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Cryopreservation of Cymbidium Finlaysonianum Lindl. by Encapsulation-Dehydration Method

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Cryopreservation of Cymbidium Finlaysonianum Lindl. by Encapsulation-Dehydration Method 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 Bueng Ngam Sub-district, Nong Phok District, Roi Et Pro- within their natural surroundings, and, in the case of cultivated vince, Thailand. The orchid capsules were surface-sterilized in species, in the surroundings where they have developed their 70% (v/v) ethyl alcohol for 5 min, followed by soaking in distinctive properties. Ex-situ conservation, on the other hand, 95% (v/v) ethyl alcohol. After flaming off the excess alcohol refers to a technique of conservation of biological diversity the capsules were dissected, and the seeds were scraped out outside its natural habitats, targeting all levels of biodiversity and cultured on solid modified New Dogashima (ND) medium such as genetics, species, and ecosystems by means of propa- (Tokuhara & Mii, 1993). The medium contained 10% (w/v) gation, micropropagation or in-vitro culture (Hirano et al., potato extract, 1% (w/v) sucrose, and 1% (w/v) agar. The pH 2005). Cryopreservation represents one of successful ex-situ of the medium was adjusted to 5.4 before sterilization by conservation methods with over 200 plant species conserved autoclaving at 121 ºC for 20 min. The cultures were kept at 25 by this method (Gámez-Pastrana et al., 2011). This techno- ± 2 ºC under a long photoperiod (16-h light/8-h dark cycle) logy is based on the long-term storage of plant materials at with a photon dose of 40 µmol/m2/s. The seeds successfully low and ultra-low temperatures, usually in liquid nitrogen at - developed into protocorms after 4 weeks of germination on 196 °C. solid modified ND medium supplemented with 1.5 mg/L Many different cryopreservation methods have been kinetin. The obtained protocorms were further cultured for 8 proposed for successful conservation of Thai orchids, includ- weeks on the same medium with subculture periods at 4-week ing vitrification (Thammasiri, 2000; Thammasiri & Soamkul, intervals, and healthy plantlets were achieved. For cryopre- 2007), encapsulation-dehydration (Hongthongkham & Bun- servation purposes, protocorms (size 2-3 mm in diameter) nag, 2014; Maneerattanarungroj et al., 2007) and encapsula- were used. tion-vitrification (Thammasiri, 2008), with plenty of protocols established for the genus Dendrobium (Bunnag et al., 2009; 2.2 Cryopreservation experiments Pimda & Bunnag, 2010; Thammasiri, 2008). Unfortunately, only a few studies have been implemented to establish simple and reliable protocols for the genus Cymbidium. Establishing 2.2.1 Preculture, encapsulation, and pretreatment a promising protocol for cryopreservation of the members of this genus is of great importance. The species Cymbidium Two-month-old protocorms (size 2-3 mm in diame- finlaysonianum is native to the central, north-eastern regions, ter) were precultured in liquid modified ND medium supple- south-eastern regions, and the peninsula of Thailand (Cribb & mented with various sucrose concentrations (0, 0.25, 0.50, Pedersen, 2014). It is currently arranged in Appendix II in the 0.75, and 1 M) for 24 h. The precultured protocorms were Checklist of CITES species and is in particular on the verge of then immersed in 3% (w/v) Ca-alginate solution and synthetic extinction due to habitat destruction and indiscriminate seeds were formed in 100 mM CaCl2 solution all of which harvesting (Islam et al., 2015). Hence, cryopreservation of this were prepared in liquid modified ND medium. After settle- orchid species is urgently needed. ment for 30 min, alginate beads were washed three times with Among available cryopreservation techniques, en- sterile distilled water. The obtained beads were then pretreated capsulation-dehydration has received much attention in recent with 1.5 M sucrose solution prepared in liquid modified ND years because of its simple, easy-to-handle and cost-effective medium for 24 h before the dehydration step. Dehydration of procedures which allow for high genetic stability of cryopre- the pretreated synthetic seeds was performed by means of air- served materials. Moreover, this technique causes no toxicity drying in a laminar air-flow cabinet for 0, 3, 6, 9, and 12 h and is even applicable to dehydration-sensitive explants (Po- prior to a 48-h cryopreservation process in liquid nitrogen. pova et al., 2016; Zainuddin et al., 2011). More importantly, Alginate beads not subjected to the preculture and pretreat- this technology is suitable for cryopreservation of plant proto- ment steps served as Control I, while those subjected to pre- corms. Therefore, cryopreservation of the C. finlaysonianum treatment with 1.5 M sucrose acted as Control II. For mea- protocorms using encapsulation-dehydration method is of surement of water content, the dehydrated beads from each particular interest. treatment were incubated at 60 ºC for 24 h and the water For this purpose, this study was carried out to content was calculated based on the weight loss of the dried establish a simple and reproducible protocol for the encap- beads using the equation: water content (in g H2O/g DW) = sulation-dehydration based cryopreservation of C. finlaysonia- (FW - DW)/DW, where FW and DW represent the fresh num protocorms. In this context, sucrose concentrations in the weight and dry weight of the beads, respectively. preculture step and the length of time required for dehydration of explants were optimized in order to yield high viability and 2.2.2 Freezing, thawing, and regrowth regrowth of the orchid protocorms following cryopreservation in liquid nitrogen. Random amplified poly-morphic DNA Following the dehydration step, 15 dehydrated (RAPD) analysis was also performed to detect the genetic beads from each treatment were transferred to a 1.8 mL cryo- stability of plantlets raised from cryopreserved protocorms. tube and the cryotubes were directly immersed into liquid nitrogen for 48 h. At a given time point, the cryotubes were 2. Materials and methods removed from the liquid nitrogen and rapidly thawed in a water bath at 40 ºC for 2 min. The cryopreserved protocorms 2.1 Plant materials and culture conditions were preliminarily analyzed for their viability by 2,3,5- triphenyltetrazolium chloride (TTC) staining or tetrazolium Seven-month-old capsules of C. finlaysonianum test. The 15 cryopreserved protocorms from each treatment were collected from Tham Pha Nam Thip Non-Hunting Area, were transferred to a tube. After addition of 1 ml of TTC 684 W. Worrachottiyanon & S.
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