Songklanakarin J. Sci. Technol. 40 (3), 682-691, May - Jun. 2018 Original Article Cryopreservation of Cymbidium finlaysonianum Lindl. by encapsulation-dehydration method Wiphusinee Worrachottiyanon1 and Sumontip Bunnag2* 1 Department of Biology, Faculty of Science, Khon Kaen University, Mueang, Khon Kaen, 40002 Thailand 2 Applied Taxonomic Research Center, Department of Biology, Faculty of Science, Khon Kaen University, Mueang, Khon Kaen, 40002 Thailand Received: 19 December 2016; Revised: 22 March 2017; Accepted: 27 March 2017 Abstract Thailand is home to a variety of wild orchids, most of which grow on trees in the forests. However, overexploitation of wild populations along with loss of habitat has accounted for the threatened status of wild orchids. Hence, conservation of wild orchids is urgently needed in the country. The specific purpose of this study was to establish a simple and reproducible protocol for the encapsulation-dehydration based cryopreservation of the orchid species Cymbidium finlaysonianum, which represents one of the threatened orchid species in Thailand. In this study, protocorms (2-3 mm in diameter) of the orchid were precultured for 24 h in liquid New Dogashima (ND) medium containing 0-1 M sucrose, encapsulated in 3% w/v Ca-alginate, pretreated for 24 h in liquid ND medium supplemented with 1.5 M sucrose and dehydrated for 0-12 h before plunging into liquid nitrogen for 48 h. The results showed that preculture of the protocorms with 0.75 M sucrose before encapsulation, followed by pretreatment with 1.5 M sucrose and dehydration for 12 h yielded the highest viability and regrowth (65.56%) of the protocorms determined following a 48 h cryopreservation process. Random amplified polymorphic DNA analysis employed for comparison of the polymorphic bands between plantlets raised from non-cryopreserved and cryopreserved protocorms revealed a similar index value of 0.998 indicating the genetic stability of the cryopreserved protocorms. Keywords: encapsulation-dehydration technique, protocorms, RAPD analysis, cryopreservation 1. Introduction cultivation, as well as cumulative environments. Among the potential threats, orchid smugglers have long been and are still Thailand is well-known as one of the leading the most serious one to epiphytic wild orchids, as evidenced growers and exporters of orchid cut-flowers and potted by the fact that Belgian authorities launched Operation Nero orchids, as well as home to numerous wild orchids. However, Wolfe in 1990, in which they seized thousands of smuggled with the increased demand for Thai native orchids, owing to orchids from Thailand. Moreover, the Department of Forestry their unique characteristics and fragrance, immense quantities of Thailand recently estimated that nearly six hundred thou- of wild orchids have been collected within the country every sand wild orchids are illegally exported each year, mainly to year leading to a rapid decline in their populations with a Japan and Europe (Orlean, 1998). Presently, the majority of tendency to be at risk of extinction. Apart from overexploita- Thai native orchids are recorded in the red list of species by tion of wild populations, Thai wild orchids have long been the Convention on International Trade in Endangered Species threatened by the extension of agricultural areas and shifting of Wild Fauna and Flora (CITES), indicating the threatened status of the orchids. Nowadays, both in-situ and ex-situ conservations *Corresponding author have been used to preserve many orchid species. In-situ con- Email address: [email protected] servation is defined as conservation of ecosystems and natural W. Worrachottiyanon & S. Bunnag / Songklanakarin J. Sci. Technol. 40 (3), 682-691, 2018 683 habitats, the maintenance of viable populations of the species vince, Thailand. The orchid capsules were surface-sterilized in within their natural surroundings, and, in the case of cultivated 70% (v/v) ethyl alcohol for 5 min, followed by soaking in species, in the surroundings where they have developed their 95% (v/v) ethyl alcohol. After flaming off the excess alcohol distinctive properties. Ex-situ conservation, on the other hand, the capsules were dissected, and the seeds were scraped out refers to a technique of conservation of biological diversity and cultured on solid modified New Dogashima (ND) medium outside its natural habitats, targeting all levels of biodiversity (Tokuhara & Mii, 1993). The medium contained 10% (w/v) such as genetics, species, and ecosystems by means of propa- potato extract, 1% (w/v) sucrose, and 1% (w/v) agar. The pH gation, micropropagation or in-vitro culture (Hirano et al., of the medium was adjusted to 5.4 before sterilization by 2005). Cryopreservation represents one of successful ex-situ autoclaving at 121 ºC for 20 min. The cultures were kept at 25 conservation methods with over 200 plant species conserved ± 2 ºC under a long photoperiod (16-h light/8-h dark cycle) by this method (Gámez-Pastrana et al., 2011). This techno- with a photon dose of 40 µmol/m2/s. The seeds successfully logy is based on the long-term storage of plant materials at developed into protocorms after 4 weeks of germination on low and ultra-low temperatures, usually in liquid nitrogen at - solid modified ND medium supplemented with 1.5 mg/L 196 °C. kinetin. The obtained protocorms were further cultured for 8 Many different cryopreservation methods have been weeks on the same medium with subculture periods at 4-week proposed for successful conservation of Thai orchids, includ- intervals, and healthy plantlets were achieved. For cryopre- ing vitrification (Thammasiri, 2000; Thammasiri & Soamkul, servation purposes, protocorms (size 2-3 mm in diameter) 2007), encapsulation-dehydration (Hongthongkham & Bun- were used. nag, 2014; Maneerattanarungroj et al., 2007) and encapsula- tion-vitrification (Thammasiri, 2008), with plenty of protocols 2.2 Cryopreservation experiments established for the genus Dendrobium (Bunnag et al., 2009; Pimda & Bunnag, 2010; Thammasiri, 2008). Unfortunately, 2.2.1 Preculture, encapsulation, and pretreatment only a few studies have been implemented to establish simple and reliable protocols for the genus Cymbidium. Establishing Two-month-old protocorms (size 2-3 mm in diame- a promising protocol for cryopreservation of the members of ter) were precultured in liquid modified ND medium supple- this genus is of great importance. The species Cymbidium mented with various sucrose concentrations (0, 0.25, 0.50, finlaysonianum is native to the central, north-eastern regions, 0.75, and 1 M) for 24 h. The precultured protocorms were south-eastern regions, and the peninsula of Thailand (Cribb & then immersed in 3% (w/v) Ca-alginate solution and synthetic Pedersen, 2014). It is currently arranged in Appendix II in the seeds were formed in 100 mM CaCl2 solution all of which Checklist of CITES species and is in particular on the verge of were prepared in liquid modified ND medium. After settle- extinction due to habitat destruction and indiscriminate ment for 30 min, alginate beads were washed three times with harvesting (Islam et al., 2015). Hence, cryopreservation of this sterile distilled water. The obtained beads were then pretreated orchid species is urgently needed. with 1.5 M sucrose solution prepared in liquid modified ND Among available cryopreservation techniques, en- medium for 24 h before the dehydration step. Dehydration of capsulation-dehydration has received much attention in recent the pretreated synthetic seeds was performed by means of air- years because of its simple, easy-to-handle and cost-effective drying in a laminar air-flow cabinet for 0, 3, 6, 9, and 12 h procedures which allow for high genetic stability of cryopre- prior to a 48-h cryopreservation process in liquid nitrogen. served materials. Moreover, this technique causes no toxicity Alginate beads not subjected to the preculture and pretreat- and is even applicable to dehydration-sensitive explants (Po- ment steps served as Control I, while those subjected to pre- pova et al., 2016; Zainuddin et al., 2011). More importantly, treatment with 1.5 M sucrose acted as Control II. For mea- this technology is suitable for cryopreservation of plant proto- surement of water content, the dehydrated beads from each corms. Therefore, cryopreservation of the C. finlaysonianum treatment were incubated at 60 ºC for 24 h and the water protocorms using encapsulation-dehydration method is of content was calculated based on the weight loss of the dried particular interest. beads using the equation: water content (in g H2O/g DW) = For this purpose, this study was carried out to (FW - DW)/DW, where FW and DW represent the fresh establish a simple and reproducible protocol for the encap- weight and dry weight of the beads, respectively. sulation-dehydration based cryopreservation of C. finlaysonia- num protocorms. In this context, sucrose concentrations in the 2.2.2 Freezing, thawing, and regrowth preculture step and the length of time required for dehydration of explants were optimized in order to yield high viability and Following the dehydration step, 15 dehydrated regrowth of the orchid protocorms following cryopreservation beads from each treatment were transferred to a 1.8 mL cryo- in liquid nitrogen. Random amplified poly-morphic DNA tube and the cryotubes were directly immersed into liquid (RAPD) analysis was also performed to detect the genetic nitrogen for 48 h. At a given time point, the cryotubes were stability of plantlets raised from cryopreserved protocorms. removed from the liquid nitrogen and rapidly thawed in a water bath at 40 ºC for 2 min. The cryopreserved protocorms 2. Materials and Methods were preliminarily analyzed for their viability by 2,3,5- triphenyltetrazolium chloride (TTC) staining or tetrazolium 2.1 Plant materials and culture conditions test. The 15 cryopreserved protocorms from each treatment were transferred to a tube. After addition of 1 ml of TTC Seven-month-old capsules of C. finlaysonianum solution, the tubes were incubated in the dark at 37 ºC for 24 were collected from Tham Pha Nam Thip Non-Hunting Area, h.