วารสารพืชศาสตร์สงขลานครินทร์ ปีที่ 6 ฉบับที่ 4 (ตุลาคม-ธันวาคม): 10-18, 2562 Songklanakarin Journal of 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 [ 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. and dead, respectively. Three replicates, each with ten protocorms, were used in this experiment. 2. The development of V cryo-plate protocol According to the result of trial study, the Statistical analysis application of precondition step followed by two- All experiments were organized using a step preculture was used. Because this procedure completely randomized design (CRD). The mean could provide more vigorous explants than that of values were subjected to analysis of variance single step of preculture (data not shown). Two- (ANOVA) and separated using DMRT at P ≤ 0.05. A month-old protocorms were preconditioned on three-way ANOVA was used to decide the solid MVW containing 0.1 M sucrose for 7 days interaction effect between two or three variables under 16/8 h photoperiod at 25±2 °C. After during cryopreservation step. The statistical test was precondition, two-step preculture was employed. analyzed by SPSS software 17.0 The preconditioned protocorms were firstly precultured (1st preculture) with MVW containing 0.2 Results and Discussion M sucrose (1 day) followed by the 2nd preculture on 1. Influence of the sucrose concentration in MVW supplemented with different concentrations preculture: viability and histological observation of sucrose (0.4 and 0.6 M) for 1 day. They were The protocorm viability test, as stained by embedded onto the aluminium cryo-plate using 3% TTC, exhibited red stained protocorms (indicated to Na-alginate solution and 1 M CaCl2 solution was as viable) whereas non-viable protocorms remained

12 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18 Soonthornkalump et al. (2019) white (Figure 1). There was no significant difference However, preculture with a high concentration of of survival percentage between protocorms sucrose (0.4-0.8 M) seemed to reduce the preculturing on MVW supplemented with 0.2 M protocorm survival (68.61±8.61% to 21.11±2.22%) sucrose (95.96±3.21%) and the control (100±0.00%). (Figure 2).

Figure 1 The TTC assay of Paphiopedilum niveum (Rchb.f.) Stein exhibiting (A) the red stained protocorm (as referred to viable) with shoot pole (arrow) and (B) non-viable protocorm with unstained color

Figure 2 Viability determination by TCC assay of Paphiopedilum niveum (Rchb.f.) Stein protocorm after preculture in various concentrations of sucrose Means±SE with different letters are significantly different at P ≤ 0.05

Histological observation demonstrated that sucrose (control). The increasing sucrose the normal histological feature of protocorm concentration exhibited the shrinkage of cell consisted of 2 parts; upper and lower (Figure.3A). membrane. The severe plasmolysis was found in The upper part contained the small cells with large 0.8 M sucrose precultured protocorm (Figure 3C). nuclei and dense stained cytoplasm indicating the These characters demonstrated a response of plant meristematic cells. While, the large parenchyma cells suffering under stress from excessive cells containing storage carbohydrate were hypertonic condition which lead to plasma observed at the lower part of protocorm (Figure 3B). membrane damage from large osmotic gradient These good features were noticed in protocorms (Waterhouse et al., 2006). In the previous study, the preculturing both in 0.2 M sucrose and 0.058 M damaged of different cellular components such as 13 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18 Soonthornkalump et al. (2019) plasmolysis, nuclear shrinkage, rupture of the cell occurring in the freezing and thawing step wall were found in cryopreserved PLBs of (Poobathy et al., 2012). Since the damaged cellular Brassidium Shooting Star without osmoprotection component was undesired, the 0.2 M sucrose was and dehydration with vitrification solution (Ahmad selected as the suitable sucrose concentration Mubbarakh et al., 2013). The cryopreserved cells of because this condition did not damage anatomical Vanda Kaseem’s Delight PLBs displayed a ruptured features and storage carbohydrate. The preculture cell wall and many cells lost their cytoplasmic medium containing 0.2 M sucrose was chosen to be contents, most likely as a result of ice crystallization the 1st preculture in the further experiment.

Figure 3 Histological characteristics (A-C) and histochemical observation by PAS reaction (D-F) of Paphiopedilum niveum (Rchb.f.) Stein protocorm. (A) The intact protocorm showing the meristematic tissues at the shoot pole (black arrow) or upper part and the parenchymatous tissues at the lower part. (B) The protocorm precultured on MVW supplemented with 0.2 M sucrose exhibiting the similar histological features to the control. (C) The precultured protocorm on MVW supplemented with high sucrose concentration presenting plasmolyzed cells indicated by the detachment plasma membrane (red arrow head). (D) The PAS reaction showing purple stained granules of storage carbohydrate (sc) and blue stained nuclei (nu) (red arrow head) in the intact, (E) 0.2 M precultured protocorm and (F) 0.6 and 0.8 M sucrose precultured protocorm

2. The development of V cryo-plate protocol The highest survival percentage of non- step (Suzuki et al., 2006). However, P. niveum cryopreserved protocorm at 60% was obtained protocorms did not allow survivor after exposure to from treatment no. 18 (Table 1). The stepwise LN. From visual observation, morphological increased of sucrose concentration in two-step characters of survived non-cryopreserved preculture gave healthier protocorm compared with protocorm showed the light green color at the the single sucrose preculture (data not shown). The shoot pole (Figure 4A). Meanwhile, cryopreserved previous study explained the role of two-step protocorm exhibiting the dead protocorm with pale preculture that the supplementation of 0.1 M white color (Figure. 4B). However, P. niveum is sucrose in medium could provide a transient known as slow grower which may need longer increased ABA level and trigger the accumulation of period in the physiological adaptation than the proline which increased further in the 2nd preculture other plant species. The result of statistical analysis

14 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18 Soonthornkalump et al. (2019) demonstrated that crucial steps affecting the osmoprotection, dehydration and the interaction survivability of protocorm were preculture, between preculture and osmoprotection (Table 2).

Table 1 Survival percentage of non-cryopreserved and cryopreserved Paphiopedilum niveum (Rchb.f.) Stein protocorm after culture on regrowth medium for 1 month Treatment Preculture Sucrose conc. PVS2 Survival (%) Precondition No. 1st 2nd in LS (30 min) (min) -LN +LNns 1 30 26.67±6.67bcd 0.00±0.00a 2 0.4 M 45 20.00±0.00cd 0.00±0.00a 3 60 26.67±0.00bcd 0.00±0.00a 4 0.4 M 30 13.33±0.00cd 0.00±0.00a 5 sucrose 0.8 M 45 0.00±0.00d 0.00±0.00a 6 (1 day) 60 15.00±0.00cd 0.00±0.00a 7 30 28.33±6.01bcd 0.00±0.00a 8 1.2 M 45 0.00±0.00d 0.00±0.00a 0.1 M 0.2 M 9 60 6.67±6.67cd 0.00±0.00a sucrose sucrose 10 30 33.33±6.67bc 0.00±0.00a (7 days) (1 day) 11 0.4 M 45 6.67±6.67cd 0.00±0.00a 12 60 13.33±6.67cd 0.00±0.00a 13 0.6 M 30 26.67±6.67bcd 0.00±0.00a 14 sucrose 0.8 M 45 26.67±6.67bcd 0.00±0.00a 15 (1 day) 60 20.00±11.55cd 0.00±0.00a 16 30 53.33±13.3ab 0.00±0.00a 17 1.2 M 45 53.33±17.64ab 0.00±0.00a 18 60 60.00±11.55a 0.00±0.00a LS: loading solution; PVS2: Plant vitrification solution 2; -LN: non-cryopreserved; +LN: cryopreserved Data shown are mean±standard error (S.E.). Comparison of the mean values was analyzed using the DMRT at P ≤ 0.05 ns: no significant

Table 2 Three-way ANOVA with interaction between all combinations of three main factors (preculture; Pre, osmoprotection; Osm and dehydration; Deh) exhibiting in the cryopreservation process of Paphiopedilum niveum (Rchb.f.) Stein protocorm using V cryo-plate method Factors df MS F P Preculture (Pre) 1 4090.74 19.636 * Osmoprotection (Osm) 2 1354.17 6.5 * Dehydration with PVS2 (Deh) 2 704.17 3.38 * Interaction Pre X Osm 2 2894.91 13.896 * Interaction Pre X Deh 2 78.24 0.376 ns Interaction Osm X Deh 4 43.75 0.21 ns Interaction Pre X Osm X Deh 4 351.16 1.686 ns Error 36 Total 54

DF: Degree of freedom; MS: Mean square; F: F-ratio * indicating significant difference at P ≤ 0.05, ns: non-significant difference

15 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18 Soonthornkalump et al. (2019)

Figure 4 Morphological characters of Paphiopedilum niveum (Rchb.f.) Stein. (A) The survive non- cryopreserved protocorm showing light green shoot tip (arrow) and (B) the pale white color presented in non-survived of cryopreserved protocorm

This early developed procedure could not 0.2 M sucrose precultured protocorm showed high provide the suitable condition to the plant cell survival percentage with undamaged cellular after exposure to LN due to the mortality of post- component and storage carbohydrate which were cryopreserved protocorm. This event may explain necessary for regrowth. This sucrose concentration by the toxic effects from high concentration of could be applied for the early developed PVS2. DMSO contained in PVS2 can enhance cell cryopreservation protocol in the 1st preculture step. membrane permeability (Chen et al., 2015). The The early developed V cryo-plate method for P. rapid penetration into the cell of PVS2 increased niveum protocorm cryopreservation gave the best excessive osmotic stress which damaged to plasma result in survival percentage of non-cryopreserved membrane (Sakai and Engelmann, 2007). The protocorms but the cryopreserved protocorm failed damaged plasma membrane resulting in ion leakage to survive. The further studies will be required for might activate the program cell death (Pegg, 2010). better understanding of cryoprotectant actions In order to avoid the toxicity of PVS2, dehydration- which can compromise toxicity from dehydration based method may be recommended in this case. and cell viability. Moreover, the dehydration-based Droplet-dehydration, encapsulation dehydration cryopreservation may give the better result in and D cryo-plate method which employed the survival percentage. dehydration by air in laminar air-flow cabinet may give the better result. These method has been Acknowledgements introduced to some orchid cryopreservation such as This research was financially supported by PLBs of Brassidium Shooting Star (Yin et al., 2011), the Graduate school of Prince of Songkla University protocorm of Arundina graminifolia (Cordova II and for research and Graduate Studies Scholarship Thammasiri, 2016) and pollinia of Dendrobium (95000201), including the Scholarship for Support signatum (Jitsopakul et al., 2019). However, the Exchange Students and International Credit intensive investigation of dehydration-based Transferred through ASEAN Community. cryopreservation on Paphiopedilum species is needed because there is never been reported. References Chen, C.Y., Song, J., Pas, J., Meijer, L.H.H. and Han, S. Conclusion 2015. DMSO induces dehydration near lipid This current study reported the effects of membrane surfaces. Biophysical Journal 109(2): sucrose preculture on histological and histochemical 330-339. changes of precultured protocorm of P. niveum. The

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18 ว. พืชศาสตร์สงขลานครินทร์ 6 (4): 10-18 Songklanakarin J. Pl. Sci., 6 (4): 10-18