วารสารพืชศาสตร์สงขลานครินทร์ ปีที่ 6 ฉบับที่ 4 (ตุลาคม-ธันวาคม): 10-18, 2562 Songklanakarin Journal of Plant Science, Vol. 6, No. 4 (October-December): 10-18, 2019 Research article The Investigation of Condition for Cryopreservation of Snow-White Venus’s Slipper Orchid Protocorm [Paphiopedilum niveum (Rchb.f.) Stein] using V cryo-plate Method Soonthornkalump, S.1* Yamamoto, S.2 Nakkanong, K.3 and Meesawat, U.1 1 Department of Biology, Faculty of Science, Prince of Songkla University, Kho Hong, Hat Yai, Songkhla, Thailand, 90110 2 Genetic Resources Center, National Agriculture and Food Research Organization (NARO), 2-1-2 Kanondai, Tsukuba, Ibaraki, Japan, 305-8518 3 Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University, Kho Hong, Hat Yai, Songkhla, Thailand, 90110 * Corresponding author: [email protected] Received 26 April 2019; Revised 24 May 2019; Accepted 11 June 2019 Abstract Paphiopedilum niveum (Rchb.f.) Stein is native orchid to Southern Thailand which has been considered as an endangered species. The over collection decreased its wild population, so conservation of its genetic material is needed. The cryopreservation is efficient long term storage method. The objective of this study focused on the investigation of sucrose concentration at the preculture step and V cryo-plate protocol development to cryopreserved protocorm of P. niveum. In order to investigate the optimal sucrose concentration in the preculture medium , two-month-old protocorms were precultured in modified Vacin and Went medium (MVW) containing 0.058, 0.2, 0.4, 0.6 and 0.8 M sucrose for 24 h. The viability determination was tested by TTC assay and anatomical observation. The result presented that precultured protocorms in 0.2 M sucrose provided the highest survival at 96% without anatomical damage which was placed as the 1st preculture step. The highest survival percentage (60%) of non-cryopreserved protocorm was obtained from procedure followed by the 1st preculture in MVW containing 0.2 M sucrose (1 day) and 0.6 M sucrose (1 day), followed by osmoprotection using loading solution (LS) containing 1.2 M sucrose (30 min). Protocorms were dehydrated by PVS2 incubation for 60 min. However, there was no survival of cryopreserved protocorm after preserved in liquid nitrogen. Keywords: Anatomical observation, endangered species, genetic resources conservation, orchid Introduction Paphiopedilum niveum (Rchb.f.) Stein is the Although wild populations have been conserving in terrestrial orchid which distributed on the shading area the national park, the illegal poaching is still threating. of limestone of the Northern Peninsular Malaysia and Recently, the ex situ conservation could be conserved Andaman archipelago (Pedersen et al., 2011). The a number of P. niveum in botanic garden as living conservation status of P. niveum was evaluated to specimens. However, the conservation using endangered (EN) by the International Union for cryopreservation may useful in long term conservation Conservation of Nature (IUCN) because the continuous program. Cryopreservation is the technique for long- decrease of wild population from human intrusions term storage under extremely low temperature. and disturbance (Pedersen et al., 2011; Rankou, 2015). Generally, the cryopreservation method usually Online open access e-journal : www. natres.psu.ac.th/department/plantScience/sjps/default.htm Published by Department of Plant Science, Faculty of Natural Resources, Prince of Songkla University. All rights reserved. For Permissions, please e-mail: [email protected]. Soonthornkalump et al. (2019) consisted of five major steps which are preculture, of P. niveum protocorm is still under development. osmoprotection, dehydration, storage in liquid This study was carried out to develop the V cryo-plate nitrogen (LN), and regrowth. In cryopreservation, the protocol for cryopreserved P. niveum protocorm. actively meristematic tissues or cells are required such as, shoot tips and somatic embryo (Sakai and Materials and methods Engelmann, 2007). Samples are generally excised from Plant material preparation mother plant into the small size which allowed the Five-month-old P. niveum capsule was good penetration and absorption of cryoprotectant surface sterilized by dip into 70% ethanol then during the cryopreservation (Niino et al., 2017). flame for a few second to diminish hairs. Flamed Preculture step induces the endogenous capsule was longitudinal excised and seeds were cryoprotectant accumulation (Kaczmarczyk et al., transferred to sterile distilled water for 2 weeks. 2012). Loading treatment containing glycerol and Then, seeds were transferred to modified liquid sucrose increase the cellular osmolality which induces Vacin and Went medium (MVW) (Vacin and Went, tolerance to freezing dehydration (Reed, 2008). Plant 1949) containing full strength VW macronutrient and vitrification solution 2 (PVS2) is a high concentrated half strength MS (Murashige and Skoog, 1962) vitrification solution which is used in dehydration micronutrient supplemented with 5 mg/L chitosan, before rapid immersion in LN (Sakai and Engelmann, 2 g/L peptone and 2% (w/v) sucrose. The pH of 2007). medium is adjusted to 5.3 with 1 N NaOH. Medium Preculture is the first important step that was sterilized with autoclave at 121 °C for 20 min. needs to determine in cryopreservation protocol. In Seeds were cultured under dark condition on 120 addition, a physiological response during preculture rpm orbital agitator at 25±2 °C. Two-month-old step can be occurred. For instance, the increasing of protocorms (0.5-1 mm diameter) with shoot pole abscisic acid (ABA) trigger the proline accumulation in were used as plant materials in the determination plant cells which enhance the desiccation tolerance of appropriated condition of the 1st preculture and (Suzuki et al., 2006). Sucrose in preculture medium V cryo-plate protocol for cryopreservation act as osmolyte which enhances frost hardiness during investigation. cold acclimatization by increase cell membrane integrity (Pinker et al., 2009a). Moreover, the absorbed 1. Influence of the sucrose concentration in sucrose could be converted to the storage form of preculture: viability and histological observation carbohydrate which important in recovery growth Two-month-old protocorms were precultured (Pinker et al., 2009b). Thus, the optimization of in 50 ml liquid MVW medium supplemented with sucrose concentration is essential to determine the various sucrose concentrations (0.058 (control), 0.2, suitable preculture condition in this study. 0.4, 0.6 and 0.8 M) for 24 h under light condition at V cryo-plate method which is developed 25±2 °C on 100 rpm agitator. After incubation, from encapsulation vitrification and droplet vitrification viability of precultured protocorm was determined is the efficient cryopreservation method (Yamamoto et using triphenyltetrazolium chloride (TTC test) which al., 2011). This method can reduce the loss of plant was slightly modified from Verleysen et al. (2004). materials during the processing and is easy to carry on TTC solution containing 0.6% TTC with 0.05% Tween (Sekizawa et al., 2011). V cryo-plate was applied to 85 in a 0.05 M Na2HPO4/KH2PO4 buffer and the pH conserve many plant species such as Clinopodium was adjusted to pH 7.4. Four protocorms were odorum (Griseb.) Harley (Engelmann-Sylvestre and placed into a centrifuge tube containing 1.5 ml of Engelmann, 2015), Dianthus caryophyllus L. (Sekizawa TTC solution and incubated overnight at room et al., 2011) and Morus spp. (Yamamoto et al., 2011). temperature. The viable protocorms showing red However, an optimized protocol for cryopreservation stained color could be visualized under microscopic 11 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18 Soonthornkalump et al. (2019) observation. Three replicates, each with ten then dropped for gel hardening (15 min). After that, protocorms, were used in each treatment. Samples the cryo-plate with embedded protocorms were of precultured protocorms were fixed with FAA II transferred to osmoprotection step by immersed (formaldehyde: glacial acetic acid: 70% ethanol; into loading solution (LS) containing 2 M glycerol 5:5:90 v/v/v) for 48 h. Fixed protocorms were rinsed with sucrose (0.4, 0.8 and 1.2 M) for 30 min. These and stored with 70% ethanol. Fixed protocorms were protocorms were dehydrated in PVS2 containing dehydrated using tertiary-butyl-alcohol (TBA) series at 30% (w/v) glycerol, 15% (w/v) ethylene glycol, 15% 70%, 85%, 95% and 100%, 2 h for each (w/v) DMSO and 0.4 M sucrose (Sakai and concentration. After that, samples were immersed in Engelmann, 2007) at different exposure times (for the mixture of paraffin oil : paraffin wax, 1:1 (v/v) 30, 45 and 60 min). The cryo-plate was put into followed by soaked in pure paraffin wax (Histoplast) cryotube and then plunged in LN at least for 1 h. for infiltration (2 h for each step at 56 °C in the hot air oven) (Ruzin, 1999). A piece of sample was Rewarming and survival determination embedded in paraffin wax. Embedded specimens Cryopreserved protocorms were rewarmed were cut into 6 µm thickness with a rotary in 1 M sucrose solution at room temperature for 15 microtome (AO, 820 SPENCER). The section was min (Sekizawa et al., 2011). The post-rewarming affixed to glass slide and stained with Delafield’s protocorms were cultured in iron-free solid MVW hematoxylin and safranin staining to observe the medium containing 0.1 mg/L 1-Naphthaleneacetic histological alteration (Johansen, 1940). Periodic acid (NAA), 0.2% (w/v) polyvinylpyrrolidone (PVP-40) acid–Schiff (PAS) reaction was employed to examine and 0.2 % (w/v) activated charcoal (AC) under the the storage carbohydrate (Ruzin, 1999). The darkness at 25±2 °C for 7 days and then transferred preculture medium containing optimal sucrose to light condition. After 1 month of culture, survival concentration that provided the high survival percentage was determined using visual percentage with the less histological and observation. The viable lutescent and non-viable histochemical changes would be used in the further browning protocorms were determined as survival experiment.