Elsevier

Biotechnology Advances

Volume 34, Issue 4, July–August 2016, Pages 380-403
Biotechnology Advances

Research review paper
Frozen beauty: The cryobiotechnology of orchid diversity

https://doi.org/10.1016/j.biotechadv.2016.01.001Get rights and content

Abstract

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 their complex floral morphology, pollinator specificity and multiple ethnobotanical uses, including as food, flavourings, medicines, ornaments, and perfumes. These important traits have stimulated world-wide collection of orchid species, often for the commercial production of hybrids and leading to frequent overexploitation. Increasing human activities 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.

Introduction

With an estimated 880 genera and over 25,000 species, the Orchidaceae is the largest family of flowering plants (Givnish 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 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 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 tradition of orchid ink painting persists even today (Fig. 1).

Regarding their use in medicine, all parts of orchids have been utilized, including 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 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 Blume,2 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 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 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 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 (e.g. Liu and Mori, 1992, Zheng et al., 1998, Shih et al., 2002, Kim et al., 2003, Morita et al., 2005, Li et al., 2011). The presence of medicinally active chemicals such as polysaccharides and secondary metabolites including alkaloids, glycosides, phenolic compounds, and many others have been also documented in orchid tissues (reviewed by Gutiérrez, 2010, Ng et al., 2012).

Beyond medicine, orchid products are widely used the food and beverage industries. Specifically, vanillin extracted from the seed pods of Vanilla planifolia 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 & M.W. Chase; (Fig. 1; Bulpitt, 2005)].

However, in the modern world, orchids are known and grown primarily as 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 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 programmes 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.

Orchids are an important part of plant biodiversity on the planet due to their high 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, 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 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, 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 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 become extremely rare (Koopowitz and Kaye, 1983, Koopowitz, 1986, Dasgupta et al., 2004). Their high specificity for insect pollinators, minute seeds (often weighing μg) 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 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 result of an increased use of weed killers and artificial fertilizers, deforestation, and land clearance, has imperilled 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 of Wild Fauna and Flora (CITES) (n.d.), 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 needed. Traditionally, botanic gardens have taken the lead role in orchid conservation, by developing and maintaining well-documented living 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 phylogenetic research (Koopowitz, 1986, Farrell and Fitzgerald, 1989, Tasker, 1989, Koopowitz and Thornhill, 1994, Kolomeitseva et al., 2011, Swarts and Dixon, 2009).

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). 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 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 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) 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 be relatively ineffective (Pritchard and Seaton, 1993, Merritt et al., 2014). For example, seeds of Phaius tankervilleae (Banks) Blume dehydrated to 5% water content (WC) 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, 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, 2012a, Dowling and Jusaitis, 2012b).

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 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 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 of orchids have been widely used for both conservation and commercial purposes (Dasgupta et al., 2004, Teixeira da Silva, 2013b). These micropropagation techniques 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), and Thai orchid exports in 2011 totalled €154 million (Lippe, 2012).

Mature seeds of some orchid species require several months of cold stratification 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 such species, immature seeds that germinate readily after sowing are considered a primary material for the initiation of tissue cultures (Hirano et al., 2005a, Hirano et al., 2005b, Nikishina et al., 2007). Based on an understanding of dry seed storage of other species, such immature seeds may not, however, be optimal for long-term storage.

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 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, Republic of 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 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, phenotypical and genetic variations in orchid materials in the course of repeated subcultures are well-documented (Tokuhara and Mii, 1998, Arditti, 2008, Khoddamzadeh 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 cryopreservation, which allows safe and long-term storage of orchid germplasm once an appropriate protocol is designed and validated.

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 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 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, 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) 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, Pritchard, 2007). It can also provide safe and cost-effective long-term conservation of large quantities of genetic material in small facilities, with 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 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 Benson, 2008, Wang et al., 2012, Wang et al., 2014). On a large-scale, cryopreservation is now being applied to back-up field collections of many crops; for example, 2291 accessions of apple at the USDA cryobank at Fort Collins, USA (Towill et al., 2004; Volk, personal communication). Also, the whole national collection of Alluim spp. (> 1150 accessions) is 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), 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 et al., 2011).

In orchids, cryopreservation has been an efficient means of conserving seeds and 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., 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 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 analyse 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.

Section snippets

Cryopreservation of pollen

Orchid pollen occurs as monads to tetrads and multiples of tetrads, included in aggregated 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 al., 2011). However, pollinia in opened flowers of four orchids

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 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,

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 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 PLB formation, the cells show cytological characteristics and have

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

Cryopreservation of orchid cell suspensions and isolated protoplasts

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

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

Cryopreservation of transgenic orchid materials

Despite a large diversity of successful protocols developed for orchid transformation (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 following a vitrification protocol (Teixeira da Silva, 2013a). Explants could be recovered and they produced new PLBs after one year of cryogenic

Systematic 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

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

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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      Citation Excerpt :

      Over the last decade, numerous studies have focused on the use of tissue culture and cryogenic methods for ex situ conservation of rare and threatened species, especially those growing in Australia, and on the implementation of successful protocols in conservation programs for the recovery of Australian biodiversity (Bunn et al., 2011; Kaczmarczyk et al., 2011, 2013; Menon et al., 2014; Funnekotter et al., 2015, 2017; Bustam et al., 2016; Whiteley et al., 2016; Broadhurst and Coates, 2017; Streczynski et al., 2019). There is also visible progress in research on this group of plants in other countries, although to a much smaller extent (Barnicoat et al., 2011; Mikuła et al., 2011a; Kiran et al., 2012; Makowski et al., 2016; Popova et al., 2016; Kodym et al., 2018; Choi et al., 2019; Popova and Kim, 2019; Edesi et al., 2020; Gladfelter et al., 2020; Seydi et al., 2020; Sharma et al., 2020). A broader approach to the conservation of threatened species makes their recovery worldwide possible.

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    Both authors contributed equally to this work.

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