminoven

(a commercially available amino acid mixture) compared to protocorms grown on

other media. Enhanced seed germination and subsequent plant growth in Habenaria

macroceratitis Willd. on modiﬁed Malmgren modiﬁed medium have been reported

(Stewart and Kane 2010). The advantage of using amino acid mixtures instead of

undeﬁned organic supplements containing nitrogen such as peptone, casein hydro-

lysate, and in particular coconut water and pineapple juice is that commercial amino

acid mixtures contain given concentrations of known amino acids. On the other

hand, unlike inorganic nitrogen sources, the recommended levels of amino acids

even at higher concentrations are not suitable for terrestrial orchid seed germination.

11 Conclusions

Since all amino acids are not required during seed germination, a combination of

selected important amino acids can be used more effectively to achieve high seed

germination ratios. It has been shown in Orchis simia Lam. that bigger protocorms

(4.5-fold bigger) were obtained on media supplemented with pineapple juice (PJ) in

combination with Aminoven (a commercially available amino acid mixture) com-

pared to protocorms grown on other media. Enhanced seed germination and subse-

quent plant growth in Habenaria macroceratitis Willd. on modiﬁed Malmgren

modiﬁed medium have been reported (Stewart and Kane 2010). The advantage of

using amino acid mix tures instead of undeﬁned organic supplements containing

nitrogen such as peptone, casein hydrolysate, and in particular coconut water and

pineapple juice is that commercial amino acid mixtures contain given concentrations

of known amino acids. On the other hand, unlike inorganic nitrogen sources, the

recommended levels of amino acids even at higher concentrations are not always

suitable for terr estrial orchid seed germination. (the fragments are repeating!)

Asymbiotic Seed Germination in Terrestrial Orchids: Problems, Progress,... 253

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Progress and Prospect of Orchid Breeding:

An Overview

Khosro Balilashaki, Zahra Dehghanian, Vahideh Gougerdchi,

Elaheh Kavusi, Fatemeh Feizi, Xiaoyun Tang, Maryam Vahedi,

and Mohammad Musharof Hossain

1 Introduction

Orchid is the general name of Orchidaceae, which belongs to perennial herbaceous

plants with unique and attractive ﬂower shapes and colors, and is the second-largest

family of ﬂowering plants with high ornamental, medicinal, and other economic

value. Orchidaceae is the most evolved, highly specialized, diverse, and widespread

plant family belonging to Monocotyledons, with about 801 genera and 28,237

species (Shriram and Kumar 2022). Orchids are virtually found on all continents

except icy Antarctica and hot deserts but their greatest variation is to be found in the

tropical and subtropical regions, mostly Asia, South America, and Central America.

K. Balilashaki (*) · F. Feizi

Department of Horticultural Science, Faculty of Agricultural Science, University of Guilan,

Rasht, Iran

e-mail: khosrobali@alumni.ut.ac.ir

Z. Dehghanian

Department of Biotechnology, Faculty of Agriculture, Azarbaijan Shahid Madani University,

Tabriz, Iran

V. Gougerdchi · E. Kavusi

Department of Plant Breeding and Biotechnology, Faculty of Agriculture, University of Tabriz,

Tabriz, Iran

X. Tang

College of Art Colleges of Landscape Architecture, Fujian Agriculture and Forestry University,

Fuzhou, China

M. Vahedi

Department of Horticultural Science, Faculty of Agricultural Sciences and Engineering, College

of Agriculture and Natural Resources, University of Tehran, Tehran, Iran

M. M. Hossain

Department of Botany, University of Chittagong, Chittagong, Bangladesh

P. Tiwari, J.-T. Chen (eds.), Advances in Orchid Biology, Biotechnology and Omics,

https://doi.org/10.1007/978-981-99-1079-3_9

261

So far, over 1,06,000 hybrids have already been registered and developed (Hossain

et al. 2013) and more than 1000 new hybrids are added per year.

262 K. Balilashaki et al.

The shape of orchids is simple and elegant, the fragrance of ﬂowers attacks

people, is one of the precious ﬂowers, deeply loved by people. As a ﬂower with

high ornamental value and miniature potted plants, orchids are widely used in trade

and commercial markets, and the current market demand is increasing (Chao et al.

2018). The world business value of orchids exceeded billion dollars, and the

countries of Thailand, Singapore, and Malaysia dominated the world orchid market.

The global orchid trade value was estimated at US$ 504 million in 2013

(Cheamuangphan et al. 2013), and the ﬁgure is undoubtedly increased many folds

recently. In addition, Gastrodia elata, Dendrobium nobile, Cypripedium henryi, and

other species of orchids are excellent Chinese herbal medicine, with great medicinal

value (Wang et al. 2020).

With the prosperity and development of the orchid market in the world, many

countries are engaged in orchid breeding, and the demand for technological renewal

and industrial upgrading of orchid breeding is increasing. At present, the breeding

methods of orchids are still mainly based on the combination of wild resource

domestication and traditional breeding. However, there are many practical problems

with traditional breeding techniques, such as prolonged bleeding times and a huge

workload. With the renewal and development of technology, several new breeding

methods have emerged. In recent years, compared with the time-consuming tradi-

tional breeding, the method of using the CRISPR/Cas9-KO system to carry out

orchid molecular breeding has produced ideal alleles in less than 20 months, which

greatly accelerates the efﬁciency of breeding (Semiarti et al. 2020). The genetic

modiﬁcation of orchids by Agrobacterium-mediated transformation and gene gun

technique has been successfully and continuously applied, which has made great

progress in the improvement of important traits of orchids, such as ﬂower color,

fragrance, cut ﬂower shelf-life, and so on (da Silva et al. 2016). This article mainly

reviews the history and methods of orchid breeding, which might be a reference for

future studies on orchid breeding.

2 The History of Breeding

As an ornamental plant, orchids have a history of more than 2000 years. Undoubt-

edly, the Chinese described orchids for medicinal use (Bulpitt 2005). Between

551 and 479 B.C., the elite people of Japan grew orchids for their beauty and

fragrance (Hossain et al. 2013). In ancient times, orchids are often found in poetry,

with the rise of private gardens, orchid cultivation is more and more extensive, and

after thousands of years of choice and utilization, the formation of a variety of orchid

varieties resulted in a wealth of orchid culture.

Orchids have a long history of cultivat ion and are extremely rich in germplasm

resources. The ancient laborers began the original breeding work by selecting the

most satisfactory or strange types. For thousands of years, a wealth of experience has

been accumulated, and many numbers of ﬁne garden plant species have been

created. Because of the characteristics of history and culture, breeding is not alone

as a technique but is usually included in cultivation and reproduction methods.

Progress and Prospect of Orchid Breeding: An Overview 263

Chinese ornamental and cultivated orchids are much earlier than those cultivated

in the West. As early as 2400 years ago in the Spring and Aut umn period, there was

already a description of orchids. The oldest book on orchids in Japan is “Igansai-

ranpin” written by Jo-an Matsuoka in 1728 A.D. in which species of Aerides,

Bletilla, Cymbidium, Dendrobium, and Neoﬁnetia were described. The Samurai

grew Neoﬁnetia falcata, the merchants grew Cymbidium, and possibly the peasants

grew Bletilla (Bulpitt 2005). In ancient times, people ﬁrst collected wild orchids, as

for artiﬁcial cultivation of orchids, until the court began. After Wei and Jin dynasties,

orchids expanded from court cultivation to private gardens of literati class and were

used to decorate gardens and beautify the environment. It was not until the Tang

Dynasty that the cultivation of orchids developed into general gardens and ﬂorists

(Deng 1990).

The cultivation of orchids became very widespread during 960–1126 AD, and

records of the descriptive features, ecology, and distribution b ecame abundant

(Schiff 2018). The Jin Zhang Lan Pu written by Zhao Shigeng of the Southern

Song Dynasty in 1233 can be said to be the earliest monograph on orchids in China,

and it is also the ﬁrst monograph on orchids in the world (Chen et al. 2011). The

book is divided into three volumes and ﬁve parts, describing the morphological

characteristics of more than 30 species of orchids. About 10 years later, Wang

Guixue wrote a book (Wang’s Treatise on Orchids) in 1247, which describ ed the

types and grades of orchid genealogy and the use of soil or soil mixture as potting

medium (Luo et al. 2012). Early articles on orchids and their cultivation were

relatively short.

The orchid cultivation in Ming Dynasty entered the prosperous period, the orchid

variety in the south of the Yangtze River increased continuously, the cultivation

experience became more and more abundant, and the orchid gradually became the

common appreciation of the general people. Qing Dynasty was the most prosperous

period of orchid cultivation in China. With the continuous emergence of genealogy

and new horticultural varieties in the past dynasties, a number of Yilan with rich

experience have emerged. Based on summing up the previous experience, they have

put forward new ideas and wrote valuable orchid Monographs. The period of the

Republic of China is also an important period of development in the history of

Chinese orchids. During this period, after 2 or 300 years of exploration, the valve

type theory of orchids has been completed, and a large number of orchid varieties

have been discovered. In the twentieth century, Chinese orchids have entered a more

prosperous period, the number of all kinds of orchid books published; the wide range

of newly developed national orchid varieties, the huge contingent of orchid enthu-

siasts, and the active orchid trading have exceeded the previous dynasties (Reinikka

1972).

Ancient horticulturists gradually mastered ﬁne cultivation and management tech-

niques, constantly used sexual reproduction combined with selection to breed new

varieties, and used asexual propagation methods to preserve special variation types

and other horticultural ideas and methods for traditional breeding. It also laid a

foundation for the formation and development of modern orchid variety groups.

264 K. Balilashaki et al.

In Europe, the cultivation of orchids only started in the late eighteenth century. In

1778, Dr. John Fotherrdill brought Cymbidium ensifolium and Phaius tanker villeae

back to England for the ﬁrst time (Reinikka 1972). In 1780, Vere Kensington

introduced Cymbidium pendulum into Europe. Since then, Orchid plants have been

found in Asia and Oceania and sent to the United Kingdom, and then spread to

Europe and the United States through the United Kingdom.

The Orchid was initially ignored in England, but it has been noticed by the British

since 1889 by the hybrid of Cymbidium eburneum and C. lowianum. From 1904 to

1905, there were many new orchids introduced from Vietnam to Europe, such as C.

insigne, C. erythrostylum, and C. eburneum. Since then, there has been more and

more crossbreeding, and new hybrids have been emerging. There were only four

hybrids in 1904 and 88 in 1908, which increased to as many as 170. One of the

parents of C. insigne is praiseworthy, it shows obvious genetic factors, leaving future

generations with good characteristics, such as growth habits, ﬂower shape and color,

and easy to cross with other orchids of the genus.

In crossbreeding, most of the parents in Europe are large ﬂower types. They are

easier to cross and obtain hybrid offspring, so they are valuable and excellent

parents. These hybrids have many ﬂowers, large ﬂowers, long ﬂowering, and

beautiful colors, so they are very popular as cut ﬂowers. As a result, the number of

new hybrid varieties has increased greatly every year, and tens of thousands of new

hybrid varieties have been increased at present. New and excellent hybrid varieties

continue to replace the inferior old varieties. The application of new cultivation

techniques, aseptic test-tube plantlets, tissue culture, and other rapid propagation

progress, so that enterprises and companies engaged in orchid cultivation all over the

world.

Since the nineteenth century, European orchid scholars have done a lot of work.

In 1833, J. Lindley (father of orchid cultivation) sorted the genus Orchid and gave

the ﬁrst classiﬁcation of orchids. He also left an unﬁnished book, “Folia

Orchidaceae” considered a classic of Bot any.. Blume established the related genus

Cyperorchis and Irdorchis. English naturalist Charles Darwin wrote the book

“Fertilisation of Orchids” in 1862, this book was the ﬁrst essential contribution to

the knowledge and comprehension of the strategies used for the speci es to ensure

propagation. Lewis Castle published another book “Orchids: Their Structure, His-

tory and Cultivation” in 1877 that offers a concise history of the orchid coupled with

simple directions for breeding. For the ﬁrst time, Reinchenbach made a comprehen-

sive summary of the genus Orchid, describing 19 species of orchids. In 1903, Rolfe,

a British scientist, ﬁrst classiﬁed Orchidaceae in China, including nine species of

orchids. In 1919, R. Schlechter, a German orchid scholar, summarized 33 species of

Orchidaceae in East Asia. In 1924, he made a taxonomic study on Cymbidium and

Cyperorchis all over the world and established the following taxa (groups). The

renowned British plant explorer and phytogeographer J.D. Hooker (1888–90)

described 1250 species of orchids from the Indian subcontinent in his famous

book “Flora of British India.”

Progress and Prospect of Orchid Breeding: An Overview 265

In Southeast Asia, the history of planting orchids is also earlier. In addition to the

cultivation of orchids produced in the region, there are also C. ensifolium and

C. sinense from China. In recent decades, with the improvement of orchid planting

technology and the continuous emergence of hybrid orchids, many orchid growing

enterprises and companies have been formed, such as Thailand and Singapore,

where commercial orchids have entered the international orchid market and have a

ﬁxed position in the world orchid industry.

3 Botany and Structure of the Flower

Orchid ﬂowers display a bewildering array of shapes, sizes, and colors, yet all have a

distinctive “orchidness” that sets them apart from other plant groups (Apriyanti et al.

2013). The ﬂower of Orchids consists of four main parts including an outer whorl of

three sepals, an inner loop of three petals, a single large column in the center, and an

enlarged bottom petal called a labellum. The overall ﬂower shape is bilaterally

symmetrical a necessity for reliable insect pollination.

The pollen of orchids is also very special. The pollen structure of orchids is called

pollen block or pollinia (a coherent mass of pollen grains). It is not the pollen that

will spread out, and it will not cause the discomfort of some pollen allergies. Pollen

block is the general name of ﬂower powder mass, pollen mass stalk, sticky disk

handle, and sticky disk connected, and it is the organ of male and female (Johnson

and Edwards 2000). This structure is an efﬁcient structure for orchids to transmit

powder to insects. When the insect comes to the orchid ﬂower to collect honey, his

body touches the sticky plate, which sticks the orchid pollen block to the insect,

takes it to the next orchid, and completes the pollination.

4 Pollination

Pollinators are resources on which plants rely and sometimes compete, hence

systems of pollination can be thought of as ecological niches . The pollinator

assembly of a plant determines its pollination niche, even though plants interacting

with the same pool of pollinators may have diverse pollination niches due to

different pollinator use (Joffard et al. 2019). Orchids have a wide range of pollination

tactics and ﬂower characteristics. One-third of these orchid species are charming, in

that they do not provide a reward for pollinators but instead use signs that pollinators

traditionally associate with food or sex promises to attract them (Reyes et al. 2021).

Pollinators have played a signiﬁcant role in the Orchidaceae family’s diversiﬁcation

and are critical for the conservation of most orchid species that rely mainly on insects

for sexual reproduction (Schatz et al. 2017). While some orchid–pollinator relation-

ships are described as highly specialized, such as in sexually deceptive or euglossine

bee-pollinated orchids, others, such as food deceptive orchids, appear to be far more

opportunistic. Pollination niche breadth and overlap may differ between pollination

tactics and maybe biogeographical zones. Pollinators of several hundreds of orchids

have been documented in detail in several places, particularly in the Europe -

Mediterranean region, where orchid–pollinator interactions have been widely

recorded (Claessens and Kleynen 2016).

266 K. Balilashaki et al.

The morphological structure of the orchid ﬂower is an obstacle to easy fertiliza-

tion and consequently, the pollen lumps can’t be carried by the wind, either.

Although the birds play a role in the pollination of some species, insects are the

most common pollinators in nature. Identifyi ng orchid ﬂowering periods and polli-

nation biology will aid in the creation of capsules, ensuring a dependable and

sufﬁcient seed source for future trials. This data is critical for determining the best

harvesting period for high-yielding seed germ ination. Pollen and the age of the

ﬂower are both important factors in pollination success (Indan et al. 2021). Reward-

based generalized food deception, pollination, autono mous self-pollination, and

Batesian food-source mimicry are the four known ways of pollination in Cymbidium

orchids (Indan et al. 2021).

4.1 Capsule Development

In orchids, pollination mechanisms are highly specialized, and many species have

species-speciﬁc pollination systems. Specialization, unfortunately, makes species

increasingly reliant on and vulnerable to the absence of mutualism partners. The

investigation of plant–pollinator interactions is important with considering

attempting to maintain self-sustaining populations in the wild over the long term.

Pollination during the early ﬂowering phase is recommended because pollen is most

receptive between the ﬁrst and seventh days after blooming, increasing the likeli-

hood of capsule development. Using immature ﬂowers that are less than a week old

ensures that the stigmatic surface is open to pollen. Flowers close after 2 weeks, and

pollen becomes brown and unrespo nsive. Hand-pollination technique increased the

success of capsule productions as well because the time between pollination and

subsequent developmental events in embryos differs enormously in both genus and

species, and the time it takes for an orchid capsule to achieve complete maturity

differs by species (Utami and Hariyanto 2019). Winter was shown to be the best

season for pollinating Phalaenopsis hybrids, resulting in an 80–88% capsule forma-

tion rate. The germination effectiveness of seeds taken from capsules of varying

maturity levels was further reduced by the pollination season. Seeds recovered from

winter pollinated capsules consistently ou tperformed seeds gathered from other

seasons in terms of germination (Balilashaki et al. 2015). In Phalaenopsis hybrids,

it was founded that seeds derived from 5-month mature capsules needed the least

amount of time to germinate than seeds derived from 3 months or 7-month mature

capsules (Balilashaki et al. 2015).

Progress and Prospect of Orchid Breeding: An Overview 267

4.2 Seed Germination

The ultimate competent system of orchid breeding is seed propagation (Shekarriz

et al. 2014). Isolation of compatible mycorrhizal fungi is required for symbiotic seed

germination. Asymbiotic seed germination, on the other han d, does not necessitate

the isolation of mycorrhizal fungi. Moreover, asymbiotic seed germination is a

comparatively simple and effective procedure. Nonetheless, there are still some

situations where symbiotic germination seedlings are preferable. If there is a prob-

ability that symbiotic seedlings will develop more quickly than asymbiotic seed-

lings, symbiotic seed germination may become the favored method for producing

orchids (Deng 1990). Orchid seeds are incredibly tiny which may be 0.1 mm

(in Oberonia) to the highest 6 mm (in Epidendrum) in size is the world’s smallest

seed. There is a nearly 400-year gap between the ﬁrst sighting of orchid seeds and

Knudson’s successful asymbiotic germination in 1921. Since then, orchid hybridi-

zation has been used for propagation and breeding all over the world. Initial growers’

hybrids, on the other hand, could not have predicted their success (Yam and Arditti

2009). Modern hybrid seedlings are usually created by crossing two superior

parental cultivars to improve and reﬁne morphological and reproductive character-

istics as well as disease resistance (Tang and Chen 2007). Typical orchid hybrids

could serve as role models for reintroduction projects, which could help endangered

and threatened species. The most important stage in this process is to ﬁgure out

effective asymbiotic germination techniques to create seedlings for further research.

There is little data on seed germination in Phalaenopsis species, and none on

asymbiotic or symbiotic seed germination parameters. The type of basal medium

and capsule age was found to affect ability (seed maturity) (Indan et al. 2021).

5 Hybridization

Crossing orchids to other species is one option for preventing genetic extinction.

Hybridization has the effect of combining the best qualities of both parents in the

hybrid offspring. In assessing the effectiveness of a hybridization procedure,

selecting a parent with high compatibility to be crossed is critical, one major block

to the successful crossing is that the crossed parents should have close genetic

closeness and in assessing the effectiveness of a crossbreeding effort (Hartati et al.

2019). Even though crossbreeding is a straightforward and effective method for

cultivating orchid hybrids, there are various things to consider when doing so,

including the hybrid combination’s fertility, qualitative analysis of goal features,

and the selection of superior hybrid offspring (Reinikka 1972). F1 progenies formed

from two parents with opposing goal features typically show signiﬁcant phenotypic

differences. However, it has been noted that in the case of Cymbidium, hybrid seeds,

particularly those of distant hybrids, are difﬁcult to cultivate because of their distant

genetic link, with the degree of difﬁculty rising in the order intraspeciﬁc intrageneric/

intergeneric. Failure of distant hybridization is caused by parents’ incompatibility

and postfertilization embryo abortion (Li et al. 2021).

268 K. Balilashaki et al.

Since the beginning of orchid collection and cultivation, natural hybrids resulting

from crossbreeding between species have been observed in the wild. Phalaenopsis

intermedia is the oldest hybrid that produces by a cross between P. aphrodite and

P. rosea (De and Bhattacharjee 2011).

5.1 Artiﬁcial Hybridization

Orchid developers around the world have experimented with many species and

hybrids, with variable degrees of success. Orchid hybrids are the progeny of a

cross between two genetically different individuals. In this group of plants, intra-

speciﬁc, intrageneric, and intergeneric hybrids have been developed. In orchids,

intergeneric crossings are very common, and many hybrids involving two, three,

four, and ﬁve genera have been registered and listed (De and Bhattacharjee 2011).

Although free breeding is prevalent in orchids, it is not possible to make hybrids

between any two genera. The majority of orchid breeding success has been attributed

to the art of paint breeding, orchid breeders’ intuition and tenacity, and, on a few

times, pure luck. Raising progeny from seed to ﬂowering stage takes several years.

Orchid seeds, unlike those of other crops, require speciﬁc care to germinate and the

maturation of seeds takes a long time. Furthermore, the number of seeds produced in

a capsule is so large that obtaining a representative sample of the progeny is

impossible. As a result, information on the ability to combine characteristics and

their inheritance in orchids is limited (De and Bhattacharjee 2011).

6 Breeding Methods

6.1 Crossbreeding

Crossbreeding is one of the most common and effective methods in orchid breeding.

Orchid interspeciﬁc and even some intergeneric crossing is easy to succeed, but

because orchid seeds do not have endosperm and organized embryo, they need to

rely on symbiotic fungi to germinate in nature. Early hybrid breeding is very difﬁcult

to obtain hybrid progenies. In 1854, RHS founded the international login system for

orchid hybrids. The ﬁrst orchid hybrid to be logged in is Calanthe dominyi (C.

furcate C. masuca). Knudson found that sugar can replace fungi to promote seed

germination, and established the technique of in vitro propagation of any plant in

pure (that is, aseptic) culture (Knudson 1922). The registration of new orchid hybrids

showed explosive growth. Several factors must be considered when performing

crossbreeding, these factors are fertility of the hybrid combination, qualitative

analysis of target traits, and the selection of superior hybrid offspring (Reinikka

1972). The F1 progenies derived from two parents with contrasting target traits (such

as a parent with large ﬂowers in size but short ﬂowering time and the other with long

ﬂowering time but small ﬂowers in size) usually exhibit large phenotypic differences

(Zhang et al. 2011). Recently embryo rescue technique has shown the light of hope

to regenerate distant hybrids effect ively. By this technique, immature embryos are

cultured in vitro and controlled embryo abortion (Luo et al. 2012).

Progress and Prospect of Orchid Breeding: An Overview 269

6.2 Ploidy Breeding

Orchids are prone to 2n gametes during meiotic, which leads to polyploidy of hybrid

progenies. Polysomaty and polyploidy are common occurrences among orchids,

represent a powerful force for evolution, and have been found in several important

genera: Paphiopedilum, Coelogyne, Cymbidium, Dendrobium, Calanthe, Oncidium,

Paphiopedilum, Vanilla, Vanda, etc. (Hossain et al. 2013). At present, the offspring

sterility produced by interspecies crossing with different chromosome sizes or ploidy

levels is one of the problems encountered by Phalaenopsis breeder s. Therefore, the

occurrence of endopolyploidy in Phalaenopsis was studied, and a simple and

effective technique was developed to determine the nuclear DNA content and double

the number of chromosomes. In addition, ﬂow cytometry has been used for endo-

polyploidy in different tissues of Phalaenopsis species. It was found that different

patterns of endopolyploidy occurred in different tissues of Phalaenopsis species at

various stages of development. According to these results, a simple and effective

protocol was developed for the production of polyploid plants by sectioning

protocorms or protocorm-like bodies (PLBs) without using anti-microtubule agents

(Chen et al. 2011). Through this technique, a series of tetraploid species of Phalae-

nopsis were developed. For example, Phal. Doris and Phal. Zada, the super parents

of Phalaenopsis breeding, are tetraploid hybrids. Among the registered Phalaenop-

sis hybrids, 90.2% have Phal. Doris lineage and 43.5% have Phal. Zada lineage.

6.3 Selection Breeding

Breeding according to selection uses the natural diversity of genotypes as the

original material for selection. After selection breeding, three important genetic

parameters must be considered: heritability, genetic correlations between traits,

and interactions of genotypes environment. The three parameters should be used

to deal with the relationship between heritability, variation, and selection given that

plant phenotypes are determined by the environment as well as by the genetic

material (Murthy et al. 2018).

270 K. Balilashaki et al.

6.4 Molecular Marker-Assisted Breeding (MMAB)

In addition, MMAB was also carried out. The application of MMAB technologies

for practical breeding and selection has several advantages as it is fast, accurate, and

free from the in ﬂuence of environmental conditions (Jiang 2015). MMAB is to

reduce linkage liability, aggregate favorable genes, speed up the breeding process,

and improve breeding efﬁciency by using molecular marker analysis closely linked

to the target gene. Among the different molecular markers, scientists and breeders

have given prevalence to RFLP, AFLP, SNP, SSR. The genetic relationship among

81 selected Dendrobium species and hybrids was studied. AFLP markers could be

used to determine the variation between materials. This study provided useful

information on the genetic diversity of some Dendrobium orchids and will likewise

be useful for monitoring the germplasm, developing new hybrids, and protecting

new plant varieties. Molecular marker-assisted breeding has been widely used in

crop breeding, but there are few reports in orchid breeding, and there is still a lack of

molecular markers closely linked to the target traits. The breeding and application of

functional genes have become a hot resear ch direction in orchid plants in recent

years. Professor Yu Hao of the National University of Singapore has successfully

established a genetic transformation system for Dendrobium, which is expected to

carry out molecular breeding and quality-oriented improvement (Sawettalake et al.

2017; Chai and Yu 2007). Recently, the point mutants of C3H and C4H genes of

Dendrobium candidum, and the mutants of MADS44, MADS36, and MADS8 of

Phalaenopsis were successfully obtained by using the CRISPR/Cas9 gene-editing

system (Tong et al. 2020).

6.5 Transgenic Breeding

To improve the important characteristics of orchids, such as new ﬂower color,

fragrance and shape, ﬂowering control, abiotic stress tolerance, disease, and pest

resistance, transgenic technology has been applied to orchids. It is often difﬁcult to

introduce new traits into orchids through mutation or conventional breeding, but

genetic transformation can be relatively easy to achieve (Nirmala et al. 2006). The

success of orchid genetic engineering, like other plants, depends on the totipotency

of plant cells, that is, the inherent ability of plant tissues to produce cells that can

regenerate fully dynamic plants (Hossain et al. 2013).

Although the ﬂowering time, ﬂower fragrance, and color of orchids can be

controlled by genetic transformation, it remains to be determined whether the

ﬂowering period is prolonged or not. Moreover, the disastrous impact of viral

diseases on the yield and quality of orchids remains a major concern for orchid

breeders and producers.

Progress and Prospect of Orchid Breeding: An Overview 271

6.6 Conventional Breeding

Flower color, shape, and smell are the main unique identiﬁers for orchids since they

are the main determinatives of customer choice. Conventional breeding techniques,

though, have resulted in the loss of perfume in many new ﬂoricultural cultivars. Cut

ﬂower and decorative orchid breeders have concentrated on generating plant s with

enhanced vase life, transportation qualities, and overall aesthetic attributes (like

color and shape). Phalaenopsis orchids have 2–3 years of growth courses. Using

conventional hybridization to transfer beneﬁcial characteristics into commercial

cultivars is a lengthy and time-consuming procedure that will require years to

complete. Furthermore, intraspeciﬁc and/or interspeciﬁc incompatibility hampers

variety enhancement work. All ﬁve Phalaenopsis subgenus has identical chromo-

some numbers (2n ¼ 2x ¼ 38), which may be classiﬁed into small, medium, and

large chromosomal groupings based on chromosome sizes and nuclear DNA content

(Chen et al. 2011). Species with short chromosomes, including P. amabilis,

P. aphrodite, and P. equestris, are the source of the majority of commercial cultivars.

P. amboinensis and P. violacea are among the species with big chromosomes and

powerful scents. Because of interspeciﬁc incompatibility, productive crosses among

species with tiny and big chromosomes are challenging. Seed germination, as an

important component of traditional breeding, is directly relevant to the performance

and efﬁciency of crossbreeding. To develop an effective germination mechanism,

in-depth research into the developmental features and germination processes of

distant hybrid seeds is very important. Because once hybrid seeds are gained, an

appropriate cultivation strategy is required to maintain the population constant or

expand it. Because orchid grains are hard to replicate in the natural environment,

in vitro propagation system is the most signiﬁcant breeding procedure for orchids.

Seed maturation, culture situations, and culture medium are all important elements of

in vitro propagation success. Many orchid species have been studied in vitro, such as

those of the genera Cymbidium, Phalaenopsis, Dendrobium, Oncidium,

Dactylorhiza, and Calanthe (Bezerra et al. 2020). At the moment, the major goals

of in vitro propagation are the production of genotype variation and reducing the

breeding course, and substantial progress has been achieved toward these goals.

Adaptation of bioreactor system in micropropagation has opened a new era in plant

propagation.

6.7 Breeding Via Mutation

Mutation breeding is suited for breeding ornamental plants because species can be

easily reproduced, simplifying the generation of spontaneous and induced mutants

(Yamaguchi 2018). Mutation provides several beneﬁts, such as a high mutation rate,

the disruption of trait relationships, efﬁcient enhancement of individual characteris-

tics, and the reduction of the breeding course (Li et al. 2021). Over time, this kind of

breeding has already been utilized to create orchids with distinct phenotypic char-

acteristics, increased medicinal component concentration, and improved adaptation

and tolerance (De et al. 2014). Polyploidization is a usual technique of mutation

breeding. Most orchid species, such as Cymbidium (Wang et al. 2011), Dendrobium

(Zhang et al. 2011), Oncidium (Cui-Cui et al. 2010), and Phalaenopsis (Chang et al.

2019), have been successful through polyploid breeding. Orchids’ high heterozy-

gosity can boost the apparent mutation rate and result in a slew of superior mutation

kinds in a short time. Unexpected mutations can occur, resulting in harmful muta-

tions, but in most cases, only single alterations are acquired (Reinikka 1972).

Furthermore, the success of mutation breeding is determined by parameters includ-

ing explant type, genotype, induced mutation technique, and optimal dose for each

mutagenic treatment.

272 K. Balilashaki et al.

6.8 Hybrid Breeding

Due to the intrinsic beauty of ﬂowers and the capacity to transfer these characteristics

to hybrids, several species have gained worldwide attention in breeding programs.

Several species are important, such as Cymbidium devonianum, C. lowianum,

C. tracyanum, C. elegans, and others (Tiwari et al. 2022).

6.9 Molecular Breeding

A research project aimed at creating a solid approach for orchid molecular breeding

utilizing the CRISPR/Cas9 knockout technology. Phalaenopsis amabilis

protocorms cultured on New Phalaenopsis medium supplemented with peptone

were utilized as the plant materials. Ti plasmids had been ﬁlled with T-DNA

construct of pRGEB32 vector carrying PDS3 sequence, and protocorm was

immersed in the Agrobacterium tumefaciens. Transformants were detected and

veriﬁed. From PDS3T2 lines, 0.96% of PDS transformants were produced. Several

transformants have paler leaves than non-transformants. The CRISPR/Cas9 system

appears to have effectively altered the target gene in orchids, indicating that it might

be used for practical gene editing in orchids (Semiarti et al. 2020). The study

determined that the Agrobacterium tumefaciens-mediated transformation method

might be used to deliver CRISPR/Cas9 to a Dendrobium macrophyllum orchid

protocorm. The T-DNA Ubi::: Cas9:: VAR2/prGEB32, and afterward the protocorm

were cultivated for 4 weeks in the Vacin and Went culture medium +6 mg/L

hygromycin antibiotics for transformants, the A. tumefaciens strain EHA 105 was

infected.

Researchers found that transformation efﬁciency was maximum (0.66%) during

the 15-min infection phase, but it dropped to 0.43% and 0.23% after 30 and 45 min.

Cas9 (402 bp), HPT (545 bp), VAR2 (723 bp), the D. macrophyllum genome, and

TrnL-F (1200 bp) were ampliﬁed. When examining the sequenc e, a substitution

mutation was observed at the target site (Setiawati et al. 2020). MMAB offers the

advantages of fast, accurate, independent of environmentally friendly settings using

molecular biotechnologies for practical breeding and breeding (Jiang 2015). As a

result of their frequency and potential, the following are the most relevant molecular

markers: RFLP AFLP, SSR, and SNP. The ﬁrst three were broadly applied with

great success for orchid reprod uction (Li et al. 2015), established a set of markers

(Gen-SSR) for the genetic connections, and the cartooning investigations of other

orchid species at Cymbidium ensifolium. When combined with functional annota-

tions of unigenes, these marker types assist to recognize candidate genes with unique

environmental roles. The sequencing of Paphiopedilum concolor root transcriptome

in a simple sequence of repeats provides critical insight into the mechanisms of the

growth and development of the roots (Li et al. 2015). The genes linked with ﬂower

color, ﬂoral shape, and resistance in Phalaenopsis, which were utilized by Chung

et al. (2017)to ﬁnd out, was a major reference point in genetic engine ering generally

for the Phalaenopsis and Orchidaceae (Chung et al. 2017). The efﬁcacy of ﬂower

color forecast for several Phalaenopsis species was tested by applying gene-speciﬁc

single-nucleotide ampliﬁed polymorphism markers to facilitate the reproduction of a

novel Phalaenopsis variety. The Phalaenopsis aphrodite genome was conﬁrmed and

integrate with an SNP-based genetic link and optical map. This has developed a

unique asset to not only increase the reproductive performanc e of horticultural

orchids but also attributed to major studies of epiphyte genomic adaptation for future

reference (Chao et al. 2018). The ﬁrst SNP integrated high-density map with large

coverage in the genome of Dendrobium was published by Lu et al. (2018). Many

QTL sites laid the basis to map more features of medicinal relevance for the future.

When it comes to gene-mining and genome studies, Bletilla striata’s EST-SSR

transcriptome has provided a solid foundation for phylogenetic and operational

gene-mining studies (Xu et al. 2019). Researchers have laid the groundwork for

ﬁne-tuning the expression quantitative trait locus (eQTL) mapping of Dendrobium

(D. nobile, D. wardianum) by RNA sequencing, eQTL analysis, and development of

high-density genetic maps (Li and Chan 2018). Wang et al. (2019) examined in vitro

the ﬂuctuation of SNP and insertion-deletion frequencies in Oncidium “Milliongold”

somaclones that had been regenerated by protocorm-like bodies (PLBs) (Wang et al.

2019). Most species lacking refere nce genome sequences might beneﬁt from SLAF-

seq, according to the study’s ﬁndings. Molecular marker technology has been

extensively applied to the study of orchid phylogeny and genetic relationships, but

only a few studies have combined molecular marker technologies with phenotypic

characteristics. Another technology (Genome-Wide Association Research) has also

been used in studies on cabbage, tomato, and tea (Deng 1990;

Xing

etal. 2019; Fei

et al. 2020), but in orchids only to a modest extent. As a result, greater study on these

characteristics is needed to offer more precise genetic data for orchid breeding.

Camellia is a genus of ﬂowering plants in the Theaceae family, and many of its

species are economically valuable. A large number of single sequence repeats

(SSRs) in the Camellia genus have been produced in the last decade, yet there are

not enough SSRs available to the public in this genus. During the investigation, a

Progress and Prospect of Orchid Breeding: An Overview 273

total of 4,63 kb of data was collected, including 28,854 putative SSRs. They

synthesized and initially screened 172 primary pairs of 10 C. japonica accessions

and found that 111 polymorphic accessions matched those depending on taxonomy

and regional categorization. Additionally, 51 polymorphic SSR markers have been

randomly selected for future genetic interactions of 89 accessions in the Camellia

region. Each C. japonica genotype was signiﬁcantly split and grouped, as demon-

strated by the genetic structure study’s clustering algorithms. For the molecular

genetic reproduction of camellias, the results give high-quality SSR resources

(Li et al. 2021). Genomic diversity was found to be widespread among the species

(PPB: 90.1%; HE: 0.3414; H: 0.5013). Despite this, genetic diversity within groups

was limited. With PPB: 76.2%; HE: 0.2966; H ¼ 0.4319, Shiko-2 was the most

variable, whereas XS was the least variable (PPB: 67.3%; HE: 0.2344; H: 0.3478).

Nei’s gene diversity statistics, Shannon’s information measure, and AMOVA (anal-

ysis of molecular variance) with 21.3%, 21.4%, and 22.5%, respectively, indicated a

very high degree of genetic differentiation among populations. The genetic and

geographic distances were shown to be signiﬁcantly related (r ¼ 0.8154, P 0.05)

(Deng 1990). An important part of developing microsatellite loci to improve com-

mercial moth orchid breeding is molecular identiﬁcation (Phalaenopsis species).

There are Microsatellite Primer Sets for the Phalaenopsis aphrodite subspecies,

which include genomically-SSR and EST-SSR primers. To better understand Phal-

aenopsis transferability, P. aphrodite subsp. formosana will be utilized. Magnetite

beads and NGS (next-generation sequencing) collected 10 or 28 polymorphic

EST-SSRs and gSSR (genomic-SSR) markers that indicate 21 Phalaenopsis species,

including several subgenus Phalaenopsis with strong transferability. They found

that these microsatellite markers differed from those found in the Phalaenopsis

subgenus. The genetic connections among species of the Phalaenopsis subgenus

may therefore be isolated and integrated. They can help to identify parentages of

Phalaenopsis and to investigate the hybridization of Phalaenopsis (Bolaños-

Villegas et al. 2021).

274 K. Balilashaki et al.

6.10 Gene Transfer Breeding

Mutation breeding and crossbreeding are generally challenging methods for intro-

ducing new traits, like new colors or disease resistance, into orchids, but transgenic

technology makes it possible (Nirmala et al. 2006). It is most usual to utilize

Agrobacterium-mediated and microprojectile techniques to breed orchids (Fig. 1).

Dendrobium (Kuehnle and Sugii 1992) were the ﬁrst orchids to undergo successful

transformations by particle bombardment. Efﬁcient transformation methods have

been devised for certain major commercial orchids, such as Phalaenopsis (Tong

et al. 2020), Vanda (Shrestha et al. 2007), Cymbidium (Chin et al. 2007),

Dendrobium (Chen et al. 2018), Cattleya (Zhang et al. 2010), Erycina pusilla

(Li and Chan 2018), etc.

Progress and Prospect of Orchid Breeding: An Overview 275

Fig. 1 Gene transfer methods to breed Phalaenopsis orchids are based on Agrobacterium-mediated

and particle bombardment techniques and their transformation pathways to introduce superior traits

in orchids species

By using particle bombardment, Yang et al. (1999) successfully transferred a

plasmid containing GUS and NPTII markers to orchids to create kanamycin-resistant

transgenic plants (Yang et al. 1999). Scent-related genes wer e identiﬁed by using

RAPD molecular markers. Transgenic Cymbidium plants were created when NPTII,

the plasmid containing the GUS marker gene, was introduced from Agrobacterium

to Cymbidium (Chin et al. 2007). As Chai and Yu (2007) review, transgenics have

become a key means for creating new genotypes of orchids and have resulted in

important progress in ﬂora, plant architecture, and biotic and abiotic resistance.

Agrobacterium tumefaciens has created the Phalaenopsis protocorm as a vector

expressive receptor material and pCAMBIA1301 (containing the GUS report gene

and the hygromycin resistant gene hpt) (Chai and Yu 2007). Researchers used the

pollen tube route as well as the ovary injection methods for the transmission of the

cbf1 resistant gene into Phalaenopsis. On Fd and OnFNR have shown substantial

impacts on soft rot and both genes can play an important role in the resistance to

Oncidium soft rot (Tong et al. 2020).

The transformation of the Oncidium PLBs via Agrobacterium-mediated transfor-

mation for temporary expression was carried to PR1 (an important downstream gene

of acquired plant resistance). Plants that were transformed became stronger (Gao

et al. 2020). In A. thaliana, the introduction of Dendrobium Chao Praya Smile

DOAP1 led to early ﬂowering as well as early termination of inﬂorescence meristem

into ﬂower meristems [4; 61].

276 K. Balilashaki et al.

Use of protocorms produced from seeds of Phalaenopsis aphrodite and Phalae-

nopsis cultivars as an alternate transformation method. eGFP was driven by

ubiquitin promoter in the T-DNA vector construct utilized for trans formation.

Hygromycin was used to select the altered protocorms, which were then effectively

regenerated. BC1 progeny demonstrated resistance to hygromycin when

backcrossed to the transgenic line, proving the transgene is heritable. It has been

shown that all backcross F1 explants that survived were positive transformants

utilizing PCR and western blot analysis (Hsing et al. 2016). With the use of particle

bombardment, Agrobacterium-based transformation systems and direct gene trans-

formation procedures for genetically modiﬁed Oncidium orchids have been devel-

oped (Li et al. 2015; You et al. 2003). When it comes to transforming Oncidium with

ferredoxin resistant to soft rot disease (You et al. 2003), for example, the following

methods are described: using the Agrobacterium system, using particle bombard-

ment to suppress the ﬂower color gene (Yee et al. 2008), and using the same

Agrobacterium system to alter the ethylene receptor gene (Raffeiner et al. 2009).

The processing of Oncidium has grown even more complicated by adding the

phosphomannose isomerase gene to the Agrobacterium-mediated transformation

system. Because of their hygromycin sensitivity and long-term regeneration, Oncid-

ium species have had a restricted number of genetic transformation studies. GFP

(Green Fluorescent Protein), phosphotransferase hygromycin (hptII), and CymMV-

CP genes were introduced into the protocorm-like bodies o f Oncidium orchids

Oncidium Gower Ramsey and Oncidium Sweet Sugar (PLBs) using a direct gene

transformation approach. Many transgenic Oncidium orchids were investigated in a

genetic study to conﬁrm the inheritance of transgenes.

It was possible to effectively transfer the AcF3H gene from Ascocenda ﬂavanone

3-hydroxylase (AcF3H) to Dendrobium 5 N white orchid plants utilizing

Agrobacterium-mediated gene transformation. A plant expression vector with the

AcF3H gene was built in the gateway cloning method. A. tumefaciens AGL1, which

carried the plant expression vector pGWB5-AcF3H, was co-constructed as a selec-

tive marker. The agroinﬁltration method was employed to temporarily express

acF3H in white Dendrobium 5 N and Anna petals Dendrobium and the ﬁndings

revealed that, according to the study, no cyanidine concentration was detected for

white petals Dendrobium 5 N after acF3H inﬁltration. On the other hand, the content

of Dendrobium Anna petals was 6% higher than cyanidin showing that AcF3H was

transitory (Khumkarjorn et al. 2017).

Tetraploid or diploid Phalaenopsis orchids have been explored with the transfers

of Agrobacterium-mediated genes wi th a construct of T-DNA vector which contains

the eGFP powered by the ubiquitin promoter. A hybrid between the pollinia of the

transgenic plants and four separate Phalaenopsis orchid varieties revealed

hygromycin and hptII positivity in PCR and GFP protein production demonstrated

by Western blotting (Hsing et al. 2016). The AcF3H (Ascocenda Flavanone

3-hydroxylases) gene has been successfully transformed into white orchid plants

of Dendrobium 5 N utilizing Agrobacterium transformation genes. An expression

medium for the AcF3H gene was produced for the ﬁrst time utilizing gateway

cloning. For the hpt gene, the protocol-like corpus (PLBs), the A. tumefaciens line

AGL1, and the PGWB5-AcF3H vector of plant expression were co-cultivated. The

highest transformation efﬁcacy was therefore obtained by cultivating PLBs with

Agrobacterium cells in 15 min (10.13%). To verify the transgenic plants, the

seedlings were rebuilt 3 months after the transformation and PCR analysis was

performed, the hpt gene and the 35S promoter region were targeted using particular

primers. Transgenic crops had about 400 and 500 bp PCR products that matched the

gene of hpt and the 35S promoter, respectively, but no non-transgenic crops,

indicating that the AcF3H gene was present in a white orchid genome. AcF3H

was temporarily expressed using agroinﬁltration procedures in the white and the

Dendrobium Anna petals of Dendrobium 5 N and discovered in the white petals of

Dendrobium 5 N after AcF3H that wasn’t cyanidin content in the sample. The

cyanidin concentration of Dendrobium Anna petals, on the other hand, rose by

around 6%, indicating temporary expression of the AcF3H gene. When PLBs

were co-cultivated with A. tumefaciens AGL1, which maintains pGWB5-AcF3h

for 15 min, the highest transformation efﬁciency (10.13%) was attained. The wild

type and mutant libraries were completely clean reading 98,988,774 and

100,188,534 bp and De Novo, constructed at 98,446 uniqueness, accordingly for

an average length of 989 bp. When transcription proﬁles were compared between the

two libraries, 18.489 were discovered to be differently expressed.

Progress and Prospect of Orchid Breeding: An Overview 277

Most of the Kyoto encyclopedia for the enrichment of genes and genomes was

used in membrane-building and ploidy-related activities, consisting of increased

ﬂowering and changed cell sizes seen in the mutant. 29 MADS-box genes were

identiﬁed as possibilities for the ﬂoral patterning of C. goeringii, as well as severa l

ﬂoral and hormone-affecting regulators and genes. A short RNE sequence revealed

that 132 miRNA families produced in C. goeringii ﬂowers were conserved, and the

multiple-tepal formation has been caused by 11 microRNAs related to 455 target

genes. The combined study of mRNA and microRNA showed two transcription/

microRNA pathways that contribute to multi-tepal characterization (Fig. 2): a

popular ﬂoral related miR156/SPL and a miR167/ARF regulations technique for

developing reproductive organs; and a multi-tepal cell-proliferation regulations

cascade that likely regulates the miR319/TCP4–miR396/GRF regulation

(Cheamuangphan et al. 2013).

Cymbidium faberi has a distinctive ﬂoral smell which boosts its commercial

worth, one of the most renowned oriental orchids. However, until this study the

molecular process of ﬂoral fragrance production was unclear. Methyl jasmonate

(MeJA) is one of C. faberi’s major organic volatile compounds (VOCs). 79,363

unigenes were selected for further examination using comparative transcriptome

analysis. 9409 genes (GDEs) of which 558 were assigned to 258 pathways led to a

transcriptome study of blooming and withered C. faberi ﬂowers (Xu et al. 2019).

The top 10 strategies for achieving a conversion of alpha-linolenic acid to MeJA

included the metabolism of α-linolenic acid, pyruvate metabolism, and fatty acid

degradation. In one of its DEG Jasmonic Carboxylic Acid Methyl Transferases

(CfJMT, unigene 79,363), ﬂora blooming C. faberi is expressed extensively but

seldom detected in the roots or leaves. While CfJMT synthesis in tomatoes did not

raise MeJA levels, the expression of internal MeJA genes, particularly for the

treatment of injuries, has changed, indicating that CfJMT may be connected with

abiological stress in the tomato. The molecular pathways for ﬂoral fragrance gener-

ation in C. faberi have been explored as part of a study that will aid in the genetic

modiﬁcation of modern varieties of commercially valuable oriental orchids (Xu et al.

2019).

278 K. Balilashaki et al.

Fig. 2 A system of miRNA/transcription factors inﬂuences the multi-tepal characteristics of

C. goeringii

The EHA105 A. tumefaciens strain, which possesses a binary plasmid, has been

shown to successfully process Erycina pusilla plants. The promoter for the plasmid

should be CaMV 35S series (CaMV 35S) (Lee et al. 2015). The hygromycin-

containing medium can be used to select explants with 6-benzylaminopurine and

naphthaleneacetic acid modiﬁcations. According to research, protocorm-like (PLBs)

at 3 months of age is the best stage for transformation. Self-pollination allowed T1

progenies to be obtained in the 18-month MV 35S series (Li and Chan 2018; Lee

et al. 2015). To stimulate protocorm development and multiplication, the self-

pollinated seed capsules of Erycina pusilla are broken under aseptic conditions

and a sterile half-strength MS medium was used to germinate seedlings in plastic

plates (Lapjit and Tseng 2015). Upon germination, the protocorms and greens

should have a diameter of 1 cm. It’s time to get back to the basics. CRISPR/Cas9

might be used to change MA DS-box genes and alter ﬂoral morphology in Erycina.

Agrobacterium-mediated RNA interference has been investigated in the past, but

with little success (Lin et al. 2016). E. pusilla has been crossed with several

important Oncidiinae orchids to produce new commercial orchid species. The

clone PSYP1 as E. pusilla “Hsingda Golden” derived from in vitro ﬂowering system

has been granted the Plant Variety Rights in Taiwan for protection (Bolaños-

Villegas et al. 2021).

Progress and Prospect of Orchid Breeding: An Overview 279

7 Prospects for Orchid Breeding

There seems to be unevenness in science-based study and practice in the application

of forwarding genetics since laborers select effective point mutations (Hall and

Richards 2013) but do not recognize how to use them, so research labs are restricted

by the lack of viable mutants to investigate. Reverse genetics, as opposed to

forwarding genetics, investigates phenotypic changes in genetically inherited mod-

iﬁcations using huge amounts of information. Omic information from orchids, which

includes genomic, proteomic, transcriptome, and metabolome sequence analysis,

has been getting more and more a direct consequence of developments in limited

sequence alignment techniques. The above ﬁndings will serve as guidelines for

genetically modiﬁed breeding and genome engineering breeding programs, laying

the groundwork for orchid breeding programs. Moreover, among the most apparent

disadvantages of reverse genetics is that it can reveal a large number of genes linked

to favorable characteristics, making it more difﬁcult to constrict the objective gene

array. To resolve this ambiguity, it is necessary to combine genomic and other omic

data, as well as breeding and morphologic records. Integrating, acquiring knowl-

edge, and investigating will aid in the discovery of gene functions linked to essential

qualities (Langridge and Fleury 2011). Besides conquering conﬂict and infertility,

mutagenesis breeding could be used to gain large differences in ﬂower color,

anatomy, and shape. As a result, merging hybridization and mutation breeding will

be a viable tactic for recognizing hybridization’s maximum capabilities throughout

orchid rearing. Attribution of speciﬁc genes is a prevent ion effort for breeding

programs, but there is presently no reliable transition servic e for orchids. Transfor-

mation is currently accomplished primarily through Agrobacterium-mediated pro-

cesses, particle bombardment, and gene silencing. Besides this, even though plants

from essential ornate species of the genus like Phalaenopsis and Dendrobium have

already undertaken genome editing, general performance is lower. As a result, it is

still important to broaden studies on CRISPR/Cas9 to support access to key orchid

phenotypes. Furthermore, while there is reportedly very little molecular genetic

information for orchids, more transcriptomic evidence has become accessible,

which will aid in the exploration of essential qualities including ﬂower color, ﬂoral

morphological characteristics, and ﬂower aromas. As a consequence, molecular

breeding is expected to become the primary method for orchid breeding. To

summarize, for certain, if researchers investigate important characteristic genetic

traits utilizing forward or reverse genetics, or if we use conventional breeding,

natural selection, or single-molecule breeding to produce great progeny to achieve

desired attributes, every method has beneﬁts and drawbacks, and if used individu-

ally, it seems to be unusual to advance reproduction. Thus, a variety of techniques

and research directions must be incorporated to enable the production of orchids

with different ﬂower sizes and morphology, new colors, and complex ﬂower

fragrances.

280 K. Balilashaki et al.

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