Introduction

Orchid cultivation is one of the most economically significant global nursery industries, constituting a multi-billion dollar market (Teixeira da Silva 2013a). Dendrobium is a large orchid genus represented by over 1,100 species distributed from India and Sri Lanka then eastward to Japan and southward through the Philippines, Malaysia, Indonesia, and New Guinea to Australia and through the Pacific Islands to New Zealand (Pridgeon and Morrison 2006; Xu et al. 2006). The name is derived from ‘Dendron’ which means tree and ‘bios’ means ‘life’ that is an epiphytic plant that exists by clinging to the branches and trunks of host trees (Pradhan et al. 2013). This genus represents one of the most important orchids either as pot plants or as cut flowers. Several Dendrobium hybrids are very important to the orchid cut-flower industry in a number of countries. As a result, the production of Dendrobium hybrids plants has spiked, with large-scale production occurring in many countries like the Netherlands, Germany, China, Taiwan, Thailand, Philippines, Unites States, and Japan (Puchooa 2004). In general, orchids rank second in potted flowering plants in the United States, with a wholesale value of US$ 200 million for the year 2011 (US Department of Agriculture 2012). However, the USDA Floriculture report of 2013 indicated an 11 % increase in the value of potted orchids in 2012 from the previous year, while all other crops in this category had their values reduced (US Department of Agriculture 2013). Among the main orchid genera produced in the US, Dendrobium ranks second as potted orchids, behind Phalaenopsis. The State of Hawaii is the main producer of Dendrobium in the US with a total sales value of US$ 5.25 million for 2011 (U.S. Department of Agriculture 2011, 2012).

In addition to their ornamental value, some native Dendrobium species have medicinal (Hou et al. 2012; Ng et al. 2012) and ethnopharmacological importance in many Asian countries. Flower extracts of the epiphytic species D. nobile possessed antimicrobial and antitumor properties in addition to antiperoxidative activity (Devi et al. 2009), and extracted polysaccharide fractions from its stem could be considered as effective natural antitumor agents (Wang et al. 2010). Several members of the Dendrobium genus have alkaloids, amino acids, trace elements, while others contain active ingredients that can enhance immunity, promote the secretion of digestive juice, inhibit platelet aggregation, lower blood pressure or hypoglycemia, or display a range of pharmacological effects such as antioxidant, anti-aging, antipyretic and analgesic (Ng et al. 2012).

There are several in vitro preservation methods available for the preservation of Dendrobium germplasm. The choice of method, however, usually depends on the storage duration required. For short-term and medium-term storage, the aim is to reduce growth and to increase the intervals between subcultures. For long-term storage, cryopreservation is the only current method available to achieve this goal (Engelmann 2011). In vitro slow growth storage techniques are routinely used for medium-term conservation of numerous species and growth reduction is generally achieved by modifying the environmental conditions and the culture medium (Kulus and Zalewska 2014). Changes in environment conditions include a reduction in temperature, while modifications of the culture medium can include dilution of mineral elements, reduction of sugar concentration, changes in the nature and/or concentration of plant growth regulators (for example, the use of sub-lethal levels of growth retardants) and addition of osmotically active compounds (Negash et al. 2001).

Conservation of Dendrobium germplasm

The reduction in wild orchid stocks by orchid collectors, over-exploitation for medicinal purposes, deforestation and destruction of habitats for urbanization, and unauthorized trade have led to a reduction in natural populations of many orchids (Swarts and Dixon 2009) leading many orchid species to become extinct and a large number of species to become rare or endangered (Yam et al. 2010). The entire Orchidaceae is included in the CITES (Convention on International Trade in Endangered Species) Appendix II (Shefferson et al. 2005). There are a number of techniques devised to conserve orchids: ex vitro, such as botanic garden collections or ex situ germplasm banks (Hou et al. 2012; Merritt et al. 2014), as well as in vitro, including seed bank development, slow growth conservation and cryopreservation of seed, meristem, tissue-cultured shoot primordia, somatic embryos, and pollen (Table 1). In Dendrobium, immature seeds have not yet been used as an explant for cryopreservation, although they have been used for Bletilla striata (Hirano et al. 2005a), Ponerorchis graminifolia (Hirano et al. 2005b), Dactylorhiza fuchsii (Nikishina et al. 2007), and Cyrtopodium hatschbachii (Surenciski et al. 2007). Cryopreservation, which can ensure the safe and cost-efficient long-term conservation of Dendrobium germplasm, has a wide applicability for orchids both from temperate and tropical origin in which seeds, shoot meristems, protocorms, somatic embryos (i.e., protocorm-like bodies, or PLBs) and cultured cells can be preserved in liquid nitrogen (LN; −196 °C) to create cryobanks for conserving rare and endangered species (Hirano et al. 2006; Hossain et al. 2013). Until 2006, three cryopreservation techniques had been applied to orchid species, namely desiccation (air-drying), vitrification and encapsulation–dehydration (Hirano et al. 2006). However, in dehydration, since naked PLBs that are very sensitive to dehydration are first dehydrated, and since vitrification uses chemicals that can be toxic (Teixeira da Silva 2013b), this list was broadened to include five methods: air-drying, encapsulation–dehydration, encapsulation–vitrification, vitrification and droplet-vitrification. Encapsulation–dehydration method may be the most suitable as it results in a high survival frequency after cryogenic storage. Table 1 indicates that from a total of 37 documented studies in the literature, 12 used encapsulation–vitrification while 9 used encapsulation–dehydration, 7 used vitrification alone, 3 reported air-drying methods and low temperature, and a single study used in vitro methods. The encapsulation of explants in alginate beads for cryopreservation has some benefits as compared to the use of non-encapsulated samples. The alginate beads provide enhanced protection of dried materials from mechanical and oxidative stress during storage and ease of handling of small samples during pre- and post-cryopreservation.

Table 1 In vitro conservation applied to Dendrobium germplasm
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Many studies examined the cryopreservation of Dendrobium PLBs including D. ‘Walter Oumae’ (Lurswijidjarus and Thammasiri 2004) and D. candidum (Chen 2000; Chen et al. 2001; Bian et al. 2002; Lin et al. 2004; Yin and Hong 2009). Seeds of D. candidum (Wang et al. 1998, 1999), D. crystallinum and D. virgineum (Huehne and Bhinja 2012) were also successfully cryopreserved. Vendrame and Faria (2011) and Galdiano et al. (2012) showed that the cryopreservation of mature seeds was possible with vitrification, while droplet-vitrification was effective for protocorms, with phloroglucinol being an important aspect of the vitrification solution. Phloroglucinol has many biological effects in vitro and has shown to enhance a wide range of organogenic processes in plants (Teixeira da Silva et al. 2013). Orchid seeds are morphophysiologically different from other plants, to some extent limiting the application of cryopreservation and thus modification and proper adjustment of the techniques are required. Cryopreservation of vegetative tissue involves several stages, specifically the establishment of in vitro cultures, conditioning of these tissues, addition of an appropriate cryoprotectant, exposure of cultures to ultra-low temperature, re-warming and regeneration of plant cells and tissues (Kulus and Zalewska 2014). Each stage, which needs to be optimized, plays an important role in determining the survival of tissue upon re-warming.

Some orchid cryopreservation papers applied deep-freezing to seeds with a moisture content of 20 % or less (for vitrification protocols), while other studies dealt with direct freezing of orchid seeds. Seed moisture is a key factor because the presence of unbound water in seeds considerably reduces their germinability causing the embryo tissues to perish because of the formation of ice crystals in their cells during freezing in LN (Sakai et al. 1991; Sakai and Engelmann 2007). In the cryopreservation of terrestrial and epiphytic orchids with a moisture level below 11 %, seed germinability did not change after cryoconservation for most species examined (Pritchard 1984; Pritchard et al. 1999). In seeds of D. candidum, a high survival rate of 95 % was also obtained when desiccated seeds with 8–19 % water content with silica gel were directly plunged into LN (Wang et al. 1998). However, mature seeds of Dendrobium commercial hybrids with comparable seed moisture (9–18 %) did not survive after direct freezing (Vendrame et al. 2007; Galdiano et al. 2012, 2014), and the desirable dehydration with plant vitrification solution #2, PVS2 [30 % (w/v) glycerol, 15 % (w/v) ethylene glycol, 15 % (w/v) dimethyl sulfoxide (DMSO) and 0.4 M sucrose; Sakai et al. (1990)] for a period of time between 1–3 h prior to cryopreservation was essential to allow proper germination of cryopreserved seeds.

Conservation through storage should be supported by an appropriate explant viability test, since the success of any cryopreservant-based protocol is determined by the recovery of viable propagules. Viability tests such as the triphenyl tetrazolium chloride (TTC) reduction assay (Singh 1981) and the fluorescein diacetate (FDA) staining technique (Pritchard 1985) have been largely explored for seeds, protocorms and PLBs. The TTC assay, qualitative for large tissues and organs, is often used for orchid seeds and embryos because TTC reduction assay and regrowth observations used in the assessment of seedlings growth or plantlets survival were correlated (Lurswijidjarus and Thammasiri 2004; Vendrame et al. 2008; Tiau et al. 2009; Antony et al. 2010, 2011b; Subramaniam et al. 2011; Ching et al. 2012; Galdiano et al. 2012, 2014; Poobathy et al. 2013a). Dehydrogenases, through respiration in mitochondria, reduce colorless TTC to red triphenylformazan or reduced TTC (Verleysen et al. 2004). Figure 1 shows the assessment of survival using the TTC test in mature seeds and 45-day-old protocorms of Dendrobium hybrid ‘Uniwai Royale’ after cryopreservation by vitrification (unpublished data).

Fig. 1
figure 1

Stained seeds and protocorms of Dendrobium hybrid ‘Uniwai Royale’ with the 2,3,5-triphenyl tetrazolium chloride (TTC) reduction assay. Viable explants show insoluble scarlet formazan because the dehydrogenase enzyme in living tissues reduces soluble colorless TTC to insoluble scarlet formazan. a Mature seeds after cryopreservation by vitrification. b 45-days-old protocorms directly cryopreserved in liquid nitrogen (left Eppendorf tube) or PVS2-treated protocorms (right Eppendorf tube). ds dead seeds, ls living seeds, us unviable seed. Bars a: 1 mm, b: 1 cm. (Unpublished photos: Renato Fernandes Galdiano Jr.)

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Cryopreservation of seeds, pollen, protocorms and PLBs of many Dendrobium orchids has been successfully attempted for long-term conservation. Vitrification and air-drying methods have resulted in both low and slow rates of regrowth of plantlets of D. candidum (Bian et al. 2002). Therefore, the cryopreservation protocol widely used in the literature is encapsulation–vitrification, followed by encapsulation–dehydration (Table 1). The encapsulation–vitrification method has been developed for apices of numerous species from tropical origin and involves the incubation of explants in a Na-alginate solution (2–5 %) and their subsequent release (immersed in a drop of alginate) into a complexation agent (50–100 mM CaCl2 solution), where bead hardening occurs within 20–30 min. Encapsulated explants are then precultured in liquid medium with a high sucrose concentration and partially desiccated before exposure to LN (Sakai et al. 2008). In encapsulation–dehydration, encapsulation protects explants against extreme treatments such as high sucrose concentrations, which allow the removal of most freezable water from the cells and thereby allowing internal solutes to enter a vitrified state when plunged into LN and subsequently preventing the formation of lethal intracellular ice (Engelmann et al. 2008).

PLBs have been the main Dendrobium explant explored for the (cryo)preservation of Dendrobium germplasm (Table 1). They are clonal in nature (i.e., they are genetically identical), they have the potential to regenerate plants with an independent root and shoot system (hence their consideration as somatic embryos in the Orchidaceae), and represent thus a suitable and reliable source of material for germplasm conservation. PLBs behave as embryogenic cultures (similar to what was found in hybrid Cymbidium; Teixeira da Silva and Tanaka 2006) and are extremely susceptible to cryoinjuries due to the high water content within the cells (Renato Fernandes Galdiano Jr., personal observation). Consequently, additional steps such as extra preculture treatment (Antony et al. 2011b) or dehydration are required prior to preserving in LN (Yin and Hong 2009; Mohanty et al. 2012). During dehydration and freezing in LN, proteins and membranes are protected when soluble sugars such as sucrose accumulate in the cytoplasm (Sakai and Engelmann 2007; Yin and Hong 2009). Using protocorms as explants, vitrification and droplet-vitrification were explored by Vendrame and Faria (2011) and Galdiano et al. (2012) for the cryopreservation of Dendrobium germplasm. Droplet-vitrification provided limited effectiveness recovery and was influenced by sucrose pretreatment. For seeds, only vitrification has been used for an endangered species D. chrysanthum (He et al. 2010), as well as commercial hybrids D. ‘Sena Red’, D. ‘Mini WRL’, D. ‘Jaquelyn Thomas’, D. ‘BFC Pink’ and D. ‘Dong Yai’ (Vendrame et al. 2007; Galdiano et al. 2012, 2014).

In short-term conservation and cryopreservation, there may be some alterations to the genome, thus the determination of genetic integrity is necessary after (cryo)storage. Those processes can cause dehydration-, osmotic pressure- or freezing-related stresses that may result in genetic instability (Hazubska-Przbyl et al. 2010). Genetic instability and somaclonal variations may cause some differences in genotype and phenotype profiles of cryopreserved plants (Harding 2004). Some papers reported the use of randomly amplified polymorphic DNA (RAPD) for evaluating the genetic stability of Dendrobium PLBs after storage (Antony et al. 2012). Mohanty and Das (2013) had also reported one such protocol, but that study was subsequently retracted due to figure manipulation, casting doubts on the veracity, reliability or reproducibility of the results. RAPD results from selected primers indicated that the genetic stability of PLBs following storage (short-term and long-term) was maintained. However, molecular markers sometimes failed to detect somaclonal variations in ornamental plants, including in orchids (Park et al. 2009). Phenotypic characteristics of plantlets regenerated from D. candidum PLBs (Yin and Hong 2009) and of greenhouse-acclimatized seedlings associated with flow cytometry (FCM) for cryopreserved Dendrobium hybrid ‘Dong Yai’ seeds (Galdiano et al. 2014) were also used to assess genetic stability. In both studies, regenerated plantlets derived from cryopreserved PLBs or seeds were similar to controls (explants not cryopreserved). The discovery and development of new molecular markers to assess diversity are also fundamental for the study of genetic stability and to establish conservation programs for Dendrobium. Ding et al. (2008) analyzed D. officinale with sequence-related amplified polymorphism (SRAP) and obtained 96 polymorphic bands from a total of 109. Hou et al. (2012) developed 15 new and highly polymorphic microsatellite loci for D. officinale to evaluate the genetic diversity of wild and ex situ conserved populations.

There are no studies on the re-introduction of Dendrobium species back into the wild for in situ conservation, nor are there any studies that examine the storage of genetically modified material. These aspects need to be the focus of future projects.

Conclusions and future perspectives

Cryopreservation is a suitable means for preservation of Dendrobium germplasm. It requires minimal storage space and maintenance, while ensuring the stability of phenotypic or genotypic characteristics. Of the total of three cryopreservation techniques that have been applied to Dendrobium hybrids and species conservation and/or preservation, vitrification is the simplest technique because it does not require expensive equipment or laborious steps, and is thus particularly useful for Dendrobium seed and pollen. However, encapsulation–vitrification and encapsulation–dehydration are most likely the most suitable methods explored for this orchid genus because they resulted in a high survival frequency after cryogenic storage of PLBs, the major explant explored. Such techniques have been explored to a great extent and future perspectives may include optimization of existing cryopreservation protocols, as well as innovative approaches to cryopreservation.