Cryopreservation of protocorm-like bodies of Vanda lilacina Teijsm. & Binn., a Thai orchid species, by V-cryo-plate and D-cryo-plate methods



Protocorm-like bodies (PLBs) of Vanda lilacina Teijsm. & Binn. were used to study for new cryopreservation techniques, V-cryo-plate and D-cryo-plate methods, on survival score and percentage of survival. For V-cryo-plate and D-cryo-plate methods, PLBs were dehydrated with PVS2 solution for 0, 20, 40, 60 and 120 min and silica gel for 0, 0.5, 1, 1.5, 2, 2.5 and 3 h, respectively. The results showed that for V-cryo-plate method, the suitable treatment for cryopreserved PLBs of V. lilacina was dehydrated with PVS2 solution for 20 min. It gave survival score at 0.31, higher than control (+LN; 0 min), and gave percentage of survival at 33.33%. Moreover, PLBs showed survival as active green PLBs, but grew and developed slower than control (-LN; 0 min). For D-cryo-plate method, the results showed that the suitable treatment for cryopreserved PLBs of V. lilacina was dehydrated with silica gel for 1 h. It gave the highest survival score at 0.78 and percentage of survival was up to 83.78%. Moreover, PLBs showed the same growth and development as control (-LN; 0 min). The suitable cryopreservation method of V. lilacina PLBs is D-cryo-plate method using silica gel for 1 h in dehydration step.


Plant cryopreservation, Cryo-plate, Dehydration, Vitrification, PVS2, Orchidaceae


DJ Mabberley. Mabberley's Plant-Book: A portable dictionary of plants, their classification and uses, vii-xviii. Third edition, Cambridge University Press, 2008, p. 1-1021.

K Thammasiri. Thai orchid genetic resources and their improvement. Horticulturae. 2016; 2(3), 9, DOI: 10.3390/horticulturae2030009.

UNEP-WCMC. Checklist of CITES species. CITES Secretariat, Geneva, Switzerland, and UNEP-WCMC, Cambridge, United Kingdom, 2014.

W Nanakorn and S watthana. Queen Sirikit Botanic Garden (Thai Native Orchids 2). Wanida Press, Chiang Mai, 2008, 328pp.

F Engelmann. Importance of desiccation for the cryopreservation of recalcitrant seed and vegetatively propagated species. Plant Genetic Resources Newsletter. 1997, 112, 9-18.

ERJ Keller and A Senula. Cryopreservation of plant Germplasm. In: Davey MR and Anthony P (eds.). Plant Cell Culture: Essential Methods. John Wiley & Sons, Ltd, Chichester, UK., 2010. doi: 10.1002/9780470686522.ch8

A Sakai, S Kobayashi and I Oiyama. Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb. var Brasiliensis Tanaka) by a simple freezing method. Plant Sci. 1991, 74(3), 243-248.

A Uragami, A Sakai, M Nagai and T Takahashi. Survival of cultured cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification. Plant Cell Rep. 1989, 8(5), 418-421.

A Uragami, A Sakai and M Nagai. Cryopreservation of dried axillary buds from plantlets of Asparagus officinalis L. grown in vitro. Plant Cell Rep. 1990, 9(4), 328-331.

T Matsumoto, A Sakai, C Takahashi and K Yamada. Cryopreservation of in vitro grown apical meristems of wasabi (Wasabia japonica) by encapsulation-vitrification method. Cryoletters. 1995, 16(2), 189-196.

A Schafer-Menuhr, HM Schumacher and G Mix-Wagner. Long-term storage of old potato varieties by cryopreservation of shoot-tips in liquid nitrogen. Plant Genetic Resources Newsletter. 1997, 111(1), 19-24.

J Fabre and J Dereuddre. Encapsulation Dehydration – A new approach to cryopreservation of Solanum shoot-tips. Cryoletters. 1990, 11(5), 413-426.

K Thammasiri. Cryopreservation of seeds of a Thai orchid (Doritis pulcherima Lindl.) by vitrification. Cryoletters. 2000, 21, 237-244.

P Mohanty, MC Das, S Kumaria and P Tandon. High-efficency cryopreserva-tion of the medicinal orchid Dendrobium nobile Lindl. Plant Cell Tiss Org. 2012, 109, 297–305.

AA Khoddamzadeh, UR Sinniah, P Lynch, MA Kadir, SB Kadzimin and M Mahmood. Cryopreservation of protocorm-like bodies (PLBs) of Phalaenopsis bellina (Rchb. f.) Christenson by encapsulation-dehydration. Plant Cell Tiss Org. 2011, 107, 471–481. DOI 10.1007/s11240-011-9997-4

N Hai-Yan and K Kondo. Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation. Plant Sci. 1996, 118(2), 195-201.

K Thammasiri and L Soamkul. Cryopreservation of Vanda coerulea Griff. ex Lindl. Seeds by Vitrification. ScienceAsia. 2007, 33, 223-227.

N Jitsopakul, K Thammasiri and K Ishikawa. Cryopreservation of Vanda coerulea protocorms by encapsulation dehydration. Cryoletters. 2008, 29(3), 253-260.

S Yamamoto, T Rafique, WS Priyantha, K Fukui, T Matsumoto and T Niino. Development of a cryopreservation procedure using aluminum cryo-plates. Cryoletters. 2011, 32(3), 256-265.

T Matsumoto. Cryopreservation of plant genetic resources: conventional and new methods. Reviews in Agricultural Science. 2017, 5, 13-20. Doi: 10.7831/ras5.13

T Niino, S Yamamoto, K Fukui, CRC Martínez, MV Arizaga, T Matsumoto and F Engelmann. Dehydration improves cryopreservation of mat rush (Juncus decipiens Nakai) basal stem buds on cryo-plate. Cryoletters. 2013, 34, 549-560.

VN Patil and M Dadlani. Tetrazolium test for seed viability and vigor, Handbook of Seed Testing. 2009, p. 209–241.

M Milosevic, M Vujakovic and D Karagic. Vigor tests as indicators of seed viability. Genetika. 2010, 42(1), 103-118.

N Jitsopakul, K Thammasiri and K Ishikawa. Cryopreservation of Vanda coerulea protocorm-like bodies by droplet-vitrification. In: 1st International Symposium: Cryopreservation in Horticultural Species, Book of Abstract. ISHS, Leuven, 2009, p. 92.

K Sopalun, K Thammasiri and K Ishikawa. Vitrification-based cryopreservation of Grammatophyllum speciosum protocorm. Cryoletters. 2010, 31(4), 347–357.

MT Gonzalez-Arnao, CE Lazaro-Vallejo, F Engelmann, R Gamez-Pastrana, YM Martinez-Ocampo, MC Pastelin-Solano and C Diaz-Ramos. Multiplication and cryopreservation of vanilla (Vanilla planifolia ‘Andrews’). In Vitro Cell Dev–Pl. 2009, 45, 574–582.

M Yin and S Hong. Cryopreservation of Dendrobium candidum Wall. ex Lindl. protocorm-like bodies by encapsulation-vitryfication. Plant Cell Tiss Org. 2009, 98, 179–185.

P Valentovic, M Luxova, L Kolarovic and O Gasparıkova. Effect of osmotic stress on compatible solutes content, membrane stability and water relations in two maize cultivars. Plant Soil Environ. 2006, 52, 186–191.

HW Bian, JH Wang, WQ Lin, N Han and MY Zhu. Accumulation of soluble sugars, heat-stable proteins and dehydrins in cryopreservation of protocorm-like bodies of Dendrobium candidum by the air-drying method. J Plant Physiol. 2002, 159(10), 1139–1145.

E Maruyama, K Ishii and I Kinoshita. Alginate encapsulation technique and cryogenic procedure for long term storage of the tropical forest tree Guazuma crinite Mart. In Vitro cultures. Jpn Agr Res Q. 1998, 32, 301–309.

MLC Vieira. Conservação de germoplasma in vitro [In vitro germplasm conservation]. Biotecnologia cienc. desenvolv. 2000, 14, 18-20.

LA Withers and JT Williams. Conservação in vitro de recursos de plantas (In vitro conservation of plant resources). In: Torres AC; Caldas LS; Buso JA (Eds.). Cultura de Tecidos e Transformação Genética de Plantas (Culture of Tissues and Genetic Transformation of Plants). Brasília: Embrapa-SPI. 1998, 1, 297-330.

LB Cordova II and K. Thammasiri. Cryopreservation on a cryo-plate of Arundina graminifolia protocorms, dehydrated with silica gel and drying beads. Cryoletters. 2016, 37(2), 68-76.

YX Zhang, JH Wang, H Bian and MY Zhu. Pregrowth-desiccation: a simple and efficient procedure for the cryopreservation of rice (Oryza sativa L.) embryogenic suspension cells. Cryoletters. 2001, 22, 221–228.

T Niino, Wunna, K Watanabe, N Nohara, T Rafique, S Yamamoto, K Fukui, MV Arizaga, CRC Martinez, T Matsumoto and F Engelmann. Cryopreservation of mat rush lateral buds by air dehydration using aluminum cryo-plate. Plant Biotechnol. 2014, 31, 281–287.


  • There are currently no refbacks.

Online ISSN: 2228-835X

Last updated: 13 February 2019