Frozen beauty: The cryobiotechnology of orchid diversity

Elena Popova, Haeng Hoon Kim, Praveen Kumar Saxena, Florent En-
gelmann, Hugh W. Pritchard

PII: S0734-9750(16)30001-5
DOI: doi: 10.1016/j.biotechadv.2016.01.001
Reference: JBA 7011

To appear in: Biotechnology Advances

Received date: 16 July 2015

Revised date: 4 January 2016
Accepted date: 8 January 2016

Please cite this article as: Popova Elena, Kim Haeng Hoon, Saxena Praveen Kumar,
Engelmann Florent, Pritchard Hugh W., Frozen beauty: The cryobiotechnology of orchid
diversity, Biotechnology Advances (2016), doi: 10.1016/j.biotechadv.2016.01.001

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FROZEN BEAUTY: THE CRYOBIOTECHNOLOGY OF ORCHID

DIVERSITY
Elena Popova1*, Haeng Hoon Kim2, Praveen Kumar Saxena1, Florent Engelmann3*, **
and Hugh W. Pritchard4*,**

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1
Gosling Research Institute for Plant Preservation, Department of Plant Agriculture,
University of Guelph, Ontario N1G 2W1, Canada.
2
Department of Well-being Resources, Sunchon National University, 225 Jungang-ro,

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Suncheon, Jeonnam 540-742, Korea
3
Institut de Recherche pour le Développement (IRD), UMR DIADE, BP 64501, 34394
Montpellier cedex 5, France

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4
Royal Botanic Gardens, Kew, Wellcome Trust Millennium Building, Wakehurst Place,
Ardingly, West Sussex RH17 6TN, U.K.

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*Corresponding authors’ E-mails: epopova@uoguelph.ca ; florent.engelmann@ird.fr ;
h.pritchard@kew.org
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**: both authors contributed equally to this work.

Abstract
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Orchids (Orchidaceae) are one of the most diverse plant groups on the planet with over
25,000 species. For over a century, scientists and horticulturalists have been fascinated by
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their complex floral morphology, pollinator specificity and multiple ethnobotanical uses,
including as food, flavourings, medicines, ornaments, and perfumes. These important traits
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have stimulated world-wide collection of orchid species, often for the commercial
production of hybrids and leading to frequent overexploitation. Increasing human activities
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and global environmental changes are also accelerating the threat of orchid extinction in
their natural habitats. In order to improve gene conservation strategies for these unique
species, innovative developments of cryopreservation methodologies are urgently needed
based on an appreciation of low temperature (cryo) stress tolerance, the stimulation of
recovery growth of plant tissues in vitro and on the ‘omics’ characterization of the targeted
cell system (biotechnology). The successful development and application of such
cryobiotechnology now extends to nearly 100 species and commercial hybrids of orchids,
underpinning future breeding and species conservation programmes. In this contribution,
we provide an overview of the progress in cryobanking of a range of orchid tissues,
including seeds, pollen, protocorms, protocorm-like bodies, apices excised from in vitro
plants, cell suspensions, rhizomes and orchid fungal symbionts. We also highlight future
research needs.
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Keywords
Orchidaceae, orchids, biodiversity conservation, cryopreservation, tissue culture, seeds,

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apices, pollen, protocorms, protocorm-like bodies, rhizomes.

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1. Introduction
1.1. Orchids and their importance in human life

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With an estimated 880 genera and over 25,000 species, the Orchidaceae is the

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largest family of flowering plants (Givnich et al., 2015). Orchids have been known,
appreciated, used, and frequently overused for centuries in different parts of the world. The
significance of orchids in human life cannot be overestimated (Fig. 1). Many orchids
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possess high medicinal, ornamental and cultural value in their countries of origin.
Cultural significance extends to symbolism in myths the orchids have inspired
works of art, literature and poetry. In ancient Japan, China and Korea the oriental
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Cymbidiums have featured in ink drawings which, when combined with elegant poetry
writing, were meant to adorn the life of noble families (Paek and Murthy, 2002). The
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tradition of orchid ink painting persists even today (Fig. 1).

Regarding their use in medicine, all parts of orchids have been utilized, including
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leaves, roots, flowers, pseudobulbs, tubers, rhizomes and whole plants (Jalal et al., 2010;
Singh and Duggal, 2009; Gutiérrez, 2010; Pant, 2013). The medicinal value of orchids first
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received recognition in ancient China (Hew et al., 1997; Pant, 2013). In traditional Chinese
medicine (TCM), mainly native Dendrobium and Cymbidium orchid species serve as
ingredients of therapeutic preparations for treating various ailments, including diabetes,
lung cancer, stomach diseases, allergies and fatigue (Hu, 1971; Ng et al., 2012; Paek and
Murthy, 2002; Liu et al., 2014). Plants of Dendrobium spp., Gastrodia elata Blume1, and
Bletilla striata Rchb.f. continue to be an important part of the Chinese herbal industry
(Bulpitt et al., 2007). Similarly, India has a long tradition of using orchids in the
“Ayurveda” system of medicine, including four species of the genera Malaxis and
Habenaria (Singh and Duggal, 2009; Jalal et al., 2010). Plants of Cypripedium, Vanilla,
Arpophyllum, Bletilla and Epidendrum genera have also been collected for medicinal use

1Wherever possible, authorities for species names are given at the time of first mention as recorded in
The Plant List, (http://www.theplantlist.org) or the International Plant Names Index
(http://www.ipni.org/)
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by different ethnic groups in North America and Mexico, while Europeans were mostly
aware of the medicinal value of terrestrial orchids, such as Orchis spp., Dactylorhiza spp.
and Epipactis spp. (Bulpitt, 2005; Pant, 2013). Other medicinal properties relate to tonic,
antibacterial, aphrodisiac, anti-tumour, anti-pyretic and wound-healing properties (Bulpitt

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et al., 2007; Singh and Duggal, 2009; Gutiérrez, 2010) as well as contributing to curing
and helping reveal the symptoms of tuberculosis, indigestion, headache, fever, fractured

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bones, stomach diseases and, even, snake bites (Bulpitt, 2005; Pant, 2013). The medicinal
value of some traditionally-used orchid species has been recently proved by clinical trials

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(e.g. Liu and Mori, 1992; Zheng et al., 1998; Shin et al., 2002; Kim et al., 2003; Morita et
al., 2005; Li et al., 2011). The presence of medicinally active chemicals such as

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polysaccharides and secondary metabolites including alkaloids, glycosides, phenolic
compounds, and many others have been also documented in orchid tissues (reviewed by
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Gutiérrez, 2010; Ng, 2012).
Beyond medicine, orchids products are widely used the food and beverage
industries. Specifically, vanillin extracted from the seed pods of Vanilla planifolia
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Andrews (now mostly produced chemically) and salep (“Sahlep“ in Arabic) made from
dried tubers of Orchis morio [now Anacamptis morio (L.) R.M.Bateman, Pridgeon &
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M.W.Chase; (Fig. 1; Bulpitt, 2005)].

However, in the modern world, orchids are known and grown primarily as
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ornamentals, mostly for their exotic, long lasting and often fragrant flowers (Fig. 1).
Orchids represent 8% of the global floriculture trade (Martin and Madassery, 2006). In
China, Taiwan, Korea and Japan, oriental Cymbidiums are popular horticultural plants with
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high commercial value. For example, a single plant of Cymbidium goeringii (Rchb.f.)
Rchb.f. can sell for US$ 10 000 (Paek and Murthy, 2002). The wholesale value of potted
orchids in the United States in 2011 reached US$ 200 million, making them the second
most popular potted flowering plant in the country (Teixeira da Silva et al., 2014). Orchid
propagation by both large and small industries has depended on traditional as well as
modern orchid breeding programs utilizing available genetic resources (Paek and Murthy,
2002; Liu et al., 2014), and interest in sourcing wild species for unique gene combinations
remains high. Hundreds of new varieties are registered annually. However, the systematic
preservation of both old and these new plant genetic resources has not been seriously
addressed.
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1.2. Orchid biodiversity and conservation strategies

Orchids are an important part of plant biodiversity on the planet due to their high

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variability among species and their habitats. The highest diversity of orchid species has
been found in the Andes of Colombia and Ecuador, tropical rainforests of Borneo,

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Sumatra, New Guinea and Madagascar (Cribb et al., 2003; Swarts and Dixon, 2009). Areas
of particular species abundance are India, SW China, temperate SW Australia, South

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Africa and Bhutan (Cribb and Govaerts, 2005). Every year botanists discover over one
hundred new orchid species (e.g. Carnevali et al., 2014; Vale et al., 2014; Kolanowska,

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2015); for example in 2013, nearly 370 new species were described (Schuiteman, 2015).
Clearly, our knowledge of orchid genetic diversity is fairly incomplete, and there is the
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prospect that many orchid species may be lost before their discovery.
Compared to other vascular plants, orchids are considered to be the most highly
evolved of vascular plants. Having once been abundant worldwide, some species have now
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become extremely rare (Koopowitz and Kaye, 1983; Koopowitz, 1986; Dasgupta et al.,
2004). Their high specificity for insect pollinators, minute seeds (often weighing µg)
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without endosperm, and a unique life cycle requiring an association with specific
mycorrhizal fungi during the early stages of development has left orchids vulnerable to
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minor biotic and abiotic changes (Arditti and Ernst, 1984; Vinogradova and Andronova,
2002; Kandavel et al., 2004). The widespread degradation of ecosystems, for example as a
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result of an increased use of weed killers and artificial fertilizers, deforestation, and land
clearance, has imperiled orchids in their natural habitats (Farrell and Fitzgerald, 1989;
Wood, 1989; Kandavel et al., 2004; Swarts and Dixon, 2009). Moreover, global warming
is predicted to produce irreversible changes in orchid communities (Seaton et al., 2010).
Such effects are likely to be the most serious in mountain and tropical regions, including
many orchid biodiversity hotspots in Asia and Latin America (Seaton and Pritchard, 2011).
All orchids are listed under Appendix II or I of the Convention on International
Trade in Endangered Species (CITES, http://www.cites.org). The approach to conserve
these “pandas of the plant world” (Wood, 1989) requires a complex integration of
preserving natural habitats (in situ conservation), developing and applying ex situ
conservation methodologies, and a better understanding of the orchid trade and
horticultural practices. Deeper insights of orchid biology, evolution and ecology are also
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needed. Traditionally, botanic gardens have taken the lead role in orchid conservation, by
developing and maintaining well-documented livings collections (e.g., about 2700 species
at the Royal Botanic Gardens, Kew, UK), storing seeds, establishing and running in vitro
propagation facilities, setting up cryobanks and, more recently, DNA banks for

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phylogenetic research (Koopowitz, 1986; Farrell and Fitzgerald, 1989; Tasker, 1989;
Koopowitz and Thornhill, 1994; Kolomeitseva et al., 2011; Swarts and Dixon, 2009).

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Seed banking has long been advocated as an effective and viable ex situ
conservation option for plants, including orchids (Stanwood, 1985; Pritchard et al., 1999).

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However, seeds of some orchids appear to be short-lived compared to those of other higher
plants (Pritchard and Dickie, 2003; Hay et al., 2010). For example, under room

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temperature and warm conditions (e.g. 20-40°C) they may lose viability in weeks or days
(Pritchard, 1986; Pritchard and Seaton, 1993; Koopowitz and Thornhill, 1994; Pritchard et
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al., 1999). Storage at moderately low temperature (-20 to 4°C) tends to prolong seed life
(Pritchard and Dickie, 2003). There is evidence that good-quality dry seeds of some
orchids have the potential to survive for decades under conventional refrigeration (ca. 5°C)
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and freezer (-20°C) conditions (see Pritchard and Seaton, 1993; Seaton and Pritchard,
2011; Hosomi et al., 2012). However, for some orchid species low temperature storage can
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be relatively ineffective (Seaton and Pritchard, 1993; Merritt et al., 2014). For example,
seeds of Phaius tankervilleae (Banks) Blume dehydrated to 5% water content (WC)
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showed a notable decrease in germination after being stored at 4°C for six months (Hirano
et al., 2009). Symbiotic germination of Caladenia arenicola Hopper & A.P.Br. and Diuris
magnifica D.L.Jones seeds was reduced significantly, compared to freshly collected seeds,
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after 12 months of storage at different temperatures ranging from 22°C to -18°C (Batty et
al. 2001). An additional complication is that orchid seed germination can be increasingly
variable with storage time (Pritchard et al., 1999; Hay et al., 2010), perhaps reflecting seed
provenance effects (Nikishina et al., 2007) or a change in the optimum germination
conditions, e.g., nutrient composition of medium or substrate for germination.
Consequently, the method of viability assessment may have a significant impact on
germination percentage and variability in the experimental data (Dowling and Jusaitis,
2012).

1.3. In vitro technology for orchid propagation and conservation

One of the most significant applications of biotechnology to orchid diversity conservation

and sustainable use is in vitro germination. Orchid seeds often require the presence of a
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fungal symbiont for efficient emergence in situ. Such conditions can be replicated ex situ.
Building on the work of Moore (1849) and Bernard (1899), Knudson (1922) developed
reliable methods for the asymbiotic germination of orchid seeds using complex nutrient
media. Thereafter, Rotor (1949), Thomale (1956) and Morel (1960) pioneered the in vitro

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culture of orchid explants (for historical reviews see Arditti, 1984; Yam and Arditti, 2009).
Since then, in vitro techniques for germination, propagation and large-scale multiplication

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of orchids have been widely used for both conservation and commercial purposes
(Dasgupta et al., 2004; Teixeira da Silva, 2013b). These micropropagation techniques

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underpin a globally significant orchid trade. For example, United States orchid potted plant
export valued was $4.3 million in 2005 (agecon.centres.ufl.edu/OrchidExport.htm#value2),

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and Thai orchid exports in 2011 totalled €154 million (Lippe, 2012).
Mature seeds of some orchid species require several months of cold stratification
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before germination (Cherevchenko and Kushnir, 1986; Nikishina et al., 2007), and the
whole process of embryo development to plantlet formation can take months to years,
particularly for temperate species (Olivia and Arditti, 1984; Nikishina et al., 2001). For
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such species, immature seeds that germinate readily after sowing are considered a primary
material for the initiation of tissue cultures (Hirano et al., 2005a; 2005b; Nikishina et al.,
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2007). Based on an understanding of dry seed storage of other species, such immature
seeds may not, however, be optimal for long-term storage.
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Other than seeds, isolated buds as well as shoot and root tips, stalk node sections,
protocorms and protocorm-like bodies (PLB), meristematic clumps, rhizomes and even
cell suspensions and isolated protoplasts have been explored for their potential to support
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orchid micropropagation (for the lists of orchids and culture procedures see Arditti and
Ernst, 1993; Arditti and Krikorian, 1996; Arditti, 2008; Teixeira da Silva, 2013b). These
experiments provided the basis for mass in vitro production of the commercially important
tropical orchid varieties (Teixeira da Silva, 2013b) and endemic species from the remote
world regions, such as temperate Cymbidium species of China, South Korea and Japan (see
e.g. Paek and Kozai, 1998; Ogura-Tsujita and Okubo, 2006). However, successful in vitro
propagation does not remove the necessity for ex situ orchid conservation. In fact, all types
of in vitro-cultured materials that have been used for mass rapid propagation of orchids
may be also utilized for conservation purposes. Recently, in vitro slow growth techniques
and storage at low positive temperatures (from 0 to 16°C) have proved effective for some
Dendrobium species (Teixeira da Silva et al., 2014). Over 90% of seed-derived in vitro
seedlings of Dendrobium officinale Kimura & Migo tolerated 12-months of storage at 4°C
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in darkness without subculture (Shi et al., 2000). In vitro plantlets of Dendrobium draconis
Rchb.f. and Ipsea malabarica Hook.f. maintained high viability during storage at 25°C for,
respectively, 6 and 27 months (Martin and Pradeep, 2003; Rangsayatorn et al., 2009).
However, in vitro conservation is relatively labour-intensive and costly; moreover,

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phenotypical and genetic variations in orchid materials in the course of repeated
subcultures are well-documented (Tokuhara and Mii, 1998; Arditti, 2008; Khoddamzadeh

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et al., 2010; Teixeira da Silva et al., 2014). These limitations have promoted the
development of less expensive and more reliable conservation methods such as

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cryopreservation, which allows safe and long-term storage of orchid germplasm once an
appropriate protocol is designed and validated.

1.4. Cryopreservation NU
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Cryopreservation is commonly defined as the storage of genetic material at temperatures
below -130°C (sometimes referred to as “cryogenic temperature”). In the majority of world
cryogenic banks the material is stored in liquid nitrogen (LN, -196°C) or in the vapour
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phase (from -150°C to -185°C) (Walters et al., 2004). At cryogenic temperatures, all
metabolic activities in living cells are stopped, and genetic material can be maintained
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without contamination and somaclonal variations for a theoretically unlimited period of

time (Harding, 2004; Benson, 2008; Benelli et al., 2013; Wang et al., 2014). In practice,
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the longest storage duration at cryogenic temperatures known so far are 28 years for
strawberry meristems and in vitro germinated pea seedlings (Caswell and Kartha, 2009)
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and 27 years for alfalfa cell culture (Volkova et al., 2015). Cryopreservation is not the
“panacea” against ageing: there is evidence to show that deterioration of biological
samples may occur even at cryogenic temperatures during long-term storage (Walters et
al., 2004). However, despite the faster than anticipated deterioration, cryogenic storage
clearly prolonged seed shelf life. For fresh lettuce seeds stored in the vapour and liquid
phases of LN half-lives were projected to be 500 and 3400 years, respectively, which is
longer than the expected duration that can be provided by any other storage regime
available (Walters et al., 2004). The preservation of plant tissues in cryobanks is the only
long-term ex situ conservation option for vegetatively propagated (clonally reproducing)
species and for species that produce desiccation-sensitive (recalcitrant) or short-lived
seeds (Engelmann, 2004; Pritchard, 1995, 2007). It can also provide safe and cost-effective
long-term conservation of large quantities of genetic material in small facilities, with
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minimum requirements for maintenance (Stanwood, 1985; Sakai and Engelmann, 2007; Li
and Pritchard, 2009).
To date, cryopreservation using different propagules (seeds, pollen) as well as
clonal and in vitro-cultured plant materials (somatic embryos, cell cultures, meristems of in

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vitro plants, winter buds, etc.) has been successful for over 200 plant species, including
staple crops, endangered plants and plants of horticultural importance (reviewed by

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Benson, 2008; Wang et al., 2012; 2014). On a large-scale, cryopreservation is now being
applied to back-up field collections of many crops; for example, 2291 genetic collections

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of apple at the USDA cryobank at Fort Collins, USA (Towill et al., 2004; Volk et al.,
2015). Also, the whole national collection of Alluim spp. (> 1150 accessions) is

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cryobanked at RDA, Suwon, Republic of Korea (Kim et al., 2012a). Other large-scale
cryobank collections include Solanum spp. (Kaczmarczyk et al., 2011; Panta et al., 2015),
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Musa spp. (Panis et al., 2005) and mulberry (Atmakuri et al., 2009; Fukui et al., 2011). The
potential of cryopreservation in supporting the sustained production of medicinal and
aromatic plants has also been clearly demonstrated (Bajaj, 1995, Dixit et al., 2004, Popova
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et al., 2011).
In orchids, cryopreservation has been an efficient means of conserving seeds and
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pollen (e.g., Pritchard, 1984; Koopowitz, 1986; Pritchard and Prendergast, 1989;
Koopowitz and Thornhill, 1994; Mweetwa et al., 2007; Hay et al., 2010; Vendrame et al.,
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2014). By contrast, attempts to cryopreserve in vitro-cultured orchid explants, such as

meristems or protocorms, have resulted in variable regrowth. Nonetheless, significant
progress in cryopreservation of such explants has been achieved in the past 15 years (see
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e.g., Wang et al., 1998; Thinh and Takagi, 2000; Kondo et al., 2001; Bian et al., 2002;
Hirano et al., 2006 - review; Yin and Hong, 2009; Sopalun et al., 2010; Vendrame and
Faria, 2011; Ivannikov, 2012; Teixeira da Silva, 2013a; Merritt et al., 2014).
Herein, we provide a historical perspective and consider recent advances in the
development of orchid cryobiotechnology. We detail and analyze the successful
methodologies used for different orchid explants, including seeds, pollen, cultured
protocorms, meristematic tissues and rhizomes. Finally, we highlight challenges in
cryopreservation of orchid genetic resources for future conservation and use.

2. Cryopreservation of pollen

Orchid pollen occurs as monads to tetrads and multiples of tetrads, included in aggregated
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structures with a stalk and viscidium, called pollinia. All species with pollinia are
suggested to have partially hydrated pollen (PHP, Pacini and Hesse, 2002), with WCs
above 30% at anthesis. Consequently, the term ‘recalcitrant’ has been applied as an
analogy with seeds that are also shed at high WC and sensitive to dehydration (Franchi et

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al., 2011). However, pollinia in opened flowers of four orchids in Anacamptis,
Dactylorhiza and Orchis genera have WC ranging from ca. 15 – 35% and all tolerate

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subsequent equilibration to ca. 35% RH [equivalent to 8 – 10% WC (Marks et al., 2014)];
clearly indicating that not all orchid pollens are recalcitrant.

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Investigations into the cryostorage of orchid pollen are few (Table 1). Ito (1965)
pioneered sub-zero storage of orchid pollen, reporting that pollen of Dendrobium nobile

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Lindl., Dendrobium cv.“Lady Hamilton” and Calanthe furcate Bateman ex Lindl. were
able to germinate after 712, 975 and 718 days at -79°C, respectively. Moreover, the
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lifespan of orchid pollen was shown to be enhanced substantially when maintained under
ultra-cold compared with cool (4°C) conditions at which pollen viability was lost in 6
months. In this work, a cryoprotective mixture consisting of glycerol and ethylene glycol
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was successfully tested for Dendrobium nobile pollen frozen at -79°C for 93 days. This
binary mixture was an early step in the development of plant vitrification solutions (PVS),
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which often consist of two to four constituents, including glycerol, dimethylsulfoxide

(DMSO) and ethylene glycol (EG), plus sucrose (see Sakai and Engelmann, 2007).
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Air-dry pollinia of Dactylorhiza fuchsii (Druce) Soó was capable of surviving 6

year’s storage at -20°C, but with reduced seed siring ability such that fewer seeds were
produced per mature capsule compared with fertilization with fresh pollen (Marks et al.,
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2014). Pritchard and Prendergast (1989) investigated the storage behaviour of pollen of
some terrestrial orchid species of UK at -20 and -196°C. When pollen of Anacamptis
pyramidalis (L.) Rich. and Gymnadenia conopsea (L.) R.Br. were cryopreserved by
programmed cooling in the presence of 0.5 M DMSO, only 1% germination was observed
for both species compared to 42 and 26% germination of controls, respectively. Moreover,
germination of Gymnadenia conopsea was reduced to 4% in response to DMSO treatment
without LN exposure. For comparison, pollen of Listera ovata R.Br., Dactylorhiza fuchsii
and Dactylorhiza maculata (L.) Soó germinated to 23, 23, and 40%, respectively after LN
exposure. Germination of Orchis mascula (L.) L. and Cymbidium elegans Lindl. pollen
decreased after cryopreservation regardless of the cryoprotective solution employed. In
contrast to fresh pollen, no decrease in germination was recorded for air-dried (to ca. 15%
WC) pollen of Dactylorhiza fuchsii and Anacamptis pyramidalis after 12-month cryogenic
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storage. Moreover, air-dried Orchis mascula pollen tolerated 10 cooling-rewarming cycles

to -196°C with 46% survival, which was comparable to 51% germination in non-
cryopreserved controls.
Vendrame et al. (2008) reported cryopreservation of pollinia from two Dendrobium

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hybrids by direct storage in LN without cryoprotective treatment or by using a PVS-based
vitrification protocol (see below). These treatments included loading for 30 min at room

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temperature followed by PVS2 either at room temperature (27°C) or precooling on ice
(0°C) for 1-4 h before immersion in LN for 48 h. Upon retrieval from cryopreservation, the

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pollinia were used to pollinate flowers of the same hybrids to check their viability and
germination. Pollen germination above 80% was observed in most treatments and capsules

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with viable seeds (90-95%) were successfully produced by both hybrids. The seeds
contained well-formed embryos and germinated to develop into healthy seedlings.
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Similarly, a successful protocol for the long-term preservation of pollinia of Luisia
macrantha Blatt. & McCann, an endemic and endangered orchid of Western Ghats, has
been devised (Ajeeshkumar and Decruse, 2013). Air-drying for 30 min followed by LN
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treatment resulted in 54% germination in vitro; moreover, the treated pollinia gave 100%
fruit set upon sub-mating. In addition, PVS-treated pollinia exhibited 51% germination
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after 668 days storage in LN. Pollinia cryopreserved by optimized vitrification method (10
min in PVS) were able to hybridize with Vanda tessellata Hook. ex G.Don to give 87%
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fruit set and 21% viable seed, capable of producing healthy seedlings (Ajeeshkumar and
Decruse, 2013).
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Overall, cryopreservation has been successfully applied to the pollens of a range of

orchids (Table 1, Fig. 2) and can be a valuable tool in breeding programmes. It is
premature, however, to generalize about pollinia cryopreservation protocols due to a
limited number of studies published so far. It is likely that air-drying and vitrification
methods are both suitable treatments to enble the cryopreservation of orchid pollen.
However, for each species, pollen WC and its initial viability should be determined before
cryopreservation.

3. Cryopreservation of seeds

Orchid seeds broadly fall into the “orthodox” group as their longevity is enhanced by
reducing water content and by lowering storage temperature (Pritchard et al., 1999).
However, seeds of some orchid species displayed sensitivity to extreme desiccation and
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reduced viability within short periods when stored dry (3-5% WC) at some high sub-zero
temperatures (e.g., -5°C to -20°C) coincidatal with those generally accepted as
conventional seed banking regimes (Cherevchenko and Kushnir, 1986; Koopowitz and
Thornhill, 1994; Wang et al., 1998; Nikishina et al., 2001; Hirano et al., 2009). For

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example, after drying in equilibrium to 15% RH and storage at -20°C for 9 – 12 months,
seed germination of Coelogyne foerstermannii Rchb.f., Coelogyne rumphii Lindl. and

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Dendrobium stratiotes Rchb.f. fell to 1 – 5% from initial values of 65-96%. In contrast,
Xylobium undulatum (Ruiz & Pav.) Rolfe seeds lost only 13% germinability during the

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same interval (Seaton et al., 2013). Such variable responses to conventional banking
conditions have reinforced the need to investigate the cryo-storage behavior of orchid

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seeds (Pritchard et al., 1999; Merritt et al., 2014) and the seeds of other species (Li and
Pritchard, 2009).
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3.1. Cryopreservation of immature and mature seeds with high WC

Immature orchid seeds are the preferred material for in vitro micropropagation, due to
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reduced risk of contamination when isolating seeds from green capsules (Arditti, 2008).
Immature embryonic material is normally highly hydrated as the developing cells are
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vacuolated.
Direct exposure of immature seeds to LN can be lethal (Hirano et al., 2005a),
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presumably as a result of intracellular ice formation (Mazur, 1984). Cryopreservation of

such hydrated material is only possible through controlled dehydration and the application
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of cryoprotectants or a combination of both (Engelmann, 2004). Samples can be

dehydrated during slow cooling in the presence of growing extracellular ice (programmed
freezing methods), osmotically in a series of concentrated cryoprotectant solutions
(vitrification-based methods) or simply under air flow or above silica-gel (desiccation-
based methods). Particularly fragile and desiccation-sensitive materials may be turned into
‘synseeds,’ i.e., encapsulated in an alginate-based matrix (Redenbaugh, 1993). Thereafter,
the beads containing the tissue can be dehydrated and/or cryoprotected and cryopreserved
(Fabre and Dereuddre, 1990; see Benson, 2008 for the detailed description of each
method). The vast majority of methods imply rapid rewarming of cryopreserved samples in
a water bath or sucrose (unloading) solution preheated to 37-40°C.
The “classic” vitrification technique (first published by Sakai et al., 1990) is the
most frequently employed method for cryopreserving immature seeds and mature seeds
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with WC (Table 2, Fig. 2). This procedure involves step-wise dehydration of samples with
highly concentrated cryoprotectant mixtures, including loading solutions (LS) and plant
vitrification solutions (PVS). In the majority of reports listed in Tables 1-4, the LS
consisted of 2.0 M glycerol+0.4 M sucrose in the culture medium (Nishizawa et al., 1993)

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and the PVS contained (w/v) 30% glycerol + 15% dimethylsulfoxide + 15% ethylene
glycol + 13.7% sucrose (PVS2, Sakai et al., 1990). Using this method, satisfactory post-

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cryogenic germination and regrowth have been achieved for immature seeds of three
orchid genera: Bletilla, onerorchis, and Vanda (Table 2). Also, 3-month old marure seeds

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of the Thai orchid Doritis pulcherrima (now Phalaenopsis pulcherrima (Lindl.) J.J.Sm.) at
31% WC and seeds of Vanda coerulea Griff. ex Lindl. at 33% WC have been successfully

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cryopreserved using a PVS2-based vitrification protocol (Thammasiri, 2000; Thammasiri
and Soamkul, 2007). Seeds of both species withstood cryopreservation following exposure
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to PVS2 for 50 and 70 min, respectively without any preliminary culture with osmolytes or
loading treatment.
Vitrification was employed for cryopreserving immature seeds and cultured zygotic
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embryos of endangered Japanese orchids (Ishikawa et al., 1997; Hirano et al., 2005a;
2005b). The highest survival after cryopreservation of these species was achieved
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following a 3-day preculture with 0.3 M sucrose, incubation with LS for 15 min, then
exposure to PVS2 at 0°C for 1-2 h. Similar protocols have been used for cryopreserving
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mature seeds of the endangered terrestrial orchid Phaius tankervilleae (32% WC) and four
Dendrobium hybrids (Vendrame et al., 2007; Hirano et al., 2009). Interestingly, in both
studies orchid seeds tolerated cryoprotectant treatment and cryopreservation even without
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preculture. More recently, Hirano et al. (2011) used PVS2 to enable the cryopreservation
of seven Cymbidium species (three terrestrial and four epiphytic). Germination of
Cymbidium finlaysonianum Lindl. and Cymbidium tracyanum L.Castle decreased by 16-
20% after cryopreservation while germination of four other species in the same genus (C.
goeringii, C. macrorhizon Lindl., C. iridioides D.Don and C. maguanense F.Y.Liu) was
stimulated post-cryopreservation or remained comparable to controls. Exposure time to
PVS2 was optimized at 60 min for terrestrial species and for 30 min for epiphytic orchids,
respectively (Hirano et al., 2011), suggesting a possible difference in permeability
properties of the seeds of the two groups. In contrast, the PVS2-based vitrification
technique reduced post-cryopreservation germination of fresh, mature seeds of Bletilla
formosana (Hayata) Schltr. to 18-41% compared to 68% germination for seeds pre-dried
with silica gel (Wu et al., 2013). Alternatively, Hu et al. (2013) reported thatno
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germination of mature Bletilla formosana seeds after cryopreservation was observed

without PVS2 treatment (). In their study, the PVS2-based vitrification improved
germination of seeds as compared to non-cryopreserved control with no significant
variations between 10 min, 1 week and 1 year`s storage (Hu et al., 2013).

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Importantly, some chemicals with cryoprotective properties have been found to be
effective at prolonging the storage of orchid seeds under non-cryogenic temperatures.

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Thus, seeds of Dendrobium crystallinum Rchb.f., Dendrobium virgineum Rchb.f. and
Vanda teres × V. hookeriana coated in meso-inositol could be stored at 4, -20 and -70°C

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for periods from 6 months to five years; however, their viability was reduced by 20-60%
compared to fresh seeds (Huehne and Bhinija, 2012). The use of mannitol and glucose as

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protective coating was less effective (Huehne and Bhinija, 2012).
To date, cryopreservation of immature and highly hydrated orchid seeds has been
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accomplished for orchid species from several genera including Bletilla, Cymbidium,
Cyrtopodium, Dendrobium, Doritis, Encyclia, Phaius, Ponerorchis and Vanda with post-
cryopreservation germination ranging from 32 to 100%. For many of these species,
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dehydration of seeds in concentrated cryoprotectant solutions before rapid immersion in

LN was critical for post-cryopreservation survival (Table 2). Cryoprotectants function by
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reducing freezing temperatures of the intracellular solutes, inhibiting ice nucleation and
growth and reducing the amount of freezable water in biological materials (Finkle et al.,
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1985; Fuller, 2004; Volk and Walters, 2006). Hydroxy groups of sugars and sugar
alcohols, e.g., glycerol, are thought to substitute for water in the hydration shells of
membranes and macromolecules, thus stabilizing their conformation during dehydration
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and freezing-thawing events and decreasing membrane phase transition temperature

(Anchordogue et al., 1987; Crowe et al., 1987, 1990; Hoekstra et al., 2001). In addition,
some cryoprotectants can act as free radical scavengers and exhibit antioxidant activities
(Benson, 2004; Fuller, 2004). Due to these and other potential protective properties,
cryoprotectant mixtures are often used for cryopreserving plant materials that may be
intolerant to severe dehydration, such as orchid protocorms, PLBs and meristematic tissues
as explained in sections 5-7.
There is evidence, however, that mature seeds of some orchid species with high
WC can tolerate direct exposure to LN. For example, Encyclia cochleata Lemée seeds
sealed in cryovials could be cryopreserved at 24% WC without any loss in viability
(Nikishina et al., 2001). Similarly, fresh mature seeds of Bletilla formosana, an endangered
Taiwanese orchid, reached 68% germination after being air-dried for 24 h to 25% WC and
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cryopreserved by direct immersion in LN (Wu et al., 2013).

3.2. Cryopreservation of mature orchid seeds: the relationship between seed WC and post-
storage viability

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Whilst the first attempts to expose agricultural seeds to cryogenic temperatures were made
around the mid-19th century (see Pritchard and Nadarajan, 2008), cryobiology studies on

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orchid seeds started about 100 years later. Svihla and Osterman (1943) discovered that

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Cattleya hybrid seed were able to survive freezing at -78°C. Ito (1965) successfully stored
seeds of Dendrobium nobile and Cattleya hybrids for up to 465 days at -79°C; and showed
that cryoprotection with 85% glycerol was beneficial for survival of D. nobile seed after

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subzero storage. Later, Bowling and Thompson (1972) reported that the seeds of 30
different tropical orchid species withstood cold storage at -10°C. Similarly, seeds of
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Encyclia vitellinum could be stored at -40°C for 35 days without considerable loss of
viability (Koopowitz and Ward, 1984).
The first successful cryogenic exposure of mature orchid seeds, in liquid nitrogen at
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-196°C involved seeds of eight terrestrial and two epiphytic orchid species from the genera
Disa, Eulophia, Gymnadenia, Orchis, Satirium, Phalaenopsis and Vanda (Pritchard,
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1984). Seeds at 5 – 11% WC, i.e. air-dry, sealed in cryo-vials tolerated direct immersion
and short-term storage (15 min) in LN. In addition, 10 cooling-warming cycles generally
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had no effect on seed germination, such that the size of the developing protocorms and the
number of rhizoids and leaf production remained unaffected after cryopreservation. In one
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species, Orchis morio (now Anacamptis morio), LN immersion increased seed

germination, possibly due to a lipid body dissociation rather than increased permeability of
the seed coat (Pritchard, 1984).
In most studies (see Table 2), air-dry or pre-dried mature orchid seeds are sealed in
cryo-vials and simply plunged into LN (direct cryopreservation) for the required period
ranging from one week to several years, with subsequent rewarming in a water-bath (37-
40°C) for approx. 1 min or by equilibrating the vials at room temperature. High post-
cryopreservation germination has been achieved in most studies suggesting that the
majority of orchid seeds at less than ca. 13% WC can be successfully cryopreserved by this
simple method. Although seeds of some species at higher WC can also tolerate direct
exposure to LN (for example, seeds of Encyclia cochleata and Bletilla formosana at 24-25
% WC, see 3.1), such behavior is uncommon.
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For fully desiccation-tolerant seeds, successful cryopreservation is achieved by

drying seeds to WCs below their high-moisture freezing limit, which has been shown to be
equivalent to ca. 75 to 85% RH across a broad range of species (Pritchard, 1995, 2007; Hor
et al., 2005). Below this limit survival tends to be independent of the cooling regime. At

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very low WC (e.g. equilibration with silica gel at ca. 5% RH), however, there is the
possibility of cumulative desiccation stress that may affect seed survival during the

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subsequent storage (Pritchard et al., 1999). Thus, recovery growth of such seeds after
cryogenic exposure may be limited by freezing damage at one end of the optimum (safe)

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WC range and by desiccation injury at the other (Wang et al., 1998; Dussert and
Engelmann, 2006; Pritchard, 2007; Popova et al., 2012). For example, freshly harvested 4-

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month old Dendrobium candidum Wall. ex Lindl. seeds at 43% WC required partial
dehydration to 12 - 19% WC for successful cryopreservation (Wang et al., 1998). Seeds
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withstood drying to 4.5% WC but had slower development and produced yellow rather
than green protocorms after cryostorage, indicating the occurrence of desiccation injury
(Wang et al., 1998).
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Interestingly, seeds of some orchids cannot withstand direct exposure to cryogenic

temperature even at relatively low WC. For example, mature seeds of Oncidium flexuosum
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(Kunth) Lindl. (11% WC) and a Dendrobium hybrid (13% WC) were unable to germinate
or germinated at low levels after direct immersion in LN (Galdiano et al., 2012, 2013).
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Seeds of these species could be cryopreserved by a vitrification method, i.e. by using

highly concentrated cryoprotectant solution PVS2 combined with rapid cooling and
rewarming (Galdiano et al., 2012, 2013, 2014). For Oncidium flexuosum, the best
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treatment included dehydration with PVS2 for 120 min with subsequent addition of 1%
phloroglucinol, which resulted in 78% post-cryopreservation recovery compared to 91%
germination of seeds not subjected to cryopreservation (Galdiano et al., 2013). PVS2
applied alone or in combination with 1% Supercool X1000, a potential ice-blocking agent,
produced lower germination after cryopreservation. This was in agreement with the
previous finding that PVS2+1% phloroglucinol improved post-cryopreservation
germination and development of mature Dendrobium seeds, while Supercool X1000 was
ineffective (Galdiano et al., 2012). Most recently, Galdiano et al. (2014) employed
vitrification for cryopreserving mature seeds of Dendrobium hybrid “Dong Yai” (12% WC)
with a highest germination of 58% achieved following 2 h exposure to PVS2 at 0°C.
From the published literature, successful cryopreservation of seeds has now been
achieved for at least 68 orchid species and commercial genotypes from 30 genera of both
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temperate and tropical origin (Tables 2, 5; Fig. 2). However, the total number of species
successfully conserved as seeds is much higher when species in the germplasm
conservation centres are included. For example, the cryopreserved collection of orchid
seeds at the Institute of Plant Physiology in Moscow, Russia, consists of nearly 100

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tropical and 20 temperate orchid species (Nikishina, personal communication). The
collection was established in 2001 using an existing cryogenic facility (Popov et al., 2006).

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The material is mainly stored as mature seeds air-dried to WC below 15% in 2-ml cryo-
vials; the number of seed lots stored varies from one to five, depending on the species. In

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general, germination of cryopreserved seeds is high (70-100%) for the tropical species.
Germination of temperate species commonly varies from 27-45% (Table 2; Nikishina et

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al., 2001; 2007). Comparatively low germination of seeds of temperate species may be
attributed to various types of seed dormancy presented in mature seeds as well as specific
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requirements for seed germination (organic additives in the medium, modified medium
composition, presence of mycorrhizal fungi, etc.) (Rasmussen, 1995; Nikishina et al.,
2007).
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3.3 Differencial scanning calorimetry (DSC) and its application in orchid seed
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cryopreservation studies
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Understanding the thermal properties of samples and cryoprotective solutions is important

for the development of effective cryopreservation procedures (Bruňáková et al., 2011; Kim
et al., 2009, 2012a; Teixeira et al., 2014). In this context, differential scanning calorimetry
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(DSC) is often used to investigate the thermal behavior of plant materials and, in
particular, to detect water- or lipid-associated phase transitions during cooling and
rewarming (e.g., Volk and Walters, 2006; Kim et al., 2008; Fang et al., 2009; Skyba et al.,
2011).
Systematic investigations of thermal properties of orchid seeds are not available.
Histochemical and anatomical studies revealed that seeds of most orchid species have
small embryos consisting of approximately 200 cells, which are rich in proteins and lipids
(Arditti, 1992; Pritchard and Seaton, 1993; Vinogradova and Andronova, 2002). Beyond
orchids it is known that the amount of unfrozen water in seeds, as well as their post-freeze
germination, is related to seed lipid content and composition (Pritchard and Nadarajan,
2008). WC optima for oilseed cryopreservation are equivalent to about 65-75 % RH
(Pritchard, 1995). Based on the behavior of Cattleya aurantiaca seeds (lipid content of
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29% DW), such that storage at 5°C was better than -18°C, it has been postulated that the
possible formation of different physical states of lipids (fluid, solid, crystalline) in the
seeds at sub-zero temperatures might contribute to their poor lifespan at certain low
temperatures (Pritchard and Seaton, 1993). Indeed, cooling and warming of mature, dry

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Cattleya aurantiaca seeds in a DSC to ca. -100°C revealed multiple exothermic
and endothermic peaks, respectively, thought to be associated with phase transitions in

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lipids.
Using the same experimental approach, a similar association has been made

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between relatively poor storage at -18°C of dry seeds of Microtis media R.Br., Pterostylis
recurva Benth. and Caladenia flava R.Br. and lipid transformations between ca. -40°C

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and -5°C (Merritt et al., 2014). Broadly similar events are known for other dry oilseeds,
e.g. soybean, sunflower and ginseng, although generally occurring at lower temperatures
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(Vertucci, 1989; Kim et al., 2008). Correlations have been made between oil crystallization
and unusual cold storage responses in dry Cuphea (non-orchid) oilseeds. It was shown that
oil-body coalescence and massive cellular disruption occur early during seed imbibition,
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which may contribute to a rapid loss in germination ability (Volk et al., 2006). Reheating
the seeds to about 45°C prior to imbibition helped to partly avoid the loss of germination
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(Volk et al., 2006). In seeds of five orchid species, though, there was no evidence of
imbibition stress regardless of rehydration rate and temperature, even though the seed
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exhibited varying degrees of cold stress when stored at high sub-zero temperatures
between -30 and -50°C (Pritchard et al., 1999). This has led to the hypothesis that optimal
oilseed storage temperatures may be species-specific in relation to the fatty acid
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composition and thermal behavior (Pritchard, 2004). Thereafter, the optimal conditions for
orchid seed storage (optimal seed WC, maturation status, etc) may differ significantly from
species to species, and more detailed studies of seed biophysics and physiology may be
required when cryopreserving new orchid species.

3.4 Biobanking of orchid seeds together with their fungal symbionts

As mentioned above, some orchids require an association between the germinating seed
and an endophytic fungus, usually from the Basidomycete family, for maximal seedling
growth (Clements et al., 1986; Muir, 1989). Based on this specificity, some researchers
adapted the existing methodologies for cryopreserving orchid seeds together with their
fungal symbionts. Wood et al. (2000) cryopreserved seeds of Dactylorhiza fuchsii and
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Orchis morio (now Anacamptis morio) encapsulated in alginate beads with the hyphae of
the basidomycete fungus Ceratobasidium cornigerum. Beads were pretreated in 0.75 M
sucrose solution for 18 h and dried to an optimal WC of 20%. Germination after
cryopreservation was up to 85% for Dactylorhiza fuchsii and 95% for Orchis morio

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(Anacamptis morio) with fungal emergence from 100% of beads. This method has also
been applied to seeds of two threatened terrestrial orchids of Australia, Pterostylis saxicola

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D.L.Jones & M.A.Clem. and Diuris arenaria D.L.Jones (Sommerville et al., 2008). When
encapsulated with their fungal symbionts, the seeds of these species recovered fullr

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germinability after 6 months of storage at -18 and -196°C, while storage at 23°C
significantly reduced fungal recovery and germination for both species after only 3 months.

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Batty et al. (2001) separately cryopreserved seeds of rare and endangered
Australian orchids of Caladenia, Diuris, Pterostylis and Thelymitra genera and their fungal
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symbionts, using different methods, and then co-cultured them after rewarming to develop
protocorms and seedlings. Post-cryopreservation survival of all fungal symbionts was
100%, with a short lag-phase in hyphal growth of 2–6 days. Cryopreservation significantly
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improved seed germination compared to control and had no deleterious effects on fungal
capacity to stimulate seed germination. Electron microphotographs showed no apparent
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external physical damage of seed testa in any of the treatments. Some conspicuous pitting
observed in Caladenia arenicola and Diuris magnifica D.L.Jones seeds appeared to be
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confined to the surface layer (Batty et al., 2001).

Recently, Ercole et al. (2013) reported conservation of 10 fungal symbionts
belonging to Tulasnella and Ceratobasidium genera isolated from roots of Mediterranean
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terrestrial orchids. All isolates retained their ability to stimulate orchid seed germination in
vitro after being stored at -80°C for 10-24 months, though the results were variable. Some
isolates performed even better after cold storage compared to their actively growing forms,
promoting formation of orchid protocorms with well-defined apex and rhizoids while no
differences in growth-promoting activity were observed for other isolates.

3.5. Effect of seed cryopreservation on protocorm and plant development and uniformity of the
regrown plants

Applying any method for ex situ plant conservation is based on the assumption that the
procedure per se does not reduce the ability of seeds or tissues to grow subsequently and
produce healthy seedlings, i.e., a normal phenotype. This perspective applies equally when
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banking under conventional conditions or via cryopreservation. Genetic stability, or “true-

to-type” status, of the plants regenerated from stored, including cryopreserved, propagules
is another important aspect of conservation strategies (Harding et al., 2004; Volk et al.,
2011).

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For some orchids cryopreservation stimulated germination of seeds and promoted
protocorm growth (Pritchard, 1984; Nikishina et al., 2001; 2007). For example, LN-treated

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mature seeds of epiphytic orchids Angraecum magdalenae Schltr. & H.Perrier and
Miltonia flavescens× Brassia longissima germinated 7–10 days earlier compared to non-

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cryopreserved seeds from the same seed lots (Nikishina et al., 2001). In these two species
and a terrestrial orchid, Calanthe gorey, four-week cryogenic storage resulted in significant

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(by 12-30 %) improvement of seed germination. In contrast, in three other species tested in
the study cryopreservation had no effect on seed germination (Nikishina et al., 2001; see
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Table 2). Studies on temperate orchids showed that cryopreservation promoted seed
germination of Dactylorhiza fuchsii and Dactylorhiza incarnate (L.) Soó, but suppressed
germination of Dactylorhiza maculata and Platanthera bifolia (L.) Rich. (Nikishina et al.,
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2007). As shown in Table 2, seed germination of many orchid species was reduced after
cryopreservation. For example, germination of both mature and immature seeds of Vanda
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tricolor Lindl. cryopreserved by direct immersion in LN decreased by 10-15% compared

to the germination of non-cryopreserved seeds (Jitsopakul et al., 2012). By contrast,
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immature seeds of Ponerorchis graminifolia Rchb.f. and cultured zygotic embryos of

Bletilla striata cryopreserved by vitrification techniques showed similar germination and
protocorm formation levels compared to untreated control propagules (Hirano et al.,
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2005a, 2005b). Cryopreservation via the encapsulation-dehydration method improved

germination of Cyrtopodium hatschbachii Pabst immature seeds to 64% compared to ca.
50% germination of control seeds (Surenciski et al., 2012). The effect of cryostorage on
seed germination was thus unique for the species and could not be predicted based on its
physiological age or terrestrial/epiphytic habit.
Popova et al. (2003) monitored the effect of seed cryopreservation on the
development in vitro of protocorms and seedlings of Miltonia flavescens × Brassia
longissima hybrid orchid. In agreement with earlier observations (Nikishina et al., 2001),
cryopreserved seeds germinated more readily and developed larger protocorms during the
first 45 days of culture compared to non-cryopreserved seeds (Popova et al., 2003). By 90
days of growth, however, differences between the control and cryopreserved treatments
were diminished; after that protocorms from control seeds developed even faster than
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protocorms from cryopreserved seeds. When the plants formed, no significant differences
in shoot length and leaf number were observed between the control and treatment (Popov
et al., 2004). Similarly, no differences in shoot and root length and fresh weight
accumulation were observed in plants regenerated from Oncidium flexuosum seeds

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cryopreserved by vitrification as compared to non-cryopreserved control (Galdiano et al.,
2013). Cryopreservation of Vanda tricolor seeds had no effect on the size and number of

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developing protocorms or the appearance of growing seedlings (Jitsopakul et al. 2012).
Plants derived from cryopreserved seeds of Bletilla striata showed a high pollen

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fertility that was equal to that of the field-grown plants (Hirano et al., 2005a). Plants
developed from PVS2-treated and cryopreserved seeds of Bletilla formosana and B. striata

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showed no abnormalities in growth and produced normal flowers (Hirano et al., 2005a; Hu
et al., 2013). Similarly, no abnormalities were found in flower shape and colour in
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Calanthe vestita Wall. ex Lindl. and Encyclia cochleata plants developed from
cryopreserved seeds (Fig. 3) (Nikishina et al., unpublished).
A very limited number of studies have assessed the stability of orchid plants
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derived from cryopreserved seeds at the cytological level. Bletilla striata and Oncidium
flexuosum plants grown from seeds cryopreserved using PVS2-based vitrification protocols
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had no change in ploidy level based on flow cytometry (FCM) analysis (Hirano et al.,
2005a; Galdiano et al., 2013). Cyrtopodium hatschbachii plants raised from encapsulated
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and cryopreserved seeds were stable at the chromosome and phenotypic level, but showed
a more limited condensation of the chromatin during the first stages of their development
(Surenciski et al., 2007). For mature seeds of Dendrobium hybrid “Dong Yai”, incubation
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time (0-6 h) in PVS2 affected post-cryopreservation seed germination, but the

morphological appearance of the emerged plantlets remained similar and the survival level
of seedlings acclimatized in the greenhouse was >80 %. The FCM analysis of greenhouse
grown plants raised from control seeds and seeds cryopreserved after 6 h PVS2 treatment
showed similar DNA profiles indicating that ploidy level of plants remained unchanged
(Galdiano et al., 2014).
Based on the results of the studies mentioned above, we conclude that plants
developed from cryopreserved seeds are likely to be morphologically and cytologically
normal. However, further comparative studies on many different species are necessary
before general conclusions can be made on the whole orchid family with respect to
phenotypic and genotypic uniformity of plants grown from cryopreserved orchid seeds.
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4. Cryopreservation of cultured protocorms and protocorm-like bodies (PLBs)

Protocorms represent a specific stage of orchid development. Originating from the seed
embryo, the protocorm represents the earliest stage of seedling formation (Vinogradova
and Andronova, 2002). PLBs resemble protocorms in shape and structure. However, unlike

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protocorms, they can be derived in vitro from both germinating embryos and somatic
tissues (Martin and Madassery, 2006; Yam and Arditti, 2009). During the early stages of

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PLB formation, the cells show cytological characteristics and have cell wall markers

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similar to those present during zygotic embryo development; consequently, PLBs are
thought to be orchid somatic embryos (Lee et al., 2013).
Both protocorms and PLBs have a potential to regenerate into plants and are

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widely used for large-scale in vitro propagation of orchids representing a valuable
recource for commercial propagation and conservation (Dasgupta et al., 2004; Teixeira da
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Silva, 2013b). A major advantage of the PLBs over seeds is their availability throughout
the year. Due to the larger size of PLBs compared to dust-like orchid seeds, it is easier to
implement the steps of vitrification-based protocols, which require manipulation of
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samples in highly viscous solutions (Thammasiri, 2000; Merritt et al, 2014). However,
cryopreservation of both protocorms and PLBs is impaired due to their generally high WC
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(80-97%) and hypersensitivity to desiccation (Cripps and McGregor, 2006; Sopalun et al.,
2010). Electron microphotographs of cultured PLBs show that they consist mostly of
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highly vacuolated cells with thin layers of peripheral cytoplasm (Miao et al., 2005).
Occasional clusters of smaller cells with relatively large nuclei, possibly with a high
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division rate, were observed only at the surface (Miao et al., 2005).
Cryopreservation of orchid protocorms was first attempted by Dubus (1980) who
employed preculture in medium containing sucrose, glucose and glycerol in various
combinations. Precultured protocorms were cryopreserved, directly or step-wise, to -30°C
or -196°C. Though exposure to cryogenic temperature was lethal for all samples, those
cooled to -30°C displayed 20-100% survival. Since then, cryopreservation of in vitro
cultured orchid protocorms and PLBs has been accomplished using air-desiccation,
vitrification solution treatment and encapsulation-based methods (Table 3, Fig. 2).
Nonetheless, post-cryopreservation regrowth above 60% has been achieved only for some
Dendrobium and Cymbidium species (Wang et al., 1998; 1999; Kondo et al., 2001; Yin
and Hong, 2009; Vendrame and Faria, 2011; Mohanty et al., 2012, 2013). With the
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majority of other species, regrowth of cryopreserved protocorms and PLBs varied from 10
to 50% (Table 3).

4.1. Comparison of cryopreservation techniques for protocorms and PLBs

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Vitrification and droplet-vitrification have been effective when cryopreserving protocorms

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and PLBs of some Cymbidium and Dendrobium genotypes (Table 3, Fig. 2). Generally, the
optimal dehydration time using PVS2 was as short as 10-20 min at room temperature

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(Kondo et al., 2001; Vendrame and Faria, 2011; Galdiano et al., 2012). In contrast, one-
hour PVS2 treatment was used for Dactylorhiza fuchsii protocorms (Nikishina et al., 2001)

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and Miltonia flavescens × Brassia longissima hybrid PLBs (Popova et al., 2010). With
Dendrobium nobile PLBs, the optimal time of PVS2 exposure was determined to be 85
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min at 25°C and 115 min at 0°C (Mohanty et al., 2012).
Encapsulation-based, synseed techniques were effective with protocorms and PLBs
of Dendrobium and Brassisium hybrids, Dendrobium gratiosissimum Rchb.f.,
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Grammatophyllum speciosum Blume, Heterotaxis sessilis (Sw.) F.Barros, Oncidium spp.,

Paralophia epipyhtica (P.J.Cribb, Du Puy & Bosser) P.J.Cribb, Phalaenopsis bellina
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(Rchb.f.) Christenson, Cleisostoma arietinum (Rchb.f.) Garay and Vanda coerulea

(Flachsland et al., 2006; Hirano et al., 2006; Maneerattanarungroj et al., 2007; Jitsopakul et
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al., 2008; Bunnag et al., 2009; Khoddamzadeh et al., 2011; Subramaniam et al., 2011;
Gogoi et al., 2013). The optimal WC of dehydrated beads for cryopreservation varied
between 20 and 43%, and moderate survival of 11 to 67% was achieved. Alternatively,
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Teixeira da Silva (2013a) found desiccation of encapsulated PLBs to have a detrimental

effect on post-cryopreservation survival, such that the optimal WC of PLB-synseeds was
ca. 60%. Preculture on ABA-containing medium followed by air-drying to an optimal WC
between 0.1 and 0.5 g H2O/gDW (10-33% FW basis) was critical to cryopreserving PLBs
of Dendrobium candidum (Bian et al., 2002). Despite the abundance of experimental data
available, rationalization on the effectiveness of these different cryopreservation
techniques is challenging since all materials would have been at different physiological
states and subjected to varying preculture and recovery treatments. To date, the most
detailed comparison of three cryopreservation techniques was undertaken by Sopalun et al.
(2010) using Grammatophyllum speciosum protocorms. The highest survival was achieved
with droplet-vitrification (38%) followed by encapsulation-dehydration (24%) and
encapsulation-vitrification (14%). Droplet-vitrification enables higher cooling (over 100
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°C s-1) and rewarming rates due to the direct contact of the samples, covered only by a
small volume of VS, with LN or the rewarming solution, respectively (Kim et al., 2009;
Teixeira et al., 2014). Rapid cooling and rewarming can be particularly important for
successful cryopreservation of partially dehydrated explants such as protocorms. The

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significant advantage of high cooling and rewarming rates allowed by foils compared to
vial-based cryopreservation has been reported for shoot tips of potato, garlic and some

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other plants (Leunufna and Keller, 2005; Panis et al., 2005; Kim et al., 2006; 2012a).
One advantage of vitrification in cryo-ampoules (cooling rate ca. 3 ˗ 7 °C s-1) over

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slower, programmed cooling (0.5 °C min-1) has been shown for Dactylorhiza fuchsii
protocorms (Nikishina et al., 2007). Ivannikov (2012) tested encapsulation-vitrification

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versus programmed cooling for cryopreserving protocorms and seedlings of Dendrobium
aphyllum (Roxb.) C.E.C.Fisch., Dendrobium nobile, Paphiopedilum insigne (Wall. ex
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Lindl.) Pfitzer, Phalaenopsis hybrid and Heterotaxis sessilis . No survival was achieved
using programmed cooling with all genotypes. Encapsulation-vitrification was found more
promising and resulted in 12% recovery of Heterotaxis sessilis protocorms. The highest
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regrowth of PLBs of medicinal orchid Dendrobium nobile after cryopreservation was

achieved by encapsulation-vitrification (76%) as compared to encapsulation-dehydration
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(50%) (Mohanty et al., 2012). Cripps and McGregor (2006) reported that protocorms of
Paralophia epiphytica (P.J.Cribb, Du Puy & Bosser) P.J.Cribb, a rare threatened epiphytic
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orchid from Southern Madagascar, could not withstand treatment with PVS2. Survival
after cryopreservation was observed only with the encapsulation–dehydration method after
protocorms had been dehydrated below their high moisture freezing limit determined by
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differencial scanning calorimetry.

The most comprehensive analysis of cryopreservation techniques as applied to in
vitro cultured PLBs was performed by Teixeira da Silva (2013a) with Cymbidium Twilight
Moon `Day Light`. The major important factors of cryopreservation procedures identified
and optimized were explant type (intact PLBs or their sections), osmotic pretreatments
with various concentrations of sucrose, mannose, PEG-6000 and DMSO, preculture
durations (0-48 h), and cryopreservation techniques (encapsulation-dehydration versus two
vitrification-based protocols). Optimum dehydration time, preculture with sucrose and
releasing PLBs from alginate beads before plating them on regrowth medium were critical
for successful cryopreservation (Teixeira da Silva, 2013a).
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4.2. The effect of osmotic pretreatment and abscisic acid (ABA) on survival of
cryopreserved orchid specimens

Preliminary culture on medium containing elevated concentrations of sucrose, sorbitol or

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ABA has proved to be essential for post-cryopreservation regrowth of orchid explants
(Tables 3 and 4). Preculture of protocorms or PLBs on medium supplemented with 0.4-

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0.75 M sucrose for 1-2 days is the most commonly employed (Table 3). Alternatively,
prolonged preculture for 14 and even 20 days on a medium with high sucrose content can

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be combined with glycerol or sorbitol, e.g., for PLBs of Cymbidium cv. “Suvaley Venus”
(Kondo et al., 2001) and Oncidium hamana “Elfin” (Miao et al., 2005). Sucrose at low

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concentration was found to be superior to other osmotic pretreatments including various
concentrations of mannose, PEG-6000 and DMSO for cryopreserving PLBs of Cymbidium
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Twilight Moon `Day Light` (Teixeira da Silva, 2013a). In contrast, high sucrose
concentration (0.7 M rather than 0.3 – 0.5 M) in the preculture medium for 20 h was
essential for cryopreservation success with encapsulated Cymbidium protocorms; with
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recovery of up to 70% compared to 3-4% for low sucrose treatment (Gogoi et al., 2013).
Step-wise preculture in a series of sucrose-enriched media was reported to be effective
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with protocorms of Oncidium bifolium Sims (Flachsland et al., 2006), Cleisostoma arietinum
(Maneerattanarungroj et al., 2007) and Dendrobium virgineum (Maneerattanarungroj,
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2009). For Dendrobium virgineum the first step of preculture with 0.25 M sucrose was
combined with 1 week cold-hardening at 4°C (Maneerattanarungroj, 2009). In the hybrid
orchid Miltonia flavescens × Brassia longissima, however, various pretreatments including
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combinations of sucrose, ABA and reduced glutathione were not beneficial for post-
cryopreservation regrowth of PLBs (Popova et al., 2010). By contrast, increasing
concentration of 6-benzylaminopurine in post-culture medium to 5.0 and 10.0 mg/l during
the first 3 weeks of post-cryopreservation recovery resulted in 10 and 20% regrowth,
respectively. Overall, the existing literature does not show any obvious specificity of the
effect of ABA treatment between tropical and temperate orchids.
The mechanisms underlying the effect of sucrose preculture on dehydration and
cryopreservation tolerance of orchid materials require further investigation. From the
reports on cryopreservation of axillary buds, meristem and cell cultures, it is known that
preculture with high sucrose levels significantly affects cell metabolism, alters gene
expression (Reinhoud et al., 2000b; Carpentier et al., 2007), induces the accumulation of
specific amino acids and soluble sugars (Suzuki et al., 2006; Zhu et al., 2006), as well as
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changes protein and fatty acid composition (Jitsuyama et al., 2002; Carpentier et al.,
2007). Indeed, in Phalaenopsis bellina, preculture of encapsulated PLBs with 0.75 M
sucrose for 3 days resulted in accumulation of sucrose up to 7 mg/g DW compared with 1
mg/g DW sucrose in control PLBs (Khoddamzadeh et al., 2011). Metabolic changes

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caused by preculture were normally accompanied by alterations in cell ultrastructure, such
as reduction of vacuoles, condensation of cytoplasm, accumulation of starch and changes

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in endoplasmic reticulum (Jitsuyama et al., 2002; Mikula et al., 2005). Similarly, electron
microphotographs of Oncidium PLB cells after a 2 week-pretreatment with sucrose and

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glycerol showed an increased number of starch grains both around the nuclei and in the
plastids (Miao et al., 2005).

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ABA has been reported to induce desiccation and cryopreservation tolerance in
cultured plant cells and organs (Chen et al., 1985; Reed, 1993; Chetverikova, 1999;
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Hoekstra et al., 2001; Lu et al., 2009). Preculture with 0.1-0.5 mg/l ABA improved post-
cryopreservation performance of Dendrobium candidum protocorms and PLBs
cryopreserved by the vitrification method (Wang et al., 1998; 1999). Bian et al. (2002)
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recorded extensive accumulation of soluble sugars, heat-stable proteins and dehydrins in

PLBs of Dendrobium candidum following pretreatment with 0.1 mg/l ABA for 3 days. The
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same protein bands could be detected in non-pretreated PLBs after air-drying to 1.0 g
H2O/g (50% WC). In their study, a 3-day pretreatment with ABA improved survival of
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PLBs desiccated to 0.5 g H2O/g (33% WC); however, there was no positive effect of ABA
treatment on survival after cryopreservation (Bian et al., 2002). Laterly, it was proposed
that ABA and sucrose in the preculture medium trigger different induction mechanisms for
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desiccation tolerance in Spathoglottis plicata Blume protocorms (Wang et al., 2003).

Preconditioning on medium containing 0.1 to 1.0 mg/l ABA proved to be essential for
successful cryopreservation of cultured shoot primordia, cell suspension and rhizomes of a
number of orchid species (Table 4). In Dendrobium gratiosissimum protocorms, preculture
in liquid medium supplemented with 1 mg/l ABA for 7 days resulted in higher survival
after cryopreservation than preculture with elevated concentrations of sucrose: 67 and 9%
survival, respectively (Bunnag et al., 2009).
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4.3. Recovery growth of protocorms and PLBs and their physiological, biochemical and
cytological responses to cryopreservation

The regrowth and physiological responses of protocorms and PLBs to various steps of

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cryopreservation including preculture, dehydration, cooling and rewarming, and unloading
treatments have been studied in several orchids.

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It is likely that regrowth of cryopreserved orchid protocorms and PLBs is mostly
associated with newly developed PLBs, which could be formed from surviving tissues

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located in the peripheral zones of cryopreserved protocorms. However, information about
the effect of cryopreservation treatment on the ability of explants to produce new PLBs is

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contradictory. One hour treatment with PVS2 enhanced emergence of new PLBs in
Dactylorhiza fuchsii protocorms (Nikishina et al., 2007). Similarly, protocorms developed
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from cryopreserved seeds of Bratonia hybrid orchid had greater potential to multiply than
protocorms from non-cryopreserved seeds (Popova et al., 2003). Ivannikov (2012)
confirmed that cryoprotective treatment resulted in the stimulation of vegetative
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propagation in protocorms of Heterotaxis sessilis and Phalaenopsis spp., by 19 and 38%,

respectively. By contrast, formation of new PLBs from both intact and segmented PLB
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explants of Cymbidium was significantly suppressed by any treatment, including

encapsulation in alginate beads, osmotic pretreatment and desiccation (Teixeira da Silva,
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2013a).
When re-growth of maternal (cryopreserved) PLBs was assessed, a 5-day lag phase
was recorded compared to non-cryopreserved explants (Yin and Hong, 2009). However,
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other phenotypic parameters measured (including length of plantlets, internode lengths,

number of leaves and number of roots) were not significantly affected by cryopreservation
(Yin and Hong, 2009). Similarly, regrowth of cryopreserved and rewarmed protocorms of
Dendrobium virgineum was slow compared to non-cryopreserved cultures but no
morphological difference in protocorms or subsequently developed plants were observed
(Maneerattanarungroj, 2009).
Chlorophyll content markedly decreases in PLBs of Dendrobium cv. Bobby
Mesinna when cryopreserved using encapsulation-dehydration (Zainuddin et al., 2011).
Similarly, total chlorophyll content in PLBs of Brassidium cv. Shooting Star decreased
from 2.817 μg/g DW in the control to 0.256 μg/g DW in cryopreserved PLBs (Yin et al.,
2011). Closer investigations of the activities of two photosystems in PLBs of Bratonia
hybrid orchid at various stages of the vitrification protocol showed that the activities of
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noncyclic electron flow and of alternative electron transport pathways related solely to PSI
were retained both after exposure to PVS2 and immediately after cryopreservation.
However, the photosynthetic activity was totally inhibited after 24 h post-culture (Bukhov
et al., 2006; Popova et al., 2010). These observations suggested that cryopreservation per

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se did not cause immediate death of PLBs, but induced the irreversible damages that
eventually resulted in death of PLBs during post-cryopreservation culture.

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In PLBs of some Dendrobium and Brassidium cultivars, cryopreservation using
encapsulation-dehydration resulted in a significant decrease in protein content and elevated

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peroxidase activity (Yin et al., 1011; Zainuddin et al., 2011). Superoxide dismutase (SOD)
activity in PLBs of Dendrobium Sonia-28 increased following preculture and PVS2-based

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dehydration, and reached 54 and 148 U.mg−1 protein after 24 h and 1 week of post-
unloading recovery, respectively compared to 30 U.mg−1 protein in control (untreated)
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PLBs (Poobathy et al., 2013c). Oxidative stress can certainly increase in plant cells
submitted to cryopreservation as the procedure involves severe dehydration and treatment
with potentially toxic chemicals (Benson and Bremner, 2004; Ren et al., 2013), depending
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on their concentrations and exposure conditions (time and temperature). Elevated

peroxidase activity in PLBs after cryopreservation may reflect the need for decomposition
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of excessive hydrogen peroxide, thus providing evidence for activation of enzyme-driven

antioxidant protection systems in these explants (Ren et al., 2013). Indeed, Liu et al. (2013)
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found a significant negative correlation between malondialdehyde content in Dendrobium

wardianum R.Warner PLBs and their survival after exposure to LN. The addition of
antioxidants (reduced glutathione, glycine betain, ascorbic acid, melatonin, etc.) at various
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stages of the cryopreservation process is known to reduce oxidative stress and enhance
post-cryopreservation regrowth of many different plant materials, such as blackberry shoot
tips (Uchendu et al., 2010), Rhodiola crenulata (Hook.f. & Thomson) H.Ohba callus (Zhao
et al., 2011), American elm shoot tips (Uchendu et al., 2013) and Arabidopsis seedlings
(Ren et al., 2014). However, in PLBs of Dendrobium Bobby Messina and Dendrobium
Sonia-28 cryopreserved by vitrification, 0.6 mM ascorbic acid added during preculture,
loading, unloading and growth recovery steps could not improve survival (Anthony et al.,
2013; Poobathy et al., 2013b). For this hybrid orchid, 16% post-cryopreservation recovery
of PLBs could be achieved by adding activated charcoal to the regrowth medium
(Poobathy et al., 2013b)
Histological observations and SEM studies of the encapsulated PLBs of
Dendrobium Bobby Messina subjected to dehydration and cryopreservation revealed
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severe damage of PLBs (Anthony et al., 2014). Accumulation of mass homogenous cell
populations and dense cytoplasm were detected in cryopreserved PLBs three months after
rewarming, while no such effects were observed in non-cryopreserved PLBs. This
indicates that plasmolysis caused by dehydration could have a stronger effect on PLB

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regrowth than cooling-rewarming per se (Anthony et al., 2014).
Only a few studies have addressed chromosome and genetic stability of protocorms

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and PLBs after cryopreservation. Ploidy level of Vanda coerulea protocorms remained
unchanged following cryopreservation by encapsulation-dehydration in combination with

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LS treatment (Jitsopakul et al., 2008). Recently, Anthony et al. (2012) reported the results
of RAPD analysis in PLBs of Dendrobium Bobby Messina following a PVS2-based

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vitrification method. Of the 10 primers with regenerated bands, six indicated genetic
stability while three primers and one primer showed polymorphism and partial
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polymorphism, respectively (Anthony et al., 2012). RAPD analysis confirmed the genetic
marker stability of regenerated Dendrobium virgineum protocorms (Maneerattanarungroj
2009) and Vanda coerulea PLBs (Jitsopakul et al., 2011) after cryopreservation by
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encapsulation-dehydration and droplet-vitrification, respectively.

Teixeira da Silva (2013a) reported a progressive decrease in survival of the hybrid
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Cymbidium PLBs as the period of their cryogenic storage increased from one week to one
month and one year. Finally, survival of PLBs stored for one year was reduced by 40-54%
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as compared to one-week storage and by 55-63% as compared to non-cryopreserved PLBs.

However, new PLBs developed from cryopreserved materials were morphologically
normal as assessed by light and electron microscopy. Shoots that formed from newly
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derived PLBs also appeared to be normal (Teixeira da Silva, 2013a).

5. Cryopreservation of in vitro shoot apices and meristematic tissues

Unlike cryopreservation of orchid seeds, protocorms and PLBs, investigations into

cryopreservation of other explants are limited (Table 4, Fig. 2). Na and Kondo (1996)
developed a very promising method for orchid cryopreservation, which yet remains under-
evaluated. Shoot tips of in vitro developed protocorms were excised and transplanted to
culture medium with predetermined combinations of growth regulators to support their
multiplication. The cultures formed new shoot primordia (meristematic clusters), which
could be further subcultured and propagated or used as starting material for
cryopreservation. Electron microphotographs showed that meristematic clusters thus
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formed consisted of a single parenchymal layer, meristematic region, and inner

parenchyma of a nodule of shoot primordia. In early experiments, tissue-cultured shoot
primordia of Vanda pumila Hook.f. were precultured with different ABA concentrations
for 1-6 days, desiccated over silica gel, placed in cryovials and cryopreserved by direct

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immersion in LN. The highest survival (65%) was observed when shoot primordia were
precultured with 1.0 mg/l ABA for 3 days and then desiccated to 24% WC (for 7 days).

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The range of optimum WCs for cryopreservation was very narrow. Though shrinkage of
samples was detected by EM upon desiccation, it was followed by rapid rehydration and

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full recovery of their original size after explants were transferred to post-culture medium.
No callus formation was observed in the experiment. The chromosome number remained

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unchanged in tissues after cryopreservation. Later Kondo et al. (2001) employed the same
protocol to cryopreserve cultured shoot primordia of Cattleya loddigesii var. hassisoniana ,
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Cattleya walkeriana Gardner and Dendrobium cv. Yukidaruma. With all species, the
method resulted in nearly 100% regrowth (Table 4).
Three cryopreservation techniques (encapsulation-dehydration, vitrification and
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droplet-vitrification) were tested for Vanilla planifolia apices dissected from in vitro grown
plantlets. The plantlets used as source material were either obtained from microcuttings or
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formed from fragmented basal tissues (Gonzalez-Arnao et al., 2009). Cryopreservation

using the encapsulation-dehydration technique was unsuccessful, regardless of the source
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material and the preculture treatments. With vitrification, various combinations of

preculture, LS, PVS2 and PVS3 treatments were tested. For apices excised from plantlets
originating from microcuttings, PVS2 was found to be highly toxic, irrespective of the LS
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employed. The highest survival (30%) and regeneration (10%) of new shoots were
obtained for those apices through a three-step droplet vitrification protocol: 1-day
preculture with 0.3 M sucrose, LS for 20˗30 min, and dehydration with PVS3 for 30 min at
room temperature. Proteomic analysis using two-dimentional electrophoresis revealed that
17 proteins changed expression in apices treated with PVS3 compared to control ones
(González-Arnao et al., 2011).
Apices isolated from in vitro cultured plants of two Cymbidium cultivars displayed
over 90% regrowth when cryopreserved using vitrification (Thinh and Takagi, 2000). The
highest survival after cryogenic exposure was obtained for explants consisting of the apical
dome partially covered with leaf primordia followed by meristems with uncovered or fully
covered apical domes. The optimal duration of LS and PVS2 exposure were determined as
20 and 10-20 min, respectively. Dendrobium Walter Oumae shoot tips cryopreserved via
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an encapsulation-dehydration protocol, following preculture with 0.3 M sucrose for 2 days,

showed 13% recovery and developed normal plants that could be successfully acclimated
in a greenhouse (Lurswijidjarus and Thammasiri, 2004). Therefore, cryopreservation of
orchid meristematic tissues depended on sufficient dehydration and application of

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cryoprotectants following pretreatment with ABA or 10% sucrose. Cryopreservation of
meristematic clusters has obvious advantage over cryopreservation of isolated shoot tips

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due to the large quantity of plant material normally required for protocol development.

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6. Cryopreservation of orchid cell suspensions and isolated protoplasts

Suspension and callus cultures of plant cells have applications for mass propagation of

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plants and tissues in bioreactors for the production of phytochemicals (Chattopadhyay et
al., 2005; Malik et al., 2011; Nosov et al., 2014; Thanh et al., 2014), and their potential for
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cryobanking has been investigated extensively (e.g. Diettrich et al., 1982; Reinhoud et al.,
2000a; Kim et al., 2001; An et al., 2003; Popova et al., 2009). Isolated protoplasts offer
unique opportunities for developing novel germplasm through somatic hybridization,
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organelle transfer, protoclonal variation, and direct insertion of DNA (Liu et al., 2003;
Jones et al., 2012). However, information on the cryopreservation of orchid cell cultures
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and isolated protoplasts is rather limited. Doritaenopsis cv. New Toyohashi suspension
cultures have been successfully cryopreserved following preculture in liquid medium with
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0.1 M sucrose and 1.0 mg/l ABA for 1 week, 15 min exposure to LS and 1-3 h dehydration
in PVS2 (Tsukazaki et al., 2000). PLBs induced from cryopreserved cells showed no
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morphological variations. Chen et al. (2001) reported the cryopreservation of Dendrobium

candidum protoplasts using a vitrification method with 48% post-thaw survival.

7. Cryopreservation of orchid rhizomes

Orchid rhizomes are widely used as the main propagation material for Asian
temperate orchids of both high ornamental value and endangered status (Paek and Murthy,
2002). Cryopreservation of in vitro propagated rhizomes of Cymbidium kanran Makino, an
endangered Asian orchid, has been attempted using two methods: preculture-desiccation
and vitrification using modified LSs and VSs (Kim et al., 2009). Vitrification resulted in
no survival regardless of the preculture treatment and composition of cryoprotective
solutions employed. DSC profiles showed that crystallization and ice melting events still
occurred in rhizome sections after 5-6 h dehydration with VS. At this step of the protocol,
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survival of rhizomes without cryopreservation had dropped to nearly 20% due to the high
toxicity of the VS. By contrast, 90% regrowth was achieved when rhizome sections were
precultured on ABA-containing medium and preconditioned with a series of sucrose
solutions of gradually increasing concentrations (up to 0.7 M), followed by desiccation to

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23-30% WC (for 17-24 h) and direct exposure to LN (Fig. 3, Popova et al., unpublished
data).

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8. Cryopreservation of transgenic orchid materials

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Despite a large diversity of successful protocols developed for orchid transformation

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(Teixeira da Silva et al., 2011), investigations on the effectiveness of cryopreservation for
orchid transgenic materials are limited. Transgenic Cymbidium PLBs derived from particle
bombardment and containing pWI-GUS plasmid vector were successfully cryopreserved
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following a vitrification protocol (Teixeira da Silva, 2013a). Explants could be recovered
and they produced new PLBs after one year of cryogenic storage, though their survival was
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lower compared to non-cryopreserved PLBs and shorter storage periods. The non-
transgenic PLB line, which was also cryopreserved in this study showed a similar tendency
of decreasing survival following the prolonged storage in liquid nitrogen (Teixeira da
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Silva, 2013a). This observation suggests that transgenic nature of the orchid tissue may not
affect the process of cryopreservation. Further studies with a range of transgenic orchid
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plants and tissues are needed to confirm this assumption.

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9. Systematic cryopreservation research on orchid cryopreservation in their countries

of origin

In most cases, successful examples of orchid cryopreservation are restricted to occasional

reports on individual species or taxon groups. However, in some countries, often rich in
orchid diversity, cryopreservation has been addressed systematically and resulted in year-
by-year development of effective methodologies. Investigations conducted at Mahidol
University, Thailand led to the successful cryopreservation of at least 13 native Thai
orchids using both seeds and protocorms (Thammasiri, 2008). This comprehensive work
included scanning electron microscopy of orchid seeds after various treatments and
addressed the details of post-cryopreservation plant development (Lurswijidjarus and
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Thammasiri, 2004; Pornchuti and Thammasiri, 2008; Tammasiri, 2008; Sopalun et al.,
2010; Jitsopakul et al., 2008; 2011; 2012).
A significant research programme has been established by Japanese researchers
who applied vitrification-based methods to orchid seeds with high WC (see Hirano et al.,

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2006). This team reported successful cryopreservation of Bletilla striata (Ishikawa et al.,
1997; Hirano et al., 2005a), Ponerorchis graminifolia (Hirano et al., 2005b), Phaius

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tankervilleae (Hirano et al., 2009) and seven Cymbidium species (Hirano et al., 2011).
In Russia, cryopreservation techniques have been developed for seeds and

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protocorms of terrestrial European orchids including Cypripedium, Dactylorhiza, Epipactis
and Platanthera spp. and over 100 species of tropical epiphytes from the collection of the

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Main Botanical Garden, Moscow (Nikishina et al., 2001; Popov et al., 2006; Nikishina et
al., 2007; Nikishina, personal communication).
Studies at King’s Park and Botanic Garden, Perth, Australia have concentrated on
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the cryopreservation of rare and endangered native orchids, including Caladenia, Diuris,
Paralophia, Pterostylis and Thelymitra spp. (Batty et al., 2001; Cripps et al., 2006;
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Watanawikkit et al., 2012).

Scientific investigations on orchid germplasm at the Royal Botanic Gardens, Kew,
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UK have tended to take a broadly comparative approach, in search for the underlying
principles that could enable the widescale conservation of species (Pritchard, 1984;
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Pritchard and Prendergast, 1989; Seaton and Pritchard, 1993; Pritchard et al., 1999; Marks
et al., 2014).
Other national efforts have been made in Brazil (Vendrame, 2007; Vendrame and
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Faria, 2011; Galdiano et al., 2012, 2013, 2014) and in the Ukraine (Ivannikov, 2012).

10. Conclusion: Strategies for future research on cryobanking for long-term

conservation of orchid diversity

The results summarized in this review highlight significant progress in orchid

cryopreservation during the past few decades. The number of species for which
cryopreservation of seeds or isolated explants has proved to be successful has increased
many-fold since the 1980s, when the first reports on orchid cryopreservation were
published (Dubus, 1980; Pritchard, 1984). However, considering the diversity of
Orchidaceae, these reports cover < 0.5% of orchid species. The summary in Table 5 clearly
demonstrates that the genetic diversity of Orchidaceae is poorly covered in
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cryopreservation research. For example, none of the species of the subfamily

Apostasioideae has been tested for cryopreservation, and subfamilies Cypripedioideae and
Vanilloideae are represented by a single species each. Most tribes are under-represented
(less than 5 species have been assessed). Similarly, the vast majority of commercial

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genotypes tested are limited to the genera Cymbidium and Dendrobium.
Despite the variety of plant cryopreservation techniques available, only a few have

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been rigourously tested with orchids so far, and a negligible part of this knowledge has
been used to create cryogenic collections of orchid germplasm. There has been significant

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success in cryopreserving a range of tissue types with high levels of survival and
regeneration, supporting biotechnology-based breeding programmes and conservation of

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many species. However, to the best of our knowledge, such an integrated program and
research facility currently does not exist for orchids globally.
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As for many other endangered plants, research on orchid cryostorage depends on
the availability of quality collections made under legally binding access and benefit
sharing agreements that comply with the requirement of the Convention on Biological
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Diversity, including the recent Nagoya Protocol. However, such recent and historical
collections in botanical gardens often lack good infra-specific variability for the species,
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for example at the ecotypic, adaptive level. As successful cryopreservation is highly

dependent on the physiological status of the specimen, characterization of a range of
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ecotypes and genotypes may be rewarding. Such an approach will help to improve the
identification of the rate limiting steps in the cryopreservation process based on inferred
robustness of the specimen to cumulative stress. A systematic approach to orchid
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cryopreservation research should focus on the most responsive materials, controlled

manipulation of specimen physiology, thermal analysis of biophysical stability, and the
optimization of cryoprotectant mixtures and cryopreservation techniques. Similar,
systematically planned research programmes have proven to be highly effective for potato
(Kaczmarczyk et al. 2011; Niino and Valle Arizaga, 2015), Allium (Kim et al., 2012a),
Musa (Panis et al., 2005), Malus (Volk et al., 2015), Wasabia (Niino and Arizaga, 2015)
and Panax (Kim et al., 2008, Kim et al., unpublished data).
A strategy and action plan for the cryobanking of orchid germplasm are proposed
in Fig. 4. A key consideration for method design and application is the choice of material,
each of which generates logistical challenges. Among all the propagules available for
orchid conservation [i.e., seeds, pollen, shoot tips, meristematic tissues, protocorms or
PLBs and rhizomes (Table 5)], seeds are the most obvious to start with. Their minute size
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is excellent for biobanking, allowing over 3,000 seeds to be stored in one 2-ml cryogenic
vial (Seaton et al., 2010; Nikishina, personal communication). As orchids are generally
outbreeding, the seeds potentially represent a large diversity of parental genotypes, which
is crucial when conserving wild species. However, orchid seed is generally short-lived, and

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may lose viability within weeks to months if poor storage conditions are applied (Thornhill
and Koopowitz, 1992; Pritchard et al., 1999; Hay et al., 2010). Nonetheless, it remains the

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case that the seed longevity of the vast majority of orchid species remains unknown.
Similarly, credible methods for seed germination have been developed for only a few of

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the most commercially important groups of species. Moreover, in vitro germination may
not work well for all species, particularly with terrestrial genotypes in which seed

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dormancy might be present (Rasmussen, 1995; Dowling and Jusaitis, 2012; Hosomi et al.,
2012; Jitsopakul et al., 2012). To reduce such potential problems, immature seeds are often
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harvested as germination is easier (Hirano et al., 2005a) and the initiation of clean cultures
from immature green pods is also easier. However, immature seeds may have high water
content, which needs to be carefully controlled, and potentially sub-optimal longevity
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when dried. Thus, research efforts should clarify any risks associated handling immature
vs mature orchid seeds vis-à-vis any trade-off between efficient seed germination and
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reduced lifespan. Optimum conditions for the long-term storage of pollen should be
pursued in support of end-users in the horticultural trade and in conservation. For example,
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stored pollen of species with exceptionally small populations could be used to cross
pollinate plants from the wild and ex situ living collections, thereby enhancing genetic
diversity and producing seeds for storage and use.
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Protocorms and PLBs are excellent starting material for cryopreservation but they
are sensitive to the toxic effects of PVS2 and PVS3. Therefore, research should further
explore alternative cryoprotectant solutions developed within the past few years (Kim et
al., 2009; 2012b; Shin et al., 2012; Vujović et al., 2015) and the use of supplements, such
as antioxidants. Many antioxidants, such as ascorbic acid, reduced glutathione, glycine
betaine and melatonin have been shown to be effective in alleviating stress in
cryopreservation (Uchendu et al., 2010; Zhao et al., 2011; Uchendu et al., 2013; Ren et al.,
2014). Thermal analysis of orchid materials during cooling and rewarming by using DSC
or other available methods (Bruňáková et al., 2011; Skyba et al., 2011; Kim et al., 2012a;
Teixeira et al., 2014) can provide useful information about the efficiency of cryoprotective
solutions and the phase behaviour of water (e.g., the unfrozen water content) during
cooling and warming. Recent techniques such as droplet-vitrification and the cryoplate
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method, are known to be effective for some plant species and specimens (Lee et al., 2011;
Shin et al., 2012; Kim et al., 2012b; Vujović et al., 2015; Yamamoto et al., 2015) and
worth testing with PLBs.
For vegetative material, shoot tips and cultured shoot primordia are considered to

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be the most preferred material for the long-term conservation due to their high genetic
stability and potential responsiveness to cryotherapy (Wang et al., 2014). However,

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experimentation with orchid shoot primordia has remained infrequent due to the limited
availability of the plant material (Kondo et al., 2001). The same is true for in vitro cultured

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rhizomes on which only a few studies have been conducted despite their usefulness in
cryopreservation of endangered as well as commercially important species (Paek and

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Murthy, 2002).
An important methodological difficulty also exists since the steps of experimental
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protocols tested on one or a few genotypes in a genus may not work with others (Reed,
2001). Nonetheless, given the enormous diversity of orchid species and materials, there are
two approaches that warrant wider testing: the preculture of tissues with ABA (Na and
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Kondo, 1996; Wang et al., 1998; Wang et al., 1999; Bunnag et al., 2009); and the use of
encapsulation techniques that allow the cryopreservation of orchid seeds with their fungal
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symbionts (Wood et al., 2000; Sommerville et al., 2008).

In conclusion, cryobanking of orchid biodiversity offers an excellent opportunity
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for fundamental and applied research on plant conservation due to the uniqueness of orchid
growth and development. Challenges remain, regarding very limited funds available for
this kind of research in the emerging economies, the difficulty of collecting samples from
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remote regions and the general lack of knowledge on seed germination in species of this
large family. Nonetheless, there is the political will and policy framework for the
acceleration of such work. The Aichi Targets (2010-20) of the Convention on Biological
Diversity emphasize the need to safeguard genetic diversity of socio-economically
important and culturally valuable species (https://www.cbd.int/sp/targets/). Orchids are
ideal match for this ambition. Orchid cryobanks can serve as backups for living
collections, and plants derived from cryogenically stored material can be propagated and
later reintroduced into their native habitats. In addition, orchids are perfect candidates for
raising public awareness of the importance of applying science for environmental,
economic and cultural impact, including through global connectivity, such as the OSSSU
(Orchid Seed Science and Sustainable Use; osssu.org) network.
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Acknowledgement. We are grateful to Prof. Vendrame, Univeristy of Florida, USA and

Dr. Wang, University of Guelph, Canada for their help in preparing this review. The Royal
Botanic Gardens, Kew receives grant-in-aid from Defra.

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ED

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SC
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NU
MA
ED
PT
CE
AC
 ACCEPTED MANUSCRIPT

Table 1. Examples of subzero and cryopreservation of pollen of some orchid species.

Species1 Precondi- Method3 Cryopro- Storage Pollen Reference

tioning2 tectants 4 temperature germination
and control/cryo,
duration %5

PT
Anacamptis Fresh Programmed 0.5 M -196°C, 42/1 Pritchard and
pyramidalis freezing DMSO 1h Prendergast,
1989

RI
Air-dried (ca. Programmed No -20°C, 60/45 Pritchard and
15%) freezing 12 months Prendergast,
1989

SC
Air-dried (ca. Programmed No -196°C, 60/55 Pritchard and
15%) freezing 12 months Prendergast,
1989

NU
Air-dried (ca. Programmed 0.5 M -20°C, 60/0 Pritchard and
15%) freezing DMSO 12 months Prendergast,
1989
Air-dried (ca. Programmed 0.5 M -196°C, 60/0 Pritchard and
MA
15%) freezing DMSO 12 months Prendergast,
1989
Calanthe Preconditioned Direct No -79°C, -/23-60 Ito (1965)
furcate at 20 to 80% 718 days Remained
ED

(Calanthe humidity fertile6

triplicata)

Cymbidium Fresh Programmed 0.5 or 1.0 M -196°C, 50/40, 44/38 Pritchard and
PT

elegans freezing DMSO 1h Prendergast,

1989
CE

0.5 or 1.0 M -196°C, 54/30, 36/19 Pritchard and

glycerol 1h Prendergast,
1989
AC

Dactylorhiza Equilibrated at Direct No -20°C, 80/64 Marks et al.,

fuchsii 33% RH 6 years 2014
overnight at
16 °C
Fresh Programmed 0.5 M -196°C, 58/23 Pritchard and
freezing DMSO 1h Prendergast,
1989

Air-dried (ca. Programmed No -20°C, 60/58 Pritchard and

15%) freezing 12 months Prendergast,
1989

Air-dried (ca. Programmed No -196°C, 61/62 Pritchard and

15%) freezing 12 months Prendergast,
1989

Air-dried (ca. Programmed 0.5 M -20°C, 60/0 Pritchard and

15%) freezing DMSO 8 months Prendergast,
1989
 ACCEPTED MANUSCRIPT

Air-dried (ca. Programmed 0.5 M -196°C, 61/0 Pritchard and

15%) freezing DMSO 8 months Prendergast,
1989

Dactylorhiza Fresh Programmed 0.5 M -196°C, 31/40 Pritchard and

maculata freezing DMSO 1h Prendergast,
1989

PT
Dendrobium Fresh or Direct No -79°C, 15/0.5-32 Ito, 1965
cv.“Lady desiccated 975 days

RI
Hamilton”

SC
Fresh Direct 10-20% -79°C, 19-39/7-22 Ito, 1965
glycerol, 10- up to 3
20% sugar months
or mixture of

NU
both

Dendrobium Desiccated Direct No -196°C, 48 h 80/>807 Vendrame et

hybrid “Mini >80/>807
MA
over silica-gel al., 2008
WRL” (5%) Vitrif PVS2 -196°C, 48 h

Dendrobium Desiccated Direct No -196°C, 48 h 60/>807 Vendrame et

hybrid “Sena over silica-gel al., 2008
ED

Red” (5%) Vitrif PVS2 -196°C, 48 h >80%/>807

Dendrobium Preconditioned Direct No -79°C, 0-26/0-78 Ito, 1965

nobile at 0 to 100% from 10 to Remained
PT

humidity 712 days fertile after 1

year of
storage6
CE

Fresh or Direct 85-100 % -79°C, -/0-32 Ito, 1965

desiccated glycerol or 10 to 93
glycerol and days
AC

ethylene
glycol
mixture

Dendrobium Preconditioned Direct No -79°C, Remained Ito, 1965

nobile var. at 0 to 60% 53 days fertile6
nobilius humidity

Gymnadenia Fresh Programmed 0.5 M -196°C, 26/1 Pritchard and

conopsea freezing DMSO 1h Prendergast,
1989

Listera ovate Fresh Programmed 0.5 M -196°C, 25/23 Pritchard and

(Neottia ovata) freezing DMSO 1h Prendergast,
1989
 ACCEPTED MANUSCRIPT

Luisia Fresh or air- Direct No -196°C, 48 h 64 or 67/54 Ajeeshkumar

macrantha drying and Decruse,
2013
Drying over Direct No -196°C, 48 h ca. 54/53
silica-gel

Fresh Vitrif PVS2 -196°C, 48 h 57/56 Ajeeshkumar

PT
Remained and Decruse,
fertile6 2013

RI
-196°C, 57/51
668 days

SC
Orchis Fresh Programmed 0.5 or 1.0 M -196°C, 22/12, 28/12 Pritchard and
mascula freezing DMSO 1h Prendergast,
1989

NU
0.5 or 1.0 M -196°C, 28/8, 21/3
glycerol 1h

1
MA
When synonyms are used in the cited papers, the accepted names of the species based
on The Plant List, (http://www.theplantlist.org) are given in brackets.
2
Preconditioning of samples, if any. In braskets: Water content, fresh weight basis (if
reported)
ED

3
Direct: Pollen were sealed in containers and plunged directly into liquid nitrogen; Vitrif:
Vitrification
4
DMSO: dimethylsulfoxide; PVS2: Plant vitrification solution 2 (30% glycerol + 15%
PT

DMSO + 15% ethylene glycol + 13.7% sucrose, w/v, prepared in liquid nutrient medium)
5
Germination of control and cryopreserved pollen. For samples frozen without
cryoprotectants, control indicates germination before freezing. For samples frozen at
CE

presence of cryoprotectants, control indicates germination after cryoprotectant treatment

but without cryopreservation. For simplicity, values are given in round numbers.
5
Remained fertile: pods with viable seeds were produced after pollinating flowers with
AC

pollen retrived from storage.

6
In the study by Vendrame et al. (2008) germination was evaluated by the success of
flower pollination with cryopreserved pollen (success = formation of seed pods with
viable seeds).
 ACCEPTED MANUSCRIPT

Table 2. Examples of successful cryopreservation of mature and immature orchid seeds

(for all species, only optimal conditions for cryopreservation and maximal regrowth
are given).
Species1 Seed WC, Method3 Storage Germination Reference
% FW2 duration control/cryo,

PT
%4

MATURE SEEDS – TERRESTRIAL ORCHIDS

RI
Anacamptis morio 25 (WC of Enc-Deh 1 day 72/95 Wood et al.,2000
+ fungal symbionts alginate

SC
beads)
Bletilla formosana 49.5 Direct 1 day 77/2 Wu et al., 2013
1.9-24.8 Direct 1 day -/68 Wu et al., 2013

NU
49.5 Vitrif 1 day 76/40 Wu et al., 2013
Not given Vitrif 10 min to 1 78/90 Hu et al., 2013
MA
year
Caladenia arenicola 6.9 Direct 1 week 31/37 Batty et al.,2001
Seeds Direct 3-24 months 70-83/55-855 Hay et al., 2010
equilibrated
ED

at 23 and
50% RH
Caladenia flava Seeds Direct 3-24 months 35-80/0-835 Hay et al., 2010
PT

equilibrated
at 5, 13, 23,
50, 62, 75
and 92%
CE

RH
Caladenia huegelii Seeds Direct 3-24 months 63-95/80-975 Hay et al., 2010
AC

equilibrated
at 23 and
50% RH
Calanthe Gorey Not given Direct 1 month 59/90 Nikishina et al.,2001
Calanthe vestita var. 13 Direct 1 month 56/69 Nikishina et al.,2001
rubrooculata Paxt.

Cymbidium goeringii Not given Vitrif 30 min 32/32 Hirano et al., 2011
Cymbidium kanran Not given Vitrif 30 min 0/0 Hirano et al., 2011
Cymbidium Not given Vitrif 30 min 78/82 Hirano et al., 2011
macrorhizon
Dactylorhiza baltica Not given Direct 1 month 33/39 Nikishina et al.,2007
Dactylorhiza fuchsii <4 Direct 1-12 months 74/516 Pritchard et al.,1999
7.9 Direct 1 month 36/44 Nikishina et al., 2007
 ACCEPTED MANUSCRIPT

+ fungal symbionts 20 (WC of Enc-Deh 1 day 72/85 Wood et al., 2000

alginate
beads)
Dactylorhiza incarnata 7.5 Direct 1 month 14/27 Nikishina et al., 2007
Dactylorhiza maculata Not given Direct 1 month 71/42 Nikishina et al., 2007

PT
6
Dactylorhiza majalis Not given Direct 1-12 months 93/63 Pritchard et al.,1999
spp. praetermissa

Disa uniflora 11 Direct 15 min 61/56 Pritchard, 1984

RI
Diuris arenaria+ fungal 11.3 (WC of Enc-Deh 6 months 22/ca.10 Sommerville et al.,
symbionts alginate 2008

SC
beads)
Diuris fragrantissima Seeds Direct 3-24 months <20/<204 Hay et al., 2010
equilibrated

NU
at 23 and
50% RH
Diuris laxiflora Seeds Direct 3-24 months 55-85/20-555 Hay et al., 2010
MA
equilibrated
at 5, 13, 23,
50, 62, 75
and 92%
RH
ED

Diuris magnifica 5.3 Direct 1 week 37/44 Batty et al., 2001

Doritis pulcherrima 31 Vitrif 1 day 91/63 Thammasiri, 2000
PT

(Phalaenopsis pulcherrima)

Eulophia alta 11 Direct 15 min 52/47 Pritchard, 1984

CE

Eulophia gonychlia 7.5 Direct 1-12 months 100/506 Pritchard et al.,1999

AC

Eulophia stenophylla 5 Direct 15 min 68/70 Pritchard, 1984

(Eulophia streptopetala)
11 Direct 15 min 63/58 Pritchard, 1984
Gymnadenia conopsea 5 Direct 15 min 45/46 Pritchard, 1984
5
Microtis media spp. Seeds Direct 3-24 months 30-67/5-63 Hay et al., 2010
media equilibrated
at 5, 13, 23,
50, 62, 75
and 92%
RH
Orchis coriophora 5 Direct 15 min 43/43 Pritchard, 1984
(Anacamptis coriophora)
Orchis morio 5 Direct 10 cycles (5 62/76 Pritchard, 1984
(Anacamptis morio) min each)
Paphiopedilum Not given Direct 1-12 months 37/236 Pritchard et al.,1999
rothschildianum

Phaius tankervilleae 32 Vitrif 12 months 68/61 Hirano et al.,2009

 ACCEPTED MANUSCRIPT

Platanthera bifolia Not given Direct 1 month 49/24 Nikishina et al., 2007
5
Pterostylis recurva Seeds Direct 3-24 months 70-85/0-90 Hay et al., 2010
equilibrated
at 5, 13, 23,
50, 62, 75
and 92%

PT
RH
Pterostylis sanguinea 8.2 Direct 1 week 49/50 Batty et al.,2001
Seeds Direct 3-24 months 85-90/25-755 Hay et al., 2010

RI
equilibrated
at 23 and
50% RH

SC
Pterostylis saxicola + 3.3 (WC of Enc-Deh 6 months 68/ca.57 Sommerville et al.,
fungal symbionts alginate 2008
beads)

NU
Satyrium nepalense 11 Direct 15 min 58/54 Pritchard, 1984
var.ciliatum
Satyrium nepalense 11 Direct 15 min 62/62 Pritchard, 1984
MA
var. nepalense

Thelymitra crinita 5.4 Direct 1 week 59/67 Batty et al.,2001

5
Seeds Direct 3-24 months 25-85/0-80 Hay et al., 2010
ED

equilibrated
at 5, 13, 23,
50, 62, 75
and 92%
PT

RH
Thelymitra macrophylla Seeds Direct 3-24 months 20/3-375 Hay et al., 2010
CE

equilibrated
at 23 and
50% RH
MATURE SEEDS – EPIPHYTIC ORCHIDS
AC

Angraecum Not given Direct 1 month 55/80 Nikishina et al.,2001

magdalenae
Cymbidium Not given Vitrif 30 min 92/76 Hirano et al., 2011
finlaysonianum
Cymbidium iridioides Not given Vitrif 30 min 85/81 Hirano et al., 2011
Cymbidium Not given Vitrif 30 min 70/73 Hirano et al., 2011
maguanense
Cymbidium tracyanum Not given Vitrif 30 min 89/69 Hirano et al., 2011
6
Dendrobium anosmum 5 Direct 1-12 months 99/25 Pritchard et al.,1999
Dendrobium candidum 12-19 Direct 1 day 98/90-95 Wang et al.,1998
(Dendrobium moniliforme)
8-18 Direct (?) -/92-95 Zhang et al., 1997
Dendrobium 28 Vitrif 89/80 He et al., 2010
chrysanthum
 ACCEPTED MANUSCRIPT

Dendrobium Not given Vitrif 1 day -/99 Thammasiri, 2008

chrysotoxum

Dendrobium cruentum Not given Vitrif 1 day -/32 Thammasiri, 2008

Dendrobium draconis Not given Vitrif 1 day -/95 Thammasiri, 2008

PT
Dendrobium Not given Vitrif 1 day -/62 Thammasiri, 2008
hercoglossum

RI
Dendrobium Swartz. 13 Vitrif 1h 80/79 Galdiano et al., 2012
hybrid “Dong Yai”

SC
12 Vitrif 1h 86/51-58 Galdiano et al., 2014

Dendrobium, four 11-19 Vitrif 2 weeks 96/64, 71/55 Vendrame et

NU
hybrids 96/60, 64/52 al.,2007

Encyclia cochleata 24 Direct 1 month 100/100 Nikishina et al.,2001

(Prosthechea cochleata)
MA
Miltonia flavescens × 8 Direct 1 month 88-100/100 Nikishina et al.,2001;
Brassia longissima Popova et al.,2003
Oncidium bifolium 19.2 (WC of Enc-Deh 1h 88/89 Flachsland et
ED

(Gomesa bifolia) alginate al.,2006

beads)
Oncidium flexuosum 11.3 Vitrif 30 min 91/78 Galdiano et al., 2013
PT

(Gomesa flexuosa)
Phalaenopsis equestris 11 Direct 15 min 84/79 Pritchard,1984
Rhynchostylis coelestis Not given Vitrif 1 day -/85 Thammasiri, 2008
CE

Trichopilia tortilis 6 Direct 1 month 83/90 Nikishina et al.,2001

Vanda coerulea 33 Vitrif 1 day 74/67 Thammasiri and
AC

Soamkul, 2007
Thammasiri, 2008
Vanda pumilla Not given Direct 15 min 94/97 Pritchard,1984

Vanda tricolor 40 Direct 1 day 11/1 Jitsopakul et al.,

2012
IMMATURE SEEDS
Bletilla striata 33-84 (age- Direct 30 min 99/33 Hirano et al.,2005a
dependent) Vitrif 30 min 99/79
PC-Vitrif 30 min 99/90
Bletilla striata Not given Vitrif 30 min -/58 Ishikiwa et al.,1997
cultured zygotic
embryos

Cyrtopodium Not given Enc-Deh 12 h Not given, Surenciski et

hatschbachii plant regrowth
mentioned al.,2007
 ACCEPTED MANUSCRIPT

Enc-Deh 12 h 50/64 Surenciski et

al.,2012

Ponerorchis Not given Vitrif 30 min 52/48 Hirano et al.,2005b

graminifolia var.
suzukiana

PT
Vanda tricolor 45 Direct 1 day 26/10 Jitsopakul et al.,
2012
Vitrif 1 day 26/14

RI
1
When synonyms are used in the cited papers, the accepted names of the species are
given in brackets.

SC
2
Seed WC: water content of seeds before cryopreservation, fresh weight basis (if reported)
3
Direct: Seeds were sealed in containers and plunged directly into liquid nitrogen; Vitrif:
Vitrification; Enc-Deh: encapsulation-dehydration; PC-Vitrif: preculture-vitrification; DL-

NU
Vitrif: droplet vitrification.
4
Germination of control/cryopreserved seeds. For seeds cryopreserved by direct
immersion in liquid nitrogen (with or without preliminary desiccation), control indicates
MA
germination of seeds before freezing. For seeds cryopreserved by using cryoprotectants
(for example, in a vitrification method), control indicates seeds without any treatment. For
simplicity, values are given in round numbers.
5
Germination was highly variable and affected by both pre-storage relative humidity (5 to
ED

92% RH) and storage duration without any particular trend identified. All data presented
were approximated from graphics.
6
For data obtained from Pritchard et al. (1999), germination of control seeds indicates
highest germination of fresh seeds before equilibration at 32% relative humidity and
PT

storage.
CE
AC
 ACCEPTED MANUSCRIPT

Table 3. Successful cryopreservation of orchid protocorms (PC) and protocorm-like-

bodies (PLBs) using various preculture conditions and cryopreservation technologies (for
all species, only optimal conditions for cryopreservation and maximum regrowth are
given).
Species1 Material Preculture Method2 Survival, % Ref.

PT
Brassia Rex hybrid PLBs 0.5 M suc (48 h) LS By TTC Shuhaida et
DMSO test3 al.,2009
Brassidium PLBs 0.8 M suc (24 h) Enc-Deh By TTC Yin et al.,2009

RI
‘Shooting Star' test2
Caladenia latifolia PC 0.8 M glycerol Vitrif Up to 100% Watanawikkit et

SC
by FDA test3 al., 2012

Cleisostoma PC 0.25 M suc (1 w) Enc-Deh 49 Maneerattanarun

NU
areitinum groj et al., 2007
then 0.75 M suc (2
days)
MA
Cymbidium PLBs 0.2 M suc + 0.3 M Vitrif 36-90 Kondo et al.,2001
“Suvaley Venus”
sorbitol (20 days)
Cymbidium Twilight PLBs 0.058 M sucrose Enc-Deh 92 Teixeira da Silva,
ED

Moon `Day Light` (12-36 h) 2013a

Vitrif 81

Cymbidium PC 0.7 M suc (20 h) Enc-Deh 72 Gogoi et al., 2013

PT

eburneum
CE

Cymbidium PC 0.7 M suc (20 h) Enc-Deh 70 Gogoi et al., 2013

hookerianum
AC

Dactylorhiza fuchsii PC No preculture Programm 0 Nikishina et

ed al.,2007
freezing
9.1
Vitrif
Dendrobium Bobby PLBs 0.6 M suc or 1.2 M Vitrif By TTC3 Anthony et
Mesinna sorbitol (24 h) al.,2010

PLBs 0.6 M suc (24 h) Vitrif By TTC3 Anthony et

al.,2011a
40 Anthony et
al.,2012
PLBs 0.2 M suc (24 h) Vitrif 40 Anthony et
al.,2013

PLBs 1.0 M suc (24 h) Enc-Deh By Evans Zainuddin et

blue assay3 al.,2011
 ACCEPTED MANUSCRIPT

PLBs 0.4 M suc (3 days) Enc-Deh By TTC2 Anthony et

al.,2011b
Dendrobium PC 0.5 mg/l ABA (3 w) Vitrif 88 Wang et al.,1998
candidum
(Dendrobium PLBs Not mentioned Desiccatio 80 Wang et al.,,1999
moniliforme) n to 30%
WC

PT
mentioned

PLBs 0.1 mg/l ABA (3 Air-Drying ~2804 Bian et al.,2002

RI
days)
PLBs 0.75 M sucrose (5 Enc-Vitrif 85 Yin and Hong,

SC
2009
days)
PLBs Not mentioned Vitrif 88 Chen et al., 2001

NU
(cited by Teixeira
da Silva et al.,
2014)
MA
Dendrobium PC 0.3 M suc (3 days) Enc-Deh 0 Pornchuti and
cariniferum Thammasiri,
2008
Enc-Vitrif 15 Thammasiri,
2008
ED

Dendrobium PLBs 0.3 M (2 days) Enc-Vitrif 60 Mohanty et al.,

chrysanthum 2013
Dendrobium PC Not mentioned Vitrif 33 Thammasiri,
PT

cruentum 2008
Enc-Deh 27
Dendrobium PC 1 mg/l ABA (1 w) Enc-Deh 67 Bunnag et al.,
CE

gratiosissimum 2009
Dendrobium nobile PC No preculture Vitrif 68 Vendrame and
Faria,2011
AC

PLBs 0.5 M suc (2 days) Enc-Deh 50 Mohanty et al.,

2012
0.5 M suc (2 days) Enc-Vitrif 76
Dendrobium ’Sonia- PLBs 0.5 M suc (24 h) Enc-Deh By TTC3 Subramaniam et
17’ al.,2011
Dendrobium Sonia- PLBs 0.25 M suc (24 h) Vitrif By TTC3 Tiau et al., 2009
28
PLBs 0.5 M suc (6 days) Enc-Vitrif By TTC3 Ching et al., 2012

PLBs 0.4 M suc (48 h) Vitrif 3 Poobathy et al.,

2013c
PLBs 0.4 M suc (48 h) Vitrif 16 Poobathy et al.,
2013b
PLBs 0.5 M suc (24 h) DL-Vitrif By TTC3 Poobathy et al.,
2013a
 ACCEPTED MANUSCRIPT

Dendrobium Swartz. PC 0.3 M suc (24 h) DL-Vitrif 17 Galdiano et al.,

hybrid “Dong Yai” 2012

Dendrobium PC 0.25 M suc (1 Enc-Deh 57 Maneerattanarun

virgineum w,4°C) then 0.75 M groj, 2009
suc (2 days)

PT
Dendrobium PC 0.8 M suc (6 days, Vitrif 20 Wu and Shen,
wardianum 2011

RI
4°C)

SC
PC Osmotic Vitrif 47 Liu et al., 2013
pretreatment and
cold acclimation

NU
Grammatophyllum PC 0.4 M suc (2 days) DL-Vitrif 38 Sopalun et
speciosum al.,2010
Enc-Deh 24
Enc-Vitrif 14
MA
Heterotaxis sessilis PC 0.4 M suc (24h) Enc-Vitrif 12 Ivannikov,2012

Miltonia flavescens PLBs No preculture Vitrif. 20 Popova et

×Brassia longissima al.,2010
ED

hybrid
Oncidium bifolium PC 0.15 M suc (24 h)  Enc-Deh 11 Flachsland et
0.25 M suc (48 h)  al.,2006
PT

(Gomesa bifolia)
0.5 M suc (24 h)
0.75 M suc (24 h)
CE

Oncidium hamana PLBs 0.438 M suc+0.109 Enc-Deh 30 Miao et al.,2005

”elfin” M gly (2 w)  0.3 M
suc O/N
AC

Paralophia PC 0.5 M suc Enc-Deh Not given Cripps and

epipyhtica McGregor,2006

Phalaenopsis bellina PLBs 0.75 M suc (3 days) Enc-Deh 30 Khoddamzadeh

et al.,2011
Rhynchostylis PC Not mentioned Vitrif 19 Thammasiri,
gigantea 2008
Seidenfadenia PC Not mentioned Vitrif 67 Thammasiri,
mitrata 2008
Vanda coerulea PC 0.7 M suc (20 h) Enc-Deh 40 Jitsopakul et
al.,2008
PLBs 0.5 M suc (1 day) DL-Vitrif 5 Jitsopakul et
al.,2011

Vanda PLBs 0.1 M suc (1 day) Vitrif By TTC3 Poobathy et al.,

Kaseem’s Deligh 2012
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When synonyms are used in the cited papers, the accepted names of the species are
given in brackets.
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LS: loading solution; Vitrif: Vitrification; Enc-Deh: encapsulation-dehydration; Enc-Vitrif:
encapsulation-vitrification; PC-Vitrif: preculture-vitrification; DL-Vitrif: droplet vitrification;
suc: sucrose; gly: glycerol; O/N: overnight; w: week
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Survival was measured only by TTC, Evans blue or FDA viability tests, recovery
percentage or conversion to plants was not given. TTC: 2,3,5-triphenyltetrazolium chloride

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assay; FDA: fluorescein diacetate assay.
4
Survival was expressed as the percentages of dry weight increase during the recovery

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period of 4 weeks.

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Table 4. Examples of successful cryopreservation of in vitro cultured orchid shoot

primordial, apices, rhizomes and cell suspensions (for all species, only optimal conditions
for cryopreservation and maximum regrowth are given).
Species Material Preculture Method1 Survival, Reference
%

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Cattleya loddigesii Shoot 0.1 or 1.0 mg/l PC-Desic Up to 100 Kondo et al.,2001
var. hassisoniana primordia ABA (7 days)

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Cattleya Shoot 0.1 or 1.0 mg/l PC-Desic Up to100 Kondo et al.,2001
walkeriana primordia ABA (7 days)

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Cymbidium cv. Apices 0.3 M suc O/N Vitrif 93 Thinh and
Suva Royal Velvet Takagi,2000
Cymbidium kanran Rhizomes 1.0 mg/l ABA PC-Desic 90 Popova and Kim

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(3 w) then 10% unpubl.
suc (1 w)
Cymbidium, 4 Apices 0.3 M suc O/N Vitrif 76-902 Thinh and
cultivars Takagi,2000
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Dendrobium Apices 0.3 M suc for 2 En-Deh 13 Lurswijidjarus
Walter Oumae days and Thammasiri,
2004
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Dendrobium cv. Shoot 0.1 or 1.0 mg/l PC-Desic Up to100 Kondo et al.,2001
“Yukidaruma” primordia ABA (7days)

Doritaenopsis cv. Cell 0.1 M suc+1.0 Vitrif 68 Tsukazaki et

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New Toyohashi suspension mg/l ABA (1 w) al.,2000

Vanda pumila Shoot 1.0 mg/l ABA for PC-Desic 65 Na and

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primordia 3 days Kondo,1996

Vanilla planifolia Apices 0.3 M suc for 1 DL-Vitrif 30 Gonzalez-Arnao

et al.,2009
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days

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Methods: Vitrif: Vitrification; PC-Desic: preculture-desiccation; DL-Vitrif: droplet
vitrification; suc: sucrose; En-Deh: encapsulation-dehydration; O/N: overnight; w: week
2
Depending on cultivar.
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Table 5. Representation of orchid subfamilies and tribes with regard to phylogeny* in the
cryopreservation studies

Subfamily Tribe Number of Number of

species commercial
genotypes/varieties

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Cryopreservation using seeds
Epidendroideae Arethuseae 2

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Collabieae 1 1
Cymbidieae 14 1

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Epidendreae 1
Podochileae 7 5
Vandeae 7

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Orchidoideae Diseae 3
Diurideae 12
Orchideae 13
Cypripedioideae Cypripedieae 1
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Cryopreservation using pollen
Epidendroideae Collabieae 1
Cymbidieae 1
Neottieae 2
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Podochileae 2 3
Orchidoideae Orchideae 5
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Cryopreservation using protocorms and PLBs

Epidendroideae Cymbidieae 5 6
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Maxillarieae 1
Podochileae 8 4
Vandeae 5
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Orchidoideae Orchideae 1
Diurideae 1 1

Cryopreservation using meristematic tissues

Epidendroideae Epidendreae 2
Cymbidieae 1 5
Podochileae 2
Vandeae 1
Vanilloideae Vanilleae 1
TOTAL: 4 13 tribes 94** 26**
subfamilies
* Based on recent phylogeny by Givnish et al., 2015 and open literature sources.
Species names were considered as they were given in the papers cited in this
review.
** Species cryopreserved by using different materials were counted only once (for
example, Dactylorhiza fuchsii was cryopreserved using seeds, pollen and
protocorms, yet it was counted as “1” for estimation of total number of
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cryopreserved species). Same approach was followed for estimating the total
number of cryopreserved commercial genotypes.

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Table 6. Different materials that can be used for cryobanking of orchid genetic resources.

Material Advantage Disadvantage Potential Preferred methods

application
Seeds (mature Allow conservation For many species: low Wild endangered Direct immersion in
and immature) of diverse germination both ex and rare species liquid nitrogen for dried
genotypes, small vitro and in vitro, seeds. Cryoprotectant-

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storage space specific requirements based methods (PVS2-
requirements due for nutrients and/or based vitrification) or
to minute size symbiosis with encapsulation-

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mycorrhizal fungi. dehydration) for seeds
Seed physiology and with high water
storage behavior are content.

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often unknown.

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Pollen Allow conservation Haploid genome, need Wild endangered Direct immersion in
of diverse a flower to pollinate to and rare species, liquid nitrogen
genotypes, small obtain seeds commercially
storage space important species
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requirements due
to minute size
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Shoot tips of in Allow conservation Labor-consuming. Commercially Vitrification, droplet-

vitro grown of the particular Shoot tips are difficult important varieties vitrification, preculture-
plants or genotype/variety to excise and to desiccation
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meristematic handle. Less effective

clumps for multiplication
compared to
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protocorms

Protocorms Very useful for Small and difficult to Commercially Vitrification, droplet-
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and PLBs mass propagation handle. Protocorms of important varieties, vitrification,

cultured in vitro of the particular many species are wild endangered encapsulation-based
genotype/variety intolerant to orchids that need to methods
desiccation and be propagated
freezing

Rhizomes Can be used for Limited research on Commercially Vitrification, droplet-

cultured in vitro rapid propagation cryopreservation important varieties vitrification, preculture-
of oriental of Asian orchids, desiccation
rhizomatous wild endangered
orchids, easy to orchids that need to
handle be propagated
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Figure captions

Figure 1. Orchids and orchid-derived products in human life. A: Food industry.

Commercial products of Salep and Vanilla powder; B: Ornamentals. Decorative flower

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arrangement using Phalaenopsis, the most popular potted orchids in USA (excibited at the
9th Asia Pacific Orchid Conference (APOC9), South Korea, 2009; photo by E. Popova); C:
Potted terrestrial Cymbidium orchid – an important plant in Asian horticultural market

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(Photo by E. Popova, 2009); D: Cultural traditions and art. Ink drawing of a terrestrial

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Cymbidium orchid by a contemporary Chinese artist (excibited at the 18th China Orchid
Fair, Wenzhou, China, 2008. Photo by E. Popova).

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Figure 2. Application of various cryopreservation methods to orchid germplasm
(according to the literature cited, see also Tables 1-4).
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Figure 3. Regrowth of orchid explants after cryopreservation in liquid nitrogen at -
196°C and plant development from cryopreserved seeds.
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A: formation of new protocorm-like bodies and shoot apices in Branotia hybrid PLBs after
cryopreservation via vitrification method using PVS2. Scale bar = 1 mm; B: regrowth of a
rhizome section of Cymbidium kanran following ABA preculture, air desiccation and
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cryopreservation; C, D: plantlets of Angraecum magdalenae (C) and Calanthe vestita (D)

developed in vitro from cryopreserved seeds; E: Calanthe vestita plants developed from
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control (left) and cryopreserved (right) seeds blooming in a greenhouse; F: Encyclia

cochleata plant developed from cryopreserved seed blooming in a greenhouse. Photos A,
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B, C and D by E. Popova and T. Nikishina. Photos E and F by G. Kolomeytseva, with

permission.

Figure 4. Proposed strategy and action for cryobanking orchid regmplasm.

Note: the database image is used as an example only and is not used in any existing
cryobanking facility.
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Fig. 1.

A B

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Fig. 2

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Fig. 3.

A B C D

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E F

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Fig. 4

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